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## **Meet the editor**

Dr. Chai received his Ph.D. from the City University of New York in 1998 and completed his post-doctoral training at Harvard University in 2001. Currently, he is a Principal Investigator of the U.S. Department of Veterans Affairs and a faculty member of the Department of Medicine at the University of California in Irvine. He directs the Laboratory of GI Injury and Cancer at the VA

Long Beach Healthcare System in California and also serves as the IACUC Chairman at this facility. Dr. Chai has published over 30 research articles and 7 book chapters in diverse areas including protozoology, arachnology, cardiology and gastroenterology. His current research focuses on acid reflux induced esophageal cancer. He holds professional membership with AGA, AHA, and ASBMB, has been an editorial board member of 6 journals and a reviewer for multiple journals and grants.

Contents

**Preface VII**

**Angiogenesis 1**

Jianyuan Chai

**Pericytes 73**

Ohsaki

Treshalina

**to Treat Cancer 175**

**Angiogenesis 47**

Chapter 1 **Transcriptional Modulation of Tumour Induced**

Jeroen Overman and Mathias François

Chapter 3 **Manipulating Redox Signaling to Block Tumor**

**Tumor Microenvironment 89**

Chapter 2 **Roles of SRF in Endothelial Cells During Hypoxia 29**

Vera Mugoni and Massimo Mattia Santoro

Chapter 4 **Accessory Cells in Tumor Angiogenesis — Tumor-Associated**

Chapter 5 **Endothelial and Accessory Cell Interactions in Neuroblastoma**

**Mesenchymal Stromal Cell Recruitment, but Inhibits Angiogenesis in a Mouse Melanoma Model 143**

A. A. Poliakov, I. N. Mikhaylova, N. V. Andronova and H. M.

Chapter 7 **The Use of Artemisinin Compounds as Angiogenesis Inhibitors**

Chapter 6 **T-Cadherin Stimulates Melanoma Cell Proliferation and**

Qigui Li, Peter Weina and Mark Hickman

Yoshinori Minami, Takaaki Sasaki, Jun-ichi Kawabe and Yoshinobu

Jill Gershan, Andrew Chan, Magdalena Chrzanowska-Wodnicka, Bryon Johnson, Qing Robert Miao and Ramani Ramchandran

K. A. Rubina, E. I. Yurlova, V. Yu. Sysoeva, E. V. Semina, N. I. Kalinina,

## Contents

#### **Preface XI**


Chapter 7 **The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer 175** Qigui Li, Peter Weina and Mark Hickman

#### Chapter 8 **3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of Postnatal Neovasculogenesis 261**

Mani T. Valarmathi, Stefanie V. Biechler and John W. Fuseler

Preface

invade other organs.

As a process of extension of the vascular network within human body, angiogenesis plays a fundamental role to support cell survival, because all cells need oxygen and nutrients to operate and blood circulation is the only way to provide them. In human adults, angiogenesis mainly takes place in two conditions, wound healing and tumor progression. During wound healing, angiogenesis supports new tissue growth to repair the wound; therefore, it is beneficial to the body and should be promoted. In tumor progression, on the other hand, angiogenesis is hi‐ jacked to serve the mutated cells for their multiplication and therefore, it should be inhibited. This book focuses on the second situation – angiogenesis in tumor progression. However, since the molecular and cellular interactions under both conditions are essentially identical, the con‐

The book includes eight chapters written by highly experienced scholars from several na‐ tions. The first chapter, **"Transcriptional modulation of tumor induced angiogenesis"**, by Overman & Francois *(University of Queensland, Australia)*, gives a comprehensive introduc‐ tion on how angiogenesis at the molecular and cellular levels is initiated and regulated dur‐ ing tumorigenesis as comparing to a normal biological system. Despite the similarity in the molecules involved in both conditions, including transcription factors, angiogenic factors, and cell proliferation/migration factors, the key difference is the balance among these mole‐ cules. In a normal biological system, angiogenesis is highly organized in a spatial-and-tem‐ poral manner. In tumors, however, the uncontrollably replicating cancer cells create an extremely hypoxic environment, which induces a persistent production of angiogenic fac‐ tors that allow angiogenesis to go on and on. As a consequence, the vasculature generated during tumorigenesis is leaky and immature because it never has the time or molecular/ cellular mass to become completed. In a way this makes metastasis easier, because the can‐ cer cells can effortlessly enter into the circulatory system through the porous vessel wall and

The imbalance of angiogenic factors during tumorigenesis starts with the disproportional acti‐ vation of transcription factors, which are reviewed in the second chapter, **"Role of serum re‐ sponse factor in endothelial cells during hypoxia"**, by Chai *(University of California, USA)*. The best known transcription factors in tumor angiogenesis are hypoxia-inducible factor (HIF) and p53. They both can be activated by oxygen shortage. While HIF activates angiogenic factors like vascular endothelial growth factor (VEGF) to promote tumor cell survival, p53 is doomed to kill the cells through up-regulation of apoptotic factors like BAX, which is why p53 is often found mutated in the vast majority of tumor cells. Although these two transcription factors appear to be the enemies to each other, sometimes they also shake hands under the table. For instance, HIF has been reported in several occasions to help p53 to induce cell death under severe hypoxia. I guess, if you can't beat them, it won't be a bad idea to join them. In addition to the commonly

tent of the book is suitable for all the readers who are interested in angiogenesis.

## Preface

Chapter 8 **3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of Postnatal**

Mani T. Valarmathi, Stefanie V. Biechler and John W. Fuseler

**Neovasculogenesis 261**

**VI** Contents

As a process of extension of the vascular network within human body, angiogenesis plays a fundamental role to support cell survival, because all cells need oxygen and nutrients to operate and blood circulation is the only way to provide them. In human adults, angiogenesis mainly takes place in two conditions, wound healing and tumor progression. During wound healing, angiogenesis supports new tissue growth to repair the wound; therefore, it is beneficial to the body and should be promoted. In tumor progression, on the other hand, angiogenesis is hi‐ jacked to serve the mutated cells for their multiplication and therefore, it should be inhibited. This book focuses on the second situation – angiogenesis in tumor progression. However, since the molecular and cellular interactions under both conditions are essentially identical, the con‐ tent of the book is suitable for all the readers who are interested in angiogenesis.

The book includes eight chapters written by highly experienced scholars from several na‐ tions. The first chapter, **"Transcriptional modulation of tumor induced angiogenesis"**, by Overman & Francois *(University of Queensland, Australia)*, gives a comprehensive introduc‐ tion on how angiogenesis at the molecular and cellular levels is initiated and regulated dur‐ ing tumorigenesis as comparing to a normal biological system. Despite the similarity in the molecules involved in both conditions, including transcription factors, angiogenic factors, and cell proliferation/migration factors, the key difference is the balance among these mole‐ cules. In a normal biological system, angiogenesis is highly organized in a spatial-and-tem‐ poral manner. In tumors, however, the uncontrollably replicating cancer cells create an extremely hypoxic environment, which induces a persistent production of angiogenic fac‐ tors that allow angiogenesis to go on and on. As a consequence, the vasculature generated during tumorigenesis is leaky and immature because it never has the time or molecular/ cellular mass to become completed. In a way this makes metastasis easier, because the can‐ cer cells can effortlessly enter into the circulatory system through the porous vessel wall and invade other organs.

The imbalance of angiogenic factors during tumorigenesis starts with the disproportional acti‐ vation of transcription factors, which are reviewed in the second chapter, **"Role of serum re‐ sponse factor in endothelial cells during hypoxia"**, by Chai *(University of California, USA)*. The best known transcription factors in tumor angiogenesis are hypoxia-inducible factor (HIF) and p53. They both can be activated by oxygen shortage. While HIF activates angiogenic factors like vascular endothelial growth factor (VEGF) to promote tumor cell survival, p53 is doomed to kill the cells through up-regulation of apoptotic factors like BAX, which is why p53 is often found mutated in the vast majority of tumor cells. Although these two transcription factors appear to be the enemies to each other, sometimes they also shake hands under the table. For instance, HIF has been reported in several occasions to help p53 to induce cell death under severe hypoxia. I guess, if you can't beat them, it won't be a bad idea to join them. In addition to the commonly

known transcription factors involved in angiogenesis, this chapter also brings a new member into the light, i.e., Serum Response Factor (SRF). This is a much more powerful regulator than either HIF or p53, and some even call it the master regulator. SRF directly controls nearly 1% of the known human genes, and through these gene derivatives SRF may have influence on a quarter of the entire human genome. This chapter presents convincing data to show that SRF regulates hypoxia-induced angiogenesis through multi-levels and therefore could be an excel‐ lent target for cancer gene therapy.

herin in melanoma angiogenesis. T-cadherin is a membrane-associated protein and its real function remains largely unknown. While its up-regulation has been associated with high grade astrocytomas, in the majority of cancers including melanoma, T-cadherin is down-regu‐ lated or completely lost. Overexpression of T-cadherin in endothelial cells correlates with a migratory phenotype, which usually suggests a positive role in angiogenesis. However, this study found in a melanoma model that the number of microvessels is reduced when T-cadher‐

Using natural products to treat chronic diseases is always the top choice in cancer therapy, because they are cheap and less toxic compared to the synthetic drugs. In the next chapter, **"The use of artemisinin compounds as angiogenesis inhibitors to treat cancer"**, Li et al *(Walter Reed Army Institute of Research, USA)* introduce such a compound, artemisinin, an extract from the plant sagewort. Artemisinin is the first line treatment recommended by WHO for malaria. However, an increasing amount of data indicates an anti-cancer effect, particularly against tumor angiogenesis. Li et al give a thorough review on artemisinin and its derivatives in cancer and non-cancer context, and provide valuable perspectives for the

The final chapter of the book, **"3-D microvascular tissue constructs for exploring concur‐ rent temporal and spatial regulation of postnatal neovasculogenesis"**, by Valarmathi et al *(University of South Carolina, USA)*, demonstrates a marvelous research technique to study neovasculogenesis in vitro, the three-dimensional collagen scaffold. Depending on the cul‐ ture medium provided, bone marrow stromal cells can differentiate into either endothelial cells or smooth muscle cells in front of your eyes and form tube-like network within the scaffold, mimicking the vasculature formation in vivo. Although the study is on neovasculo‐ genesis, meaning generating microvessels from stem cells, the technique can be easily ap‐ plied to angiogenesis studies using differentiated endothelial cells. The beautiful images generated from confocal immunostaining, transmission and scanning electron microscope

**Jianyuan Chai, Ph.D.**

University of California Long Beach, California

USA

Preface IX

Laboratory of GI Injury and Cancer VA Long Beach Healthcare System and

in is expressed, supporting an argument that T-cadherin might inhibit angiogenesis.

future research direction.

provide a perfect end for this book.

The activation of HIF not only initiates VEGF production, the best known angiogenic stimu‐ lator, but also directs the gene transcription of two other molecules, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), both responsible for the gener‐ ation of nitric oxide (NO-), one of the key reactive oxygen species (ROS) in the body. The next chapter, **"Manipulating REDOX signaling to block tumor angiogenesis"**, by Mugoni & Santoro *(University of Torino, Italy)*, summarizes all the known ROS and dissects how they influence tumor angiogenesis. The level of ROS in the tumor microenvironment can be a determining factor for the fate of a tumor. A moderate amount of these free radicals can help to maintain normal blood pressure, protect endothelial cell integrity, and support angiogen‐ esis, while high level of ROS can cause endothelial cell death and thereby stop tumor angio‐ genesis. Therefore, manipulation of ROS level could be an alternative approach to control tumor progression.

Although angiogenesis is performed by endothelial cells, other cells also contribute to the process. In Chapter 4, **"Accessory cells in tumor angiogenesis"**, Minami et al *(Asahikawa Medical University, Japan)* introduce a major helper of endothelial cells during angiogenesis, the pericytes. Endothelial cells form the inner lining of the blood vessels, while pericytes wrap around the endothelial cells from the outside and provide molecular and cellular sup‐ port to stabilize the newly formed microvasculature. Although pericytes are usually absent in tumor vasculature due to the accelerating angiogenic activities, this chapter provides sev‐ eral strategies to increase the local population of pericytes to counteract the tumor angiogen‐ esis, which may be advanced to promising therapeutic approaches in the near future.

In the following chapter, **"Endothelial and accessory cell interactions in neuroblastoma tu‐ mor microenvironment"**, Gershan et al *(Medical College of Wisconsin, USA)* present a special case of tumor biology – neuroblastoma, and give a thorough review on its development, molecular and cellular interactions, and therapeutic strategies. Of particular interest is the point that "tumors are wounds that never heal", which precisely reflects the truth about tu‐ mors. From molecular and cellular point of view, these two events are almost identical. Mol‐ ecules up-regulated during wound healing are often found elevated in a tumor microenvironment. Wound healing requires cell proliferation, migration and differentiation, and so does tumor progression. Angiogenesis provides fundamental support for wound healing as well as for tumor growth. The only difference, as the team points out, is that wound healing is a highly orchestrated event in which the activations of cells and molecules are regulated spatially and temporally. Once the wound is healed, all of these molecular and cellular activities return to their normal physiological levels. Tumors, on the other hand, sustain the high molecular and cellular activities eternally, which is like an open wound.

In the next chapter, **"T-cadherin stimulates melanoma cell proliferation and mesenchymal stromal cell recruitment, but inhibits angiogenesis in a mouse melanoma model"**, Rubina et al *(M.V. Lomonosov Moscow State University, Russia)*present original data on the role of T-cad‐

herin in melanoma angiogenesis. T-cadherin is a membrane-associated protein and its real function remains largely unknown. While its up-regulation has been associated with high grade astrocytomas, in the majority of cancers including melanoma, T-cadherin is down-regu‐ lated or completely lost. Overexpression of T-cadherin in endothelial cells correlates with a migratory phenotype, which usually suggests a positive role in angiogenesis. However, this study found in a melanoma model that the number of microvessels is reduced when T-cadher‐ in is expressed, supporting an argument that T-cadherin might inhibit angiogenesis.

known transcription factors involved in angiogenesis, this chapter also brings a new member into the light, i.e., Serum Response Factor (SRF). This is a much more powerful regulator than either HIF or p53, and some even call it the master regulator. SRF directly controls nearly 1% of the known human genes, and through these gene derivatives SRF may have influence on a quarter of the entire human genome. This chapter presents convincing data to show that SRF regulates hypoxia-induced angiogenesis through multi-levels and therefore could be an excel‐

The activation of HIF not only initiates VEGF production, the best known angiogenic stimu‐ lator, but also directs the gene transcription of two other molecules, endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), both responsible for the gener‐ ation of nitric oxide (NO-), one of the key reactive oxygen species (ROS) in the body. The next chapter, **"Manipulating REDOX signaling to block tumor angiogenesis"**, by Mugoni & Santoro *(University of Torino, Italy)*, summarizes all the known ROS and dissects how they influence tumor angiogenesis. The level of ROS in the tumor microenvironment can be a determining factor for the fate of a tumor. A moderate amount of these free radicals can help to maintain normal blood pressure, protect endothelial cell integrity, and support angiogen‐ esis, while high level of ROS can cause endothelial cell death and thereby stop tumor angio‐ genesis. Therefore, manipulation of ROS level could be an alternative approach to control

Although angiogenesis is performed by endothelial cells, other cells also contribute to the process. In Chapter 4, **"Accessory cells in tumor angiogenesis"**, Minami et al *(Asahikawa Medical University, Japan)* introduce a major helper of endothelial cells during angiogenesis, the pericytes. Endothelial cells form the inner lining of the blood vessels, while pericytes wrap around the endothelial cells from the outside and provide molecular and cellular sup‐ port to stabilize the newly formed microvasculature. Although pericytes are usually absent in tumor vasculature due to the accelerating angiogenic activities, this chapter provides sev‐ eral strategies to increase the local population of pericytes to counteract the tumor angiogen‐

esis, which may be advanced to promising therapeutic approaches in the near future.

In the following chapter, **"Endothelial and accessory cell interactions in neuroblastoma tu‐ mor microenvironment"**, Gershan et al *(Medical College of Wisconsin, USA)* present a special case of tumor biology – neuroblastoma, and give a thorough review on its development, molecular and cellular interactions, and therapeutic strategies. Of particular interest is the point that "tumors are wounds that never heal", which precisely reflects the truth about tu‐ mors. From molecular and cellular point of view, these two events are almost identical. Mol‐ ecules up-regulated during wound healing are often found elevated in a tumor microenvironment. Wound healing requires cell proliferation, migration and differentiation, and so does tumor progression. Angiogenesis provides fundamental support for wound healing as well as for tumor growth. The only difference, as the team points out, is that wound healing is a highly orchestrated event in which the activations of cells and molecules are regulated spatially and temporally. Once the wound is healed, all of these molecular and cellular activities return to their normal physiological levels. Tumors, on the other hand, sustain the high molecular and cellular activities eternally, which is like an open wound. In the next chapter, **"T-cadherin stimulates melanoma cell proliferation and mesenchymal stromal cell recruitment, but inhibits angiogenesis in a mouse melanoma model"**, Rubina et al *(M.V. Lomonosov Moscow State University, Russia)*present original data on the role of T-cad‐

lent target for cancer gene therapy.

tumor progression.

VIII Preface

Using natural products to treat chronic diseases is always the top choice in cancer therapy, because they are cheap and less toxic compared to the synthetic drugs. In the next chapter, **"The use of artemisinin compounds as angiogenesis inhibitors to treat cancer"**, Li et al *(Walter Reed Army Institute of Research, USA)* introduce such a compound, artemisinin, an extract from the plant sagewort. Artemisinin is the first line treatment recommended by WHO for malaria. However, an increasing amount of data indicates an anti-cancer effect, particularly against tumor angiogenesis. Li et al give a thorough review on artemisinin and its derivatives in cancer and non-cancer context, and provide valuable perspectives for the future research direction.

The final chapter of the book, **"3-D microvascular tissue constructs for exploring concur‐ rent temporal and spatial regulation of postnatal neovasculogenesis"**, by Valarmathi et al *(University of South Carolina, USA)*, demonstrates a marvelous research technique to study neovasculogenesis in vitro, the three-dimensional collagen scaffold. Depending on the cul‐ ture medium provided, bone marrow stromal cells can differentiate into either endothelial cells or smooth muscle cells in front of your eyes and form tube-like network within the scaffold, mimicking the vasculature formation in vivo. Although the study is on neovasculo‐ genesis, meaning generating microvessels from stem cells, the technique can be easily ap‐ plied to angiogenesis studies using differentiated endothelial cells. The beautiful images generated from confocal immunostaining, transmission and scanning electron microscope provide a perfect end for this book.

> **Jianyuan Chai, Ph.D.** Laboratory of GI Injury and Cancer VA Long Beach Healthcare System and University of California Long Beach, California USA

**Chapter 1**

**Transcriptional Modulation of**

**Tumour Induced Angiogenesis**

Jeroen Overman and Mathias François

http://dx.doi.org/10.5772/54055

**1. Introduction**

sel growth.

Additional information is available at the end of the chapter

**2. Blood vessel development in the embryo**

tional blood- and lymphatic vascular system.

This chapter provides a summary of the current literature addressing key processes and transcriptional regulators of endothelial cell fate during embryonic blood vascular and lymphatic vascular development, and discusses the implications of these processes/regu‐ lators during tumour vascularization. First, we will address normal embryonic develop‐ ment of the vascular systems at the molecular and cellular level. With these fundamental processes recognized, the second part the chapter will focus on how these regulators face dysregulation during tumorigenesis and how they consequently facilitate abnormal ves‐

During embryogenesis, the development of the vasculature occurs prior to the onset of blood circulation, and is initiated by *de novo* formation of endothelial cells (EC) from meso‐ derm derived precursor cells. In a succession of morphogenic events, intricate transcription‐ al programs orchestrate the further differentiation, proliferation and migration of blood endothelial cells (BECs) to establish the vascular systems (fig. 1). This includes assembly of individual ECs into linear structures and the formation of lumen to facilitate the flow of blood; the designation of arterial, venous, capillary and later lymphatic endothelial cell iden‐ tity; and the remodelling, coalescence and maturation of the primary vascular plexus to form large heterogeneous interlaced structures, that warrants a contiguous and fully func‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Overman and François; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Chapter 1**

## **Transcriptional Modulation of Tumour Induced Angiogenesis**

Jeroen Overman and Mathias François

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54055

#### **1. Introduction**

This chapter provides a summary of the current literature addressing key processes and transcriptional regulators of endothelial cell fate during embryonic blood vascular and lymphatic vascular development, and discusses the implications of these processes/regu‐ lators during tumour vascularization. First, we will address normal embryonic develop‐ ment of the vascular systems at the molecular and cellular level. With these fundamental processes recognized, the second part the chapter will focus on how these regulators face dysregulation during tumorigenesis and how they consequently facilitate abnormal ves‐ sel growth.

#### **2. Blood vessel development in the embryo**

During embryogenesis, the development of the vasculature occurs prior to the onset of blood circulation, and is initiated by *de novo* formation of endothelial cells (EC) from meso‐ derm derived precursor cells. In a succession of morphogenic events, intricate transcription‐ al programs orchestrate the further differentiation, proliferation and migration of blood endothelial cells (BECs) to establish the vascular systems (fig. 1). This includes assembly of individual ECs into linear structures and the formation of lumen to facilitate the flow of blood; the designation of arterial, venous, capillary and later lymphatic endothelial cell iden‐ tity; and the remodelling, coalescence and maturation of the primary vascular plexus to form large heterogeneous interlaced structures, that warrants a contiguous and fully func‐ tional blood- and lymphatic vascular system.

© 2013 Overman and François; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **2.1. Embryonic blood vessel morphogenesis**

#### *2.1.1. Endothelial specification and initial blood vessel formation*

*De novo* generation of the first EC precursors in mammals occurs in the extra-embryonic meso‐ derm. The mesoderm is a hotbed for cell specification in the embryo, and the pluripotent hae‐ mangioblast ancestor of EC precursors (angioblasts) also gives rise to haematopoietic lineages and ostensibly even smooth muscle cells (SMC)[1-5]. In addition, ECs have been shown to share a common precursor with mesenchymal stem/stromal cells (MSC), the so-called mesen‐ chymoangioblast[6], and it has been suggested that other precursors can propagate endothelial cell lineages in the yolk sac. Together these observations signify the differentiation potential of these precursor cells, and impending consequences for plasticity during later remodelling and pathologies[7-9]. During vasculogenesis, defined as *de novo* generation of embryonic blood vessels, these pluripotent mesodermal progenitor cells acquire an endothelial cell (EC) precur‐ sor- or blood cell (BC) precursor- phenotype, and subsequently co-localize and aggregate in the mesoderm to form blood islands[10-12], with the EC precursors flattened around the edges and the BC precursors in the centre to generate the haematopoietic lineages[11-13].

#### *2.1.2. Blood vascular lumen formation*

To initiate the formation of actual vessel-like structures, the angioblasts assemble into arteri‐ al and venous cords, and in doing so form the primitive vascular plexus. These nascent rope-like threads have a solid core and are consequently not yet able to facilitate the flow of blood. This functional feature requires the heart of the cord to be tunnelled out, to give way to a central continuous lumen along the length of the nascent vessel. The transition of EC cords into vascular tubes is a process that necessitates defined EC-polarity, and a delicate interplay between adhesion and contractility. Polarity is essential for the distribution of membrane junction proteins and the definition of apical/luminal (inside) and basal/ablumi‐ nal (outside) surfaces. This is harmonized by the interplay between adhesion and contractili‐ ty, through the regulating of physical force propensity that accounts for the EC-flattening against the extracellular matrix[14-16].

Two principal cellular mechanisms have been described to explain for the formation of *de novo* blood vascular lumen: cord hollowing and cell hollowing[13, 16, 17]. Both mechanisms rely on the accumulation of vacuoles, but a fundamental difference between them is re‐ vealed in the distinct nature and location of vacuole accumulation, which is usually deter‐ mined by vessel type and size. Cord hollowing is characterized by the creation of an extracellular luminal space within a cylindrical EC-cord. This involves the loss of apical cell adhesion between the central- but not peripheral- ECs, and results in a lumen diameter that is enclosed by multiple ECs[14-16, 18, 19]. Cell hollowing on the other hand involves the in‐ tracellular fusion of vacuoles within a single EC to give rise to a cytoplasmic lumen that spans the length of the cell, and typically results in vessels that have single-EC lining[17, 20]. The aorta in the mouse embryo for example relies on extracellular lumen formation as do most major vessels[15], while intracellular lumen formation is generally the designated mechanism for smaller vessels.

**Figure 1.** *Embryonic morphogenesis of the blood vasculature.* Mesodermal progenitor cells give rise the vascular endo‐ thelium through a series of steps that progressively specify ECs. In the mesoderm, angioblasts (EC-precursors) are formed and aggregate into cords or blood island, which later arrange into the primitive vascular plexus. Angiogenic remodelling of the primary plexus gives rise to a functional vascular network, from where the lymphatic vascular sys‐

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

3

tem eventually develops.

Transcriptional Modulation of Tumour Induced Angiogenesis http://dx.doi.org/10.5772/54055 3

**2.1. Embryonic blood vessel morphogenesis**

2 Research Directions in Tumor Angiogenesis

*2.1.2. Blood vascular lumen formation*

against the extracellular matrix[14-16].

mechanism for smaller vessels.

*2.1.1. Endothelial specification and initial blood vessel formation*

*De novo* generation of the first EC precursors in mammals occurs in the extra-embryonic meso‐ derm. The mesoderm is a hotbed for cell specification in the embryo, and the pluripotent hae‐ mangioblast ancestor of EC precursors (angioblasts) also gives rise to haematopoietic lineages and ostensibly even smooth muscle cells (SMC)[1-5]. In addition, ECs have been shown to share a common precursor with mesenchymal stem/stromal cells (MSC), the so-called mesen‐ chymoangioblast[6], and it has been suggested that other precursors can propagate endothelial cell lineages in the yolk sac. Together these observations signify the differentiation potential of these precursor cells, and impending consequences for plasticity during later remodelling and pathologies[7-9]. During vasculogenesis, defined as *de novo* generation of embryonic blood vessels, these pluripotent mesodermal progenitor cells acquire an endothelial cell (EC) precur‐ sor- or blood cell (BC) precursor- phenotype, and subsequently co-localize and aggregate in the mesoderm to form blood islands[10-12], with the EC precursors flattened around the edges and

To initiate the formation of actual vessel-like structures, the angioblasts assemble into arteri‐ al and venous cords, and in doing so form the primitive vascular plexus. These nascent rope-like threads have a solid core and are consequently not yet able to facilitate the flow of blood. This functional feature requires the heart of the cord to be tunnelled out, to give way to a central continuous lumen along the length of the nascent vessel. The transition of EC cords into vascular tubes is a process that necessitates defined EC-polarity, and a delicate interplay between adhesion and contractility. Polarity is essential for the distribution of membrane junction proteins and the definition of apical/luminal (inside) and basal/ablumi‐ nal (outside) surfaces. This is harmonized by the interplay between adhesion and contractili‐ ty, through the regulating of physical force propensity that accounts for the EC-flattening

Two principal cellular mechanisms have been described to explain for the formation of *de novo* blood vascular lumen: cord hollowing and cell hollowing[13, 16, 17]. Both mechanisms rely on the accumulation of vacuoles, but a fundamental difference between them is re‐ vealed in the distinct nature and location of vacuole accumulation, which is usually deter‐ mined by vessel type and size. Cord hollowing is characterized by the creation of an extracellular luminal space within a cylindrical EC-cord. This involves the loss of apical cell adhesion between the central- but not peripheral- ECs, and results in a lumen diameter that is enclosed by multiple ECs[14-16, 18, 19]. Cell hollowing on the other hand involves the in‐ tracellular fusion of vacuoles within a single EC to give rise to a cytoplasmic lumen that spans the length of the cell, and typically results in vessels that have single-EC lining[17, 20]. The aorta in the mouse embryo for example relies on extracellular lumen formation as do most major vessels[15], while intracellular lumen formation is generally the designated

the BC precursors in the centre to generate the haematopoietic lineages[11-13].

**Figure 1.** *Embryonic morphogenesis of the blood vasculature.* Mesodermal progenitor cells give rise the vascular endo‐ thelium through a series of steps that progressively specify ECs. In the mesoderm, angioblasts (EC-precursors) are formed and aggregate into cords or blood island, which later arrange into the primitive vascular plexus. Angiogenic remodelling of the primary plexus gives rise to a functional vascular network, from where the lymphatic vascular sys‐ tem eventually develops.

#### *2.1.3. Angiogenesis and blood vessel maturation*

The institution of a continuous blood vascular lumen is a milestone for the developing vas‐ cular system and paramount for further vascular development, as it permits the flow of blood. The nascent blood vessels that constitute this primitive vascular network will subse‐ quently expand, and then functionalize, into an extensive and more intricate systemic vascu‐ lature, in two processes respectively known as angiogenesis and vessel maturation. Angiogenesis describes the processes of branching, expansion and remodelling of the primi‐ tive vasculature in response to pro-angiogenic signals. This is different from vasculogenesis in that the ECs are not generated by *de novo* differentiation of stem cells, but rather depend on the proliferation and migration of pre-existing vascular ECs. Vessel maturation on the other hand describes the functionalization of nascent blood vessels, and is characterized by mural cell ensheathment of the vessel walls. The continuous mêlée between angiogenesis and vessel maturation – wherein vessel maturation blocks angiogenic growth, and visa ver‐ sa – ensures optimal systemic blood vascular performance.

as a means to maintain vessel perfusion and tissue oxygenation in a dynamic milieu. Proangiogenic signals can, for example, originate from inflammation and hypoxia as a transient cue, or from a more broadly encompassing and tenacious source such as a neoplasm. The latter type of molecular (dys-) regulation results in abnormal vessel formation, and will be discussed later in this chapter, once the transcriptional basis for EC specification and angio‐

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

5

The complexity and significance of the numerous morphological events contributing to blood vessel formation, as are highlighted above, underline the necessity for scrupulous reg‐ ulation to ensure that these processes occur in a spatiotemporally controlled fashion with a high level of precision over EC behaviour (fig. 2). Copious amounts of transcription factors are at the foundation of these coordinating programs, to guide the dynamic gene expression profiles at different stages of embryonic EC fate determination and vascular development (fig. 1), which are later – at least partially – recapitulated during vessel growth in the adult.

*2.2.1. Ets transcription factors regulate mesodermal specification of endothelial and haematopoietic*

The E-twenty-six (ETS) family is a large group of proteins, with close to thirty members in human and mouse, that achieves transcriptional regulation by binding clusters of ETS bind‐ ing motifs on gene enhancers and promoters[22]. In itself, this conserved core DNA se‐ quence, 5'-GGA(A/T)-3', offers little binding specificity between Ets members, and is by no means exclusive to endothelial-associated genes. Similarly, Ets expression extends beyond the vascular endothelium. Even so, multiple Ets members are of crucial importance for vas‐ cular development by regulating endothelial gene transcription. The way this is accomplish‐ ed despite these seemingly ubiquitous features, is illustrated by the presence of multiple ETS motifs in large number of enhancers and promoters that regulate specific EC gene tran‐ scription. There is also a combination of distinct Ets members being expressed in cells that are programmed to attain or maintain an EC phenotype. It is thus proposed that the combi‐ natorial effort of these transcription factors accounts for the tight control over EC differentia‐ tion[23, 24]. Complementary to interaction within the Ets family, recent studies indicate that Ets members also affiliate with other partner proteins to this end, and that multiple Ets members form a transcriptional network with associated partner proteins such as Tal1 and GATA-2 to regulate EC differentiation[25]. Another method by which specificity and func‐ tion is thought to be regulated is post-translational modification, such as phosphorylation, sumoylation and acetylation[26], while regions flanking the ETS motif on the DNA have al‐

**2.2. Transcriptional basis of blood vascular endothelial cell differentiation**

so been shown to affect the binding specificity of some Ets members[22].

The exact mechanisms by which the individual or combinatorial Ets expression profiles ach‐ ieve endothelial gene regulation remain largely unknown, but several Ets members have been identified in recent years to be critical at different stages during EC specification, vas‐ culogenesis and angiogenic remodelling. For example, mouse null-embryos for the ETS translocation variant 2 (Etv2/Er71/Etsrp71) transcription factor do not form blood island due

genesis has been established.

*lineages*

Vascular remodelling conventionally occurs through sprouting- and intussusception angio‐ genesis, and together with vessel maturation gives rise to organ specific vascular beds. Intus‐ susception angiogenesis is a process of vessel invagination wherein vessels ultimate divide and split – which requires appreciably high levels of polarization and localized en masse loss of cell junctions. Sprouting angiogenesis is visibly distinct from intussusception, and unsurpris‐ ingly involves the sprouting of a subset of ECs from the vascular wall to protrude into a primed ECM. In this discrete set of ECs, the cell-cell contacts are loosened to promote a motile pheno‐ type. The actual stromal invasion requires enzymatic degradation of the basement membrane and ECM. There is a remarkably strict hierarchy amongst the distinct EC-types in angiogenic sprouts, as a single tip-cell (TC) leads the way, and a host of stalk-cells (SC) follow[21]. Filopo‐ dia protrude from the TC that sense the microenvironment for attractive and repulsive signals to guide their migration, and to eventually fuse with adjacent vessels (anastomosis), while SCs contribute principally to the recruitment of pericytes and lumen preservation, while at the same time maintaining the connection between the TC and parent vessel.

Once the newly formed blood vasculature has extended and webbed to an appropriate level, the temporal pro-angiogenic signal will fade and the nascent vessel will be dis‐ posed to maturation. Blood vessels maturation primarily requires the recruitment of peri‐ cytes and SMCs, to ensheath and stabilize the vessel wall. This mural cell coverage strengthens the cell-cell contacts, decreases vessel permeability, and assures control over vessel diameter and therefore blood flow. Also, pericytes supress EC proliferation and promote EC survival, resulting in a long EC life and a quiescent state, which is typical for mature and functional vessels. Pericytes also subsidize the construction of the vessel basement membrane and deposit various ECM components into the stroma, to generate an angiogenesis incompetent milieu.

The whole process of vessel maturation is strikingly dynamic and intermittently reversible. Mature ECs can, conversely to quiescence, be activated by pro-angiogenic signals, upon which pericytes detach, cell-junctions are loosened, and the ECM is primed for angiogenic growth. In the adult, these processes are recapitulated during pathophysiological conditions as a means to maintain vessel perfusion and tissue oxygenation in a dynamic milieu. Proangiogenic signals can, for example, originate from inflammation and hypoxia as a transient cue, or from a more broadly encompassing and tenacious source such as a neoplasm. The latter type of molecular (dys-) regulation results in abnormal vessel formation, and will be discussed later in this chapter, once the transcriptional basis for EC specification and angio‐ genesis has been established.

#### **2.2. Transcriptional basis of blood vascular endothelial cell differentiation**

*2.1.3. Angiogenesis and blood vessel maturation*

4 Research Directions in Tumor Angiogenesis

sa – ensures optimal systemic blood vascular performance.

same time maintaining the connection between the TC and parent vessel.

an angiogenesis incompetent milieu.

The institution of a continuous blood vascular lumen is a milestone for the developing vas‐ cular system and paramount for further vascular development, as it permits the flow of blood. The nascent blood vessels that constitute this primitive vascular network will subse‐ quently expand, and then functionalize, into an extensive and more intricate systemic vascu‐ lature, in two processes respectively known as angiogenesis and vessel maturation. Angiogenesis describes the processes of branching, expansion and remodelling of the primi‐ tive vasculature in response to pro-angiogenic signals. This is different from vasculogenesis in that the ECs are not generated by *de novo* differentiation of stem cells, but rather depend on the proliferation and migration of pre-existing vascular ECs. Vessel maturation on the other hand describes the functionalization of nascent blood vessels, and is characterized by mural cell ensheathment of the vessel walls. The continuous mêlée between angiogenesis and vessel maturation – wherein vessel maturation blocks angiogenic growth, and visa ver‐

Vascular remodelling conventionally occurs through sprouting- and intussusception angio‐ genesis, and together with vessel maturation gives rise to organ specific vascular beds. Intus‐ susception angiogenesis is a process of vessel invagination wherein vessels ultimate divide and split – which requires appreciably high levels of polarization and localized en masse loss of cell junctions. Sprouting angiogenesis is visibly distinct from intussusception, and unsurpris‐ ingly involves the sprouting of a subset of ECs from the vascular wall to protrude into a primed ECM. In this discrete set of ECs, the cell-cell contacts are loosened to promote a motile pheno‐ type. The actual stromal invasion requires enzymatic degradation of the basement membrane and ECM. There is a remarkably strict hierarchy amongst the distinct EC-types in angiogenic sprouts, as a single tip-cell (TC) leads the way, and a host of stalk-cells (SC) follow[21]. Filopo‐ dia protrude from the TC that sense the microenvironment for attractive and repulsive signals to guide their migration, and to eventually fuse with adjacent vessels (anastomosis), while SCs contribute principally to the recruitment of pericytes and lumen preservation, while at the

Once the newly formed blood vasculature has extended and webbed to an appropriate level, the temporal pro-angiogenic signal will fade and the nascent vessel will be dis‐ posed to maturation. Blood vessels maturation primarily requires the recruitment of peri‐ cytes and SMCs, to ensheath and stabilize the vessel wall. This mural cell coverage strengthens the cell-cell contacts, decreases vessel permeability, and assures control over vessel diameter and therefore blood flow. Also, pericytes supress EC proliferation and promote EC survival, resulting in a long EC life and a quiescent state, which is typical for mature and functional vessels. Pericytes also subsidize the construction of the vessel basement membrane and deposit various ECM components into the stroma, to generate

The whole process of vessel maturation is strikingly dynamic and intermittently reversible. Mature ECs can, conversely to quiescence, be activated by pro-angiogenic signals, upon which pericytes detach, cell-junctions are loosened, and the ECM is primed for angiogenic growth. In the adult, these processes are recapitulated during pathophysiological conditions The complexity and significance of the numerous morphological events contributing to blood vessel formation, as are highlighted above, underline the necessity for scrupulous reg‐ ulation to ensure that these processes occur in a spatiotemporally controlled fashion with a high level of precision over EC behaviour (fig. 2). Copious amounts of transcription factors are at the foundation of these coordinating programs, to guide the dynamic gene expression profiles at different stages of embryonic EC fate determination and vascular development (fig. 1), which are later – at least partially – recapitulated during vessel growth in the adult.

#### *2.2.1. Ets transcription factors regulate mesodermal specification of endothelial and haematopoietic lineages*

The E-twenty-six (ETS) family is a large group of proteins, with close to thirty members in human and mouse, that achieves transcriptional regulation by binding clusters of ETS bind‐ ing motifs on gene enhancers and promoters[22]. In itself, this conserved core DNA se‐ quence, 5'-GGA(A/T)-3', offers little binding specificity between Ets members, and is by no means exclusive to endothelial-associated genes. Similarly, Ets expression extends beyond the vascular endothelium. Even so, multiple Ets members are of crucial importance for vas‐ cular development by regulating endothelial gene transcription. The way this is accomplish‐ ed despite these seemingly ubiquitous features, is illustrated by the presence of multiple ETS motifs in large number of enhancers and promoters that regulate specific EC gene tran‐ scription. There is also a combination of distinct Ets members being expressed in cells that are programmed to attain or maintain an EC phenotype. It is thus proposed that the combi‐ natorial effort of these transcription factors accounts for the tight control over EC differentia‐ tion[23, 24]. Complementary to interaction within the Ets family, recent studies indicate that Ets members also affiliate with other partner proteins to this end, and that multiple Ets members form a transcriptional network with associated partner proteins such as Tal1 and GATA-2 to regulate EC differentiation[25]. Another method by which specificity and func‐ tion is thought to be regulated is post-translational modification, such as phosphorylation, sumoylation and acetylation[26], while regions flanking the ETS motif on the DNA have al‐ so been shown to affect the binding specificity of some Ets members[22].

The exact mechanisms by which the individual or combinatorial Ets expression profiles ach‐ ieve endothelial gene regulation remain largely unknown, but several Ets members have been identified in recent years to be critical at different stages during EC specification, vas‐ culogenesis and angiogenic remodelling. For example, mouse null-embryos for the ETS translocation variant 2 (Etv2/Er71/Etsrp71) transcription factor do not form blood island due to lack of EC and HPC specification, and are embryonic lethal with severe blood and vascu‐ lar defects[27, 28]. Friend leukemia integration 1 (Fli-1), another Ets member, has alterna‐ tively been shown to be essential during the establishment of the vascular plexus but not for endothelial specification[29]. Phylogenetically and functionally close to Fli-1 is ETS related gene (Erg)[30]. This particular Ets member acts slightly later during vascular development and is associated predominantly with angiogenesis, by controlling a host of processes such as EC junction dynamics and migration[31, 32].

motif is prevalent in endothelial enhancers and appreciably regulate endothelial gene tran‐ scription. In support of this, forced activity of both Etv2 and Foxc2 induces ectopic expres‐ sion of vascular markers VEGFR-2, Tie2, Tal1, NOTCH4 and VE-cadherin, while conversely, a mutation in the FOX:ETS motif disrupts Etv2/FoxC2 function and ablates endothelial spe‐

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

7

Upstream regulation of Etv2 has been an additional focus of recent studies, to further under‐ stand the mechanisms whereby endocardial and endothelial fate is determined and to trace back the transcriptional programs even further. In mice, the homeobox transcription factor Nkx2-5 has been shown to directly bind the Etv2 promoter and transactivate its expression in endothelial progenitor cells within the heart *in vitro* and *in vivo*[27]. In zebrafish, Etsrp was identified to be downstream of Foxc1a/b (FoxC1/C2 homologues found in zebrafish) in angio‐ blast development[38]. These factors were shown to be able to bind the upstream Etsrp enhanc‐ er *up1*, and the knockdown of Foxc1a/b results in loss of *up1* enhancer activity to drive transcription[38]. This supports the collaborative role of forkhead transcription factors and Etv2 in endothelial gene expression, and adds a dimension to the transcriptional network.

**Figure 2.** Transcriptional hierarchy orchestrating embryonic vascular development. Endothelial cell specification is an intricate process that relies on extensive crosstalk between transcription factors. Downstream of their transcriptional

regulation are signalling molecules that shape the cells and define EC identity and morphogenesis.

cific LacZ expression in mice[24].

Etv2 has in recent years arisen as the master transcriptional regulator of endothelial cell fate in mouse and zebrafish, because its function is absolutely critical for endothelial specification, with Etv2-null embryos failing to express vital endothelial markers and being devoid of ECs. Expression patterns have shown that Etv2 mainly functions in the embryonic mesoderm and blood islands at around 7.5 dpc (days post coitum) in mice, and is transiently present in larger vessels until at least 9.5 dpc[28, 33]. Mesodermally expressed Etv2 does not only direct specifi‐ cation towards EC lineages, but is also indispensible for the development of haematopoietic cells. In support of this, the endodermal stem cell precursors common to HPCs and ECs, halt differentiating towards haematopoietic or EC lineages prematurely in Etv2-null mice, in vas‐ cular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells [28]. The vascular endothelial growth factor receptor-2 (VEGFR-2/Flk1), receptor to VEGF-A and considered to be one of the most potent transducers of pro-angiogenic signalling, is thus not regulated by Etv2 in the mouse embryo. By contras, it has previously been reported that the zebrafish ortho‐ logue of Etv2, Etsrp, is required for the expression of the zebrafish VEGFR-2 orthologue, kdr[33], and the VEGFR-2 enhancer contains an ETS motif[34].

Other endothelial genes have been shown to be transcriptionally regulated by Etv2, confirm‐ ing its essential role in early vasculogenesis (refer to table 1). For example, the angiopoietin (Ang) receptor tyrosine kinase with immunoglobin-like and EGF-like domains-1 (Tie2) gene is a direct target of Etv2, and is an important vascular marker that regulates angiogene‐ sis[27]. Endothelial transcription factor GATA-2 is also a likely downstream target of Etv2[23, 28]. Similar to Etv2, GATA-2 is involved in both haemangioblast and endothelial development, and GATA-2 is severely downregulated in Etv2-null embryos[28]. Down‐ stream targets of GATA-2 include VEGFR-2[35] and ANG-2[36], and several other genes that encode endothelial proteins, such as Kruppel-like factor-2 (KLF2), Ets variant- (Etv6) and myocyte enhancer factor-2 (MEF2C), have been identified to be occupied by transcrip‐ tion factor GATA-2[37], hence might be indirectly affected by Etv2 loss of function.

The bulk of transcriptional regulation by Etv2, however, is though to be achieved through recognition of the composite FOX:ETS motif, which is exclusive to endothelial-specific en‐ hancers, and is present in approximately 23% of all endothelial genes[24]. Members of both the forkhead and Ets transcription factor families, in particular the forkhead box protein C2 (FoxC2) and Etv2, synergistically bind this motif to activate endothelial gene expression[24]. In vivo studies in Xenopus and zebrafish embryos have identified this motif within the en‐ hancer of 11 important endothelial genes, being Mef2c, VEGFR-2, Tal1, Tie2, VE-cadherin (Cdh5), ECE1, VEGFR-3 (Flt-4), PDGFRβ, FoxP1, NRP1 and NOTCH4[24]. Not all of these molecular players are individually discussed in this chapter, but it is clear that the FOX:ETS motif is prevalent in endothelial enhancers and appreciably regulate endothelial gene tran‐ scription. In support of this, forced activity of both Etv2 and Foxc2 induces ectopic expres‐ sion of vascular markers VEGFR-2, Tie2, Tal1, NOTCH4 and VE-cadherin, while conversely, a mutation in the FOX:ETS motif disrupts Etv2/FoxC2 function and ablates endothelial spe‐ cific LacZ expression in mice[24].

to lack of EC and HPC specification, and are embryonic lethal with severe blood and vascu‐ lar defects[27, 28]. Friend leukemia integration 1 (Fli-1), another Ets member, has alterna‐ tively been shown to be essential during the establishment of the vascular plexus but not for endothelial specification[29]. Phylogenetically and functionally close to Fli-1 is ETS related gene (Erg)[30]. This particular Ets member acts slightly later during vascular development and is associated predominantly with angiogenesis, by controlling a host of processes such

Etv2 has in recent years arisen as the master transcriptional regulator of endothelial cell fate in mouse and zebrafish, because its function is absolutely critical for endothelial specification, with Etv2-null embryos failing to express vital endothelial markers and being devoid of ECs. Expression patterns have shown that Etv2 mainly functions in the embryonic mesoderm and blood islands at around 7.5 dpc (days post coitum) in mice, and is transiently present in larger vessels until at least 9.5 dpc[28, 33]. Mesodermally expressed Etv2 does not only direct specifi‐ cation towards EC lineages, but is also indispensible for the development of haematopoietic cells. In support of this, the endodermal stem cell precursors common to HPCs and ECs, halt differentiating towards haematopoietic or EC lineages prematurely in Etv2-null mice, in vas‐ cular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells [28]. The vascular endothelial growth factor receptor-2 (VEGFR-2/Flk1), receptor to VEGF-A and considered to be one of the most potent transducers of pro-angiogenic signalling, is thus not regulated by Etv2 in the mouse embryo. By contras, it has previously been reported that the zebrafish ortho‐ logue of Etv2, Etsrp, is required for the expression of the zebrafish VEGFR-2 orthologue,

Other endothelial genes have been shown to be transcriptionally regulated by Etv2, confirm‐ ing its essential role in early vasculogenesis (refer to table 1). For example, the angiopoietin (Ang) receptor tyrosine kinase with immunoglobin-like and EGF-like domains-1 (Tie2) gene is a direct target of Etv2, and is an important vascular marker that regulates angiogene‐ sis[27]. Endothelial transcription factor GATA-2 is also a likely downstream target of Etv2[23, 28]. Similar to Etv2, GATA-2 is involved in both haemangioblast and endothelial development, and GATA-2 is severely downregulated in Etv2-null embryos[28]. Down‐ stream targets of GATA-2 include VEGFR-2[35] and ANG-2[36], and several other genes that encode endothelial proteins, such as Kruppel-like factor-2 (KLF2), Ets variant- (Etv6) and myocyte enhancer factor-2 (MEF2C), have been identified to be occupied by transcrip‐

tion factor GATA-2[37], hence might be indirectly affected by Etv2 loss of function.

The bulk of transcriptional regulation by Etv2, however, is though to be achieved through recognition of the composite FOX:ETS motif, which is exclusive to endothelial-specific en‐ hancers, and is present in approximately 23% of all endothelial genes[24]. Members of both the forkhead and Ets transcription factor families, in particular the forkhead box protein C2 (FoxC2) and Etv2, synergistically bind this motif to activate endothelial gene expression[24]. In vivo studies in Xenopus and zebrafish embryos have identified this motif within the en‐ hancer of 11 important endothelial genes, being Mef2c, VEGFR-2, Tal1, Tie2, VE-cadherin (Cdh5), ECE1, VEGFR-3 (Flt-4), PDGFRβ, FoxP1, NRP1 and NOTCH4[24]. Not all of these molecular players are individually discussed in this chapter, but it is clear that the FOX:ETS

as EC junction dynamics and migration[31, 32].

6 Research Directions in Tumor Angiogenesis

kdr[33], and the VEGFR-2 enhancer contains an ETS motif[34].

Upstream regulation of Etv2 has been an additional focus of recent studies, to further under‐ stand the mechanisms whereby endocardial and endothelial fate is determined and to trace back the transcriptional programs even further. In mice, the homeobox transcription factor Nkx2-5 has been shown to directly bind the Etv2 promoter and transactivate its expression in endothelial progenitor cells within the heart *in vitro* and *in vivo*[27]. In zebrafish, Etsrp was identified to be downstream of Foxc1a/b (FoxC1/C2 homologues found in zebrafish) in angio‐ blast development[38]. These factors were shown to be able to bind the upstream Etsrp enhanc‐ er *up1*, and the knockdown of Foxc1a/b results in loss of *up1* enhancer activity to drive transcription[38]. This supports the collaborative role of forkhead transcription factors and Etv2 in endothelial gene expression, and adds a dimension to the transcriptional network.

**Figure 2.** Transcriptional hierarchy orchestrating embryonic vascular development. Endothelial cell specification is an intricate process that relies on extensive crosstalk between transcription factors. Downstream of their transcriptional regulation are signalling molecules that shape the cells and define EC identity and morphogenesis.

#### *2.2.2. Fox transcription factors regulate arteriovenous specification and angiogenesis*

It is clear that forkhead transcription factor FoxC2 has an important role during EC specifi‐ cation, through the collaboration with Etv2 at early stages of embryogenesis. Notably, FoxO1 is also able to operate synergistically with Etv2 by binding the FOX:ETS motif[24]. However, not unlike Etv2, FoxO and FoxC transcription factors also direct FOX:ETS inde‐ pendent endothelial gene transcription, which is crucial for vascular development.

vascular stability during vascular development in zebrafish, with FoxC1 morphants having

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

9

The divergent roles of FoxC1/2 are not limited to orchestration blood vascular development, and concomitantly also control the development of the lymphatic vascular system. Natural‐ ly occurring mutations in the human FoxC2 gene are associated with hereditary lymphede‐ ma-distichiasis (LD) syndrome, an autosomal dominant disorder which is characterized by accumulation of interstitial flood leading to swelling (lymphedema), and aberrant eyelash growth (distichiasis)[47]. Clinical studies have revealed that patient with LD have impaired lymphatic valve function[48], and *in vivo* mouse studies have shown that lymphatic valves do not form properly in FoxC2-nul mutants[49]. Also, the smooth muscle coverage of lym‐ phatic collector vessels is increased in FoxC2 heterozygous mice, which is inherent to LD, owing to an increased expression of platelet derived growth factor β (Pdgfβ) *in vivo*[49]. Hence, it has been suggested that FoxC2 regulates lymphatic vessel maturation, and possi‐ bly lymphatic sprouting, by interacting with growth factors and transcription factors that regulate lymphatic development. Notably, the lymphatic endothelial cell (LEC) receptor VEGFR3 is thought to be upstream of FoxC2, linking pro-lymphangiogenic VEGF signalling to FoxC2 activity[49], which supports the observation that FoxC2 mutants have increased vSMC-mediated LEC maturation. FoxC2 has since been shown to cooperate with the master regulator of LEC commitment prospero homeobox protein 1 (Prox1) during lymphatic valve formation in controlling the activity of gap junction protein connexin37 (Cx37) and nuclear factor of activated T-cells cytoplastmic-1 (NFATc1)[50]. In this context, NFATc1 activity is controlled by VEGF-C that leads to FoxC2 interaction[51]. Compound FoxC1 heterozygous; FoxC2 homozygous mice further have lymphatic sprouting defects during the earliest stages

Taken together, this suggests that FoxC signalling has critical roles during lymphangiogene‐ sis and lymphatic maturation in addition to A/V specification and angiogenesis, through co‐

*2.2.3. Members SOXF transcription factors determine A/V specification and lymphangiogenenic*

The three members of the SOXF group – SOX7, SOX17 and SOX18 – are all endogenously expressed in ECs during vascular development[52], and several key functions of these tran‐ scription factors have been described over the years. This includes regulation of A/V specifi‐ cation, angiogenesis, lymphangiogenesis and red blood cell specification, but also other roles perceivably not associated with the blood or lymphatic vasculature, such as hair folli‐

SOXF transcription factors belong to the SRY-box (SOX) family that is comprised of 20 mem‐ bers. SOX members are all characterized and identified by their highly homologous 79 ami‐ no acid high-mobility group (HMG) domain, which was first discovered in their founding member sex-determining region Y (SRY)[53]. This typical SOX element binds the heptameric consensus sequence 5'-(A/T)(A/T)CAA(A/T)G-3'[54], to induce DNA bending and regulate the expression of a broad collection of genes during embryonic development[55]. Specificity

severe basement membrane defects similar to that reported for lama1[46].

of lymphangiogenesis[43].

*switch*

operation with lymphatic specific transcription factors.

cle development and endoderm differentiation.

Endothelial cells are specified in FoxO1-null mice, and thus differentiate beyond the VEGFR2+ stage of Etv2-null embryos. However, embryonic lethality occurs only slightly lat‐ er due to a severe angiogenic defect, characterized by disorganized and few vessels by E9.5, with low expression of some crucial vascular markers[39]. Amongst those downregulated is the arterial marker Eprin-B2, a key regulator of VEGFR3 receptor internalization and trans‐ ducer of VEGF-C/PI3K/Akt signalling, so it is hypothesized that FoxO1 regulates angiogene‐ sis by controlling VEGF responsiveness[39-41]. What further underlines the importance of FoxO1 is the elaborate control over its the transcriptional activity, which is regulated on many levels by posttranscriptional modifications, interaction with co-activators or co-re‐ pressors, and absolute FoxO1 protein levels, to regulate localization, DNA-binding activity, and function[42].

FoxC1 and FoxC2 are, in addition to their role in Etv2-mediated endothelial specification, re‐ quired for endothelial cells to acquire an arterial cell phenotype[43]. Both FoxC transcription factors directly activate the transcription of the arterial cell fate promoters Notch1 and Deltalike 4 (Dll4), and overexpression of FoxC genes results in concomitant induction of Notch and Dll4 expression *in vitro*[43]. Notch signalling has been shown to be essential for arterio‐ venous (A/V) specification, by mediating the transcription of Hairy/enhancer-of-split related with YRPW motif protein 1 and 2 (Hey1/2). Null-mice for either Notch1 or Hey1/2 have se‐ vere vascular defects, with impaired remodelling and general loss of arterial markers such as Ephrin-B2[44]. These arteriovenous malformations are also observed in FoxC1/2 double homozygous knockout mice, with loss of Notch1, Notch4, Dll4, Hey2 and ephrinB2, while transcription of the venous marker chicken ovalbumin upstream promoter transcription fac‐ tor 2 (COUP-TFII/NR2F2) and the pan-endothelial marker VEGFR2 is not affected[43].

FoxC1 has recently been shown to control ECM composition and basement membrane integ‐ rity, by regulating the expression of several matrix metalloproteinases (MMPs)[45], and ge‐ netically interacting with laminin α-1(lama1)[46], respectively. The homeostasis of these factors directly influences the vasculature's microenvironment, and is of great relevance to angiogenesis. In the mouse corneal stroma, MMP1a, MMP3, MMP9, MMP12 and MMP12 are upregulated in absence of FoxC1, which is associated with induced angiogenesis by the excessive degradation of the ECM and increased bioavailability of VEGF[45]. The crosstalk between VEGF signalling and forkhead transcription factors is thus a recurring observation, although it is unclear if and how they physically interact. Expression levels of collagens Col1a1, Col3a1, Col4a1 seem unaffected by loss of FoxC1[45], suggesting that FoxC1 does not directly contribute so structural basement membrane or stromal components. However, as mentioned, FoxC1 does interact with lama1 to support basement membrane integrity and vascular stability during vascular development in zebrafish, with FoxC1 morphants having severe basement membrane defects similar to that reported for lama1[46].

*2.2.2. Fox transcription factors regulate arteriovenous specification and angiogenesis*

pendent endothelial gene transcription, which is crucial for vascular development.

VEGFR2+

8 Research Directions in Tumor Angiogenesis

and function[42].

It is clear that forkhead transcription factor FoxC2 has an important role during EC specifi‐ cation, through the collaboration with Etv2 at early stages of embryogenesis. Notably, FoxO1 is also able to operate synergistically with Etv2 by binding the FOX:ETS motif[24]. However, not unlike Etv2, FoxO and FoxC transcription factors also direct FOX:ETS inde‐

Endothelial cells are specified in FoxO1-null mice, and thus differentiate beyond the

er due to a severe angiogenic defect, characterized by disorganized and few vessels by E9.5, with low expression of some crucial vascular markers[39]. Amongst those downregulated is the arterial marker Eprin-B2, a key regulator of VEGFR3 receptor internalization and trans‐ ducer of VEGF-C/PI3K/Akt signalling, so it is hypothesized that FoxO1 regulates angiogene‐ sis by controlling VEGF responsiveness[39-41]. What further underlines the importance of FoxO1 is the elaborate control over its the transcriptional activity, which is regulated on many levels by posttranscriptional modifications, interaction with co-activators or co-re‐ pressors, and absolute FoxO1 protein levels, to regulate localization, DNA-binding activity,

FoxC1 and FoxC2 are, in addition to their role in Etv2-mediated endothelial specification, re‐ quired for endothelial cells to acquire an arterial cell phenotype[43]. Both FoxC transcription factors directly activate the transcription of the arterial cell fate promoters Notch1 and Deltalike 4 (Dll4), and overexpression of FoxC genes results in concomitant induction of Notch and Dll4 expression *in vitro*[43]. Notch signalling has been shown to be essential for arterio‐ venous (A/V) specification, by mediating the transcription of Hairy/enhancer-of-split related with YRPW motif protein 1 and 2 (Hey1/2). Null-mice for either Notch1 or Hey1/2 have se‐ vere vascular defects, with impaired remodelling and general loss of arterial markers such as Ephrin-B2[44]. These arteriovenous malformations are also observed in FoxC1/2 double homozygous knockout mice, with loss of Notch1, Notch4, Dll4, Hey2 and ephrinB2, while transcription of the venous marker chicken ovalbumin upstream promoter transcription fac‐ tor 2 (COUP-TFII/NR2F2) and the pan-endothelial marker VEGFR2 is not affected[43].

FoxC1 has recently been shown to control ECM composition and basement membrane integ‐ rity, by regulating the expression of several matrix metalloproteinases (MMPs)[45], and ge‐ netically interacting with laminin α-1(lama1)[46], respectively. The homeostasis of these factors directly influences the vasculature's microenvironment, and is of great relevance to angiogenesis. In the mouse corneal stroma, MMP1a, MMP3, MMP9, MMP12 and MMP12 are upregulated in absence of FoxC1, which is associated with induced angiogenesis by the excessive degradation of the ECM and increased bioavailability of VEGF[45]. The crosstalk between VEGF signalling and forkhead transcription factors is thus a recurring observation, although it is unclear if and how they physically interact. Expression levels of collagens Col1a1, Col3a1, Col4a1 seem unaffected by loss of FoxC1[45], suggesting that FoxC1 does not directly contribute so structural basement membrane or stromal components. However, as mentioned, FoxC1 does interact with lama1 to support basement membrane integrity and

stage of Etv2-null embryos. However, embryonic lethality occurs only slightly lat‐

The divergent roles of FoxC1/2 are not limited to orchestration blood vascular development, and concomitantly also control the development of the lymphatic vascular system. Natural‐ ly occurring mutations in the human FoxC2 gene are associated with hereditary lymphede‐ ma-distichiasis (LD) syndrome, an autosomal dominant disorder which is characterized by accumulation of interstitial flood leading to swelling (lymphedema), and aberrant eyelash growth (distichiasis)[47]. Clinical studies have revealed that patient with LD have impaired lymphatic valve function[48], and *in vivo* mouse studies have shown that lymphatic valves do not form properly in FoxC2-nul mutants[49]. Also, the smooth muscle coverage of lym‐ phatic collector vessels is increased in FoxC2 heterozygous mice, which is inherent to LD, owing to an increased expression of platelet derived growth factor β (Pdgfβ) *in vivo*[49]. Hence, it has been suggested that FoxC2 regulates lymphatic vessel maturation, and possi‐ bly lymphatic sprouting, by interacting with growth factors and transcription factors that regulate lymphatic development. Notably, the lymphatic endothelial cell (LEC) receptor VEGFR3 is thought to be upstream of FoxC2, linking pro-lymphangiogenic VEGF signalling to FoxC2 activity[49], which supports the observation that FoxC2 mutants have increased vSMC-mediated LEC maturation. FoxC2 has since been shown to cooperate with the master regulator of LEC commitment prospero homeobox protein 1 (Prox1) during lymphatic valve formation in controlling the activity of gap junction protein connexin37 (Cx37) and nuclear factor of activated T-cells cytoplastmic-1 (NFATc1)[50]. In this context, NFATc1 activity is controlled by VEGF-C that leads to FoxC2 interaction[51]. Compound FoxC1 heterozygous; FoxC2 homozygous mice further have lymphatic sprouting defects during the earliest stages of lymphangiogenesis[43].

Taken together, this suggests that FoxC signalling has critical roles during lymphangiogene‐ sis and lymphatic maturation in addition to A/V specification and angiogenesis, through co‐ operation with lymphatic specific transcription factors.

#### *2.2.3. Members SOXF transcription factors determine A/V specification and lymphangiogenenic switch*

The three members of the SOXF group – SOX7, SOX17 and SOX18 – are all endogenously expressed in ECs during vascular development[52], and several key functions of these tran‐ scription factors have been described over the years. This includes regulation of A/V specifi‐ cation, angiogenesis, lymphangiogenesis and red blood cell specification, but also other roles perceivably not associated with the blood or lymphatic vasculature, such as hair folli‐ cle development and endoderm differentiation.

SOXF transcription factors belong to the SRY-box (SOX) family that is comprised of 20 mem‐ bers. SOX members are all characterized and identified by their highly homologous 79 ami‐ no acid high-mobility group (HMG) domain, which was first discovered in their founding member sex-determining region Y (SRY)[53]. This typical SOX element binds the heptameric consensus sequence 5'-(A/T)(A/T)CAA(A/T)G-3'[54], to induce DNA bending and regulate the expression of a broad collection of genes during embryonic development[55]. Specificity

and functional differentiation between SOX-groups and individual members is accomplish‐ ed by additional operative elements on the SOX transcription factors, and through associa‐ tion with partner proteins[54, 56, 57]. Their coexpression and HMG domain homology, however, does suggest that functional redundancies or cooperative roles apply for members within the same SOX group. However, of the SOXF group only SOX18 is endogenously ex‐ pressed during lymphatic vascular development in LEC precursors[58].

lymph sacs and lymphatic vasculature[68, 69]. However, after the initial LEC specification, Prox1 expression becomes independent of SOX18, and later COUP-TFII, but itself remains

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

11

Tumour cells are characterized by chronic proliferation and immortality, due to mutations in genes that regulate cell cycle, homeostasis and cell death[70]. As a solid tumour grows, it is evident that the need for oxygen and nutrients increases correspondingly, and waste ma‐ terials need to be carried off in escalating amounts, which rationalizes the commonly ob‐ served tumour-induced neo-vascularisation. To accomplish this remarkable feat, tumour cells exploit many of the vascular signalling pathways that are activated during embryogen‐ esis, but without tight spatiotemporal control (fig. 3). Vascular architecture and integrity is therefore often compromised, promoting malign features of progressive tumours, such as

Due to the high oxygen demand and great metabolic activity of tumour cells, the peritumor‐ al region usually becomes hypervascularised. However, this does not truly solve the prob‐ lem for tumour cells, as in their gluttony they induce constitutive pro-angiogenic signalling that fails to generate a functional vascular network (fig. 3ab). The balance between pro-an‐ giogenic signalling and the subsequent maturation of the newly formed nascent vessels is key for proper circulation and perfusion. Typically, vessel maturation is inadequate in tu‐ mour tissue, owing to persistent presence of pro-angiogenic factors. The overabundance of pro-angiogenic signalling originates in part from the tumour directly, but is also a result of the chronic hypoxic and acidic state of the tumour microenvironment. In addition, tumours often trigger and maintain a chronic inflammatory response, wherein cells of the innate and adaptive immune system – mostly macrophages, neutrophils, mast cells and lymphocytes – infiltrate the tumour stoma and crosstalk with ECs to activate quiescent ECs and sustain pro-angiogenic signalling. Although an immune response can in fact reject certain tumours, malignant tumours and their microenvironment can generally evade immune cell mediated

However, tumour angiogenesis proceeds in an unorganized tempest of random sprouting because the guiding signals in the stroma are disorganized, and sprouting cells are unable to filter out any consistent cues. Abnormal shunts, including arteriovenous anastomoses, are commonly observed due to abrogated intervascular communication leading to bi-directional blood flow and impaired perfusion[72]. Tumours are highly diverse due to their tissue of origin and the heterogeneity of the mutations underlying their tumorigenic state. The type and degree of tumour vessel abnormality is correspondingly context dependent, but there are some general traits that tumour vessels share. These regard to overall vascular organiza‐

destruction, and instead recruit them to their angiogenic campaign[70, 71].

critical for lymphatic remodelling and maintenance of LEC identity[68, 69].

**3. Blood vessel development in solid tumours**

**3.1. Characteristics of the tumour vasculature**

metastatic behaviour.

SOX18 function in vascular development has received considerable attention since the natu‐ rally occurring *ragged* mouse mutation, the mural counterpart of the human syndrome hy‐ potrichosis-lymphedema-telangiecstasia (HLT) and underlying cause of severe cardiovascular and hair follicle defects, was identified in the Sox18 gene (Sox18Ra)[59]. This mutation produces a truncated form of SOX18 that acts in a dominant negative fashion and fails to recruit essential co-factors, and is therefor unable to induce target gene transcrip‐ tion[56, 59]. The defects in the *ragged* mice are much more severe than the observed pheno‐ type of Sox18-null mice[59], as truncated SOX18 competes with redundant SOXF members to occupy the same site on the DNA. This supports the notion that redundancies exist amongst SOXF transcription factors, and in fact it has been shown that SOX7 and SOX17 can activate SOX18 targets by binding to SOX18 promoter elements[58].

In the zebrafish embryo, individual knockdown of either SOX7 or SOX18 causes no obvious vascular defects, while the SOX7/18 double knockdown is characterized by partial loss of circulation, ectopic shunts between the main artery and vein, cardiac oedema, blood pool‐ ing, and a general loss of A/V specification[60, 61]. Indeed, SOX7 and SOX18 were found to be coexpressed in ECs and their precursors, and their combined loss of function resulted in reduction of arterial markers Ephrin-B2, notch3 and Dll4 and ectopic expression of the ve‐ nous endothelial marker VEGFR3 in the dorsal aorta (DA)[60, 61].

Several direct SOX18 vascular target genes have been described, notably the genes encoding the tight junction component claudin-5[62] and the vascular adhesion molecule VCAM-1[63], which are both essential for vascular integrity and endothelial activation dur‐ ing angiogenesis. SOX18 also directly activates the expression of MMP7, EphrinB2, interleu‐ kin receptor 7 (IL-7R)[64] and Robo4[65] *in vitro*. Robo4 expression *in vivo* is correspondingly under control of Sox7/18 activity in the mouse caudal vein, and in the intersegmental vessels (ISV) of zebrafish embryos[65]. Archetypically, Robo4 functions in axon guidance, but has more recently been identified as an important coordinator of EC migration during spouting angiogenesis in zebrafish[66]. *In vitro* assays have further shown that compound SOX17 het‐ erozygous; SOX18-null primary ECs have a sprouting and vascular remodelling defect[67].

SOX18-null mice, although devoid of any obvious blood vascular defects, are characterized by the lack of lymphatic vasculature. This is inherent to the *Ragged* mouse, and describes a nonredundant role for SOX18 in mouse lymphatic endothelial differentiation[68]. At the on‐ set of lymphangiogenesis, SOX18 is coexpressed with COUP-TFII and drives the expression of Prox1 in a subset of endothelial cells lining the wall of the CV. These LECs form the basis of the lymphatic vasculature, and absolutely require transient SOX18 and COUP-TFII activi‐ ty to induce Prox1 transcription[68, 69].. SOX18-null and COUP-TFII-null mice do not ex‐ press Prox1 in the embryonic CV, are devoid of LECs, and consequently have a total lack of lymph sacs and lymphatic vasculature[68, 69]. However, after the initial LEC specification, Prox1 expression becomes independent of SOX18, and later COUP-TFII, but itself remains critical for lymphatic remodelling and maintenance of LEC identity[68, 69].

#### **3. Blood vessel development in solid tumours**

and functional differentiation between SOX-groups and individual members is accomplish‐ ed by additional operative elements on the SOX transcription factors, and through associa‐ tion with partner proteins[54, 56, 57]. Their coexpression and HMG domain homology, however, does suggest that functional redundancies or cooperative roles apply for members within the same SOX group. However, of the SOXF group only SOX18 is endogenously ex‐

SOX18 function in vascular development has received considerable attention since the natu‐ rally occurring *ragged* mouse mutation, the mural counterpart of the human syndrome hy‐ potrichosis-lymphedema-telangiecstasia (HLT) and underlying cause of severe cardiovascular and hair follicle defects, was identified in the Sox18 gene (Sox18Ra)[59]. This mutation produces a truncated form of SOX18 that acts in a dominant negative fashion and fails to recruit essential co-factors, and is therefor unable to induce target gene transcrip‐ tion[56, 59]. The defects in the *ragged* mice are much more severe than the observed pheno‐ type of Sox18-null mice[59], as truncated SOX18 competes with redundant SOXF members to occupy the same site on the DNA. This supports the notion that redundancies exist amongst SOXF transcription factors, and in fact it has been shown that SOX7 and SOX17 can

In the zebrafish embryo, individual knockdown of either SOX7 or SOX18 causes no obvious vascular defects, while the SOX7/18 double knockdown is characterized by partial loss of circulation, ectopic shunts between the main artery and vein, cardiac oedema, blood pool‐ ing, and a general loss of A/V specification[60, 61]. Indeed, SOX7 and SOX18 were found to be coexpressed in ECs and their precursors, and their combined loss of function resulted in reduction of arterial markers Ephrin-B2, notch3 and Dll4 and ectopic expression of the ve‐

Several direct SOX18 vascular target genes have been described, notably the genes encoding the tight junction component claudin-5[62] and the vascular adhesion molecule VCAM-1[63], which are both essential for vascular integrity and endothelial activation dur‐ ing angiogenesis. SOX18 also directly activates the expression of MMP7, EphrinB2, interleu‐ kin receptor 7 (IL-7R)[64] and Robo4[65] *in vitro*. Robo4 expression *in vivo* is correspondingly under control of Sox7/18 activity in the mouse caudal vein, and in the intersegmental vessels (ISV) of zebrafish embryos[65]. Archetypically, Robo4 functions in axon guidance, but has more recently been identified as an important coordinator of EC migration during spouting angiogenesis in zebrafish[66]. *In vitro* assays have further shown that compound SOX17 het‐ erozygous; SOX18-null primary ECs have a sprouting and vascular remodelling defect[67]. SOX18-null mice, although devoid of any obvious blood vascular defects, are characterized by the lack of lymphatic vasculature. This is inherent to the *Ragged* mouse, and describes a nonredundant role for SOX18 in mouse lymphatic endothelial differentiation[68]. At the on‐ set of lymphangiogenesis, SOX18 is coexpressed with COUP-TFII and drives the expression of Prox1 in a subset of endothelial cells lining the wall of the CV. These LECs form the basis of the lymphatic vasculature, and absolutely require transient SOX18 and COUP-TFII activi‐ ty to induce Prox1 transcription[68, 69].. SOX18-null and COUP-TFII-null mice do not ex‐ press Prox1 in the embryonic CV, are devoid of LECs, and consequently have a total lack of

pressed during lymphatic vascular development in LEC precursors[58].

10 Research Directions in Tumor Angiogenesis

activate SOX18 targets by binding to SOX18 promoter elements[58].

nous endothelial marker VEGFR3 in the dorsal aorta (DA)[60, 61].

Tumour cells are characterized by chronic proliferation and immortality, due to mutations in genes that regulate cell cycle, homeostasis and cell death[70]. As a solid tumour grows, it is evident that the need for oxygen and nutrients increases correspondingly, and waste ma‐ terials need to be carried off in escalating amounts, which rationalizes the commonly ob‐ served tumour-induced neo-vascularisation. To accomplish this remarkable feat, tumour cells exploit many of the vascular signalling pathways that are activated during embryogen‐ esis, but without tight spatiotemporal control (fig. 3). Vascular architecture and integrity is therefore often compromised, promoting malign features of progressive tumours, such as metastatic behaviour.

#### **3.1. Characteristics of the tumour vasculature**

Due to the high oxygen demand and great metabolic activity of tumour cells, the peritumor‐ al region usually becomes hypervascularised. However, this does not truly solve the prob‐ lem for tumour cells, as in their gluttony they induce constitutive pro-angiogenic signalling that fails to generate a functional vascular network (fig. 3ab). The balance between pro-an‐ giogenic signalling and the subsequent maturation of the newly formed nascent vessels is key for proper circulation and perfusion. Typically, vessel maturation is inadequate in tu‐ mour tissue, owing to persistent presence of pro-angiogenic factors. The overabundance of pro-angiogenic signalling originates in part from the tumour directly, but is also a result of the chronic hypoxic and acidic state of the tumour microenvironment. In addition, tumours often trigger and maintain a chronic inflammatory response, wherein cells of the innate and adaptive immune system – mostly macrophages, neutrophils, mast cells and lymphocytes – infiltrate the tumour stoma and crosstalk with ECs to activate quiescent ECs and sustain pro-angiogenic signalling. Although an immune response can in fact reject certain tumours, malignant tumours and their microenvironment can generally evade immune cell mediated destruction, and instead recruit them to their angiogenic campaign[70, 71].

However, tumour angiogenesis proceeds in an unorganized tempest of random sprouting because the guiding signals in the stroma are disorganized, and sprouting cells are unable to filter out any consistent cues. Abnormal shunts, including arteriovenous anastomoses, are commonly observed due to abrogated intervascular communication leading to bi-directional blood flow and impaired perfusion[72]. Tumours are highly diverse due to their tissue of origin and the heterogeneity of the mutations underlying their tumorigenic state. The type and degree of tumour vessel abnormality is correspondingly context dependent, but there are some general traits that tumour vessels share. These regard to overall vascular organiza‐ tion and hierarchy as a network, immediate manifestation of maturation deficiencies, and morphology of vascular ECs.

way is that which leads to proliferation of a pre-existing vasculature, as it occurs in embry‐ onic remodelling and normal vascularization in the adult. However, tumours also promote the mobilization and specification of bone marrow derived cells (BMDCs). In addition, tu‐ mour cells themselves can transdifferentiate into ECs to be incorporated into the tumour

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

13

**Figure 3.** Tumour vascularization strategies originating from TF-dysregulation. **(A)** As it grows, a tumour adapts sever‐ al techniques to induce vascularization, either though proliferation of preexcisting peritumoral vessels or by promot‐ ing differentiation of non-EC into vascular endothelium. **(B)** The peritumoral and intratumoral regions get hypervascularized by the pro-angiogenic and pro-vasculogenic signals that the tumour instigates, which facilitates vessel intravasation metastatis through the vasculature. **(C)** Transciptional dysregulation underlies the angiogenic and

Proliferation of the existing vasculature proceeds for a large part through VEGF signalling. The VEGF signalling axis controls angiogenic- and lymphangiogenic sprouting through reg‐ ulation of cell proliferation and migration, with a set of several VEGF ligands and VEGFR receptors. VEGF-A is particularly angiogenic, while VEGF-C and VEGF-D are primarily lymphangiogenic. The downstream effect however is much dependant on the VEGFR they bind, with several possible combinations and dynamic receptor homodimerization, hetero‐ dymerization or co-receptor (NRPs) interaction adding to the complexity. In general, VEGF-A binds to VEGFR1 or VEGFR2 with the former interaction being anti-angiogenic to due high affinity but low downstream tyrosine kinase activity, and the latter being pro-angio‐

vasculature (fig. 3)[82].

vasculogenic signalling that tumour emanate.

While the dysregulation of angiogenesis causes overall hypervascularization, vessels are distributed unevenly throughout the peritumoral region, with very low vascular density in some areas. Moreover, large tumours instigate high tissue pressure that can compress and constrict vessels, and vessel diameter thus becomes independent of blood flow rate[73]. Nor‐ mally, high interstitial pressure is an important queue for lymphatic vessel to drain off the excess fluid, but this function is perturbed in tumour tissue and extravasated fluid is not the sole cause of pressure rise[74, 75]. Where larger blood vessels in normal tissue branch into gradually decreasing size vessels and eventually thin-walled capillaries, this obvious hierar‐ chy is often lost in tumour vasculature, and heterogeneous vessel subtypes are randomly distributed throughout the tumour vascular bed[76, 77]. This affects, but not truly reflects, their functional status.

Where normal vascular endothelial cells line up in the vessel wall to create a continuous bar‐ rier to maintain tissue fluid homeostasis and allow the selective diffusion and transport of certain molecules, the tumour vasculature is characterized by loss of EC polarity and cellcell adhesion that results in an incontinuous and leaky vessel wall. This is aggravated by the loosening of EC-associated mural cells, who fail attach tightly to ECs in the presence of con‐ stitutive pro-angiogenic signalling, which in turn leads to reduced vessel stability and inco‐ herent deposition of basement membrane- and ECM components[78, 79]. These resultant vessels cannot maintain a trans-vascular pressure gradient, because excessive amounts of fluid leak into the interstitial space through the porous vessels. Furthermore, tumour cells can gain entrance to the vascular system, for either transport throughout the circulation, or incorporation into the vessel wall.

The entry of tumour cells into the vasculature is a primary facilitator of distant metastasis formation, and is importantly applicable for both blood vessel and lymphatic vessels (fig 3b). It is of note that the lymphatic system is specifically designed to not only transport im‐ mune cells, but also to absorb, and drain off, fluid and larger molecules. Therefore, lymphat‐ ic capillaries are inadvertently effective in the uptake of tumour cells, and regional lymph node metastasis is a common indication of malignant tumour progression that is used a prognostic tool in human cancer patients[80, 81].

Overall, tumour cells seem to be able to initiate a chronic state of angiogenesis and lym‐ phangiogenesis, but in doing so fail to create normal functional vascular networks. The sig‐ nalling programmes that underlie these tumour-induced malformations may often have their foundation at a transcription level, with balance in transcriptional networks tipped to‐ wards proliferation of both tumour- and vascular EC proliferation and migration.

#### **3.2. Cellular origin of the tumour derived endothelium**

The vascular expansion that rapid growing tumours induce requires great numbers of vas‐ cular EC to form these structures. Tumours engage in three distinct strategies to wheel in these recruits and promote angiogenesis. The most obvious pro-angiogenic signalling path‐ way is that which leads to proliferation of a pre-existing vasculature, as it occurs in embry‐ onic remodelling and normal vascularization in the adult. However, tumours also promote the mobilization and specification of bone marrow derived cells (BMDCs). In addition, tu‐ mour cells themselves can transdifferentiate into ECs to be incorporated into the tumour vasculature (fig. 3)[82].

tion and hierarchy as a network, immediate manifestation of maturation deficiencies, and

While the dysregulation of angiogenesis causes overall hypervascularization, vessels are distributed unevenly throughout the peritumoral region, with very low vascular density in some areas. Moreover, large tumours instigate high tissue pressure that can compress and constrict vessels, and vessel diameter thus becomes independent of blood flow rate[73]. Nor‐ mally, high interstitial pressure is an important queue for lymphatic vessel to drain off the excess fluid, but this function is perturbed in tumour tissue and extravasated fluid is not the sole cause of pressure rise[74, 75]. Where larger blood vessels in normal tissue branch into gradually decreasing size vessels and eventually thin-walled capillaries, this obvious hierar‐ chy is often lost in tumour vasculature, and heterogeneous vessel subtypes are randomly distributed throughout the tumour vascular bed[76, 77]. This affects, but not truly reflects,

Where normal vascular endothelial cells line up in the vessel wall to create a continuous bar‐ rier to maintain tissue fluid homeostasis and allow the selective diffusion and transport of certain molecules, the tumour vasculature is characterized by loss of EC polarity and cellcell adhesion that results in an incontinuous and leaky vessel wall. This is aggravated by the loosening of EC-associated mural cells, who fail attach tightly to ECs in the presence of con‐ stitutive pro-angiogenic signalling, which in turn leads to reduced vessel stability and inco‐ herent deposition of basement membrane- and ECM components[78, 79]. These resultant vessels cannot maintain a trans-vascular pressure gradient, because excessive amounts of fluid leak into the interstitial space through the porous vessels. Furthermore, tumour cells can gain entrance to the vascular system, for either transport throughout the circulation, or

The entry of tumour cells into the vasculature is a primary facilitator of distant metastasis formation, and is importantly applicable for both blood vessel and lymphatic vessels (fig 3b). It is of note that the lymphatic system is specifically designed to not only transport im‐ mune cells, but also to absorb, and drain off, fluid and larger molecules. Therefore, lymphat‐ ic capillaries are inadvertently effective in the uptake of tumour cells, and regional lymph node metastasis is a common indication of malignant tumour progression that is used a

Overall, tumour cells seem to be able to initiate a chronic state of angiogenesis and lym‐ phangiogenesis, but in doing so fail to create normal functional vascular networks. The sig‐ nalling programmes that underlie these tumour-induced malformations may often have their foundation at a transcription level, with balance in transcriptional networks tipped to‐

The vascular expansion that rapid growing tumours induce requires great numbers of vas‐ cular EC to form these structures. Tumours engage in three distinct strategies to wheel in these recruits and promote angiogenesis. The most obvious pro-angiogenic signalling path‐

wards proliferation of both tumour- and vascular EC proliferation and migration.

morphology of vascular ECs.

12 Research Directions in Tumor Angiogenesis

their functional status.

incorporation into the vessel wall.

prognostic tool in human cancer patients[80, 81].

**3.2. Cellular origin of the tumour derived endothelium**

**Figure 3.** Tumour vascularization strategies originating from TF-dysregulation. **(A)** As it grows, a tumour adapts sever‐ al techniques to induce vascularization, either though proliferation of preexcisting peritumoral vessels or by promot‐ ing differentiation of non-EC into vascular endothelium. **(B)** The peritumoral and intratumoral regions get hypervascularized by the pro-angiogenic and pro-vasculogenic signals that the tumour instigates, which facilitates vessel intravasation metastatis through the vasculature. **(C)** Transciptional dysregulation underlies the angiogenic and vasculogenic signalling that tumour emanate.

Proliferation of the existing vasculature proceeds for a large part through VEGF signalling. The VEGF signalling axis controls angiogenic- and lymphangiogenic sprouting through reg‐ ulation of cell proliferation and migration, with a set of several VEGF ligands and VEGFR receptors. VEGF-A is particularly angiogenic, while VEGF-C and VEGF-D are primarily lymphangiogenic. The downstream effect however is much dependant on the VEGFR they bind, with several possible combinations and dynamic receptor homodimerization, hetero‐ dymerization or co-receptor (NRPs) interaction adding to the complexity. In general, VEGF-A binds to VEGFR1 or VEGFR2 with the former interaction being anti-angiogenic to due high affinity but low downstream tyrosine kinase activity, and the latter being pro-angio‐ genic. VEGF-C and VEGF-D on the other hand primarily bind the lymphangiogenic VEGFR3 receptor or VEGFR2-3 heterodimers to promote lymphangiogenesis. Hence, VEGFs, their receptors, and regulatory proteins upstream of VEGF – or signalling molecules that crosstalk with VEGF – are beguiling (lymph-)angiogenic players[83, 84].

bers of the ternary complex factor (TCF) subfamily. These transcription factors have been shown to be overexpressed in tumour cells of divergent cancer types, and to facilitate tu‐ mour progression, vascularization and invasion by regulation of growth factor responsive‐

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

15

With the recently discovery of tumour associated vascular ECs, however, it is imminent that key players of cell fate determination contribute to tumour induced neo-vascularization. The master regulator of endothelial and haematopoietic cell specification, Etv2, is only transient‐ ly expressed during embryonic development, as further angiogenesis generally occurs through proliferation of pre-existing vasculature. As Etv2 activity is absolutely critical for the specification of ECs, it is conceivable that transdifferentiation of tumour cells and specifi‐ cation and/or mobilization of bone marrow derived progenitors, requires Etv2 activity in tu‐

Although little is known about the actual expression levels of Etv2 in tumour cells or their microenvironment, several direct target genes or other downstream Etv2 targets are upregu‐ lated in tumour tissue. The Ang-2/Tie-2 system, for example, is often strongly activated in endothelial cells of tumour associated remodelling vessels, leading to increased angiogene‐ sis and proliferation[93, 113-115]. MMPs are known to facilitate a broad range of vascular events by ECM remodelling and paving the tumour stroma to promote angiogenesis, and MMP overexpression is instrumental to progression of distinct cancer types[116, 117]. Etv2 can also directly activate the MMP-1 promoter, and MMP-1 is often overexpressed in cancer

Other Etv2 targets, many of which carry the FOX:ETS motif in their promoter, are ubiqui‐ tously dysregulated during tumour angiogenesis[122-126]. It is not clear whether this is Etv2-dependent, but it has been shown that Etv2 activity can induce ectopic expression of these genes in embryonic development, and it is conceivable that Etv2 function is recapitu‐ lated and exploited in tumour vasculogenesis and angiogenesis. This could explain the transdifferentiation capacity of tumour cells that contribute to the vascular progenitor popu‐ lation, and the recruitment of BMDCs as Etv2 activity specifies EC and haematopoietic line‐ ages from stem cells in the mesoderm. In addition, putative Etv2 targets during tumour angiogenesis have extensive crosstalk with growth factor signalling, which further endorses

The presence and role of FoxC2 in tumour angiogenesis has been fairly well character‐ ized over the past few years, and it has been shown that the expression of FoxC2 in tu‐ mour endothelium coincides with neovascularization. This further supports the notion of Etv2 recurrence during tumour vascularization because of the synergistic function be‐ tween these transcription factors in regulating endothelial genes expression through the

FoxC2 overexpression is associated with aggressive human cancers, and has been shown to be overexpressed in mammary breast cancer cells *in vitro* where it directly promotes a meta‐

the suggested role and significance of Etv2 in this process[127].

ness and MMP expression [107-112] (fig. 3C).

mour angiogenesis[91] (fig. 3c).

as are many others[118-121].

*3.3.2. Forkhead transcription factors*

FOX:ETS motif.

Recently, light has been shed on tumour signalling to neighbouring endothelium, which convolutes this classical growth factor signalling. Microvesicles released from tumour cells can transport genetic material and signalling molecules directly into endothelial (progenitor) cells that can make epigenetic modification to regulatory genes and otherwise alter expres‐ sion patterns[85-88]. These microvesicles can also originate from non-tumour cells, such as EPC, to activate angiogenic programmes in vascular ECs[89, 90]. This demonstrates that cells residing in the tumour stroma are altered at a more fundamental level to contribute to tumour vascularization.

Although angiogenesis is the prevailing concept that accounts for tumour vascularization, it is becoming ever more prevalent that vasculogenesis has a significant contribution to vessel formation in tumours. EPCs, and other BMDCs such as tumour associated macrophages (TAMs), mesenchymal progenitor cells (MPC), monocytes, are thought to participate in tu‐ mour vascularization in varying degrees, and are common components of the tumour stro‐ ma [91-95]. These cells can actively be recruited to the site of neovascularization [96], and reside there to promote angiogenesis or differentiate into vascular EC themselves. This proc‐ ess is further propagated by chronic inflammation of the tumour microenvironment[97]. Furthermore, tissue resident stem cells may contribute to angiogenesis as was shown to be the case in renal cancinoma's[98].

Adding to the mechanism of vasculogenesis and the role of stem cells, is an active role for tumour cells themselves. A heterogeneous malignant tumour is often characterized by sub‐ populations of cancer stem cells (CSCs) that have great self-renewal and differentiation ca‐ pacity, similar to normal stem cells[99, 100]. These CMCs have the ability to acquire an endothelial progenitor phenotype, and function as vascular ECs, which benefits tumour vas‐ cularization and proliferation[101, 102]. This practise is generally dependent on conditions such as hypoxia, where tumour cells find themselves in acute need of supply and transdif‐ ferentiate in vascular progenitors[103-105]. Vascular mimicry is a remarkable demonstration of this CSC-trait. Tumour cells in this process align into channel-like structures, gain EC gene expression, acquire and EC phenotype, and roughly function as blood vessel (fig. 3B). Suggested mechanisms by which tumour cells can differentiate into vascular progenitor in‐ clude signalling through VEGF and IKKβ [102, 106].

#### **3.3. Dysregulation of transcriptional angiogenic pathways**

#### *3.3.1. Ets transcription factors*

Many Ets transcription factors have a suggested or confirmed role in tumour angiogenesis and progression. Probably the most obvious Ets members to be involved in tumorigenesis are Fli1 and ERG, which have been acknowledged for their role in embryonic angiogenesis and vasculogenesis in a previous section of this chapter, but also ETS1/2 and several mem‐ bers of the ternary complex factor (TCF) subfamily. These transcription factors have been shown to be overexpressed in tumour cells of divergent cancer types, and to facilitate tu‐ mour progression, vascularization and invasion by regulation of growth factor responsive‐ ness and MMP expression [107-112] (fig. 3C).

With the recently discovery of tumour associated vascular ECs, however, it is imminent that key players of cell fate determination contribute to tumour induced neo-vascularization. The master regulator of endothelial and haematopoietic cell specification, Etv2, is only transient‐ ly expressed during embryonic development, as further angiogenesis generally occurs through proliferation of pre-existing vasculature. As Etv2 activity is absolutely critical for the specification of ECs, it is conceivable that transdifferentiation of tumour cells and specifi‐ cation and/or mobilization of bone marrow derived progenitors, requires Etv2 activity in tu‐ mour angiogenesis[91] (fig. 3c).

Although little is known about the actual expression levels of Etv2 in tumour cells or their microenvironment, several direct target genes or other downstream Etv2 targets are upregu‐ lated in tumour tissue. The Ang-2/Tie-2 system, for example, is often strongly activated in endothelial cells of tumour associated remodelling vessels, leading to increased angiogene‐ sis and proliferation[93, 113-115]. MMPs are known to facilitate a broad range of vascular events by ECM remodelling and paving the tumour stroma to promote angiogenesis, and MMP overexpression is instrumental to progression of distinct cancer types[116, 117]. Etv2 can also directly activate the MMP-1 promoter, and MMP-1 is often overexpressed in cancer as are many others[118-121].

Other Etv2 targets, many of which carry the FOX:ETS motif in their promoter, are ubiqui‐ tously dysregulated during tumour angiogenesis[122-126]. It is not clear whether this is Etv2-dependent, but it has been shown that Etv2 activity can induce ectopic expression of these genes in embryonic development, and it is conceivable that Etv2 function is recapitu‐ lated and exploited in tumour vasculogenesis and angiogenesis. This could explain the transdifferentiation capacity of tumour cells that contribute to the vascular progenitor popu‐ lation, and the recruitment of BMDCs as Etv2 activity specifies EC and haematopoietic line‐ ages from stem cells in the mesoderm. In addition, putative Etv2 targets during tumour angiogenesis have extensive crosstalk with growth factor signalling, which further endorses the suggested role and significance of Etv2 in this process[127].

#### *3.3.2. Forkhead transcription factors*

genic. VEGF-C and VEGF-D on the other hand primarily bind the lymphangiogenic VEGFR3 receptor or VEGFR2-3 heterodimers to promote lymphangiogenesis. Hence, VEGFs, their receptors, and regulatory proteins upstream of VEGF – or signalling molecules

Recently, light has been shed on tumour signalling to neighbouring endothelium, which convolutes this classical growth factor signalling. Microvesicles released from tumour cells can transport genetic material and signalling molecules directly into endothelial (progenitor) cells that can make epigenetic modification to regulatory genes and otherwise alter expres‐ sion patterns[85-88]. These microvesicles can also originate from non-tumour cells, such as EPC, to activate angiogenic programmes in vascular ECs[89, 90]. This demonstrates that cells residing in the tumour stroma are altered at a more fundamental level to contribute to

Although angiogenesis is the prevailing concept that accounts for tumour vascularization, it is becoming ever more prevalent that vasculogenesis has a significant contribution to vessel formation in tumours. EPCs, and other BMDCs such as tumour associated macrophages (TAMs), mesenchymal progenitor cells (MPC), monocytes, are thought to participate in tu‐ mour vascularization in varying degrees, and are common components of the tumour stro‐ ma [91-95]. These cells can actively be recruited to the site of neovascularization [96], and reside there to promote angiogenesis or differentiate into vascular EC themselves. This proc‐ ess is further propagated by chronic inflammation of the tumour microenvironment[97]. Furthermore, tissue resident stem cells may contribute to angiogenesis as was shown to be

Adding to the mechanism of vasculogenesis and the role of stem cells, is an active role for tumour cells themselves. A heterogeneous malignant tumour is often characterized by sub‐ populations of cancer stem cells (CSCs) that have great self-renewal and differentiation ca‐ pacity, similar to normal stem cells[99, 100]. These CMCs have the ability to acquire an endothelial progenitor phenotype, and function as vascular ECs, which benefits tumour vas‐ cularization and proliferation[101, 102]. This practise is generally dependent on conditions such as hypoxia, where tumour cells find themselves in acute need of supply and transdif‐ ferentiate in vascular progenitors[103-105]. Vascular mimicry is a remarkable demonstration of this CSC-trait. Tumour cells in this process align into channel-like structures, gain EC gene expression, acquire and EC phenotype, and roughly function as blood vessel (fig. 3B). Suggested mechanisms by which tumour cells can differentiate into vascular progenitor in‐

Many Ets transcription factors have a suggested or confirmed role in tumour angiogenesis and progression. Probably the most obvious Ets members to be involved in tumorigenesis are Fli1 and ERG, which have been acknowledged for their role in embryonic angiogenesis and vasculogenesis in a previous section of this chapter, but also ETS1/2 and several mem‐

that crosstalk with VEGF – are beguiling (lymph-)angiogenic players[83, 84].

tumour vascularization.

14 Research Directions in Tumor Angiogenesis

the case in renal cancinoma's[98].

*3.3.1. Ets transcription factors*

clude signalling through VEGF and IKKβ [102, 106].

**3.3. Dysregulation of transcriptional angiogenic pathways**

The presence and role of FoxC2 in tumour angiogenesis has been fairly well character‐ ized over the past few years, and it has been shown that the expression of FoxC2 in tu‐ mour endothelium coincides with neovascularization. This further supports the notion of Etv2 recurrence during tumour vascularization because of the synergistic function be‐ tween these transcription factors in regulating endothelial genes expression through the FOX:ETS motif.

FoxC2 overexpression is associated with aggressive human cancers, and has been shown to be overexpressed in mammary breast cancer cells *in vitro* where it directly promotes a meta‐ stasis phenotype[128]. More recently, FoxC2 was detected in the tumour ECs of human and mouse melanomas, and it therefore hypothesized that FoxC2 directly contributes to tumour angiogenesis[129]. In a B16 melanoma mouse model, the high expression level of FoxC2 in tumour cells and endothelium correlates with the induced expression of a set of angiogenic factors, such as Notch ligand Dll4, MMP-2, Pdgfβ and VEGF. Deleting one copy of FoxC2 causes reduction of their expression levels, and these FoxC2 heterozygous mutants also dis‐ play reduced angiogenesis and correspondingly perturbed tumour growth with signs of tu‐ mour necrosis [129]. This is in line with the roles of the suggested targets of FoxC2 in tumour neovascularization[127, 130], and the pro-migratory and angiogenic phenotype of FoxC2 overexpressing ECs[129, 131] (fig 3c).

*3.3.3. SoxF transcription factors*

improved by overexpressing functional SOX18[52].

tive tumours [146].

**4. Concluding remarks**

ulators that can flick cell fate switches.

SOXF is expressed transiently in the developing endothelium and then again during patho‐ logical conditions, such as wound healing where SOX18 is reexpressed in the capillary endo‐ thelium[145], and in tumorigenesis where SOX18 is reexpressed in the tumour stroma[146], including the blood and lymphatic vasculature[52, 147]. Recently, SOXF transcription factors have emerged as novel prognostic markers during gastric cancer progression, as SOX7, SOX17 and particularly SOX18 are frequently overexpressed in gastric tumour tissue of hu‐ man cancer patients, and survival rates are considerably lower for patients with SOX18 posi‐

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

17

The role of Sox18 in tumour angiogenesis has been studied in SOX18-null, and SOXF loss of function (SOX18 dominant negative mutant-) mice. These studies revealed that melanoma tumours grow more slowly in absence of SOX18 protein or function *in vivo*, with a corre‐ sponding reduction in tumour associated microvessel density[52] (fig. 3c). This was further illustrated *in vitro*, where ECs and human breast cancer cells with the dominant negative form of SOX18 proliferate poorly, and tube formation of ECs is impaired, which could be

SOX18 has also been shown to directly facilitate the metastatic spread of tumour cells to the sentinel lymph node in mice[147]. This is likely to be achieved by promoting neolymphan‐ giogenesis in the tumour microenvironment and thereby paving the way for tumour cell mi‐ gration towards the draining lymph node. During tumour growth, SOX18 has been shown to be reexpressed in LECs and is suggested to promote lymphatic vascular expansion[147]. Indeed, SOX18 heterozygous mutant mice have reduced lymphatic vessel density, which is accompanied by a decrease in lymphatic drainage and sentinel lymph node metastasis[147]. Taken together, these observations allocate an important role to SOX18 and possibly other SOXF transcription factors in regulation tumour vascularization. A recent finding descibes that SOX18 expression in tumour tissue is regulated on an epigenetic level by multiple states of promoter-methylation, which underlines the intricacy and divergency of transcriptional programmes in tumours[148]. With a role for SOXF members in arteriovenous specification, angiogenesis and lymphangiogenesis, their dysregulation in tumour settings might be a pa‐

rameter influencing the heterogeneity and overabundance of tumour vasculature.

The blood and lymphatic vascular systems are crucial in higher vertebrates for the transport of fluids, oxygen, signalling molecules, immune cells, waste material and other components that maintain homeostasis in the body. These systems develop very early on during embry‐ onic development and are orchestrated by a finely tuned combination of transcriptional reg‐

The transcriptional networks that underlie EC specification are usually transient or at least very well ordered in the embryo, but this all changes in tumour settings where they are dis‐

Tumour-induced endothelial to mesenchymal transition can promote FoxC2 expression, which feeds back into further mesenchymal differentiation[128, 132]. This can for a part ex‐ plain the pro-tumorigenic character of FoxC2, as it increases the ability of tumour ECs to mi‐ grate and proliferate, and prevents entry of tumour ECs into a quiescent state. Interestingly, FoxC2 heterozygous mutant mice indeed show a reduced amount of tumour-associated fi‐ broblasts, corroborating this hypothesis[129]. FoxC2 may further contribute to tumour an‐ giogenesis by recruiting mesenchymal stem cells[133], or endothelial progenitor cells [134], although this has yet to be determined.

Interestingly, FoxC1 is also upregulated in some tumour but its role in in tumour angiogene‐ sis is unclear, as deletion of one copy of FoxC 1in mice does not seem to affect melanoma tumour growth or angiogenesis[129]. Also, neither FoxC1 nor FoxC2 explicitly affect tumour lymphangiogenesis as lymphatic marker Lyve-1 and Prox1 expression levels are independ‐ ent of FoxC1/2 activity in melanoma tumours[129].

FoxO transcription factors operate, in contrast to FoxC1, as tumour suppres‐ sors[135-137]. Their function in mediating PI3K-AKT and HIF signalling make them key regulators of cell cycle and apoptosis, and therefore, inactivation of FoxO's is frequently observed in cancer[136, 138-141]. Mouse studies have revealed that FoxOs display func‐ tional redundancy in tumour suppression and vascular homeostasis, and triple FoxO knockout (FoxO1, FoxO3, FoxO4) mice develop aggressive tumours with a poor surviv‐ al rate, and have widely altered expression levels of EC-survival and vascular genes[137]. FoxO1 is required for embryonic vascular development, and its inactivation in cancer has repercussions on tumour vascularization, which is confirmed by vascular remodelling defects in FoxO1-null mice and their established crosstalk with VEGF-sig‐ nalling[39]. This instigates a paradox wherein tumour cells gain 'immortality' through FoxO inactivation, and simultaneously seem to lose vessel functionalization via the same mechanism[39, 141-143].

On a particular note, FoxO3 depletion in tumour cells can attenuate migration due to reduction in MMP expression, leading to decreased tumour size[144]. Henceforth, the compound FoxO alterations in tumours, and modifications to specific Fox members, must be further explored to fully appreciate the contexts dependent roles of these tran‐ scription factors.

#### *3.3.3. SoxF transcription factors*

stasis phenotype[128]. More recently, FoxC2 was detected in the tumour ECs of human and mouse melanomas, and it therefore hypothesized that FoxC2 directly contributes to tumour angiogenesis[129]. In a B16 melanoma mouse model, the high expression level of FoxC2 in tumour cells and endothelium correlates with the induced expression of a set of angiogenic factors, such as Notch ligand Dll4, MMP-2, Pdgfβ and VEGF. Deleting one copy of FoxC2 causes reduction of their expression levels, and these FoxC2 heterozygous mutants also dis‐ play reduced angiogenesis and correspondingly perturbed tumour growth with signs of tu‐ mour necrosis [129]. This is in line with the roles of the suggested targets of FoxC2 in tumour neovascularization[127, 130], and the pro-migratory and angiogenic phenotype of

Tumour-induced endothelial to mesenchymal transition can promote FoxC2 expression, which feeds back into further mesenchymal differentiation[128, 132]. This can for a part ex‐ plain the pro-tumorigenic character of FoxC2, as it increases the ability of tumour ECs to mi‐ grate and proliferate, and prevents entry of tumour ECs into a quiescent state. Interestingly, FoxC2 heterozygous mutant mice indeed show a reduced amount of tumour-associated fi‐ broblasts, corroborating this hypothesis[129]. FoxC2 may further contribute to tumour an‐ giogenesis by recruiting mesenchymal stem cells[133], or endothelial progenitor cells [134],

Interestingly, FoxC1 is also upregulated in some tumour but its role in in tumour angiogene‐ sis is unclear, as deletion of one copy of FoxC 1in mice does not seem to affect melanoma tumour growth or angiogenesis[129]. Also, neither FoxC1 nor FoxC2 explicitly affect tumour lymphangiogenesis as lymphatic marker Lyve-1 and Prox1 expression levels are independ‐

FoxO transcription factors operate, in contrast to FoxC1, as tumour suppres‐ sors[135-137]. Their function in mediating PI3K-AKT and HIF signalling make them key regulators of cell cycle and apoptosis, and therefore, inactivation of FoxO's is frequently observed in cancer[136, 138-141]. Mouse studies have revealed that FoxOs display func‐ tional redundancy in tumour suppression and vascular homeostasis, and triple FoxO knockout (FoxO1, FoxO3, FoxO4) mice develop aggressive tumours with a poor surviv‐ al rate, and have widely altered expression levels of EC-survival and vascular genes[137]. FoxO1 is required for embryonic vascular development, and its inactivation in cancer has repercussions on tumour vascularization, which is confirmed by vascular remodelling defects in FoxO1-null mice and their established crosstalk with VEGF-sig‐ nalling[39]. This instigates a paradox wherein tumour cells gain 'immortality' through FoxO inactivation, and simultaneously seem to lose vessel functionalization via the

On a particular note, FoxO3 depletion in tumour cells can attenuate migration due to reduction in MMP expression, leading to decreased tumour size[144]. Henceforth, the compound FoxO alterations in tumours, and modifications to specific Fox members, must be further explored to fully appreciate the contexts dependent roles of these tran‐

FoxC2 overexpressing ECs[129, 131] (fig 3c).

16 Research Directions in Tumor Angiogenesis

although this has yet to be determined.

same mechanism[39, 141-143].

scription factors.

ent of FoxC1/2 activity in melanoma tumours[129].

SOXF is expressed transiently in the developing endothelium and then again during patho‐ logical conditions, such as wound healing where SOX18 is reexpressed in the capillary endo‐ thelium[145], and in tumorigenesis where SOX18 is reexpressed in the tumour stroma[146], including the blood and lymphatic vasculature[52, 147]. Recently, SOXF transcription factors have emerged as novel prognostic markers during gastric cancer progression, as SOX7, SOX17 and particularly SOX18 are frequently overexpressed in gastric tumour tissue of hu‐ man cancer patients, and survival rates are considerably lower for patients with SOX18 posi‐ tive tumours [146].

The role of Sox18 in tumour angiogenesis has been studied in SOX18-null, and SOXF loss of function (SOX18 dominant negative mutant-) mice. These studies revealed that melanoma tumours grow more slowly in absence of SOX18 protein or function *in vivo*, with a corre‐ sponding reduction in tumour associated microvessel density[52] (fig. 3c). This was further illustrated *in vitro*, where ECs and human breast cancer cells with the dominant negative form of SOX18 proliferate poorly, and tube formation of ECs is impaired, which could be improved by overexpressing functional SOX18[52].

SOX18 has also been shown to directly facilitate the metastatic spread of tumour cells to the sentinel lymph node in mice[147]. This is likely to be achieved by promoting neolymphan‐ giogenesis in the tumour microenvironment and thereby paving the way for tumour cell mi‐ gration towards the draining lymph node. During tumour growth, SOX18 has been shown to be reexpressed in LECs and is suggested to promote lymphatic vascular expansion[147]. Indeed, SOX18 heterozygous mutant mice have reduced lymphatic vessel density, which is accompanied by a decrease in lymphatic drainage and sentinel lymph node metastasis[147].

Taken together, these observations allocate an important role to SOX18 and possibly other SOXF transcription factors in regulation tumour vascularization. A recent finding descibes that SOX18 expression in tumour tissue is regulated on an epigenetic level by multiple states of promoter-methylation, which underlines the intricacy and divergency of transcriptional programmes in tumours[148]. With a role for SOXF members in arteriovenous specification, angiogenesis and lymphangiogenesis, their dysregulation in tumour settings might be a pa‐ rameter influencing the heterogeneity and overabundance of tumour vasculature.

#### **4. Concluding remarks**

The blood and lymphatic vascular systems are crucial in higher vertebrates for the transport of fluids, oxygen, signalling molecules, immune cells, waste material and other components that maintain homeostasis in the body. These systems develop very early on during embry‐ onic development and are orchestrated by a finely tuned combination of transcriptional reg‐ ulators that can flick cell fate switches.

The transcriptional networks that underlie EC specification are usually transient or at least very well ordered in the embryo, but this all changes in tumour settings where they are dis‐ torted and exploited to induce chronic angiogenesis and vasculogenesis. Although most at‐ tention in therapeutic cancer research over the years has gone to growth factor signalling or other downstream players of proliferation, migration and morphogenesis there seems to be an emerging paradigm shift in studying both prognostic and therapeutic potential of funda‐ mental transcription factors. The ETS, Forkhead, and SOXF transcription factors discussed in this overview are in many ways associated with tumour proliferation and vascularization. Studies in developmental biology have laid the groundwork for further study of transcrip‐ tion factors dysregulation in tumours. Remarkably, there is a high level over crosstalk with traditional VEGF signalling either through increased VEGF bio-availability, transduction, or responsiveness within these transcriptional networks.

[7] Friedl, P. and S. Alexander, *Cancer invasion and the microenvironment: plasticity and*

Transcriptional Modulation of Tumour Induced Angiogenesis

http://dx.doi.org/10.5772/54055

19

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In the years to come, these transcription factors will expectantly further develop as prognos‐ tic tools for tumorigenesis and possibly arise as molecular targets for treatment of malignant tumours. At the very least, studying these fundamental regulators in cancer will add to our understanding of tumour origins and the tools they utilize to achieve proliferation, angio‐ genesis, and malignancy.

#### **Author details**

Jeroen Overman and Mathias François

\*Address all correspondence to: m.francois@imb.uq.edu.au

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

#### **References**


[7] Friedl, P. and S. Alexander, *Cancer invasion and the microenvironment: plasticity and reciprocity.* Cell, 2011. 147(5): p. 992-1009.

torted and exploited to induce chronic angiogenesis and vasculogenesis. Although most at‐ tention in therapeutic cancer research over the years has gone to growth factor signalling or other downstream players of proliferation, migration and morphogenesis there seems to be an emerging paradigm shift in studying both prognostic and therapeutic potential of funda‐ mental transcription factors. The ETS, Forkhead, and SOXF transcription factors discussed in this overview are in many ways associated with tumour proliferation and vascularization. Studies in developmental biology have laid the groundwork for further study of transcrip‐ tion factors dysregulation in tumours. Remarkably, there is a high level over crosstalk with traditional VEGF signalling either through increased VEGF bio-availability, transduction, or

In the years to come, these transcription factors will expectantly further develop as prognos‐ tic tools for tumorigenesis and possibly arise as molecular targets for treatment of malignant tumours. At the very least, studying these fundamental regulators in cancer will add to our understanding of tumour origins and the tools they utilize to achieve proliferation, angio‐

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Australia

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**Chapter 2**

**Roles of SRF in Endothelial Cells During Hypoxia**

Oxygen is a basic need for human life. Maintaining adequate oxygen supply is essential for proper cellular functions. In normal tissue, the oxygen supply usually matches metabolic requirements, and even if there is a brief oxygen shortage, the body can overcome it by an increase in the oxygen extracted from the blood or an increase in local blood flow. In advanced solid tumors, however, due to uncontrolled cell proliferation, the oxygen consumption rate often exceeds the oxygen available around the area, resulting in local hypoxia. The diffusion distance from blood vessels to surrounding tissues is usually no more than 100-200 µm; therefore, the further into the center of the tumors, the lower the oxygen level gets. As measured by Eppendorf probe, pO2 in normal tissue is between 17 and 65 mm Hg, while in

As a result of oxygen deficiency, two things can happen to the suffering cells. Cells can either stop proliferation and die of apoptosis or necrosis, or fight back by taking adaptive processes that lead to increased proliferation, migration and tissue reorganization. While the ultimate fate of the cells varies with tissue type, the severity and duration of hypoxia play critical roles in choosing the direction. In moderate oxygen decline (~ 2-7 mm Hg), the cells in oxygen starvation and the cells carrying oxygen (red blood cells) run towards each other. Cancer cells can move away from their original locations to where oxygen is sufficient, while endothelial cells in the blood vessels can also take an action to move out to form new vessels to bring oxygen towards the center of hypoxia. The former process is known as metastasis, and the latter is angiogenesis. Angiogenesis and metastasis support cancer cells to survive through hypoxic crisis and allow malignant progression. Under severe hypoxic condition (< 1 mm Hg), however, cells are prone to die of apoptosis if glycolytic ATP available, otherwise, die of

> © 2013 Chai; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

wide range of tumors, pO2 can go down to 2 mm or even to zero.

Jianyuan Chai

**1. Introduction**

necrosis.

http://dx.doi.org/10.5772/52182

## **Roles of SRF in Endothelial Cells During Hypoxia**

#### Jianyuan Chai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52182

#### **1. Introduction**

Oxygen is a basic need for human life. Maintaining adequate oxygen supply is essential for proper cellular functions. In normal tissue, the oxygen supply usually matches metabolic requirements, and even if there is a brief oxygen shortage, the body can overcome it by an increase in the oxygen extracted from the blood or an increase in local blood flow. In advanced solid tumors, however, due to uncontrolled cell proliferation, the oxygen consumption rate often exceeds the oxygen available around the area, resulting in local hypoxia. The diffusion distance from blood vessels to surrounding tissues is usually no more than 100-200 µm; therefore, the further into the center of the tumors, the lower the oxygen level gets. As measured by Eppendorf probe, pO2 in normal tissue is between 17 and 65 mm Hg, while in wide range of tumors, pO2 can go down to 2 mm or even to zero.

As a result of oxygen deficiency, two things can happen to the suffering cells. Cells can either stop proliferation and die of apoptosis or necrosis, or fight back by taking adaptive processes that lead to increased proliferation, migration and tissue reorganization. While the ultimate fate of the cells varies with tissue type, the severity and duration of hypoxia play critical roles in choosing the direction. In moderate oxygen decline (~ 2-7 mm Hg), the cells in oxygen starvation and the cells carrying oxygen (red blood cells) run towards each other. Cancer cells can move away from their original locations to where oxygen is sufficient, while endothelial cells in the blood vessels can also take an action to move out to form new vessels to bring oxygen towards the center of hypoxia. The former process is known as metastasis, and the latter is angiogenesis. Angiogenesis and metastasis support cancer cells to survive through hypoxic crisis and allow malignant progression. Under severe hypoxic condition (< 1 mm Hg), however, cells are prone to die of apoptosis if glycolytic ATP available, otherwise, die of necrosis.

© 2013 Chai; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hypoxia-induced apoptosis proceeds through the mitochondrial pathway, as the mitochon‐ dria are the primary site of oxygen consumption in a cell. Under normoxic conditions, the mitochondria consume about 90% of available oxygen in the generation of ATP through oxidative phosphorylation in order to meet the metabolic needs of the cell [1, 2]. When there is not sufficient oxygen to support this process, mitochondrial damage occurs, which leads to apoptotic cell death.

dominantly expressed on endothelial cells [25]. In addition, VEGF can also bind to three other transmembrane proteins: VEGFR-3 (Flt-4), which is expressed mainly on lymphatic endothelial cells and only responds to VEGF-C and -D, Neuropilin-1 and Neuropilin-2, which work as co-receptors with VEGFR-2 [26]. Hypoxia-induced VEGF up-regulation is considered to be the major driving force for angiogenesis during tumor progression [27]. Tremendous effort has been made in cancer chemotherapy to inhibit this process and has achieved some significant results, but some expectations have not been met. In addition to VEGF, HIF also regulates several other angiogenic factors such as placenta-like growth

Roles of SRF in Endothelial Cells During Hypoxia

http://dx.doi.org/10.5772/52182

31

Like HIF, p53 is expressed at a low level under normal oxygen conditions and degraded constantly by MDM2 through ubiquitination [29, 30]. Under cellular stress like hypoxia, however, ATM/ATR kinases become active and phosphorylate p53 at its N terminus, which disrupts its interaction with MDM2 and thus, p53 becomes stabilized and moves into the nucleus to activate pro-apoptotic genes [31]. As mentioned above, hypoxia indu‐ ces apoptosis through mitochondrial damage. The mitochondrial integrity is guarded by Bcl-2 family proteins which include anti-apoptotic members like Bcl-2 and Bcl-XL, and al‐ so pro-apoptotic members, such as Bax and Bak. The balance between these two teams is critical to the fate of a cell. Bcl-2 is an integral membrane protein that targets the outer mitochondrial membrane, and it can form homodimers with each other or heterodimers with Bax. Bax, on the other hand, can do the same. When Bcl-2 predominates, mitochon‐ dria stay intact and cells are protected. However, while Bax is in excess, Bax homodim‐ ers become dominant, the cells are susceptible to apoptosis. Bax expression is regulated by p53; therefore, p53 activation increases the ratio of Bax to Bacl-2 and reduces the chance of Bcl-2 and Bax association. It has been postulated that 50% reduction in the for‐ mation of Bcl-2/Bax complexes can drive the cells toward apoptosis [32]. When Bax in‐ serts into the outer mitochondrial membrane, it opens pores to allow the molecules sequestered in between outer and inner mitochondrial membrane to leak out into the cy‐ tosol. One of the released molecules is cytochrome c, which can bind to the apoptotic protease activating factor-1 (APAF-1] and promote it to form an apoptosome. The apop‐ tosome then binds caspase-9 and activates it to cleave two other caspases, caspase-3 and -7. These two caspases orchestrate apoptosis through cleavage of key substrates within

p53 and HIF1α are an odd couple, one supporting cell death and the other supporting cell survival. These two transcription factors can interact with each other directly because HIF1α contains two p53-binding sites within its ODD domain [33]. Unlike HIF, p53 appears to be less sensitive to oxygen level change. Under moderate hypoxic conditions, HIF1α binds to HIF1β to activate genes that promote cell survival, while p53 still remains inactive. Some *in vitro* studies even showed that in such a situation p53 actually promotes HDM2-mediated

factor, platelet-derived growth factor, angiopoietin-1 and -2 [28].

**3. p53**

the cell, resulting in cell death.

To live or to die for a cell under hypoxia is all regulated through different expression and activation of transcription factors. A number of transcription factors have been reported to respond to oxygen deficiency, including AP-1 [3], FOS [4], JUN [4], CREB/ATF [5], DEC1 [6], EGR1 [7], ETS1 [8], GADD153 [9], GATA2 [10], MASH2 [11], NF-IL-6 [12], NFĸB [13], RTEF-1 [14], SMADs [15], SP1 [16], STAT5 [17], and of course, the most popular ones, HIF [18] and p53 [19].

#### **2. Hypoxia inducible factor**

Hypoxia inducible factor (HIF) is the best studied transcription factor in hypoxia. When‐ ever there is a discussion about hypoxia, HIF is always an inevitable topic. HIF is com‐ posed of two subunits, α and β. While HIFβ is constitutively expressed, HIFα functions more like an oxygen sensor, varying in response to oxygen level [20]. HIFα has an ex‐ tremely short half-life under normoxic conditions due to ubiquitination by von Hippel-Lindau factor (VHL). Hypoxia does not change HIFα expression per se but stabilizes it by inhibiting hydroxylation at prolines 402 and 564 so that VHL can no longer bind to HIFα to cause proteasomal degradation. Instead, it enables HIFα to bind to HIFβ in the nucleus, generating a functional heterodimeric transcription factor that is able to activate genes that contain hypoxia-response elements (5'-RCGTG-3'), such as genes coding for glucose transporters, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and erythropoietin (EPO) [21, 22]. In normal tissue, the expression of such genes is to counteract the detrimental impact of hypoxia and to help cells to sur‐ vive through oxygen crisis. In cancer, however, this role of HIF is abused to support tu‐ mor growth and resistance to chemotherapy. Up to date, there are three members in HIF family. HIF-1α is most ubiquitously expressed, while HIF-2α, which shares 48% identity and similar functions with HIF1α, is more restricted to endothelial cells [23]. HIF-3α is the least characterized but may function as a negative regulator of hypoxia, as its dimer with the β subunit has no transcriptional activity [24].

The most prominent role of HIF during hypoxia is to support angiogenesis through tran‐ scriptional activation of VEGF. VEGF belongs to a family that contains VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta-like growth factor. VEGF-A, the first growth factor that was identified to have special effects on endothelial cells, further splits into five isoforms. VEGF is mainly produced by endothelial cells, macrophages, fibroblasts, and smooth muscle cells. It promotes endothelial cell migration, proliferation and surviv‐ al through its receptors, VEGFR-1 (Flt-1) and/or VEGFR-2 (Flk-1/KDR), which are pre‐ dominantly expressed on endothelial cells [25]. In addition, VEGF can also bind to three other transmembrane proteins: VEGFR-3 (Flt-4), which is expressed mainly on lymphatic endothelial cells and only responds to VEGF-C and -D, Neuropilin-1 and Neuropilin-2, which work as co-receptors with VEGFR-2 [26]. Hypoxia-induced VEGF up-regulation is considered to be the major driving force for angiogenesis during tumor progression [27]. Tremendous effort has been made in cancer chemotherapy to inhibit this process and has achieved some significant results, but some expectations have not been met. In addition to VEGF, HIF also regulates several other angiogenic factors such as placenta-like growth factor, platelet-derived growth factor, angiopoietin-1 and -2 [28].

#### **3. p53**

Hypoxia-induced apoptosis proceeds through the mitochondrial pathway, as the mitochon‐ dria are the primary site of oxygen consumption in a cell. Under normoxic conditions, the mitochondria consume about 90% of available oxygen in the generation of ATP through oxidative phosphorylation in order to meet the metabolic needs of the cell [1, 2]. When there is not sufficient oxygen to support this process, mitochondrial damage occurs, which leads to

To live or to die for a cell under hypoxia is all regulated through different expression and activation of transcription factors. A number of transcription factors have been reported to respond to oxygen deficiency, including AP-1 [3], FOS [4], JUN [4], CREB/ATF [5], DEC1 [6], EGR1 [7], ETS1 [8], GADD153 [9], GATA2 [10], MASH2 [11], NF-IL-6 [12], NFĸB [13], RTEF-1 [14], SMADs [15], SP1 [16], STAT5 [17], and of course, the most popular ones, HIF [18] and p53

Hypoxia inducible factor (HIF) is the best studied transcription factor in hypoxia. When‐ ever there is a discussion about hypoxia, HIF is always an inevitable topic. HIF is com‐ posed of two subunits, α and β. While HIFβ is constitutively expressed, HIFα functions more like an oxygen sensor, varying in response to oxygen level [20]. HIFα has an ex‐ tremely short half-life under normoxic conditions due to ubiquitination by von Hippel-Lindau factor (VHL). Hypoxia does not change HIFα expression per se but stabilizes it by inhibiting hydroxylation at prolines 402 and 564 so that VHL can no longer bind to HIFα to cause proteasomal degradation. Instead, it enables HIFα to bind to HIFβ in the nucleus, generating a functional heterodimeric transcription factor that is able to activate genes that contain hypoxia-response elements (5'-RCGTG-3'), such as genes coding for glucose transporters, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and erythropoietin (EPO) [21, 22]. In normal tissue, the expression of such genes is to counteract the detrimental impact of hypoxia and to help cells to sur‐ vive through oxygen crisis. In cancer, however, this role of HIF is abused to support tu‐ mor growth and resistance to chemotherapy. Up to date, there are three members in HIF family. HIF-1α is most ubiquitously expressed, while HIF-2α, which shares 48% identity and similar functions with HIF1α, is more restricted to endothelial cells [23]. HIF-3α is the least characterized but may function as a negative regulator of hypoxia, as its dimer

The most prominent role of HIF during hypoxia is to support angiogenesis through tran‐ scriptional activation of VEGF. VEGF belongs to a family that contains VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E and placenta-like growth factor. VEGF-A, the first growth factor that was identified to have special effects on endothelial cells, further splits into five isoforms. VEGF is mainly produced by endothelial cells, macrophages, fibroblasts, and smooth muscle cells. It promotes endothelial cell migration, proliferation and surviv‐ al through its receptors, VEGFR-1 (Flt-1) and/or VEGFR-2 (Flk-1/KDR), which are pre‐

apoptotic cell death.

30 Research Directions in Tumor Angiogenesis

**2. Hypoxia inducible factor**

with the β subunit has no transcriptional activity [24].

[19].

Like HIF, p53 is expressed at a low level under normal oxygen conditions and degraded constantly by MDM2 through ubiquitination [29, 30]. Under cellular stress like hypoxia, however, ATM/ATR kinases become active and phosphorylate p53 at its N terminus, which disrupts its interaction with MDM2 and thus, p53 becomes stabilized and moves into the nucleus to activate pro-apoptotic genes [31]. As mentioned above, hypoxia indu‐ ces apoptosis through mitochondrial damage. The mitochondrial integrity is guarded by Bcl-2 family proteins which include anti-apoptotic members like Bcl-2 and Bcl-XL, and al‐ so pro-apoptotic members, such as Bax and Bak. The balance between these two teams is critical to the fate of a cell. Bcl-2 is an integral membrane protein that targets the outer mitochondrial membrane, and it can form homodimers with each other or heterodimers with Bax. Bax, on the other hand, can do the same. When Bcl-2 predominates, mitochon‐ dria stay intact and cells are protected. However, while Bax is in excess, Bax homodim‐ ers become dominant, the cells are susceptible to apoptosis. Bax expression is regulated by p53; therefore, p53 activation increases the ratio of Bax to Bacl-2 and reduces the chance of Bcl-2 and Bax association. It has been postulated that 50% reduction in the for‐ mation of Bcl-2/Bax complexes can drive the cells toward apoptosis [32]. When Bax in‐ serts into the outer mitochondrial membrane, it opens pores to allow the molecules sequestered in between outer and inner mitochondrial membrane to leak out into the cy‐ tosol. One of the released molecules is cytochrome c, which can bind to the apoptotic protease activating factor-1 (APAF-1] and promote it to form an apoptosome. The apop‐ tosome then binds caspase-9 and activates it to cleave two other caspases, caspase-3 and -7. These two caspases orchestrate apoptosis through cleavage of key substrates within the cell, resulting in cell death.

p53 and HIF1α are an odd couple, one supporting cell death and the other supporting cell survival. These two transcription factors can interact with each other directly because HIF1α contains two p53-binding sites within its ODD domain [33]. Unlike HIF, p53 appears to be less sensitive to oxygen level change. Under moderate hypoxic conditions, HIF1α binds to HIF1β to activate genes that promote cell survival, while p53 still remains inactive. Some *in vitro* studies even showed that in such a situation p53 actually promotes HDM2-mediated HIF1α degradation [34]. Under severe oxygen poverty, however, HIF1α becomes dephos‐ phorylated and may choose to help p53 to induce cell death [35].

#### **4. Hypoxia activates SRF**

Although many transcription factors have been studied extensively under hypoxia [36], the reaction of serum response factor (SRF) to oxygen shortage has rarely been discussed.

SRF regulates numerous genes that are involved in cellular responses to mitogenic stimu‐ li as well as cellular stress [37-39]. These genes fall into many diversified categories, in‐ cluding immediate early genes (FOS, EGR1, etc.), cytoskeletal genes (ACTB, CFL1, DES, DSTN, TTN, KRT17, etc.), muscle-related genes (ACTA2, MYH6, MYH11, SM22α, TNNT1, ATP2A1, etc.), growth factors (IGF2, FGF10, FGFR3, TGFB1i1, etc.), extracellular matrix proteins (CCN1, CTGF, etc.), cell adhesion molecules (ITGA1, ITGA5, ITGB1, etc.), intercellular junctional molecules (TJP1, CDH5, CDH11, etc.), neuronal receptors (NR4A1, NR4A2, etc.), and apoptosis regulators (BCL2). This list is still growing. All these genes contain a common DNA sequence, CC(A/T)6GG, so-called CArG box or serum response element (SRE), which SRF recognizes. Some of these genes contain multiple CArG boxes, for example, EGR1 has six and CCN1 has five, and even SRF itself has four SRE sequen‐ ces [40], indicating a tight regulation by SRF. In addition to the hundreds of genes that SRF directly regulates, a growing number of genes that do not contain SRE have been found to respond to SRF activition [41, 42].

Hypoxia is a form of stress to the cells; therefore, it triggers a response from SRF undoubtedly. As shown in Figure 1, under hypoxic condition, there is not only an increase in the level of SRF expression (Figure 1A), but also an increase in SRF phosphorylation (Figure 1A), which enhances SRF binding activity to SRE (Figure 1B). Moreover, this activation of SRF is inde‐ pendent from either HIF or p53, because neither shut down of HIF with its specific inhibitor Dimethyl Bisphenol A (DBA) (Figure 1C), nor inhibition of p53 with Pifithrin-α (Figure 1D) has impact on hypoxia-induced SRF activation.

**Figure 1.** Hypoxia activates SRF in mouse brain endothelial cells (bEND3) regardless HIF and p53 status. A. Cells were cultured in a hypoxic chamber (5% CO2 : 94% N2 : 1% O2) at 37˚C for 2, 6 and 24 hours. Total protein was isolated and immune-precipitated with an antibody against SRF. Total and phosphorylated SRF were detected by Western blot analysis. B. SRF protein activity was analyzed by electrophoretic mobility shift assay with P32-labeled consensus SRE (SRE) and mutant SRE (mSRE) oligos. SRF to SRE binding activity was increased by hypoxic treatment. The lack of bind‐ ing ability to the mutant SRE probe as well as the super shift with the SRF antibody (anti-SRF) confirmed the binding specificity. C. In the presence of Dimethyl Bisphenol A (DBA), a specific inhibitor for HIF, hypoxic treatment failed to stabilize HIF1α, but did not affect SRF activation. D. Incubation with p53 inhibitor Pifithrin-α suppressed p53 activation

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Here we show that hypoxia-induced angiogenic activity in brain endothelial cells is completely lost when SRF is knocked down by RNA interference (Figure 2), indicating that SRF is essential to hypoxia-induced angiogenesis. On the other hand, when extra copies of SRF gene are introduced into these cells, hypoxia-induced angiogenic activity is enhanced. It has been postulated that hypoxia induces angiogenesis through HIF-VEGF-MAPK/Rho-SRF pathway [45]. From our previous study [43], we know that VEGF does activate SRF through MAPK and Rho pathways. However, this is just one side of a coin. As shown above (Figure 1), the increase of VEGF signaling during hypoxia is due to HIF activation, while hypoxia activates SRF independently from HIF and therefore, independently from VEGF as well. SRF responds to hypoxia directly as other transcription factors like HIF and p53. In addition, SRF also serves

by hypoxia but did not affect SRF either.

#### **5. SRF supports hypoxia-induced angiogenesis**

Previously, we have shown that SRF is required for VEGF-induced *in vitro* angiogenesis, and without SRF, VEGF cannot induce endothelial cell proliferation and migration, which are essential for angiogenesis [43]. Our findings were confirmed and extended later by an *in vivo* study on mouse embryonic development, which demonstrated that knockout of SRF in endothelial cells impairs sprouting angiogenesis from arteries to veins [44]. Transcriptional analysis showed that SRF deficiency not only had negative impact on genes responsible for endothelial connection (e.g. VE-cadherin) and adhesion (e.g. integrin α5 and β1), but also suppressed angiogenic factors like VEGF and angiopoietin-1 and -2.

HIF1α degradation [34]. Under severe oxygen poverty, however, HIF1α becomes dephos‐

Although many transcription factors have been studied extensively under hypoxia [36], the reaction of serum response factor (SRF) to oxygen shortage has rarely been discussed.

SRF regulates numerous genes that are involved in cellular responses to mitogenic stimu‐ li as well as cellular stress [37-39]. These genes fall into many diversified categories, in‐ cluding immediate early genes (FOS, EGR1, etc.), cytoskeletal genes (ACTB, CFL1, DES, DSTN, TTN, KRT17, etc.), muscle-related genes (ACTA2, MYH6, MYH11, SM22α, TNNT1, ATP2A1, etc.), growth factors (IGF2, FGF10, FGFR3, TGFB1i1, etc.), extracellular matrix proteins (CCN1, CTGF, etc.), cell adhesion molecules (ITGA1, ITGA5, ITGB1, etc.), intercellular junctional molecules (TJP1, CDH5, CDH11, etc.), neuronal receptors (NR4A1, NR4A2, etc.), and apoptosis regulators (BCL2). This list is still growing. All these genes contain a common DNA sequence, CC(A/T)6GG, so-called CArG box or serum response element (SRE), which SRF recognizes. Some of these genes contain multiple CArG boxes, for example, EGR1 has six and CCN1 has five, and even SRF itself has four SRE sequen‐ ces [40], indicating a tight regulation by SRF. In addition to the hundreds of genes that SRF directly regulates, a growing number of genes that do not contain SRE have been

Hypoxia is a form of stress to the cells; therefore, it triggers a response from SRF undoubtedly. As shown in Figure 1, under hypoxic condition, there is not only an increase in the level of SRF expression (Figure 1A), but also an increase in SRF phosphorylation (Figure 1A), which enhances SRF binding activity to SRE (Figure 1B). Moreover, this activation of SRF is inde‐ pendent from either HIF or p53, because neither shut down of HIF with its specific inhibitor Dimethyl Bisphenol A (DBA) (Figure 1C), nor inhibition of p53 with Pifithrin-α (Figure 1D)

Previously, we have shown that SRF is required for VEGF-induced *in vitro* angiogenesis, and without SRF, VEGF cannot induce endothelial cell proliferation and migration, which are essential for angiogenesis [43]. Our findings were confirmed and extended later by an *in vivo* study on mouse embryonic development, which demonstrated that knockout of SRF in endothelial cells impairs sprouting angiogenesis from arteries to veins [44]. Transcriptional analysis showed that SRF deficiency not only had negative impact on genes responsible for endothelial connection (e.g. VE-cadherin) and adhesion (e.g. integrin α5 and β1), but also

phorylated and may choose to help p53 to induce cell death [35].

**4. Hypoxia activates SRF**

32 Research Directions in Tumor Angiogenesis

found to respond to SRF activition [41, 42].

has impact on hypoxia-induced SRF activation.

**5. SRF supports hypoxia-induced angiogenesis**

suppressed angiogenic factors like VEGF and angiopoietin-1 and -2.

**Figure 1.** Hypoxia activates SRF in mouse brain endothelial cells (bEND3) regardless HIF and p53 status. A. Cells were cultured in a hypoxic chamber (5% CO2 : 94% N2 : 1% O2) at 37˚C for 2, 6 and 24 hours. Total protein was isolated and immune-precipitated with an antibody against SRF. Total and phosphorylated SRF were detected by Western blot analysis. B. SRF protein activity was analyzed by electrophoretic mobility shift assay with P32-labeled consensus SRE (SRE) and mutant SRE (mSRE) oligos. SRF to SRE binding activity was increased by hypoxic treatment. The lack of bind‐ ing ability to the mutant SRE probe as well as the super shift with the SRF antibody (anti-SRF) confirmed the binding specificity. C. In the presence of Dimethyl Bisphenol A (DBA), a specific inhibitor for HIF, hypoxic treatment failed to stabilize HIF1α, but did not affect SRF activation. D. Incubation with p53 inhibitor Pifithrin-α suppressed p53 activation by hypoxia but did not affect SRF either.

Here we show that hypoxia-induced angiogenic activity in brain endothelial cells is completely lost when SRF is knocked down by RNA interference (Figure 2), indicating that SRF is essential to hypoxia-induced angiogenesis. On the other hand, when extra copies of SRF gene are introduced into these cells, hypoxia-induced angiogenic activity is enhanced. It has been postulated that hypoxia induces angiogenesis through HIF-VEGF-MAPK/Rho-SRF pathway [45]. From our previous study [43], we know that VEGF does activate SRF through MAPK and Rho pathways. However, this is just one side of a coin. As shown above (Figure 1), the increase of VEGF signaling during hypoxia is due to HIF activation, while hypoxia activates SRF independently from HIF and therefore, independently from VEGF as well. SRF responds to hypoxia directly as other transcription factors like HIF and p53. In addition, SRF also serves as a downstream regulator in cell proliferation, adhesion and migration, thus any mitogenic factor that aims to stimulate such cellular activities requires SRF involvement.

Activation of eNOS, iNOS and SRF is dependent on Rho GTPase-regulated actin dynamics. Actin de-polymerization activates eNOS [62, 63] and iNOS [64, 65] but suppresses SRF, resulting in apoptosis [66, 67]. Conversely, actin polymerization activates SRF but suppresses

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**Figure 3.** Knockdown of SRF in brain endothelial cells increases hypoxia-induced apoptosis. bEND3 cells were cultured on cover slips under a hypoxic condition for 2, 6 and 24 hours. TUNEL assays were performed to detect apoptosis. Ap‐ parently, overexpression of SRF (SRF+) promoted cell survival, while knockdown of SRF (SRF-) made cells more vulnera‐

Moderate hypoxia induces cell adaptation but not apoptosis. However, when SRF is insuffi‐ cient (SRF-), cells become vulnerable to cellular stress and even a brief oxygen shortage can trigger apoptotic cell death (Figure 3). On the other hand, forced overexpression of SRF (SRF +) in these cells can make them more resistant to hypoxic damage and able to survive through even more harsh oxygen crisis. The advantage of SRF over HIF is its broad involvement in the molecular regulation of the cell machinery. Once SRF is activated, it not only promotes cell survival through up-regulation of growth factors and cytoskeletal components, but also protects mitochondrial integrity through up-regulation of anti-apoptotic proteins like Bcl-2 [68]. In another word, SRF supports cell survival at multiple levels. Up-regulation of growth factors stimulates cell proliferation and migration, which require adequate supplies of cytoskeletal proteins, because without cytoskeleton to provide the platform, cells cannot proliferate or migrate, and SRF makes sure these molecules available at the time of need. Finally, SRF also makes sure mitochondria intact so that they can provide the energy that cell proliferation and migration need. Mitochondrial integrity depends on the balance between pro-apoptotic and anti-apoptotic proteins, typically, BAX versus Bcl-2. Severe hypoxia activates p53, which drives up-regulation of BAX, pushing cells toward apoptosis, as BAX gene contains four binding sites for p53. On the other hand, hypoxia also activates SRF (as shown above), which drives up-regulation of Bcl-2, pushing cells toward survival, as Bcl-2 gene contains two SREs in its promoter [68]. The BAX and Bcl-2 fight turns into a wrestle between

eNOS and iNOS, supporting cell survival.

ble to hypoxic damage.

**Figure 2.** Knockdown of SRF in brain endothelial cells (bEND3) prevents hypoxia-induced angiogenesis. bEND3 cells were cultured in collagen gel matrix under a hypoxic condition. The collagen gel matrix was made of 50% type I colla‐ gen in HEPES (pH 8.5) Hanks buffer balanced growth medium. The mixture was solidified in 12-well plates at 37˚C for 20 minutes. Cells were mixed in the liquid gel, plated on top of the solidified gel in the 12-well plates and incubated at 37˚C for additional 20 minutes. More layers of cells were plated in the wells by repeating this step. Eventually, growth medium was added to the top of the solidified gel containing endothelial cells and the plates were incubated at 37˚C for a week. The control cells formed a cobblestone monolayer at the end, while SRF over-expressing cells (SRF+) moved vertically and horizontally within the gel matrix. The cells with SRF knockdown (SRF-), on the other hand, stayed inactively. Under hypoxia, both control and SRF+ cells formed cable-like structure, an indication of angiogenic activity, while the SRF- cells showed sign of death.

#### **6. SRF protects endothelial cells against hypoxia-induced apoptosis**

Several studies indicate that hypoxia-induced apoptosis is solely dependent on the mitochon‐ drial pathway [46-48], which is regulated by Bcl-2 family members [49, 50]. Hypoxia induces an increase in the ratio of the pro-apoptotic protein Bax to the anti-apoptotic protein(s) Bcl-2 and/or Bcl-XL, thereby increases mitochondrial permeability and enables release of cytochrome c to cytoplasm [51]. Cytochrome c released into the cytoplasm forms complexes with Apaf-1 and triggers a caspase cascade to execute apoptotic cell death [52, 53]. It has been demonstrated in neuronal cells that hypoxia-induced Bax expression and DNA fragmentation are mediated through induction of nitric oxide (NO) [54, 55]. NO in endothelial cells is generated by both the endothelial and inducible isoforms of nitric oxide synthase (eNOS and iNOS) via oxidation of the substrate, L-arginine. Hypoxia can induce both iNOS and eNOS expression because the iNOS gene promoter has the hypoxia response element for HIF1 [56, 57] and the eNOS gene promoter has binding sites for HIF2 [58]. NO has a dual action on the vascular endothelium: at low concentrations (nM), as are present under basal conditions, it protects cells against apoptotic stimuli [59]. When its levels become elevated (µM), as in the case of severe ischemia/ hypoxia, NO also initiates apoptosis in both endothelial and non-endothelial cells [60, 61]. Activation of eNOS, iNOS and SRF is dependent on Rho GTPase-regulated actin dynamics. Actin de-polymerization activates eNOS [62, 63] and iNOS [64, 65] but suppresses SRF, resulting in apoptosis [66, 67]. Conversely, actin polymerization activates SRF but suppresses eNOS and iNOS, supporting cell survival.

as a downstream regulator in cell proliferation, adhesion and migration, thus any mitogenic

**Figure 2.** Knockdown of SRF in brain endothelial cells (bEND3) prevents hypoxia-induced angiogenesis. bEND3 cells were cultured in collagen gel matrix under a hypoxic condition. The collagen gel matrix was made of 50% type I colla‐ gen in HEPES (pH 8.5) Hanks buffer balanced growth medium. The mixture was solidified in 12-well plates at 37˚C for 20 minutes. Cells were mixed in the liquid gel, plated on top of the solidified gel in the 12-well plates and incubated at 37˚C for additional 20 minutes. More layers of cells were plated in the wells by repeating this step. Eventually, growth medium was added to the top of the solidified gel containing endothelial cells and the plates were incubated at 37˚C for a week. The control cells formed a cobblestone monolayer at the end, while SRF over-expressing cells (SRF+) moved vertically and horizontally within the gel matrix. The cells with SRF knockdown (SRF-), on the other hand, stayed inactively. Under hypoxia, both control and SRF+ cells formed cable-like structure, an indication of angiogenic

**6. SRF protects endothelial cells against hypoxia-induced apoptosis**

Several studies indicate that hypoxia-induced apoptosis is solely dependent on the mitochon‐ drial pathway [46-48], which is regulated by Bcl-2 family members [49, 50]. Hypoxia induces an increase in the ratio of the pro-apoptotic protein Bax to the anti-apoptotic protein(s) Bcl-2 and/or Bcl-XL, thereby increases mitochondrial permeability and enables release of cytochrome c to cytoplasm [51]. Cytochrome c released into the cytoplasm forms complexes with Apaf-1 and triggers a caspase cascade to execute apoptotic cell death [52, 53]. It has been demonstrated in neuronal cells that hypoxia-induced Bax expression and DNA fragmentation are mediated through induction of nitric oxide (NO) [54, 55]. NO in endothelial cells is generated by both the endothelial and inducible isoforms of nitric oxide synthase (eNOS and iNOS) via oxidation of the substrate, L-arginine. Hypoxia can induce both iNOS and eNOS expression because the iNOS gene promoter has the hypoxia response element for HIF1 [56, 57] and the eNOS gene promoter has binding sites for HIF2 [58]. NO has a dual action on the vascular endothelium: at low concentrations (nM), as are present under basal conditions, it protects cells against apoptotic stimuli [59]. When its levels become elevated (µM), as in the case of severe ischemia/ hypoxia, NO also initiates apoptosis in both endothelial and non-endothelial cells [60, 61].

activity, while the SRF- cells showed sign of death.

34 Research Directions in Tumor Angiogenesis

factor that aims to stimulate such cellular activities requires SRF involvement.

**Figure 3.** Knockdown of SRF in brain endothelial cells increases hypoxia-induced apoptosis. bEND3 cells were cultured on cover slips under a hypoxic condition for 2, 6 and 24 hours. TUNEL assays were performed to detect apoptosis. Ap‐ parently, overexpression of SRF (SRF+) promoted cell survival, while knockdown of SRF (SRF-) made cells more vulnera‐ ble to hypoxic damage.

Moderate hypoxia induces cell adaptation but not apoptosis. However, when SRF is insuffi‐ cient (SRF-), cells become vulnerable to cellular stress and even a brief oxygen shortage can trigger apoptotic cell death (Figure 3). On the other hand, forced overexpression of SRF (SRF +) in these cells can make them more resistant to hypoxic damage and able to survive through even more harsh oxygen crisis. The advantage of SRF over HIF is its broad involvement in the molecular regulation of the cell machinery. Once SRF is activated, it not only promotes cell survival through up-regulation of growth factors and cytoskeletal components, but also protects mitochondrial integrity through up-regulation of anti-apoptotic proteins like Bcl-2 [68]. In another word, SRF supports cell survival at multiple levels. Up-regulation of growth factors stimulates cell proliferation and migration, which require adequate supplies of cytoskeletal proteins, because without cytoskeleton to provide the platform, cells cannot proliferate or migrate, and SRF makes sure these molecules available at the time of need. Finally, SRF also makes sure mitochondria intact so that they can provide the energy that cell proliferation and migration need. Mitochondrial integrity depends on the balance between pro-apoptotic and anti-apoptotic proteins, typically, BAX versus Bcl-2. Severe hypoxia activates p53, which drives up-regulation of BAX, pushing cells toward apoptosis, as BAX gene contains four binding sites for p53. On the other hand, hypoxia also activates SRF (as shown above), which drives up-regulation of Bcl-2, pushing cells toward survival, as Bcl-2 gene contains two SREs in its promoter [68]. The BAX and Bcl-2 fight turns into a wrestle between p53 and SRF. As shown in Figure 4, manipulation of SRF expression can change BAX/Bcl-2 ratio, and ultimately, change the fate of the cells under hypoxia.

**Figure 4.** SRF promotes Bcl-2 but suppresses Bax. A. Western blot analysis showed an increase of Bax and a decrease of Bcl-2 in bEND3 cells due to SRF deficiency. B. Immunocytochemistry showed a similar effect.

**Figure 5.** Knockdown of SRF in brain endothelial cells increases mitochondrial permeability during hypoxia. bEND3 cells were cultured on cover slips under a hypoxic condition and stained with a cationic dye. The dye fluoresces differ‐ ently in healthy vs. apoptotic cells. In healthy cells, the dye accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. While in apoptotic cells, the dye cannot aggregate in the mitochondria due to the altered mitochondrial transmembrane potential, and thus it remains in the cytoplasm in its monomer form, fluorescing green.

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Mitochondrial permeability is reflected by the opposite movement of BAX and cytochrome c. Normally, BAX remains in the cytoplasm at a low level, while cytochrome c hides in between the inner and outer membranes of the mitochondria. When cells suffer from an oxygen shortage, BAX jumps, moving toward mitochondria. The insertion of BAX into the outer mitochondrial membrane opens pores to let cytochrome c leak out. Cytoplasmic cytochrome

The impact of SRF on mitochondrial integrity during hypoxia is not only reflected at the molecular level, but it can also be visualized at the subcellular level. As shown in Figure 5, incubation of brain endothelial cells under a hypoxic condition induces mitochondrial leakage, as reflected by the color change of a fluorescent dye. The longer the hypoxic exposure goes, the fewer intact mitochondria exist. However, forced overexpression of SRF in these cells can reverse the effect of hypoxia, protecting mitochondria against hypoxic damage. Conversely, knockdown of SRF can lower the threshold of mitochondrial tolerance to oxygen deprivation, so that a short hypoxic exposure can cause a massive mitochondrial leakage.

p53 and SRF. As shown in Figure 4, manipulation of SRF expression can change BAX/Bcl-2

**Figure 4.** SRF promotes Bcl-2 but suppresses Bax. A. Western blot analysis showed an increase of Bax and a decrease

The impact of SRF on mitochondrial integrity during hypoxia is not only reflected at the molecular level, but it can also be visualized at the subcellular level. As shown in Figure 5, incubation of brain endothelial cells under a hypoxic condition induces mitochondrial leakage, as reflected by the color change of a fluorescent dye. The longer the hypoxic exposure goes, the fewer intact mitochondria exist. However, forced overexpression of SRF in these cells can reverse the effect of hypoxia, protecting mitochondria against hypoxic damage. Conversely, knockdown of SRF can lower the threshold of mitochondrial tolerance to oxygen deprivation,

of Bcl-2 in bEND3 cells due to SRF deficiency. B. Immunocytochemistry showed a similar effect.

so that a short hypoxic exposure can cause a massive mitochondrial leakage.

ratio, and ultimately, change the fate of the cells under hypoxia.

36 Research Directions in Tumor Angiogenesis

**Figure 5.** Knockdown of SRF in brain endothelial cells increases mitochondrial permeability during hypoxia. bEND3 cells were cultured on cover slips under a hypoxic condition and stained with a cationic dye. The dye fluoresces differ‐ ently in healthy vs. apoptotic cells. In healthy cells, the dye accumulates and aggregates in the mitochondria, giving off a bright red fluorescence. While in apoptotic cells, the dye cannot aggregate in the mitochondria due to the altered mitochondrial transmembrane potential, and thus it remains in the cytoplasm in its monomer form, fluorescing green.

Mitochondrial permeability is reflected by the opposite movement of BAX and cytochrome c. Normally, BAX remains in the cytoplasm at a low level, while cytochrome c hides in between the inner and outer membranes of the mitochondria. When cells suffer from an oxygen shortage, BAX jumps, moving toward mitochondria. The insertion of BAX into the outer mitochondrial membrane opens pores to let cytochrome c leak out. Cytoplasmic cytochrome c binds to Apaf-1 and triggers caspase cascade, leading to apoptotic cell death. During this event, the level of SRF is a determining factor for the fate of the cell. As illustrated in Figure 6, as oxygen crisis prolongs the opposite movement of BAX and cytochrome c increases, and cells prone to die. Manipulation of SRF level can either facilitate this process or reverse it, depending on what we desire.

been reported that HIF-deficient embryonic stem cells resist to hypoxia-induced p53 activation and apoptosis [70]. A similar observation was also reported in neuronal cells where HIF helps p53 to endorse cell death [71]. For this reason, treatments targeting HIF do not always achieve

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39

Unlike HIF, SRF promotes cell survival through multi-level and fundamental regulations. Level 1 – growth factors: as discussed above, SRF is not only activated by growth factors, but also turns around to stimulate growth factor expression. This positive feedback reinforces the signal for cell survival. Level 2 – cytoskeletal components: no matter it is for cancer cells to move away from their primary location to look for new places with better oxygen and nutrient supply, or for cancer cells to allure endothelial cells with chemicals to form new vessels to bring oxygen and nutrients to the tumors, cytoskeletal regeneration and rearrangement are essential requirements. The molecules involved in these processes are tightly controlled by SRF. As shown in our previous study [43], without SRF, even the most potent angiogenic factor VEGF cannot stimulate angiogenesis. Level 3 – anti-apoptosis: hypoxia induces apoptosis through disrupting mitochondrial outer membrane, while mitochondrial integrity is guarded by Bcl-2, which is controlled by SRF. Therefore, SRF should be a better candidate for cancer gene therapy, and a treatment targeting SRF, instead of HIF, should achieve better results.

This work is supported by the Department of Veterans Affairs of the United States.

VA Long Beach Healthcare System, Long Beach and University of California, Irvine, USA

[1] Rolfe DF, Brown GC. Cellular energy utilization and molecular origin of standard metabolic rate in mammals. Physiological Reviews. 1997 July 1, 1997;77(3):731-58.

[2] Brown GC, Borutaite V. There is no evidence that mitochondria are the main source of reactive oxygen species in mammalian cells. Mitochondrion. 2012;12(1):1-4.

[3] Yao KS, Xanthoudakis S, Curran T, O'Dwyer PJ. Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia. Molecular

and Cellular Biology. 1994 September 1, 1994;14(9):5997-6003.

inhibition of tumor angiogenesis.

**Acknowledgements**

**Author details**

Jianyuan Chai

**References**

**Figure 6.** SRF protects mitochondrial integrity. As oxygen deprivation extends, more and Bax binds to mitochondria and opens up channels to allow cytochrome c to escape from mitochondria into cytoplasm, where it forms complexes with Apaf-1 and triggers caspase cascade. With overexpression of SRF, cells can reverse Bcl-2/Bax ratio decrease caused by hypoxia and prevent cytochrome c leakage, while lack of SRF accelerates mitochondrial breakdown.

#### **7. Conclusions**

Due to unregulated proliferation of malignant cells, oxygen deficiency is common in tumor development. Cancer cells have learned a few tricks to survive through oxygen crisis, and one of them is to stimulate endothelial cells to build new vessels extending oxygen toward the hypoxic area. However, depending on the severity of hypoxia, endothelial cells may follow the cue to support tumor growth by engaging in angiogenesis, or commit a suicide by engaging in apoptosis and leave the tumor cells to die. It is our interest to guide the endothelial cells to choose the second path. The best known players in the battle against hypoxia are HIF and p53. In general, HIF up-regulates angiogenic factors to promote angiogenesis, while p53 upregulates pro-apoptotic genes to induce apoptosis. However, the relationship between HIF and p53 is not always a bull-and-bear fight; sometimes they can also join forces to become friends. HIF can bind to MDM2 to stabilize p53 and thereby to promote apoptosis [69]. It has been reported that HIF-deficient embryonic stem cells resist to hypoxia-induced p53 activation and apoptosis [70]. A similar observation was also reported in neuronal cells where HIF helps p53 to endorse cell death [71]. For this reason, treatments targeting HIF do not always achieve inhibition of tumor angiogenesis.

Unlike HIF, SRF promotes cell survival through multi-level and fundamental regulations. Level 1 – growth factors: as discussed above, SRF is not only activated by growth factors, but also turns around to stimulate growth factor expression. This positive feedback reinforces the signal for cell survival. Level 2 – cytoskeletal components: no matter it is for cancer cells to move away from their primary location to look for new places with better oxygen and nutrient supply, or for cancer cells to allure endothelial cells with chemicals to form new vessels to bring oxygen and nutrients to the tumors, cytoskeletal regeneration and rearrangement are essential requirements. The molecules involved in these processes are tightly controlled by SRF. As shown in our previous study [43], without SRF, even the most potent angiogenic factor VEGF cannot stimulate angiogenesis. Level 3 – anti-apoptosis: hypoxia induces apoptosis through disrupting mitochondrial outer membrane, while mitochondrial integrity is guarded by Bcl-2, which is controlled by SRF. Therefore, SRF should be a better candidate for cancer gene therapy, and a treatment targeting SRF, instead of HIF, should achieve better results.

#### **Acknowledgements**

This work is supported by the Department of Veterans Affairs of the United States.

#### **Author details**

#### Jianyuan Chai

c binds to Apaf-1 and triggers caspase cascade, leading to apoptotic cell death. During this event, the level of SRF is a determining factor for the fate of the cell. As illustrated in Figure 6, as oxygen crisis prolongs the opposite movement of BAX and cytochrome c increases, and cells prone to die. Manipulation of SRF level can either facilitate this process or reverse it,

**Figure 6.** SRF protects mitochondrial integrity. As oxygen deprivation extends, more and Bax binds to mitochondria and opens up channels to allow cytochrome c to escape from mitochondria into cytoplasm, where it forms complexes with Apaf-1 and triggers caspase cascade. With overexpression of SRF, cells can reverse Bcl-2/Bax ratio decrease caused by hypoxia and prevent cytochrome c leakage, while lack of SRF accelerates mitochondrial breakdown.

Due to unregulated proliferation of malignant cells, oxygen deficiency is common in tumor development. Cancer cells have learned a few tricks to survive through oxygen crisis, and one of them is to stimulate endothelial cells to build new vessels extending oxygen toward the hypoxic area. However, depending on the severity of hypoxia, endothelial cells may follow the cue to support tumor growth by engaging in angiogenesis, or commit a suicide by engaging in apoptosis and leave the tumor cells to die. It is our interest to guide the endothelial cells to choose the second path. The best known players in the battle against hypoxia are HIF and p53. In general, HIF up-regulates angiogenic factors to promote angiogenesis, while p53 upregulates pro-apoptotic genes to induce apoptosis. However, the relationship between HIF and p53 is not always a bull-and-bear fight; sometimes they can also join forces to become friends. HIF can bind to MDM2 to stabilize p53 and thereby to promote apoptosis [69]. It has

depending on what we desire.

38 Research Directions in Tumor Angiogenesis

**7. Conclusions**

VA Long Beach Healthcare System, Long Beach and University of California, Irvine, USA

#### **References**


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**Chapter 3**

and

**Manipulating Redox Signaling**

**to Block Tumor Angiogenesis**

Vera Mugoni and Massimo Mattia Santoro

Additional information is available at the end of the chapter

for the induction and regulation of angiogenesis.

A tumor consists of a population of rapidly dividing and growing cancer cells. Cancer cells have lost their ability to divide in a controlled fashion and as a consequence they rapidly accumulate mutations. In such way cancer cells (or sub-populations of cancer cells within a tumor) will acquire stronger proliferative capacity [1]. Tumors cannot grow beyond a certain size due to a lack of oxygen and other essential nutrients. Tumors cells have then acquired a specific feature that is to induce blood vessel growth, a process called tumor angiogenesis. Tumor angiogenesis is a necessary and required step for transition from a small harmless cluster of cells to a large tumor [2]. The early induction of tumor vasculature is termed "angiogenic switch", that occurs when a tumor mass reaches about dimensions of 2 mm2

moves towards progression. The "angiogenic switch" is a rate-limiting step for tumor growth that is not limited at earliest stages, but occurs also at different stages of tumor-progression. The angiogenic switch induces angiogenic sprouting and new vessels formation and matura‐ tion. Activation of angiogenesis in premalignant lesions and dormant metastasis is mandatory for tumor survival. The fact that tumor mass is depending on angiogenesis has driven the medical research towards the characterization of molecular pathways and cellular dynamics

Tumor angiogenesis is regulated by several growth factors (EGF, TGFα, bFGF, VEGF). Induction of these angiogenic factors is triggered by various stresses [3]. For instance, tissue hypoxia exerts its pro-angiogenic action through various angiogenic factors, the most notable is VEGF (vascular endothelial growth factor), which has been mainly associated with initiating the process of angiogenesis through the recruitment and proliferation of endothelial cells [4]. Recently, reactive oxygen species (ROS) have been found to stimulate angiogenic response in the normal and pathological angiogenesis. ROS can cause tissue injury in one hand and

and reproduction in any medium, provided the original work is properly cited.

© 2013 Mugoni and Santoro; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

http://dx.doi.org/10.5772/54593

**1. Introduction**

## **Manipulating Redox Signaling to Block Tumor Angiogenesis**

Vera Mugoni and Massimo Mattia Santoro

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54593

#### **1. Introduction**

A tumor consists of a population of rapidly dividing and growing cancer cells. Cancer cells have lost their ability to divide in a controlled fashion and as a consequence they rapidly accumulate mutations. In such way cancer cells (or sub-populations of cancer cells within a tumor) will acquire stronger proliferative capacity [1]. Tumors cannot grow beyond a certain size due to a lack of oxygen and other essential nutrients. Tumors cells have then acquired a specific feature that is to induce blood vessel growth, a process called tumor angiogenesis. Tumor angiogenesis is a necessary and required step for transition from a small harmless cluster of cells to a large tumor [2]. The early induction of tumor vasculature is termed "angiogenic switch", that occurs when a tumor mass reaches about dimensions of 2 mm2 and moves towards progression. The "angiogenic switch" is a rate-limiting step for tumor growth that is not limited at earliest stages, but occurs also at different stages of tumor-progression. The angiogenic switch induces angiogenic sprouting and new vessels formation and matura‐ tion. Activation of angiogenesis in premalignant lesions and dormant metastasis is mandatory for tumor survival. The fact that tumor mass is depending on angiogenesis has driven the medical research towards the characterization of molecular pathways and cellular dynamics for the induction and regulation of angiogenesis.

Tumor angiogenesis is regulated by several growth factors (EGF, TGFα, bFGF, VEGF). Induction of these angiogenic factors is triggered by various stresses [3]. For instance, tissue hypoxia exerts its pro-angiogenic action through various angiogenic factors, the most notable is VEGF (vascular endothelial growth factor), which has been mainly associated with initiating the process of angiogenesis through the recruitment and proliferation of endothelial cells [4]. Recently, reactive oxygen species (ROS) have been found to stimulate angiogenic response in the normal and pathological angiogenesis. ROS can cause tissue injury in one hand and

© 2013 Mugoni and Santoro; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

promote tissue repair in another hand by promoting angiogenesis. It thus appears that after causing injury to the cells, ROS promptly initiate the tissue repair process by triggering angiogenic response. Recently, it has been reported that redox signaling may influence pathological angiogenesis as well [5,6].

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important in regulation of cell survival. In general, moderate levels of ROS/RNS functions as signals to promote cell proliferation and survival, whereas severe increase of ROS/RNS can induce cell death. Under physiologic conditions, the balance between generation and elimination of ROS/RNS main‐ tains the proper function of redox-sensitive signaling proteins. Normally, the redox homeo‐ stasis ensures that cells respond properly to endogenous and exogenous stimuli. However, when the redox homeostasis is disturbed, oxidative stress may lead to aberrant cell death and

Manipulating Redox Signaling to Block Tumor Angiogenesis

http://dx.doi.org/10.5772/54593

49

Reactive species are highly reactive chemical molecules or ions, characterized by unpaired electrons that react with other molecules in order to stabilize their electron configuration and gain a more stable state. Consequently, the reaction of ROS/RNS with cellular molecules is a damaging reaction of oxidation. Oxidized molecules are dysfunctional and may induce cell death. Initially, the presence of ROS/RNS was linked only to cellular damage and cell degen‐ erative processes. However, accumulating evidences derived from the characterization of mechanisms for buffering and regulating reactive species opened the possibility that oxidative species are important for cellular homeostasis. Reactive species had been also described as second messenger molecules and their interaction with molecules is identified as a posttranslational modification (i.e. S- nitrosylation of proteins) that can trigger a specific intracel‐ lular signal. At the present, the evidence is that a tight regulation of pro-oxidative species levels is essential for cellular homeostasis and that such regulatory mechanism is fundamental to

maintain a safe redox state and activate related redox signaling pathways [10].

In vascular beds, the redox state is mainly modulated by oxygen concentration and by mechanical forces (i.e. shear stress caused by blood flow) [11]. In normal conditions oxygen levels are constant and essential to guarantee sufficient provision for tissues oxygenation. Mechanisms for sensing oxygen tension are based on redox-mediated signaling. During normoxic conditions the transcription factor HIF1α (hypoxia inducible factor) is degraded in a ROS-dependent manner, while during hypoxia the concentration of oxygen is lower and ROS levels are differentially modulated. Consequently, HIF1α couples with HIF1β and activates transcription of genes involved in angiogenesis, vascular remodeling and cell

Redox signaling events are also activated in endothelial cells during normal angiogenesis for sensing mechanical forces. Shear forces are constantly present on endothelial cells where regulate cell proliferation, survival and migration. Vascular forces exercise a mechanical stimulus that is perceived by endothelial cells and translated into intracellular molecular pathways. Therefore, concomitant to shear forces there is an upregulation in production of RNS and ROS. In adult ECs, the mechanical oscillatory shear stress induces the activation of specific antioxidant enzymes or proteins like peroxiredoxins (Prx) that act as "mechanosensitive antioxidants" [13]. Moreover, specific antioxidant and protective genes are induced. Shear stress causes upregulation of specific "antioxidant transcriptional factors" Nrf2 and ATF in developing embryonic vasculature as well as in adult ECs [14]. Most of the molecules with oxidative properties that modulate endothelial cell homeostasis in normal conditions are included in redox molecular pathways that are altered in pathogenic angiogenesis [15]. There

contribute to disease development [9].

proliferation [12].

#### **2. Redox signaling in normal and pathological angiogenesis**

**Redox signaling** is a biochemical communication by free radicals, reactive oxygen species (ROS), and other electronically activated species such as nitric oxide and other oxides of nitrogen acting as biological messengers [7]. Pro- and anti-oxidative species act as second messengers. Pro-oxidative species are physiologically produced by cells and tightly regulated with antioxidant systems. Down-regulation of antioxidant system or up-regulation in pro‐ duction of pro-oxidative species leads to oxidative stress state. This condition is reported as dangerous for cells since it conveys macromolecules damage. Importantly, it has been reported that oxidative stress plays a key role in the regulation of tumor angiogenesis [8]. The complex molecular network that regulates endothelial cells homeostasis during angiogenesis includes molecules sensitive to redox state of biological environment. The redox state is determined by the relative abundance of highly chemically reactive species derived from oxygen (ROS: Reactive Oxygen Species) or nitrogen (RNS: Reactive Nitrogen Species) (Table 1).


**Table 1.** List of oxygen (ROS) and nitrogen (RNS) reactive species commonly found in normal and pathological tissues.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are important in regulation of cell survival. In general, moderate levels of ROS/RNS functions as signals to promote cell proliferation and survival, whereas severe increase of ROS/RNS can induce cell death. Under physiologic conditions, the balance between generation and elimination of ROS/RNS main‐ tains the proper function of redox-sensitive signaling proteins. Normally, the redox homeo‐ stasis ensures that cells respond properly to endogenous and exogenous stimuli. However, when the redox homeostasis is disturbed, oxidative stress may lead to aberrant cell death and contribute to disease development [9].

promote tissue repair in another hand by promoting angiogenesis. It thus appears that after causing injury to the cells, ROS promptly initiate the tissue repair process by triggering angiogenic response. Recently, it has been reported that redox signaling may influence

**Redox signaling** is a biochemical communication by free radicals, reactive oxygen species (ROS), and other electronically activated species such as nitric oxide and other oxides of nitrogen acting as biological messengers [7]. Pro- and anti-oxidative species act as second messengers. Pro-oxidative species are physiologically produced by cells and tightly regulated with antioxidant systems. Down-regulation of antioxidant system or up-regulation in pro‐ duction of pro-oxidative species leads to oxidative stress state. This condition is reported as dangerous for cells since it conveys macromolecules damage. Importantly, it has been reported that oxidative stress plays a key role in the regulation of tumor angiogenesis [8]. The complex molecular network that regulates endothelial cells homeostasis during angiogenesis includes molecules sensitive to redox state of biological environment. The redox state is determined by the relative abundance of highly chemically reactive species derived from oxygen (ROS:

**2. Redox signaling in normal and pathological angiogenesis**

Reactive Oxygen Species) or nitrogen (RNS: Reactive Nitrogen Species) (Table 1).

**Table 1.** List of oxygen (ROS) and nitrogen (RNS) reactive species commonly found in normal and pathological tissues.

pathological angiogenesis as well [5,6].

48 Research Directions in Tumor Angiogenesis

Reactive species are highly reactive chemical molecules or ions, characterized by unpaired electrons that react with other molecules in order to stabilize their electron configuration and gain a more stable state. Consequently, the reaction of ROS/RNS with cellular molecules is a damaging reaction of oxidation. Oxidized molecules are dysfunctional and may induce cell death. Initially, the presence of ROS/RNS was linked only to cellular damage and cell degen‐ erative processes. However, accumulating evidences derived from the characterization of mechanisms for buffering and regulating reactive species opened the possibility that oxidative species are important for cellular homeostasis. Reactive species had been also described as second messenger molecules and their interaction with molecules is identified as a posttranslational modification (i.e. S- nitrosylation of proteins) that can trigger a specific intracel‐ lular signal. At the present, the evidence is that a tight regulation of pro-oxidative species levels is essential for cellular homeostasis and that such regulatory mechanism is fundamental to maintain a safe redox state and activate related redox signaling pathways [10].

In vascular beds, the redox state is mainly modulated by oxygen concentration and by mechanical forces (i.e. shear stress caused by blood flow) [11]. In normal conditions oxygen levels are constant and essential to guarantee sufficient provision for tissues oxygenation. Mechanisms for sensing oxygen tension are based on redox-mediated signaling. During normoxic conditions the transcription factor HIF1α (hypoxia inducible factor) is degraded in a ROS-dependent manner, while during hypoxia the concentration of oxygen is lower and ROS levels are differentially modulated. Consequently, HIF1α couples with HIF1β and activates transcription of genes involved in angiogenesis, vascular remodeling and cell proliferation [12].

Redox signaling events are also activated in endothelial cells during normal angiogenesis for sensing mechanical forces. Shear forces are constantly present on endothelial cells where regulate cell proliferation, survival and migration. Vascular forces exercise a mechanical stimulus that is perceived by endothelial cells and translated into intracellular molecular pathways. Therefore, concomitant to shear forces there is an upregulation in production of RNS and ROS. In adult ECs, the mechanical oscillatory shear stress induces the activation of specific antioxidant enzymes or proteins like peroxiredoxins (Prx) that act as "mechanosensitive antioxidants" [13]. Moreover, specific antioxidant and protective genes are induced. Shear stress causes upregulation of specific "antioxidant transcriptional factors" Nrf2 and ATF in developing embryonic vasculature as well as in adult ECs [14]. Most of the molecules with oxidative properties that modulate endothelial cell homeostasis in normal conditions are included in redox molecular pathways that are altered in pathogenic angiogenesis [15]. There are specific oxidized products or redox sensitive proteins that behave differentially. ROSactivated factors play different role in context of pathologic angiogenesis or normal angio‐ genesis. The ATM kinase protein, which is involved in regulation of endothelial cells survival and proliferation is activated in tumor condition under upregulation of ROS and promotes new vessel formation, while it is not activated in normal vasculature [16]. Oxidative stress triggered by inflammation in tumor conditions (i.e. human melanoma) causes lipid peroxida‐ tion with consequent accumulation of an oxidized compound: ω-(2-carboxyethyl)-pyrrole (CEP). The CEP acts as a ligand for Toll-like receptor 2 (TLR2) and induces angiogenesis independently from VEGF [17]. Similarly, oxidized lipid (carboxyalkyl pyrroles, CAPs) molecules bind to their TLRs receptors and activate angiogenesis in some specific pathological conditions such us age related macular degeneration [18].

vascular smooth muscle cells, monocytes and macrophages. NOX1, NOX2, NOX4 and NOX5 are constitutively expressed in the endothelial cells and their functionality is regulated by several vascular conditions like shear stress, hypoxia or stimuli as hormones, cytokines, proangiogenic factors [21]. It has been demonstrated that NADPH oxidase are sensitive to proangiogenic vascular endothelial growth factor (VEGF) activation and it seems probable that reactive oxygen species derived from their oxidase activity may sustain activated VEGFR2 and promote endothelial cells migration and proliferation [22]. In endothelium the regulation of NOX activity is tightly associated with redox balance, since it has been demonstrated that NOX are involved with a series of cardiovascular disease like hypertension, atherosclerosis or ischemia/reperfusion injury [23]. A specific role of NADPH oxidase activity is also reported during the angiogenesis process [24]. NOX1 activity mediates the interaction between leukocyte cells and endothelium and is involved in the initiation of cell migration. NOX1 levels are sensitive to oscillatory shear stress conditions and are positively regulated by HIF-1 and PDGF. Also NOX1 is involved in angiogenic switch by sustaining VEGF signaling and upregulation of matrix metalloproteinase production [25]. NOX2 is also sensitive to vascular pro-angiogenic factors and is reported to be involved in the regulation of ROS signaling for cytoskeleton organization in ECs migration [26]. NOX4 is the most abundant isoform in endothelial tissues and is responsible for basal superoxide production. As a matter of fact the role of NOX4 in vascular tissues is still far from been understood [27]. NOX5 isoform is present in mammalian cells, but its function can be substituted by other isoforms (i.e. DUOX in rodents). In vitro studies report upregulation of NOX5 stimulates endothelial cells prolifera‐ tion and organization in microvascular tubules. Also NOX5 is sensitive to pro-angiogenic stimuli like angiopoietins [28]. Considering vascular pro-angiogenic factors tightly regulate by NADPH oxidases several inhibitors have been developed as possible approach to modulate redox signaling in tumor angiogenesis. The most studied NADPH oxidase inhibitors are apocyanin and diphenyleneiodonium (DPI). They are quite non-specific inhibitors since they block assembly of enzyme or electron flow. Emerging new inhibitors for endothelial NOX isoforms are triazolopyrimidines inhibitors such as VAS2870 and VAS3947, whose preliminary in vitro and in vivo studies have been reported beneficial for endothelium dysfunctions under

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51

**Cyclooxygenases (COX).** Cyclooxygenases act in the rate-limiting step of prostanoids biosynthesis. There are two kinds of enzymes: cyclooxygenase-1 (COX-1) and cyclooxyge‐ nase-2, also known as prostaglandin endoperoxide H synthase -1 and -2 (PGHS-1 and PGHS-2). Recently, a cyclooxygenase-1 isoenzyme was identified as COX-3 [30]. Prostanoids are lipid molecules that are produced from all animal cells in response to specific stimuli, like hormones. After activation COX produce prostanoids from free fatty acids, typically from arachidonic acid (AA). In particular COX catalyze the bis-oxygenation of AA into the prostaglandin endoperoxide PGG2, an intermediate molecule that is subsequently converted into different kinds of prostanoids by specific enzymes that are downstream COX. There are five main categories of prostanoid molecules and their specific receptors: 1) prostaglandin D2 (PGD2) whose receptors are named DP1-DP-2; 2) prostaglandin E2 (PGE2) whose receptors are named EP1-EP4, 3) prostaglandin F2α (PGF2α) whose receptors are named FP, 4) prostaglandin I2

oxidative stress [29].

In the following three different paragraphs we will define the cellular systems regulating redox signaling and how they control molecules and factors clearly involved in angiogenesis. In addition, here we plan to present paragraphs about main sources for production of oxidative species and systems for counteract their products and maintenance of an equilibrated cell redox state. Finally, we will describe molecules sensitive to redox signaling that are known for being part of established pathway for tumor angiogenesis signaling.

#### **3. Molecules generating oxidative species in endothelial cells**

In endothelial cells the endogenous production of pro-oxidative species is mainly generated by four different enzymes: NADPH oxidases (NOX), Cyclooxygenases (COX), Xanthine oxidoreductase (XOR), and dysfunctional endothelial NOS (eNOS).

**NADPH oxidases (NOX).** NADPH oxidases are a family of enzymes composed by seven members: NOX1, NOX2, NOX3, NOX4, NOX5 and two homologues DUOX1, DUOX2. All of them are transmembrane proteins containing a NADPH-binding site, a FAD binding region, heme-binding sites and several subunits that function in the regulation and maturation of enzymes. p22phox, DUOX activator 1 (DUAX1) and DUOX activator 2 (DUOX2) are important factors for NOX and DUOX maturation. Among factors important for NOX and DUOX enzyme activation we found p67phox, NOX activator1 (NOXA1), small GTPase (RAC1 and RAC2). On the contrary specific regulator of NOX4 is polymerase δ-interacting protein 2 (POLDIP2) and Ca2+ ions are specific activators of NOX5 and DUOX1/DUOX2 isoforms. These enzymes are also associated to spatial regulator subunits p40phox, p47phox and NOX organizer1 (NOXO1) that are important for the enzyme complex structure [19]. NADPH oxidase catalytic activity consists in the generation of superoxide anions (O<sup>2</sup> - ) through an electrons transfer cycle from an electron donor (NADPH) to FAD subunit, heme groups and to a final electron acceptor that is a molecule of oxygen. Activation of NOX4, DUOX1 and DUOX2 results mainly in the release of hydrogen peroxide instead of superoxide anions. The specific role of this enzyme family consists in the production and release of pro-oxidative species. Such class of enzymes is considered one of the main player in the redox signaling in cardiovascular system [20]. NADPH oxidases are expressed in various types of cells along the vascular wall, including vascular smooth muscle cells, monocytes and macrophages. NOX1, NOX2, NOX4 and NOX5 are constitutively expressed in the endothelial cells and their functionality is regulated by several vascular conditions like shear stress, hypoxia or stimuli as hormones, cytokines, proangiogenic factors [21]. It has been demonstrated that NADPH oxidase are sensitive to proangiogenic vascular endothelial growth factor (VEGF) activation and it seems probable that reactive oxygen species derived from their oxidase activity may sustain activated VEGFR2 and promote endothelial cells migration and proliferation [22]. In endothelium the regulation of NOX activity is tightly associated with redox balance, since it has been demonstrated that NOX are involved with a series of cardiovascular disease like hypertension, atherosclerosis or ischemia/reperfusion injury [23]. A specific role of NADPH oxidase activity is also reported during the angiogenesis process [24]. NOX1 activity mediates the interaction between leukocyte cells and endothelium and is involved in the initiation of cell migration. NOX1 levels are sensitive to oscillatory shear stress conditions and are positively regulated by HIF-1 and PDGF. Also NOX1 is involved in angiogenic switch by sustaining VEGF signaling and upregulation of matrix metalloproteinase production [25]. NOX2 is also sensitive to vascular pro-angiogenic factors and is reported to be involved in the regulation of ROS signaling for cytoskeleton organization in ECs migration [26]. NOX4 is the most abundant isoform in endothelial tissues and is responsible for basal superoxide production. As a matter of fact the role of NOX4 in vascular tissues is still far from been understood [27]. NOX5 isoform is present in mammalian cells, but its function can be substituted by other isoforms (i.e. DUOX in rodents). In vitro studies report upregulation of NOX5 stimulates endothelial cells prolifera‐ tion and organization in microvascular tubules. Also NOX5 is sensitive to pro-angiogenic stimuli like angiopoietins [28]. Considering vascular pro-angiogenic factors tightly regulate by NADPH oxidases several inhibitors have been developed as possible approach to modulate redox signaling in tumor angiogenesis. The most studied NADPH oxidase inhibitors are apocyanin and diphenyleneiodonium (DPI). They are quite non-specific inhibitors since they block assembly of enzyme or electron flow. Emerging new inhibitors for endothelial NOX isoforms are triazolopyrimidines inhibitors such as VAS2870 and VAS3947, whose preliminary in vitro and in vivo studies have been reported beneficial for endothelium dysfunctions under oxidative stress [29].

are specific oxidized products or redox sensitive proteins that behave differentially. ROSactivated factors play different role in context of pathologic angiogenesis or normal angio‐ genesis. The ATM kinase protein, which is involved in regulation of endothelial cells survival and proliferation is activated in tumor condition under upregulation of ROS and promotes new vessel formation, while it is not activated in normal vasculature [16]. Oxidative stress triggered by inflammation in tumor conditions (i.e. human melanoma) causes lipid peroxida‐ tion with consequent accumulation of an oxidized compound: ω-(2-carboxyethyl)-pyrrole (CEP). The CEP acts as a ligand for Toll-like receptor 2 (TLR2) and induces angiogenesis independently from VEGF [17]. Similarly, oxidized lipid (carboxyalkyl pyrroles, CAPs) molecules bind to their TLRs receptors and activate angiogenesis in some specific pathological

In the following three different paragraphs we will define the cellular systems regulating redox signaling and how they control molecules and factors clearly involved in angiogenesis. In addition, here we plan to present paragraphs about main sources for production of oxidative species and systems for counteract their products and maintenance of an equilibrated cell redox state. Finally, we will describe molecules sensitive to redox signaling that are known for

In endothelial cells the endogenous production of pro-oxidative species is mainly generated by four different enzymes: NADPH oxidases (NOX), Cyclooxygenases (COX), Xanthine

**NADPH oxidases (NOX).** NADPH oxidases are a family of enzymes composed by seven members: NOX1, NOX2, NOX3, NOX4, NOX5 and two homologues DUOX1, DUOX2. All of them are transmembrane proteins containing a NADPH-binding site, a FAD binding region, heme-binding sites and several subunits that function in the regulation and maturation of enzymes. p22phox, DUOX activator 1 (DUAX1) and DUOX activator 2 (DUOX2) are important factors for NOX and DUOX maturation. Among factors important for NOX and DUOX enzyme activation we found p67phox, NOX activator1 (NOXA1), small GTPase (RAC1 and RAC2). On the contrary specific regulator of NOX4 is polymerase δ-interacting protein 2 (POLDIP2) and Ca2+ ions are specific activators of NOX5 and DUOX1/DUOX2 isoforms. These enzymes are also associated to spatial regulator subunits p40phox, p47phox and NOX organizer1 (NOXO1) that are important for the enzyme complex structure [19]. NADPH oxidase catalytic activity


an electron donor (NADPH) to FAD subunit, heme groups and to a final electron acceptor that is a molecule of oxygen. Activation of NOX4, DUOX1 and DUOX2 results mainly in the release of hydrogen peroxide instead of superoxide anions. The specific role of this enzyme family consists in the production and release of pro-oxidative species. Such class of enzymes is considered one of the main player in the redox signaling in cardiovascular system [20]. NADPH oxidases are expressed in various types of cells along the vascular wall, including

) through an electrons transfer cycle from

conditions such us age related macular degeneration [18].

50 Research Directions in Tumor Angiogenesis

being part of established pathway for tumor angiogenesis signaling.

oxidoreductase (XOR), and dysfunctional endothelial NOS (eNOS).

consists in the generation of superoxide anions (O<sup>2</sup>

**3. Molecules generating oxidative species in endothelial cells**

**Cyclooxygenases (COX).** Cyclooxygenases act in the rate-limiting step of prostanoids biosynthesis. There are two kinds of enzymes: cyclooxygenase-1 (COX-1) and cyclooxyge‐ nase-2, also known as prostaglandin endoperoxide H synthase -1 and -2 (PGHS-1 and PGHS-2). Recently, a cyclooxygenase-1 isoenzyme was identified as COX-3 [30]. Prostanoids are lipid molecules that are produced from all animal cells in response to specific stimuli, like hormones. After activation COX produce prostanoids from free fatty acids, typically from arachidonic acid (AA). In particular COX catalyze the bis-oxygenation of AA into the prostaglandin endoperoxide PGG2, an intermediate molecule that is subsequently converted into different kinds of prostanoids by specific enzymes that are downstream COX. There are five main categories of prostanoid molecules and their specific receptors: 1) prostaglandin D2 (PGD2) whose receptors are named DP1-DP-2; 2) prostaglandin E2 (PGE2) whose receptors are named EP1-EP4, 3) prostaglandin F2α (PGF2α) whose receptors are named FP, 4) prostaglandin I2 (PGI2) whose receptors are named IP and 5) thromboxane A2 (TXA2) whose receptors are named TP. It is also been reported that some categories of prostanoids can bind peroxisome proliferator-activated receptors (PPARs). PPARs are key activators of prostanoids signaling, the binding to their specific G-protein linked receptor activates an intracellular second messenger (i.e. IP3/cAMP/DAG/Ca2+) that starts a molecular pathway, which is characteristic for each kind of ligand-receptor. Prostanoids are a class of short-life molecules: immediately after their production they are released outside from cells by specific receptors (prostaglandin transporters, PTG), allowing them to act in a paracrine or autocrine way. Prostanoids are implicated in the regulation of several physiological states (i.e. renal system, kidney functions) such us pathological states (i.e. inflammation, cancer). In the cardiovascular system this class of molecules is relevant for the homeostasis of the vasculature. Prostanoids differentially modulate vascular remodeling by direct action on endothelial cells and their progenitors (endothelial progenitor cells, EPCs) as well as on platelets and smooth muscle cells. Mainly prostacyclin PGI2 and thromboxane (TXA2) are involved in the regulation of cardiovascular system homeostasis, even though they act in a different way. PGI2 is synthetized from COX-2 and is a local vasodilator. It also regulates vascular relation by modulation of smooth muscle cells. Moreover PGI2 limits the aggregation of platelets and favorites angiogenesis by exerting a direct effect on cellular pathways of EPCs [31,32]. There are contradictory studies on PGI2 action during tumor angiogenesis, it is reported PGI2 induces tumor angiogenesis by binding to peroxisomes proliferator-activated receptor – δ (PPAR- δ)[33], on the other hand it is also been reported that healthy tissues have higher levels of PGI synthases than tumor cells. So, it has been speculated that tumor cells might induce PGI2 in neighboring endothelial cells and so they take advantage of its angiogenic property for growth. On the contrary, TXA2 is synthetized from COX-1 in platelets and promotes vasoconstriction and platelets aggregates. TXA2 plays an important role in tissue repair as well as on pathological conditions by favouring atherogenesis. Consequently, the ratio between TXA2 and PGI2 is fundamental for the main‐ tenance of physiological homeostasis [34]. In tumor conditions there are also prostanoids PGE2 and PGF2α, PGD2. Signaling of PGE2 and its receptor is involved in tumor angiogenesis. PGE2 induces upregulation of metallopeptidase 9 and activates the fibroblast factor receptor type 1 (FGFR1) [35]. Also PGF2α is considered a prostanoids molecule that sustains tumor angiogenesis by inducing activation of EGR-1, HIF-1a and VEGF. Regarding PGD2 and its receptor DP1 there are evidences for their signaling implication in vessels homeostasis, but there are opposing reports about the role of PGD2 for normal and tumor angiogenesis [36]. While molecular pathways regarding prostanoids signaling in the regulation of vessels proliferation are not fully described, their metabolism is considered of significant importance for development of anti-angiogenic drugs. Drugs for modulation of prostanoids levels are divided into two classes of molecules: 1) inhibitors of prostanoids biosynthetic enzymes (e.g. limiting prostanoids biosynthesis) and 2) antagonists of prostanoids receptors (e.g. blocking prostanoids 'cellular signaling). The most important drugs of the first class molecules are COX inhibitors like NSAIDs (aspirin, non-selective COX inhibitor) and COXIBs (selective COX-2 inhibitor) whose evidence as chemo preventive agents is yet reported in preclinical studies. Among inhibitor molecules, there are also available inhibitors of terminal prostaglandin

synthetize (tPGSs), and in particular for mPGES-1 (microsomal prostaglandin E synthetize -1). Inhibitors of mPGES-1, like AF3442e, are now at the beginning of clinical trials. Regarding the second class of drugs, there are many selective and isoform specific molecules. In particular EP antagonists have been successfully tested for limiting angiogenesis in different kinds of pathologies: the ONO8711 (EP1 antagonist) has been tested for inhibitory effects on metastasis and invasion in hepatocellular carcinoma, the EP3 antagonist ONOA23240 has been tested for limiting metastasis in Lewis lung carcinoma, the EP4 antagonists ONOA23208 and AH23848 have been tested for limiting angiogenesis and metastasis in skin melanoma, colorectal adenomas, lung carcinoma and ovarian carcinoma. The limits in application of EP antagonists is the high level of specificity of action: EP isoform antagonists effects are mediated by signaling related to a specific EP isoform relative expression in a tumor, that is always tissue and tumor dependent [37]. An alternative approach that is still needs to be validated is the application of drugs that modulate the activation of PPAR. Even though prostanoids are a heterogeneous class of molecules whose metabolism and signaling still needs to be largely characterized in

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53

tumors, they are involved in angiogenesis processes and targetable from drugs.

anion (O2


oxide and generation of ONOO-

lipid peroxidation) and improving NO bioavailability [43].

**Xanthine oxidoreductase (XOR).** Xanthine oxidoreductase is molybdenum-iron-sulfur flavin hydrolase and, consequently, its essential cofactors are molybdopterin (Mo-Co), two ironsulfur centers (Fe2-S2) and flavin adenine dinucleotide (FAD). XOR shifts between two interconvertible forms: Xanthine oxidase (XO; EC 1.1.3.22) and Xanthine Dehydrogenase (XDH; EC 1.17.14). XOR enzyme works in the purine degradation pathway where it converts hypoxan‐ thine and xanthine to uric acid. The catalytic reaction consists of an electron flow from precursor molecules to electron acceptors (cofactors). In the first part of reaction xanthine reduces XOR at the Mo-Co core and subsequently the Fe2-S2 coordination core mediates the re-oxidation of XOR by reducing FAD into FADH2. In order to restore FAD+, electrons are shifted to NAD+ and in turns directly to oxygen. Consequently, the re-oxidation reaction of XOR yields to two molecules of hydrogen peroxide (H2O2) and two molecules of superoxide

) [38]. XOR enzyme is expressed at highest levels in the gut and in the liver. However,

species [41] or by regulating myofilaments sensitivity to

it has been also detected in the heart and in endothelial cells [39]. Behind purine metabolism, XOR is reported to be important for redox signaling since superoxide radicals generated as side products have shown in pathological conditions of cardiovascular system. In vitro overexpression of XOR in cultured endothelial cells reduces cell viability, proliferation and ability to generate vascular tubes due to upregulation of ROS levels [40]. It has also been described that XOR-generated ROS affect heart cardiac contractility by reacting with nitric

Ca2+ [42]. Moreover XOR activity seems to play a role in oxidative state of infusion /reperfusion injury as well as in myocardial infarction. Treatments with inhibitors of XOR as allopurinol and oxypurinol are reported as benefic for cardiovascular pathologies related to XORgenerated ROS overload. Clinical studies have demonstrated that XOR-inhibition diminishes endothelium dysfunctions by limiting oxidation of molecules and in particular of lipids by favoring vaso-relaxation. In particular allopurinol treatment improves endothelium functions in patients with congestive heart failure by reducing plasma levels of malondyaldehyde (i.e. synthetize (tPGSs), and in particular for mPGES-1 (microsomal prostaglandin E synthetize -1). Inhibitors of mPGES-1, like AF3442e, are now at the beginning of clinical trials. Regarding the second class of drugs, there are many selective and isoform specific molecules. In particular EP antagonists have been successfully tested for limiting angiogenesis in different kinds of pathologies: the ONO8711 (EP1 antagonist) has been tested for inhibitory effects on metastasis and invasion in hepatocellular carcinoma, the EP3 antagonist ONOA23240 has been tested for limiting metastasis in Lewis lung carcinoma, the EP4 antagonists ONOA23208 and AH23848 have been tested for limiting angiogenesis and metastasis in skin melanoma, colorectal adenomas, lung carcinoma and ovarian carcinoma. The limits in application of EP antagonists is the high level of specificity of action: EP isoform antagonists effects are mediated by signaling related to a specific EP isoform relative expression in a tumor, that is always tissue and tumor dependent [37]. An alternative approach that is still needs to be validated is the application of drugs that modulate the activation of PPAR. Even though prostanoids are a heterogeneous class of molecules whose metabolism and signaling still needs to be largely characterized in tumors, they are involved in angiogenesis processes and targetable from drugs.

(PGI2) whose receptors are named IP and 5) thromboxane A2 (TXA2) whose receptors are named TP. It is also been reported that some categories of prostanoids can bind peroxisome proliferator-activated receptors (PPARs). PPARs are key activators of prostanoids signaling, the binding to their specific G-protein linked receptor activates an intracellular second messenger (i.e. IP3/cAMP/DAG/Ca2+) that starts a molecular pathway, which is characteristic for each kind of ligand-receptor. Prostanoids are a class of short-life molecules: immediately after their production they are released outside from cells by specific receptors (prostaglandin transporters, PTG), allowing them to act in a paracrine or autocrine way. Prostanoids are implicated in the regulation of several physiological states (i.e. renal system, kidney functions) such us pathological states (i.e. inflammation, cancer). In the cardiovascular system this class of molecules is relevant for the homeostasis of the vasculature. Prostanoids differentially modulate vascular remodeling by direct action on endothelial cells and their progenitors (endothelial progenitor cells, EPCs) as well as on platelets and smooth muscle cells. Mainly prostacyclin PGI2 and thromboxane (TXA2) are involved in the regulation of cardiovascular system homeostasis, even though they act in a different way. PGI2 is synthetized from COX-2 and is a local vasodilator. It also regulates vascular relation by modulation of smooth muscle cells. Moreover PGI2 limits the aggregation of platelets and favorites angiogenesis by exerting a direct effect on cellular pathways of EPCs [31,32]. There are contradictory studies on PGI2 action during tumor angiogenesis, it is reported PGI2 induces tumor angiogenesis by binding to peroxisomes proliferator-activated receptor – δ (PPAR- δ)[33], on the other hand it is also been reported that healthy tissues have higher levels of PGI synthases than tumor cells. So, it has been speculated that tumor cells might induce PGI2 in neighboring endothelial cells and so they take advantage of its angiogenic property for growth. On the contrary, TXA2 is synthetized from COX-1 in platelets and promotes vasoconstriction and platelets aggregates. TXA2 plays an important role in tissue repair as well as on pathological conditions by favouring atherogenesis. Consequently, the ratio between TXA2 and PGI2 is fundamental for the main‐ tenance of physiological homeostasis [34]. In tumor conditions there are also prostanoids PGE2 and PGF2α, PGD2. Signaling of PGE2 and its receptor is involved in tumor angiogenesis. PGE2 induces upregulation of metallopeptidase 9 and activates the fibroblast factor receptor type 1 (FGFR1) [35]. Also PGF2α is considered a prostanoids molecule that sustains tumor angiogenesis by inducing activation of EGR-1, HIF-1a and VEGF. Regarding PGD2 and its receptor DP1 there are evidences for their signaling implication in vessels homeostasis, but there are opposing reports about the role of PGD2 for normal and tumor angiogenesis [36]. While molecular pathways regarding prostanoids signaling in the regulation of vessels proliferation are not fully described, their metabolism is considered of significant importance for development of anti-angiogenic drugs. Drugs for modulation of prostanoids levels are divided into two classes of molecules: 1) inhibitors of prostanoids biosynthetic enzymes (e.g. limiting prostanoids biosynthesis) and 2) antagonists of prostanoids receptors (e.g. blocking prostanoids 'cellular signaling). The most important drugs of the first class molecules are COX inhibitors like NSAIDs (aspirin, non-selective COX inhibitor) and COXIBs (selective COX-2 inhibitor) whose evidence as chemo preventive agents is yet reported in preclinical studies. Among inhibitor molecules, there are also available inhibitors of terminal prostaglandin

52 Research Directions in Tumor Angiogenesis

**Xanthine oxidoreductase (XOR).** Xanthine oxidoreductase is molybdenum-iron-sulfur flavin hydrolase and, consequently, its essential cofactors are molybdopterin (Mo-Co), two ironsulfur centers (Fe2-S2) and flavin adenine dinucleotide (FAD). XOR shifts between two interconvertible forms: Xanthine oxidase (XO; EC 1.1.3.22) and Xanthine Dehydrogenase (XDH; EC 1.17.14). XOR enzyme works in the purine degradation pathway where it converts hypoxan‐ thine and xanthine to uric acid. The catalytic reaction consists of an electron flow from precursor molecules to electron acceptors (cofactors). In the first part of reaction xanthine reduces XOR at the Mo-Co core and subsequently the Fe2-S2 coordination core mediates the re-oxidation of XOR by reducing FAD into FADH2. In order to restore FAD+, electrons are shifted to NAD+ and in turns directly to oxygen. Consequently, the re-oxidation reaction of XOR yields to two molecules of hydrogen peroxide (H2O2) and two molecules of superoxide anion (O2 - ) [38]. XOR enzyme is expressed at highest levels in the gut and in the liver. However, it has been also detected in the heart and in endothelial cells [39]. Behind purine metabolism, XOR is reported to be important for redox signaling since superoxide radicals generated as side products have shown in pathological conditions of cardiovascular system. In vitro overexpression of XOR in cultured endothelial cells reduces cell viability, proliferation and ability to generate vascular tubes due to upregulation of ROS levels [40]. It has also been described that XOR-generated ROS affect heart cardiac contractility by reacting with nitric oxide and generation of ONOO species [41] or by regulating myofilaments sensitivity to Ca2+ [42]. Moreover XOR activity seems to play a role in oxidative state of infusion /reperfusion injury as well as in myocardial infarction. Treatments with inhibitors of XOR as allopurinol and oxypurinol are reported as benefic for cardiovascular pathologies related to XORgenerated ROS overload. Clinical studies have demonstrated that XOR-inhibition diminishes endothelium dysfunctions by limiting oxidation of molecules and in particular of lipids by favoring vaso-relaxation. In particular allopurinol treatment improves endothelium functions in patients with congestive heart failure by reducing plasma levels of malondyaldehyde (i.e. lipid peroxidation) and improving NO bioavailability [43].

**Endothelial Nitric Oxide Synthase (eNOS).** eNOS is one of the three isoform of nitric oxide synthase family and it is constitutively expressed in endothelial cells. eNOS is important since it is the major source of endothelial nitric oxide (NO). NOS enzymes work as homodimers with support of several cofactors. One monomer is linked to flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN) and binds the second monomer at the oxygenase domain that contains a prosthetic heme group. The second monomer is also linked to cofactors: tetrahydrobiopterin (BH4) and molecular oxygen. The catalytic activity consists in an electron flow between NOS cofactors where the substrate: L-arginine is oxidized to L-citrulline with concomitant production of NO. Moreover calmodulin (CaM) and Ca2+ are essential for functional enzyme [44]. When eNOS is not able to produce NO due the absence of specific cofactors the reduction of oxygen and concomitant production of NO are uncoupled. As consequence of uncoupled eNOS the superoxide anion is produced instead of NO [45]. The importance of eNOS for endothelium homeostasis is related to NO as well as to its side product O2 - . In physiological conditions NO function is widely characterized as regulator molecule for vasorelaxation and maintenance of healthy vascular beds. However the level of NO is critical for vascular homeostasis. Low and medium NO levels are involved in cellular signaling, while high NO levels are related to apoptosis and cell damage. NO is a gas that diffuse among tissues. NO is a very reactive molecule that spontaneously reacts with free radicals (i.e. superoxide anions) generating reactive nitrogen species (RNS) among which the most common is peroxynitrate (ONOO-). Peroxynitrate is a potent pro-oxidative radical that cause intracellular damage by nitration and S-nitrosylation of proteins, lipid and DNA [46]. Excessive cellular damage causes severe endothelium dysfunctions as reported in multiple cases of cardiovascular disease as diabetes and hyper‐ tensions or inflammation [47,48]. eNOS expression and NO are important players for angio‐ genesis not only in physiological conditions, but also in tumor conditions. NO contributes to angiogenesis by activating intracellular molecular pathway such as the mitogen activated kinases (MAPK), cyclic GMP (cGMP), and by regulating expression of fibroblast growth factor (FGF-2) and controlling the balance between metalloprotease (MMP) and their inhibitors in surrounding tissues. In tumor conditions it has been reported that tumor cells can upregulate NO levels by induction of specific intracellular NOS isoforms (iNOS and nNOS) in order to activate NO-dependent angiogenic signaling. Also eNOS is normally expressed by endothelial tumor cells and is sensitive to multiple factors present in the tumor microenvironment. Proangiogenic factors such as vascular endothelial growth factor (VEGF), sex hormones or angiopoietins activate eNOS and positively regulate eNOS in endothelial cells through specific molecular pathways such as 1) Akt-phosphoinositide3 (PI3K) pathway, 2) phospholipase Cγ (PLCγ)-diacylglicerol (DAG)/Ca2+ 3) adenilate cyclase (AC) -protein kinase A (PKA). Upregu‐ lation of eNOS triggers NO-specific intracellular signaling not only through cGMP, but also leading to post-translational modifications of proteins to form S-nitrosothiol and, thus, generating a specific oxidative signaling mediated by S-nitrosylation. An example of such mechanism is the nitrosylation of caspase 3 that inhibits apoptosis or the nitrosylation of p21Ras that enforces cGMP signaling by increasing endothelial cell proliferation [49,50]. Supporting evidences for NO involvement in tumor progression come from in vivo studies

with NOS inhibitors that have demonstrated a peculiar role for NO in sustaining tumor growth. Anti-metastatic effects have been reported in several kinds of tumors under treatment with NOS inhibitors NG-methyl-L-arginine (NMMA) and NG – Nitro-L-arginine methyl ester

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Together with these enzymes the mitochondrial electron transport chain (ETC) has been recognized as responsible for pro-oxidative species production. The mitochondrial respirato‐ ry chain is one of the first sources of pro-oxidative species to have been characterized in cells. Mechanism through witch oxidative species are produced in mitochondria are widely described as side products of ETC [53,54]. As it has been described ETC consists in an electron flow among different protein complexes in the inner mitochondria membranes. Electrons from NADPH are transferred NADPH-ubiquinone oxidoreductase complex I which consequently transfer electrons downstream to complex II. Then, electrons according to electrochemical gradients flow to complexes III and IV. The final step of the chain is the reduction of oxygen to water, however it has been quantified that about 1-4% of oxygen fails to be properly reduced and superoxide is produced as consequence. Dysfunctional ETC leads to high levels of ROS in mitochondria that are reported as cytotoxic, however this condi‐ tion has been also associated with induction of pro-angiogenic signaling [55]. In vitro and in vivo treatments with inhibitors of ETC (i.e. rotenone) inhibits VEGF -induced signaling and vascular walls remodeling [56] suggesting that ETC may play a role in redox signaling in

**4. Cellular systems for counterbalance oxidative species in angiogenesis:**

In order to limit oxidative stress levels cells are armed with a series of enzymes and molecules. Important enzymes for degradation of hydrogen peroxide and superoxide are family of superoxide dismutase (SOD), catalase (CAT), peroxiredoxins (PRX), thioredoxin (TRX) and gluthatione peroxidase (GPx). All these enzymes play a critical role in modulation redox

**Superoxide dismutase** (SOD) is the most important cellular mechanism of protection against

The catalytic reaction of SOD involves metal cations (i.e. Cu, Zn, Mn) as cofactors that continuously shift between reduced and oxidized forms in the active site of the enzymes. In humans there are three isoforms of superoxide dismutase enzymes that are distinguished for their cellular localization: SOD1 (CuZn-SOD) which is localized essentially in the cytosol, SOD2 (Mn-SOD) which is localized in the mitochondria and SOD3 (CuZn-SOD, also known as ec-SOD) which is localized in the extracellular matrix. All three isoforms catalyze the same reaction, which is important, not only to scavenge the cytotoxic effects of superoxide anion accumulation (i.e. oxidation and inactivation of proteins), but also to prevent the reaction of



(L-NAME) [51,52].

normal and pathological angiogenesis.

**4.1. Antioxidant enzymes**

signaling.

superoxide anion (O2

**Natural antioxidants and scavenging systems**

with NOS inhibitors that have demonstrated a peculiar role for NO in sustaining tumor growth. Anti-metastatic effects have been reported in several kinds of tumors under treatment with NOS inhibitors NG-methyl-L-arginine (NMMA) and NG – Nitro-L-arginine methyl ester (L-NAME) [51,52].

Together with these enzymes the mitochondrial electron transport chain (ETC) has been recognized as responsible for pro-oxidative species production. The mitochondrial respirato‐ ry chain is one of the first sources of pro-oxidative species to have been characterized in cells. Mechanism through witch oxidative species are produced in mitochondria are widely described as side products of ETC [53,54]. As it has been described ETC consists in an electron flow among different protein complexes in the inner mitochondria membranes. Electrons from NADPH are transferred NADPH-ubiquinone oxidoreductase complex I which consequently transfer electrons downstream to complex II. Then, electrons according to electrochemical gradients flow to complexes III and IV. The final step of the chain is the reduction of oxygen to water, however it has been quantified that about 1-4% of oxygen fails to be properly reduced and superoxide is produced as consequence. Dysfunctional ETC leads to high levels of ROS in mitochondria that are reported as cytotoxic, however this condi‐ tion has been also associated with induction of pro-angiogenic signaling [55]. In vitro and in vivo treatments with inhibitors of ETC (i.e. rotenone) inhibits VEGF -induced signaling and vascular walls remodeling [56] suggesting that ETC may play a role in redox signaling in normal and pathological angiogenesis.

#### **4. Cellular systems for counterbalance oxidative species in angiogenesis: Natural antioxidants and scavenging systems**

#### **4.1. Antioxidant enzymes**

**Endothelial Nitric Oxide Synthase (eNOS).** eNOS is one of the three isoform of nitric oxide synthase family and it is constitutively expressed in endothelial cells. eNOS is important since it is the major source of endothelial nitric oxide (NO). NOS enzymes work as homodimers with support of several cofactors. One monomer is linked to flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN) and binds the second monomer at the oxygenase domain that contains a prosthetic heme group. The second monomer is also linked to cofactors: tetrahydrobiopterin (BH4) and molecular oxygen. The catalytic activity consists in an electron flow between NOS cofactors where the substrate: L-arginine is oxidized to L-citrulline with concomitant production of NO. Moreover calmodulin (CaM) and Ca2+ are essential for functional enzyme [44]. When eNOS is not able to produce NO due the absence of specific cofactors the reduction of oxygen and concomitant production of NO are uncoupled. As consequence of uncoupled eNOS the superoxide anion is produced instead of NO [45]. The importance of eNOS for endothelium homeostasis is


widely characterized as regulator molecule for vasorelaxation and maintenance of healthy vascular beds. However the level of NO is critical for vascular homeostasis. Low and medium NO levels are involved in cellular signaling, while high NO levels are related to apoptosis and cell damage. NO is a gas that diffuse among tissues. NO is a very reactive molecule that spontaneously reacts with free radicals (i.e. superoxide anions) generating reactive nitrogen species (RNS) among which the most common is peroxynitrate (ONOO-). Peroxynitrate is a potent pro-oxidative radical that cause intracellular damage by nitration and S-nitrosylation of proteins, lipid and DNA [46]. Excessive cellular damage causes severe endothelium dysfunctions as reported in multiple cases of cardiovascular disease as diabetes and hyper‐ tensions or inflammation [47,48]. eNOS expression and NO are important players for angio‐ genesis not only in physiological conditions, but also in tumor conditions. NO contributes to angiogenesis by activating intracellular molecular pathway such as the mitogen activated kinases (MAPK), cyclic GMP (cGMP), and by regulating expression of fibroblast growth factor (FGF-2) and controlling the balance between metalloprotease (MMP) and their inhibitors in surrounding tissues. In tumor conditions it has been reported that tumor cells can upregulate NO levels by induction of specific intracellular NOS isoforms (iNOS and nNOS) in order to activate NO-dependent angiogenic signaling. Also eNOS is normally expressed by endothelial tumor cells and is sensitive to multiple factors present in the tumor microenvironment. Proangiogenic factors such as vascular endothelial growth factor (VEGF), sex hormones or angiopoietins activate eNOS and positively regulate eNOS in endothelial cells through specific molecular pathways such as 1) Akt-phosphoinositide3 (PI3K) pathway, 2) phospholipase Cγ (PLCγ)-diacylglicerol (DAG)/Ca2+ 3) adenilate cyclase (AC) -protein kinase A (PKA). Upregu‐ lation of eNOS triggers NO-specific intracellular signaling not only through cGMP, but also leading to post-translational modifications of proteins to form S-nitrosothiol and, thus, generating a specific oxidative signaling mediated by S-nitrosylation. An example of such mechanism is the nitrosylation of caspase 3 that inhibits apoptosis or the nitrosylation of p21Ras that enforces cGMP signaling by increasing endothelial cell proliferation [49,50]. Supporting evidences for NO involvement in tumor progression come from in vivo studies

. In physiological conditions NO function is

related to NO as well as to its side product O2

54 Research Directions in Tumor Angiogenesis

In order to limit oxidative stress levels cells are armed with a series of enzymes and molecules. Important enzymes for degradation of hydrogen peroxide and superoxide are family of superoxide dismutase (SOD), catalase (CAT), peroxiredoxins (PRX), thioredoxin (TRX) and gluthatione peroxidase (GPx). All these enzymes play a critical role in modulation redox signaling.

**Superoxide dismutase** (SOD) is the most important cellular mechanism of protection against superoxide anion (O2 -). SOD catalyzes the dismutation of O2 - into hydrogen peroxide (H2O2). The catalytic reaction of SOD involves metal cations (i.e. Cu, Zn, Mn) as cofactors that continuously shift between reduced and oxidized forms in the active site of the enzymes. In humans there are three isoforms of superoxide dismutase enzymes that are distinguished for their cellular localization: SOD1 (CuZn-SOD) which is localized essentially in the cytosol, SOD2 (Mn-SOD) which is localized in the mitochondria and SOD3 (CuZn-SOD, also known as ec-SOD) which is localized in the extracellular matrix. All three isoforms catalyze the same reaction, which is important, not only to scavenge the cytotoxic effects of superoxide anion accumulation (i.e. oxidation and inactivation of proteins), but also to prevent the reaction of O2 – with nitric oxide (NO) to generate peroxynitrate. In this way these enzymes guarantee the metabolism of H2O2, important for redox signaling [57]. In the vasculature the signaling of H2O2 produced by SODs activates multiple pathways important for angiogenesis. The H2O2 generated by SOD3 in the extracellular space favorites VEGFR2 signaling and consequently modulates angiogenesis. H2O2 produced by SOD3 under conditions of ischemic injury, protects tissues and promotes neovascularization by enhancing Ras-ERK, PI3kinase-Akt pathways and VEGF expression [58].

in order to down-regulate intracellular ROS levels and make tumor cells more sensitive to

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**Peroxiredoxins (PRX)** are a family of ubiquitous antioxidant enzymes constituted by several isoforms. PRX catalytic activity consists in the reduction of cellular hydrogen peroxide and for some aspects it overlaps with enzyme activity of other antioxidant enzymes (i.e. catalase and GPx) [67]. However, PRXs differentiate from other antioxidant enzymes for their mechanism of activity. Their enzymatic active site is constituted by cysteine amminoacids that metabolize H2O2 by cycling between oxidation and reduction reactions. When a molecule of hydrogen peroxide enters, the active Cys-SH oxides into Cys-SOH. This intermediate form can be further oxidized to Cys-SO2H. The recycling of cysteine is mediated by glutathione, ascorbic acid or sulfiredoxins. According with the setting of the active site PRX are divided into three groups. The first two groups are Typical 2-Cys-PRX and Atypical 2-Cys-PRX, according with folding structure, and both contains two residues of Cys in their active site are. The third class, 1-Cys-PRX contains only one Cys in the active site. Besides the difference in the number of active cysteine, all PRXs act as intracellular H2O2 scavengers. PRXs are localized primarily in the cytosol, but they are localized also in intracellular organelles (peroxisomes, mitochondria) where they take part to regulation of H2O2 levels and redox signaling [68,69]. In vivo knockout mice for PRX-VI are more sensitive to oxidative stress under hyperoxia exposure, while knockout mice for PRX-I and PRX-II develop severe blood cells disease (hemolytic anemia and hematopoietic cancer) [70,71]. The role of PRXs in redox signaling in cardiovascular system is

**Thioredoxins (TRX)** are a small class of antioxidant enzymes composed of two isoforms: TRX1, which is primarily localized in the cytosol and nucleus, and TRX, which is found in mitochon‐ dria. All TRX enzymes are ubiquitously expressed and are characterized by a dithiol-disulfide site. The active site of TRX contains a specific and highly conserved motif with two residues of Cysteine that are essentials to reduce oxidized proteins and buffer ROS. TRX can be continuously reconverted from oxidized form into reduced form thanks to thioredoxin reductase enzymes activity. The TRX system is modulated by an endogenous inhibitor protein, called TXNIP (TRX-interacting protein), that prevents TRX to form disulfides [72]. The TRX system has been shown to be essential for life since the knockout mice of either isoform is lethal for embryo development [73,74]. Moreover endothelium specific *Trx2* transgenic mice as well as mice overexpressing *Trx1* demonstrate a crucial role of this class of enzymes in buffering oxidative stress in endothelial cells. TRX1 can modulate different cellular processes involved with endothelial cell homeostasis and angiogenesis. In endothelial cells TRX1 prevents degradation of HIF1α and consequently modulate VEGF expression facilitating pro-angio‐ genic processes. Moreover, TRX1 can regulate proliferation and migration of endothelial cells by modulation of NF-kB activity and upregulation of matrix metalloproteases (MMPs). TRX2 have been demonstrated to play also a specific role in endothelial cells by inducing angiogen‐ esis and arteriogenesis in pathological conditions (i.e. murine model of ischemia) [75]. The importance of this antioxidant system for promoting angiogenesis has been considered for development of anticancer drugs. In vitro studies performed with TRX inhibitors (i.e. PMX464, AJM290) confirm the pivotal role of this class of enzymes in preventing endothelial cell

therapy (paclitaxel, etoposide and arsenic triosside) [66].

still not clear.

proliferation and differentiation [76].

The H2O2 generated by SOD1 is actively produced in endosomes under inflammation signals and activates NF-kB. Moreover such H2O2 generated by SOD1 is particularly important in endothelial cells where acts as endothelium–derived hyperpolarization factor (EDHF). It has been demonstrated that in the tumorigenic context, SOD1 overexpression promotes angio‐ genesis and tumor growth. Also the H2O2 generated by SOD2 is important for endothelium. It has been demonstrated that SOD2 overexpression favorites Akt pathway activation and enhances vessels formations *in vivo* by favoring endothelial cells sprouting. On the contrary, SOD2 deficiency causes increased mitochondrial O2 – that results in mitochondria damage (i.e. mtDNA and mitochondrial proteins oxidation) and endothelial dysfunctions [59,60]. Addi‐ tionally, all SODs enzymes modulate vessels homeostasis by influencing EPCs. SOD3-/- mice show EPCs failure in physiological processes of migration and differentiation. A recent report indicate that SOD1-deficient EPCs show shortages in migration and ability to generate small vessels networks [61]. Thus, SODs enzymes play their role in redox signaling by regulating angiogenesis through H2O2 and protecting EPCs from excess of O2 –.

**Catalase (CAT)** catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen by helping the antioxidant machinery in cells. The active site of the enzyme is made by four porphyry heme groups, that are essential to catalyze the flow of electrons between atoms. The mechanism of reaction is not jet fully characterized however it is supposed to occur into two steps: in the first step the iron is reduced with concomitant production of water, in the second step another molecule of hydrogen peroxide enters into the active site and allows the re-oxidation of iron with contemporary production of water and oxygen [62]. The balance of the reaction consists of two molecules of H2O2 that are decomposed into two molecules of water and one molecule of oxygen. Catalase is intracellular localized, mainly in peroxisomes of animal and vegetal cells. In addition, there are also data supporting its localization in the mitochondria and cytosol. Catalase is an enzyme shared between all organisms. All cells contain catalase, however knock-out animal models do not display severe phenotypes. Also human patients showing reduced levels of catalase enzymes do not display severe health disorder [63]. It is supposed that the lack of catalytic activity of catalase may be replaced by multiple alternative antioxidant systems. The catalase plays an important role in the redox signaling since it regulates H2O2 levels, which is important for homeostasis of vascular beds. Catalase of endothelial cells protects smooth muscle cells from oxidative damage of luminal peroxide [64] and is involved in mechanisms of vessels relaxation [65]. Moreover, it has been also reported that catalase in combination with SOD play a synergistic role in the regulation of endothelium permeability. Overexpression of catalase was also applied to breast cancer cells in order to down-regulate intracellular ROS levels and make tumor cells more sensitive to therapy (paclitaxel, etoposide and arsenic triosside) [66].

O2 –

pathways and VEGF expression [58].

56 Research Directions in Tumor Angiogenesis

SOD2 deficiency causes increased mitochondrial O2

angiogenesis through H2O2 and protecting EPCs from excess of O2

 with nitric oxide (NO) to generate peroxynitrate. In this way these enzymes guarantee the metabolism of H2O2, important for redox signaling [57]. In the vasculature the signaling of H2O2 produced by SODs activates multiple pathways important for angiogenesis. The H2O2 generated by SOD3 in the extracellular space favorites VEGFR2 signaling and consequently modulates angiogenesis. H2O2 produced by SOD3 under conditions of ischemic injury, protects tissues and promotes neovascularization by enhancing Ras-ERK, PI3kinase-Akt

The H2O2 generated by SOD1 is actively produced in endosomes under inflammation signals and activates NF-kB. Moreover such H2O2 generated by SOD1 is particularly important in endothelial cells where acts as endothelium–derived hyperpolarization factor (EDHF). It has been demonstrated that in the tumorigenic context, SOD1 overexpression promotes angio‐ genesis and tumor growth. Also the H2O2 generated by SOD2 is important for endothelium. It has been demonstrated that SOD2 overexpression favorites Akt pathway activation and enhances vessels formations *in vivo* by favoring endothelial cells sprouting. On the contrary,

mtDNA and mitochondrial proteins oxidation) and endothelial dysfunctions [59,60]. Addi‐ tionally, all SODs enzymes modulate vessels homeostasis by influencing EPCs. SOD3-/- mice show EPCs failure in physiological processes of migration and differentiation. A recent report indicate that SOD1-deficient EPCs show shortages in migration and ability to generate small vessels networks [61]. Thus, SODs enzymes play their role in redox signaling by regulating

**Catalase (CAT)** catalyzes the decomposition of hydrogen peroxide (H2O2) into water and oxygen by helping the antioxidant machinery in cells. The active site of the enzyme is made by four porphyry heme groups, that are essential to catalyze the flow of electrons between atoms. The mechanism of reaction is not jet fully characterized however it is supposed to occur into two steps: in the first step the iron is reduced with concomitant production of water, in the second step another molecule of hydrogen peroxide enters into the active site and allows the re-oxidation of iron with contemporary production of water and oxygen [62]. The balance of the reaction consists of two molecules of H2O2 that are decomposed into two molecules of water and one molecule of oxygen. Catalase is intracellular localized, mainly in peroxisomes of animal and vegetal cells. In addition, there are also data supporting its localization in the mitochondria and cytosol. Catalase is an enzyme shared between all organisms. All cells contain catalase, however knock-out animal models do not display severe phenotypes. Also human patients showing reduced levels of catalase enzymes do not display severe health disorder [63]. It is supposed that the lack of catalytic activity of catalase may be replaced by multiple alternative antioxidant systems. The catalase plays an important role in the redox signaling since it regulates H2O2 levels, which is important for homeostasis of vascular beds. Catalase of endothelial cells protects smooth muscle cells from oxidative damage of luminal peroxide [64] and is involved in mechanisms of vessels relaxation [65]. Moreover, it has been also reported that catalase in combination with SOD play a synergistic role in the regulation of endothelium permeability. Overexpression of catalase was also applied to breast cancer cells

–

that results in mitochondria damage (i.e.

–.

**Peroxiredoxins (PRX)** are a family of ubiquitous antioxidant enzymes constituted by several isoforms. PRX catalytic activity consists in the reduction of cellular hydrogen peroxide and for some aspects it overlaps with enzyme activity of other antioxidant enzymes (i.e. catalase and GPx) [67]. However, PRXs differentiate from other antioxidant enzymes for their mechanism of activity. Their enzymatic active site is constituted by cysteine amminoacids that metabolize H2O2 by cycling between oxidation and reduction reactions. When a molecule of hydrogen peroxide enters, the active Cys-SH oxides into Cys-SOH. This intermediate form can be further oxidized to Cys-SO2H. The recycling of cysteine is mediated by glutathione, ascorbic acid or sulfiredoxins. According with the setting of the active site PRX are divided into three groups. The first two groups are Typical 2-Cys-PRX and Atypical 2-Cys-PRX, according with folding structure, and both contains two residues of Cys in their active site are. The third class, 1-Cys-PRX contains only one Cys in the active site. Besides the difference in the number of active cysteine, all PRXs act as intracellular H2O2 scavengers. PRXs are localized primarily in the cytosol, but they are localized also in intracellular organelles (peroxisomes, mitochondria) where they take part to regulation of H2O2 levels and redox signaling [68,69]. In vivo knockout mice for PRX-VI are more sensitive to oxidative stress under hyperoxia exposure, while knockout mice for PRX-I and PRX-II develop severe blood cells disease (hemolytic anemia and hematopoietic cancer) [70,71]. The role of PRXs in redox signaling in cardiovascular system is still not clear.

**Thioredoxins (TRX)** are a small class of antioxidant enzymes composed of two isoforms: TRX1, which is primarily localized in the cytosol and nucleus, and TRX, which is found in mitochon‐ dria. All TRX enzymes are ubiquitously expressed and are characterized by a dithiol-disulfide site. The active site of TRX contains a specific and highly conserved motif with two residues of Cysteine that are essentials to reduce oxidized proteins and buffer ROS. TRX can be continuously reconverted from oxidized form into reduced form thanks to thioredoxin reductase enzymes activity. The TRX system is modulated by an endogenous inhibitor protein, called TXNIP (TRX-interacting protein), that prevents TRX to form disulfides [72]. The TRX system has been shown to be essential for life since the knockout mice of either isoform is lethal for embryo development [73,74]. Moreover endothelium specific *Trx2* transgenic mice as well as mice overexpressing *Trx1* demonstrate a crucial role of this class of enzymes in buffering oxidative stress in endothelial cells. TRX1 can modulate different cellular processes involved with endothelial cell homeostasis and angiogenesis. In endothelial cells TRX1 prevents degradation of HIF1α and consequently modulate VEGF expression facilitating pro-angio‐ genic processes. Moreover, TRX1 can regulate proliferation and migration of endothelial cells by modulation of NF-kB activity and upregulation of matrix metalloproteases (MMPs). TRX2 have been demonstrated to play also a specific role in endothelial cells by inducing angiogen‐ esis and arteriogenesis in pathological conditions (i.e. murine model of ischemia) [75]. The importance of this antioxidant system for promoting angiogenesis has been considered for development of anticancer drugs. In vitro studies performed with TRX inhibitors (i.e. PMX464, AJM290) confirm the pivotal role of this class of enzymes in preventing endothelial cell proliferation and differentiation [76].

**Glutathione peroxidases (GPx)** are a family of enzymes that catalyze the reduction of hydrogen peroxide and organic hydroperoxides to water. The reaction consists in the oxidation of monomeric glutathione to glutathione disulfide with the involvement of a selenic acid group. Oxidized glutathione molecules are then reduced by a specific glutathione reductase [77]. In humans there are eight isoforms of glutathione peroxidases with different intracellular localizations and different relative abundance in tissues. Human GPx1, GPx2, GPx3, GPx4 and Gpx6 are different from other isoforms for containing seleno-cysteines in their catalytic sites, which identifies them as seleno-proteins. All GPxs play a fundamental role in the antioxidant molecular network as peroxide scavenging enzymes, however specific notes are reported for different isoforms. GPx4 has been identified mainly as phospholipid hydroperoxidase since it not only reduces peroxides but it is also efficient in reducing phospholipids, cholesterol and lipoproteins hydroperoxide [78]. In pig livers GPx4 activity was reported for inhibition of lipid peroxidation [79] and curiously crucial for sperm maturation [80]. GPx3 is produced in the tubules of kidney and secreted in extracellular fluids as well as in the plasma, but its antioxi‐ dant activity does not seem to be essential since GPx3-/- mice do not show abnormal phenotype [81]. Recent studies on GPx3 promoter regulation suggest that its expressivity is implicated in epithelial tumor development but a specific role needs to be addressed [82]. GPx2 is expressed in the gastrointestinal system and is supposed to play a key role as antioxidant enzyme in the gut. Also, GPx2-/- mice do not show abnormal phenotype [83] but in vitro and in vivo data regarding loss of GPx2 expression report a role for GPx2 in regulation of inflammationmediated carcinogenesis and for supporting growth of established tumors. Among the GPxs isoforms the most studied and characterized is GPx1, which is also the most abundant one. It is ubiquitously expressed and it is localized mainly in the cytosol and in mitochondria. GPx1 is believed to be the most important peroxide scavenger in the family [84], even tough also GPx1-/- mice are not lethal and develop normally [85]. In vivo data indicate that the loss of this enzyme is correlated with high oxidative damage condition. Loss of GPx1 in condition of cerebral inflammation increases pro-oxidative species level and favors interactions between leukocytes and endothelial cells of cerebral microvasculature [86]. Loss of GPx1 in human microvascular cells as well as in GPx1-/- mice favors endothelium response to lipo-polisaccaride pro-oxidant stimuli favoring intracellular reactive oxygen species accumulation and altering expression of adhesion molecules. Levels of GPx1 are also reported to modulate angiogenic endothelial progenitor cells (EPCs) in correlation with aging. EPCs of old subjects, that have impaired GPx1 levels, are more sensitive to oxidative damage [87].

correlate black tea assumption with beneficial effects on endothelium dysfunctions in indi‐ viduals with chronic heart disease and hypercholesterolemia [89]. In vitro studies on endo‐ thelial cells demonstrate polyphenols modulate redox signaling by regulation of arachidonic acid cascade. In particular, it has been reported that polyphenols from virgin olive oil and red wine reduce significantly angiogenesis by inhibition of cyclooxigenase2 (COX2) and activation of redox sensitive NF-kB pathway [90]. Microarray data and RT-PCR analyses show that treatment of endothelial cells (HUVEC) with resveratrol (contained in red wine) can upregu‐ late eNOS and decrease the levels of endothelin-1, suggesting a protective role against endothelium contractions. Moreover, resveratrol exerts a protective effects on endothelium as assed also under pro-oxidative state (in presence of H2O2). Together with anthocyanin, also polyphenols (contained in berries that have red pigments) are reported to have an antioxidant positive effect for cardiovascular system. Recently, anthocyanins from six berries extracts have been mixed in a formula (OptiBerry), which in vitro exhibits anti-angiogenic properties on human microvascular endothelial cells and also in vivo impairs endothelioma cells for tumor

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Among **lipids** there are also very important antioxidant molecules. They are naturally present in plants like carotenes (retinol and b-carotene), alpha-tocopherol (also known as Vitamin E) or synthetized by animal cells, like CoenzymeQ10. The characteristic lipid character of these molecules allows them to localize in cell membranes (intracellular organelles and plasma membrane) where they can buffer lipid radicals and prevent reactions of peroxidation. Antioxidant Vitamin E properties for lipid peroxidation were efficiently assayed in GPx4-/ mice [92]. Carotenoids are tetraterpenoid pigments contained exclusively in plant cells that can be assumed with diet and act as terminal antioxidant molecules, once oxidized they can not be "re-used" from cells [93]. CoenzymeQ10 (CoQ10) is a terpenoid molecule whose antioxidant activity has been reported for maintenance of healthy cardiovascular system. Recent clinical trials have also show the use of CoQ10 for lowering blood pressure. At the present there are not evidences regarding the involvement of lipid antioxidant molecules in

Several other **antioxidant genes** are normally induced in cells to shield against dangerous deregulation of redox balance. Among those which play a key role in angiogenesis we can find heme-oxygenase-1 (HMOX-1) and nuclear factor erythroid 2 (NRF2) [95]. Upregulation of these genes are correlates to tumor metastasis and progression suggesting how oxidative stress

**Vascular Endothelial Growth Factor (VEGF)** family encloses six glycoproteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D AND VEGF-E, all of them belong to a superfamily of growth factors. Endothelial cells have three types of specific VEGF receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR, Flk-1), VEGFR-3 (Flt-4). Signaling mediated by VEGF and respective receptors has been characterized as one of the most powerful factors for induction and maintenance of

growth [91].

conditions of tumor angiogenesis [94].

is a condition implicated in tumor angiogenesis [96].

**5. Angiogenic molecules regulated by redox signaling**

#### **4.2. Antioxidant molecules**

Recent evidence suggests that many natural **antioxidant molecules** contained in foods or plants have beneficial effects against tumor progression. Polyphenols as well as terpenoids act on overall oxidative stress levels. By modulation of cytokines, metabolizing enzymes, growth factors and various molecules in redox signaling, antioxidants regulate pathways for tumor angiogenesis. Natural **polyphenols** are a class of compounds constituted by molecules containing repetitive units of phenols that characterize them with antioxidant properties. Polyphenols are naturally present in vegetal derived foods (i.e. fruits, tea, red wine, honey, olive oil) and can be assumed directly with the diet [88]. Data regarding alimentary habits correlate black tea assumption with beneficial effects on endothelium dysfunctions in indi‐ viduals with chronic heart disease and hypercholesterolemia [89]. In vitro studies on endo‐ thelial cells demonstrate polyphenols modulate redox signaling by regulation of arachidonic acid cascade. In particular, it has been reported that polyphenols from virgin olive oil and red wine reduce significantly angiogenesis by inhibition of cyclooxigenase2 (COX2) and activation of redox sensitive NF-kB pathway [90]. Microarray data and RT-PCR analyses show that treatment of endothelial cells (HUVEC) with resveratrol (contained in red wine) can upregu‐ late eNOS and decrease the levels of endothelin-1, suggesting a protective role against endothelium contractions. Moreover, resveratrol exerts a protective effects on endothelium as assed also under pro-oxidative state (in presence of H2O2). Together with anthocyanin, also polyphenols (contained in berries that have red pigments) are reported to have an antioxidant positive effect for cardiovascular system. Recently, anthocyanins from six berries extracts have been mixed in a formula (OptiBerry), which in vitro exhibits anti-angiogenic properties on human microvascular endothelial cells and also in vivo impairs endothelioma cells for tumor growth [91].

**Glutathione peroxidases (GPx)** are a family of enzymes that catalyze the reduction of hydrogen peroxide and organic hydroperoxides to water. The reaction consists in the oxidation of monomeric glutathione to glutathione disulfide with the involvement of a selenic acid group. Oxidized glutathione molecules are then reduced by a specific glutathione reductase [77]. In humans there are eight isoforms of glutathione peroxidases with different intracellular localizations and different relative abundance in tissues. Human GPx1, GPx2, GPx3, GPx4 and Gpx6 are different from other isoforms for containing seleno-cysteines in their catalytic sites, which identifies them as seleno-proteins. All GPxs play a fundamental role in the antioxidant molecular network as peroxide scavenging enzymes, however specific notes are reported for different isoforms. GPx4 has been identified mainly as phospholipid hydroperoxidase since it not only reduces peroxides but it is also efficient in reducing phospholipids, cholesterol and lipoproteins hydroperoxide [78]. In pig livers GPx4 activity was reported for inhibition of lipid peroxidation [79] and curiously crucial for sperm maturation [80]. GPx3 is produced in the tubules of kidney and secreted in extracellular fluids as well as in the plasma, but its antioxi‐ dant activity does not seem to be essential since GPx3-/- mice do not show abnormal phenotype [81]. Recent studies on GPx3 promoter regulation suggest that its expressivity is implicated in epithelial tumor development but a specific role needs to be addressed [82]. GPx2 is expressed in the gastrointestinal system and is supposed to play a key role as antioxidant enzyme in the gut. Also, GPx2-/- mice do not show abnormal phenotype [83] but in vitro and in vivo data regarding loss of GPx2 expression report a role for GPx2 in regulation of inflammationmediated carcinogenesis and for supporting growth of established tumors. Among the GPxs isoforms the most studied and characterized is GPx1, which is also the most abundant one. It is ubiquitously expressed and it is localized mainly in the cytosol and in mitochondria. GPx1 is believed to be the most important peroxide scavenger in the family [84], even tough also GPx1-/- mice are not lethal and develop normally [85]. In vivo data indicate that the loss of this enzyme is correlated with high oxidative damage condition. Loss of GPx1 in condition of cerebral inflammation increases pro-oxidative species level and favors interactions between leukocytes and endothelial cells of cerebral microvasculature [86]. Loss of GPx1 in human microvascular cells as well as in GPx1-/- mice favors endothelium response to lipo-polisaccaride pro-oxidant stimuli favoring intracellular reactive oxygen species accumulation and altering expression of adhesion molecules. Levels of GPx1 are also reported to modulate angiogenic endothelial progenitor cells (EPCs) in correlation with aging. EPCs of old subjects, that have

impaired GPx1 levels, are more sensitive to oxidative damage [87].

Recent evidence suggests that many natural **antioxidant molecules** contained in foods or plants have beneficial effects against tumor progression. Polyphenols as well as terpenoids act on overall oxidative stress levels. By modulation of cytokines, metabolizing enzymes, growth factors and various molecules in redox signaling, antioxidants regulate pathways for tumor angiogenesis. Natural **polyphenols** are a class of compounds constituted by molecules containing repetitive units of phenols that characterize them with antioxidant properties. Polyphenols are naturally present in vegetal derived foods (i.e. fruits, tea, red wine, honey, olive oil) and can be assumed directly with the diet [88]. Data regarding alimentary habits

**4.2. Antioxidant molecules**

58 Research Directions in Tumor Angiogenesis

Among **lipids** there are also very important antioxidant molecules. They are naturally present in plants like carotenes (retinol and b-carotene), alpha-tocopherol (also known as Vitamin E) or synthetized by animal cells, like CoenzymeQ10. The characteristic lipid character of these molecules allows them to localize in cell membranes (intracellular organelles and plasma membrane) where they can buffer lipid radicals and prevent reactions of peroxidation. Antioxidant Vitamin E properties for lipid peroxidation were efficiently assayed in GPx4-/ mice [92]. Carotenoids are tetraterpenoid pigments contained exclusively in plant cells that can be assumed with diet and act as terminal antioxidant molecules, once oxidized they can not be "re-used" from cells [93]. CoenzymeQ10 (CoQ10) is a terpenoid molecule whose antioxidant activity has been reported for maintenance of healthy cardiovascular system. Recent clinical trials have also show the use of CoQ10 for lowering blood pressure. At the present there are not evidences regarding the involvement of lipid antioxidant molecules in conditions of tumor angiogenesis [94].

Several other **antioxidant genes** are normally induced in cells to shield against dangerous deregulation of redox balance. Among those which play a key role in angiogenesis we can find heme-oxygenase-1 (HMOX-1) and nuclear factor erythroid 2 (NRF2) [95]. Upregulation of these genes are correlates to tumor metastasis and progression suggesting how oxidative stress is a condition implicated in tumor angiogenesis [96].

#### **5. Angiogenic molecules regulated by redox signaling**

**Vascular Endothelial Growth Factor (VEGF)** family encloses six glycoproteins: VEGF-A, VEGF-B, VEGF-C, VEGF-D AND VEGF-E, all of them belong to a superfamily of growth factors. Endothelial cells have three types of specific VEGF receptors: VEGFR-1 (Flt-1), VEGFR-2 (KDR, Flk-1), VEGFR-3 (Flt-4). Signaling mediated by VEGF and respective receptors has been characterized as one of the most powerful factors for induction and maintenance of angiogenesis in a series of physiological as well as pathological (i.e. tumor) angiogenesis [97,98]. Among the multiple mechanisms of VEGF signaling regulation we can also found its redox state. Modulators of redox state as the concentration of oxygen directly regulates VEGF levels. VEGF promoter contains a hypoxia responsive element that can be bound by transcrip‐ tion factors HIF-1α- HIF1β under conditions of low oxygen concentration in vascular vessels (hypoxia). In vitro studies by using human genome array on pulmonary artery endothelial cells maintained in hypoxia for 24hours report up-regulation of expression of VEGF genes. Accordingly, in vivo hypoxia conditions induce VEGF and VEGFR-1/2 expression [99,100]. It has been also reported that modulation of redox state by NO levels regulates angiogenesis and tumor progression through modulation of VEGF–VEGFR signaling. In vitro treatments of human tumor cells with NO-donor (i.e. SNAP) or NO-generating compounds upregulate VEGF expression and stimulate angiogenesis [101]. Further specifications about VEGF modulation by NO are related to endothelial NOS activity.

of NF-kB is considered an alternative approach to block pathological angiogenesis. Among inhibitors of NF-kB it has been reported evidence for antioxidant molecules. In vitro treatments of cells with pro-oxidative species (hydrogen peroxide, LPS, TNF-α ) activates NF-kB, while contemporary antioxidants addiction inhibit its response [113,114]. NF-kB activation can also be impaired by N-acetylcysteine (that acts in the NO pathway), terpenoids (vitamin E) and mitochondria-specific antioxidant (rotenone) [115,116]. At the present it is not fully clarified as antioxidants interact with NF-kB and inactivate it. It is supposed they can act indirectly by altering different molecules that interact with NF-kB on redox pathways or directly by

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**6. Conclusion: Manipulating redox signaling as anti-tumor angiogenesis**

Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells, and recent studies suggest that this biochemical property of cancer cells can be exploited for therapeutic benefits. Cancer cells in advanced stage tumors frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it might be possible to preferentially eliminate these cells by pharmacological ROS insults [118]. Reactive oxygen species (ROS) might function as a double-edged sword in endothelial cells. A moderate increase of ROS may promote cell proliferation and survival. However, when the increase of ROS reaches a certain level (the toxic threshold), it may overwhelm the antioxidant capacity of the cell and trigger cell death. Under physiological conditions, normal endothelial cells maintain redox homeostasis with a low level of basal ROS by controlling the balance between ROS generation (pro-oxidants) and elimination (antioxidant capacity). Endothelial cells in normal vessels can tolerate a certain level of exogenous oxidative stress owing to their 'reserve' antioxidant capacity, which can be mobilized to prevent the ROS level from reaching the cell-death threshold. In In endothelial cells of tumor vessels the increase in ROS generation from metabolic abnormalities and oncogenic signaling may trigger a redox adaption response. This response leads to an upregulation of antioxidant capacity and a shift of redox dynamics that maintain the ROS levels below the toxic threshold. As such, tumor angiogenic cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS-generating agents or compounds that inhibit the antioxidant system. A further increase of ROS stress in these cancer cells using exogenous ROS-modulating agents is likely to cause elevation of ROS above the threshold level, leading to cell death. This might constitute a biochemical basis to design therapeutic strategies to selectively kill tumor

The role of redox signaling in tumor angiogenesis is not yet completely characterized. Although converse mechanisms are postulated about how oxidative species recruit new blood vessels for tumor progression, it is well established redox signaling modulates angiogenesis. Analysis and characterization of molecules that sustain redox signaling is a new opportunity

inhibition of IKK kinase activity [117].

angiogenic cells using ROS-mediated mechanisms [119-121].

for set up innovative strategies of anti-cancer therapy (Figure 1).

**therapy**

**Angiopoietins (Ang)** are a group of four growth factors (Ang-1, Ang-2, Ang-3, Ang-4) involved in blood vessels formation. Signaling mediated by angiopoietins and their spe‐ cific receptors (TIE, tyrosine kinases receptor) has been characterized as key factors in an‐ giogenesis [102]. They are particularly sensitive to endothelium environment since angiopoietins are modulated by pro-oxidative species. In vitro and in vivo studies in en‐ dothelial cells report Ang1 is induced by hydrogen peroxide and abrogation of catalase activity relates to low ability in cell migration and vessels formation [103]. Also, angio‐ poietin-like proteins (ANGPTL) are involved in redox signaling in tumor conditions. Up‐ regulation of expression of ANGPL4 promotes NADPH oxidases activity causing an alteration in relative abundance of superoxide anion over hydrogen peroxide. Finally, such redox alteration induces tumor cells escape from anoikis and promotes survival via specific activation of PI3K/PKBα/ERK pathway [104].

**Vascular Endothelial (VE)-Cadherin** is an adhesion protein in the adherent junction complexes of endothelial cells and has been characterized as the major system for con‐ trolling endothelial cells junctions [105]. VE-Cadherin controls vascular permeability and remodeling of blood vessels also under mechanical stimuli (shear forces) [106]. VE-Cad‐ herin regulation is sensitive to pro-angiogenic factors, in particular to VEGF [107]. VE-Cadherin is also directly regulated by redox signaling pathway. In vitro assays showed that resveratrol promotes proliferation and migration of cerebral endothelial cells by modulation of VE-cadherin as result of activation of MAPK/ERK pathway and NO upre‐ gulation [108]. It has also been demonstrated that resveratrol control initiation of arterio‐ genesis by blocking oxidative stress dependent phosphorylation of VE-Cadherin [109]. Interestingly, it is also reported that nitrate concentration contributes to control VE-Cad‐ herin stability in adherent junction of human primary endothelial cells (HUVEC) and prevents blood vessel leakage [110].

**Nuclear factor-kB (NF-kB)** is a transcriptional factor that promotes tumor growth and invasiveness by activation of angiogenic molecules in endothelial cells [111]. Using a zebrafish animal model it has been shown that in vivo NF-kB inhibition causes loss of vascular integrity and interferes with physiologic vessels morphology [112]. Inhibition or negative modulation of NF-kB is considered an alternative approach to block pathological angiogenesis. Among inhibitors of NF-kB it has been reported evidence for antioxidant molecules. In vitro treatments of cells with pro-oxidative species (hydrogen peroxide, LPS, TNF-α ) activates NF-kB, while contemporary antioxidants addiction inhibit its response [113,114]. NF-kB activation can also be impaired by N-acetylcysteine (that acts in the NO pathway), terpenoids (vitamin E) and mitochondria-specific antioxidant (rotenone) [115,116]. At the present it is not fully clarified as antioxidants interact with NF-kB and inactivate it. It is supposed they can act indirectly by altering different molecules that interact with NF-kB on redox pathways or directly by inhibition of IKK kinase activity [117].

angiogenesis in a series of physiological as well as pathological (i.e. tumor) angiogenesis [97,98]. Among the multiple mechanisms of VEGF signaling regulation we can also found its redox state. Modulators of redox state as the concentration of oxygen directly regulates VEGF levels. VEGF promoter contains a hypoxia responsive element that can be bound by transcrip‐ tion factors HIF-1α- HIF1β under conditions of low oxygen concentration in vascular vessels (hypoxia). In vitro studies by using human genome array on pulmonary artery endothelial cells maintained in hypoxia for 24hours report up-regulation of expression of VEGF genes. Accordingly, in vivo hypoxia conditions induce VEGF and VEGFR-1/2 expression [99,100]. It has been also reported that modulation of redox state by NO levels regulates angiogenesis and tumor progression through modulation of VEGF–VEGFR signaling. In vitro treatments of human tumor cells with NO-donor (i.e. SNAP) or NO-generating compounds upregulate VEGF expression and stimulate angiogenesis [101]. Further specifications about VEGF

**Angiopoietins (Ang)** are a group of four growth factors (Ang-1, Ang-2, Ang-3, Ang-4) involved in blood vessels formation. Signaling mediated by angiopoietins and their spe‐ cific receptors (TIE, tyrosine kinases receptor) has been characterized as key factors in an‐ giogenesis [102]. They are particularly sensitive to endothelium environment since angiopoietins are modulated by pro-oxidative species. In vitro and in vivo studies in en‐ dothelial cells report Ang1 is induced by hydrogen peroxide and abrogation of catalase activity relates to low ability in cell migration and vessels formation [103]. Also, angio‐ poietin-like proteins (ANGPTL) are involved in redox signaling in tumor conditions. Up‐ regulation of expression of ANGPL4 promotes NADPH oxidases activity causing an alteration in relative abundance of superoxide anion over hydrogen peroxide. Finally, such redox alteration induces tumor cells escape from anoikis and promotes survival via

**Vascular Endothelial (VE)-Cadherin** is an adhesion protein in the adherent junction complexes of endothelial cells and has been characterized as the major system for con‐ trolling endothelial cells junctions [105]. VE-Cadherin controls vascular permeability and remodeling of blood vessels also under mechanical stimuli (shear forces) [106]. VE-Cad‐ herin regulation is sensitive to pro-angiogenic factors, in particular to VEGF [107]. VE-Cadherin is also directly regulated by redox signaling pathway. In vitro assays showed that resveratrol promotes proliferation and migration of cerebral endothelial cells by modulation of VE-cadherin as result of activation of MAPK/ERK pathway and NO upre‐ gulation [108]. It has also been demonstrated that resveratrol control initiation of arterio‐ genesis by blocking oxidative stress dependent phosphorylation of VE-Cadherin [109]. Interestingly, it is also reported that nitrate concentration contributes to control VE-Cad‐ herin stability in adherent junction of human primary endothelial cells (HUVEC) and

**Nuclear factor-kB (NF-kB)** is a transcriptional factor that promotes tumor growth and invasiveness by activation of angiogenic molecules in endothelial cells [111]. Using a zebrafish animal model it has been shown that in vivo NF-kB inhibition causes loss of vascular integrity and interferes with physiologic vessels morphology [112]. Inhibition or negative modulation

modulation by NO are related to endothelial NOS activity.

60 Research Directions in Tumor Angiogenesis

specific activation of PI3K/PKBα/ERK pathway [104].

prevents blood vessel leakage [110].

#### **6. Conclusion: Manipulating redox signaling as anti-tumor angiogenesis therapy**

Increased generation of reactive oxygen species (ROS) and an altered redox status have long been observed in cancer cells, and recent studies suggest that this biochemical property of cancer cells can be exploited for therapeutic benefits. Cancer cells in advanced stage tumors frequently exhibit multiple genetic alterations and high oxidative stress, suggesting that it might be possible to preferentially eliminate these cells by pharmacological ROS insults [118].

Reactive oxygen species (ROS) might function as a double-edged sword in endothelial cells. A moderate increase of ROS may promote cell proliferation and survival. However, when the increase of ROS reaches a certain level (the toxic threshold), it may overwhelm the antioxidant capacity of the cell and trigger cell death. Under physiological conditions, normal endothelial cells maintain redox homeostasis with a low level of basal ROS by controlling the balance between ROS generation (pro-oxidants) and elimination (antioxidant capacity). Endothelial cells in normal vessels can tolerate a certain level of exogenous oxidative stress owing to their 'reserve' antioxidant capacity, which can be mobilized to prevent the ROS level from reaching the cell-death threshold. In In endothelial cells of tumor vessels the increase in ROS generation from metabolic abnormalities and oncogenic signaling may trigger a redox adaption response. This response leads to an upregulation of antioxidant capacity and a shift of redox dynamics that maintain the ROS levels below the toxic threshold. As such, tumor angiogenic cells would be more dependent on the antioxidant system and more vulnerable to further oxidative stress induced by exogenous ROS-generating agents or compounds that inhibit the antioxidant system. A further increase of ROS stress in these cancer cells using exogenous ROS-modulating agents is likely to cause elevation of ROS above the threshold level, leading to cell death. This might constitute a biochemical basis to design therapeutic strategies to selectively kill tumor angiogenic cells using ROS-mediated mechanisms [119-121].

The role of redox signaling in tumor angiogenesis is not yet completely characterized. Although converse mechanisms are postulated about how oxidative species recruit new blood vessels for tumor progression, it is well established redox signaling modulates angiogenesis. Analysis and characterization of molecules that sustain redox signaling is a new opportunity for set up innovative strategies of anti-cancer therapy (Figure 1).

**Acknowledgements**

**Author details**

**References**

Vera Mugoni and Massimo Mattia Santoro\*

Center, University of Torino, Italy

pubmed/22850752

407(6801):249– 257.

19217420

\*Address all correspondence to: massimo.santoro@unito.it

Rev Eukaryot Gene Expr 2008; 18(1):35-45.

We apologize to the many researchers whose work was not cited in this review due to space limitations. We would like to thank all members of Santoro lab for support and discussion.

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63

Department of Molecular Biotechnology and Health Sciencesm, Molecular Biotechnology

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MMS is supported by grants from HFSP, Marie Curie IRG, Telethon and AIRC.

**Figure 1. Schematic representation of redox mechanisms in tumor angiogenesis.** Multiple stimuli coming from tumor microenvironment (growth factor, prostanoids, oxygen tension, mechanical forces) induce specific activation of intracellular pro-oxidant enzymes (NADPH oxidase, xantine oxidoreductase, uncoupled eNOS). Consequently, raising levels of oxygen and nitrogen pro-oxidant species (ROS/RNS) modulate the activation of multiple cellular pathways by acting on molecular and transcriptional factors. Signaling induced by oxidative species results mainly in endothelial cells motility and proliferation towards vascular remodeling and formation of new blood vessels. HIF-1α hypoxia-indu‐ cible transcription factor; ETS E-twenty six family transcription factor; AP-I activator protein 1; p53 tumor suppressor protein; NF-kB nuclear factor – kB. PKC protein kinase C; PI3K phosphatidylinositol3-OH kinase; PTP protein tyrosine phosphatase; SRC tyrosine protein kinase; p38MAPK p38 mitogen-activated protein kinase; Akt serine/threonine-spe‐ cific protein kinase; ERK1/2 extracellular signal-regulated kinases; MMP matrix metalloproteinase; VE–Cadherin: vas‐ cular endothelial-cadherin; VEGFR vascular endothelial growth factor receptor; ICAM intracellular adhesion molecule 1; VACAM vascular cell adhesion molecule 1.

#### **Acknowledgements**

We apologize to the many researchers whose work was not cited in this review due to space limitations. We would like to thank all members of Santoro lab for support and discussion. MMS is supported by grants from HFSP, Marie Curie IRG, Telethon and AIRC.

#### **Author details**

Vera Mugoni and Massimo Mattia Santoro\*

\*Address all correspondence to: massimo.santoro@unito.it

Department of Molecular Biotechnology and Health Sciencesm, Molecular Biotechnology Center, University of Torino, Italy

#### **References**

**Figure 1. Schematic representation of redox mechanisms in tumor angiogenesis.** Multiple stimuli coming from tumor microenvironment (growth factor, prostanoids, oxygen tension, mechanical forces) induce specific activation of intracellular pro-oxidant enzymes (NADPH oxidase, xantine oxidoreductase, uncoupled eNOS). Consequently, raising levels of oxygen and nitrogen pro-oxidant species (ROS/RNS) modulate the activation of multiple cellular pathways by acting on molecular and transcriptional factors. Signaling induced by oxidative species results mainly in endothelial cells motility and proliferation towards vascular remodeling and formation of new blood vessels. HIF-1α hypoxia-indu‐ cible transcription factor; ETS E-twenty six family transcription factor; AP-I activator protein 1; p53 tumor suppressor protein; NF-kB nuclear factor – kB. PKC protein kinase C; PI3K phosphatidylinositol3-OH kinase; PTP protein tyrosine phosphatase; SRC tyrosine protein kinase; p38MAPK p38 mitogen-activated protein kinase; Akt serine/threonine-spe‐ cific protein kinase; ERK1/2 extracellular signal-regulated kinases; MMP matrix metalloproteinase; VE–Cadherin: vas‐ cular endothelial-cadherin; VEGFR vascular endothelial growth factor receptor; ICAM intracellular adhesion molecule

1; VACAM vascular cell adhesion molecule 1.

62 Research Directions in Tumor Angiogenesis


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**Chapter 4**

**Accessory Cells in Tumor Angiogenesis**

**— Tumor-Associated Pericytes**

Jun-ichi Kawabe and Yoshinobu Ohsaki

Additional information is available at the end of the chapter

In contrast to the normal tissue vasculature, tumor vessels are structurally and functionally abnormal [1-3]. These abnormal tumor vessels are characterized by an irregular, disorganized, and tortuous architecture with a highly dysfunctional and leaky endothelial cell (EC) layer [1, 3]. ECs are often loosely connected with each other and are covered by fewer and abnormal

Research into the molecular mechanisms and physiology of PCs associated with tumor an‐ giogenesis is a critical field in cancer research. In this chapter, we will focus on the patho‐ physiology of PCs in tumor angiogenesis, the role of PCs in resistance to anti-angiogenesis

Despite the increasing evidence that PCs plays important roles in the angiogenic proc‐ ess, the origin of PCs is still not fully understood. They are commonly described as orig‐ inating from various types of progenitors depending on their anatomical location in the body. For example, epicardial, mesenchymal, and neural crest cells are believed to be a source for pericytes in the cardiac coronary vasculature, dorsal aorta, and cardiac out‐

Pericytes play an important role in stabilizing blood vessels in the microvasculature [6, 7]. A feature of pericyte function is their ability to provide vascular stability through crosstalk be‐

and reproduction in any medium, provided the original work is properly cited.

© 2013 Minami et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Yoshinori Minami, Takaaki Sasaki,

http://dx.doi.org/10.5772/54523

**1. Introduction**

mural pericytes (PCs) [2-4].

flow tract, respectively [5].

therapy, and PCs as a therapeutic target.

**2. Pathophysiology of pericytes in tumor angiogenesis**


**Chapter 4**

## **Accessory Cells in Tumor Angiogenesis — Tumor-Associated Pericytes**

Yoshinori Minami, Takaaki Sasaki, Jun-ichi Kawabe and Yoshinobu Ohsaki

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54523

#### **1. Introduction**

oxide-mediated regulation of vascular endothelial growth factor and metalloprotei‐

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fifteen years later. Biochem Pharmacol 2006;72(11):1493-505.

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72 Research Directions in Tumor Angiogenesis

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In contrast to the normal tissue vasculature, tumor vessels are structurally and functionally abnormal [1-3]. These abnormal tumor vessels are characterized by an irregular, disorganized, and tortuous architecture with a highly dysfunctional and leaky endothelial cell (EC) layer [1, 3]. ECs are often loosely connected with each other and are covered by fewer and abnormal mural pericytes (PCs) [2-4].

Research into the molecular mechanisms and physiology of PCs associated with tumor an‐ giogenesis is a critical field in cancer research. In this chapter, we will focus on the patho‐ physiology of PCs in tumor angiogenesis, the role of PCs in resistance to anti-angiogenesis therapy, and PCs as a therapeutic target.

#### **2. Pathophysiology of pericytes in tumor angiogenesis**

Despite the increasing evidence that PCs plays important roles in the angiogenic proc‐ ess, the origin of PCs is still not fully understood. They are commonly described as orig‐ inating from various types of progenitors depending on their anatomical location in the body. For example, epicardial, mesenchymal, and neural crest cells are believed to be a source for pericytes in the cardiac coronary vasculature, dorsal aorta, and cardiac out‐ flow tract, respectively [5].

Pericytes play an important role in stabilizing blood vessels in the microvasculature [6, 7]. A feature of pericyte function is their ability to provide vascular stability through crosstalk be‐

tween PCs and endothelial cells (ECs). PCs deposit matrix or releasing factors that can promote EC differentiation or quiescence [8].

gen for pericytes and fibroblasts. PDGF consists of A, B, C, and D polypeptide chains, and it forms the homodimers PDGF-AA, BB, CC, and DD, and the heterodimer PDGF-AB [14]. The specific tyrosine kinase receptors of the PDGFR family consist of PDGFR-α and PDGFR-β [15, 16]. PDGFR-α binds to PDGF-AA, BB, AB, and CC, whereas PDGFR-

Accessory Cells in Tumor Angiogenesis — Tumor-Associated Pericytes

http://dx.doi.org/10.5772/54523

75

Previous studies have shown that a <90% reduction in pericyte coverage in mice is compatible with postnatal survival [18], whereas loss of >95% of pericytes is lethal [18, 19], suggesting that a rather low threshold of pericyte density is required for basal function of microvasculature.

Activated ECs secrete PDGF-BB to attract PCs and PC progenitors, which are either tissueresident cells and/or cells derived from bone marrow, and express PDGFRs [20], suggesting a paracrine signaling circuit [12, 18]. Pericyte deficiency, seen in knockout mice lacking PDGF-BB and its receptor, PDGFR-β, resulted in various changes in microvasculature, including endothelial hyperplasia, vessel dilation, tortuosity, leakage, and rupture, leading to wide‐

Studies of implanted tumors have shown that pericytes initially accumulate at the interface of tumor and host tissue and later around new blood vessels, exhibiting close contacts with ECs. Maturation of the tumor-associated vasculature is accompanied quantitatively by a reduced PC volume and qualitatively by morphological changes in whichPCs become flattened and

There is evidence that overexpression of PDGF-BB in tumor cells dramatically increases the PC coverage [23]. Moreover, Song et al. have also shown that tumor-derived PDGF-BB increases tumor PC coverage by activation of stromal-derived factor 1 alpha (SDF-1α) [24]. Thus, PDGF-BB appears to be a critical player in the recruitment of PCs to newly

The angiopoietin (Ang) family consists of several members including Ang-1, Ang-2, Ang-3 (murine specific), and Ang-4 (human specific), which have two tyrosine kinase receptors, Tie-1

Ang-1 was initially identified as an activating ligand for Tie-2, which is expressed by perivas‐ cular cells [26]. Genetic deletion of Ang-1 resulted in prenatal lethality, due to severe heart and vascular defects, very similar in phenotype to Tie-2-deficient mice [27]. Ang-1 is predominantly secreted by PCs and can bind with Tie-2 on ECs in a paracrine pattern. Ang-1 enhances PC-EC interactions, represses the proliferation and migration of ECs, and promotes the maturation of newly formed blood vessels [27, 28]. Constitutive Ang-1/Tie-2 signaling is required to main‐

Ang-2 was initially identified as a homologue of Ang-1 [32]. Ang-2 was found to bind to Tie-2 with an affinity similar to that of Ang-1. However, unlike Ang-1, exogenous Ang-2 produces only a very weak activation of Tie-2 on ECs. When ECs are activated by tumor-derived proangiogenic factors, Ang-2 acts as an autocrine antagonist of Ang-1/Tie-2 signaling [33]. More‐

spread and lethal microhemorrhages and edema in late gestation [19, 21].

β binds with BB and DD [17].

elongated [22].

formed vessels [25].

and Tie-2.

*2.1.2. Angiopoietin/Tie family*

tain the quiescent vasculature [29-31] (Fig. 1).

#### **2.1. Crosstalk between ECs and PCs**

In blood vessels, the crosstalk between ECs and PCs plays a critical role in the regulation of vascular formation, maturation, remodeling, stabilization and function [9]. PCs communicate with ECs by direct physical contact and paracrine signaling pathways.

Gap junctions provide direct contact between PCs and ECs that enable the exchange of ions and small molecules. Adhesion plaques anchor PCs to ECs, while peg-and-socket junctions enable the cells to penetrate the vascular basement membrane [10].

A variety of signaling factors mediate PC–EC interactions, including platelet-derived growth factor subunit B (PDGFB) and angiopoietin/Tie 2 [11].

**Figure 1.** Crosstalk between endothelial cells and pericytes

#### *2.1.1. PDGF/PDGFR family*

Pericyte homeostasis in normal biology is regulated in significant part by signaling through the PDGF ligand and receptor system (Fig. 1) [12, 13]. PDGF is a potent mito‐ gen for pericytes and fibroblasts. PDGF consists of A, B, C, and D polypeptide chains, and it forms the homodimers PDGF-AA, BB, CC, and DD, and the heterodimer PDGF-AB [14]. The specific tyrosine kinase receptors of the PDGFR family consist of PDGFR-α and PDGFR-β [15, 16]. PDGFR-α binds to PDGF-AA, BB, AB, and CC, whereas PDGFRβ binds with BB and DD [17].

Previous studies have shown that a <90% reduction in pericyte coverage in mice is compatible with postnatal survival [18], whereas loss of >95% of pericytes is lethal [18, 19], suggesting that a rather low threshold of pericyte density is required for basal function of microvasculature.

Activated ECs secrete PDGF-BB to attract PCs and PC progenitors, which are either tissueresident cells and/or cells derived from bone marrow, and express PDGFRs [20], suggesting a paracrine signaling circuit [12, 18]. Pericyte deficiency, seen in knockout mice lacking PDGF-BB and its receptor, PDGFR-β, resulted in various changes in microvasculature, including endothelial hyperplasia, vessel dilation, tortuosity, leakage, and rupture, leading to wide‐ spread and lethal microhemorrhages and edema in late gestation [19, 21].

Studies of implanted tumors have shown that pericytes initially accumulate at the interface of tumor and host tissue and later around new blood vessels, exhibiting close contacts with ECs. Maturation of the tumor-associated vasculature is accompanied quantitatively by a reduced PC volume and qualitatively by morphological changes in whichPCs become flattened and elongated [22].

There is evidence that overexpression of PDGF-BB in tumor cells dramatically increases the PC coverage [23]. Moreover, Song et al. have also shown that tumor-derived PDGF-BB increases tumor PC coverage by activation of stromal-derived factor 1 alpha (SDF-1α) [24]. Thus, PDGF-BB appears to be a critical player in the recruitment of PCs to newly formed vessels [25].

#### *2.1.2. Angiopoietin/Tie family*

tween PCs and endothelial cells (ECs). PCs deposit matrix or releasing factors that can promote

In blood vessels, the crosstalk between ECs and PCs plays a critical role in the regulation of vascular formation, maturation, remodeling, stabilization and function [9]. PCs communicate

Gap junctions provide direct contact between PCs and ECs that enable the exchange of ions and small molecules. Adhesion plaques anchor PCs to ECs, while peg-and-socket junctions

A variety of signaling factors mediate PC–EC interactions, including platelet-derived growth

Pericyte homeostasis in normal biology is regulated in significant part by signaling through the PDGF ligand and receptor system (Fig. 1) [12, 13]. PDGF is a potent mito‐

with ECs by direct physical contact and paracrine signaling pathways.

enable the cells to penetrate the vascular basement membrane [10].

factor subunit B (PDGFB) and angiopoietin/Tie 2 [11].

**Figure 1.** Crosstalk between endothelial cells and pericytes

*2.1.1. PDGF/PDGFR family*

EC differentiation or quiescence [8].

74 Research Directions in Tumor Angiogenesis

**2.1. Crosstalk between ECs and PCs**

The angiopoietin (Ang) family consists of several members including Ang-1, Ang-2, Ang-3 (murine specific), and Ang-4 (human specific), which have two tyrosine kinase receptors, Tie-1 and Tie-2.

Ang-1 was initially identified as an activating ligand for Tie-2, which is expressed by perivas‐ cular cells [26]. Genetic deletion of Ang-1 resulted in prenatal lethality, due to severe heart and vascular defects, very similar in phenotype to Tie-2-deficient mice [27]. Ang-1 is predominantly secreted by PCs and can bind with Tie-2 on ECs in a paracrine pattern. Ang-1 enhances PC-EC interactions, represses the proliferation and migration of ECs, and promotes the maturation of newly formed blood vessels [27, 28]. Constitutive Ang-1/Tie-2 signaling is required to main‐ tain the quiescent vasculature [29-31] (Fig. 1).

Ang-2 was initially identified as a homologue of Ang-1 [32]. Ang-2 was found to bind to Tie-2 with an affinity similar to that of Ang-1. However, unlike Ang-1, exogenous Ang-2 produces only a very weak activation of Tie-2 on ECs. When ECs are activated by tumor-derived proangiogenic factors, Ang-2 acts as an autocrine antagonist of Ang-1/Tie-2 signaling [33]. More‐ over, Ang-2 activates the downstream pathways including Pl3K/Akt, and thus functions as a promoter of angiogenesis [32]. Nasarre et al. have shown that tumors implanted into geneti‐ cally Ang-2-ablated mice grew more slowly than those implanted into wild-type mice [34], which suggests that Ang-2 is a potent target for anti-tumor therapies (Fig. 1).

sections (Table 1). Desmin is a muscle-specific class III intermediate filament found in mature

**Molecular Marker Alternative name Mouse Human**

pericytes in tumor vasculature

Tyrosine kinase receptor + +

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+ +

http://dx.doi.org/10.5772/54523

77

+ +

+ +

+ +

+ +

+ +


contractile filaments

developed pericyte contractile filaments

Tyrosine kinase receptor Expressed in pericytes in early stages of angiogenesis

Novel marker for pericytes and vascular smooth muscle cells GTPase-activating protein

ganglioside

Bone marrow–derived hematopoietic cells expressing the PC marker NG2 were identified in close contact with tumor blood vessels in animal models of melanoma [46], pancreatic islet

skeletal, cardiac, and smooth-muscle cells.

**PDGFR-β** Platelet-derived growth factor β

**NG2** Neuron-glial 2 (chondroitin

**MMP9** Matrix metalloproteinase-9,

**VEGFR1** vascular endothelial growth

**RGS5** Regulator of G-protein

sulfate proteoglycan) High-molecular-weight melanoma-associated antigen

(HMWMAA)

gelatinase B

factor receptor-1

**Table 1.** Markers of pericytes for microscopic imaging (antibody availability)

**3G5 Ganglioside antigen** - Specific for a pericyte surface

signaling-5

**2.4. Role of bone marrow-derived PC progenitors**

**a-SMA** α-Smooth muscle actin Expressed only locally by

**Desmin** Reactive to developing and

**Nestin** - + + **Smooth muscle myosin** - + + **Tropomyosin** - + +

**Aminopeptidase A** CD249, BP1 + + **Aminopeptidase N** CD13 + +

**Sulphatide** 3'-sulphogalactosylceramide - +

Tie-2 receptor expression recently has been identified in mesenchymal cells that are present in the stroma, implicating a repository for tumor vessel pericytes [35].

#### **2.2. PCs in tumor angiogenesis**

Many tumors express the pro-angiogenic vascular endothelial growth factor (VEGF) at high levels [36]. In contrast to ECs in normal tissues, ECs in the tumor vasculature are dependent on VEGF for survival [37]. Excessive VEGF signaling through VEGF receptor 2 (VEGFR2) loosens tight junctions of ECs, increasing permeability in the interstitial tumor microenviron‐ ment. Interestingly, in tumors with reduced levels of VEGF and other angiogenic regulatory factors, tumor vessels are less torturous, with normalized blood flow due to improved PC coverage, the so-called ''vascular normalization'' [3, 38, 39].

PCs stabilize ECs and mediate EC survival and maturation in normal vasculature, through both direct cell contact with ECs and paracrine signaling. It was reported that PCs in tumor vasculature are abnormal [40]. Low PC coverage correlates with poor clinical outcome in sev‐ eral different tumor types [41-43], but so far, the active involvement of PCs in tumor progres‐ sion remains unclear. PCs are usually absent in tumor vasculature or have loose associations with ECs, leaving most of the tumor microvessels immature, the significance of which has been revealed in studies in which genetic or pharmacologic ablation of PC coverage facilitates metastatic dissemination of tumor cells [43, 44].

Activated PCs loosely attach to microvessels and develop cytoplasmic extensions into the tumor parenchyma [45]. Compared to quiescent PCs, activated PCs can change their genomic expression profiles [9], leading to phenotypes that are highly proliferative with the pluripotency to differentiate into other PCs, matrix-forming cells, smooth muscle cells, or adipocytes.

#### **2.3. Molecular marker of PCs**

The challenges of defining a PC have not been made easier by the fact that a general pan-PC molecular marker has not been found. Because of the diverse characteristics, functions, and locations of PCs in various organs, it probably never will be discovered. There are, however, a few dynamic molecular markers that are present in PCs, albeit not exclusively, and that are commonly used for their detection. The expression patterns of these markers can vary in a tissue-specific manner or be dependent on the developmental or angiogenic stage of a blood vessel. Desmin and alpha-smooth-muscle actin (α-SMA) are contractile filaments, and regu‐ lator of G protein signaling 5 (RGS-5) is a GTPase-activating protein; all three are intracellular proteins. Neuron-glial 2 (NG2), a chondroitin sulfate proteoglycan, and platelet-derived growth factor receptor beta (PDGFRβ), a tyrosine-kinase receptor, are cell-surface proteins. Antibodies against these proteins (except RGS-5) are commonly used to identify PCs in tissue sections (Table 1). Desmin is a muscle-specific class III intermediate filament found in mature skeletal, cardiac, and smooth-muscle cells.

over, Ang-2 activates the downstream pathways including Pl3K/Akt, and thus functions as a promoter of angiogenesis [32]. Nasarre et al. have shown that tumors implanted into geneti‐ cally Ang-2-ablated mice grew more slowly than those implanted into wild-type mice [34],

Tie-2 receptor expression recently has been identified in mesenchymal cells that are present in

Many tumors express the pro-angiogenic vascular endothelial growth factor (VEGF) at high levels [36]. In contrast to ECs in normal tissues, ECs in the tumor vasculature are dependent on VEGF for survival [37]. Excessive VEGF signaling through VEGF receptor 2 (VEGFR2) loosens tight junctions of ECs, increasing permeability in the interstitial tumor microenviron‐ ment. Interestingly, in tumors with reduced levels of VEGF and other angiogenic regulatory factors, tumor vessels are less torturous, with normalized blood flow due to improved PC

PCs stabilize ECs and mediate EC survival and maturation in normal vasculature, through both direct cell contact with ECs and paracrine signaling. It was reported that PCs in tumor vasculature are abnormal [40]. Low PC coverage correlates with poor clinical outcome in sev‐ eral different tumor types [41-43], but so far, the active involvement of PCs in tumor progres‐ sion remains unclear. PCs are usually absent in tumor vasculature or have loose associations with ECs, leaving most of the tumor microvessels immature, the significance of which has been revealed in studies in which genetic or pharmacologic ablation of PC coverage facilitates

Activated PCs loosely attach to microvessels and develop cytoplasmic extensions into the tumor parenchyma [45]. Compared to quiescent PCs, activated PCs can change their genomic expression profiles [9], leading to phenotypes that are highly proliferative with the pluripotency to differentiate into other PCs, matrix-forming cells, smooth muscle

The challenges of defining a PC have not been made easier by the fact that a general pan-PC molecular marker has not been found. Because of the diverse characteristics, functions, and locations of PCs in various organs, it probably never will be discovered. There are, however, a few dynamic molecular markers that are present in PCs, albeit not exclusively, and that are commonly used for their detection. The expression patterns of these markers can vary in a tissue-specific manner or be dependent on the developmental or angiogenic stage of a blood vessel. Desmin and alpha-smooth-muscle actin (α-SMA) are contractile filaments, and regu‐ lator of G protein signaling 5 (RGS-5) is a GTPase-activating protein; all three are intracellular proteins. Neuron-glial 2 (NG2), a chondroitin sulfate proteoglycan, and platelet-derived growth factor receptor beta (PDGFRβ), a tyrosine-kinase receptor, are cell-surface proteins. Antibodies against these proteins (except RGS-5) are commonly used to identify PCs in tissue

which suggests that Ang-2 is a potent target for anti-tumor therapies (Fig. 1).

the stroma, implicating a repository for tumor vessel pericytes [35].

coverage, the so-called ''vascular normalization'' [3, 38, 39].

metastatic dissemination of tumor cells [43, 44].

cells, or adipocytes.

**2.3. Molecular marker of PCs**

**2.2. PCs in tumor angiogenesis**

76 Research Directions in Tumor Angiogenesis


**Table 1.** Markers of pericytes for microscopic imaging (antibody availability)

#### **2.4. Role of bone marrow-derived PC progenitors**

Bone marrow–derived hematopoietic cells expressing the PC marker NG2 were identified in close contact with tumor blood vessels in animal models of melanoma [46], pancreatic islet carcinomas [47], and brain tumors [48, 49]. Thus, PC progenitor cells appear to be recruited to sites of angiogenesis from the bone marrow niche; however, intravenously injected PC pro‐ genitor cells may fail to migrate and integrate into the tumor vasculature [50].

Preclinical and clinical studies have largely focused on the role of tumor PCs in promoting EC survival and stabilizing the tumor vasculature through a variety of signaling networks. As noted earlier, PC recruitment to tumor neovessels is dependent on signaling through the

PDGF-BB/PDGFRβ signaling appears to be critical for maintaining the PC–EC contacts needed for vessel stabilization. Vascular regression could also lead to the normalization of tumor mi‐ crovessels and the opening of previously collapsed vessels [66] via decreased interstitial fluid pressure [67]. These data suggest that PDGF/PDGFR pathway inhibition is a potent target for

**Drug Name Target Type Clinical stage**

**Axitinib (Inlyta)** PDGFRs, VEGFRs, cKit Small molecule inhibitor Approved for metastatic RCC

PDGFRs, VEGFRs, cKit Small molecule inhibitor Phase III

**Motesanib** PDGFRs, VEGFRs, cKit Small molecule inhibitor Phase III

**Tivozanib** PDGFRs, VEGFRs, cKit Small molecule inhibitor Phase III

**Table 2.** PDGF/PDGFR inhibitors that are approved and/or in clinical development

PDGFRs, VEGFRs, cKit Small molecule inhibitor Approved for metastatic RCC

PDGFRs, VEGFRs, EGFR Small molecule inhibitor Approved for metastatic medullary

Small molecule inhibitor Phase III

Small molecule inhibitor Phase III

Combining PDGFRβ tyrosine kinase inhibition with VEGF inhibition more efficiently blocked tumor angiogenesis than VEGF inhibition alone in several experimental models [63, 71-74]. Bergers et al. have shown that combined treatment by anti-PDGFR agents together with anti-VEGF significantly reduces PC coverage and increases the success of anti-tumor treatment in the RIP1-TAG2 mouse model [63]. Similarly, PDGF inhibition disrupts PC support and sen‐ sitizes ECs to anti-angiogenic chemotherapy, resulting in regression of pre-existing tumor

Small molecule inhibitor Approved for metastatic RCC,

Small molecule inhibitor Approved for metastatic RCC, HPCC

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79

thyroid cancer

imatinib-resistant GIST, PNET

PDGF-BB/PDGFRβ and Ang-1/Tie-2 networks.

**4.1. Targeting PDGF-BB/ PDGFRβ signaling**

**Sunitinib (Sutent)** PDGFRs, VEGFRs, FLT-3, CSF1R

cKit

**Cabozantinib** PDGFRs, VEGFRs, cMet, RET, cKit

**Regorafenib** PDGFRs, VEGFRs, Raf, cKit

PDGFRs, VEGFRs, Raf,

**Sorafenib (Nexavar)**

**Pazopanib (Votrient)**

**Vandetanib (Caprelsa)**

**Cediranib (Recentin)**

anti-tumor therapies by leading to improved drug delivery [68-70].

Tumor hypoxia due to the vascular regression following anti-angiogenic therapy appears to induce recruitment of various bone marrow-derived cells to the tumor microenvironment [51]. Rajantie et al. demonstrated the significant contribution of bone marrow–derived cells using an inducible hypoxia-inducible factor 1 alpha subunit (HIF1-α) animal model. In response to hypoxia in glioblastomas [52, 53], not only Tie-2-, VEGFR1-, CD11b-, and F4/80-positive cells but ECs and PC progenitor cells are released into the circulation from the bone marrow through the HIF1-α signal pathway. Then, they contribute to the neovascularization of glioblastoma [51]. In an HIF1-α knock-down mouse model, fewer bone marrow-derived cells are recruited to the tumors, which severely impairs tumor growth. These data suggest paradoxical induction of tumor angiogenesis via bone marrow-derived vessel progenitor cells after anti-angiogenic therapy.

#### **3. Role of PCs in resistance to anti-angiogenic therapies**

Although an anti-VEGF therapy, bevacizumab, has shown clinical efficacy in the treatment of several tumor types, its efficacy will ultimately be limited by acquired drug resistance. [54]. Putative mechanisms of resistance to anti-VEGF therapy include (1) activation and/or up-reg‐ ulation of alternative pro-angiogenic pathways including PDGF/PDGFR signaling in the tu‐ mor [55], (2) recruitment of bone marrow-derived pro-angiogenic cells that differentiate into PCs, and (3) increased PC coverage of tumor microvasculature partially mediated by PDGFR signaling [56, 57].

Studies have shown that vessels without PC coverage are more dependent on VEGF signaling for survival [9] and that inhibition of VEGF leads to increased PC coverage of the tumor vas‐ culature [58]. PCs may protect ECs from VEGF withdrawal, leading to PC-mediated resistance to anti-angiogenic therapies.

#### **4. Targeting PCs as an anti-angiogenic therapy**

Although a series of anti-angiogenic strategies targeting VEGF or its receptor VEGFR2 have been shown to efficiently prevent the growth of many types of tumors [59, 60], reports have shown that targeting VEGF signaling alone is often ineffective at inducing vascular regression or preventing the rapid regrowth of tumor vessels [58, 61-63]. One possible explanation for this failure is that the anti-angiogenic inhibitors mainly target immature ECs lacking PCs cov‐ erage, while showing a limited effect on the PC-associated mature vessels [63-65].

Although tumor PCs are less abundant and more loosely attached to vessels than those in healthy tissues, they have emerged as a critical therapeutic target for anti-angiogenic therapy. Preclinical and clinical studies have largely focused on the role of tumor PCs in promoting EC survival and stabilizing the tumor vasculature through a variety of signaling networks. As noted earlier, PC recruitment to tumor neovessels is dependent on signaling through the PDGF-BB/PDGFRβ and Ang-1/Tie-2 networks.

#### **4.1. Targeting PDGF-BB/ PDGFRβ signaling**

carcinomas [47], and brain tumors [48, 49]. Thus, PC progenitor cells appear to be recruited to sites of angiogenesis from the bone marrow niche; however, intravenously injected PC pro‐

Tumor hypoxia due to the vascular regression following anti-angiogenic therapy appears to induce recruitment of various bone marrow-derived cells to the tumor microenvironment [51]. Rajantie et al. demonstrated the significant contribution of bone marrow–derived cells using an inducible hypoxia-inducible factor 1 alpha subunit (HIF1-α) animal model. In response to hypoxia in glioblastomas [52, 53], not only Tie-2-, VEGFR1-, CD11b-, and F4/80-positive cells but ECs and PC progenitor cells are released into the circulation from the bone marrow through the HIF1-α signal pathway. Then, they contribute to the neovascularization of glioblastoma [51]. In an HIF1-α knock-down mouse model, fewer bone marrow-derived cells are recruited to the tumors, which severely impairs tumor growth. These data suggest paradoxical induction of tumor angiogenesis via bone marrow-derived vessel progenitor cells after anti-angiogenic

Although an anti-VEGF therapy, bevacizumab, has shown clinical efficacy in the treatment of several tumor types, its efficacy will ultimately be limited by acquired drug resistance. [54]. Putative mechanisms of resistance to anti-VEGF therapy include (1) activation and/or up-reg‐ ulation of alternative pro-angiogenic pathways including PDGF/PDGFR signaling in the tu‐ mor [55], (2) recruitment of bone marrow-derived pro-angiogenic cells that differentiate into PCs, and (3) increased PC coverage of tumor microvasculature partially mediated by PDGFR

Studies have shown that vessels without PC coverage are more dependent on VEGF signaling for survival [9] and that inhibition of VEGF leads to increased PC coverage of the tumor vas‐ culature [58]. PCs may protect ECs from VEGF withdrawal, leading to PC-mediated resistance

Although a series of anti-angiogenic strategies targeting VEGF or its receptor VEGFR2 have been shown to efficiently prevent the growth of many types of tumors [59, 60], reports have shown that targeting VEGF signaling alone is often ineffective at inducing vascular regression or preventing the rapid regrowth of tumor vessels [58, 61-63]. One possible explanation for this failure is that the anti-angiogenic inhibitors mainly target immature ECs lacking PCs cov‐

Although tumor PCs are less abundant and more loosely attached to vessels than those in healthy tissues, they have emerged as a critical therapeutic target for anti-angiogenic therapy.

erage, while showing a limited effect on the PC-associated mature vessels [63-65].

genitor cells may fail to migrate and integrate into the tumor vasculature [50].

**3. Role of PCs in resistance to anti-angiogenic therapies**

**4. Targeting PCs as an anti-angiogenic therapy**

therapy.

signaling [56, 57].

to anti-angiogenic therapies.

78 Research Directions in Tumor Angiogenesis

PDGF-BB/PDGFRβ signaling appears to be critical for maintaining the PC–EC contacts needed for vessel stabilization. Vascular regression could also lead to the normalization of tumor mi‐ crovessels and the opening of previously collapsed vessels [66] via decreased interstitial fluid pressure [67]. These data suggest that PDGF/PDGFR pathway inhibition is a potent target for anti-tumor therapies by leading to improved drug delivery [68-70].


**Table 2.** PDGF/PDGFR inhibitors that are approved and/or in clinical development

Combining PDGFRβ tyrosine kinase inhibition with VEGF inhibition more efficiently blocked tumor angiogenesis than VEGF inhibition alone in several experimental models [63, 71-74]. Bergers et al. have shown that combined treatment by anti-PDGFR agents together with anti-VEGF significantly reduces PC coverage and increases the success of anti-tumor treatment in the RIP1-TAG2 mouse model [63]. Similarly, PDGF inhibition disrupts PC support and sen‐ sitizes ECs to anti-angiogenic chemotherapy, resulting in regression of pre-existing tumor vasculature in a mouse model [13]. Long-term blockade of PDGF signaling by anti-PDGFRβ antibody reduces the concentration of PCs within the tumor tissue and also increases the apoptosis of ECs [73].

affected circulating serum Ang-2 levels. This agent is being evaluated in combination with sunitinib in renal cell carcinoma. Currently, a phase II trial for kidney and a phase I trial for

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http://dx.doi.org/10.5772/54523

81

Other agents in development include monoclonal antibodies directed against Ang-2 (MEDI-3617, AMG 780, REGN910) and multi-targeted tyrosine kinase inhibitors inhibiting

At least two alternative therapeutic approaches appear plausible given the role of PCs in promoting tumor angiogenesis. The first approach is to promote excessive PC recruit‐ ment, thereby causing vessel stabilization and restricting vessel sprouting. This approach may limit tumor angiogenesis in blood vessels with normal PC investment of the EC and may prevent the dissemination of tumor cells into the circulation by reducing the leakiness of intratumoral blood vessels and, perhaps, by also blocking extravasation of

The second approach involves the use of PC progenitor cells as a cellular vehicle for gene delivery. This idea is supported by previous work using progenitor ECs [85-87], and more

Neither of these approaches promoting PC recruitment to the tumor vasculature has been tested in preclinical models or clinical trials; both are highly speculative and no proof-of-prin‐

Based on the crucial role of PCs in microvessel maturity and the concomitant histological evaluation of EC-PC interactions and tumor microvessel morphology, combining different chemotherapeutic agents and anti-angiogenic treatments that normalize tumor vasculature seems to be inevitable. Many new angiogenic inhibitors target pathways that are involved in the recruitment of PCs to tumor microvessels. Therefore, it is essential to assess PCs in parallel with ECs when studying tumor vasculature. This evaluation, which can be performed in a diagnostic pathology laboratory, can be used as a decision-making tool to select patients who

Y. Ohsaki was supported by a Grant-in-aid for scientific research (C) #20590910 for the study

of cancer treatment by regulating bone marrow-derived endothelial progenitor cells.

recently, PCs [50] to deliver anti-angiogenic gene therapy.

ciple studies have been conducted in animal models.

might benefit from anti-angiogenic therapies.

other solid tumors are underway.

Tie-2 (CEP11981, ARRY614) [84].

**4.3. Other approaches**

circulating tumor cells.

**5. Conclusions**

**Acknowledgements**

Several studies have tested the effects of combining anti-tumor agents with anti-PC agents that target PDGF or other PC markers, such as NG2 proteoglycan [75]. Involve‐ ment of the SDF-1α/CXCR4 axis in PC recruitment within PDGF-BB–overexpressing tu‐ mors suggests that a blockade of this axis may provide an additional target in antiangiogenic tumor therapy [24].

Most recently, treatment of primary tumors in an animal model of breast cancer with combi‐ nation VEGF and PDGF receptor therapy led to decreased PC coverage and an increased number of metastases. The observed promotion of metastasis by imatinib is consistent with previous reports demonstrating the key role of PDGFRβ signaling in PC recruitment and the importance of PCs in limiting tumor cell metastasis [43]. These findings provide the mecha‐ nistic basis for the differential effects these agents have on metastasis promotion.

However, a human clinical trial for renal carcinoma showed that inhibition of both the VEGF and PDGF pathways resulted in no therapeutic benefit when compared to inhibition of the VEGF pathway alone; in fact, the combined regimen exhibited toxicity [76]. Given these results, further preclinical studies are needed to clarify the mechanism(s) by which PDGF-targeted agents affect PC–EC interactions, and additional clinical studies are needed to clarify the po‐ tential benefits and risks associated with anti-PC tumor therapy.

#### **4.2. Targeting Ang/Tie signaling**

PCs have been shown to stabilize blood vessels and provide EC survival signals through the Ang-1/Tie-2 pathway [73, 77]. Therefore, by targeting tumor PCs it may be possible to over‐ come PC-mediated resistance to VEGF pathway inhibition and achieve more effective tumor vessel destabilization through disruption of the PC–EC association or directly through PC loss.

Trebananib (AMG 386) is a peptide-Fc fusion protein that inhibits angiogenesis by neutralizing the interaction between the Tie-2 receptor and Ang-1 and Ang-2 [78]. In phase I testing, it was found to be well tolerated in combination with chemotherapy [79] and to reduce tumor blood flow or permeability [80]. In a phase II trial of trebananib in combination with paclitaxel in patients with recurrent ovarian cancer, although a statistically significant improvement in progression-free survival for the treatment arm was not observed, the objective response rates and progression-free survival at the higher dose are suggestive of an antitumor effect [81]. The toxicity profile, including peripheral edema but not bowel perforations, is consistent with a mechanism distinct from that of VEGF inhibitors. Trebananib plus paclitaxel is now being investigated in an ongoing phase III study (TRINOVA-1 [Trial in Ovarian Cancer-1]) for the treatment of recurrent ovarian cancer. Phase II trials in breast, colorectal, kidney, stomach, and liver cancers are underway.

CVX060 (PF-04856884) is a recombinant humanized monoclonal antibody fused to two Ang-2 binding peptides [82, 83]. In preclinical studies, CVX-060 was anti-angiogenic and decreased tumor proliferation. In phase I testing, this agent significantly decreased tumor blood flow and affected circulating serum Ang-2 levels. This agent is being evaluated in combination with sunitinib in renal cell carcinoma. Currently, a phase II trial for kidney and a phase I trial for other solid tumors are underway.

Other agents in development include monoclonal antibodies directed against Ang-2 (MEDI-3617, AMG 780, REGN910) and multi-targeted tyrosine kinase inhibitors inhibiting Tie-2 (CEP11981, ARRY614) [84].

#### **4.3. Other approaches**

vasculature in a mouse model [13]. Long-term blockade of PDGF signaling by anti-PDGFRβ antibody reduces the concentration of PCs within the tumor tissue and also increases the

Several studies have tested the effects of combining anti-tumor agents with anti-PC agents that target PDGF or other PC markers, such as NG2 proteoglycan [75]. Involve‐ ment of the SDF-1α/CXCR4 axis in PC recruitment within PDGF-BB–overexpressing tu‐ mors suggests that a blockade of this axis may provide an additional target in anti-

Most recently, treatment of primary tumors in an animal model of breast cancer with combi‐ nation VEGF and PDGF receptor therapy led to decreased PC coverage and an increased number of metastases. The observed promotion of metastasis by imatinib is consistent with previous reports demonstrating the key role of PDGFRβ signaling in PC recruitment and the importance of PCs in limiting tumor cell metastasis [43]. These findings provide the mecha‐

However, a human clinical trial for renal carcinoma showed that inhibition of both the VEGF and PDGF pathways resulted in no therapeutic benefit when compared to inhibition of the VEGF pathway alone; in fact, the combined regimen exhibited toxicity [76]. Given these results, further preclinical studies are needed to clarify the mechanism(s) by which PDGF-targeted agents affect PC–EC interactions, and additional clinical studies are needed to clarify the po‐

PCs have been shown to stabilize blood vessels and provide EC survival signals through the Ang-1/Tie-2 pathway [73, 77]. Therefore, by targeting tumor PCs it may be possible to over‐ come PC-mediated resistance to VEGF pathway inhibition and achieve more effective tumor vessel destabilization through disruption of the PC–EC association or directly through PC loss. Trebananib (AMG 386) is a peptide-Fc fusion protein that inhibits angiogenesis by neutralizing the interaction between the Tie-2 receptor and Ang-1 and Ang-2 [78]. In phase I testing, it was found to be well tolerated in combination with chemotherapy [79] and to reduce tumor blood flow or permeability [80]. In a phase II trial of trebananib in combination with paclitaxel in patients with recurrent ovarian cancer, although a statistically significant improvement in progression-free survival for the treatment arm was not observed, the objective response rates and progression-free survival at the higher dose are suggestive of an antitumor effect [81]. The toxicity profile, including peripheral edema but not bowel perforations, is consistent with a mechanism distinct from that of VEGF inhibitors. Trebananib plus paclitaxel is now being investigated in an ongoing phase III study (TRINOVA-1 [Trial in Ovarian Cancer-1]) for the treatment of recurrent ovarian cancer. Phase II trials in breast, colorectal, kidney, stomach, and

CVX060 (PF-04856884) is a recombinant humanized monoclonal antibody fused to two Ang-2 binding peptides [82, 83]. In preclinical studies, CVX-060 was anti-angiogenic and decreased tumor proliferation. In phase I testing, this agent significantly decreased tumor blood flow and

nistic basis for the differential effects these agents have on metastasis promotion.

tential benefits and risks associated with anti-PC tumor therapy.

apoptosis of ECs [73].

80 Research Directions in Tumor Angiogenesis

angiogenic tumor therapy [24].

**4.2. Targeting Ang/Tie signaling**

liver cancers are underway.

At least two alternative therapeutic approaches appear plausible given the role of PCs in promoting tumor angiogenesis. The first approach is to promote excessive PC recruit‐ ment, thereby causing vessel stabilization and restricting vessel sprouting. This approach may limit tumor angiogenesis in blood vessels with normal PC investment of the EC and may prevent the dissemination of tumor cells into the circulation by reducing the leakiness of intratumoral blood vessels and, perhaps, by also blocking extravasation of circulating tumor cells.

The second approach involves the use of PC progenitor cells as a cellular vehicle for gene delivery. This idea is supported by previous work using progenitor ECs [85-87], and more recently, PCs [50] to deliver anti-angiogenic gene therapy.

Neither of these approaches promoting PC recruitment to the tumor vasculature has been tested in preclinical models or clinical trials; both are highly speculative and no proof-of-prin‐ ciple studies have been conducted in animal models.

#### **5. Conclusions**

Based on the crucial role of PCs in microvessel maturity and the concomitant histological evaluation of EC-PC interactions and tumor microvessel morphology, combining different chemotherapeutic agents and anti-angiogenic treatments that normalize tumor vasculature seems to be inevitable. Many new angiogenic inhibitors target pathways that are involved in the recruitment of PCs to tumor microvessels. Therefore, it is essential to assess PCs in parallel with ECs when studying tumor vasculature. This evaluation, which can be performed in a diagnostic pathology laboratory, can be used as a decision-making tool to select patients who might benefit from anti-angiogenic therapies.

#### **Acknowledgements**

Y. Ohsaki was supported by a Grant-in-aid for scientific research (C) #20590910 for the study of cancer treatment by regulating bone marrow-derived endothelial progenitor cells.

#### **Author details**

Yoshinori Minami1 , Takaaki Sasaki1\*, Jun-ichi Kawabe2 and Yoshinobu Ohsaki1

\*Address all correspondence to: takaaki6@asahikawa-med.ac.jp

1 Respiratory Center, Asahikawa Medical University, Asahikawa, Japan

2 Department of Cardiovascular Regeneration and Innovation, Asahikawa Medical Univer‐ sity, Asahikawa, Japan

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Accessory Cells in Tumor Angiogenesis — Tumor-Associated Pericytes

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**Author details**

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Yoshinori Minami1

sity, Asahikawa, Japan

2641-58.

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112(8): p. 1134-6.

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2 Department of Cardiovascular Regeneration and Innovation, Asahikawa Medical Univer‐

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**Chapter 5**

**Endothelial and Accessory Cell Interactions in**

Early childhood tumors that originate from the adrenal medulla and sympathetic nervous system are classified as neuroendocrine tumors [2]. Based on immunohistological criteria, neuroendocrine tumors can be broadly categorized as either neural or epithelial. As the name implies, tumors of the neural subtype display various degrees of neuronal differentiation and they stain positive for the neuroendocrine markers, synaptophysin and chromogranin A [3, 4]. Less well-differentiated or more primitive neural tumors are referred to as neuroblastoma (NB) while tumors with more differentiated features, such as ganglion and nerve bundles, are referred to as ganglioneuroblastoma and ganglioneuroma. This chapter focuses on NB, a form of cancer that occurs in infants and young children. NB is by far the most common cancer in infants, and the fourth most common type of cancer in children [5]. There are approximately 650 new cases each year in the United States, and NB accounts for 15% of all cancer deaths in children. At present, NB patients have limited options for therapy and there is a pressing need to find better treatment options. To develop better treatment options, it is critical to understand the origins of this disease, and mechanisms involved in disease progression. The first section of this chapter is dedicated to a review of neuroendocrine embryology in order to shed some light on the cell that may be responsible for NB. The exact NB progenitor cell has not been identified, however there is evidence that these cells are derived from the neural crest (NC). Understanding the differentiation of NC to cell types that constitute the peripheral nervous system, and the mechanisms utilized during this process is critical to our knowledge of NB

> © 2013 Gershan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

**Neuroblastoma Tumor Microenvironment**

Jill Gershan, Andrew Chan,

Ramani Ramchandran

http://dx.doi.org/10.5772/55486

**1. Introduction**

Magdalena Chrzanowska-Wodnicka, Bryon Johnson, Qing Robert Miao and

Additional information is available at the end of the chapter


## **Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment**

Jill Gershan, Andrew Chan, Magdalena Chrzanowska-Wodnicka, Bryon Johnson, Qing Robert Miao and Ramani Ramchandran

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55486

**1. Introduction**

[84] Cascone, T. and J.V. Heymach, Targeting the Angiopoietin/Tie2 Pathway: Cutting Tumor Vessels With a Double-Edged Sword? J Clin Oncol, 2012. 30(4): p. 441-444. [85] Dudek, A.Z., et al., Systemic inhibition of tumour angiogenesis by endothelial cell-

[86] Somani, A., et al., The establishment of murine blood outgrowth endothelial cells and

[87] Milbauer, L.C., et al., Blood outgrowth endothelial cell migration and trapping in

observations relevant to gene therapy. Transl Res, 2007. 150(1): p. 30-9.

vivo: a window into gene therapy. Transl Res, 2009. 153(4): p. 179-89.

based gene therapy. Br J Cancer, 2007. 97(4): p. 513-22.

88 Research Directions in Tumor Angiogenesis

Early childhood tumors that originate from the adrenal medulla and sympathetic nervous system are classified as neuroendocrine tumors [2]. Based on immunohistological criteria, neuroendocrine tumors can be broadly categorized as either neural or epithelial. As the name implies, tumors of the neural subtype display various degrees of neuronal differentiation and they stain positive for the neuroendocrine markers, synaptophysin and chromogranin A [3, 4]. Less well-differentiated or more primitive neural tumors are referred to as neuroblastoma (NB) while tumors with more differentiated features, such as ganglion and nerve bundles, are referred to as ganglioneuroblastoma and ganglioneuroma. This chapter focuses on NB, a form of cancer that occurs in infants and young children. NB is by far the most common cancer in infants, and the fourth most common type of cancer in children [5]. There are approximately 650 new cases each year in the United States, and NB accounts for 15% of all cancer deaths in children. At present, NB patients have limited options for therapy and there is a pressing need to find better treatment options. To develop better treatment options, it is critical to understand the origins of this disease, and mechanisms involved in disease progression. The first section of this chapter is dedicated to a review of neuroendocrine embryology in order to shed some light on the cell that may be responsible for NB. The exact NB progenitor cell has not been identified, however there is evidence that these cells are derived from the neural crest (NC). Understanding the differentiation of NC to cell types that constitute the peripheral nervous system, and the mechanisms utilized during this process is critical to our knowledge of NB

© 2013 Gershan et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

progression. In particular, migration of NC cells along the dorsal-ventral axis of the developing embryo, and the role of matrix in this process is likely to benefit our understanding of mechanisms of cancer metastasis in general. Subsequent sections of this chapter will address NB progression and the many factors (including genetic alterations such as *N-Myc* (*MYCN*) amplification) and cues from the surrounding microenvironment that determine tumor cell proliferation, survival, migration and angiogenesis. The tumor microenvironment is com‐ posed of endothelial cells, immune cells, and stromal cells, and, based on their phenotype, either contribute or prevent the progression or metastasis of tumor. We will focus on the contributions of Schwann cells, extracellular matrix, endothelial and immune cells to NB progression and pathogenesis to highlight the intricacies of how the microenvironment affects tumor development.

such as the carotid aorta, vagus nerve, bladder and prostate in addition to the adrenal medulla. The origin of these chromaffin cells has been attributed to a common precursor population called sympathoadrenal (SA) cells that give rise to both sympathetic neurons and chromaffin cells. Because NB is often associated with the SA cell or its progenitors [8], the development of these cells in embryogenesis provides clues to the disease inception and progression. During early PNS development there are three overlapping stages in which NBs could arise [9]. These are (1) the formation and fate specification of NC into sympathoadrenal (SA) progenitors, (2) bilateral migration and differentiation of SA cells and their coalescence near the aorta, and (3) differentiation of PNS neurons into fully developed ganglia and the establishment of synaptic

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

http://dx.doi.org/10.5772/55486

91

Since chromaffin cells and neurons of the SA system arise from neural crest (NC) cells, it has been proposed that NC cells may be the origin for NB. The NC is a transient embryonic population of cells that arise from the dorsal region of the newly formed neural tube [10]. NC cells undergo epithelial-to-mesenchymal (EMT) transition, and begin to migrate through the developing mesenchyme to differentiate into the craniofacial skeleton, melanocytes as well as

**a.** Formation and fate specification of NC into SA progenitors: The NC cells form at the border between neural and non-neural tissue in the vertebrate embryo. As the neural fold elevates, cells induced to become NC are located in the dorsal neural tube. The specifica‐ tion of NC cells is intricately linked to neural induction since these two processes also dictate the neural-non-neural boundary. It is well accepted from evidence in multiple model systems that loss of bone morphogenetic protein (BMP) signaling coincides with neural induction and thereby NC induction. BMP signaling alone is not sufficient for NC induction, as members of the Wnt and fibroblast growth factor (FGF) families have also been associated with NC induction [11]. Studies in mice, chicken, frog and zebrafish have implicated a cascade of transcription factors that confer NC cell identity. These include NC specifier genes such as Slug, Zic5, Sox9, Sox10, FoxD3, c-Myc and AP2 [12]. These factors are expressed in premigratory, and, or early migratory NC cells and are likely involved in the induction and survival of these cells. The differentiation of NC cells into the SA progenitor pathway is poorly understood. Single cell labeling studies in zebrafish [13] support the premise that Neurogenin-2 in pre- and early migrating NC cells promotes the sensory neuron differentiation at the expense of sympathetic neurons [13]. These data imply that the SA lineage is specified at an early migratory stage; however, it is unclear which molecular mechanisms trigger expression of Neurogenin-2 in a subset of NC cells. Cells during this early less differentiated stage could contribute to NB since alteration in the transcriptional signaling cascade may lead to precocious precursor cell proliferation or lack of further differentiation of these cells into the next phase of NC development.

**b.** Bilateral migration and differentiation of SA cells: Once specified, NC cells must delami‐ nate from the neural tube in order to migrate to their final destination (Figure 1). Delami‐ nation is a process of tissue splitting into separate populations regardless of cellular

**2.2. Signaling mechanisms guiding neural crest development**

connections.

the SA system.

#### **2. Neuroblastoma developmental mechanisms**

Two branching networks that often develop side-by-side during embryonic development include nerves and blood vessels [6]. During embryogenesis, the neural network comprised of both the central nervous system (CNS) and peripheral nervous system (PNS) develops first, and is composed of specialized cells called neurons that relay and transmit signals across different parts of the body [7]. The CNS includes the brain, spinal cord and retina while the PNS consists of sensory neurons, ganglia and the interconnecting nerves that connect to the CNS. Neurons project long cable like cellular extensions called axons that, via electrochemical waves, transmit signals by the release of neurotransmitters at axonal junctions or synapses.

#### **2.1. NB: A peripheral nervous system tumor**

NB is a PNS tumor derived from embryonic neural precursor cells. To understand the ontogeny of NB, the development and differentiation of neural precursor cells that are involved in PNS development will be discussed to obtain a better appreciation of the cells, signaling pathways and mechanisms involved in NB. Within the PNS there are somatic and visceral neurons. The somatic neurons innervate skin, bone joints and muscles, and their cell bodies often lie in the dorsal root ganglia of the spinal cord. The visceral neurons innervate internal organs, blood vessels and glands. The visceral component of the PNS is called the autonomic nervous system (ANS), and consists of two parts: the sympathetic nervous system (SNS) and the parasympa‐ thetic nervous system (PSNS). Both the SNS and PSNS often work in complementary but opposite fashions to maintain homeostasis in most organs. Two types of neurons, namely the pre-and post-ganglion, represent the majority of ANS, and are responsible for regulating the function of target organs. The pre-ganglionic neurons of the SNS are short while those of the PSNS are long. As a general principle, neurotransmitters are secreted at a synapse that usually occurs at the junction of two axons emerging from two neurons. One exception to this rule is observed in the chromaffin cells of the adrenal medulla. These neuroendocrine cells do not possess axons and directly release neurotransmitters (catecholamines, noradrenalin, adrena‐ line) into systemic circulation thereby affecting multiple organs. The chromaffin cells play an important role in the fight-or-flight response and are found in small numbers in structures such as the carotid aorta, vagus nerve, bladder and prostate in addition to the adrenal medulla. The origin of these chromaffin cells has been attributed to a common precursor population called sympathoadrenal (SA) cells that give rise to both sympathetic neurons and chromaffin cells. Because NB is often associated with the SA cell or its progenitors [8], the development of these cells in embryogenesis provides clues to the disease inception and progression. During early PNS development there are three overlapping stages in which NBs could arise [9]. These are (1) the formation and fate specification of NC into sympathoadrenal (SA) progenitors, (2) bilateral migration and differentiation of SA cells and their coalescence near the aorta, and (3) differentiation of PNS neurons into fully developed ganglia and the establishment of synaptic connections.

#### **2.2. Signaling mechanisms guiding neural crest development**

progression. In particular, migration of NC cells along the dorsal-ventral axis of the developing embryo, and the role of matrix in this process is likely to benefit our understanding of mechanisms of cancer metastasis in general. Subsequent sections of this chapter will address NB progression and the many factors (including genetic alterations such as *N-Myc* (*MYCN*) amplification) and cues from the surrounding microenvironment that determine tumor cell proliferation, survival, migration and angiogenesis. The tumor microenvironment is com‐ posed of endothelial cells, immune cells, and stromal cells, and, based on their phenotype, either contribute or prevent the progression or metastasis of tumor. We will focus on the contributions of Schwann cells, extracellular matrix, endothelial and immune cells to NB progression and pathogenesis to highlight the intricacies of how the microenvironment affects

Two branching networks that often develop side-by-side during embryonic development include nerves and blood vessels [6]. During embryogenesis, the neural network comprised of both the central nervous system (CNS) and peripheral nervous system (PNS) develops first, and is composed of specialized cells called neurons that relay and transmit signals across different parts of the body [7]. The CNS includes the brain, spinal cord and retina while the PNS consists of sensory neurons, ganglia and the interconnecting nerves that connect to the CNS. Neurons project long cable like cellular extensions called axons that, via electrochemical waves, transmit signals by the release of neurotransmitters at axonal junctions or synapses.

NB is a PNS tumor derived from embryonic neural precursor cells. To understand the ontogeny of NB, the development and differentiation of neural precursor cells that are involved in PNS development will be discussed to obtain a better appreciation of the cells, signaling pathways and mechanisms involved in NB. Within the PNS there are somatic and visceral neurons. The somatic neurons innervate skin, bone joints and muscles, and their cell bodies often lie in the dorsal root ganglia of the spinal cord. The visceral neurons innervate internal organs, blood vessels and glands. The visceral component of the PNS is called the autonomic nervous system (ANS), and consists of two parts: the sympathetic nervous system (SNS) and the parasympa‐ thetic nervous system (PSNS). Both the SNS and PSNS often work in complementary but opposite fashions to maintain homeostasis in most organs. Two types of neurons, namely the pre-and post-ganglion, represent the majority of ANS, and are responsible for regulating the function of target organs. The pre-ganglionic neurons of the SNS are short while those of the PSNS are long. As a general principle, neurotransmitters are secreted at a synapse that usually occurs at the junction of two axons emerging from two neurons. One exception to this rule is observed in the chromaffin cells of the adrenal medulla. These neuroendocrine cells do not possess axons and directly release neurotransmitters (catecholamines, noradrenalin, adrena‐ line) into systemic circulation thereby affecting multiple organs. The chromaffin cells play an important role in the fight-or-flight response and are found in small numbers in structures

tumor development.

90 Research Directions in Tumor Angiogenesis

**2. Neuroblastoma developmental mechanisms**

**2.1. NB: A peripheral nervous system tumor**

Since chromaffin cells and neurons of the SA system arise from neural crest (NC) cells, it has been proposed that NC cells may be the origin for NB. The NC is a transient embryonic population of cells that arise from the dorsal region of the newly formed neural tube [10]. NC cells undergo epithelial-to-mesenchymal (EMT) transition, and begin to migrate through the developing mesenchyme to differentiate into the craniofacial skeleton, melanocytes as well as the SA system.


mechanisms [14, 15]. With reference to NC, delamination is often used interchangeably with epithelial-to-mesenchymal transition. EMT is a series of molecular events that orchestrate changes from an epithelial cell phenotype into a mesenchymal (migratory) phenotype [16]. Several EMT-inducing transcription factors such as Snail, SoxE and Foxd3 function during multiple steps of NC development. In addition, EMT induces changes in junctional proteins such as N-cadherin and cadherin6B. These processes are reminiscent of events during general tumorigenesis whereby cancer cells undergo EMT and lose the ability to adhere to substratum leading to loss of contact-mediated inhibition. Therefore, NC cells provide a relevant model to investigate different aspects of tumorigenesis and metastasis especially with respect to NB.

As NC cells delaminate from the neural tube, tissues surrounding the neural tube produce both positive and negative cues that guide NC along defined pathways [17]. Trunk NC migration is guided by signals emerging from adjacent somites [18], which falls under three distinct phases, (1) Directed migration resulting from contact with the ectoderm and cues from the microenvironment, (2) Contact-mediated guidance facilitating homing to the target site, and (3) Contact-inhibition of movement upon entry and colonization of the target site (i.e. the trunk for SA) [15]. These migratory behaviors occur as streams of cells, and once in the trunk, NC cells migrate either ventromedially or dorsolaterally (Figure 1) [8]. NC cells migrating via the ventromedial route (without invading the somite) become neurons and glia of the sym‐ pathetic ganglia and adrenal chromaffin cells. NC cells that take the ventromedial route and invade and remain in the sclerotome coalesce to form Schwann cells, and the sensory neurons and glia of the dorsal root ganglia (DRG). We will discuss the role of Schwann cells in NB Pathogenesis later in this chapter. NC cells that take a dorsolateral route, in between the dorsal Ectoderm and the dermamyotome, differentiate into melanocytes (Figure 1) [17]. Because NBs often emerge in the sympathetic ganglia, it is conceivable that during the migratory process, NC cells that carry mutations in critical genes implicated in NBs, such as MYCN, may lose contact inhibition and prematurely proliferate in response to molecular signals that emanate from surrounding tissue. In terms of molecular cues, trunk NC cells that migrate via the ventromedial route enter the somite via attraction cues from CXCR4/CXCL12 signaling, which has also been implicated in breast cancer metastasis [19, 20]. These NC cells are confined to the rostral sclerotome by Neuropilin2/Semaphorin3F repulsion molecules working in concert with Eph/ephrin signalling, F-spondin and proteoglycans, which, reinforce this migration route. Similarly, signaling through CXCR4/CXCl12, ErbB2 and 3/Neuregulin, and GFRá3/artemin mediate NC cell attraction past the somite and work in concert with Neuropilin1/Semaphor‐ in4A repulsion cues from surrounding tissues that restricts NC cell migration to the dorsal

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**c.** Differentiation of PNS neurons into fully developed ganglia: Once the migrated partially differentiated NC cells (SA progenitors) reach the vicinity of dorsal aorta, bone morpho‐ genetic proteins secreted by SA cells trigger a molecular signaling cascade that is essential and sufficient to initiate the differentiation into both the noradrenergic sympathetic neurons and the cholinergic parasympathetic neurons of the ANS. Interestingly, BMP signaling is used at the first (NC induction) and third (NC differentiation into PNS neurons) stage of NC development implying its critical role in this pathway, and perhaps in NB. BMP-2 treatment of human NB cell lines (RTBM1 and SH-SY5Y) leads to growth arrest and differentiation [21]. BMP receptor IA expressed on SA progenitors responds to BMP inducing the expression of the proneural gene mammalian achaete-scute homolog (*Mash-1*) and the paired-like homebox2B (*PHOX2B*) transcription factors. PHOX2B is essential for maintaining Mash-1 expression and proliferation of SA progenitors. Human NB cell lines show high Hash-1 expression, and retinoic acid treatment decreases Hash-1 expression and promotes neurite extension [22, 23]. Germline mutations of *PHOX2B* in both a familial case of NB and in a patient with a genetically determined congenital malformation of NC-derived cells-namely, Hirschsprung disease (HSCR) exemplifies the underlying contribution of late stage genes in NC development to NB pathogenesis [24].

aorta [17].

**Figure 1. Neural crest cell development in zebrafish.** A high power image of the trunk neural crest cell migration is depicted. A cartoon of the cross-section of the trunk region is indicated. Black cells overlying the ectoderm are neural crest cells that specify from the dorsal roof plate (yellow region) of the neural tube (NT). NC cells that migrate dorso‐ ventrally (teal color) will differentiate into melanocytes (black shaped cells). NC cells that migrate ventromedially dif‐ ferentiate into sympathetic ganglia (purple cells). NC cells (red) that migrate through the somite form Schwann cells and sensory neurons and glia of DRG. NC cells that migrate between the somite and the neural tube as indicated dif‐ ferentiate into sympathetic ganglia.and chromaffin cells. Inhibitory signals (inverted T) and activation signals (arrows) guide the migration of the NC cells from the dorsal to ventral region. DA: dorsal aorta, NO: notochord, and blue struc‐ tures are somites.

As NC cells delaminate from the neural tube, tissues surrounding the neural tube produce both positive and negative cues that guide NC along defined pathways [17]. Trunk NC migration is guided by signals emerging from adjacent somites [18], which falls under three distinct phases, (1) Directed migration resulting from contact with the ectoderm and cues from the microenvironment, (2) Contact-mediated guidance facilitating homing to the target site, and (3) Contact-inhibition of movement upon entry and colonization of the target site (i.e. the trunk for SA) [15]. These migratory behaviors occur as streams of cells, and once in the trunk, NC cells migrate either ventromedially or dorsolaterally (Figure 1) [8]. NC cells migrating via the ventromedial route (without invading the somite) become neurons and glia of the sym‐ pathetic ganglia and adrenal chromaffin cells. NC cells that take the ventromedial route and invade and remain in the sclerotome coalesce to form Schwann cells, and the sensory neurons and glia of the dorsal root ganglia (DRG). We will discuss the role of Schwann cells in NB Pathogenesis later in this chapter. NC cells that take a dorsolateral route, in between the dorsal Ectoderm and the dermamyotome, differentiate into melanocytes (Figure 1) [17]. Because NBs often emerge in the sympathetic ganglia, it is conceivable that during the migratory process, NC cells that carry mutations in critical genes implicated in NBs, such as MYCN, may lose contact inhibition and prematurely proliferate in response to molecular signals that emanate from surrounding tissue. In terms of molecular cues, trunk NC cells that migrate via the ventromedial route enter the somite via attraction cues from CXCR4/CXCL12 signaling, which has also been implicated in breast cancer metastasis [19, 20]. These NC cells are confined to the rostral sclerotome by Neuropilin2/Semaphorin3F repulsion molecules working in concert with Eph/ephrin signalling, F-spondin and proteoglycans, which, reinforce this migration route. Similarly, signaling through CXCR4/CXCl12, ErbB2 and 3/Neuregulin, and GFRá3/artemin mediate NC cell attraction past the somite and work in concert with Neuropilin1/Semaphor‐ in4A repulsion cues from surrounding tissues that restricts NC cell migration to the dorsal aorta [17].

mechanisms [14, 15]. With reference to NC, delamination is often used interchangeably with epithelial-to-mesenchymal transition. EMT is a series of molecular events that orchestrate changes from an epithelial cell phenotype into a mesenchymal (migratory) phenotype [16]. Several EMT-inducing transcription factors such as Snail, SoxE and Foxd3 function during multiple steps of NC development. In addition, EMT induces changes in junctional proteins such as N-cadherin and cadherin6B. These processes are reminiscent of events during general tumorigenesis whereby cancer cells undergo EMT and lose the ability to adhere to substratum leading to loss of contact-mediated inhibition. Therefore, NC cells provide a relevant model to investigate different aspects of tumorigenesis and

**Figure 1. Neural crest cell development in zebrafish.** A high power image of the trunk neural crest cell migration is depicted. A cartoon of the cross-section of the trunk region is indicated. Black cells overlying the ectoderm are neural crest cells that specify from the dorsal roof plate (yellow region) of the neural tube (NT). NC cells that migrate dorso‐ ventrally (teal color) will differentiate into melanocytes (black shaped cells). NC cells that migrate ventromedially dif‐ ferentiate into sympathetic ganglia (purple cells). NC cells (red) that migrate through the somite form Schwann cells and sensory neurons and glia of DRG. NC cells that migrate between the somite and the neural tube as indicated dif‐ ferentiate into sympathetic ganglia.and chromaffin cells. Inhibitory signals (inverted T) and activation signals (arrows) guide the migration of the NC cells from the dorsal to ventral region. DA: dorsal aorta, NO: notochord, and blue struc‐

metastasis especially with respect to NB.

92 Research Directions in Tumor Angiogenesis

tures are somites.

**c.** Differentiation of PNS neurons into fully developed ganglia: Once the migrated partially differentiated NC cells (SA progenitors) reach the vicinity of dorsal aorta, bone morpho‐ genetic proteins secreted by SA cells trigger a molecular signaling cascade that is essential and sufficient to initiate the differentiation into both the noradrenergic sympathetic neurons and the cholinergic parasympathetic neurons of the ANS. Interestingly, BMP signaling is used at the first (NC induction) and third (NC differentiation into PNS neurons) stage of NC development implying its critical role in this pathway, and perhaps in NB. BMP-2 treatment of human NB cell lines (RTBM1 and SH-SY5Y) leads to growth arrest and differentiation [21]. BMP receptor IA expressed on SA progenitors responds to BMP inducing the expression of the proneural gene mammalian achaete-scute homolog (*Mash-1*) and the paired-like homebox2B (*PHOX2B*) transcription factors. PHOX2B is essential for maintaining Mash-1 expression and proliferation of SA progenitors. Human NB cell lines show high Hash-1 expression, and retinoic acid treatment decreases Hash-1 expression and promotes neurite extension [22, 23]. Germline mutations of *PHOX2B* in both a familial case of NB and in a patient with a genetically determined congenital malformation of NC-derived cells-namely, Hirschsprung disease (HSCR) exemplifies the underlying contribution of late stage genes in NC development to NB pathogenesis [24].

#### **2.3. Model system contribution to neuroblastoma pathogenesis**

Much information contributing to the pathogenesis of NB has been generated from in vitro studies performed on cell lines derived from patients [8]. Similar to other tumors, oncogene amplification or allelic loss has been linked to NB progression. Proto-oncogenes v-myc myelocytomatosis viral related oncogene (*MYCN)*, anaplastic lymphoma kinase (*ALK*) and, more recently, transforming tyrosine kinase receptor type A *(TrkA)* [25] have been widely suspected as likely contributors in NB pathogenesis. In fact, *MYCN* amplification is one of the few predictors of a poor clinical outcome for patients with NB. Tumors without *MYCN* amplification that correlate with poor survival frequently show an aberrant up regulation of genes in the *MYC* pathway, and down regulation of genes in the SA lineage differentiation pathway emphasizing the importance of MYC signaling in NB pathogenesis. BMP signal transducers in SA cells, namely transcription factors *PHOX2A* and *PHOX2B*, bind and activate noradrenergic marker genes, tyrosine hydroxylase (TH) and dopamine-β-hydroxylase that are essential enzymes for noradrenaline production. Evidence shows that *MYCN* overexpression under the tyrosine hydroxylase promoter in mice drives tumor formation that resembles human NB [26]. Recently, a dopamine-β-hydroxylase promoter driving *MYCN* and *ALK* genes in zebrafish [27] also resulted in NB formation. In this fish model, the authors demonstrated that upregulated *MYCN* mediated sympathetic neuroblast proliferation, which is eventually mitigated by a developmentally timed apoptosis of neuroblast cells. The concomitant activa‐ tion of ALK blocks the neuroblast apoptosis at a critical window in development thereby establishing a novel synergistic mechanism for these two oncogenes in NB pathogenesis. These tumor models clearly imply that turning on oncogenes in cells that are undergoing transitions during NC development is at the heart of NB ontogeny and progression.

**3. Role of Schwann cells in NB tumor microenvironment**

detail here.

of NC cells [35, 36].

**3.1. Schwann cells in normal development and NB**

NB is characterized by the co-existence of both stromal Schwann cells and neuroblastic tumor cells. The NC origin of Schwann cells suggests that they may co-evolve with tumor cells from common neural progenitors. However, the origin and functional relevance of tumorassociated Schwann cells remain controversial. Although widely assumed to be infiltrating normal Schwann cells, the finding of common genetic alterations shared with neuroblastic tumor cells argues for the same origin as tumor cells. It is well established that stroma-rich NB is associated with differentiated tumors of favorable prognosis. On the contrary, stromapoor NB is associated with metastatic diseases and poor outcomes. Among the various organs, a high fraction of NB disseminates to the bone and bone marrow. How Schwannian stroma affect tumor dissemination has not been extensively studied. To this end, several soluble factors have been isolated from Schwann cells that have proliferative, survival and angiogen‐ ic activities in the tumor microenvironment. These include Chemokine C-X-C motif ligand 13 (CXCL13), Secreted Protein Acidic and Rich in Cysteine (SPARC), and Pigment Epithelium-Derived Factor (PEDF). Determining their roles in NB progression will aid in future development of novel treatments for this childhood malignancy, and will be discussed in

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In the PNS, Schwann cells serve as the major glial cell type for individual neurons. During development, NC progenitors differentiate into multiple lineages including neurons, glial cells, pigment cells, endocrine cells and mesenchymal cells [28]. Based on the hierarchical organization of lineage segregation, NC-derived stem cells (NCSC) first undergo gliogene‐ sis to generate a pool of Schwann cell precursors (SCP)(Figure 2). The helix-loop-helix transcription factor, Sox10, is required for this event by promoting the survival of NCSC and glial cell specification [29-31]. Sox10 also plays an instructive role in determining how NCSC response to neuregulin-1 (NRG-1)[32, 33]. In the PNS, NRG-1 stimulates Schwann cell proliferation, migration, and myelination [34]. NRG-1 also regulates the migratory behavior

The maturation of SCP gives rise to immature Schwann cells, which in E15 mouse embryos, encapsulate bundles of axons through a process of radial sorting [37-39]. At this stage the ratio of Schwann cells to axons is 1:1 and this fine balance is partly achieved by axon-driven proliferation of immature Schwann cells. A host of factors are implicated that includes NRG-1 [40], transforming growth factor-β (TGF-β) [41-44], and laminins. Further differentiation of immature Schwann cells generates myelinating Schwann cells which surround large diameter axons, while smaller diameter axons are covered with nonmyelinating Schwann cells [45]. The functional importance of Schwann cells in neuronal survival is well established. For example, mice lacking the NRG-1 receptor, ErbB3, are devoid of SCP and have extensive neuronal cell death in the dorsal root ganglia [46]. Apart from NRG-1, additional trophic factors implicated in neuronal survival include brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and hepatocyte growth factor (HGF) [47].

**Figure 2. Schwann cell development.** Schematic representation of different developmental stages of Schwann cells showing the transitory cell types being involved during embryogenesis and after birth.

#### **3. Role of Schwann cells in NB tumor microenvironment**

**2.3. Model system contribution to neuroblastoma pathogenesis**

94 Research Directions in Tumor Angiogenesis

during NC development is at the heart of NB ontogeny and progression.

**Figure 2. Schwann cell development.** Schematic representation of different developmental stages of Schwann cells

showing the transitory cell types being involved during embryogenesis and after birth.

Much information contributing to the pathogenesis of NB has been generated from in vitro studies performed on cell lines derived from patients [8]. Similar to other tumors, oncogene amplification or allelic loss has been linked to NB progression. Proto-oncogenes v-myc myelocytomatosis viral related oncogene (*MYCN)*, anaplastic lymphoma kinase (*ALK*) and, more recently, transforming tyrosine kinase receptor type A *(TrkA)* [25] have been widely suspected as likely contributors in NB pathogenesis. In fact, *MYCN* amplification is one of the few predictors of a poor clinical outcome for patients with NB. Tumors without *MYCN* amplification that correlate with poor survival frequently show an aberrant up regulation of genes in the *MYC* pathway, and down regulation of genes in the SA lineage differentiation pathway emphasizing the importance of MYC signaling in NB pathogenesis. BMP signal transducers in SA cells, namely transcription factors *PHOX2A* and *PHOX2B*, bind and activate noradrenergic marker genes, tyrosine hydroxylase (TH) and dopamine-β-hydroxylase that are essential enzymes for noradrenaline production. Evidence shows that *MYCN* overexpression under the tyrosine hydroxylase promoter in mice drives tumor formation that resembles human NB [26]. Recently, a dopamine-β-hydroxylase promoter driving *MYCN* and *ALK* genes in zebrafish [27] also resulted in NB formation. In this fish model, the authors demonstrated that upregulated *MYCN* mediated sympathetic neuroblast proliferation, which is eventually mitigated by a developmentally timed apoptosis of neuroblast cells. The concomitant activa‐ tion of ALK blocks the neuroblast apoptosis at a critical window in development thereby establishing a novel synergistic mechanism for these two oncogenes in NB pathogenesis. These tumor models clearly imply that turning on oncogenes in cells that are undergoing transitions

NB is characterized by the co-existence of both stromal Schwann cells and neuroblastic tumor cells. The NC origin of Schwann cells suggests that they may co-evolve with tumor cells from common neural progenitors. However, the origin and functional relevance of tumorassociated Schwann cells remain controversial. Although widely assumed to be infiltrating normal Schwann cells, the finding of common genetic alterations shared with neuroblastic tumor cells argues for the same origin as tumor cells. It is well established that stroma-rich NB is associated with differentiated tumors of favorable prognosis. On the contrary, stromapoor NB is associated with metastatic diseases and poor outcomes. Among the various organs, a high fraction of NB disseminates to the bone and bone marrow. How Schwannian stroma affect tumor dissemination has not been extensively studied. To this end, several soluble factors have been isolated from Schwann cells that have proliferative, survival and angiogen‐ ic activities in the tumor microenvironment. These include Chemokine C-X-C motif ligand 13 (CXCL13), Secreted Protein Acidic and Rich in Cysteine (SPARC), and Pigment Epithelium-Derived Factor (PEDF). Determining their roles in NB progression will aid in future development of novel treatments for this childhood malignancy, and will be discussed in detail here.

#### **3.1. Schwann cells in normal development and NB**

In the PNS, Schwann cells serve as the major glial cell type for individual neurons. During development, NC progenitors differentiate into multiple lineages including neurons, glial cells, pigment cells, endocrine cells and mesenchymal cells [28]. Based on the hierarchical organization of lineage segregation, NC-derived stem cells (NCSC) first undergo gliogene‐ sis to generate a pool of Schwann cell precursors (SCP)(Figure 2). The helix-loop-helix transcription factor, Sox10, is required for this event by promoting the survival of NCSC and glial cell specification [29-31]. Sox10 also plays an instructive role in determining how NCSC response to neuregulin-1 (NRG-1)[32, 33]. In the PNS, NRG-1 stimulates Schwann cell proliferation, migration, and myelination [34]. NRG-1 also regulates the migratory behavior of NC cells [35, 36].

The maturation of SCP gives rise to immature Schwann cells, which in E15 mouse embryos, encapsulate bundles of axons through a process of radial sorting [37-39]. At this stage the ratio of Schwann cells to axons is 1:1 and this fine balance is partly achieved by axon-driven proliferation of immature Schwann cells. A host of factors are implicated that includes NRG-1 [40], transforming growth factor-β (TGF-β) [41-44], and laminins. Further differentiation of immature Schwann cells generates myelinating Schwann cells which surround large diameter axons, while smaller diameter axons are covered with nonmyelinating Schwann cells [45]. The functional importance of Schwann cells in neuronal survival is well established. For example, mice lacking the NRG-1 receptor, ErbB3, are devoid of SCP and have extensive neuronal cell death in the dorsal root ganglia [46]. Apart from NRG-1, additional trophic factors implicated in neuronal survival include brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), and hepatocyte growth factor (HGF) [47].

In early childhood, tumors originating from the adrenal medulla and sympathetic nervous system are classified as neuroendocrine tumors. Based on immunohistological criteria, neuroendocrine tumors can be broadly categorized into two types – neural and epithelial. As its name implies, tumors of the neural subtype display various degrees of neuronal differen‐ tiation and they stained positive for neuroendocrine markers, synaptophysin and chromog‐ ranin A [3, 4]. Less well-differentiated or more primitive neural tumors are referred to as NB while tumors with more differentiated features, such as ganglion and nerve bundles, are referred to as ganglioneuroblastoma (GNB) and ganglioneuroma (GN) (Figure 3A). Overall, GNB and GN show greater immunoreactivities towards the three neurofilament (NF) phos‐ phoisoforms, NF-L (light), NF-M (medium), and NF-H (heavy)[48]. Also, well-differentiated tumors have higher expression of neuronal markers such as microtubule associated proteins (MAPS) and tau. Furthermore, in GNB and GN, both glial cell markers, glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) are detected, providing evidence of differen‐ tiation into non-myelinating and myelinating Schwann cells, respectively [48].

90-100% survival survival. Interestingly, patients with the focal nodular subtype has the poorest survival of only 18%. Thus, tumors with good prognosis are the favorable stroma-poor and well-differentiated or intermixed stroma-rich. These tumors have non-advanced staging. In contrast, unfavorable stroma-poor and focal nodular stroma-rich lead topoor prognosis, and

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At the molecular level, stroma-poor tumors have a higher frequency (24%) of *MYCN* gene amplification as opposed to 10% for stroma-rich tumors [49, 50]. In this case, the overexpression of *MYCN* most likely leads to the expansion of the NC progenitor population. Indeed, silencing *MYCN* in NB cell lines promotes differentiation [51-53]. *MYCN* expression appears to be differentially regulated in neuroblastic and Schwannian S-type cells. For instance, LA1-55n, a neuroblastic tumor subline, has readily detectable *MYCN* expression while this oncoprotein was not present in the S-type counterpart, LA1-5s [54]. Similarly, ALK mutant protein can be detected in the neuroblastic subline of SK-N-SH while absent in several S-type sublines [55]. Thus, while *MYCN* amplification drives the expansion neuronal progenitors [56], this onco‐ genic event does not appear to impede differentiation into either neuronal or Schwann cell

The common NC origin of Schwann cells and neurons would argue that Schwannian stroma in NB is derived from a common cancer initiating cell [59]. However, this assertion is not without controversy. An earlier study using cytogenetic analysis of 19 NBs demonstrated that 18 of these tumors displayed near-triploidy while no chromosomal aberrations were detected in Schwann cells [60]. This leads to the conclusion that Schwann cells in tumor stroma are reactive in nature and most likely from infiltrating normal Schwann cells. With the advent of laser-capture microdissection and allelotyping techniques, Mora *et. al.* have demonstrated in 27 of 28 NBs, S100-positive Schwann cells have identical allelic compositions as the neuro‐ blastic tumor cells [59, 61]. Also, Schwannian stromal cells isolated from bone metastases have identical marker chromosomes as neuroblastic tumor cells [62]. Finally, the Schwannian S-type cell line, SH-EP1, harbors a F1174L mutation in the *ALK* gene that is also present in the neuroblastic N-type tumor cell line, SH-SY5Y (author's unpublished results)(Figure 3B). Both cell lines are derived from the widely used SK-N-SH NB cells [63]. All these data provide

they are frequently stage III and IV diseases.

evidence that Schwann cells are tumor-derived.

**3.3. The role of trophic factors in Schwannian stromal and NB pathogenesis**

Since Schwannian stroma-rich tumors are associated with favorable prognosis, it is logical to assume that Schwann cells harbor tumor-suppressing properties. To this end, experiments aiming to address the biological relevance of Schwann cells in NB are limited and confined mostly to *in vitro* studies. By co-cultivation experiments, neuroblastic tumor cells have been shown to stimulate the proliferation of Schwann cells [64]. This observation may explain the rapid expansion of Schwannian stromal during NB maturation. In the same study, Schwann cells also promote neurite outgrowth in neuroblastic tumor cells. Similar survival and differentiation promoting activities were also reported when Schwann cell conditioned medium was tested on four NB cell lines [55, 65]. These results are consistent with the differentiated histology associated with stroma-rich tumors. One caveat is that Schwann cells

lineages [57, 58].

**Figure 3. Neural crest tumor typesA.** The different histologic groups and subtypes of neural crest tumors with their characteristics depicted [1]. **B.** Brightfield photomicrographs of neuroblastic SH-SY5Y and Schwannian SH-EP1 cell lines (gifts from Dr. Robert A. Ross).

#### **3.2. Histology and origin of Schwannian stroma in NB**

The relevance of Schwannian stroma in the diagnosis and prognosis of NB has been addressed by the seminal study by Shimada *et. al.* [1]. In general, NB can be subdivided into stroma-poor and stroma-rich groups. Stroma-poor tumors have diffuse growth patterns of neuroblastic tumor cells divided by thin septa of fibrovascular tissues. This subgroup represents the classical NB and has either an undifferentiated or differentiating histology with various degrees of mitoses and karyorrhexis or nuclear fragmentation (MKI). In general, stroma-poor tumors are considered as favorable if diagnosed <1.5 yr old, with a low MKI and a differenti‐ ating histology. This group has a survival rate of 84%. On the contrary, stroma-poor tumors that are unfavorable have a high MKI (for <1.5 yr old), undifferentiated histology (1.5-5 yr old) and occur in patients greater than 5 years of age. This group has a survival rate of only 4.5%.

Tumors of the stroma-rich group have extensive Schwannian stroma and are representative of the GNB and GN histological types. This group can be further classified into three histo‐ logical subtypes – well-differentiated, intermixed and focal nodular (Figure 3A). The overall survival for stroma-rich tumors is 67% as compared to 47% for stroma-poor counterparts. Expectedly, patients with stroma-rich tumors that are well-differentiated or intermixed have 90-100% survival survival. Interestingly, patients with the focal nodular subtype has the poorest survival of only 18%. Thus, tumors with good prognosis are the favorable stroma-poor and well-differentiated or intermixed stroma-rich. These tumors have non-advanced staging. In contrast, unfavorable stroma-poor and focal nodular stroma-rich lead topoor prognosis, and they are frequently stage III and IV diseases.

In early childhood, tumors originating from the adrenal medulla and sympathetic nervous system are classified as neuroendocrine tumors. Based on immunohistological criteria, neuroendocrine tumors can be broadly categorized into two types – neural and epithelial. As its name implies, tumors of the neural subtype display various degrees of neuronal differen‐ tiation and they stained positive for neuroendocrine markers, synaptophysin and chromog‐ ranin A [3, 4]. Less well-differentiated or more primitive neural tumors are referred to as NB while tumors with more differentiated features, such as ganglion and nerve bundles, are referred to as ganglioneuroblastoma (GNB) and ganglioneuroma (GN) (Figure 3A). Overall, GNB and GN show greater immunoreactivities towards the three neurofilament (NF) phos‐ phoisoforms, NF-L (light), NF-M (medium), and NF-H (heavy)[48]. Also, well-differentiated tumors have higher expression of neuronal markers such as microtubule associated proteins (MAPS) and tau. Furthermore, in GNB and GN, both glial cell markers, glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) are detected, providing evidence of differen‐

tiation into non-myelinating and myelinating Schwann cells, respectively [48].

lines (gifts from Dr. Robert A. Ross).

96 Research Directions in Tumor Angiogenesis

**3.2. Histology and origin of Schwannian stroma in NB**

**Figure 3. Neural crest tumor typesA.** The different histologic groups and subtypes of neural crest tumors with their characteristics depicted [1]. **B.** Brightfield photomicrographs of neuroblastic SH-SY5Y and Schwannian SH-EP1 cell

The relevance of Schwannian stroma in the diagnosis and prognosis of NB has been addressed by the seminal study by Shimada *et. al.* [1]. In general, NB can be subdivided into stroma-poor and stroma-rich groups. Stroma-poor tumors have diffuse growth patterns of neuroblastic tumor cells divided by thin septa of fibrovascular tissues. This subgroup represents the classical NB and has either an undifferentiated or differentiating histology with various degrees of mitoses and karyorrhexis or nuclear fragmentation (MKI). In general, stroma-poor tumors are considered as favorable if diagnosed <1.5 yr old, with a low MKI and a differenti‐ ating histology. This group has a survival rate of 84%. On the contrary, stroma-poor tumors that are unfavorable have a high MKI (for <1.5 yr old), undifferentiated histology (1.5-5 yr old) and occur in patients greater than 5 years of age. This group has a survival rate of only 4.5%.

Tumors of the stroma-rich group have extensive Schwannian stroma and are representative of the GNB and GN histological types. This group can be further classified into three histo‐ logical subtypes – well-differentiated, intermixed and focal nodular (Figure 3A). The overall survival for stroma-rich tumors is 67% as compared to 47% for stroma-poor counterparts. Expectedly, patients with stroma-rich tumors that are well-differentiated or intermixed have At the molecular level, stroma-poor tumors have a higher frequency (24%) of *MYCN* gene amplification as opposed to 10% for stroma-rich tumors [49, 50]. In this case, the overexpression of *MYCN* most likely leads to the expansion of the NC progenitor population. Indeed, silencing *MYCN* in NB cell lines promotes differentiation [51-53]. *MYCN* expression appears to be differentially regulated in neuroblastic and Schwannian S-type cells. For instance, LA1-55n, a neuroblastic tumor subline, has readily detectable *MYCN* expression while this oncoprotein was not present in the S-type counterpart, LA1-5s [54]. Similarly, ALK mutant protein can be detected in the neuroblastic subline of SK-N-SH while absent in several S-type sublines [55]. Thus, while *MYCN* amplification drives the expansion neuronal progenitors [56], this onco‐ genic event does not appear to impede differentiation into either neuronal or Schwann cell lineages [57, 58].

The common NC origin of Schwann cells and neurons would argue that Schwannian stroma in NB is derived from a common cancer initiating cell [59]. However, this assertion is not without controversy. An earlier study using cytogenetic analysis of 19 NBs demonstrated that 18 of these tumors displayed near-triploidy while no chromosomal aberrations were detected in Schwann cells [60]. This leads to the conclusion that Schwann cells in tumor stroma are reactive in nature and most likely from infiltrating normal Schwann cells. With the advent of laser-capture microdissection and allelotyping techniques, Mora *et. al.* have demonstrated in 27 of 28 NBs, S100-positive Schwann cells have identical allelic compositions as the neuro‐ blastic tumor cells [59, 61]. Also, Schwannian stromal cells isolated from bone metastases have identical marker chromosomes as neuroblastic tumor cells [62]. Finally, the Schwannian S-type cell line, SH-EP1, harbors a F1174L mutation in the *ALK* gene that is also present in the neuroblastic N-type tumor cell line, SH-SY5Y (author's unpublished results)(Figure 3B). Both cell lines are derived from the widely used SK-N-SH NB cells [63]. All these data provide evidence that Schwann cells are tumor-derived.

#### **3.3. The role of trophic factors in Schwannian stromal and NB pathogenesis**

Since Schwannian stroma-rich tumors are associated with favorable prognosis, it is logical to assume that Schwann cells harbor tumor-suppressing properties. To this end, experiments aiming to address the biological relevance of Schwann cells in NB are limited and confined mostly to *in vitro* studies. By co-cultivation experiments, neuroblastic tumor cells have been shown to stimulate the proliferation of Schwann cells [64]. This observation may explain the rapid expansion of Schwannian stromal during NB maturation. In the same study, Schwann cells also promote neurite outgrowth in neuroblastic tumor cells. Similar survival and differentiation promoting activities were also reported when Schwann cell conditioned medium was tested on four NB cell lines [55, 65]. These results are consistent with the differentiated histology associated with stroma-rich tumors. One caveat is that Schwann cells used in these studies were isolated from normal human peripheral nerves. It will be of interest to compare tumor-derived versus normal Schwann cells in their abilities to promote differen‐ tiation and survival of neuroblastic tumor cells. Several trophic factors have been implicated in neuronal homeostasis. These include NGF, BDNF, LIF, and CNTF [66]. The biological effects of conditioned medium mentioned above are most likely the results of a combination of these soluble factors. Clearly defining their specific biological activities, for example, differentiation versus survival may have therapeutic implications. For instance, factors that only promote differentiation but not growth can have therapeutic effects in stroma-poor tumors. Alterna‐ tively, targeting the receptors for survival promoting factors such as the TrkB receptor for BDNF may be a plausible treatment strategy [25].

a more motile or myogenic phenotype [81, 82]. In addition, CAFs also confer a pro-tumorigen‐ ic microenvironment by remodeling the extracellular matrices and producing pro-angiogen‐ ic and pro-mitogenic trophic factors. In one study, an evaluation of 60 NBs revealed an inverse correlation between the existence of CAFs and Schwannian stroma [83]. In stroma-rich GNs, alpha-smooth muscle actin (α-sma)-positive and h-Caldesmon-negative CAFs are rarely detected. On the contrary, ~90% of stromal cells in Schwannian stroma-poor NB stained positive for CAFs. Indeed, the presence of CAFs in NB is associated with microvascular proliferation. All these findings reiterate the role of Schwann cells in conferring homeosta‐ sis in NB tumor microenvironment and this may be achieved by blocking the expansion of CAFs. However, it is unclear how the relative fractions of Schwann cells versus CAFs are being regulated and whether neuroblastic tumor cells can play an instructive role in these

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

http://dx.doi.org/10.5772/55486

99

One plausible link between Schwann cells and CAFs is the well-established role of bone marrow-derived human mesenchymal stem cells (hMSCs) in the formation of tumor stroma. hMSCs are pluripotent and can differentiate into multiple cell types such as bone, cartilages, and adipose tissues [84]. hMSCs when co-mixed with weakly metastatic breast cancer cells greatly enhance their metastatic potential [85]. Interestingly, hMSCs co-mixed with NB undergo a conversion to a cell type expressing the Schwann cell markers, S100 and Egr-2 [86]. Similarly, prolonged exposure of hMSCs to tumor-derived conditioned media also results in their transition to myofibroblasts [87]. Thus, it is plausible that neuroblastic tumor cells may dictate the composition of tumor stroma by instructing hMSCs to differentiate into either Schwann cells or CAFs. Another interesting aspect of hMSCs in NB is that bone marrow is a common site of metastatic spread [88]. The ability of the chemokine, stromal-derived factor (SDF-1/CXCL12), in bone marrow homing by binding to its receptor, CXCR4, on neuroblastic tumor cells has been reported [89]. Following seeding in the bone marrow, neuroblastic tumor cells may instruct hMSCs to differentiate into Schwann cells, thereby creating a favorable

An additional intriguing finding is that Schwann cells isolated from quail sciatic nerves can undergo transdifferentiation into myofibroblasts [90]. In vitro, TGF-β drastically enhanced the

into the first branchial arch of E2 chick embryos, these Schwann cells incorporate into the

it is tempting to speculate that neuroblastic tumor cells secrete TGF-β to remodel tumor stromal by converting Schwannian-rich to a CAF-rich tumor microenvironment. In summary, this level of plasticity in stromal remodeling may allow tumor cells to adapt to local hypoxic environ‐

From a treatment standpoint, NB in infants has a more favorable prognosis with low-grade tumors that resolved spontaneously. However, the overall survival for patients greater than 4 year old remains around 40%. Also, there are few options once tumors are refractory to conventional chemo- and radiation-therapies. How can studying the role of Schwann cells in

myofibroblasts. When transplanted

cells [90]. Based on these observations,

metastatic niche in an otherwise non-permissive environment.

conversion of cultured Schwann cells to α-sma<sup>+</sup> and sox10+

perivascular space of developing vessel walls as α-sma<sup>+</sup>

**3.5. The role of Schwannian stromal in NB therapy**

ment or in seeding of metastatic cells.

events.

The paracrine effects of trophic factors produced by Schwann cells are not restricted to neuroblastic tumor cells. For instance, three factors secreted by Schwann cells are known to inhibit angiogenesis. These include tissue inhibitor of metalloproteinase-2 (TIMP-2)[67], PEDF [68] and SPARC [69]. TIMP-2 was identified as a potential anti-angiogenic mediator in the conditioned medium of Schwann cells derived from both adult nerves and stroma-rich GN [67]. The negative effects of TIMP-2 on angiogenesis are independent of its ability to inhibit metalloproteinase (MMP) activities [70]. Instead TIMP-2 binds directly to endothelial cells through α3β1 integrin and dampens β1-mediated signaling and cell proliferation. PEDF, on the other hand, is a 50 kDa glycoprotein that belongs to the SERPIN family of serine protease inhibitors [71], and it binds to a PLA2/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) receptor [72]. PEDF suppresses angiogenesis by inducing apoptosis in endothelial cells, blocking motility and tube formation [73]. In NB, PEDF enhances Schwann cell growth and inhibits basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) induced endothelial cell migration [68]. Consistent with these activities, the least differentiated NB show weak staining for PEDF while high levels are observed in welldifferentiated GNB and GN. Finally, SPARC is a matricellular protein implicated in adipo‐ genesis [74]. Surface receptors such as Stabilin-1 and α5β3 integrin have been implicated in mediating SPARC biological activities [75, 76]. Its anti-angiogenic activities are mediated by the direct binding to a host of angiogenic mediators such as VEGF, and platelet-derived growth factor (PDGF)[77, 78]. High levels of SPARC are associated with favorable outcomes in NB [69]. *In vivo* experimental proof further supports the anti-tumorigenic role of Schwannian stroma. Using an NB xenotransplant model, NB cells implanted in sciatic nerve have greater number of infiltrating Schwann cells, more differentiated neuroblasts and reduced vascularity when compared to tumor cells injected outside of the sciatic nerves [79]. All these findings reinforce the notion that the favorable prognosis in stroma-rich NB is the consequence of a host of antiangiogenic factors produced by the Schwannian stromal compartment.

#### **3.4. Plasticity of Schwannian stroma**

During NB progression, there is evidence of dynamic remodeling of the Schwannian tumor microenvironment that involves additional stromal cell types. One such cell type is cancerassociated fibroblasts (CAFs). CAFs are frequently detected in epithelial tumors such as breast carcinomas [80]. CAFs are "reactive" in nature and differ from normal fibroblasts by having a more motile or myogenic phenotype [81, 82]. In addition, CAFs also confer a pro-tumorigen‐ ic microenvironment by remodeling the extracellular matrices and producing pro-angiogen‐ ic and pro-mitogenic trophic factors. In one study, an evaluation of 60 NBs revealed an inverse correlation between the existence of CAFs and Schwannian stroma [83]. In stroma-rich GNs, alpha-smooth muscle actin (α-sma)-positive and h-Caldesmon-negative CAFs are rarely detected. On the contrary, ~90% of stromal cells in Schwannian stroma-poor NB stained positive for CAFs. Indeed, the presence of CAFs in NB is associated with microvascular proliferation. All these findings reiterate the role of Schwann cells in conferring homeosta‐ sis in NB tumor microenvironment and this may be achieved by blocking the expansion of CAFs. However, it is unclear how the relative fractions of Schwann cells versus CAFs are being regulated and whether neuroblastic tumor cells can play an instructive role in these events.

One plausible link between Schwann cells and CAFs is the well-established role of bone marrow-derived human mesenchymal stem cells (hMSCs) in the formation of tumor stroma. hMSCs are pluripotent and can differentiate into multiple cell types such as bone, cartilages, and adipose tissues [84]. hMSCs when co-mixed with weakly metastatic breast cancer cells greatly enhance their metastatic potential [85]. Interestingly, hMSCs co-mixed with NB undergo a conversion to a cell type expressing the Schwann cell markers, S100 and Egr-2 [86]. Similarly, prolonged exposure of hMSCs to tumor-derived conditioned media also results in their transition to myofibroblasts [87]. Thus, it is plausible that neuroblastic tumor cells may dictate the composition of tumor stroma by instructing hMSCs to differentiate into either Schwann cells or CAFs. Another interesting aspect of hMSCs in NB is that bone marrow is a common site of metastatic spread [88]. The ability of the chemokine, stromal-derived factor (SDF-1/CXCL12), in bone marrow homing by binding to its receptor, CXCR4, on neuroblastic tumor cells has been reported [89]. Following seeding in the bone marrow, neuroblastic tumor cells may instruct hMSCs to differentiate into Schwann cells, thereby creating a favorable metastatic niche in an otherwise non-permissive environment.

An additional intriguing finding is that Schwann cells isolated from quail sciatic nerves can undergo transdifferentiation into myofibroblasts [90]. In vitro, TGF-β drastically enhanced the conversion of cultured Schwann cells to α-sma<sup>+</sup> and sox10+ myofibroblasts. When transplanted into the first branchial arch of E2 chick embryos, these Schwann cells incorporate into the perivascular space of developing vessel walls as α-sma<sup>+</sup> cells [90]. Based on these observations, it is tempting to speculate that neuroblastic tumor cells secrete TGF-β to remodel tumor stromal by converting Schwannian-rich to a CAF-rich tumor microenvironment. In summary, this level of plasticity in stromal remodeling may allow tumor cells to adapt to local hypoxic environ‐ ment or in seeding of metastatic cells.

#### **3.5. The role of Schwannian stromal in NB therapy**

used in these studies were isolated from normal human peripheral nerves. It will be of interest to compare tumor-derived versus normal Schwann cells in their abilities to promote differen‐ tiation and survival of neuroblastic tumor cells. Several trophic factors have been implicated in neuronal homeostasis. These include NGF, BDNF, LIF, and CNTF [66]. The biological effects of conditioned medium mentioned above are most likely the results of a combination of these soluble factors. Clearly defining their specific biological activities, for example, differentiation versus survival may have therapeutic implications. For instance, factors that only promote differentiation but not growth can have therapeutic effects in stroma-poor tumors. Alterna‐ tively, targeting the receptors for survival promoting factors such as the TrkB receptor for

The paracrine effects of trophic factors produced by Schwann cells are not restricted to neuroblastic tumor cells. For instance, three factors secreted by Schwann cells are known to inhibit angiogenesis. These include tissue inhibitor of metalloproteinase-2 (TIMP-2)[67], PEDF [68] and SPARC [69]. TIMP-2 was identified as a potential anti-angiogenic mediator in the conditioned medium of Schwann cells derived from both adult nerves and stroma-rich GN [67]. The negative effects of TIMP-2 on angiogenesis are independent of its ability to inhibit metalloproteinase (MMP) activities [70]. Instead TIMP-2 binds directly to endothelial cells through α3β1 integrin and dampens β1-mediated signaling and cell proliferation. PEDF, on the other hand, is a 50 kDa glycoprotein that belongs to the SERPIN family of serine protease inhibitors [71], and it binds to a PLA2/nutrin/patatin-like phospholipase domain-containing 2 (PNPLA2) receptor [72]. PEDF suppresses angiogenesis by inducing apoptosis in endothelial cells, blocking motility and tube formation [73]. In NB, PEDF enhances Schwann cell growth and inhibits basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) induced endothelial cell migration [68]. Consistent with these activities, the least differentiated NB show weak staining for PEDF while high levels are observed in welldifferentiated GNB and GN. Finally, SPARC is a matricellular protein implicated in adipo‐ genesis [74]. Surface receptors such as Stabilin-1 and α5β3 integrin have been implicated in mediating SPARC biological activities [75, 76]. Its anti-angiogenic activities are mediated by the direct binding to a host of angiogenic mediators such as VEGF, and platelet-derived growth factor (PDGF)[77, 78]. High levels of SPARC are associated with favorable outcomes in NB [69]. *In vivo* experimental proof further supports the anti-tumorigenic role of Schwannian stroma. Using an NB xenotransplant model, NB cells implanted in sciatic nerve have greater number of infiltrating Schwann cells, more differentiated neuroblasts and reduced vascularity when compared to tumor cells injected outside of the sciatic nerves [79]. All these findings reinforce the notion that the favorable prognosis in stroma-rich NB is the consequence of a host of anti-

angiogenic factors produced by the Schwannian stromal compartment.

During NB progression, there is evidence of dynamic remodeling of the Schwannian tumor microenvironment that involves additional stromal cell types. One such cell type is cancerassociated fibroblasts (CAFs). CAFs are frequently detected in epithelial tumors such as breast carcinomas [80]. CAFs are "reactive" in nature and differ from normal fibroblasts by having

**3.4. Plasticity of Schwannian stroma**

BDNF may be a plausible treatment strategy [25].

98 Research Directions in Tumor Angiogenesis

From a treatment standpoint, NB in infants has a more favorable prognosis with low-grade tumors that resolved spontaneously. However, the overall survival for patients greater than 4 year old remains around 40%. Also, there are few options once tumors are refractory to conventional chemo- and radiation-therapies. How can studying the role of Schwann cells in NB can translate into better treatment? As mentioned above, the ability of NB to differentiate into neurons and Schwann cells even in the presence of *MYCN* gene amplification can be explored in the clinical settings. Resident cancer initiating (stem) cells or "intermediate I-type" cell lines such as NUB-7 and BE(2)-C can be differentiated into neurons by retinoic acid (RA) exposure or into Schwann cells by 5-bromo-2'-deoxyuridine (BrdU) exposure [58, 91, 92]. The current standard therapy for high risk NB includes initial induction chemotherapy, followed by autologous hematopoietic stem cell transplantation, and residual disease is treated with a maintenance dose of 13-cis-RA [93, 94]. Under this aggressive treatment regimen, only onethird of patients survived [95]. It will be of interest to test if a combination of RA and BrdU is more effective in differentiating residual NB. In fact, the role of BrdU as a radiosensitizing agent is well established [96, 97].

**4. Cell-matrix and cell-cell molecular interactions in the neuroblastoma**

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101

In 1986 Harold F. Dvorak coined the phrase: "Tumors are wounds that never heal". His comment was based on similarities in the content of new blood vessels, lymphocytes, macro‐ phages, and connective tissue components (including cellular and extracellular matrix elements) present in healing wounds and tissue surrounding tumor cells [104]. During tumor (parenchyma) development, the wound repair resolution stage fails, resulting in a microen‐ vironment (stroma) that never "heals". Multiple factors in the "wounded" tumor microenvir‐ onment promote NB progression. In this section, we highlight the role of the extracellular

The NB tumor microenvironment provides biochemical and mechanical signals similar to the microenvironments of other tumor types, but there is specificity in how NB tumor cells respond to these signals. It is well recognized that the interaction of tumor with stroma occurs via biochemical signaling and that the ECM provides a source of signals that instruct cellular behavior. Our understanding of how biomechanical signaling generated by shear stress, compression, and tension affect survival, proliferation, migration, and gene expression is increasing [105]. Changes in tension homeostasis occur in cancer, with breast cancer as one of the best studied examples [106]. Mechanical cues from the ECM may influence retinoic acidmediated differentiation, which in turn may regulate clinically relevant aspects of NB biology. Recent studies show that ECM stiffness provides a physical cue that reduces NB proliferation and promotes differentiation [107]. Increasing ECM stiffness enhances neurite extension (neuritogenesis) and suppresses cell proliferation. Increased ECM stiffness also reduces expression of the oncogenic *MYCN* transcription factor. Furthermore, the addition of RA enhances ECM stiffness. Together, the data suggest that the mechanical signals from the cellular microenvironment influence NB differentiation in synergy with the RA biochemical

One of the matrix proteins with a documented role in tumor progression is SPARC (osteonectin or BM-40). SPARC is a 34 kDa calcium-binding glycoprotein shown to associate with the cell membrane and membrane receptors [108, 109]. SPARC appears to have a cancer-type specific effect on tumor metastasis. In prostate cancer, SPARC is linked with increased migration and prostate cancer metastasis to bone. This occurs via activation of integrins αvβ3 and αvβ5 expressed on tumor cells [110]. In contrast, SPARC appears to act as a tumor suppressor in NB. This tumor suppressor effect has been studied in the context of radiation therapy. Irradiation of NB tumor cells was shown to inhibit SPARC expression. Interestingly, SPARC expression was significantly decreased in radiation-therapy resistant cancer cells [111]. Exogenous

**4.1. Biochemical and biophysical cues from the extracellular matrix**

**tumor microenvironment**

matrix (ECM) in this process.

differentiation factor [107].

*4.1.2. SPARC and cell survival*

*4.1.1. ECM stiffness conveys differentiation signals*

Another treatment modality is inhibition of angiogenesis. Bevacizumab (Avastin), a human‐ ized monoclonal antibody against VEGF has been shown to enhance the efficacy of topotecan in a NB xenograft model [98]. It has moderate toxicity with overall severe adverse events of 17% [99]. Extensive clinical trial data of Bevacizumab for NB is lacking and its therapeutic efficacy in treating this pediatric tumor is yet to be determined. Nevertheless, the fact that Schwann cells secrete a host of soluble anti-angiogenic factors can be harnessed for therapeutic use. For example, PEDF is effective in blocking growth in a wide variety of tumors [73, 100, 101]. In fact, the delivery of PEDF by adenoviral-mediated gene transfer in NB suppresses angiogenesis and blocks tumor growth [102].

One of the overarching concerns in treatment-resistant high risk NB is the involvement of developmental plasticity inSchwann cells. Indeed, Schwann cells have the capacity to dedif‐ ferentiate into less mature progenitors *in vivo* under regenerative conditions. This level of plasticity in Schwann cells has been observed in injured axons wherethis activity requires an active Raf kinase [103]. One scenario is that following intense chemotherapies, while most hyperproliferative neuroblastic tumor cells are expected to be eradicated, residual stromal cells survive and undergo dedifferentiation into neural progenitors to repopulate the primary tumor site. Alternatively, as reported by our group, treatment of the ALK-positive tumor cell line SK-N-SH with an ALK inhibitor leads to the outgrowth of S-type cell populations while N-type cells are mostly eliminated [55]. Conditioned media from these Schwann-like cells confer striking survival toward N-type cells. Thus, tumor-associated Schwann cells or CAFs may provide a chemoresistant niche to support tumor recurrence from the few neuroblastic tumor cells that survive.

In summary, while Schwannian stroma have been considered as a benign byproduct of maturing NB, their presence is intimately linked to the survival and differentiation of neuro‐ blastic tumor cells. The development of transgenic animal models that can recapitulate features of stroma-rich and stroma-poor tumors will be necessary to better understand this interaction. These *in vivo* models will be useful for deciphering the biological effects of Schwannian stroma on tumor cells, the paracrine factors involved and their intracellular signaling. Although Schwannian stroma is an attractive target for NB therapy, the NB tumor stroma/microenvir‐ onment, which is composed of the extracellular matrix plays an equally important role in NB pathogenesis, which is discussed next.

### **4. Cell-matrix and cell-cell molecular interactions in the neuroblastoma tumor microenvironment**

In 1986 Harold F. Dvorak coined the phrase: "Tumors are wounds that never heal". His comment was based on similarities in the content of new blood vessels, lymphocytes, macro‐ phages, and connective tissue components (including cellular and extracellular matrix elements) present in healing wounds and tissue surrounding tumor cells [104]. During tumor (parenchyma) development, the wound repair resolution stage fails, resulting in a microen‐ vironment (stroma) that never "heals". Multiple factors in the "wounded" tumor microenvir‐ onment promote NB progression. In this section, we highlight the role of the extracellular matrix (ECM) in this process.

#### **4.1. Biochemical and biophysical cues from the extracellular matrix**

#### *4.1.1. ECM stiffness conveys differentiation signals*

NB can translate into better treatment? As mentioned above, the ability of NB to differentiate into neurons and Schwann cells even in the presence of *MYCN* gene amplification can be explored in the clinical settings. Resident cancer initiating (stem) cells or "intermediate I-type" cell lines such as NUB-7 and BE(2)-C can be differentiated into neurons by retinoic acid (RA) exposure or into Schwann cells by 5-bromo-2'-deoxyuridine (BrdU) exposure [58, 91, 92]. The current standard therapy for high risk NB includes initial induction chemotherapy, followed by autologous hematopoietic stem cell transplantation, and residual disease is treated with a maintenance dose of 13-cis-RA [93, 94]. Under this aggressive treatment regimen, only onethird of patients survived [95]. It will be of interest to test if a combination of RA and BrdU is more effective in differentiating residual NB. In fact, the role of BrdU as a radiosensitizing

Another treatment modality is inhibition of angiogenesis. Bevacizumab (Avastin), a human‐ ized monoclonal antibody against VEGF has been shown to enhance the efficacy of topotecan in a NB xenograft model [98]. It has moderate toxicity with overall severe adverse events of 17% [99]. Extensive clinical trial data of Bevacizumab for NB is lacking and its therapeutic efficacy in treating this pediatric tumor is yet to be determined. Nevertheless, the fact that Schwann cells secrete a host of soluble anti-angiogenic factors can be harnessed for therapeutic use. For example, PEDF is effective in blocking growth in a wide variety of tumors [73, 100, 101]. In fact, the delivery of PEDF by adenoviral-mediated gene transfer in NB suppresses

One of the overarching concerns in treatment-resistant high risk NB is the involvement of developmental plasticity inSchwann cells. Indeed, Schwann cells have the capacity to dedif‐ ferentiate into less mature progenitors *in vivo* under regenerative conditions. This level of plasticity in Schwann cells has been observed in injured axons wherethis activity requires an active Raf kinase [103]. One scenario is that following intense chemotherapies, while most hyperproliferative neuroblastic tumor cells are expected to be eradicated, residual stromal cells survive and undergo dedifferentiation into neural progenitors to repopulate the primary tumor site. Alternatively, as reported by our group, treatment of the ALK-positive tumor cell line SK-N-SH with an ALK inhibitor leads to the outgrowth of S-type cell populations while N-type cells are mostly eliminated [55]. Conditioned media from these Schwann-like cells confer striking survival toward N-type cells. Thus, tumor-associated Schwann cells or CAFs may provide a chemoresistant niche to support tumor recurrence from the few neuroblastic

In summary, while Schwannian stroma have been considered as a benign byproduct of maturing NB, their presence is intimately linked to the survival and differentiation of neuro‐ blastic tumor cells. The development of transgenic animal models that can recapitulate features of stroma-rich and stroma-poor tumors will be necessary to better understand this interaction. These *in vivo* models will be useful for deciphering the biological effects of Schwannian stroma on tumor cells, the paracrine factors involved and their intracellular signaling. Although Schwannian stroma is an attractive target for NB therapy, the NB tumor stroma/microenvir‐ onment, which is composed of the extracellular matrix plays an equally important role in NB

agent is well established [96, 97].

100 Research Directions in Tumor Angiogenesis

tumor cells that survive.

pathogenesis, which is discussed next.

angiogenesis and blocks tumor growth [102].

The NB tumor microenvironment provides biochemical and mechanical signals similar to the microenvironments of other tumor types, but there is specificity in how NB tumor cells respond to these signals. It is well recognized that the interaction of tumor with stroma occurs via biochemical signaling and that the ECM provides a source of signals that instruct cellular behavior. Our understanding of how biomechanical signaling generated by shear stress, compression, and tension affect survival, proliferation, migration, and gene expression is increasing [105]. Changes in tension homeostasis occur in cancer, with breast cancer as one of the best studied examples [106]. Mechanical cues from the ECM may influence retinoic acidmediated differentiation, which in turn may regulate clinically relevant aspects of NB biology. Recent studies show that ECM stiffness provides a physical cue that reduces NB proliferation and promotes differentiation [107]. Increasing ECM stiffness enhances neurite extension (neuritogenesis) and suppresses cell proliferation. Increased ECM stiffness also reduces expression of the oncogenic *MYCN* transcription factor. Furthermore, the addition of RA enhances ECM stiffness. Together, the data suggest that the mechanical signals from the cellular microenvironment influence NB differentiation in synergy with the RA biochemical differentiation factor [107].

#### *4.1.2. SPARC and cell survival*

One of the matrix proteins with a documented role in tumor progression is SPARC (osteonectin or BM-40). SPARC is a 34 kDa calcium-binding glycoprotein shown to associate with the cell membrane and membrane receptors [108, 109]. SPARC appears to have a cancer-type specific effect on tumor metastasis. In prostate cancer, SPARC is linked with increased migration and prostate cancer metastasis to bone. This occurs via activation of integrins αvβ3 and αvβ5 expressed on tumor cells [110]. In contrast, SPARC appears to act as a tumor suppressor in NB. This tumor suppressor effect has been studied in the context of radiation therapy. Irradiation of NB tumor cells was shown to inhibit SPARC expression. Interestingly, SPARC expression was significantly decreased in radiation-therapy resistant cancer cells [111]. Exogenous overexpression of SPARC significantly suppressed the activity of AKT. This suppression was accompanied by an increase in the PTEN tumor suppressor protein both in vitro and in vivo, [112] and sensitized NB cells to radiation by inhibiting irradiation-induced cell cycle arrest. Therefore, SPARC expression restored NB radiosensitivity. In addition to this function, SPARC expressed by NB cells appears to affect endothelial cells in the immediate vicinity. Interest‐ ingly, SPARC overexpression and secretion by NB cells induced endothelial cell apoptosis, inhibited angiogenesis and suppressed expression of the pro-angiogenic molecules, VEGF, FGF, PDGF and MMP-9 in endothelial cells. This suppressed expression of growth factors was mediated by inhibition of the Notch signaling pathway [113]. Therefore, promoting SPARC expression may be a plausible anti-NB therapy.

*4.2.3. Reelin signaling in NB*

Reelin is an extracellular secreted protein of the Cajal-Retzius cells located in the marginal zone of the developing cerebral cortex, and is required for the organization of the cortex into layers of neurons [135]. In the absence of reelin, neurons exhibit a broader and irregular pattern of positioning[136].Althoughreelininteractswithintegrins andcadherins, signals fromreelinare transduced by cell membrane receptors: ApoER2 and Very Low Density Lipoprotein Receptor (VLDLR) and by the intracellular regulatory protein disabled-1 (Dab1) [137, 138]. Down‐ stream signaling involves adapter protein crk and the small GTPase Rap1 [139]. Reelin trig‐ gers theactivationofRap1inmigratingcerebralcorticalneuronswhentheyaremidwaythrough theirmigrationpath(fromtheventricularzonetowardthecorticalplate).ThisactivationofRap1 by reelin is critical for neuronal multipolar polarization and migration along glia, and there‐ fore, normal cerebral cortex organization [140-143]. However,reelin expression is notlimitedto thenormaltissues suchasbrain,butisalsodetectedinseveraldifferenttumorpathologieswhere it has been linked with tumor aggressiveness [144]. A recent study suggests that reelin signal‐ ing regulates a migratory switch promoting metastasis in NB [145]. Reelin expression is negatively regulated by miR-128, a brain-enriched microRNA. miR-128 is downregulated in untreated NB patients, and ectopic miR-128 overexpression reduced NB cell motility and invasiveness and impaired cell growth. Furthermore, a small series of primary human NBs showed an association between high levels of miR-128 expression and favorable features, such as a favorable stage score based on the International Neuroblastoma Staging System Classifica‐ tion (Shimada category, [146]). In addition to the autocrine function in differentiating tumors, reelin acts as a chemoattractant for several NB cell lines. It is also expressed in blood vessels in several NB cell lines, but not in normal tissue. Therefore, it is postulated that in addition to the autocrine function, paracrine reelin presented by NB blood vessels may act as a chemoattrac‐ tant and promote hematogenic and lymphogenic dissemination in NB progression [145].

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*4.2.4. Gap junctions – Cellular connectivity and suppression of growth*

increased GJIC and cell-cell adhesion [148].

Cell-cell interactions are mediated by specialized connections between membranes of adjacent cells called gap junctions. Gap junctions form by connecting two hemichannels (connexons) on neighboring cells, with each hemichannel comprised of a hexamer of connexin. Of the 20 known connexins, connexin 43 is the most ubiquitously expressed [147]. Gap junctional coupling in NB is negatively regulated by protein kinase C (PKC) [148]. PKC isozymes regulate various aspects of proliferation and PKC inhibitors are under study in clinical trials as potential anti-cancer therapy. Tamoxifen, an estrogen receptor antagonist, exerts some of its anti-tumor effects via PKC signaling [149, 150]. However, the exact cellular mechanisms targeted by PKC inhibitors are not known. Recently, it was shown that inhibition of PKC in NB cell lines increases GJIC via a mechanism that does not depend on the redistribution of connexin 43 or its phosphorylation [148]. Furthermore, PKC inhibition promoted cell-cell adhesion, a finding that suggests that suppression of tumor growth by PKC inhibition may be due to effects on

Overall, these studies suggest that the extracellular matrix and CAMs play an important role in the biochemical and biophysical regulation of NB. The careful examination of NB environ‐

#### **4.2. Role of cell adhesion molecules in NB progression**

#### *4.2.1. NCAM and NB progression*

Intercellular communication is a fundamental biological property that is regulated during cellular growth and differentiation. In general terms, abnormalities in gap junction intracel‐ lular communication (GJIC) and cell-cell adhesion correlate with poor prognosis for cancer treatment [114-117]. Loss of either cell-cell adhesion or GJIC occurs in cancers, and gain of communication or adhesion suppresses tumor growth [118, 119]. Cell adhesion molecules (CAMs) have been reported to regulate tumor progression and metastasis, acting as oncogenes or tumor suppressors [120-122], and one such molecule namely Neural cell adhesion molecule (NCAM) is of particular importance for both, normal brain development [123] and NB regulation. NCAM is the main protein carrier of polysialic acid (polySia), a major regulator of cell-cell interactions in the developing nervous system that is required for neuronal plasticity. Studies in NCAM knockout mice showed that the effects of polySia occur via the expression of NCAM [123]. During normal neuronal differentiation or upon RA induced differentiation of a NB cell line, NCAM appears in non-polysialated form. This allows for its hemophilic interactions, and in turn triggers enhanced ERK signaling and MAPK-dependent neuritogen‐ esis [124]. Therefore, it can be expected that inhibition of polysialation will promote neuronal differentiation and may inhibit NB progression.

#### *4.2.2. N-cadherin and NB progression*

Clinical studies suggest that tumor invasiveness, not the ability to detach from the primary tumor are determinants of the progression to metastasis [125]. In epithelial-derived tumors, metastasis is often preceded by the loss of E-cadherin cell-cell adhesion [126, 127]. The loss of E-cadherin is often accompanied by de novo expression of N-cadherin, which promotes cell motility and migration; a phenomenon called "the cadherin switch" [128-130]. Further, Ncadherin homophilic interactions between tumor cells and surrounding tissue such as tumor vessel endothelium and stroma facilitate the transit and survival of tumor cells in distant organs [131-133]. N-cadherin thus may play a role in preventing metastasis in NB through such homotypic and heterotypic cell-cell interactions. In line with this hypothesis, N-cadherins are expressed on various NB tumors and NB cell lines, with lowest levels in patients under‐ going metastasis. Therefore, its expression negatively correlates with metastasis [134].

#### *4.2.3. Reelin signaling in NB*

overexpression of SPARC significantly suppressed the activity of AKT. This suppression was accompanied by an increase in the PTEN tumor suppressor protein both in vitro and in vivo, [112] and sensitized NB cells to radiation by inhibiting irradiation-induced cell cycle arrest. Therefore, SPARC expression restored NB radiosensitivity. In addition to this function, SPARC expressed by NB cells appears to affect endothelial cells in the immediate vicinity. Interest‐ ingly, SPARC overexpression and secretion by NB cells induced endothelial cell apoptosis, inhibited angiogenesis and suppressed expression of the pro-angiogenic molecules, VEGF, FGF, PDGF and MMP-9 in endothelial cells. This suppressed expression of growth factors was mediated by inhibition of the Notch signaling pathway [113]. Therefore, promoting SPARC

Intercellular communication is a fundamental biological property that is regulated during cellular growth and differentiation. In general terms, abnormalities in gap junction intracel‐ lular communication (GJIC) and cell-cell adhesion correlate with poor prognosis for cancer treatment [114-117]. Loss of either cell-cell adhesion or GJIC occurs in cancers, and gain of communication or adhesion suppresses tumor growth [118, 119]. Cell adhesion molecules (CAMs) have been reported to regulate tumor progression and metastasis, acting as oncogenes or tumor suppressors [120-122], and one such molecule namely Neural cell adhesion molecule (NCAM) is of particular importance for both, normal brain development [123] and NB regulation. NCAM is the main protein carrier of polysialic acid (polySia), a major regulator of cell-cell interactions in the developing nervous system that is required for neuronal plasticity. Studies in NCAM knockout mice showed that the effects of polySia occur via the expression of NCAM [123]. During normal neuronal differentiation or upon RA induced differentiation of a NB cell line, NCAM appears in non-polysialated form. This allows for its hemophilic interactions, and in turn triggers enhanced ERK signaling and MAPK-dependent neuritogen‐ esis [124]. Therefore, it can be expected that inhibition of polysialation will promote neuronal

Clinical studies suggest that tumor invasiveness, not the ability to detach from the primary tumor are determinants of the progression to metastasis [125]. In epithelial-derived tumors, metastasis is often preceded by the loss of E-cadherin cell-cell adhesion [126, 127]. The loss of E-cadherin is often accompanied by de novo expression of N-cadherin, which promotes cell motility and migration; a phenomenon called "the cadherin switch" [128-130]. Further, Ncadherin homophilic interactions between tumor cells and surrounding tissue such as tumor vessel endothelium and stroma facilitate the transit and survival of tumor cells in distant organs [131-133]. N-cadherin thus may play a role in preventing metastasis in NB through such homotypic and heterotypic cell-cell interactions. In line with this hypothesis, N-cadherins are expressed on various NB tumors and NB cell lines, with lowest levels in patients under‐ going metastasis. Therefore, its expression negatively correlates with metastasis [134].

expression may be a plausible anti-NB therapy.

differentiation and may inhibit NB progression.

*4.2.2. N-cadherin and NB progression*

*4.2.1. NCAM and NB progression*

102 Research Directions in Tumor Angiogenesis

**4.2. Role of cell adhesion molecules in NB progression**

Reelin is an extracellular secreted protein of the Cajal-Retzius cells located in the marginal zone of the developing cerebral cortex, and is required for the organization of the cortex into layers of neurons [135]. In the absence of reelin, neurons exhibit a broader and irregular pattern of positioning[136].Althoughreelininteractswithintegrins andcadherins, signals fromreelinare transduced by cell membrane receptors: ApoER2 and Very Low Density Lipoprotein Receptor (VLDLR) and by the intracellular regulatory protein disabled-1 (Dab1) [137, 138]. Down‐ stream signaling involves adapter protein crk and the small GTPase Rap1 [139]. Reelin trig‐ gers theactivationofRap1inmigratingcerebralcorticalneuronswhentheyaremidwaythrough theirmigrationpath(fromtheventricularzonetowardthecorticalplate).ThisactivationofRap1 by reelin is critical for neuronal multipolar polarization and migration along glia, and there‐ fore, normal cerebral cortex organization [140-143]. However,reelin expression is notlimitedto thenormaltissues suchasbrain,butisalsodetectedinseveraldifferenttumorpathologieswhere it has been linked with tumor aggressiveness [144]. A recent study suggests that reelin signal‐ ing regulates a migratory switch promoting metastasis in NB [145]. Reelin expression is negatively regulated by miR-128, a brain-enriched microRNA. miR-128 is downregulated in untreated NB patients, and ectopic miR-128 overexpression reduced NB cell motility and invasiveness and impaired cell growth. Furthermore, a small series of primary human NBs showed an association between high levels of miR-128 expression and favorable features, such as a favorable stage score based on the International Neuroblastoma Staging System Classifica‐ tion (Shimada category, [146]). In addition to the autocrine function in differentiating tumors, reelin acts as a chemoattractant for several NB cell lines. It is also expressed in blood vessels in several NB cell lines, but not in normal tissue. Therefore, it is postulated that in addition to the autocrine function, paracrine reelin presented by NB blood vessels may act as a chemoattrac‐ tant and promote hematogenic and lymphogenic dissemination in NB progression [145].

#### *4.2.4. Gap junctions – Cellular connectivity and suppression of growth*

Cell-cell interactions are mediated by specialized connections between membranes of adjacent cells called gap junctions. Gap junctions form by connecting two hemichannels (connexons) on neighboring cells, with each hemichannel comprised of a hexamer of connexin. Of the 20 known connexins, connexin 43 is the most ubiquitously expressed [147]. Gap junctional coupling in NB is negatively regulated by protein kinase C (PKC) [148]. PKC isozymes regulate various aspects of proliferation and PKC inhibitors are under study in clinical trials as potential anti-cancer therapy. Tamoxifen, an estrogen receptor antagonist, exerts some of its anti-tumor effects via PKC signaling [149, 150]. However, the exact cellular mechanisms targeted by PKC inhibitors are not known. Recently, it was shown that inhibition of PKC in NB cell lines increases GJIC via a mechanism that does not depend on the redistribution of connexin 43 or its phosphorylation [148]. Furthermore, PKC inhibition promoted cell-cell adhesion, a finding that suggests that suppression of tumor growth by PKC inhibition may be due to effects on increased GJIC and cell-cell adhesion [148].

Overall, these studies suggest that the extracellular matrix and CAMs play an important role in the biochemical and biophysical regulation of NB. The careful examination of NB environ‐ ment-specific cues to fully define their effects on NB tumor progression offers an opportunity for NB targeted therapy.

genesis inhibitors, such as activin A, interleukin-6 and leukemia inhibitory factor [169, 170]. Activin A represses NB growth, endothelial cell proliferation and angiogenesis [171, 172]. In addition, highly expressed activin A in differentiated NB strongly correlates with a favorable NB outcome [173]. Interestingly, inhibition of PI3K/rapamycin results in the degradation of

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A second gene family implicated in NB is the TRK family of neurotrophin receptors (NTRK) that play critical roles in the development of the CNS and PNS [25, 175]. The 3 characterized members are TrkA (NTRK1), TrkB (NTRK2) and TrkC (NTRK3) with nerve growth factor (NGF), BDNF and neurotrophin-3 (NT-3) as their primary ligands, respectively [175]. The sequential Trk expression is important for complete differentiation of normal sympathetic neurons, and the *Trk* genes expressed reflect the stage of neuronal differentiation [176]. High expression of TrkA and TrkC are associated with the ability for NB to differentiate and spontaneous regress, and are predominately found in clinically favorable NB. One mechanism that could explain this is that high expression of TrkA reduces the expression of angiogenic factors in NB cells and suppresses NB tumor xenograft growth associated with reduced angiogenic factor expression and vascularization of tumors [177]. In contrast, TrkB and its ligand, BDNF, are highly expressed in aggressive NB associated with increased cell survival,

**5.3. Anti-angiogenesis treatments in NB - conventional anti-VEGF/VEGFR2 signaling**

Although targeting of the tumor vasculature represents a promising tool for cancer therapy, there are no current clinical trials of anti-angiogenesis therapy for NB [157-159]. There are several pre-clinical studies in NB animal models [157-159], and depending on the unique aspects of NB, several different approaches for anti-angiogenesis therapy is feasible. VEGF and its cognate receptor 2 (VEGFR2) are major regulators of angiogenesis. Anti-VEGF/VEGFR2 signaling pathways and inhibition of endothelial cell proliferation and migration are the most common anti-angiogenesis therapeutic approaches. The recently approved anti-angiogenesis drug, bevacizumab (Avastin), is a recombinant monoclonal antibody that binds VEGF-A and subsequently blocks the activation of its receptors. Bevacizumab reduces NB tumor growth by reducing angiogenesis [178]. In addition, treatment with bevacizumab can transiently induce tumor vasculature remodeling allowing for improved delivery and efficacy of chemotherapy in NB tumor xenografts [98]. A VEGFR-2 tyrosine kinase inhibitor Sugen 5416 (SU5416, Semoxinal) is a specific VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) tyrosine kinase inhibitor that has shown efficacy in inhibiting angiogenesis in vivo models of NB [179]. Efficacy of inhibiting tumor growth was increased when SU5416 was given in combination with irradiation or chemotherapy [180, 181]. In addition to VEGF inhibitors, other angiogenesis inhibitors have shown efficacy on NB tumor angiogenesis and growth, which is discussed in detail elsewhere [157, 159, 182]. TNP-470 is a synthetic analog of fumagillin, an antibiotic isolated from the fungus Aspergillus fumigatus fresenius with antineoplastic activity. TNP-470 is a potent selective inhibitor of Methionine aminopeptidase-2 (MetAP-2) resulting in endothelial cell cycle arrest late in G1 phase and leading to inhibition of tumor angiogenesis [183]. TNP-470

*MYCN* in NB tumor cells and results in blockage of angiogenesis indirectly [174].

angiogenesis and drug resistance [25, 175].

**pathways**

#### **5. Role of endothelial cells in NB microenvironment pathogenesis**

#### **5.1. Role of angiogenesis in NB pathogenesis**

In 1962, Dr. Judah Folkman described the seminal observation that tumor angiogenesis is dependent on de novo blood vessel formation [151]. The sprouting of new blood vessels from pre-existing ones is a multi-step process consisting of endothelial cell proliferation, migration and tube formation [152]. Tumor angiogenesis is not only induced by growth factors and cytokines secreted from tumor cells [153], but also modulated by cell-cell interaction [152]. Aberrant angiogenesis is associated with excessive growth-promoting signals and a lack of sufficient "pruning," cues that spatially and temporally modulate vessel growth, remodeling and stabilization [152]. As compared to normal blood vessels, tumor vessels are more dilated and tortuous, form arteriovenous shunts, and lack the normal artery-capillary-vein hierarchy [154]. Tumor vasculature not only provides oxygen and nutrients to promote tumor prolifer‐ ation and progression, but also facilitates tumor metastatic spreading. Thus, tumor angiogen‐ esis represents an attractive new target for tumor therapy because it is well accepted that new blood vessel formation promotes tumor growth and metastatic spread [152, 155, 156].

In terms of NB, current evidence suggests that advanced and aggressive stages of NB are dependent on angiogenesis [157-159]. Meitar et al [160] demonstrated the association of the tumor angiogenesis and poor outcome in human NB. Like most solid tumors, several wellknown pro-angiogenic growth factors such as VEGF-A, VEGF-B, bFGF, angiopoietin-2 (Ang-2), transforming growth factor alpha (TGF-α) and PDGF were found in advanced-stage NB tumors [161]. Human NBs produce extracellular matrix-degrading enzymes, that induce endothelial cell proliferation and are angiogenic in vivo [162]. Integrins αvβ3 and αvβ5 are more highly expressed in blood vessels of high-risk versus low-risk NB tumors [163]. In addition, lymphatic vessels are observed in NB [164] with higher expression of the VEGF-C lymphangiogenesis growth factor observed in advanced stage of NB [161]. These evidences suggest that tumor angiogenesis likely contributes to NB pathogenesis.

#### **5.2. Contributions of** *MYCN* **amplification and trks-mediated signaling pathways to NB tumor angiogenesis**

NB is an embryonic tumor that is derived from cells of the primitive NC [165]. In general, genetic abnormalities play a key role in determining tumor phenotype, [165, 166]. *MYCN* amplification is one of the first identified genetic defects in NB, and high levels of *MYCN* are associated with aggressive tumor behavior and poor survival [167]. *MYCN* is member of the MYC family of basic helix-loop-helix transcription factors that control a broad regulatory network implicated in cell cycle, DNA damage response, differentiation and apoptosis [168]. There is evidence that *MYCN* amplification is also associated with tumor angiogenesis. Several studies demonstrated that *MYCN* amplification in NB suppressed the expression of angio‐ genesis inhibitors, such as activin A, interleukin-6 and leukemia inhibitory factor [169, 170]. Activin A represses NB growth, endothelial cell proliferation and angiogenesis [171, 172]. In addition, highly expressed activin A in differentiated NB strongly correlates with a favorable NB outcome [173]. Interestingly, inhibition of PI3K/rapamycin results in the degradation of *MYCN* in NB tumor cells and results in blockage of angiogenesis indirectly [174].

ment-specific cues to fully define their effects on NB tumor progression offers an opportunity

In 1962, Dr. Judah Folkman described the seminal observation that tumor angiogenesis is dependent on de novo blood vessel formation [151]. The sprouting of new blood vessels from pre-existing ones is a multi-step process consisting of endothelial cell proliferation, migration and tube formation [152]. Tumor angiogenesis is not only induced by growth factors and cytokines secreted from tumor cells [153], but also modulated by cell-cell interaction [152]. Aberrant angiogenesis is associated with excessive growth-promoting signals and a lack of sufficient "pruning," cues that spatially and temporally modulate vessel growth, remodeling and stabilization [152]. As compared to normal blood vessels, tumor vessels are more dilated and tortuous, form arteriovenous shunts, and lack the normal artery-capillary-vein hierarchy [154]. Tumor vasculature not only provides oxygen and nutrients to promote tumor prolifer‐ ation and progression, but also facilitates tumor metastatic spreading. Thus, tumor angiogen‐ esis represents an attractive new target for tumor therapy because it is well accepted that new

**5. Role of endothelial cells in NB microenvironment pathogenesis**

blood vessel formation promotes tumor growth and metastatic spread [152, 155, 156].

suggest that tumor angiogenesis likely contributes to NB pathogenesis.

In terms of NB, current evidence suggests that advanced and aggressive stages of NB are dependent on angiogenesis [157-159]. Meitar et al [160] demonstrated the association of the tumor angiogenesis and poor outcome in human NB. Like most solid tumors, several wellknown pro-angiogenic growth factors such as VEGF-A, VEGF-B, bFGF, angiopoietin-2 (Ang-2), transforming growth factor alpha (TGF-α) and PDGF were found in advanced-stage NB tumors [161]. Human NBs produce extracellular matrix-degrading enzymes, that induce endothelial cell proliferation and are angiogenic in vivo [162]. Integrins αvβ3 and αvβ5 are more highly expressed in blood vessels of high-risk versus low-risk NB tumors [163]. In addition, lymphatic vessels are observed in NB [164] with higher expression of the VEGF-C lymphangiogenesis growth factor observed in advanced stage of NB [161]. These evidences

**5.2. Contributions of** *MYCN* **amplification and trks-mediated signaling pathways to NB**

NB is an embryonic tumor that is derived from cells of the primitive NC [165]. In general, genetic abnormalities play a key role in determining tumor phenotype, [165, 166]. *MYCN* amplification is one of the first identified genetic defects in NB, and high levels of *MYCN* are associated with aggressive tumor behavior and poor survival [167]. *MYCN* is member of the MYC family of basic helix-loop-helix transcription factors that control a broad regulatory network implicated in cell cycle, DNA damage response, differentiation and apoptosis [168]. There is evidence that *MYCN* amplification is also associated with tumor angiogenesis. Several studies demonstrated that *MYCN* amplification in NB suppressed the expression of angio‐

for NB targeted therapy.

104 Research Directions in Tumor Angiogenesis

**tumor angiogenesis**

**5.1. Role of angiogenesis in NB pathogenesis**

A second gene family implicated in NB is the TRK family of neurotrophin receptors (NTRK) that play critical roles in the development of the CNS and PNS [25, 175]. The 3 characterized members are TrkA (NTRK1), TrkB (NTRK2) and TrkC (NTRK3) with nerve growth factor (NGF), BDNF and neurotrophin-3 (NT-3) as their primary ligands, respectively [175]. The sequential Trk expression is important for complete differentiation of normal sympathetic neurons, and the *Trk* genes expressed reflect the stage of neuronal differentiation [176]. High expression of TrkA and TrkC are associated with the ability for NB to differentiate and spontaneous regress, and are predominately found in clinically favorable NB. One mechanism that could explain this is that high expression of TrkA reduces the expression of angiogenic factors in NB cells and suppresses NB tumor xenograft growth associated with reduced angiogenic factor expression and vascularization of tumors [177]. In contrast, TrkB and its ligand, BDNF, are highly expressed in aggressive NB associated with increased cell survival, angiogenesis and drug resistance [25, 175].

#### **5.3. Anti-angiogenesis treatments in NB - conventional anti-VEGF/VEGFR2 signaling pathways**

Although targeting of the tumor vasculature represents a promising tool for cancer therapy, there are no current clinical trials of anti-angiogenesis therapy for NB [157-159]. There are several pre-clinical studies in NB animal models [157-159], and depending on the unique aspects of NB, several different approaches for anti-angiogenesis therapy is feasible. VEGF and its cognate receptor 2 (VEGFR2) are major regulators of angiogenesis. Anti-VEGF/VEGFR2 signaling pathways and inhibition of endothelial cell proliferation and migration are the most common anti-angiogenesis therapeutic approaches. The recently approved anti-angiogenesis drug, bevacizumab (Avastin), is a recombinant monoclonal antibody that binds VEGF-A and subsequently blocks the activation of its receptors. Bevacizumab reduces NB tumor growth by reducing angiogenesis [178]. In addition, treatment with bevacizumab can transiently induce tumor vasculature remodeling allowing for improved delivery and efficacy of chemotherapy in NB tumor xenografts [98]. A VEGFR-2 tyrosine kinase inhibitor Sugen 5416 (SU5416, Semoxinal) is a specific VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1) tyrosine kinase inhibitor that has shown efficacy in inhibiting angiogenesis in vivo models of NB [179]. Efficacy of inhibiting tumor growth was increased when SU5416 was given in combination with irradiation or chemotherapy [180, 181]. In addition to VEGF inhibitors, other angiogenesis inhibitors have shown efficacy on NB tumor angiogenesis and growth, which is discussed in detail elsewhere [157, 159, 182]. TNP-470 is a synthetic analog of fumagillin, an antibiotic isolated from the fungus Aspergillus fumigatus fresenius with antineoplastic activity. TNP-470 is a potent selective inhibitor of Methionine aminopeptidase-2 (MetAP-2) resulting in endothelial cell cycle arrest late in G1 phase and leading to inhibition of tumor angiogenesis [183]. TNP-470 treatment in a NB tumor xenograft model reduced the tumor growth rate and decreased capillary density [184-188], and increased the efficacy of chemotherapy [181]. Taken together, these results suggest that anti-angiogenesis is an effective approach for reducing NB growth and burden. In addition to direct approaches targeting the vasculature in NB, indirect antiangiogenesis approaches have also shown efficacy in NB. For the most part, these approaches rely on the induction of differentiation of NB. For example, retinoids have been shown to exert their effects by inducing differentiation of NB cells. Retinoids and fenretinide, a synthetic retinoid, have demonstrated anti-angiogenic effects in NB tumor xenografts [189, 190]. The inhibitory effects were mediated by retinoic acid induced expression of thrombospondin-1 (TSP-1) in NB cells. TSP-1 is an important endogenous angiogenesis inhibitor that inhibits endothelial cell proliferation and migration. Interestingly, TSP-1 is silenced in a subset of undifferentiated advanced-stage NB tumors and NB cell lines due to promoter methylation [191]. Remarkably, ABT-510, a peptide derived from TSP-1, suppressed the growth of NB tumor xenografts [192]. In combination with valproic acid, ABT-510 showed potent inhibitory effects on the growth of NB tumor xenografts. Taken together, these results suggest that both direct and indirect approaches of targeting angiogenesis are feasible therapeutic approaches for NB.

the differentiation state of immune cells which ultimately determines whether or not these cells can be activated to contribute to anti-tumor immunity. This section highlights how immune cells are affected by factors in the tumor microenvironment to become both tolero‐ genic and pro-angiogenic with an emphasis on the interconnection between angiogenesis and tumor immunity. In addition, future prospects for treating NB with combinations of antiangiogenic agents and immune-based therapies as a strategy to reverse the immune suppres‐

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**Figure 4. The contribution of immune cells in the tumor microenvironment.** Pro M: pro-monocyte; MM: myelo‐ monocytic stem cell; M: monocyte; M1 and M2: type 1 and 2 macrophages; MDSC: myeloid-derived suppressor cell; DC: dendritic cell; iDC: immature dendritic cell; VLC: vascular leukocyte cell; Tregs: T regulatory cells; IFN-γ: interferon gamma; TNF-α: tumor necrosis factor alpha; TGF-β: transforming growth factor beta; VEGF: vascular endothelial growth factor; bFGF: basic fibroblast growth factor; PIGF: placental growth factor; and MMP9: matrix metalloprotei‐

Innate immune cells of the myeloid lineage, including monocytes, macrophages and dendritic cells have been implicated as drivers of angiogenesis (Figure 4). Of these cells, the contribution to angiogenesis has been best characterized for macrophages. Studies in both human tumors

sion in the tumor microenvironment is discussed.

nase 9.

**6.1. Myeloid cells**

#### **6. Molecular and cellular mechanistic interface between endothelial and immune cells in NB**

The statement that "tumors are wounds that never heal" [21] has relevance for which pheno‐ type of immune cells are present in the tumor microenvironment, and whether these cells interact to promote or prevent tumor. During the initial stage of wound healing there is an inflammatory response that is produced by an influx of immune cells that release inflammatory mediators. The next stage of tissue remodeling is characterized by a down-regulation of the immune response, cell proliferation, and revascularization of the wound via angiogenesis [193-195]. In the resolution stage of tissue remodeling, cell proliferation is halted and vessels are stabilized. In the tumor microenvironment, there is a perpetual state of inflammation, cell proliferation and angiogenesis similar to an unhealed wound. Chronic hypoxia in the tumor microenvironment is a contributing factor as to why tumors are wounds that never heal. The cellular response to hypoxia is controlled by the expression of hypoxia inducible factors (HIF) [196]. Low oxygen tension prevents the ubiquitination and subsequent proteosomal degrada‐ tion of HIF-α proteins allowing them to translocate to the nucleus and dimerize with HIF-β forming functional transcription factors (HIF-1α/HIF-β or HIF-2α/HIF-β) that promote upregulation of angiogenic target genes. There is also evidence that HIF-1α regulates energy homeostasis and plays a role in the differentiation of immune cells that can have pro or antitumor effects [197]. Notably, HIF-2α expression is required to maintain an undifferentiated state of NB tumor-initiating cells, and expression of HIF-2α is associated with poor outcome in NB [197, 198]. Hypoxia and chronic inflammation are key characteristics of the tumor microenvironment that promote immune suppression and vascularization. In NB as well as other solid tumors, cytokine and chemokine mediators as well as angiogenic factors influence the differentiation state of immune cells which ultimately determines whether or not these cells can be activated to contribute to anti-tumor immunity. This section highlights how immune cells are affected by factors in the tumor microenvironment to become both tolero‐ genic and pro-angiogenic with an emphasis on the interconnection between angiogenesis and tumor immunity. In addition, future prospects for treating NB with combinations of antiangiogenic agents and immune-based therapies as a strategy to reverse the immune suppres‐ sion in the tumor microenvironment is discussed.

**Figure 4. The contribution of immune cells in the tumor microenvironment.** Pro M: pro-monocyte; MM: myelo‐ monocytic stem cell; M: monocyte; M1 and M2: type 1 and 2 macrophages; MDSC: myeloid-derived suppressor cell; DC: dendritic cell; iDC: immature dendritic cell; VLC: vascular leukocyte cell; Tregs: T regulatory cells; IFN-γ: interferon gamma; TNF-α: tumor necrosis factor alpha; TGF-β: transforming growth factor beta; VEGF: vascular endothelial growth factor; bFGF: basic fibroblast growth factor; PIGF: placental growth factor; and MMP9: matrix metalloprotei‐ nase 9.

#### **6.1. Myeloid cells**

treatment in a NB tumor xenograft model reduced the tumor growth rate and decreased capillary density [184-188], and increased the efficacy of chemotherapy [181]. Taken together, these results suggest that anti-angiogenesis is an effective approach for reducing NB growth and burden. In addition to direct approaches targeting the vasculature in NB, indirect antiangiogenesis approaches have also shown efficacy in NB. For the most part, these approaches rely on the induction of differentiation of NB. For example, retinoids have been shown to exert their effects by inducing differentiation of NB cells. Retinoids and fenretinide, a synthetic retinoid, have demonstrated anti-angiogenic effects in NB tumor xenografts [189, 190]. The inhibitory effects were mediated by retinoic acid induced expression of thrombospondin-1 (TSP-1) in NB cells. TSP-1 is an important endogenous angiogenesis inhibitor that inhibits endothelial cell proliferation and migration. Interestingly, TSP-1 is silenced in a subset of undifferentiated advanced-stage NB tumors and NB cell lines due to promoter methylation [191]. Remarkably, ABT-510, a peptide derived from TSP-1, suppressed the growth of NB tumor xenografts [192]. In combination with valproic acid, ABT-510 showed potent inhibitory effects on the growth of NB tumor xenografts. Taken together, these results suggest that both direct and indirect approaches of targeting angiogenesis are feasible therapeutic approaches

**6. Molecular and cellular mechanistic interface between endothelial and**

The statement that "tumors are wounds that never heal" [21] has relevance for which pheno‐ type of immune cells are present in the tumor microenvironment, and whether these cells interact to promote or prevent tumor. During the initial stage of wound healing there is an inflammatory response that is produced by an influx of immune cells that release inflammatory mediators. The next stage of tissue remodeling is characterized by a down-regulation of the immune response, cell proliferation, and revascularization of the wound via angiogenesis [193-195]. In the resolution stage of tissue remodeling, cell proliferation is halted and vessels are stabilized. In the tumor microenvironment, there is a perpetual state of inflammation, cell proliferation and angiogenesis similar to an unhealed wound. Chronic hypoxia in the tumor microenvironment is a contributing factor as to why tumors are wounds that never heal. The cellular response to hypoxia is controlled by the expression of hypoxia inducible factors (HIF) [196]. Low oxygen tension prevents the ubiquitination and subsequent proteosomal degrada‐ tion of HIF-α proteins allowing them to translocate to the nucleus and dimerize with HIF-β forming functional transcription factors (HIF-1α/HIF-β or HIF-2α/HIF-β) that promote upregulation of angiogenic target genes. There is also evidence that HIF-1α regulates energy homeostasis and plays a role in the differentiation of immune cells that can have pro or antitumor effects [197]. Notably, HIF-2α expression is required to maintain an undifferentiated state of NB tumor-initiating cells, and expression of HIF-2α is associated with poor outcome in NB [197, 198]. Hypoxia and chronic inflammation are key characteristics of the tumor microenvironment that promote immune suppression and vascularization. In NB as well as other solid tumors, cytokine and chemokine mediators as well as angiogenic factors influence

for NB.

**immune cells in NB**

106 Research Directions in Tumor Angiogenesis

Innate immune cells of the myeloid lineage, including monocytes, macrophages and dendritic cells have been implicated as drivers of angiogenesis (Figure 4). Of these cells, the contribution to angiogenesis has been best characterized for macrophages. Studies in both human tumors and murine tumor models have shown that the presence of tumor associated macrophages (TAMs) correlates with enhanced vessel density, tumor progression and metastasis [199]. During inflammation, monocytes are attracted by chemo-attractants to damaged tissues, where they differentiate into macrophages. These macrophages are phenotypically plastic, and depending on the environmental signals within wounds or tumors, they differentiate into functional subsets with different activation states [200]. At sites of inflammation, interferon gamma (IFN-γ) and TNF-α facilitate macrophage differentiation into cytotoxic "M1" cells that secrete pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IL-12 and IL-23). M1 macrophages are phagocytic, sustain tissue inflammation, and promote a T helper-1 (TH1) anti-tumor immune response [201, 202]. Alternatively, when induced in the presence of IL-4, IL-13, IL-10 and TGFβ, macrophages differentiate into "M2" cells that secrete IL-10 and participate in tissue remodeling and immune suppression. M2 macrophages also produce angiogenic factors. These factors include VEGF, placental growth factor (PIGF), arginase and the Tie2 angiopoietin cell surface receptor [203, 204]. Monocytes/macrophages that express Tie2 (referred to as TEMS) are a source of VEGF and have been found in human and murine spontaneous and orthotopic tumors [205, 206]. TEMS reside in close proximity to the tumor vasculature and are possibly recruited by angiopoietin-2-expressing endothelial cells [199].

CD34+

pro-angiogenic factors [224].

secrete matrix metalloproteases.

systemic decrease in T cells.

**6.2. T cells**

 bone marrow progenitor cells limits differentiation along the DC lineage [210], and engagement of VEGFR-2 inhibits the maturation of DCs [210-212]. Furthermore, high levels of tumor-derived VEGF are associated with the presence of DCs with decreased co-stimulatory molecule expression [213]. There is evidence that tumor-infiltrating immature DCs also promote angiogenesis by secreting VEGF and bFGF [214, 215], and that immature DCs participate in de novo formation of blood vessels or neovascularization in the tumor micro‐ environment. Under the influence of VEGF or angiogenic factors, immature mDCs transdifferentiate into endothelial-like DCs (called vascular leukocytes, VLC) expressing both DC and endothelial markers such as von Willebrand factor, VEGFR-2, and VE-cadherin (CD31) [216]. Remarkably, human VLC were able to form perfusable blood vessels when transplanted

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

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109

into immune-deficient mice, indicating a potential to support neovascularization.

In addition to macrophages and DCs, neutrophils, eosinophils and mast cells can contribute to tumor angiogenesis. Tumor-infiltrating neutrophils and mast cells secrete VEGF and MMP-9 [217]. Secretion of MMP-9 facilitates the availability of pro-angiogenic factors through a remodeling of the extracellular matrix. Interestingly, an increase in the number of neutrophils in the tumor microenvironment correlates with increased micro-vessel density [218]. The presence of mast cells in murine models of melanoma [219], squamous cell carcinoma [220] and pancreatic islet tumors [221] has been associated with increased angiogenesis. Mast cells are present in the tumor microenvironment prior to vessel formation [222], and they congre‐ gate near tumor-derived vessels [220, 223]. Since mast cells contain pro-angiogenic factors in their secretory granules, it has been hypothesized that secretion of these factors by mast cells promotes tumor angiogenesis [199]. There is indirect evidence that eosinophils promote tumor angiogenesis, as they have been detected in human tumors [199], and they are also a source of

Myeloid-derived suppressor cells (MDSC) are immature myeloid progenitors of monocytes, neutrophils and DCs. As tumor-resident cells, MDSC facilitate tumor progression by their immunosuppressive properties. However, these cells may also have a role in promoting angiogenesis. Studies have shown that tumor angiogenesis is decreased when the MDSC chemo-attractant, BV8 (PROK2), was neutralized [225], and tumor blood vessel density increased when MDSC were co-injected with colorectal cancer cells into mice [226]. MDSC also

Cancer patients have a decrease in immune function that can be attributed in part to the tolerogenic differentiation of innate immune cells. However, there is evidence that VEGF also interferes with T cell development. Effective T cells have the ability to specifically recognize and kill tumors. In fact, the most significant predictor of survival from solid tumors is the presence of CD8 T cells in the tumor core and invasive margins [227]. In vivo administration of a supraphysiologic concentration of recombinant VEGF blocks bone marrow-derived progenitor cells from seeding in the thymus reducing T cell production [228]. These data imply that VEGF secreted from tumors or cells in the tumor microenvironment may contribute to a

Using a physiologic model of skin wound repair, CCR2hi/VEGF-expressing macrophages were shown to initiate vascular sprouts during the early stages of tissue repair [195]. During the early repair period, macrophages with both M1 and M2 gene profiles were present, but cells with a M2 phenotype predominated during the later stages of repair. Results of this study imply that VEGF-expressing macrophages initiate wound-tissue vascularization. The presence of both M1 and M2 macrophages during early repair may be a reflection of the presence of M1 cells during the resolution of inflammation and the presence of M2 cells associated with initiation of an immune-suppressive tissue repair program. The data obtained from this physiologic model of wound healing parallels the process that occurs within the tumor microenvironment. In tumors, M1 cells are often found in sites of chronic inflamma‐ tion, simulating the inflammatory stage of wound healing, while M2 cells are associated with vascularization, immune suppression and tissue repair [207]. However, this paradigm of tissue repair is not absolute for tumors as demonstrated by an aggressive inflammatory form of breast cancer where there is up-regulation of both VEGF and the IL-6 pro-inflammatory (M1) cytokine [208].

Dendritic cells (DCs) are professional antigen-presenting cells by nature, and they are inti‐ mately involved in the activation of tumor-specific T cells. DCs originate from CD34+ bone marrow precursors, and they differentiate into heterogeneous subsets due to differentiation plasticity. Within this heterogeneity there are functionally 2 major distinct subtypes of dendritic cells classified as myeloid DC (mDCs) and plasmacytoid DC (pDCs). Plasmacytoid DCs produce anti-angiogenic type I interferons [209], and mDC have the capacity to function as potent antigen-presenting cells. The maturation state of DCs adds another layer of com‐ plexity as immature DCs have high endocytic activity, but they lack expression of the costimulatory molecules that are necessary for T cell activation. Based on these properties, immature DCs are considered as immune-tolerogenic rather than immune-activating. VEGF affects the development and maturation of DCs. Binding of VEGF to the VEGFR-1 receptor on CD34+ bone marrow progenitor cells limits differentiation along the DC lineage [210], and engagement of VEGFR-2 inhibits the maturation of DCs [210-212]. Furthermore, high levels of tumor-derived VEGF are associated with the presence of DCs with decreased co-stimulatory molecule expression [213]. There is evidence that tumor-infiltrating immature DCs also promote angiogenesis by secreting VEGF and bFGF [214, 215], and that immature DCs participate in de novo formation of blood vessels or neovascularization in the tumor micro‐ environment. Under the influence of VEGF or angiogenic factors, immature mDCs transdifferentiate into endothelial-like DCs (called vascular leukocytes, VLC) expressing both DC and endothelial markers such as von Willebrand factor, VEGFR-2, and VE-cadherin (CD31) [216]. Remarkably, human VLC were able to form perfusable blood vessels when transplanted into immune-deficient mice, indicating a potential to support neovascularization.

In addition to macrophages and DCs, neutrophils, eosinophils and mast cells can contribute to tumor angiogenesis. Tumor-infiltrating neutrophils and mast cells secrete VEGF and MMP-9 [217]. Secretion of MMP-9 facilitates the availability of pro-angiogenic factors through a remodeling of the extracellular matrix. Interestingly, an increase in the number of neutrophils in the tumor microenvironment correlates with increased micro-vessel density [218]. The presence of mast cells in murine models of melanoma [219], squamous cell carcinoma [220] and pancreatic islet tumors [221] has been associated with increased angiogenesis. Mast cells are present in the tumor microenvironment prior to vessel formation [222], and they congre‐ gate near tumor-derived vessels [220, 223]. Since mast cells contain pro-angiogenic factors in their secretory granules, it has been hypothesized that secretion of these factors by mast cells promotes tumor angiogenesis [199]. There is indirect evidence that eosinophils promote tumor angiogenesis, as they have been detected in human tumors [199], and they are also a source of pro-angiogenic factors [224].

Myeloid-derived suppressor cells (MDSC) are immature myeloid progenitors of monocytes, neutrophils and DCs. As tumor-resident cells, MDSC facilitate tumor progression by their immunosuppressive properties. However, these cells may also have a role in promoting angiogenesis. Studies have shown that tumor angiogenesis is decreased when the MDSC chemo-attractant, BV8 (PROK2), was neutralized [225], and tumor blood vessel density increased when MDSC were co-injected with colorectal cancer cells into mice [226]. MDSC also secrete matrix metalloproteases.

#### **6.2. T cells**

and murine tumor models have shown that the presence of tumor associated macrophages (TAMs) correlates with enhanced vessel density, tumor progression and metastasis [199]. During inflammation, monocytes are attracted by chemo-attractants to damaged tissues, where they differentiate into macrophages. These macrophages are phenotypically plastic, and depending on the environmental signals within wounds or tumors, they differentiate into functional subsets with different activation states [200]. At sites of inflammation, interferon gamma (IFN-γ) and TNF-α facilitate macrophage differentiation into cytotoxic "M1" cells that secrete pro-inflammatory cytokines (TNF-α, IL-1, IL-6, IL-12 and IL-23). M1 macrophages are phagocytic, sustain tissue inflammation, and promote a T helper-1 (TH1) anti-tumor immune response [201, 202]. Alternatively, when induced in the presence of IL-4, IL-13, IL-10 and TGFβ, macrophages differentiate into "M2" cells that secrete IL-10 and participate in tissue remodeling and immune suppression. M2 macrophages also produce angiogenic factors. These factors include VEGF, placental growth factor (PIGF), arginase and the Tie2 angiopoietin cell surface receptor [203, 204]. Monocytes/macrophages that express Tie2 (referred to as TEMS) are a source of VEGF and have been found in human and murine spontaneous and orthotopic tumors [205, 206]. TEMS reside in close proximity to the tumor vasculature and are

Using a physiologic model of skin wound repair, CCR2hi/VEGF-expressing macrophages were shown to initiate vascular sprouts during the early stages of tissue repair [195]. During the early repair period, macrophages with both M1 and M2 gene profiles were present, but cells with a M2 phenotype predominated during the later stages of repair. Results of this study imply that VEGF-expressing macrophages initiate wound-tissue vascularization. The presence of both M1 and M2 macrophages during early repair may be a reflection of the presence of M1 cells during the resolution of inflammation and the presence of M2 cells associated with initiation of an immune-suppressive tissue repair program. The data obtained from this physiologic model of wound healing parallels the process that occurs within the tumor microenvironment. In tumors, M1 cells are often found in sites of chronic inflamma‐ tion, simulating the inflammatory stage of wound healing, while M2 cells are associated with vascularization, immune suppression and tissue repair [207]. However, this paradigm of tissue repair is not absolute for tumors as demonstrated by an aggressive inflammatory form of breast cancer where there is up-regulation of both VEGF and the IL-6 pro-inflammatory

Dendritic cells (DCs) are professional antigen-presenting cells by nature, and they are inti‐ mately involved in the activation of tumor-specific T cells. DCs originate from CD34+ bone marrow precursors, and they differentiate into heterogeneous subsets due to differentiation plasticity. Within this heterogeneity there are functionally 2 major distinct subtypes of dendritic cells classified as myeloid DC (mDCs) and plasmacytoid DC (pDCs). Plasmacytoid DCs produce anti-angiogenic type I interferons [209], and mDC have the capacity to function as potent antigen-presenting cells. The maturation state of DCs adds another layer of com‐ plexity as immature DCs have high endocytic activity, but they lack expression of the costimulatory molecules that are necessary for T cell activation. Based on these properties, immature DCs are considered as immune-tolerogenic rather than immune-activating. VEGF affects the development and maturation of DCs. Binding of VEGF to the VEGFR-1 receptor on

possibly recruited by angiopoietin-2-expressing endothelial cells [199].

(M1) cytokine [208].

108 Research Directions in Tumor Angiogenesis

Cancer patients have a decrease in immune function that can be attributed in part to the tolerogenic differentiation of innate immune cells. However, there is evidence that VEGF also interferes with T cell development. Effective T cells have the ability to specifically recognize and kill tumors. In fact, the most significant predictor of survival from solid tumors is the presence of CD8 T cells in the tumor core and invasive margins [227]. In vivo administration of a supraphysiologic concentration of recombinant VEGF blocks bone marrow-derived progenitor cells from seeding in the thymus reducing T cell production [228]. These data imply that VEGF secreted from tumors or cells in the tumor microenvironment may contribute to a systemic decrease in T cells.

As previously described, cells of the innate immune system have an important pro-angiogenic role in the tumor microenvironment. However, cells that mediate adaptive immunity also contribute to angiogenesis. The tumor microenvironment is immune suppressive due to the presence of multiple tolerogenic mechanisms. One of the most potent immune suppressive mediators arises through the differentiation of CD4+ CD25- T cells into CD4+ CD25+ FoxP3+ regulatory T cells (Tregs) [229]. In addition to promoting tumorigenesis through immunosup‐ pression, there is evidence that Tregs contribute to tumor angiogenesis. Accumulation of Tregs in the tumor microenvironment is associated with increased angiogenesis and increased microvessel density [230]. CD4+ CD25+ Tregs secrete higher amounts of VEGF than CD4+ CD25- CD4 T cells, and when Tregs are depleted from the tumor microenvironment there is less VEGF and angiogenesis present [231]. Therefore, elimination of Tregs as a form of tumor immunotherapy may provide two benefits: a release from immune suppression and decreased angiogenesis.

TAMs expressing CD68 and IL-6, as well as IL-6-expressing CD33+

is recognized as key factor in the failure of effective anti-tumor immunity.

in progress.

survival of treated patients [232].

in the bone marrow, are indicators of poor survival [234]. Expression of inflammationassociated genes (IL-6, IL-6R, IL-10 and TGFβ1) also correlates with a poor 5-year event-free survival [235]. In search of new therapies aimed at targeting these high-risk factors, both preclinical and clinical studies designed to test either immune or anti-angiogenic therapies are

The goal of immune therapy is to summon immune effector cells to the tumor microenviron‐ ment and promote activation against the tumor. Much of the effort in cancer immunotherapy has focused on the activation of effector T cells, but in order to achieve an effective anti-tumor T cell response, tumor antigen, mature antigen-presenting DCs and tumor antigen-reactive T cells must be present [236]. Autologous or allogeneic whole tumor cell vaccines, tumor lysate vaccines, antigen–primed DC vaccines, and induction of endogenous tumor cell lysis are all strategies to provide a source of tumor antigens. Agents such as GM-CSF, toll-like receptor (TLR) ligands, or agonistic anti-CD40 antibody are administered to promote DC migration and maturation. Blockade of T cell inhibitory receptors with anti-CTLA-4 or anti-PD-1 antibodies, administration of T cell survival chemokines (IL-2, IL-12 or IL-15), Treg blockade, or adoptive transfer of immune cells are therapies that can promote T cell activation. Recent attention has been directed to targeting immune suppressive factors in the tumor microenvironment using molecular inhibitors or antibodies. As previously noted, the functional complexity of immune cells, and their modulation by the tumor microenvironment to become immune suppressive,

For NB, the efficacy of several different immune therapies has been examined in both preclin‐ ical murine tumor models (Table 1) and clinical trials (Table 2). Preclinical therapies include whole-cell tumor vaccines secreting immune activating cytokines (GM-CSF, IFN-γ, IL-21) or expressing immune co-stimulatory molecules (CD54 (ICAM), CD80, CD86, CD137L). In the N2a murine tumor model, our laboratory and others have shown that depletion of Tregs using anti-CD4 or anti-CD25 mAbs increases vaccine-induced anti-tumor immunity [242, 246]. Another immune therapy designed to augment the number of anti-tumor T cells involves the adoptive cell transfer (ACT) of lymphocytes or T cells. For this therapy, autologous T cells are expanded ex vivo and returned to the patient after they have been activated against tumor antigens. Tumor-specific T cell receptors genetically attached to T cell activating domains (chimeric antigen receptors or CARS) have been transduced into T cells as a method to increase the anti-tumor cytolytic activity provided by ACT. In a preclinical model, adoptive transfer of T cells expressing an anti-GD2 CAR and the CCR2b chemokine receptor promoted trafficking of T cells to the tumor and resulted in tumor regression [245]. Of 11 patients enrolled in a clinical trial testing ACT of Epstein-Barr virus (EBV)-specific T cells expressing the anti-GD2 CAR, 2 patients had tumor regression and 2 patients experience stable disease (Table 2). One of the earliest pre-clinical strategies used a combination of anti-human GD2 antibody and IL-2 treatment in a human-mouse NB xenograft model [237]. After over a decade of study, a combination of anti-GD2, IL-2, GM-CSF and cis-retinoic acid, given in the context of autolo‐ gous hematopoietic stem cell transplantation, has now been shown to improve the event-free

CD14+

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

myelomonocytic cells

111

http://dx.doi.org/10.5772/55486

To summarize the pro-tumorigenic role of immune cells in the tumor microenvironment, there is now convincing evidence that suppressive immune cells can contribute to tumor angiogenesis. This angiogenic activity may be a reflection of the natural wound healing process, as wounds naturally switch from an immune-activating acute inflammatory environment to one that is immune suppressive and pro-angiogenic. As an unhealed wound, the tumor microenvironment may continually cycle between one of inflammation and immune suppression. An understanding of how immune cells, tumor cells, endothelial cells and other cells in the microenvironment contribute to immune suppression and angiogene‐ sis is key in order to devise therapies that can reprogram cells in this environment to be both immune activating and anti-angiogenic. Given the parallels between suppressed antitumor immunity and angiogenesis, therapies designed to relieve anti-tumor immune suppression may halt the angiogenic program, or vice versa. Studies to test synergy between immune-based and anti-angiogenic therapies have recently emerged; however, for NB, this field is in its infancy.

#### **6.3. Anti-angiogenic and immune therapies to treat NB**

The current standard of care for high-risk NB patients includes myeloablative chemotherapy followed by autologous hematopoietic stem cell transplant (AHSCT) and isotretinoin (13-*cis*retinoic acid). While these treatments have improved the survival of patients with high-risk disease, approximately 60% of these patients will relapse and die of their disease. Recently, immune therapy has been added to standard treatment protocols as a strategy to improve survival. Post-transplant treatment with an antibody that targets the highly expressed GD2 disialoganglioside on NB tumor cells, in combination with interleukin-2 (IL-2) and granulocyte macrophage colony-stimulating factor (GM-CSF), has resulted in a 2-year 20% increase in event-free survival compared to patients treated with standard therapy alone [232]. Despite this multimodal therapy, the mortality rate remains high for patients with metastatic NB. Indicators of disease associated with a poor prognosis include a paucity of stromal Schwann cells, *MYCN* amplification, expression of the TrkB receptor tyrosine kinase and a high vascular index [233]. Infiltration of immune cell subsets has also been associated with high-risk disease. TAMs expressing CD68 and IL-6, as well as IL-6-expressing CD33+ CD14+ myelomonocytic cells in the bone marrow, are indicators of poor survival [234]. Expression of inflammationassociated genes (IL-6, IL-6R, IL-10 and TGFβ1) also correlates with a poor 5-year event-free survival [235]. In search of new therapies aimed at targeting these high-risk factors, both preclinical and clinical studies designed to test either immune or anti-angiogenic therapies are in progress.

As previously described, cells of the innate immune system have an important pro-angiogenic role in the tumor microenvironment. However, cells that mediate adaptive immunity also contribute to angiogenesis. The tumor microenvironment is immune suppressive due to the presence of multiple tolerogenic mechanisms. One of the most potent immune suppressive

regulatory T cells (Tregs) [229]. In addition to promoting tumorigenesis through immunosup‐ pression, there is evidence that Tregs contribute to tumor angiogenesis. Accumulation of Tregs in the tumor microenvironment is associated with increased angiogenesis and increased

is less VEGF and angiogenesis present [231]. Therefore, elimination of Tregs as a form of tumor immunotherapy may provide two benefits: a release from immune suppression and decreased

To summarize the pro-tumorigenic role of immune cells in the tumor microenvironment, there is now convincing evidence that suppressive immune cells can contribute to tumor angiogenesis. This angiogenic activity may be a reflection of the natural wound healing process, as wounds naturally switch from an immune-activating acute inflammatory environment to one that is immune suppressive and pro-angiogenic. As an unhealed wound, the tumor microenvironment may continually cycle between one of inflammation and immune suppression. An understanding of how immune cells, tumor cells, endothelial cells and other cells in the microenvironment contribute to immune suppression and angiogene‐ sis is key in order to devise therapies that can reprogram cells in this environment to be both immune activating and anti-angiogenic. Given the parallels between suppressed antitumor immunity and angiogenesis, therapies designed to relieve anti-tumor immune suppression may halt the angiogenic program, or vice versa. Studies to test synergy between immune-based and anti-angiogenic therapies have recently emerged; however, for NB, this

The current standard of care for high-risk NB patients includes myeloablative chemotherapy followed by autologous hematopoietic stem cell transplant (AHSCT) and isotretinoin (13-*cis*retinoic acid). While these treatments have improved the survival of patients with high-risk disease, approximately 60% of these patients will relapse and die of their disease. Recently, immune therapy has been added to standard treatment protocols as a strategy to improve survival. Post-transplant treatment with an antibody that targets the highly expressed GD2 disialoganglioside on NB tumor cells, in combination with interleukin-2 (IL-2) and granulocyte macrophage colony-stimulating factor (GM-CSF), has resulted in a 2-year 20% increase in event-free survival compared to patients treated with standard therapy alone [232]. Despite this multimodal therapy, the mortality rate remains high for patients with metastatic NB. Indicators of disease associated with a poor prognosis include a paucity of stromal Schwann cells, *MYCN* amplification, expression of the TrkB receptor tyrosine kinase and a high vascular index [233]. Infiltration of immune cell subsets has also been associated with high-risk disease.

CD4 T cells, and when Tregs are depleted from the tumor microenvironment there

CD25+

CD25-

T cells into CD4+

Tregs secrete higher amounts of VEGF than

CD25+

FoxP3+

mediators arises through the differentiation of CD4+

**6.3. Anti-angiogenic and immune therapies to treat NB**

microvessel density [230]. CD4+

110 Research Directions in Tumor Angiogenesis

CD4+

CD25-

angiogenesis.

field is in its infancy.

The goal of immune therapy is to summon immune effector cells to the tumor microenviron‐ ment and promote activation against the tumor. Much of the effort in cancer immunotherapy has focused on the activation of effector T cells, but in order to achieve an effective anti-tumor T cell response, tumor antigen, mature antigen-presenting DCs and tumor antigen-reactive T cells must be present [236]. Autologous or allogeneic whole tumor cell vaccines, tumor lysate vaccines, antigen–primed DC vaccines, and induction of endogenous tumor cell lysis are all strategies to provide a source of tumor antigens. Agents such as GM-CSF, toll-like receptor (TLR) ligands, or agonistic anti-CD40 antibody are administered to promote DC migration and maturation. Blockade of T cell inhibitory receptors with anti-CTLA-4 or anti-PD-1 antibodies, administration of T cell survival chemokines (IL-2, IL-12 or IL-15), Treg blockade, or adoptive transfer of immune cells are therapies that can promote T cell activation. Recent attention has been directed to targeting immune suppressive factors in the tumor microenvironment using molecular inhibitors or antibodies. As previously noted, the functional complexity of immune cells, and their modulation by the tumor microenvironment to become immune suppressive, is recognized as key factor in the failure of effective anti-tumor immunity.

For NB, the efficacy of several different immune therapies has been examined in both preclin‐ ical murine tumor models (Table 1) and clinical trials (Table 2). Preclinical therapies include whole-cell tumor vaccines secreting immune activating cytokines (GM-CSF, IFN-γ, IL-21) or expressing immune co-stimulatory molecules (CD54 (ICAM), CD80, CD86, CD137L). In the N2a murine tumor model, our laboratory and others have shown that depletion of Tregs using anti-CD4 or anti-CD25 mAbs increases vaccine-induced anti-tumor immunity [242, 246]. Another immune therapy designed to augment the number of anti-tumor T cells involves the adoptive cell transfer (ACT) of lymphocytes or T cells. For this therapy, autologous T cells are expanded ex vivo and returned to the patient after they have been activated against tumor antigens. Tumor-specific T cell receptors genetically attached to T cell activating domains (chimeric antigen receptors or CARS) have been transduced into T cells as a method to increase the anti-tumor cytolytic activity provided by ACT. In a preclinical model, adoptive transfer of T cells expressing an anti-GD2 CAR and the CCR2b chemokine receptor promoted trafficking of T cells to the tumor and resulted in tumor regression [245]. Of 11 patients enrolled in a clinical trial testing ACT of Epstein-Barr virus (EBV)-specific T cells expressing the anti-GD2 CAR, 2 patients had tumor regression and 2 patients experience stable disease (Table 2). One of the earliest pre-clinical strategies used a combination of anti-human GD2 antibody and IL-2 treatment in a human-mouse NB xenograft model [237]. After over a decade of study, a combination of anti-GD2, IL-2, GM-CSF and cis-retinoic acid, given in the context of autolo‐ gous hematopoietic stem cell transplantation, has now been shown to improve the event-free survival of treated patients [232].


In addition to infiltration of specific immune cellular subsets, a high NB vascular index also correlates with aggressive disease [255]. High expression of pro-angiogenic factors (HIF-2α, VEGF-A, bFGF, TGF-α, PDGF-A, angiopoietin-2, MMP-2, MMP-9, and integrins αvβ3 and αvβ5) as well as down-regulation the endothelial cell growth inhibitor, activin A, have been reported in advance stage or high risk NB [256-260]. Given these findings, studies have been designed to target angiogenesis with (1) Agents that directly target endothelial cells (endosta‐ tin, thrombospondin-1, thalidomide), (2) Agents that indirectly block the production or activity of pro-angiogenic molecules (antibodies to VEGF or VEGF receptors), or (3) Or agents that target both endothelial and tumor cells (receptor tyrosine kinase inhibitors (RTK) and inter‐ feron alpha (IFN-α)). For a complete review, refer to [233]. TPN-470 is an agent that inhibits the proliferation of endothelial cells by inactivating methionine aminopeptidase; however, biochemical instability may limit its application [261]. Fenretinide (N-(4-hydroxyphenyl or 4HPR) is a synthetic analog of retinoic acid that represses endothelial cell proliferation and is

**Therapy Response Reference**

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

Of 10 patients, 1CR, 1PR and 3SD; 4 patients

Of 21 patients with relapsed or refractory disease: 2CR 1PR; increased NK cytolytic

Of 27 patients there were no CR or PR, 3 patients had anti-tumor activity

In patients with minimal disease there was a rise in circulating CD4 and CD8 T cells specific

Of 23 patients with non-bulky tumor there

Of 11 patients 2CR and 2SD [253]

[248]

http://dx.doi.org/10.5772/55486

113

[248]

[249]

[250]

[252]

[254]

[232]

IL-2 No objective tumor response. [247]

with anti-tumor CTLs

line; 1PR, 7SD and 4PD

Anti-LI-CAM CAR Of 6 patients, 1 with limited disease had a PR [251]

for autologous tumor

Improved event-free survival Incorporated into standard of care

were 5CR

Allogeneic NB secreting IL-2 No cytotoxicity against the vaccinating cell

activity

Autologous NB transfected to produce

Allogeneic NB secreting IL-2 and

Anti-GD2 (hu14.18)/IL-2 fusion

Autologous IL-2-secreting tumor

ACT of EBV-specific T cells transduced

Anti-GD2 (ch14.18) GM-CSF, IL-2 and

**Table 2.** Immunotherapies for neuroblastoma (clinical)

Anti-GD2 (hu14.18)/IL-2 fusion

cis-retinoic acid following myeloablative conditioning and

IL-2

lymphotactin

protein

vaccine

protein

AHSCT

with anti-GD2 CAR

**Table 1.** Immunotherapies for neuroblastoma (pre-clinical)


**Model Therapy Response References**

Suppressed dissemination of human SK-N-AS NB injected under the splenic capsule

against N2a and prolonged survival of N2a-bearing mice

Protection from AGN2a tumor

Enhanced protection to AGN2a

Protective anti-tumor immunity and detection of survivin-

tumor site and reduced tumor

Induced tyrosine hydroxylasespecific CTLs and eradicated

Reduction in growth of huNB xenograft and increased trafficking of CD2 CAR CCR2b T

Reduced dissemination of intravenous inoculated N2a

Increase in survival of AGN2a-

Regression of tumor in retroperitoneal inoculated N2a-

bearing mice

challenge

tumor challenge

specific CTLs

growth

primary tumor

cells to the tumor

tumor.

bearing mice

[237]

[238]

[239]

[240]

[241]

[242]

[243]

[244]

[245]

[242]

[246]

Human/mouse chimeric anti-GD2 and IL-2 (ch14.8-IL-2) plus IL-2-activated human PBMCs

N2a syngeneic mouse IL-2-secreting N2a tumor vaccine Induced protective immunity

secreting N2a tumor vaccine

Anti-CD25 mAb followed by AGN2a CD80, CD86-expressing

NXS2 syngeneic mouse Survivin DNA minigene vaccine Increase in CD8 T cells at the

tumor vaccine

minigene vaccine

GD2 CAR and CCR2b

vaccine and anti-CD4 mAb

following TBI and HSCT and AGN2a tumor vaccine expressing CD54, CD80, CD86, and CD137L

vaccine

AGN2a tumor vaccine transiently transfected to express CD54, CD80, CD86 and CD137L

N2a syngeneic mouse GM-CSF or GM-CSF and IFN-γ

N2a syngeneic mouse IL-21-secreting AGN2a tumor

NXS2 Tyrosine hydroxylase DNA

SCID ACT of T cells expressing anti-

N2a IL-21-secreting N2a tumor

AGN2a (N2a subclone) ACT of CD25-depleted T cells

**Table 1.** Immunotherapies for neuroblastoma (pre-clinical)

SCID (immune deficient)

112 Research Directions in Tumor Angiogenesis

AGN2a (N2a subclone) syngeneic mouse

AGN2a (N2a subclone) syngeneic mouse

mouse

In addition to infiltration of specific immune cellular subsets, a high NB vascular index also correlates with aggressive disease [255]. High expression of pro-angiogenic factors (HIF-2α, VEGF-A, bFGF, TGF-α, PDGF-A, angiopoietin-2, MMP-2, MMP-9, and integrins αvβ3 and αvβ5) as well as down-regulation the endothelial cell growth inhibitor, activin A, have been reported in advance stage or high risk NB [256-260]. Given these findings, studies have been designed to target angiogenesis with (1) Agents that directly target endothelial cells (endosta‐ tin, thrombospondin-1, thalidomide), (2) Agents that indirectly block the production or activity of pro-angiogenic molecules (antibodies to VEGF or VEGF receptors), or (3) Or agents that target both endothelial and tumor cells (receptor tyrosine kinase inhibitors (RTK) and inter‐ feron alpha (IFN-α)). For a complete review, refer to [233]. TPN-470 is an agent that inhibits the proliferation of endothelial cells by inactivating methionine aminopeptidase; however, biochemical instability may limit its application [261]. Fenretinide (N-(4-hydroxyphenyl or 4HPR) is a synthetic analog of retinoic acid that represses endothelial cell proliferation and is associated with a reduction in VEGFR-2 and FGF-2R-2 receptor expression on endothelial cells [262]. Retinoids are promising anti-tumor agents because they also induce the differentiation of NB cells and promote the survival of tumor-reactive CD8 T cells [233, 263]. As mentioned previously, the isotretinoin retinoid has recently been added to standard care protocols for the treatment of refractory NB. Bevacizumab is an anti-VEGF monoclonal antibody that binds to VEGF receptors, blocking signaling through these receptors. A VEGF Trap decoy is another agent used to block VEGFR. This agent is composed of VEGFR-1 and VEGFR-2 segments fused to an IgG1 molecule [264]. The receptor tyrosine kinase inhibitors, SUGEN, axitinib, imatinib mesylate, sunitinib, sorafenib and ZD6474 differentially target various receptors including PDGFR, VEGFR, the stem cell factor receptor (c-KIT), the FMS-related tyrosine kinase 3, epidermal growth factor receptor (EGFR) and RET on endothelial and tumor cells [265, 266]. Preclinical studies testing the effects of these agents on human-mouse NB xenografts have been performed. For these studies, human NB cell lines were grafted (orthotopically or subcutane‐ ously) into immune compromised mice. Tumor growth, apoptosis of tumor and endothelial cells, and tumor vascularization were examined after treatment with the anti-angiogenic agent(s). It is important to note that these mouse models cannot accurately assess impact of the immune system on tumor growth because they lack an intact human immune system. A summary of NB anti-angiogenic preclinical studies is shown in Table 3.

**Model Therapy Response Reference**

ZD6474 RTK Inhibited tumor growth and

Sunitinib and sorafenib Inhibited angiogenesis and

Axitinib Tumor growth delay, but no

Since the infiltration of immune-suppressive cells and a high vascular index both correlate with aggressive NB, interventions designed to reverse both immune suppression and angio‐ genesis represent promising treatment approaches. However, studies testing such combina‐ tion therapies for the treatment of NB or other cancers are relatively scarce (Table 4). One ongoing phase I NB trial combines immune therapy with anti-angiogenic therapy. For this study, an iodine 131I-conjugated anti-GD2 monoclonal antibody is administered in combination with bevacizumab [276]. Combination therapies for other cancers (renal cell carcinoma) have included treatment with bevacizumab and IFN-α [277] or IL-2 [278]. Surprisingly, combina‐ tions of anti-tumor immune and anti-angiogenic therapies have not been tested preclinically in NB, and there are relatively few preclinical studies in other tumor models (Table 4). However, there is evidence that these therapies can act synergistically to elicit anti-tumor responses: (1) Combinations of cytokine-secreting tumor cell-based vaccines and agents that block VEGFR signaling were tested in melanoma and breast cancer models; (2) Immuneactivating cytokines, endostatin, and pigment epithelium-derived factor were tested in a hepatocellular carcinoma model; (3) Adoptive transfer of tumor-antigen specific T cells with anti-VEGF and IL-2 was tested in a melanoma tumor model; and (4) Vaccination with a viral vector encoding immune stimulatory molecules and treatment with sunitinib was tested in a

While it is almost certain that a combination of therapies (chemotherapy, radiation, targeted therapies, immune and/or anti-angiogenic) will be required to mount an effective anti-tumor response, the appropriate combination will likely vary among the different cancer types. For

Inhibited growth of NB xenografts and stabilized the growth of large tumors

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

induced endothelial cell

Synergistic anti-tumor effect [274]

apoptosis

tumor growth

regression

[272]

http://dx.doi.org/10.5772/55486

115

[266]

[273]

[275]

Combined treatment with a thrombospondin-1 peptide and valproic acid histone deacetylase inhibitor

Bevacizumab and cyclophosphamide

**Table 3.** Neuroblastoma pre-clinical anti-angiogenic therapies

colon cancer transgenic mouse model.



**Table 3.** Neuroblastoma pre-clinical anti-angiogenic therapies

associated with a reduction in VEGFR-2 and FGF-2R-2 receptor expression on endothelial cells [262]. Retinoids are promising anti-tumor agents because they also induce the differentiation of NB cells and promote the survival of tumor-reactive CD8 T cells [233, 263]. As mentioned previously, the isotretinoin retinoid has recently been added to standard care protocols for the treatment of refractory NB. Bevacizumab is an anti-VEGF monoclonal antibody that binds to VEGF receptors, blocking signaling through these receptors. A VEGF Trap decoy is another agent used to block VEGFR. This agent is composed of VEGFR-1 and VEGFR-2 segments fused to an IgG1 molecule [264]. The receptor tyrosine kinase inhibitors, SUGEN, axitinib, imatinib mesylate, sunitinib, sorafenib and ZD6474 differentially target various receptors including PDGFR, VEGFR, the stem cell factor receptor (c-KIT), the FMS-related tyrosine kinase 3, epidermal growth factor receptor (EGFR) and RET on endothelial and tumor cells [265, 266]. Preclinical studies testing the effects of these agents on human-mouse NB xenografts have been performed. For these studies, human NB cell lines were grafted (orthotopically or subcutane‐ ously) into immune compromised mice. Tumor growth, apoptosis of tumor and endothelial cells, and tumor vascularization were examined after treatment with the anti-angiogenic agent(s). It is important to note that these mouse models cannot accurately assess impact of the immune system on tumor growth because they lack an intact human immune system. A

**Model Therapy Response Reference**

proliferation and migration

endothelial cell proliferation

suppressed PDGFR and c-Kit

tumor growth and increased

and angiogenesis

formation and vessel

remodeling

phosphorylation

apoptosis of NB

vessel density, tumor growth and angiogenesis [267]

[268]

[264]

[269]

[265]

[270, 271]

TNP-470 (AMG-1470) Inhibited endothelial cell

Fenretinide Prevented the induction of

High dose VEGF Trap decoy Disrupted early vessel

Imatinib mesylate (Gleevec) Inhibited NB growth and

SUGEN (SU11657) Reduced angiogenesis,

Bevacizumab Decrease in tumor micro-

summary of NB anti-angiogenic preclinical studies is shown in Table 3.

NB xenografts into

114 Research Directions in Tumor Angiogenesis

compromised (Nude, SCID, NOD-SCID)

immune-

Since the infiltration of immune-suppressive cells and a high vascular index both correlate with aggressive NB, interventions designed to reverse both immune suppression and angio‐ genesis represent promising treatment approaches. However, studies testing such combina‐ tion therapies for the treatment of NB or other cancers are relatively scarce (Table 4). One ongoing phase I NB trial combines immune therapy with anti-angiogenic therapy. For this study, an iodine 131I-conjugated anti-GD2 monoclonal antibody is administered in combination with bevacizumab [276]. Combination therapies for other cancers (renal cell carcinoma) have included treatment with bevacizumab and IFN-α [277] or IL-2 [278]. Surprisingly, combina‐ tions of anti-tumor immune and anti-angiogenic therapies have not been tested preclinically in NB, and there are relatively few preclinical studies in other tumor models (Table 4). However, there is evidence that these therapies can act synergistically to elicit anti-tumor responses: (1) Combinations of cytokine-secreting tumor cell-based vaccines and agents that block VEGFR signaling were tested in melanoma and breast cancer models; (2) Immuneactivating cytokines, endostatin, and pigment epithelium-derived factor were tested in a hepatocellular carcinoma model; (3) Adoptive transfer of tumor-antigen specific T cells with anti-VEGF and IL-2 was tested in a melanoma tumor model; and (4) Vaccination with a viral vector encoding immune stimulatory molecules and treatment with sunitinib was tested in a colon cancer transgenic mouse model.

While it is almost certain that a combination of therapies (chemotherapy, radiation, targeted therapies, immune and/or anti-angiogenic) will be required to mount an effective anti-tumor response, the appropriate combination will likely vary among the different cancer types. For


NB, the ideal combination is yet to be determined. Bevacizumab (Avastin®) is FDA-approved for other solid tumors and represents a promising addition to augment immune and chemo‐ therapeutic anti-tumor efficacy for NB. Receptor tyrosine kinase inhibitors, including imatinib mesylate (Gleevec®), sorafenib (Nexavar®), and sunitinib (Sutent®) have shown some antitumor efficacy in NB preclinical studies, and these agents are also FDA-approved for the treatment of some solid tumors. The results from studies using combined anti-angiogenic and anti-tumor immune therapy are encouraging and offer a new avenue to explore more effective eradication of NB and other cancers. Given the multiple types of immunotherapy and antiangiogenic agents, as well as different platforms of delivery, more studies using combinations

Endothelial and Accessory Cell Interactions in Neuroblastoma Tumor Microenvironment

http://dx.doi.org/10.5772/55486

117

NB is an enigmatic childhood cancer that has developmental origins in NC cell lineage. MYCN, ALK and TRKA are the key target genes for NB prognosis. Extracellular matrix and cell adhesion molecules that participate in interactions and signaling across endothelial cells, immune and Schwann cells in the NB microenvironment have potential for targeting. The future for NB biology and therapy looks bright and multiple modalities affecting various cell

ALK: anaplastic lymphoma kinase; ANS: autonomic nervous system; Ang-2: angiopoie‐ tin-2; ACT: adoptive cell transfer; BMP: bone morphogenic protein; BDNF: brain-derived neurotrophic factor; bFGF: basic fibroblast growth factor; BrdU: 5-bromo-2'-deoxyuridine; CNS: central nervous system; CNTF: ciliary neurotrophic factor; CAFs: cancer-associated fibroblasts; CAM: cell adhesion molecules; CAR: chimeric antigen receptor; CR: complete response; DRG: dorsal root ganglia; DC: dendritic cell; EMT: epithelial to mesenchymal transition; ECM: extracellular matrix; EBV: Epstein-Barr virus; EGFR: epidermal growth factor receptor; FDA: Federal Drug Administration; FGF: fibroblast growth factor; GNB: ganglio‐ neuroblastoma; GN: ganglioneuroma; GFAP: glial fibrillary acidic protein; GJIC: gap junction intracellular communication; GM-CSF: granulocyte macrophage colony-stimulating factor; HGF: hepatocyte growth factor; hMSC: human mesenchymal stem cells; HIF: hypoxia inducible factor; IFN-γ: interferon gamma; IFN-α: interferon alpha; LIF: leukemia inhibito‐ ry factor; MYCN: v-myc myelocytomatosis viral-related protein; MAPs: microtubule associated proteins; MBP: myelin basic protein; MKI: mitosis-karyorrhexis index; MMP: metalloproteinase; mDC: myeloid dendritic cell; MDSC: myeloid-derived suppressor cell; NB: neuroblastoma; NC: neural crest; NCSC: neural crest-derived stem cell; NRG-1 neuregu‐ lin-1; NF: neurofilament; NCAM: neural cell adhesion molecule; NGF: nerve growth factor; NT-3: neurotropin-3; PBMC: peripheral blood mononuclear cell; PNS: peripheral nervous

of these therapies are warranted.

types and signals in NB microenvironment are anticipated.

**7. Conclusion**

**Nomenclature**

**Table 4.** Combined immune and anti-angiogenic therapy

NB, the ideal combination is yet to be determined. Bevacizumab (Avastin®) is FDA-approved for other solid tumors and represents a promising addition to augment immune and chemo‐ therapeutic anti-tumor efficacy for NB. Receptor tyrosine kinase inhibitors, including imatinib mesylate (Gleevec®), sorafenib (Nexavar®), and sunitinib (Sutent®) have shown some antitumor efficacy in NB preclinical studies, and these agents are also FDA-approved for the treatment of some solid tumors. The results from studies using combined anti-angiogenic and anti-tumor immune therapy are encouraging and offer a new avenue to explore more effective eradication of NB and other cancers. Given the multiple types of immunotherapy and antiangiogenic agents, as well as different platforms of delivery, more studies using combinations of these therapies are warranted.

#### **7. Conclusion**

**System Therapy Response Reference**

Renal cell carcinoma Bevacizumab and IL-2 No clinical benefit [278]

progression-free survival compared to IFN-α alone

A significant increase in tumor-free survival associated with a reduction in tumor-infiltrating immature DC and Tregs and an increase in effector

In non-tolerant WT syngeneic mice there was accelerated tumor regression associated with expansion of CD4 and CD8 T cells. In tolerant neu transgeneic mice there was delayed tumor growth, but

no regression

anti-VEGF was administered prior to irradiation and immune

therapy

Regression of large tumor (>8,000 mm2) required infusion of all vectors

There was a significant increase in survival in tumor-bearing mice when

Treatment with sunitinib prior to vaccination resulted in a significant reduction in tumor growth

T cells

In progress Clinicaltrials.gov

[277]

[279]

[280]

[281]

[282]

[283]

131I-labeled anti-GD2 mAb and bevacizumab

Renal cell carcinoma Bevacizumab and IFN-α Significant increase in

vaccine with a recombinant adenoassociated virus vector expressing a soluble VEGF

receptor

CSF secreting tumor vaccine in combination with anti-VEGFR-1, DC101 mAb

Adenovirus vectors encoding IL-12, GM-CSF, endostatin and pigment epithelium-derived factor

Pmel-1 transgenic T cells with anti-VEGF, a tumor vaccine expressing melanoma tumor antigen, gp100, and IL-2 after non-myeloablative total body irradiation

Sunitinib plus primary vaccination with CD80, ICAM1, LFA-3 and CEA expressing vaccinia virus and a boost with fowlpox

virus

**Table 4.** Combined immune and anti-angiogenic therapy

B16F10 melanoma GM-CSF secreting tumor

Her2/neu breast cancer Her2/neu expressing GM-

B-16 melanoma Adoptive transfer of

**Neuroblastoma clinical**

116 Research Directions in Tumor Angiogenesis

**Clinical trials in other tumor models**

**Preclinical Studies in other tumor models**

Woodchuck

MC38-CEA colon carcinoma in CEAtransgenic mice

hepatocellular carcinoma

**trial**

NB is an enigmatic childhood cancer that has developmental origins in NC cell lineage. MYCN, ALK and TRKA are the key target genes for NB prognosis. Extracellular matrix and cell adhesion molecules that participate in interactions and signaling across endothelial cells, immune and Schwann cells in the NB microenvironment have potential for targeting. The future for NB biology and therapy looks bright and multiple modalities affecting various cell types and signals in NB microenvironment are anticipated.

#### **Nomenclature**

ALK: anaplastic lymphoma kinase; ANS: autonomic nervous system; Ang-2: angiopoie‐ tin-2; ACT: adoptive cell transfer; BMP: bone morphogenic protein; BDNF: brain-derived neurotrophic factor; bFGF: basic fibroblast growth factor; BrdU: 5-bromo-2'-deoxyuridine; CNS: central nervous system; CNTF: ciliary neurotrophic factor; CAFs: cancer-associated fibroblasts; CAM: cell adhesion molecules; CAR: chimeric antigen receptor; CR: complete response; DRG: dorsal root ganglia; DC: dendritic cell; EMT: epithelial to mesenchymal transition; ECM: extracellular matrix; EBV: Epstein-Barr virus; EGFR: epidermal growth factor receptor; FDA: Federal Drug Administration; FGF: fibroblast growth factor; GNB: ganglio‐ neuroblastoma; GN: ganglioneuroma; GFAP: glial fibrillary acidic protein; GJIC: gap junction intracellular communication; GM-CSF: granulocyte macrophage colony-stimulating factor; HGF: hepatocyte growth factor; hMSC: human mesenchymal stem cells; HIF: hypoxia inducible factor; IFN-γ: interferon gamma; IFN-α: interferon alpha; LIF: leukemia inhibito‐ ry factor; MYCN: v-myc myelocytomatosis viral-related protein; MAPs: microtubule associated proteins; MBP: myelin basic protein; MKI: mitosis-karyorrhexis index; MMP: metalloproteinase; mDC: myeloid dendritic cell; MDSC: myeloid-derived suppressor cell; NB: neuroblastoma; NC: neural crest; NCSC: neural crest-derived stem cell; NRG-1 neuregu‐ lin-1; NF: neurofilament; NCAM: neural cell adhesion molecule; NGF: nerve growth factor; NT-3: neurotropin-3; PBMC: peripheral blood mononuclear cell; PNS: peripheral nervous system; PSNS: parasympathetic nervous system; PEDF: pigment epithelium-derived factor; PDGF: platelet-derived growth factor; PolySia: polysialic acid; PIGF: placental growth factor; pDC: plasmacytoid dendritic cell; PD: progressive disease; PR: partial response; RA: retinoic acid; RTK: receptor tyrosine kinase; SA: sympathoadrenal; SPARC: Secreted Protein Acidic and Rich in Cysteine; SC: Schwann cell; SCP: Schwann cell precursor; SAE: severe adverse effect; SD: stable disease; Trk: tyrosine kinase receptor; TH: tyrosine hydroxylase; TGF-β : transforming growth factor-beta; TIMP-2: tissue inhibitor of metalloproteinase-2; TGF-α: transforming growth factor-alpha; TSP-1: thrombospondin-1; TAMs: tumor-associated macrophages; TNF-α: tumor necrosis factor-alpha; TH1: T helper-1; TEMS: Tie2 monocytes/ macrophages; Tregs: T regulatory cells; TLR: toll-like receptor; VLC: vascular leukocytes; VEGF: vascular endothelial growth factor

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#### **Acknowledgements**

We thank members of our respective labs for supporting our programs in developmental, vascular, tumor and immune biology. We also thank Marjorie Siebert Aylen Foundation for supporting RR's research on NB. MC-W is supported by NHLBI grant HL111582. BJ is supported by NIH grant CA100030. RR is supported by NHLBI grant HL090712, and seed funds from Children's Research Institute. QRM is supported by NHLBI grant HL108938 and start-up funds from MCW. We thank MACC fund and the BloodCenter of Wisconsin for their generous contribution to the research programs of the authors (BJ, AC and JG; MC-W).

#### **Author details**

Jill Gershan1 , Andrew Chan2 , Magdalena Chrzanowska-Wodnicka3 , Bryon Johnson4 , Qing Robert Miao5 and Ramani Ramchandran6\*

\*Address all correspondence to: rramchan@mcw.edu

1 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

2 School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong Kong

3 Blood Research Institute, Milwaukee, WI, USA

4 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

5 Divisions of Pediatric Surgery and Pediatric Pathology, Departments of Surgery and Path‐ ology, Medical College of Wisconsin, Milwaukee, WI, USA

6 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

#### **References**

system; PSNS: parasympathetic nervous system; PEDF: pigment epithelium-derived factor; PDGF: platelet-derived growth factor; PolySia: polysialic acid; PIGF: placental growth factor; pDC: plasmacytoid dendritic cell; PD: progressive disease; PR: partial response; RA: retinoic acid; RTK: receptor tyrosine kinase; SA: sympathoadrenal; SPARC: Secreted Protein Acidic and Rich in Cysteine; SC: Schwann cell; SCP: Schwann cell precursor; SAE: severe adverse effect; SD: stable disease; Trk: tyrosine kinase receptor; TH: tyrosine hydroxylase; TGF-β : transforming growth factor-beta; TIMP-2: tissue inhibitor of metalloproteinase-2; TGF-α: transforming growth factor-alpha; TSP-1: thrombospondin-1; TAMs: tumor-associated macrophages; TNF-α: tumor necrosis factor-alpha; TH1: T helper-1; TEMS: Tie2 monocytes/ macrophages; Tregs: T regulatory cells; TLR: toll-like receptor; VLC: vascular leukocytes;

We thank members of our respective labs for supporting our programs in developmental, vascular, tumor and immune biology. We also thank Marjorie Siebert Aylen Foundation for supporting RR's research on NB. MC-W is supported by NHLBI grant HL111582. BJ is supported by NIH grant CA100030. RR is supported by NHLBI grant HL090712, and seed funds from Children's Research Institute. QRM is supported by NHLBI grant HL108938 and start-up funds from MCW. We thank MACC fund and the BloodCenter of Wisconsin for their generous contribution to the research programs of the authors (BJ, AC and JG; MC-W).

, Magdalena Chrzanowska-Wodnicka3

2 School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, NT, Hong

5 Divisions of Pediatric Surgery and Pediatric Pathology, Departments of Surgery and Path‐

, Bryon Johnson4

,

VEGF: vascular endothelial growth factor

, Andrew Chan2

and Ramani Ramchandran6\*

1 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

4 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

6 Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA

\*Address all correspondence to: rramchan@mcw.edu

3 Blood Research Institute, Milwaukee, WI, USA

ology, Medical College of Wisconsin, Milwaukee, WI, USA

**Acknowledgements**

118 Research Directions in Tumor Angiogenesis

**Author details**

Qing Robert Miao5

Jill Gershan1

Kong


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**Chapter 6**

**T-Cadherin Stimulates Melanoma Cell Proliferation and**

Melanocytes are special pigment cells that reside predominantly in the skin and eyes. In the skin, melanocytes are located in the bottom layer (the stratum basale) of the skin's epidermis and in the hair follicles (Gray-Schopfer et al., 2001). Melanocytes produce melanins responsible for skin and hair color and perform protection function of the basal keratinocytes from ultraviolet light through synthesis and donation of melanin (Gray-Schopfer et al., 2001). Melanocytes maintain constant contact with the basal layer of the epidermis through direct interaction with basal keratinocytes and via secretion of soluble factors. Upon ultraviolet radiation, keratinocytes produce factors that control melanocyte proliferation, differentiation and motility (Gray-Schopfer et al., 2007). Melanocytes maintain during a lifetime a stable-ratio

Initially, cutaneous melanocytes originate from neural crest cells and migrate into the skin during embryonic development. Neural crest cells start their migration from the neural tube shortly after the closure of the neural tube. These cells migrate along several well-defined pathways in a ventral direction from the neural tube through the somites. As the epithelial somites undergo a transition to form the dermatome (presumptive dermis), myotome (pre‐ sumptive muscle), and sclerotome (presumptive vertebrae), most ventrally migrating neural crest cells invade the rostral half of each sclerotome and avoid the caudal (posterior) part of

> © 2013 Rubina et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Mesenchymal Stromal Cell Recruitment, but Inhibits**

**Angiogenesis in a Mouse Melanoma Model**

K. A. Rubina, E. I. Yurlova, V. Yu. Sysoeva, E. V. Semina, N. I. Kalinina, A. A. Poliakov, I. N. Mikhaylova, N. V. Andronova and

Additional information is available at the end of the chapter

of 1:5 with basal keratinocytes (Fitzpatrick et al., 1979).

H. M. Treshalina

**1. Introduction**

http://dx.doi.org/10.5772/53350


## **T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits Angiogenesis in a Mouse Melanoma Model**

K. A. Rubina, E. I. Yurlova, V. Yu. Sysoeva, E. V. Semina, N. I. Kalinina, A. A. Poliakov, I. N. Mikhaylova, N. V. Andronova and H. M. Treshalina

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53350

#### **1. Introduction**

[280] Manning EA, Ullman JG, Leatherman JM, Asquith JM, Hansen TR, Armstrong TD, Hicklin DJ, Jaffee EM, Emens LA: A vascular endothelial growth factor receptor-2 in‐ hibitor enhances antitumor immunity through an immune-based mechanism. Clini‐ cal cancer research : an official journal of the American Association for Cancer

[281] Huang KW, Wu HL, Lin HL, Liang PC, Chen PJ, Chen SH, Lee HI, Su PY, Wu WH, Lee PH et al: Combining antiangiogenic therapy with immunotherapy exerts better therapeutical effects on large tumors in a woodchuck hepatoma model. Proceedings of the National Academy of Sciences of the United States of America 2010; 107(33)

[282] Chinnasamy D, Yu Z, Kerkar SP, Zhang L, Morgan RA, Restifo NP, Rosenberg SA: Local delivery of interleukin-12 using T cells targeting VEGF receptor-2 eradicates multiple vascularized tumors in mice. Clinical cancer research : an official journal of

[283] Farsaci B, Higgins JP, Hodge JW: Consequence of dose scheduling of sunitinib on host immune response elements and vaccine combination therapy. International

the American Association for Cancer Research 2012; 18(6) 1672-1683.

journal of cancerJournal international du cancer 2012; 130(8) 1948-1959.

Research 2007; 13(13) 3951-3959.

14769-14774.

142 Research Directions in Tumor Angiogenesis

Melanocytes are special pigment cells that reside predominantly in the skin and eyes. In the skin, melanocytes are located in the bottom layer (the stratum basale) of the skin's epidermis and in the hair follicles (Gray-Schopfer et al., 2001). Melanocytes produce melanins responsible for skin and hair color and perform protection function of the basal keratinocytes from ultraviolet light through synthesis and donation of melanin (Gray-Schopfer et al., 2001). Melanocytes maintain constant contact with the basal layer of the epidermis through direct interaction with basal keratinocytes and via secretion of soluble factors. Upon ultraviolet radiation, keratinocytes produce factors that control melanocyte proliferation, differentiation and motility (Gray-Schopfer et al., 2007). Melanocytes maintain during a lifetime a stable-ratio of 1:5 with basal keratinocytes (Fitzpatrick et al., 1979).

Initially, cutaneous melanocytes originate from neural crest cells and migrate into the skin during embryonic development. Neural crest cells start their migration from the neural tube shortly after the closure of the neural tube. These cells migrate along several well-defined pathways in a ventral direction from the neural tube through the somites. As the epithelial somites undergo a transition to form the dermatome (presumptive dermis), myotome (pre‐ sumptive muscle), and sclerotome (presumptive vertebrae), most ventrally migrating neural crest cells invade the rostral half of each sclerotome and avoid the caudal (posterior) part of

© 2013 Rubina et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

each sclerotome (Rickmann et. al., 1985; Teillet et al., 1987; Serbedzua et al., 1989; Ranscht and Bronner, 1991).

er, 2005) and are involved in tissue and organ development durig embryogenesis and maintainance of the normal cell arrangement in the adult organism. The regulation of cadherin expression patterns and their activity at the neural crest-forming area plays a critical role in emigration of melanocyte precursors - neural crest cells from the neural tube (Nakagawa and

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits…

http://dx.doi.org/10.5772/53350

145

Cadherins play an important role in specific cell-cell adhesion, cell recognition and signaling (Angst et al., 2001; Gumbiner, 2005; Wheelock and Johnson, 2003). Cadherins are transmem‐ brane glycoproteins with their extracellular part responsible for homotypic binding between the neighboring cells, while intracellular part is involved in anchoring cadherins to the cytoske‐ leton. Cadherins interact with the cytoskeleton via catenins (alfa and beta catenins and p120) and form a multicomponent complex which also comprises a number of regulatory molecules such as protein tyrosine kinase, protein tyrosine phosphatase and small GTPases (Perez-Moreno et al., 2003; Sallee et al., 2006; Gumbiner, 2005; Vincent et al., 2004; Rubina et al., 2007). Disruption of cadherin adherent junctions and their dysfunction has been associated with tumor cell invasion and metastasis (Takeichi, 1993). In human carcinoma the loss of E-cadherin expression leads to dedifferentiation and increased invasiveness of carcinoma cells (Frixen et al., 1991). The change in the expression pattern of cadherins in melanocytes from E-cadherin, P-cadherin and desmoglein to N-cadherin is associated with melanocyte transformation and metastasis (Bonitsis et al., 2006). In normal skin, melanocytes project multiple extensions to keratinocytes and form cadherin adhesive contacts with the basal keratinocytes, which control maintain proliferation and correct positioning of melanocytes (Haas and Herlyng, 2005; Haass et al., 2005). E-cadherin is mainly responsible fot these cell-cell interaction. The loss of func‐ tional E-cadherin or its downregulation let melanocytes escape form keratinocyte control and correlates with high invasiveness and metastasis of the overlying melanoma cells (Hsu et al.,

Downregulation of E-cadherin and/or its dysfunction is one of the earliest steps in the development of metastases in cutaneous melanoma (Johnson, 1999). E-cadherin expression is detected in cultured melanocytes and naevus cells, while its expression is often lost in cultured melanoma cells from the primary tumors or metastasis (Danen et al., 1996). In the radial growth phase melanoma cells could retain expression of membrane E-cadherin (Sanders et al., 1999). It was even noted that there is a correlation between E-cadherin expression and the depth of the primary tumor (Andersen et al., 2004). Experiments on re-expression of E-cadherin using adenoviral transfer of full length E-cadherin cDNA showed the reduction in tumorigenicity and decrease in proliferation rate of melanoma cells (Hsu et al., 2000). The mechanism of Ecadherin transcriptional downregulation in melanomas involves gene silencing by methyla‐ tion or transrepression by the Snail protein from a superfamily of zinc-finger transcription factors (Tsutsumida et al., 2004). Thus it was suggested that E-cadherin could play an impor‐ tant role in the preventing the melanocytes transformation and limiting their proliferation

Catenins are a group of cadherin-associated molecules and they were also suggested to be involved in malignant transformation of melanocytes. The change in catenin expression pattern was found in melanocytic naevus and melanomas, where the expression of alfa and

Takeichi, 1998).

1996; Silye et al., 1998).

(cited in Bonitsis et al., 2006).

Originated from a population of highly motile neural crest progenitors, melanocytes protect basal keratinocytes, in the skin. At the same time, they could become precursors of the most dangerous form of cancer - melanoma. Skin cancer including the most frequently occurring forms such as basal cell carcinoma, squamous cell carcinoma and melanoma, is one of the most common human malignancies. Today melanoma is one the fastest growing malignancies. The high propensity of melanoma to form metastasis is the most important feature that distin‐ guishes melanoma from other types of skin cancers. According to the World Health Organi‐ zation, melanoma accounts for only 25% of skin cancers. However, it is the most dangerous form of skin cancer leading to high mortality. If diagnosed early it can be successfully removed by surgical resection and about 80% of cases are cured this way (Gray-Schopfer et al., 2001). However, at progressed metastatic stages melanoma is highly resistant to currently existing therapies and has a very poor prognosis. This area requires future research to understand melanoma biology and develop new therapeutic solutions.

Melanoma begins as a benign naevus but can quickly progress to the malignant stage (Bar-Eli., 1997; Luca and Bar-Eli., 1998). Herewith melanocytes start to proliferate and spread, which can be limited to the epidermis (junctional naevus), or the dermis (dermal naevus) or both (compound naevus) (Gray-Schopfer et al., 2001). Naevi can progress to the radial-growthphase (RGP) melanoma which is an intra-epidermal lesion with sporadic local microinvasion into the underlying derma. However, RGP melanoma can transform into the vertical-growth phase (VGP) melanoma with a higher invasive potential in which melanoma cells from tumor nodules or nests invade the underlying derma. Finally, melanoma can develop metastases after the vertical growth phase (Clark et al., 1984). Not all melanomas pass through each of these phases and can progress from isolated melanocytes or naevi, while both, RGP or VGP, can develop directly into metastatic malignant melanoma (Miller et al., 2006). Four main clinical subtypes of melanoma are described (Clark et al., 1984). Superficial spreading melanoma (SSM) is the most common form and it is associated with severe sunburns, especially at an early age (Ishihara et al., 2001; Gilchrest et al., 1999). In most cases, SSM is flat, with intraepidermal microinvasion, particularly at the edges of the lesion. Nodular melanoma comprises raised nodules and has almost no flat parts. Acral lentiginous melanoma (ALM) is not linked to UV exposure and is usually found on the palms of the hands, soles of the feet and in the nail bed (Kuchelmeister et al., 2000). Lentigo maligna appears to be flat and is associated with chronic sun exposure in elderly people.

#### **2. Cadherin-mediated adhesion in melanoma progression**

Tumor progression is characterized by uncontrolled cell proliferation, high invasive potential into surrounding tissue and metastasis to distant organs. It is believed that this is largely due to disruption or dysfunction of intercellular contacts (Hanahan and Weinberg, 2000). Cadher‐ ins comprise a large family of Ca2+-dependent adhesion molecules (Angst et al., 2001; Gumbin‐ er, 2005) and are involved in tissue and organ development durig embryogenesis and maintainance of the normal cell arrangement in the adult organism. The regulation of cadherin expression patterns and their activity at the neural crest-forming area plays a critical role in emigration of melanocyte precursors - neural crest cells from the neural tube (Nakagawa and Takeichi, 1998).

each sclerotome (Rickmann et. al., 1985; Teillet et al., 1987; Serbedzua et al., 1989; Ranscht and

Originated from a population of highly motile neural crest progenitors, melanocytes protect basal keratinocytes, in the skin. At the same time, they could become precursors of the most dangerous form of cancer - melanoma. Skin cancer including the most frequently occurring forms such as basal cell carcinoma, squamous cell carcinoma and melanoma, is one of the most common human malignancies. Today melanoma is one the fastest growing malignancies. The high propensity of melanoma to form metastasis is the most important feature that distin‐ guishes melanoma from other types of skin cancers. According to the World Health Organi‐ zation, melanoma accounts for only 25% of skin cancers. However, it is the most dangerous form of skin cancer leading to high mortality. If diagnosed early it can be successfully removed by surgical resection and about 80% of cases are cured this way (Gray-Schopfer et al., 2001). However, at progressed metastatic stages melanoma is highly resistant to currently existing therapies and has a very poor prognosis. This area requires future research to understand

Melanoma begins as a benign naevus but can quickly progress to the malignant stage (Bar-Eli., 1997; Luca and Bar-Eli., 1998). Herewith melanocytes start to proliferate and spread, which can be limited to the epidermis (junctional naevus), or the dermis (dermal naevus) or both (compound naevus) (Gray-Schopfer et al., 2001). Naevi can progress to the radial-growthphase (RGP) melanoma which is an intra-epidermal lesion with sporadic local microinvasion into the underlying derma. However, RGP melanoma can transform into the vertical-growth phase (VGP) melanoma with a higher invasive potential in which melanoma cells from tumor nodules or nests invade the underlying derma. Finally, melanoma can develop metastases after the vertical growth phase (Clark et al., 1984). Not all melanomas pass through each of these phases and can progress from isolated melanocytes or naevi, while both, RGP or VGP, can develop directly into metastatic malignant melanoma (Miller et al., 2006). Four main clinical subtypes of melanoma are described (Clark et al., 1984). Superficial spreading melanoma (SSM) is the most common form and it is associated with severe sunburns, especially at an early age (Ishihara et al., 2001; Gilchrest et al., 1999). In most cases, SSM is flat, with intraepidermal microinvasion, particularly at the edges of the lesion. Nodular melanoma comprises raised nodules and has almost no flat parts. Acral lentiginous melanoma (ALM) is not linked to UV exposure and is usually found on the palms of the hands, soles of the feet and in the nail bed (Kuchelmeister et al., 2000). Lentigo maligna appears to be flat and is associated with

melanoma biology and develop new therapeutic solutions.

chronic sun exposure in elderly people.

**2. Cadherin-mediated adhesion in melanoma progression**

Tumor progression is characterized by uncontrolled cell proliferation, high invasive potential into surrounding tissue and metastasis to distant organs. It is believed that this is largely due to disruption or dysfunction of intercellular contacts (Hanahan and Weinberg, 2000). Cadher‐ ins comprise a large family of Ca2+-dependent adhesion molecules (Angst et al., 2001; Gumbin‐

Bronner, 1991).

144 Research Directions in Tumor Angiogenesis

Cadherins play an important role in specific cell-cell adhesion, cell recognition and signaling (Angst et al., 2001; Gumbiner, 2005; Wheelock and Johnson, 2003). Cadherins are transmem‐ brane glycoproteins with their extracellular part responsible for homotypic binding between the neighboring cells, while intracellular part is involved in anchoring cadherins to the cytoske‐ leton. Cadherins interact with the cytoskeleton via catenins (alfa and beta catenins and p120) and form a multicomponent complex which also comprises a number of regulatory molecules such as protein tyrosine kinase, protein tyrosine phosphatase and small GTPases (Perez-Moreno et al., 2003; Sallee et al., 2006; Gumbiner, 2005; Vincent et al., 2004; Rubina et al., 2007).

Disruption of cadherin adherent junctions and their dysfunction has been associated with tumor cell invasion and metastasis (Takeichi, 1993). In human carcinoma the loss of E-cadherin expression leads to dedifferentiation and increased invasiveness of carcinoma cells (Frixen et al., 1991). The change in the expression pattern of cadherins in melanocytes from E-cadherin, P-cadherin and desmoglein to N-cadherin is associated with melanocyte transformation and metastasis (Bonitsis et al., 2006). In normal skin, melanocytes project multiple extensions to keratinocytes and form cadherin adhesive contacts with the basal keratinocytes, which control maintain proliferation and correct positioning of melanocytes (Haas and Herlyng, 2005; Haass et al., 2005). E-cadherin is mainly responsible fot these cell-cell interaction. The loss of func‐ tional E-cadherin or its downregulation let melanocytes escape form keratinocyte control and correlates with high invasiveness and metastasis of the overlying melanoma cells (Hsu et al., 1996; Silye et al., 1998).

Downregulation of E-cadherin and/or its dysfunction is one of the earliest steps in the development of metastases in cutaneous melanoma (Johnson, 1999). E-cadherin expression is detected in cultured melanocytes and naevus cells, while its expression is often lost in cultured melanoma cells from the primary tumors or metastasis (Danen et al., 1996). In the radial growth phase melanoma cells could retain expression of membrane E-cadherin (Sanders et al., 1999). It was even noted that there is a correlation between E-cadherin expression and the depth of the primary tumor (Andersen et al., 2004). Experiments on re-expression of E-cadherin using adenoviral transfer of full length E-cadherin cDNA showed the reduction in tumorigenicity and decrease in proliferation rate of melanoma cells (Hsu et al., 2000). The mechanism of Ecadherin transcriptional downregulation in melanomas involves gene silencing by methyla‐ tion or transrepression by the Snail protein from a superfamily of zinc-finger transcription factors (Tsutsumida et al., 2004). Thus it was suggested that E-cadherin could play an impor‐ tant role in the preventing the melanocytes transformation and limiting their proliferation (cited in Bonitsis et al., 2006).

Catenins are a group of cadherin-associated molecules and they were also suggested to be involved in malignant transformation of melanocytes. The change in catenin expression pattern was found in melanocytic naevus and melanomas, where the expression of alfa and beta catenins was reduced or altered, while beta catenin was often overexpressed (Zhang and Hersey., 1999). The loss of E-cadherin in melanocytes may also indirectly influence the βcatenin cytoplasmic content and affects the β-catenin/wnt signaling pathways. Namely, the reduction in the membranous E-cadherin resulted in the accumulation of free cytoplasmic βcatenin which did not degrade in proteasomes and was translocated to the nucleus. Nuclear β-catenin could be involved in regulation of gene expression responsible for growth and metastasis control via β-catenin/wnt signaling pathways (McGary et al., 2002). Also the presence of the functional E-cadherin in melanocytes ensured their correct adhesion to keratinocytes and limited their motility and proliferation (Gruss et al., 2001; Tang et al., 1994).

appeared at a very early stage of vascular development in mesodermal cells of yolk sac mesenchyme; it was also expressed in progenitor cells during the early angioblast differentia‐ tion and endocardial development (Dejana et al., 2000; Cavallaro et al., 2006). At the later stages, VE-cadherin expression was restricted to the peripheral layer of blood islands that give rise to

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits…

http://dx.doi.org/10.5772/53350

147

VE-cadherin was also shown to be important for melanoma cell invasion and metastasis. At the beginning of diapedesis, endothelial cells located below the attached melanoma cells disassemble their VE-cadherin-mediated adhesion contacts. This allowed melanoma cell to penetrate through the VE-cadherin-negative regions in the endothelial cell monolayer and intercalate between endothelial cells. Subsequently, the endothelial cells surrounding the melanoma cells extended processes and spread over melanoma. The leading edges of the projections of endothelial cell expressed high levels of N-cadherin but not VE-cadherin. VEcadherin expression was restored when the endothelial cells met and reformed cell-cell contacts above the melanoma cell (Voura et al., 1998). Highly aggressive human cutaneous and uveal melanoma cells were also found to express VE-cadherin in contrast to less aggressive cells (Hendrix et al., 2001). This expression contributed to the ability of melanoma cells to mimic endothelial cells and form patterned networks of vascular channels (Hendrix et al., 2001; Hendrix et al., 2003). These data indicated that melanoma cells cooperate with the endothelial cells in the process of invasion and that the regulated changes in the expression of cadherins

Thus, further studies are required to elucidate the biochemical and cellular mechanism of melanoma transformation and progression and the role of cadherins in this process. Lately, an atypical member of the cadherin superfamily – T-cadherin was was shown to be involved in melanoma progression. However, its role and mechanism of action were not completely

T-cadherin is a unique member of cadherin superfamily because it lacks the transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosyl-phosphatidy‐ linositol (GPI) moiety (Fredette and Ranscht, 1994; Fredette et al., 1996; Ranscht and Dours-Zimmermann 1991). Although T-cadherin contains five Ca2+–binding domains on its Nterminal end it does not have the His-Ala-Val motif responsible for the recognition and binding of the classical cadherins. It was shown that T-cadherin can mediate week homophilic adhesion in aggregation assays *in vitro* (Vestal and Ranscht, 1992; Resink et al., 1999) but the lack of intracellular domain strongly suggested that Т-cadherin is not involved in stable сеll-сеll adhesion. Moreover, T-cadherin was show to be absent from the adherent junctions and was located within lipid rafts in the plasma membrane (Philippova et al., 1998); was redistributed to the leading edge in migrating cells (Philippova et al., 2003). These imply T-cadherin as a navigation receptor involved in transduction of extracellular cues in migrating cells rather than

endothelial cells (Dejana et al., 2000).

understood.

**3. T-cadherin in cancer**

an adhesion molecule.

played an essential role in melanoma growth and metastasis.

Despite the majority of studies showed a correlation between the decreased E-cadherin expressionandtumorigenicityofmelanomacells,thereweredatasuggestintgthatinsomecases E-cadherin expression could be retained or even elevated in melanoma (Ruiter and van Muijen, 1998; Nishizawa et al., 2005). It was reported that membranous E-cadherin was present in the metastasizing melanomas and their corresponding lymph node metastasis (Silye et al., 1998).

The loss of E-cadherin expression in melanomas correlates with the increase in N-cadherin expression. This change contributed to the survival advantage of melanoma cells and their invasive and migratory properties (McGary et al., 2002). The shift in cadherin expression pattern was found both in vivo and in vitro (Hsu et al., 1996; Hsu et al., 2000). It was suggested that melanoma cells form N-cadherin adhesion contacts with fibroblasts, vascular endothelial cells, and adjacent melanoma cells (Li and Herlyn, 2000). The N-cadherin-mediated adhesion facilitated migration of melanoma cells over dermal fibroblasts and their transmigration into the vascular system (Haass and Herlyn, 2005; Li et al., 2002) and induced formation of communication gap junction between melanoma cells and the surrounding stroma (McGary et al., 2002). Anti-N-cadherin antibodies retarded the transendothelial migration of melanoma cells and induced their apoptosis, which linked the N-cadherin in the ability of melanoma cells for diapedesis (Li et al., 2002; Sandig et al., 1997; Voura et al., 1998). Surprisingly, adenoviral gene transfer of E-cadherin inhibited N-cadherin expression in melanoma cells and their survival and migration (Hsu et al., 2000). At the same time N-cadherin overexpression did not affect the endogenous E-cadherin expression (Li et al., 2001).

Little is known about the role of P-cadherin in the progression of malignant melanomas. Pcadherin is expressed in basal keratinocytes, melanocytes, in the cells of the basal and outer layers of skin appendages (Klymkowsky and Parr, 1995). As Similarly to E-cadherin, Pcadherin was thought to be involved in the regulation of melanocyte proliferation and migration (Klymkowsky and Parr, 1995). It was found that the soluble form of P-cadherin missing the transmembrane and the cytoplasmic part was expressed in melanoma cells (Bauer et al., 2005) and was associated with increasing tumor thickness and metastasis and reduced patient survival (Bachmann et al., 2005).

VE-cadherin is another member of the cadherin superfamily, which was shown to be involved in melanoma progression. VE-cadherin was found to be exclusively expressed on endothelial cells in normal vessels and mediated homophilic contacts between neighboring cells regulating endothelial barrier function (Dejana, 1996). VE-cadherin is essential for both the development and the maintenance of blood vessels in the adult organism. In the embryo, VE-cadherin appeared at a very early stage of vascular development in mesodermal cells of yolk sac mesenchyme; it was also expressed in progenitor cells during the early angioblast differentia‐ tion and endocardial development (Dejana et al., 2000; Cavallaro et al., 2006). At the later stages, VE-cadherin expression was restricted to the peripheral layer of blood islands that give rise to endothelial cells (Dejana et al., 2000).

VE-cadherin was also shown to be important for melanoma cell invasion and metastasis. At the beginning of diapedesis, endothelial cells located below the attached melanoma cells disassemble their VE-cadherin-mediated adhesion contacts. This allowed melanoma cell to penetrate through the VE-cadherin-negative regions in the endothelial cell monolayer and intercalate between endothelial cells. Subsequently, the endothelial cells surrounding the melanoma cells extended processes and spread over melanoma. The leading edges of the projections of endothelial cell expressed high levels of N-cadherin but not VE-cadherin. VEcadherin expression was restored when the endothelial cells met and reformed cell-cell contacts above the melanoma cell (Voura et al., 1998). Highly aggressive human cutaneous and uveal melanoma cells were also found to express VE-cadherin in contrast to less aggressive cells (Hendrix et al., 2001). This expression contributed to the ability of melanoma cells to mimic endothelial cells and form patterned networks of vascular channels (Hendrix et al., 2001; Hendrix et al., 2003). These data indicated that melanoma cells cooperate with the endothelial cells in the process of invasion and that the regulated changes in the expression of cadherins played an essential role in melanoma growth and metastasis.

Thus, further studies are required to elucidate the biochemical and cellular mechanism of melanoma transformation and progression and the role of cadherins in this process. Lately, an atypical member of the cadherin superfamily – T-cadherin was was shown to be involved in melanoma progression. However, its role and mechanism of action were not completely understood.

#### **3. T-cadherin in cancer**

beta catenins was reduced or altered, while beta catenin was often overexpressed (Zhang and Hersey., 1999). The loss of E-cadherin in melanocytes may also indirectly influence the βcatenin cytoplasmic content and affects the β-catenin/wnt signaling pathways. Namely, the reduction in the membranous E-cadherin resulted in the accumulation of free cytoplasmic βcatenin which did not degrade in proteasomes and was translocated to the nucleus. Nuclear β-catenin could be involved in regulation of gene expression responsible for growth and metastasis control via β-catenin/wnt signaling pathways (McGary et al., 2002). Also the presence of the functional E-cadherin in melanocytes ensured their correct adhesion to keratinocytes and limited their motility and proliferation (Gruss et al., 2001; Tang et al., 1994). Despite the majority of studies showed a correlation between the decreased E-cadherin expressionandtumorigenicityofmelanomacells,thereweredatasuggestintgthatinsomecases E-cadherin expression could be retained or even elevated in melanoma (Ruiter and van Muijen, 1998; Nishizawa et al., 2005). It was reported that membranous E-cadherin was present in the metastasizing melanomas and their corresponding lymph node metastasis (Silye et al., 1998). The loss of E-cadherin expression in melanomas correlates with the increase in N-cadherin expression. This change contributed to the survival advantage of melanoma cells and their invasive and migratory properties (McGary et al., 2002). The shift in cadherin expression pattern was found both in vivo and in vitro (Hsu et al., 1996; Hsu et al., 2000). It was suggested that melanoma cells form N-cadherin adhesion contacts with fibroblasts, vascular endothelial cells, and adjacent melanoma cells (Li and Herlyn, 2000). The N-cadherin-mediated adhesion facilitated migration of melanoma cells over dermal fibroblasts and their transmigration into the vascular system (Haass and Herlyn, 2005; Li et al., 2002) and induced formation of communication gap junction between melanoma cells and the surrounding stroma (McGary et al., 2002). Anti-N-cadherin antibodies retarded the transendothelial migration of melanoma cells and induced their apoptosis, which linked the N-cadherin in the ability of melanoma cells for diapedesis (Li et al., 2002; Sandig et al., 1997; Voura et al., 1998). Surprisingly, adenoviral gene transfer of E-cadherin inhibited N-cadherin expression in melanoma cells and their survival and migration (Hsu et al., 2000). At the same time N-cadherin overexpression did not

affect the endogenous E-cadherin expression (Li et al., 2001).

patient survival (Bachmann et al., 2005).

146 Research Directions in Tumor Angiogenesis

Little is known about the role of P-cadherin in the progression of malignant melanomas. Pcadherin is expressed in basal keratinocytes, melanocytes, in the cells of the basal and outer layers of skin appendages (Klymkowsky and Parr, 1995). As Similarly to E-cadherin, Pcadherin was thought to be involved in the regulation of melanocyte proliferation and migration (Klymkowsky and Parr, 1995). It was found that the soluble form of P-cadherin missing the transmembrane and the cytoplasmic part was expressed in melanoma cells (Bauer et al., 2005) and was associated with increasing tumor thickness and metastasis and reduced

VE-cadherin is another member of the cadherin superfamily, which was shown to be involved in melanoma progression. VE-cadherin was found to be exclusively expressed on endothelial cells in normal vessels and mediated homophilic contacts between neighboring cells regulating endothelial barrier function (Dejana, 1996). VE-cadherin is essential for both the development and the maintenance of blood vessels in the adult organism. In the embryo, VE-cadherin T-cadherin is a unique member of cadherin superfamily because it lacks the transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosyl-phosphatidy‐ linositol (GPI) moiety (Fredette and Ranscht, 1994; Fredette et al., 1996; Ranscht and Dours-Zimmermann 1991). Although T-cadherin contains five Ca2+–binding domains on its Nterminal end it does not have the His-Ala-Val motif responsible for the recognition and binding of the classical cadherins. It was shown that T-cadherin can mediate week homophilic adhesion in aggregation assays *in vitro* (Vestal and Ranscht, 1992; Resink et al., 1999) but the lack of intracellular domain strongly suggested that Т-cadherin is not involved in stable сеll-сеll adhesion. Moreover, T-cadherin was show to be absent from the adherent junctions and was located within lipid rafts in the plasma membrane (Philippova et al., 1998); was redistributed to the leading edge in migrating cells (Philippova et al., 2003). These imply T-cadherin as a navigation receptor involved in transduction of extracellular cues in migrating cells rather than an adhesion molecule.

Little is known about the biological role and underlying mechanisms of T-cadherin in malig‐ nant transformation and tumor progression. In some reports, T-cadherin was regarded as a tumor suppressor and its downregulation was associated with tumor progression. Downre‐ gulation of T-cadherin was shown to be associated also with tumorogenicity in breast (Riener et al., 2008), lung (Sato et al., 1998), and gallbladder cancers (Adachi et al., 2009). However, in other cancers such as ovarian, endometrial (Widschwendter et al., 2004; Suehiro et al., 2008) and osteosarcoma (Zucchini et al., 2004), decreased expression of T-cadherin positively correlated with patient survival. T-cadherin was upregulated in human invasive hepatocel‐ lular carcinomas (Riou et al., 2006) and astrocytomas (Gutmann et al., 2001).

The role of T-cadherin in melanoma progression and vascularization was addressed in a few studies and the results were contradictory. T-cadherin was expressed in normal human skin melanocytes (Kuphal et al., 2009; Bosserhoff et al., 2011). However, it was shown that the precursors of melanocytes did not express T-cadherin and invaded T-cadherin negative rostral parts of sclerotomes avoiding T-cadherin positive caudal parts of sclerotomes during neural crest cell migration (Ranscht and Bronner-Fraser, 1991). These results led to a hypothesis that T-cadherin is a navigating receptor that provides topographic guidance for migrating mela‐ nocyte precursors, and that de-differentiated or transformed melanocytes may loose Tcadherin expression. Indeed, T-cadherin expression was found to be diminished in melanocytes induced to de-differentiate to melanoblast-related cells and T-cadherin expres‐ sion was undetectable in about 80% of human melanoma cell lines (Kuphal et al., 2009; Bosserhoff et al., 2011). While T-cadherin was expressed in benign naevus nests, its expression was lost in most tissue samples of human primary melanoma, lymph and visceral melanoma metastasis indicating the potential role of T-cadherin in melanoma progression (Kuphal et al., 2009). In addition, T-cadherin re-expression by stable transfection in human melanoma cells reduced the rate of tumor growth in the *nu/nu* mouse tumor model, decreased cell capacity for anchorage-independent growth, and for migration and invasion *in vitro* (Kuphal et al., 2009). However, it was shown in other studies that T-cadherin overexpression in endothelial cells stimulated intratumoral angiogenesis in tumor co-culture spheroid model with melano‐ ma cells *in vitro* (Ghosh et al., 2007). Despite T-cadherin expression is lost in the majority of melanoma cell lines, 20% of melanomas still express T-cadherin (Kuphal et al., 2009; Bosserhoff

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To gain insights into the function of T-cadherin in melanoma progression and growth we first examined T-cadherin expression in the normal human skin melanocytes, melanoma cells and the blood vessels of the primary melanomas and melanoma metastasis in human samples and

We performed a comparative study of T-cadherin expression in normal skin and in melanoma samples. Tissue samples of primary human melanoma and metastasis obtained from patients undergoing surgical treatment were immediately frozen with liquid nitrogen and stored at

Human skin biopsies from 6 healthy donors, 10 tissue samples of primary human melanoma and 12 samples of visceral melanoma metastasis were obtained from Blokhin Russian Cancer Research Center of Russian Academy of Medical Sciences. Consequent cryosections of the samples (7µm thick) were fixed in 4% paraformaldehyde (PRS Panreac, Spain), whashed and then incubated in a mixture of primary antibodies against T-cadherin (rabbit anti-human, ProSci, USA) or endothelial cells marker vWF (Von Willebrand factor, mouse anti-human, BD

C. Human skin biopsies from healthy donors and melanoma samples of patients were immunostained with antibodies against T-cadherin and vascular cell markers and analyzed

et al., 2011) and possess invasive and metastatic potential.

**4. T-cadherin expression in human melanoma**

in the experimental models.

using fluorescent microscope.


T-cadherin is expressed in the basal layer of keratinocytes, in melanocytes and in vascular cells of the dermal blood vessels in the normal skin (Zhou et al., 2002; Kuphal et al., 2009; Rubina et al., 2012). However, its expression was consequently lost upon cell transformation and tumor progression in pre-malignant skin lesions and in non-melanoma skin cancer. In skin lesions, such as actinic keratosis T-cadherin was abundantly expressed in the atypical keratinocytes, while its expression varied in Bowen disease and was weaker than in the normal skin (Pfaff et al., 2010). Expression of T-cadherin was reduced in psoriatic samples (Zhou et al., 2003) and was down-regulated or completely absent in invasive cutaneous squamous cell carcinoma (Takeuchi et al., 2002a) and in basal cell carcinoma of the skin (Takeuchi et al., 2002b). Data obtained in our lab supported these findings and confirmed that T-cadherin expression was not changed if cells maintained the attachment to the basal membrane in the lesions charac‐ terized by slow or controlled keratinocytes growth (keratoacanthoma, psoriasis, actinic keratosis and superficial basalioma) (Rubina et al., 2012). However, T-cadherin expression in keratinocytes was downregulated upon tumor progression in basosquamous cell carcinoma, squamous cell carcinoma and in some cases of basal cell carcinoma, i.e. tumors with high proliferative, invasive, and metastatic potential (Rubina et al., 2012).

Apart from regulation of keratinocyte proliferation, T-cadherin may affect tumor progression through its direct involvement in neovascularization. While in the normal blood vessels Tcadherin was abundantly expressed in endothelial and mural cells (Ivanov et al., 2001), its expression was altered in tumor vessels in Lewis carcinoma lung metastasis and F9 endoder‐ mal teratocarcinoma and in human xenografts PC-3 prostate cancer or A673 rhabdomyosar‐ coma (Riou et al., 2006; Wyder et al., 2000). Inactivation of T-cadherin gene limited mammary tumor vascularization and reduced tumor growth in the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyV-mT) transgenic model (Hebbard et al., 2008). In human hepatocellular carcinoma (НСС) T-cadherin was also upregulated in intratumoral capillary endothelial cells and this increase соrrеlаtеd with tumor growth and metastasis (Adachi et al., 2006). Data obtained in our lab indicated that in pre-malignant skin lesions all blood vessels uniformly expressed T-cadherin (Rubina et al., 2012). The aberrant expression of T-cadherin and vascular markers was detected in aggressively developing skin tumors such as basosqu‐ amous cell carcinoma, squamous cell carcinoma and in some cases of basal cell carcinoma. We suggested that the high level of expression of T-cadherin in the normal keratinocytes and in benign tumors regulates the growth of blood vessels. Upon malignant transformation expression of T-cadherin was lost in tumor cells and altered on vascular cells. This caused the abnormality and excessive vascularization of the tumors (Rubina et al., 2012).

The role of T-cadherin in melanoma progression and vascularization was addressed in a few studies and the results were contradictory. T-cadherin was expressed in normal human skin melanocytes (Kuphal et al., 2009; Bosserhoff et al., 2011). However, it was shown that the precursors of melanocytes did not express T-cadherin and invaded T-cadherin negative rostral parts of sclerotomes avoiding T-cadherin positive caudal parts of sclerotomes during neural crest cell migration (Ranscht and Bronner-Fraser, 1991). These results led to a hypothesis that T-cadherin is a navigating receptor that provides topographic guidance for migrating mela‐ nocyte precursors, and that de-differentiated or transformed melanocytes may loose Tcadherin expression. Indeed, T-cadherin expression was found to be diminished in melanocytes induced to de-differentiate to melanoblast-related cells and T-cadherin expres‐ sion was undetectable in about 80% of human melanoma cell lines (Kuphal et al., 2009; Bosserhoff et al., 2011). While T-cadherin was expressed in benign naevus nests, its expression was lost in most tissue samples of human primary melanoma, lymph and visceral melanoma metastasis indicating the potential role of T-cadherin in melanoma progression (Kuphal et al., 2009). In addition, T-cadherin re-expression by stable transfection in human melanoma cells reduced the rate of tumor growth in the *nu/nu* mouse tumor model, decreased cell capacity for anchorage-independent growth, and for migration and invasion *in vitro* (Kuphal et al., 2009). However, it was shown in other studies that T-cadherin overexpression in endothelial cells stimulated intratumoral angiogenesis in tumor co-culture spheroid model with melano‐ ma cells *in vitro* (Ghosh et al., 2007). Despite T-cadherin expression is lost in the majority of melanoma cell lines, 20% of melanomas still express T-cadherin (Kuphal et al., 2009; Bosserhoff et al., 2011) and possess invasive and metastatic potential.

To gain insights into the function of T-cadherin in melanoma progression and growth we first examined T-cadherin expression in the normal human skin melanocytes, melanoma cells and the blood vessels of the primary melanomas and melanoma metastasis in human samples and in the experimental models.

#### **4. T-cadherin expression in human melanoma**

Little is known about the biological role and underlying mechanisms of T-cadherin in malig‐ nant transformation and tumor progression. In some reports, T-cadherin was regarded as a tumor suppressor and its downregulation was associated with tumor progression. Downre‐ gulation of T-cadherin was shown to be associated also with tumorogenicity in breast (Riener et al., 2008), lung (Sato et al., 1998), and gallbladder cancers (Adachi et al., 2009). However, in other cancers such as ovarian, endometrial (Widschwendter et al., 2004; Suehiro et al., 2008) and osteosarcoma (Zucchini et al., 2004), decreased expression of T-cadherin positively correlated with patient survival. T-cadherin was upregulated in human invasive hepatocel‐

T-cadherin is expressed in the basal layer of keratinocytes, in melanocytes and in vascular cells of the dermal blood vessels in the normal skin (Zhou et al., 2002; Kuphal et al., 2009; Rubina et al., 2012). However, its expression was consequently lost upon cell transformation and tumor progression in pre-malignant skin lesions and in non-melanoma skin cancer. In skin lesions, such as actinic keratosis T-cadherin was abundantly expressed in the atypical keratinocytes, while its expression varied in Bowen disease and was weaker than in the normal skin (Pfaff et al., 2010). Expression of T-cadherin was reduced in psoriatic samples (Zhou et al., 2003) and was down-regulated or completely absent in invasive cutaneous squamous cell carcinoma (Takeuchi et al., 2002a) and in basal cell carcinoma of the skin (Takeuchi et al., 2002b). Data obtained in our lab supported these findings and confirmed that T-cadherin expression was not changed if cells maintained the attachment to the basal membrane in the lesions charac‐ terized by slow or controlled keratinocytes growth (keratoacanthoma, psoriasis, actinic keratosis and superficial basalioma) (Rubina et al., 2012). However, T-cadherin expression in keratinocytes was downregulated upon tumor progression in basosquamous cell carcinoma, squamous cell carcinoma and in some cases of basal cell carcinoma, i.e. tumors with high

Apart from regulation of keratinocyte proliferation, T-cadherin may affect tumor progression through its direct involvement in neovascularization. While in the normal blood vessels Tcadherin was abundantly expressed in endothelial and mural cells (Ivanov et al., 2001), its expression was altered in tumor vessels in Lewis carcinoma lung metastasis and F9 endoder‐ mal teratocarcinoma and in human xenografts PC-3 prostate cancer or A673 rhabdomyosar‐ coma (Riou et al., 2006; Wyder et al., 2000). Inactivation of T-cadherin gene limited mammary tumor vascularization and reduced tumor growth in the mouse mammary tumor virus (MMTV)-polyoma virus middle T (PyV-mT) transgenic model (Hebbard et al., 2008). In human hepatocellular carcinoma (НСС) T-cadherin was also upregulated in intratumoral capillary endothelial cells and this increase соrrеlаtеd with tumor growth and metastasis (Adachi et al., 2006). Data obtained in our lab indicated that in pre-malignant skin lesions all blood vessels uniformly expressed T-cadherin (Rubina et al., 2012). The aberrant expression of T-cadherin and vascular markers was detected in aggressively developing skin tumors such as basosqu‐ amous cell carcinoma, squamous cell carcinoma and in some cases of basal cell carcinoma. We suggested that the high level of expression of T-cadherin in the normal keratinocytes and in benign tumors regulates the growth of blood vessels. Upon malignant transformation expression of T-cadherin was lost in tumor cells and altered on vascular cells. This caused the

lular carcinomas (Riou et al., 2006) and astrocytomas (Gutmann et al., 2001).

148 Research Directions in Tumor Angiogenesis

proliferative, invasive, and metastatic potential (Rubina et al., 2012).

abnormality and excessive vascularization of the tumors (Rubina et al., 2012).

We performed a comparative study of T-cadherin expression in normal skin and in melanoma samples. Tissue samples of primary human melanoma and metastasis obtained from patients undergoing surgical treatment were immediately frozen with liquid nitrogen and stored at -80o C. Human skin biopsies from healthy donors and melanoma samples of patients were immunostained with antibodies against T-cadherin and vascular cell markers and analyzed using fluorescent microscope.

Human skin biopsies from 6 healthy donors, 10 tissue samples of primary human melanoma and 12 samples of visceral melanoma metastasis were obtained from Blokhin Russian Cancer Research Center of Russian Academy of Medical Sciences. Consequent cryosections of the samples (7µm thick) were fixed in 4% paraformaldehyde (PRS Panreac, Spain), whashed and then incubated in a mixture of primary antibodies against T-cadherin (rabbit anti-human, ProSci, USA) or endothelial cells marker vWF (Von Willebrand factor, mouse anti-human, BD Biosciences, USA ), or melanoma (gp100) Ab-3 (Ab-3 is a mixture of Ab-1 (HMB45) and Ab-2 (HMB50) antibodies which are extremely sensitive and recognize differentiating melanocytes and melanomas, Lab Vision- Neomarkers, USA) – for 1 hour and subsequent extensive washing in PBS. Then sections were incubated in a mixture of secondary antibodies Alexa488 conjugated donkey anti-mouse and Alexa594-conjugated donkey anti-rabbit or Alexa488 conjugated donkey anti-rabbit and Alexa594-conjugated donkey anti-mouse (Molecular Probes, USA) (1µg/ml in PBS). Cell nuclei were counterstained with DAPI (Molecular Probes, USA). Images were obtained using Zeiss Axiovert 200M microscope equipped with CCD camera AxioCam HRc and Axiovision software (Zeiss, Germany) and further processed using Adobe PhotoShop software (Adobe Systems, USA).

The immunofluorescent staining with gp 100 antibodies demonstrated that differentiating melanocytes were located beneath the layer of epidermal basal keratinocytes and extended their processes over keratinocytes (Fig. 2A). We were able to show the expression of T-cadherin in differentiating melanocytes by the overlay of green and red fluorescence (Fig. 2A, B and C) and in mature melanocytes by mapping the red fluorescence that revealed T-cadherin expression (Fig. 2C) with a phase-contrast image where the dark cells corresponded to mature

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**B**

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**C**

**D**

**20 µm** 

**50 µm** 

**Figure 2.** Double immunofluorescent staining of normal skin samples with antibodies against T-cadherin (red) (A, B, C) and gp 100 (green) (A, B). Figure D depicts phase contrast image. Figures B, C, D represent parallel frozen sections of the same sample. Differentiating melanocytes (green fluorescence in A, B) and mature melanocytes (phase contrast image with dark cells in D and corresponding immunofluorescent staining against T-cadherin – red fluorescence in C) all express T-cadherin. Nuclei were counterstained with DAPI (blue). Colocalization of T-cadherin and gp 100 is

Previous studies demonstrated that T-cadherin expression is reduced during cancer progres‐ sion. Thus, we examined T-cadherin expression in melanoma samples of patients undergoing surgical treatment. Consequent cryosections of 10 tissue samples of the primary human melanoma and 12 samples of visceral metastasis, including two cases of primary melanoma that developed metastasis within a year were double immunostained with antibodies against

melanocytes (Fig. 2C and D).

showed by the arrow. Bars, 100 µm.

**A**

We assessed the areas occupied by the T-cadherin-positive melanoma cells using MetaMorph 5.0 (Universal Imaging) and Adobe PhotoShop software (Adobe Systems). In a field of view, we determined the T-cadherin-positive areas and normalized to the DAPI-stained area unit of each section. For the quantification, T-cadherin-positive areas were counted in 4-5 fields of view (1.107 mm2 ) on 10 random sections for each sample at 100x total magnification (10x objective).

In the normal skin T-cadherin was abundantly expressed in basal keratinocytes, in differenti‐ ating keratinocytes, in the stromal cells and in all blood vessels located in the underlying derma (Fig.1).

**Figure 1.** Double immunofluorescent staining of normal skin samples with antibodies against T-cadherin (red) and vWF (green). T-cadherin expression was detected in the basal keratinocytes, suprabasal layers, stromal cells and in the blood vessels (colocalization of T-cadherin and vWF is showed by the arrow). Nuclei were counterstained with DAPI (blue). Bars, 100 µm.

The immunofluorescent staining with gp 100 antibodies demonstrated that differentiating melanocytes were located beneath the layer of epidermal basal keratinocytes and extended their processes over keratinocytes (Fig. 2A). We were able to show the expression of T-cadherin in differentiating melanocytes by the overlay of green and red fluorescence (Fig. 2A, B and C) and in mature melanocytes by mapping the red fluorescence that revealed T-cadherin expression (Fig. 2C) with a phase-contrast image where the dark cells corresponded to mature melanocytes (Fig. 2C and D).

Biosciences, USA ), or melanoma (gp100) Ab-3 (Ab-3 is a mixture of Ab-1 (HMB45) and Ab-2 (HMB50) antibodies which are extremely sensitive and recognize differentiating melanocytes and melanomas, Lab Vision- Neomarkers, USA) – for 1 hour and subsequent extensive washing in PBS. Then sections were incubated in a mixture of secondary antibodies Alexa488 conjugated donkey anti-mouse and Alexa594-conjugated donkey anti-rabbit or Alexa488 conjugated donkey anti-rabbit and Alexa594-conjugated donkey anti-mouse (Molecular Probes, USA) (1µg/ml in PBS). Cell nuclei were counterstained with DAPI (Molecular Probes, USA). Images were obtained using Zeiss Axiovert 200M microscope equipped with CCD camera AxioCam HRc and Axiovision software (Zeiss, Germany) and further processed using

We assessed the areas occupied by the T-cadherin-positive melanoma cells using MetaMorph 5.0 (Universal Imaging) and Adobe PhotoShop software (Adobe Systems). In a field of view, we determined the T-cadherin-positive areas and normalized to the DAPI-stained area unit of each section. For the quantification, T-cadherin-positive areas were counted in 4-5 fields of

In the normal skin T-cadherin was abundantly expressed in basal keratinocytes, in differenti‐ ating keratinocytes, in the stromal cells and in all blood vessels located in the underlying derma

**Figure 1.** Double immunofluorescent staining of normal skin samples with antibodies against T-cadherin (red) and vWF (green). T-cadherin expression was detected in the basal keratinocytes, suprabasal layers, stromal cells and in the blood vessels (colocalization of T-cadherin and vWF is showed by the arrow). Nuclei were counterstained with DAPI

) on 10 random sections for each sample at 100x total magnification (10x

Adobe PhotoShop software (Adobe Systems, USA).

view (1.107 mm2

150 Research Directions in Tumor Angiogenesis

(blue). Bars, 100 µm.

objective).

(Fig.1).

**Figure 2.** Double immunofluorescent staining of normal skin samples with antibodies against T-cadherin (red) (A, B, C) and gp 100 (green) (A, B). Figure D depicts phase contrast image. Figures B, C, D represent parallel frozen sections of the same sample. Differentiating melanocytes (green fluorescence in A, B) and mature melanocytes (phase contrast image with dark cells in D and corresponding immunofluorescent staining against T-cadherin – red fluorescence in C) all express T-cadherin. Nuclei were counterstained with DAPI (blue). Colocalization of T-cadherin and gp 100 is showed by the arrow. Bars, 100 µm.

Previous studies demonstrated that T-cadherin expression is reduced during cancer progres‐ sion. Thus, we examined T-cadherin expression in melanoma samples of patients undergoing surgical treatment. Consequent cryosections of 10 tissue samples of the primary human melanoma and 12 samples of visceral metastasis, including two cases of primary melanoma that developed metastasis within a year were double immunostained with antibodies against melanoma cell marker gp 100, T-cadherin, endothelial cell marker vWF and analyzed using fluorescent microscope.

et al., 1999). This vasculogenic mimicry adds challenges to the practical surgery and requires

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Our data indicate that the areas devoid of T-cadherin in the primary melanomas were more intensively vascularized than the areas with high level of T-cadherin expression. 80% of vWFpositive blood vessels also expressed T-cadherin (arrows in Fig.3A), while no T-cadherin could be detected in the rest of the blood vessels (arrows in Fig.3A, 4A). Besides vWF-positive blood vessels in the primary melanomas (Fig.4A) and in the visceral metastasis, vascular channels devoid of endothelial cells were found. Interestingly, vascular channels were lined up by the

**50 µm 50 µm** 

new cancer diagnostic and treatment strategies.

cells expressing T-cadherin (Fig.4A, C).

**B C**

respectively). Nuclei were counterstained with DAPI (blue). Bars, 50 µm.

**50 µm** 

**Figure 4.** Double immunofluorescent staining of the primary melanoma (A) and visceral metastasis samples (B, C) with antibodies against T-cadherin (red) and vWF (green). (A) Large lacuna can be seen in the center of the image of the primary melanoma (showed by an arrow). The walls of the lacunas in the primary melanomas (A) and metastasis (B, C) were lined up by T-cadherin positive melanoma cells (red). Blood vessels of the primary melanomas and metastasis were heterogeneous. In (A) vWF-positive blood vessels with no T-cadherin expression could be seen located in the immediate vicinity of the lacuna (green) fluorescence, marked by an empty arrow in A): in (B and asterisk in C) both variants of vWF-positive blood vessels are presented: T-cadherin positive and T-cadherin negative (arrows in A and C,

The tissue staining with subsequent image analysis showed that in the primary melanoma 60% of the sections was occupied by melanoma cells uniformly expressing T-cadherin (Fig.3A), while 30% of the sections contained areas with heterogeneous, mosaic pattern of T-cadherin expression where some melanoma cells were T-cadherin positive and some cells - T-cadherin negative (Fig.3B and 3C), and the rest 10% exhibited no T-cadherin expression.

**Figure 3.** Double immunofluorescent staining of the primary melanoma sample with antibodies against T-cadherin (red) (A, B, C) and vWF (green) (A, B) or gp 100 (green) (C). Nuclei were counterstained with DAPI (blue). Blood vessels that expressed T-cadherin are showed by the arrow in A, blood vessels with no T-cdherin are marked by an empty arrow in B. Bars, 50 µm.

Tumor growth and progression require blood supply. There are three mechanisms by which solid tumors acquire their blood supply. First, tumor and the surrounding stromal cells secrete angiogenic factors that stimulate vessel growth and recruitment into the tumor from the preexisting vessels in a well described process of angiogenesis (Folkman et al., 1971; Folkman et al., 1992, Folkman 1995). Asahara and colleagues demonstrated the incorporation of circulating endothelial progenitors form the peripheral blood into sites of ischemia-induced angiogenesis (Asahara et al., 1997). Beyond these two mechanisms, aggressive primary and metastatic melanomas are capable of generating microcirculatory channels composed of extracellular matrix and lined by tumor cells that express VE-cadherin (Hendrix et al., 2001; Maniotis et al., 1999). These vascular channels (lacunas) allow blood flow. However, lacunas are not strictly vasculogenic or angiogenic because they are formed by melanoma cells (Folberg et al., 2002; Maniotis et al., 1999). Therefore the name "vasculogenic mimicry" was assigned to the process by which melanoma cells generate non-endothelial cell-lined channels (Maniotis et al., 1999). This vasculogenic mimicry adds challenges to the practical surgery and requires new cancer diagnostic and treatment strategies.

melanoma cell marker gp 100, T-cadherin, endothelial cell marker vWF and analyzed using

The tissue staining with subsequent image analysis showed that in the primary melanoma 60% of the sections was occupied by melanoma cells uniformly expressing T-cadherin (Fig.3A), while 30% of the sections contained areas with heterogeneous, mosaic pattern of T-cadherin expression where some melanoma cells were T-cadherin positive and some cells - T-cadherin

**B C**

**Figure 3.** Double immunofluorescent staining of the primary melanoma sample with antibodies against T-cadherin (red) (A, B, C) and vWF (green) (A, B) or gp 100 (green) (C). Nuclei were counterstained with DAPI (blue). Blood vessels that expressed T-cadherin are showed by the arrow in A, blood vessels with no T-cdherin are marked by an empty

Tumor growth and progression require blood supply. There are three mechanisms by which solid tumors acquire their blood supply. First, tumor and the surrounding stromal cells secrete angiogenic factors that stimulate vessel growth and recruitment into the tumor from the preexisting vessels in a well described process of angiogenesis (Folkman et al., 1971; Folkman et al., 1992, Folkman 1995). Asahara and colleagues demonstrated the incorporation of circulating endothelial progenitors form the peripheral blood into sites of ischemia-induced angiogenesis (Asahara et al., 1997). Beyond these two mechanisms, aggressive primary and metastatic melanomas are capable of generating microcirculatory channels composed of extracellular matrix and lined by tumor cells that express VE-cadherin (Hendrix et al., 2001; Maniotis et al., 1999). These vascular channels (lacunas) allow blood flow. However, lacunas are not strictly vasculogenic or angiogenic because they are formed by melanoma cells (Folberg et al., 2002; Maniotis et al., 1999). Therefore the name "vasculogenic mimicry" was assigned to the process by which melanoma cells generate non-endothelial cell-lined channels (Maniotis

**50 µm** 

negative (Fig.3B and 3C), and the rest 10% exhibited no T-cadherin expression.

fluorescent microscope.

152 Research Directions in Tumor Angiogenesis

arrow in B. Bars, 50 µm.

Our data indicate that the areas devoid of T-cadherin in the primary melanomas were more intensively vascularized than the areas with high level of T-cadherin expression. 80% of vWFpositive blood vessels also expressed T-cadherin (arrows in Fig.3A), while no T-cadherin could be detected in the rest of the blood vessels (arrows in Fig.3A, 4A). Besides vWF-positive blood vessels in the primary melanomas (Fig.4A) and in the visceral metastasis, vascular channels devoid of endothelial cells were found. Interestingly, vascular channels were lined up by the cells expressing T-cadherin (Fig.4A, C).

**Figure 4.** Double immunofluorescent staining of the primary melanoma (A) and visceral metastasis samples (B, C) with antibodies against T-cadherin (red) and vWF (green). (A) Large lacuna can be seen in the center of the image of the primary melanoma (showed by an arrow). The walls of the lacunas in the primary melanomas (A) and metastasis (B, C) were lined up by T-cadherin positive melanoma cells (red). Blood vessels of the primary melanomas and metastasis were heterogeneous. In (A) vWF-positive blood vessels with no T-cadherin expression could be seen located in the immediate vicinity of the lacuna (green) fluorescence, marked by an empty arrow in A): in (B and asterisk in C) both variants of vWF-positive blood vessels are presented: T-cadherin positive and T-cadherin negative (arrows in A and C, respectively). Nuclei were counterstained with DAPI (blue). Bars, 50 µm.

Our data indicated that T-cadherin expression in human melanoma was gradually reduced upon melanoma progression. While nearly 60% of melanoma cells in the primary melano‐ mas expressed T-cadherin, 30% of the sections exhibited a mosaic pattern of T-cadherin expression in melanoma cells and 10% of the sections were T-cadherin negative. In metasta‐ sis, 60% of the sections were occupied by melanoma cells with heterogeneous T-cadherin expression (mosaic pattern) and the number of T-cadherin-positive cells was reduced to 30% (Fig. 5). These results were in agreement with the data obtained by Kuphal (Kuphal et al., 2009) who detected T-cadherin expression in melanocytes in the healthy skin and intratumor‐ al capillaries of the primary melanoma samples while T-cadherin expression was lost in melanoma cells.

**5. The effect of T-cadherin expression on melanoma cell proliferation and**

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To assess the effect of T-cadherin expression on melanoma cell proliferation we generated stable cell lines of murine malignant melanoma B16F10 cells (ATCC® №CRL-6475™) by transfecting the cells with pcDNA-Tcad using Lipofectamine™ 2000 reagent (Invitrogen, USA. As a control, luciferase cDNA fragment in the antisense orientation was cloned into the pcDNA 3.1 vector. The cells were cultured and transfected as described before (Yurlova et al., 2010). For stable cell line generation transfected cells were cloned and selected by incubation with 2 mg/ml G418 (Invitrogen, USA). For some experiments we also used the polyclonal mouse melanoma B16F10 cell cultures obtained after transfection and subsequent selection with G418 without cloning. Expression of T-cadherin in polyclonal mouse melanoma cell cultures and melanoma cell clones was examined by Western blotting. Three clones of B16F10 melanoma cells with different level of T-cadherin expression was chosen: a control clone with no Tcadherin (T-), clone with low T-cadherin expression (T+) and clone with high T-cadherin

To investigate the effect of T-cadherin expression on melanoma cell proliferation, cells were seeded on 6-well plates and harvested by trypsinization after 24, 48 and 72 h in culture and counted with the Countess® (Invitrogen, USA). After 3 days, the number of T-cadherinexpressing cells (T+ and T++) was 44-48% higher compared to the control (Fig. 6A). We obtained similar results using the impedance measurement with xCELLigence system: the cell index was continuously monitored with 1 min interval for 5 hrs and with 10 min interval for 96 hrs. (Fig. 6B). The xCELLigence system (Roche, USA) monitors cellular events in real time and measures electrical impedance across microelectrodes integrated on the bottom of E-Plates. Thus, our data indicated that T-cadherin expression in mouse B16F10 melanoma cells in‐

**Figure 6.** T-cadherin expression stimulated proliferation of B16F10 cells *in vitro*. (A) Cells were incubated in RPMI 1640 medium containing 10% FBS for indicated periods of time, harvested and counted using CellCounter. (B) The cell pro‐ liferation assay was performed with xCELLigence system. Attachment of 5000 cells per chamber was monitored with the impedance measurement of the xCELLigence System for 96 hrs. The cell indexes of B16F10 T- (control cells, black), T+ (cells with low level of T-cadherin, blue) and T++ (cells with high level of T-cadherin, red) are shown. Results are

**apoptosis**

expression (T++) (Yurlova et al., in press).

creased their proliferation *in vitro*.

presented as the means ± SEM of three independent experiments.

The same pattern was previously described for pre-cancer skin lesions and non-melanoma skin cancer: T-cadherin expression was gradually lost upon malignization. The aberrant expression of T-cadherin was also detected in the blood vessels and correlated with the histological features and invasive behavior of more aggressive tumors (Rubina et al., 2012).

**Figure 5.** Loss of the expression of T-cadherin in melanocytes during metastasis.

To elucidate the potential role of T-cadherin in melanoma progression and growth, we used an established model of highly aggressive murine melanoma B16F10 in BDF1 mice. The murine melanoma B16F10 with lung metastases is considered as an adequate model for testing the efficacy of new anticancer therapies in preclinical evaluations and the forecasting model of human disseminated melanoma in anticancer screening (Teicher and Andrews, 2004).

### **5. The effect of T-cadherin expression on melanoma cell proliferation and apoptosis**

Our data indicated that T-cadherin expression in human melanoma was gradually reduced upon melanoma progression. While nearly 60% of melanoma cells in the primary melano‐ mas expressed T-cadherin, 30% of the sections exhibited a mosaic pattern of T-cadherin expression in melanoma cells and 10% of the sections were T-cadherin negative. In metasta‐ sis, 60% of the sections were occupied by melanoma cells with heterogeneous T-cadherin expression (mosaic pattern) and the number of T-cadherin-positive cells was reduced to 30% (Fig. 5). These results were in agreement with the data obtained by Kuphal (Kuphal et al., 2009) who detected T-cadherin expression in melanocytes in the healthy skin and intratumor‐ al capillaries of the primary melanoma samples while T-cadherin expression was lost in

The same pattern was previously described for pre-cancer skin lesions and non-melanoma skin cancer: T-cadherin expression was gradually lost upon malignization. The aberrant expression of T-cadherin was also detected in the blood vessels and correlated with the histological features and invasive behavior of more aggressive tumors (Rubina et al., 2012).

**Figure 5.** Loss of the expression of T-cadherin in melanocytes during metastasis.

To elucidate the potential role of T-cadherin in melanoma progression and growth, we used an established model of highly aggressive murine melanoma B16F10 in BDF1 mice. The murine melanoma B16F10 with lung metastases is considered as an adequate model for testing the efficacy of new anticancer therapies in preclinical evaluations and the forecasting model of human disseminated melanoma in anticancer screening (Teicher and Andrews, 2004).

melanoma cells.

154 Research Directions in Tumor Angiogenesis

To assess the effect of T-cadherin expression on melanoma cell proliferation we generated stable cell lines of murine malignant melanoma B16F10 cells (ATCC® №CRL-6475™) by transfecting the cells with pcDNA-Tcad using Lipofectamine™ 2000 reagent (Invitrogen, USA. As a control, luciferase cDNA fragment in the antisense orientation was cloned into the pcDNA 3.1 vector. The cells were cultured and transfected as described before (Yurlova et al., 2010). For stable cell line generation transfected cells were cloned and selected by incubation with 2 mg/ml G418 (Invitrogen, USA). For some experiments we also used the polyclonal mouse melanoma B16F10 cell cultures obtained after transfection and subsequent selection with G418 without cloning. Expression of T-cadherin in polyclonal mouse melanoma cell cultures and melanoma cell clones was examined by Western blotting. Three clones of B16F10 melanoma cells with different level of T-cadherin expression was chosen: a control clone with no Tcadherin (T-), clone with low T-cadherin expression (T+) and clone with high T-cadherin expression (T++) (Yurlova et al., in press).

To investigate the effect of T-cadherin expression on melanoma cell proliferation, cells were seeded on 6-well plates and harvested by trypsinization after 24, 48 and 72 h in culture and counted with the Countess® (Invitrogen, USA). After 3 days, the number of T-cadherinexpressing cells (T+ and T++) was 44-48% higher compared to the control (Fig. 6A). We obtained similar results using the impedance measurement with xCELLigence system: the cell index was continuously monitored with 1 min interval for 5 hrs and with 10 min interval for 96 hrs. (Fig. 6B). The xCELLigence system (Roche, USA) monitors cellular events in real time and measures electrical impedance across microelectrodes integrated on the bottom of E-Plates. Thus, our data indicated that T-cadherin expression in mouse B16F10 melanoma cells in‐ creased their proliferation *in vitro*.

**Figure 6.** T-cadherin expression stimulated proliferation of B16F10 cells *in vitro*. (A) Cells were incubated in RPMI 1640 medium containing 10% FBS for indicated periods of time, harvested and counted using CellCounter. (B) The cell pro‐ liferation assay was performed with xCELLigence system. Attachment of 5000 cells per chamber was monitored with the impedance measurement of the xCELLigence System for 96 hrs. The cell indexes of B16F10 T- (control cells, black), T+ (cells with low level of T-cadherin, blue) and T++ (cells with high level of T-cadherin, red) are shown. Results are presented as the means ± SEM of three independent experiments.

To evaluate the effect of T-cadherin expression on apoptosis of B16F10 cells we performed staining with Annexin V. The B16F10 cell clones were resuspended in 200 µl of Annexin V binding buffer (BD Biosciences, USA), incubated with 5 µl of Annexin V-Phycoerythrin (BD Biosciences, USA) and 10 µl of 7-AAD (BD Biosciences, USA) for 15 min in the dark. The percentage of apoptotic cells was evaluated by flow cytometry (FACSCanto II™, BD Bioscien‐ ces, USA). The fraction and absolute number (data not shown) of apoptotic and dead cells in T-cadherin-expressing clones were not significantly different from the control.

control B16F10 cells, correspondingly (Fig. 7A). Difference between the control and Tcadherin-expressing tumor volumes was statistically significant. The histological analysis showed that tumors formed by the control and T-cadherin-expressing B16F10 clones had different morphology. The tumors generated by the T-cadherin-expressing melanoma clones

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We also compared the spontaneous metastatic potential of the B16F10 clones. 28 days after injection, tumor-bearing mice were sacrificed and their lungs were analyzed. We found that T-cadherin overexpression enhanced spontaneous metastatic activity of B16F10 cells. The B16F10 clones with high level of T-cadherin (T++) formed metastasis in 54.5% cases in com‐ parison to 18.2% and 9.1% formed by T+ and T- cells, respectively. Taken together, these results suggested that the expression of T-cadherin stimulated growth of the primary tumors *in vivo*

)

**Figure 7.** T-cadherin expression in B16F10 cells stimulates tumor growth *in vivo*. (A) 1х10<sup>6</sup> of B16F10 cells (T-, T+, or T+ +) were injected subcutaneously in BDF1 mice (n=11). The tumor volume was measured every 3 day after the tumor cell inoculation. Data represent the mean ± SEM. (B) The necrosis area was calculated as the number of cryosections

Because T-cadherin was shown to inhibit angiogenesis in some models (Rubina et al., 2007) we examined the effect of T-cadherin on the neovascularization of B16F10 primary melanoma sites. For that, cryosections of primary melanomas were stained with anti-CD31 antibody to visualize endothelial cells in the blood vessels penetrating the tumor. The quantitative evaluation revealed a 1.3-1.5-fold decrease in the number of medium size vessels and 1.5-2 fold reduction in capillaries in T-cadherin-expressing primary melanomas compared to the control (Fig. 8). There was no detected difference in the amount of large or stable vessels using double immunofluorescent staining with anti-CD31 and anti-NG2 (marker of pericytes and

containing necrosis foci normalized to the total number of sections. Results are the means ± SEM, p<0.05.

**7. Effect of T-cadherin expression on tumor vascularization**

smooth muscle cells) antibodies (data not shown).

contained wider areas of necrosis (Fig. 7B).

and enhanced their invasion and metastatic potential.

(a) (b)

Thus, in contrast to the earlier study by Kuphal (Kuphal et al., 2009) who found no effect of Tcadherin expression on human melanoma cell proliferation, the current study showed that the expression of T-cadherin in mouse melanoma cells stimulated their proliferation *in vitro* and had no effect on the apoptosis.

### **6. Effect of T-cadherin expression on tumor growth and metastatic potentials of B16F10 clones in vivo**

Animal studies were conducted according to the guidelines of the Institutional Animal Care and Use committee of Cardiology Research Center (permit number 385.06.2009). In order to establish a relationship between T-cadherin expression level and increased proliferation of melanoma cells and melanoma growth in vivo, we injected the clones of B16F10 cells with different level of T-cadherin expression into BDF1 mice. 1х10<sup>6</sup> B16F10 cells (T-, T+, or T++ clones) in 0.3 ml of serum-free media were injected subcutaneously in BDF1 mice (n=11 in each group). 28 days after the injection, animals were sacrificed and primary tumors and lungs were collected. The measurement of the tumors was performed 4 times a week over 28 days. The tissue samples were embedded in Tissue-Tek (Sakura, USA), frozen in liquid nitrogen and stored at -80°C. The cryosections (6 µm) were fixed in 4% formaldehyde. To analyze the necrosis areas, cryosections were stained with Mayer's hematoxylin. Vascularization and stroma content was analyzed using the following primary antibodies: anti-T-cadherin, anti-CD31 to visualize endothelial cells (1:100, BD Biosciences, USA), anti-NG2 to visualize pericytes (1:100, Abcam, USA), anti-CD90 to visualize MSCs (1:100, BD Biosciences, USA). For negative controls, non-specific IgGs were used in similar concentration. Cell nuclei were counterstained with DAPI (Sigma-Aldrich, USA). The sections were analyzed using Leica AF6000 microscope and MetaMorph 5.0 (Universal Imaging, USA). For statistics, five view fields on five random sections for each tumor sample were used. The blood vessels were separated into three groups: capillaries (CD31-positive vessels without lumen and with length <20 µm); medium vessels (CD31-positive vessels with length 20-40 µm), and large vessels (with diameter >40 µm). The contribution of the stroma into the growth of primary tumor was determined as the area of CD90-positive cells in a field normalized to the DAPI-stained area of each cryosection. To evaluate the necrosis area we calculated the number of cryosections containing necrosis foci and normalized it to the total number of assessed sections.

The growth kinetic of tumors was compared within the next 4 weeks and then the histopa‐ thology was studied. The T+ and T++ B16F10 cells generated tumors 2.5 and 3 times larger than control B16F10 cells, correspondingly (Fig. 7A). Difference between the control and Tcadherin-expressing tumor volumes was statistically significant. The histological analysis showed that tumors formed by the control and T-cadherin-expressing B16F10 clones had different morphology. The tumors generated by the T-cadherin-expressing melanoma clones contained wider areas of necrosis (Fig. 7B).

To evaluate the effect of T-cadherin expression on apoptosis of B16F10 cells we performed staining with Annexin V. The B16F10 cell clones were resuspended in 200 µl of Annexin V binding buffer (BD Biosciences, USA), incubated with 5 µl of Annexin V-Phycoerythrin (BD Biosciences, USA) and 10 µl of 7-AAD (BD Biosciences, USA) for 15 min in the dark. The percentage of apoptotic cells was evaluated by flow cytometry (FACSCanto II™, BD Bioscien‐ ces, USA). The fraction and absolute number (data not shown) of apoptotic and dead cells in

Thus, in contrast to the earlier study by Kuphal (Kuphal et al., 2009) who found no effect of Tcadherin expression on human melanoma cell proliferation, the current study showed that the expression of T-cadherin in mouse melanoma cells stimulated their proliferation *in vitro* and

Animal studies were conducted according to the guidelines of the Institutional Animal Care and Use committee of Cardiology Research Center (permit number 385.06.2009). In order to establish a relationship between T-cadherin expression level and increased proliferation of melanoma cells and melanoma growth in vivo, we injected the clones of B16F10 cells with different level of T-cadherin expression into BDF1 mice. 1х10<sup>6</sup> B16F10 cells (T-, T+, or T++ clones) in 0.3 ml of serum-free media were injected subcutaneously in BDF1 mice (n=11 in each group). 28 days after the injection, animals were sacrificed and primary tumors and lungs were collected. The measurement of the tumors was performed 4 times a week over 28 days. The tissue samples were embedded in Tissue-Tek (Sakura, USA), frozen in liquid nitrogen and stored at -80°C. The cryosections (6 µm) were fixed in 4% formaldehyde. To analyze the necrosis areas, cryosections were stained with Mayer's hematoxylin. Vascularization and stroma content was analyzed using the following primary antibodies: anti-T-cadherin, anti-CD31 to visualize endothelial cells (1:100, BD Biosciences, USA), anti-NG2 to visualize pericytes (1:100, Abcam, USA), anti-CD90 to visualize MSCs (1:100, BD Biosciences, USA). For negative controls, non-specific IgGs were used in similar concentration. Cell nuclei were counterstained with DAPI (Sigma-Aldrich, USA). The sections were analyzed using Leica AF6000 microscope and MetaMorph 5.0 (Universal Imaging, USA). For statistics, five view fields on five random sections for each tumor sample were used. The blood vessels were separated into three groups: capillaries (CD31-positive vessels without lumen and with length <20 µm); medium vessels (CD31-positive vessels with length 20-40 µm), and large vessels (with diameter >40 µm). The contribution of the stroma into the growth of primary tumor was determined as the area of CD90-positive cells in a field normalized to the DAPI-stained area of each cryosection. To evaluate the necrosis area we calculated the number of cryosections

T-cadherin-expressing clones were not significantly different from the control.

**6. Effect of T-cadherin expression on tumor growth and metastatic**

containing necrosis foci and normalized it to the total number of assessed sections.

The growth kinetic of tumors was compared within the next 4 weeks and then the histopa‐ thology was studied. The T+ and T++ B16F10 cells generated tumors 2.5 and 3 times larger than

had no effect on the apoptosis.

156 Research Directions in Tumor Angiogenesis

**potentials of B16F10 clones in vivo**

We also compared the spontaneous metastatic potential of the B16F10 clones. 28 days after injection, tumor-bearing mice were sacrificed and their lungs were analyzed. We found that T-cadherin overexpression enhanced spontaneous metastatic activity of B16F10 cells. The B16F10 clones with high level of T-cadherin (T++) formed metastasis in 54.5% cases in com‐ parison to 18.2% and 9.1% formed by T+ and T- cells, respectively. Taken together, these results suggested that the expression of T-cadherin stimulated growth of the primary tumors *in vivo* and enhanced their invasion and metastatic potential.

**Figure 7.** T-cadherin expression in B16F10 cells stimulates tumor growth *in vivo*. (A) 1х10<sup>6</sup> of B16F10 cells (T-, T+, or T+ +) were injected subcutaneously in BDF1 mice (n=11). The tumor volume was measured every 3 day after the tumor cell inoculation. Data represent the mean ± SEM. (B) The necrosis area was calculated as the number of cryosections containing necrosis foci normalized to the total number of sections. Results are the means ± SEM, p<0.05.

#### **7. Effect of T-cadherin expression on tumor vascularization**

Because T-cadherin was shown to inhibit angiogenesis in some models (Rubina et al., 2007) we examined the effect of T-cadherin on the neovascularization of B16F10 primary melanoma sites. For that, cryosections of primary melanomas were stained with anti-CD31 antibody to visualize endothelial cells in the blood vessels penetrating the tumor. The quantitative evaluation revealed a 1.3-1.5-fold decrease in the number of medium size vessels and 1.5-2 fold reduction in capillaries in T-cadherin-expressing primary melanomas compared to the control (Fig. 8). There was no detected difference in the amount of large or stable vessels using double immunofluorescent staining with anti-CD31 and anti-NG2 (marker of pericytes and smooth muscle cells) antibodies (data not shown).

Similar results were obtained using melanoma primary tumors formed by polyclonal mouse melanoma cell culture (data not shown). We concluded that the effects of T-cadherin were not related to the individual features of the selected clones.

**Figure 8.** T-cadherin expression in B16F10 cells caused the reduction in tumor vascularization. 28 days after the mela‐ noma cell inoculation, sections of primary tumors formed by the B16F10 T-, T+ or T++ cells were stained with anti-CD31 and anti-T-cadherin antibodies. The quantitative assessment of the blood vessels from 30 random fields of five independent tumors is presented. The results are the means ± SEM of three independent experiments, p<0.05.

These results suggested that T-cadherin overexpression in B16F10 melanoma cells suppresses tumor neovascularization by limiting tumor neoangiogenesis.

#### **8. Effect of T-cadherin expression on host stroma**

Over the past decade it was discovered that heterogeneous population of progenitor cells known as multipotent stromal cells or mesenchymal stem cells (MSCs) derived from the bone marrow or adipose tissue exhibited a marked tropism for tumors (Klopp et al., 2011). Circu‐ lating in the blood stream, MSC from the bone marrow or resident mesenchymal stromal cells could engraft within the tumor microenvironment and incorporate into the stroma of solid tumors as tumor-associated fibroblasts and contribute to the growth of the primary tumor sites (Mishra et al., 2008; Spaeth et al., 2008). MSCs can also act as pericytes-like cells and potentiate tumor growth, vascularization and metastasis. The mechanism by which MSCs support the tumor growth and progression is in the intercellular interactions with tumor cells and the release of the paracrine signals (Spaeth et al., 2009). MSCs themselves are likely to respond to chemoattractants similar to many immune cells that migrate to injury or inflammation site (Spaeth et al., 2008).

To examine whether T-cadherin expression influences the recruitment/proliferation of stromal cells in the model of mouse melanoma growth and progression, cryosections of primary tumor sites were stained with anti-CD90 antibody to visualize the activated stroma (Campioni et al., 2008) (Fig. 9A). Immunofluorescent analysis revealed a 2.4–2.9-fold increase in the CD90

\*

\*

\*

\*

positive areas in the T-cadherin-expressing tumor samples compared to the controls (Fig. 9B). CD90-positive cells were arranged in a form of cell aggregates among the tumor cells or located perivascular around CD31-positive vessels structures. Thus, for the first time we revealed that the expression of T-cadherin stimulated the recruitment of CD90-positive cells to the primary tumor site. The CD90-positive cells were represented mainly by MSCs. However, some populations of neutrophils, T cells and monocytes could also express CD90

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To support the *in vivo* data on the T-cadherin-mediated recruitment of the stromal cells to the growing tumor, we established a transwell assay in which MSCs were co-cultured with the melanoma clones with different T-cadherin expression. The transwell system allowed exchange of the medium in the absence of direct interaction between cells in the lower and upper chambers. The MSCs were isolated from subcutaneous adipose tissue of the ingui‐ nal region of CBA/C57BL male mice and cultured until the 2nd passage as described before (Rubina et al., 2009). To assess the effect of T-cadherin expression in melanoma cells on their ability to induce MSC migration, MSCs were seeded in the upper chamber and melanoma cell clones were seeded in the lower chamber. The MSCs were allowed to migrate across the collagene-covered membrane and the conditioned medium from the B16F10 clones served as a chemoattractant. We found that migration of MSCs towards the conditioned medium from the T++ B16F10 cells was at least 1.5-fold higher than towards the T+ or T- B16F10 cells

We did not detect the shedding of T-cadherin into the conditioned medium from the Tcadherin expressing B16F10 clones using Western blotting (data not shown). Thus, we suggested that the observed effects on MSCs migration were mediated by secretion of chemoattractants and growth factors into the conditioned medium by the T-cadherin express‐ ing melanoma cells. To prove that we studied the expression of angiogenec factors, extracel‐ lular matrix proteins, adhesion molecules and chemokines using the PCR Array assay

**9. T-cadherin expression changes the gene expression pattern in B16F10**

For PCR Array assay, total RNA was isolated from B16F10 clones using RNeasy Mini kit. 1 µg of total RNA was treated with DNase, cDNA was prepared using RT2 First Strand kit (SABiosciences, USA). For each experiment, cDNA sample was mixed with RT2 qPCR Master mix and distributed across the PCR array 96-well plates. After cycling with real-time PCR (IQ5 PCR platform, Bio Rad, USA), obtained amplification data (fold-changes in Ct values) was analyzed with SABiosciences software. RNA expression of each gene was normalized using 5 housekeeping genes. The relative expression of each gene, compared to expression in the control B16F10 clone was calculated on the website using ∆∆Ct method. A gene was considered as differentially regulated if the difference was >2-fold compared with the control

and be recruited to the tumors (Rege and Hagood, 2006).

(SABiosciences, USA) and quantitative PCR.

(Fig. 10).

**melanoma cells**

clone.

positive areas in the T-cadherin-expressing tumor samples compared to the controls (Fig. 9B). CD90-positive cells were arranged in a form of cell aggregates among the tumor cells or located perivascular around CD31-positive vessels structures. Thus, for the first time we revealed that the expression of T-cadherin stimulated the recruitment of CD90-positive cells to the primary tumor site. The CD90-positive cells were represented mainly by MSCs. However, some populations of neutrophils, T cells and monocytes could also express CD90 and be recruited to the tumors (Rege and Hagood, 2006).

Similar results were obtained using melanoma primary tumors formed by polyclonal mouse melanoma cell culture (data not shown). We concluded that the effects of T-cadherin were not

> B16F10 T-B16F10 T+ B16F10 T++

median vessels capillaries

tumor neovascularization by limiting tumor neoangiogenesis.

**8. Effect of T-cadherin expression on host stroma**

**Figure 8.** T-cadherin expression in B16F10 cells caused the reduction in tumor vascularization. 28 days after the mela‐ noma cell inoculation, sections of primary tumors formed by the B16F10 T-, T+ or T++ cells were stained with anti-CD31 and anti-T-cadherin antibodies. The quantitative assessment of the blood vessels from 30 random fields of five independent tumors is presented. The results are the means ± SEM of three independent experiments, p<0.05.

These results suggested that T-cadherin overexpression in B16F10 melanoma cells suppresses

Over the past decade it was discovered that heterogeneous population of progenitor cells known as multipotent stromal cells or mesenchymal stem cells (MSCs) derived from the bone marrow or adipose tissue exhibited a marked tropism for tumors (Klopp et al., 2011). Circu‐ lating in the blood stream, MSC from the bone marrow or resident mesenchymal stromal cells could engraft within the tumor microenvironment and incorporate into the stroma of solid tumors as tumor-associated fibroblasts and contribute to the growth of the primary tumor sites (Mishra et al., 2008; Spaeth et al., 2008). MSCs can also act as pericytes-like cells and potentiate tumor growth, vascularization and metastasis. The mechanism by which MSCs support the tumor growth and progression is in the intercellular interactions with tumor cells and the release of the paracrine signals (Spaeth et al., 2009). MSCs themselves are likely to respond to chemoattractants similar to many immune cells that migrate to injury or inflammation site

To examine whether T-cadherin expression influences the recruitment/proliferation of stromal cells in the model of mouse melanoma growth and progression, cryosections of primary tumor sites were stained with anti-CD90 antibody to visualize the activated stroma (Campioni et al., 2008) (Fig. 9A). Immunofluorescent analysis revealed a 2.4–2.9-fold increase in the CD90

related to the individual features of the selected clones.

(Spaeth et al., 2008).

Blood vessel number / field

158 Research Directions in Tumor Angiogenesis

To support the *in vivo* data on the T-cadherin-mediated recruitment of the stromal cells to the growing tumor, we established a transwell assay in which MSCs were co-cultured with the melanoma clones with different T-cadherin expression. The transwell system allowed exchange of the medium in the absence of direct interaction between cells in the lower and upper chambers. The MSCs were isolated from subcutaneous adipose tissue of the ingui‐ nal region of CBA/C57BL male mice and cultured until the 2nd passage as described before (Rubina et al., 2009). To assess the effect of T-cadherin expression in melanoma cells on their ability to induce MSC migration, MSCs were seeded in the upper chamber and melanoma cell clones were seeded in the lower chamber. The MSCs were allowed to migrate across the collagene-covered membrane and the conditioned medium from the B16F10 clones served as a chemoattractant. We found that migration of MSCs towards the conditioned medium from the T++ B16F10 cells was at least 1.5-fold higher than towards the T+ or T- B16F10 cells (Fig. 10).

We did not detect the shedding of T-cadherin into the conditioned medium from the Tcadherin expressing B16F10 clones using Western blotting (data not shown). Thus, we suggested that the observed effects on MSCs migration were mediated by secretion of chemoattractants and growth factors into the conditioned medium by the T-cadherin express‐ ing melanoma cells. To prove that we studied the expression of angiogenec factors, extracel‐ lular matrix proteins, adhesion molecules and chemokines using the PCR Array assay (SABiosciences, USA) and quantitative PCR.

### **9. T-cadherin expression changes the gene expression pattern in B16F10 melanoma cells**

For PCR Array assay, total RNA was isolated from B16F10 clones using RNeasy Mini kit. 1 µg of total RNA was treated with DNase, cDNA was prepared using RT2 First Strand kit (SABiosciences, USA). For each experiment, cDNA sample was mixed with RT2 qPCR Master mix and distributed across the PCR array 96-well plates. After cycling with real-time PCR (IQ5 PCR platform, Bio Rad, USA), obtained amplification data (fold-changes in Ct values) was analyzed with SABiosciences software. RNA expression of each gene was normalized using 5 housekeeping genes. The relative expression of each gene, compared to expression in the control B16F10 clone was calculated on the website using ∆∆Ct method. A gene was considered as differentially regulated if the difference was >2-fold compared with the control clone.

\*

\*

\*

\*

**Figure 9.** The effect of T-cadherin expression in B16F10 cells on MSC recruitment. (A) 28 days after melanoma cell inoculation, sections of the primary tumor formed by the B16F10 T- (left), T+ (middle), or T++ (right) cells were stained with anti-CD90 (red) and with anti-T-cadherin (green) antibody. Bars - 100 µm. (B) The contribution of the stroma into the growth of primary tumor was determined as an area of CD90-positive cells in a field normalized to the DAPIstained area unit of each cryosection using program MetaMorph 5.0. Results are the means ± SEM of three independ‐ ent experiments, p<0.05.

For quantitative PCR the RNeasy Mini Kit (Qiagen, Germany) was used to extract the total RNA. cDNA were prepared using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, USA). The primers were obtained from Sintol (Russia). Real-time qPCR analysis was performed with SYBR Green I on Rotor – Gene™ 3000 (Corbett Research, UK). The gene expression was normalized to the expression of β-actin and GAPDH. The primer specificity was confirmed by melting curve analysis. The qRT-PCR was repeated five times.

We performed quantitative PCR and PCR Array Assay of melanoma cells and revealed that T-cadherin expression in B16F10 melanoma cells resulted the increase in expression of chemokines CXCL10, CCL5, CXCL11 and CCL7, which were earlier implicated in the growth and metastasis of different neuroectodermal tumors (Somasundaram and Herlyn, 2009). In a screen of several human and mouse melanoma cell lines, it was detected that the expression of chemokine receptors CCR7, CCR10, CXCR1, CXCR2 and CXCR4 could dramatically increase the rate of metastases (Longo-Imedio et al., 2005; Simonetti et al., 2006; Singh et al., 2009; Wiley et al., 2001). In the present study, the up-regulation of CXCL10, CCL5, CXCL11 and CCL7 genes was found to be correlated with the increase in spontaneous metastatic activity in the B16F10 mouse melanoma model.

In normal conditions, the blood vessel growth is strictly controlled by the balance between pro-angiogenic and and anti-angiogenic factors. At the same time, tumor progression is accompanied by neoangiogenesis due to enhanced production of pro-angiogenic molecules by tumor and stromal cells (Hanahan and Folkman, 1996). To elucidate the mechanisms

**Gene Annealing**

**temperature**

**Table 1.** Sequences of primers used in qRT-PCR.

Migration cell number

MSCs/ 10%FBS

MSCs/ NIH3Т3

are the means ± SEM of three independent experiments performed in duplicates, p<0.05.

**Forward 5'-3'**

MSCs/ B16F10 T-

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits…

**Figure 10.** The effect of conditioned medium from the B16F10 cell clones with different T-cadherin expression on mi‐ gration of MSCs. Migrated MSCs were quantified after fixation and hematoxylin staining of the membrane. The results

GAPDH 60 GACCCCTTCATTGACCTCAACTAC TGGTGGTGCAGGATGCATTGCTGA β-actin 61 AGTGTGACGTTGACATCCGTA GCCAGAGCAGTAATCTCCTTCT VEGF А 60 AGAGCAGAAGTCCCATGAAGTGA TCAATCGGACGGCAGTAGCT PDGF В 58,5 TCTCTGCTGCTACCTGCGTCTGG GTGTGCTCGGGTCATGTTCAAGTC HGF 60 TCATTGGTAAAGGAGGCAGCTATA CTGGCATTTGATGCCACTCTTA MMP2 53 AGTTCCCGTTCCGCTTCC GACACATGGGGCACCTTCTG EGF 60 CCTGCCCCCTTCCTAGTTTTC CTCCGTTCTGTTGGTCTACCC uPAR 60 CGTTACCTCGAGTGTGCGTCCTG AGCCTCGGGTGTAGTCCTCATCCT c-Met 60 CAACGAGAGCTGTACCTTGACCTTA GCGGGACCAACTGTGCAT uPA 53 GAATGCGCCTGCTGTC AGGGTCGCTTCTGGTTGTC MMP9 61 GCGGTGTGGGGCGAGGTG CCAGGGGGAAAGGCGTGTG

MSCs/ B16F10 T+

MSCs/ B16F10 T++

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161

**Reverse 5'-3'**

\*

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits… http://dx.doi.org/10.5772/53350 161

**Figure 10.** The effect of conditioned medium from the B16F10 cell clones with different T-cadherin expression on mi‐ gration of MSCs. Migrated MSCs were quantified after fixation and hematoxylin staining of the membrane. The results are the means ± SEM of three independent experiments performed in duplicates, p<0.05.


**Table 1.** Sequences of primers used in qRT-PCR.

For quantitative PCR the RNeasy Mini Kit (Qiagen, Germany) was used to extract the total RNA. cDNA were prepared using the RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas, USA). The primers were obtained from Sintol (Russia). Real-time qPCR analysis was performed with SYBR Green I on Rotor – Gene™ 3000 (Corbett Research, UK). The gene expression was normalized to the expression of β-actin and GAPDH. The primer specificity

**Figure 9.** The effect of T-cadherin expression in B16F10 cells on MSC recruitment. (A) 28 days after melanoma cell inoculation, sections of the primary tumor formed by the B16F10 T- (left), T+ (middle), or T++ (right) cells were stained with anti-CD90 (red) and with anti-T-cadherin (green) antibody. Bars - 100 µm. (B) The contribution of the stroma into the growth of primary tumor was determined as an area of CD90-positive cells in a field normalized to the DAPIstained area unit of each cryosection using program MetaMorph 5.0. Results are the means ± SEM of three independ‐

B16F10 T- B16F10 T+ B16F10 T++

We performed quantitative PCR and PCR Array Assay of melanoma cells and revealed that T-cadherin expression in B16F10 melanoma cells resulted the increase in expression of chemokines CXCL10, CCL5, CXCL11 and CCL7, which were earlier implicated in the growth and metastasis of different neuroectodermal tumors (Somasundaram and Herlyn, 2009). In a screen of several human and mouse melanoma cell lines, it was detected that the expression of chemokine receptors CCR7, CCR10, CXCR1, CXCR2 and CXCR4 could dramatically increase the rate of metastases (Longo-Imedio et al., 2005; Simonetti et al., 2006; Singh et al., 2009; Wiley et al., 2001). In the present study, the up-regulation of CXCL10, CCL5, CXCL11 and CCL7 genes was found to be correlated with the increase in spontaneous metastatic activity

was confirmed by melting curve analysis. The qRT-PCR was repeated five times.

in the B16F10 mouse melanoma model.

(a)

160 Research Directions in Tumor Angiogenesis

(b)

ent experiments, p<0.05.

In normal conditions, the blood vessel growth is strictly controlled by the balance between pro-angiogenic and and anti-angiogenic factors. At the same time, tumor progression is accompanied by neoangiogenesis due to enhanced production of pro-angiogenic molecules by tumor and stromal cells (Hanahan and Folkman, 1996). To elucidate the mechanisms \*

responsible for T-cadherin-mediated suppression of angiogenesis in primary melanoma we performed qRT PCR and PCR Arrays. No difference in the mRNA expression level of the main angiogenic growth factors such as VEGF А, HGF, bFGF, EGF, PDGF B, TGFβ be‐ tween control and T-cadherin-expressing clones was revealed. However, PCR Array analysis revealed that T-cadherin expression in B16F10 cells resulted in upregulation of mRNA of such antiangiogenic molecules as CXCL 10 (Strieter et al., 1995); angiopoietin 2 (Cao et al., 2007); procollagen type XVIIIα1 - a precursor of the angiogenesis inhibitor endostatin (O'Reilly et al., 1997) and chromogranin A - a precursor of angiogenesis inhibitor vasosta‐ tin-1 (Belloni et al., 2007). Angiopoietin 2 acts together with VEGF A in initiating blood vessel growth through inhibition of the interactions between endothelial and perivascular cells and destabiliziation of blood vessels. However, in the absence of VEGF A, angiopoietin 2 suppresses angiogenesis and promotes vessel regression (Holash et al., 1999). Since in the present study VEGF A level was not changed after the expression of T-cadherin, the elevated angiopoietin 2 expression could act in reducing the number of newly formed vessels. We also found that the T-cadherin-expressing B16F10 cells demonstrated decreased expression of angiogenic molecules TGFα (Leker et al., 2009) and Tie 1 (Sato et al., 1995). Thus, the PCR Array analysis indicated that the balance between the pro-angiogenic and anti-angiogenic factors was shifted towards the latter, which could reduce the number of medium size vessels and capillaries in vivo.

It was shown that the overexpression of some integrins can induce matrix metalloproteinases (MMPs) expression in melanoma cells or their activation (Khatib et al., 2001; Sil et al., 2011). Several MMPs including MMP-1, -2, -3, -7, -9, -13, -14, -15, -16 as well as uPA were implicated in human melanoma progression, invasion and metastasis (Bianchini et al., 2006; Ria et al., 2010). Using qRT-PCR we examined the expression of mRNA of MMPs with gelatinase activity and uPA. No differences in MMP2, MMP9 and uPA expression in the control and T-cadhering positive B16F10 cell clones were revealed. Further PCR The array analysis established that MMP14 was the only protease with enhanced expression detected in the T-cadherin expressing

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits…

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163

Melanoma cells express multiple isoforms of laminin that were shown to mediate cell attach‐ ment and invasion via integrin receptors using laminin as a substrate (Oikawa et al., 2011). In addition, expression of fibronectin was correlated with the acquisition of invasive and metastatic behavior of human melanoma (Gaggioli et al., 2007). Using PCR Array analysis we found that T-cadherin overexpressing melanoma cells exhibited the elevated level of fibro‐ nectin 1 and laminin α3 expression suggesting their role in the increased metastatic potential of these cells. The obtained data indicated that T-cadherin expression affects the expression of

In contrast to the results obtained in the present study, the re-expression of T-cadherin by stable transfection in human melanoma cells reduced the rate of tumor growth in the nu/nu mouse tumor model, decreased cell capacity for anchorage-independent growth, migration and invasion in vitro, while cell proliferation was not affected (Kuphal et al., 2009).This discrepancy could be due to the differences in the experimental conditions of the models (highly aggressive murine melanoma B16F10 in the BDF1 mice versus human melanoma cells injected into immunodeficient nu/nu mice). The difference could also be explained by the distinct signaling pathways and spectrum growth factors and receptors expressed by mouse and human

In the present study, we found that T-cadherin is expressed in normal epidermal keratinocytes, vascular cells of the dermal blood vessels and melanocytes in the human skin. However, upon malignant transformation we observed mosaic pattern of T-cadherin expression in primary melanomas and partial or complete loss of T-cadherin in melanoma metastasis. These data are in accordance with the earlier published results and confirmed the correlation between tumor progression and the loss of T-cadherin expression. It was previously reported that 80% of the human melanoma cell lines did not express T-cadherin and re-expression of T-cadherin reduced the tumorigenicity of these cell lines in nu/nu mouse model. However, 20% of human melanoma cell lines abundantly expressed T-cadherin and possessed invasive and metastatic potential. This prompted us to use a well-described model of highly aggressive murine melanoma B16F10 in BDF1 mice and examine the effect of T-cadherin expression in melanoma cells on their proliferation, tumor growth, invasive and metastatic potential and neovascula‐

certain genes involved in regulation of melanoma growth and progression.

clone T++.

melanoma.

**10. Conclusions**

It is well known that MSCs secrete many growth factors and cytokines and their production increases in hypoxic conditions (Rubina et al., 2009; Martin-Rendon 2007). Among them is HGF/SF, which is a potent stimulator of DNA synthesis and growth in normal human melanocytes and melanoma cells (Halaban et al., 1993). Thus, it was shown that overexpression of the proto-oncogene c-Met (HGFR - HGF receptor) is tightly correlated with human mela‐ noma progression from the radial to the vertical stage (Natali et al., 1993). In the present study we found that control B16F10 melanoma cells expressed low levels of c-Met and no HGF. Our quantitative PCR analysis demonstrated that T-cadherin overexpression resulted in 6-fold increase in the content of c-Met mRNA in melanoma cells. This data suggested that one of the mechanisms by which T-cadherin could be able to affect the growth of B16F10 melanoma cells is the regulation of c-Met/HGF signaling pathway. We speculated that the melanoma cells expressing T-cadherin could secrete the high levels of chemokines resulting in MSCs recruit‐ ment to the primary tumor site. MSCs in hypoxic conditions are known to increase the production of HGF (Rubina et al., 2009). Thus, in hypoxic conditions of the primary tumor the recruited MSCs could produce the high levels of HGF, which upon binding to c-Met on melanoma cells could cause their increased proliferation and invasion

Apparently, invasive and metastasizing cancers are characterized by the change in integrin expression pattern (Makrilia et al., 2009). Thus, the overexpressions of integrins such as α3, α5 and α1 or their single subunits were shown to be involved in melanoma growth and progression (Kuphal et al., 2005). So we compared the expression level of certain integrins in the control and T-cadherin expressing melanoma cells using PCR Array. We revealed the upregulation of mRNA expression of α5, αV, αE, and β3 integrins upon T-cadherinsion. This correlated with the increase in the metastatic activity of those cells and possibly contributed to melanoma progression.

It was shown that the overexpression of some integrins can induce matrix metalloproteinases (MMPs) expression in melanoma cells or their activation (Khatib et al., 2001; Sil et al., 2011). Several MMPs including MMP-1, -2, -3, -7, -9, -13, -14, -15, -16 as well as uPA were implicated in human melanoma progression, invasion and metastasis (Bianchini et al., 2006; Ria et al., 2010). Using qRT-PCR we examined the expression of mRNA of MMPs with gelatinase activity and uPA. No differences in MMP2, MMP9 and uPA expression in the control and T-cadhering positive B16F10 cell clones were revealed. Further PCR The array analysis established that MMP14 was the only protease with enhanced expression detected in the T-cadherin expressing clone T++.

Melanoma cells express multiple isoforms of laminin that were shown to mediate cell attach‐ ment and invasion via integrin receptors using laminin as a substrate (Oikawa et al., 2011). In addition, expression of fibronectin was correlated with the acquisition of invasive and metastatic behavior of human melanoma (Gaggioli et al., 2007). Using PCR Array analysis we found that T-cadherin overexpressing melanoma cells exhibited the elevated level of fibro‐ nectin 1 and laminin α3 expression suggesting their role in the increased metastatic potential of these cells. The obtained data indicated that T-cadherin expression affects the expression of certain genes involved in regulation of melanoma growth and progression.

In contrast to the results obtained in the present study, the re-expression of T-cadherin by stable transfection in human melanoma cells reduced the rate of tumor growth in the nu/nu mouse tumor model, decreased cell capacity for anchorage-independent growth, migration and invasion in vitro, while cell proliferation was not affected (Kuphal et al., 2009).This discrepancy could be due to the differences in the experimental conditions of the models (highly aggressive murine melanoma B16F10 in the BDF1 mice versus human melanoma cells injected into immunodeficient nu/nu mice). The difference could also be explained by the distinct signaling pathways and spectrum growth factors and receptors expressed by mouse and human melanoma.

#### **10. Conclusions**

responsible for T-cadherin-mediated suppression of angiogenesis in primary melanoma we performed qRT PCR and PCR Arrays. No difference in the mRNA expression level of the main angiogenic growth factors such as VEGF А, HGF, bFGF, EGF, PDGF B, TGFβ be‐ tween control and T-cadherin-expressing clones was revealed. However, PCR Array analysis revealed that T-cadherin expression in B16F10 cells resulted in upregulation of mRNA of such antiangiogenic molecules as CXCL 10 (Strieter et al., 1995); angiopoietin 2 (Cao et al., 2007); procollagen type XVIIIα1 - a precursor of the angiogenesis inhibitor endostatin (O'Reilly et al., 1997) and chromogranin A - a precursor of angiogenesis inhibitor vasosta‐ tin-1 (Belloni et al., 2007). Angiopoietin 2 acts together with VEGF A in initiating blood vessel growth through inhibition of the interactions between endothelial and perivascular cells and destabiliziation of blood vessels. However, in the absence of VEGF A, angiopoietin 2 suppresses angiogenesis and promotes vessel regression (Holash et al., 1999). Since in the present study VEGF A level was not changed after the expression of T-cadherin, the elevated angiopoietin 2 expression could act in reducing the number of newly formed vessels. We also found that the T-cadherin-expressing B16F10 cells demonstrated decreased expression of angiogenic molecules TGFα (Leker et al., 2009) and Tie 1 (Sato et al., 1995). Thus, the PCR Array analysis indicated that the balance between the pro-angiogenic and anti-angiogenic factors was shifted towards the latter, which could reduce the number of medium size vessels

It is well known that MSCs secrete many growth factors and cytokines and their production increases in hypoxic conditions (Rubina et al., 2009; Martin-Rendon 2007). Among them is HGF/SF, which is a potent stimulator of DNA synthesis and growth in normal human melanocytes and melanoma cells (Halaban et al., 1993). Thus, it was shown that overexpression of the proto-oncogene c-Met (HGFR - HGF receptor) is tightly correlated with human mela‐ noma progression from the radial to the vertical stage (Natali et al., 1993). In the present study we found that control B16F10 melanoma cells expressed low levels of c-Met and no HGF. Our quantitative PCR analysis demonstrated that T-cadherin overexpression resulted in 6-fold increase in the content of c-Met mRNA in melanoma cells. This data suggested that one of the mechanisms by which T-cadherin could be able to affect the growth of B16F10 melanoma cells is the regulation of c-Met/HGF signaling pathway. We speculated that the melanoma cells expressing T-cadherin could secrete the high levels of chemokines resulting in MSCs recruit‐ ment to the primary tumor site. MSCs in hypoxic conditions are known to increase the production of HGF (Rubina et al., 2009). Thus, in hypoxic conditions of the primary tumor the recruited MSCs could produce the high levels of HGF, which upon binding to c-Met on

Apparently, invasive and metastasizing cancers are characterized by the change in integrin expression pattern (Makrilia et al., 2009). Thus, the overexpressions of integrins such as α3, α5 and α1 or their single subunits were shown to be involved in melanoma growth and progression (Kuphal et al., 2005). So we compared the expression level of certain integrins in the control and T-cadherin expressing melanoma cells using PCR Array. We revealed the upregulation of mRNA expression of α5, αV, αE, and β3 integrins upon T-cadherinsion. This correlated with the increase in the metastatic activity of those cells and possibly contributed

melanoma cells could cause their increased proliferation and invasion

and capillaries in vivo.

162 Research Directions in Tumor Angiogenesis

to melanoma progression.

In the present study, we found that T-cadherin is expressed in normal epidermal keratinocytes, vascular cells of the dermal blood vessels and melanocytes in the human skin. However, upon malignant transformation we observed mosaic pattern of T-cadherin expression in primary melanomas and partial or complete loss of T-cadherin in melanoma metastasis. These data are in accordance with the earlier published results and confirmed the correlation between tumor progression and the loss of T-cadherin expression. It was previously reported that 80% of the human melanoma cell lines did not express T-cadherin and re-expression of T-cadherin reduced the tumorigenicity of these cell lines in nu/nu mouse model. However, 20% of human melanoma cell lines abundantly expressed T-cadherin and possessed invasive and metastatic potential. This prompted us to use a well-described model of highly aggressive murine melanoma B16F10 in BDF1 mice and examine the effect of T-cadherin expression in melanoma cells on their proliferation, tumor growth, invasive and metastatic potential and neovascula‐ rization. We showed that overexpression of T-cadherin in melanoma B16F10 cells resulted in the increased tumor growth and metastasi as well as the recruitment of MSC into the primary site. We suggested that in response to the chemoattractants (chemokines) produced by the Tcadherin-expressing tumors, the stromal cells migrated into the primary site and produced HGF. In return, HGF triggered the HGF/c-Met signaling cascade in T-cadherin-expressing melanoma cells that could lead to their increased proliferation and metastasis. The elevated expression level of prooncogenic integrins, the extracellular matrix components and MMP14 in these cells could be contributing factors to enhanced metastatic and invasive potential. However, T-cadherin expression in melanoma cells exerted inhibitory effect on vascularization of the primary tumors, which is likely to be due to the switch in the balance of pro- and antiangiogenic molecules. The established link between the expression of T-cadherin and pathological processes that trigger neovascularization and tumor progression are particularly important in search for new approaches for inhibiting metastasis of the less curable tumors such as disseminated melanoma of the skin. Further investigations studies are needed to identify the role of T-cadherin in the initiation of tumor progression associated with the regulation of neoangiogenesis. Our studies provided new evidence on the role of tumor microenvironment and will help to identify the critical points for suppressing the blood supply at the early stages of tumor progression. We concluded that the expression of T-cadherin in melanoma cells underlies a novel mechanism of stem cell tropism to malignant solid tumors, which may be important for the development of the optimal stem cell-based therapy. Inves‐ tigation of such mechanisms is an important task in finding new targets for cancer treatment.

3 N.N. Blokhin Russian Cancer Research Center of Russian Academy of Medical Sciences,

T-Cadherin Stimulates Melanoma Cell Proliferation and Mesenchymal Stromal Cell Recruitment, but Inhibits…

http://dx.doi.org/10.5772/53350

165

4 Division of Developmental Neurobiology, MRC National Institute for Medical Research,

[1] Adachi Y, Takeuchi T, Nagayama T, Ohtsuki Y, & Furihata M (2009) Zeb1-mediated T-cadherin repression increases the invasive potential of gallbladder cancer. FEBS

[2] Adachi Y, Takeuchi T, Sonobe H & Ohtsuki Y (2006) An adiponectin receptor, T-cad‐ herin, was selectively expressed in intratumoral capillary endothelial cells in hepato‐ cellular carcinoma: possible cross talk between T-cadherin and FGF-2 pathways.

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London, UK

**References**

#### **Grant support**

This work was supported by a grant (№ 16.512.12.2005) of the Ministry for Education and Science of Russian Federation and grant (№ 08-04-01024-а) of Russian Foundation for Basic Research.

#### **Author details**

K. A. Rubina1\*, E. I. Yurlova<sup>1</sup> , V. Yu. Sysoeva<sup>1</sup> , E. V. Semina2 , N. I. Kalinina1 , A. A. Poliakov4 , I. N. Mikhaylova3 , N. V. Andronova3 and H. M. Treshalina3

\*Address all correspondence to: rkseniya@mail.ru

1 Faculty of Basic Medicine, M.V. Lomonosov Moscow State University, Moscow, Russian Federation

2 Institute of Experimental Cardiology, Cardiology Research Center of Russia, Moscow, Russian Federation

3 N.N. Blokhin Russian Cancer Research Center of Russian Academy of Medical Sciences, Moscow, Russian Federation

4 Division of Developmental Neurobiology, MRC National Institute for Medical Research, London, UK

#### **References**

rization. We showed that overexpression of T-cadherin in melanoma B16F10 cells resulted in the increased tumor growth and metastasi as well as the recruitment of MSC into the primary site. We suggested that in response to the chemoattractants (chemokines) produced by the Tcadherin-expressing tumors, the stromal cells migrated into the primary site and produced HGF. In return, HGF triggered the HGF/c-Met signaling cascade in T-cadherin-expressing melanoma cells that could lead to their increased proliferation and metastasis. The elevated expression level of prooncogenic integrins, the extracellular matrix components and MMP14 in these cells could be contributing factors to enhanced metastatic and invasive potential. However, T-cadherin expression in melanoma cells exerted inhibitory effect on vascularization of the primary tumors, which is likely to be due to the switch in the balance of pro- and antiangiogenic molecules. The established link between the expression of T-cadherin and pathological processes that trigger neovascularization and tumor progression are particularly important in search for new approaches for inhibiting metastasis of the less curable tumors such as disseminated melanoma of the skin. Further investigations studies are needed to identify the role of T-cadherin in the initiation of tumor progression associated with the regulation of neoangiogenesis. Our studies provided new evidence on the role of tumor microenvironment and will help to identify the critical points for suppressing the blood supply at the early stages of tumor progression. We concluded that the expression of T-cadherin in melanoma cells underlies a novel mechanism of stem cell tropism to malignant solid tumors, which may be important for the development of the optimal stem cell-based therapy. Inves‐ tigation of such mechanisms is an important task in finding new targets for cancer treatment.

This work was supported by a grant (№ 16.512.12.2005) of the Ministry for Education and Science of Russian Federation and grant (№ 08-04-01024-а) of Russian Foundation for Basic

, E. V. Semina2

and H. M. Treshalina3

1 Faculty of Basic Medicine, M.V. Lomonosov Moscow State University, Moscow, Russian

2 Institute of Experimental Cardiology, Cardiology Research Center of Russia, Moscow,

, N. I. Kalinina1

, A. A. Poliakov4

,

, V. Yu. Sysoeva<sup>1</sup>

, N. V. Andronova3

\*Address all correspondence to: rkseniya@mail.ru

**Grant support**

164 Research Directions in Tumor Angiogenesis

**Author details**

I. N. Mikhaylova3

Russian Federation

Federation

K. A. Rubina1\*, E. I. Yurlova<sup>1</sup>

Research.


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**Chapter 7**

**The Use of Artemisinin Compounds as**

Qigui Li, Peter Weina and Mark Hickman

http://dx.doi.org/10.5772/54109

**1. Introduction**

Additional information is available at the end of the chapter

**Angiogenesis Inhibitors to Treat Cancer**

Angiogenesis takes place during development, and vascular remodeling is a controlled ser‐ ies of events leading to neovascularization, which supports changing tissue requirements. Blood vessels and stromal components are responsive to pro- and anti-angiogenic factors that allow vascular remodeling during development, wound healing and pregnancy. In pathological situations such as cancer, however, the same angiogenic signaling pathways are induced and exploited. Cancer angiogenesis is a requirement for the development and growth of solid tumors beyond 2–3 mm3 (Cao et al., 2011). Several angiogenic activators in‐ cluding members of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) gene families and various inhibitors of angiogenesis have been described. In steady-state conditions, the balance between angiogenic activators and inhibitors results in very limited new blood vessel growth in the majority of tissues. The balance tilts in favor of the angiogenic stimulators, however, in a variety of proliferative processes. It is now gener‐ ally accepted that angiogenesis is a rate-limiting process in tumor growth. Without new blood vessels to supply nutrients and dispose of catabolic products, tumor cells cannot sus‐ tain proliferation and thus are likely to remain dormant (Ferrara, 2010; Daniele et al., 2012).

Survival and proliferation of cancer depends on angiogenesis, which could be a target of can‐ cer therapy. Angiogenesis is a complex physiological process. One example of this is found in the signaling pathways associated with the stimulus of various pro-angiogenic factors, VEGF and its receptors (VEGFR) which represents one of the best-validated signaling pathways in angiogenesis. A number of drugs approved by the FDA on market have been shown to inhibit anti-angiogenic pathway of VEGF. These agents include bevacizumab, a humanized anti-VEGF-A monoclonal antibody (Ferrara 2010), and two small molecule inhibitors targeting VEGFR2, sorafenib and sunitinib (Bergers and Hanahan 2008; Ellis and Hicklin 2008; Escudier

> © 2013 Li et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

#### **Chapter 7**

## **The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer**

Qigui Li, Peter Weina and Mark Hickman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54109

#### **1. Introduction**

[116] Zucchini C, Bianchini M, Valvassori L, Perdichizzi S, Benini S, Manara MC, Solmi R, Strippoli P, Picci P, Carinci P & Scotlandi K (2004) Identification of candidate genes involved in the reversal of malignant phenotype of osteosarcoma cells transfected

with the liver/bone/kidney alkaline phosphatase gene. Bone, 34(4):672-679.

174 Research Directions in Tumor Angiogenesis

Angiogenesis takes place during development, and vascular remodeling is a controlled ser‐ ies of events leading to neovascularization, which supports changing tissue requirements. Blood vessels and stromal components are responsive to pro- and anti-angiogenic factors that allow vascular remodeling during development, wound healing and pregnancy. In pathological situations such as cancer, however, the same angiogenic signaling pathways are induced and exploited. Cancer angiogenesis is a requirement for the development and growth of solid tumors beyond 2–3 mm3 (Cao et al., 2011). Several angiogenic activators in‐ cluding members of the vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) gene families and various inhibitors of angiogenesis have been described. In steady-state conditions, the balance between angiogenic activators and inhibitors results in very limited new blood vessel growth in the majority of tissues. The balance tilts in favor of the angiogenic stimulators, however, in a variety of proliferative processes. It is now gener‐ ally accepted that angiogenesis is a rate-limiting process in tumor growth. Without new blood vessels to supply nutrients and dispose of catabolic products, tumor cells cannot sus‐ tain proliferation and thus are likely to remain dormant (Ferrara, 2010; Daniele et al., 2012).

Survival and proliferation of cancer depends on angiogenesis, which could be a target of can‐ cer therapy. Angiogenesis is a complex physiological process. One example of this is found in the signaling pathways associated with the stimulus of various pro-angiogenic factors, VEGF and its receptors (VEGFR) which represents one of the best-validated signaling pathways in angiogenesis. A number of drugs approved by the FDA on market have been shown to inhibit anti-angiogenic pathway of VEGF. These agents include bevacizumab, a humanized anti-VEGF-A monoclonal antibody (Ferrara 2010), and two small molecule inhibitors targeting VEGFR2, sorafenib and sunitinib (Bergers and Hanahan 2008; Ellis and Hicklin 2008; Escudier

© 2013 Li et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

et al., 2007; Motzer et al., 2007). Not all cancer patients, however, benefit from such anti-angio‐ genic therapies, and some that do benefit initially have been shown to become less responsive during the treatment as well as show some adverse effects over time (Bergers and Hanahan 2008; Chen and Cleck, 2009; Ellis and Hicklin 2008). Over the last few decades, numerous antiangiogenic agents have been developed, and some of them have been tested in clinical settings. Angiogenesis includes a complex and multistep process, however, that has not been sufficient‐ ly elucidated. Hence, there is an urgent need to investigate the mechanisms that mediate resist‐ ance to anti-angiogenic agents. Recent advances have been made in identifying a number of novel alternate processes involved in angiogenesis. If these new findings of alternate mecha‐ nisms are confirmed, cancer therapy strategies may also be affected.

cally relevant. In particular, cancer angiogenesis plays a key role in the growth, invasion, and metastasis of cancers. After more than 30 years of intensive study, many agents, in‐ cluding novel candidate of ARTs, that target angiogenesis as cancer therapy and preven‐ tion of metastasis of existing tumors have been translated from the laboratory to the bedside. Therefore, ARTs-induced inhibition of angiogenesis could be a promising thera‐ peutic strategy for treatment of cancer and prevention of metastasis. Various clinical tri‐ als using ARTs for anti-cancer therapy have been guided by the anti-angiogenesis research of ARTs that has been conducted anti-cancer. Since new and alternative angio‐ genesis mechanisms have been found, further research on the mechanism of anti-angio‐ genesis could lead us to understand more deeply the possibilities inherent in the

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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177

The new strategies for the development of ARTs for cancer therapy and metastasis preven‐ tion should include a plan for increasing their anti-angiogenic activity through a variety of approaches ranging from medicinal chemistry approaches to develop more potent ART-ana‐ logues to changes in formulation and/or dosing. The real potential and benefits of the ART drug class for cancer treatment and metastasis prevention remain yet to be discovered. Giv‐ en the interest in using ARTs for cancer therapy, the door has been opened for challenging research in this area, which is likely to yield new cancer therapies that now do not exist. The aim of this chapter is to provide an overview of the recent advances and new development

Significant antitumor activity of ART and licensed semisynthetic its derivatives has been documented *in vitro, in vivo* and through clinical trials considerable research has been focused on the most active compounds, namely, artesunate (AS) and dihydroarte‐

ART and its derivatives are lactonic sesquiterpenoid compounds first discovered in China. A crude extract of the wormwood plant *Artemisia annua* (qinghao) was first used as an anti‐ pyretic 2000 years ago. The antipyretic therapy dates back to the third century B.C. in the "Handbook of Prescriptions for Emergency Treatment" edited by Ge Hong (281-340 B.C.) where he recommended tea-brewed leaves of the wormwood plant to treat fever and chills. The specific effect of ART on the fever of malaria was reported in the 16th century in the "Compendium of *Materia Medica*" published by Li Shizen in 1596 cited Ge Hong's prescrip‐ tion (Li and Weina, 2011). The active constituent of the extract was identified and purified in the 1970s, and named qinghaosu, or artemisinin (ART). Although ART proved effective in clinical trials in the 1980s, a number of semi-synthetic derivatives were developed to im‐ prove the drug's pharmacological properties and antimalarial potency (Li et al., 2007). The structure of ART, which includes an endoperoxide bridge (C-O-O-C), is unique among anti‐

development of ARTs for cancer therapy (Li and Hickman, 2011).

of this class of drugs as potential anti-angiogenic agents.

misinin (DHA).

**2.1. ART and its derivatives**

**2. Activities of artemisinins (ARTs) as anti-cancer agents**

Artemisinin (ART) is a natural product of the plant *Artemisia annua L.* Reduction of ART yields the more active dihydroartemisinin (DHA), a compound which can be further converted to dif‐ ferent derivatives, including, artesunate (AS) and artemether (AM), which are generally refer‐ red to as artemisinins (ARTs). ARTs are widely known for their potent antimalarial activity, but also been potential anti-cancer activity both *in vitro* and *in vivo* over the past few years. ARTs have inhibitory effects on cancer cell growth and also inhibit angiogenesis. Several stud‐ ies have revealed that ART inhibits the growth of many transformed cell lines and has a selec‐ tive cytotoxic effect. In one study, ART was shown to be more toxic to cancer than normal cells. In most of the systems, preloading of cancer cells with iron or iron-saturated holotransferrin triggers ART cytotoxicity with an increase in the activity of ARTs by 100-fold in some cell lines. It has been hypothesized that iron-activated ARTs induce damage by release of highly alkylat‐ ing carbon-centered radicals and radical oxygen species (ROS). Radicals may play a role in the cell alterations reported in ARTs-treated cancer cells such as enhanced apoptosis, arrest of growth, inhibition of angiogenesis, and DNA damage. More studies have demonstrated that ART and its derivatives possess an anti-angiogenic activity (Li and Hickman, 2011).

ARTs inhibit angiogenesis which is a vital process in metastasis. AS and DHA inhibit cho‐ rioallantoic membrane angiogenesis at low concentrations and decrease the levels of two major VEGF receptors on human umbilical vein endothelial cells (ECs). AS inhibits prolifer‐ ation and differentiation of human microvascular dermal ECs in a dose-dependent manner and reduces Flt-1 and KDR/flk-1 expression. Conditioned media from K562 cells pretreated with AS and DHA inhibits VEGF expression and secretion in chronic myeloid leukemia K562 cells, leading to a decrease in genetic activity associated with angiogenesis. ARTs in‐ hibit cell migration and concomitantly decrease the expression of matrix metalloproteinase proteins such as MMP2 and the avß3 integrins in human melanoma cells. ARTs also regu‐ late the levels of urokinase plasminogen activator (u-PA), and the matrix metalloproteinases MMP2, MMP7 and MMP9 all of which are related to metastasis. Also, ARTs have been shown to increase production of reactive oxygen species and also inhibits the hypoxia in‐ duced production of a transcription factor, hypoxia inducible factor-1α (HIF1α). The HIF1α transcription factor increases tumor angiogenesis to support the survival of poorly nourish‐ ed cancer cells. ARTs have shown pleiotropic effects through different experimental studies.

Definitely, ART compounds exhibit a wide spectrum of biological activities, including, for example, anti-angiogenic, anti-tumorigenic and even anti-viral, all of which are medi‐ cally relevant. In particular, cancer angiogenesis plays a key role in the growth, invasion, and metastasis of cancers. After more than 30 years of intensive study, many agents, in‐ cluding novel candidate of ARTs, that target angiogenesis as cancer therapy and preven‐ tion of metastasis of existing tumors have been translated from the laboratory to the bedside. Therefore, ARTs-induced inhibition of angiogenesis could be a promising thera‐ peutic strategy for treatment of cancer and prevention of metastasis. Various clinical tri‐ als using ARTs for anti-cancer therapy have been guided by the anti-angiogenesis research of ARTs that has been conducted anti-cancer. Since new and alternative angio‐ genesis mechanisms have been found, further research on the mechanism of anti-angio‐ genesis could lead us to understand more deeply the possibilities inherent in the development of ARTs for cancer therapy (Li and Hickman, 2011).

The new strategies for the development of ARTs for cancer therapy and metastasis preven‐ tion should include a plan for increasing their anti-angiogenic activity through a variety of approaches ranging from medicinal chemistry approaches to develop more potent ART-ana‐ logues to changes in formulation and/or dosing. The real potential and benefits of the ART drug class for cancer treatment and metastasis prevention remain yet to be discovered. Giv‐ en the interest in using ARTs for cancer therapy, the door has been opened for challenging research in this area, which is likely to yield new cancer therapies that now do not exist. The aim of this chapter is to provide an overview of the recent advances and new development of this class of drugs as potential anti-angiogenic agents.

#### **2. Activities of artemisinins (ARTs) as anti-cancer agents**

Significant antitumor activity of ART and licensed semisynthetic its derivatives has been documented *in vitro, in vivo* and through clinical trials considerable research has been focused on the most active compounds, namely, artesunate (AS) and dihydroarte‐ misinin (DHA).

#### **2.1. ART and its derivatives**

et al., 2007; Motzer et al., 2007). Not all cancer patients, however, benefit from such anti-angio‐ genic therapies, and some that do benefit initially have been shown to become less responsive during the treatment as well as show some adverse effects over time (Bergers and Hanahan 2008; Chen and Cleck, 2009; Ellis and Hicklin 2008). Over the last few decades, numerous antiangiogenic agents have been developed, and some of them have been tested in clinical settings. Angiogenesis includes a complex and multistep process, however, that has not been sufficient‐ ly elucidated. Hence, there is an urgent need to investigate the mechanisms that mediate resist‐ ance to anti-angiogenic agents. Recent advances have been made in identifying a number of novel alternate processes involved in angiogenesis. If these new findings of alternate mecha‐

Artemisinin (ART) is a natural product of the plant *Artemisia annua L.* Reduction of ART yields the more active dihydroartemisinin (DHA), a compound which can be further converted to dif‐ ferent derivatives, including, artesunate (AS) and artemether (AM), which are generally refer‐ red to as artemisinins (ARTs). ARTs are widely known for their potent antimalarial activity, but also been potential anti-cancer activity both *in vitro* and *in vivo* over the past few years. ARTs have inhibitory effects on cancer cell growth and also inhibit angiogenesis. Several stud‐ ies have revealed that ART inhibits the growth of many transformed cell lines and has a selec‐ tive cytotoxic effect. In one study, ART was shown to be more toxic to cancer than normal cells. In most of the systems, preloading of cancer cells with iron or iron-saturated holotransferrin triggers ART cytotoxicity with an increase in the activity of ARTs by 100-fold in some cell lines. It has been hypothesized that iron-activated ARTs induce damage by release of highly alkylat‐ ing carbon-centered radicals and radical oxygen species (ROS). Radicals may play a role in the cell alterations reported in ARTs-treated cancer cells such as enhanced apoptosis, arrest of growth, inhibition of angiogenesis, and DNA damage. More studies have demonstrated that

ART and its derivatives possess an anti-angiogenic activity (Li and Hickman, 2011).

ARTs inhibit angiogenesis which is a vital process in metastasis. AS and DHA inhibit cho‐ rioallantoic membrane angiogenesis at low concentrations and decrease the levels of two major VEGF receptors on human umbilical vein endothelial cells (ECs). AS inhibits prolifer‐ ation and differentiation of human microvascular dermal ECs in a dose-dependent manner and reduces Flt-1 and KDR/flk-1 expression. Conditioned media from K562 cells pretreated with AS and DHA inhibits VEGF expression and secretion in chronic myeloid leukemia K562 cells, leading to a decrease in genetic activity associated with angiogenesis. ARTs in‐ hibit cell migration and concomitantly decrease the expression of matrix metalloproteinase proteins such as MMP2 and the avß3 integrins in human melanoma cells. ARTs also regu‐ late the levels of urokinase plasminogen activator (u-PA), and the matrix metalloproteinases MMP2, MMP7 and MMP9 all of which are related to metastasis. Also, ARTs have been shown to increase production of reactive oxygen species and also inhibits the hypoxia in‐ duced production of a transcription factor, hypoxia inducible factor-1α (HIF1α). The HIF1α transcription factor increases tumor angiogenesis to support the survival of poorly nourish‐ ed cancer cells. ARTs have shown pleiotropic effects through different experimental studies.

Definitely, ART compounds exhibit a wide spectrum of biological activities, including, for example, anti-angiogenic, anti-tumorigenic and even anti-viral, all of which are medi‐

nisms are confirmed, cancer therapy strategies may also be affected.

176 Research Directions in Tumor Angiogenesis

ART and its derivatives are lactonic sesquiterpenoid compounds first discovered in China. A crude extract of the wormwood plant *Artemisia annua* (qinghao) was first used as an anti‐ pyretic 2000 years ago. The antipyretic therapy dates back to the third century B.C. in the "Handbook of Prescriptions for Emergency Treatment" edited by Ge Hong (281-340 B.C.) where he recommended tea-brewed leaves of the wormwood plant to treat fever and chills. The specific effect of ART on the fever of malaria was reported in the 16th century in the "Compendium of *Materia Medica*" published by Li Shizen in 1596 cited Ge Hong's prescrip‐ tion (Li and Weina, 2011). The active constituent of the extract was identified and purified in the 1970s, and named qinghaosu, or artemisinin (ART). Although ART proved effective in clinical trials in the 1980s, a number of semi-synthetic derivatives were developed to im‐ prove the drug's pharmacological properties and antimalarial potency (Li et al., 2007). The structure of ART, which includes an endoperoxide bridge (C-O-O-C), is unique among anti‐ malarial drugs. Semisynthetic ARTs are obtained from dihydroartemisinin (DHA), which is the reduced lactol derivative of ART, the main active metabolite of ARTs (Li et al., 1998). The first generation of semisynthetic ARTs includes the lipophilic arts, arteether (AE) and artemether (AM), while artesunate (AS) is the water soluble derivative (Li and Weina, 2011).

O

O

H

O O

O

arteether (AE), artesunate (AS) and artemisone

*2.2.1. WHO policies in malaria treatments*

**3.** Low toxicity (excellent safety profile)

H

CH3

CH3

O O H

O

H

O

CH3

H H O

O

CH2 CH3 C

**1.** Rapid action and high efficacy against multi-drug resistant *P. falciparum*

**2.** Evidence of ART drug resistance confirmed on the Cambodia-Thailand border

**4.** Gametocidal effect (prevents the transmission of malaria from person to person)

To treat uncomplicated malaria, the objective is to cure the infection. This is important as it will help prevent progression to severe disease and prevent additional morbidity associated with treatment failure. Cure of the infection translates to eradication of the parasite from the body. In treatment evaluations in all settings, emerging evidence indicates that it is necessa‐ ry to follow patients for enough time to document a clinical cure. In assessing drug efficacy in high-transmission settings, temporary suppression of infection for 14 days has not been considered sufficient. The public health goal of treatment is to reduce transmission of the in‐ fection to others, i.e. to reduce the infectious reservoir. A secondary but equally important objective of treatment is to prevent the emergence and spread of resistance to antimalarials. Tolerability, the adverse effect profile and the speed of therapeutic response are also impor‐

H

CH3

O O

O

H

CH3

O O H

OH

Artemisinin (ART) Dihydroartemisinin (DHA) Artemether (AM)

CH3

H H

CH3

O

CH3

H H O

O

H

O O

O

H

CH3

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

CH3

CH2 CH2 COONa

O O H

O

H

N

O O

CH3

179

http://dx.doi.org/10.5772/54109

H H

CH3

CH3

CH3

H H

CH3

O

CH3

O

H

O

Arteether (AE) Artesunate (AS) Artemisone <sup>S</sup>

**Figure 1.** Chemical structures of artemisinin (ART) and its five derivatives, dihydroartemisinin (DHA), artemether (AM),

The pharmacological and clinical evaluations of ART group of drugs have been taken place

CH3

CH3

H H

CH3

for 30 years and four advantages have been evaluated.

O

AS and its bioactive metabolite, DHA, have been the topic of considerable research attention in recent years for both anti-cancer and antimalarial indications. The key structural feature in all of the ART-related molecules that mediates their antimalarial activity, and some of their anti-cancer activities, is an endoperoxide bridge. The endoperoxides are a promising class of antimalarial drugs which may meet the dual challenges posed by drug-resistant par‐ asites and the rapid progression of malarial illness. Of the available derivatives, AS has the most favorable pharmacological profile for use in ART-based combination therapy treat‐ ment of uncomplicated malaria and intravenous therapy of severe malaria (Li and Weina, 2010a). The effectiveness of AS has been mostly attributed to its rapid and extensive hydrol‐ ysis to DHA (Batty et al., 1998b; Davis et al., 2001; Li et al., 2009; Navaratnam et al., 2000).

Artemisone, a second-generation ART which is not metabolized to DHA, has shown im‐ proved pharmacokinetic properties including a longer half-life and lower toxicity (D'Ales‐ sandro et al., 2007; Schmuck et al., 2009) (Figure 1). Fully synthetic ART derivatives have also been designed by preserving the peroxide moiety which confers potent drug activity. These compounds are easily synthesized from simple starting materials; accordingly, these compounds are currently under intense development (Creek et al., 2008; Jefford 2007; Ram‐ irez et al., 2009; Taylor et al., 2004). Hundreds of these compounds have been made; many resemble ART, but only one of these compounds, arteflene, has been taken beyond preclini‐ cal development (Radloff et al., 1996).

ART and its active derivatives have been widely used as antimalarial drugs for more than 30 years, and they have also been shown recently to be effective in killing cancer cells (Li et al., 2011). A number of studies demonstrated that ART and its bioactive derivatives exhibit po‐ tent anti-cancer effects in a variety of human cancer cell model systems. Recently, the antiangiogenic activity of ARTs has been demonstrated, and these compounds have been shown to be potential anti-cancer agents (Crespo-Ortiz and Wei, 2012).

#### **2.2. ARTs as first-line therapies for treatments of malaria**

Global malaria control is being threatened on an unprecedented scale by rapidly growing resistance of *P. falciparum* to conventional monotherapies such as chloroquine, sulfadoxinepyrimethamine (SP) and amodiaquine. Multi-drug resistant *falciparum* malaria is widely prevalent in South-East Asia and South America. Now Africa, the continent with highest burden of malaria is also being seriously affected by drug resistance. A significant advant‐ age of ART and its derivatives in malaria treatment shows early evidence of cross-resist‐ ance to other antimalarial drugs. As a response to the rising tide of antimalarial drug resistance, WHO issued new Guideline for the Treatment of Malaria (WHO 2006; 2008) and recommends that treatment policies for *falciparum* malaria in all countries experiencing re‐ sistance to monotherapies should be combination therapies, preferably those containing an ART derivative.

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer http://dx.doi.org/10.5772/54109 179

**Figure 1.** Chemical structures of artemisinin (ART) and its five derivatives, dihydroartemisinin (DHA), artemether (AM), arteether (AE), artesunate (AS) and artemisone

#### *2.2.1. WHO policies in malaria treatments*

malarial drugs. Semisynthetic ARTs are obtained from dihydroartemisinin (DHA), which is the reduced lactol derivative of ART, the main active metabolite of ARTs (Li et al., 1998). The first generation of semisynthetic ARTs includes the lipophilic arts, arteether (AE) and artemether (AM), while artesunate (AS) is the water soluble derivative (Li and Weina, 2011). AS and its bioactive metabolite, DHA, have been the topic of considerable research attention in recent years for both anti-cancer and antimalarial indications. The key structural feature in all of the ART-related molecules that mediates their antimalarial activity, and some of their anti-cancer activities, is an endoperoxide bridge. The endoperoxides are a promising class of antimalarial drugs which may meet the dual challenges posed by drug-resistant par‐ asites and the rapid progression of malarial illness. Of the available derivatives, AS has the most favorable pharmacological profile for use in ART-based combination therapy treat‐ ment of uncomplicated malaria and intravenous therapy of severe malaria (Li and Weina, 2010a). The effectiveness of AS has been mostly attributed to its rapid and extensive hydrol‐ ysis to DHA (Batty et al., 1998b; Davis et al., 2001; Li et al., 2009; Navaratnam et al., 2000). Artemisone, a second-generation ART which is not metabolized to DHA, has shown im‐ proved pharmacokinetic properties including a longer half-life and lower toxicity (D'Ales‐ sandro et al., 2007; Schmuck et al., 2009) (Figure 1). Fully synthetic ART derivatives have also been designed by preserving the peroxide moiety which confers potent drug activity. These compounds are easily synthesized from simple starting materials; accordingly, these compounds are currently under intense development (Creek et al., 2008; Jefford 2007; Ram‐ irez et al., 2009; Taylor et al., 2004). Hundreds of these compounds have been made; many resemble ART, but only one of these compounds, arteflene, has been taken beyond preclini‐

ART and its active derivatives have been widely used as antimalarial drugs for more than 30 years, and they have also been shown recently to be effective in killing cancer cells (Li et al., 2011). A number of studies demonstrated that ART and its bioactive derivatives exhibit po‐ tent anti-cancer effects in a variety of human cancer cell model systems. Recently, the antiangiogenic activity of ARTs has been demonstrated, and these compounds have been shown

Global malaria control is being threatened on an unprecedented scale by rapidly growing resistance of *P. falciparum* to conventional monotherapies such as chloroquine, sulfadoxinepyrimethamine (SP) and amodiaquine. Multi-drug resistant *falciparum* malaria is widely prevalent in South-East Asia and South America. Now Africa, the continent with highest burden of malaria is also being seriously affected by drug resistance. A significant advant‐ age of ART and its derivatives in malaria treatment shows early evidence of cross-resist‐ ance to other antimalarial drugs. As a response to the rising tide of antimalarial drug resistance, WHO issued new Guideline for the Treatment of Malaria (WHO 2006; 2008) and recommends that treatment policies for *falciparum* malaria in all countries experiencing re‐ sistance to monotherapies should be combination therapies, preferably those containing an

cal development (Radloff et al., 1996).

178 Research Directions in Tumor Angiogenesis

ART derivative.

to be potential anti-cancer agents (Crespo-Ortiz and Wei, 2012).

**2.2. ARTs as first-line therapies for treatments of malaria**

The pharmacological and clinical evaluations of ART group of drugs have been taken place for 30 years and four advantages have been evaluated.


To treat uncomplicated malaria, the objective is to cure the infection. This is important as it will help prevent progression to severe disease and prevent additional morbidity associated with treatment failure. Cure of the infection translates to eradication of the parasite from the body. In treatment evaluations in all settings, emerging evidence indicates that it is necessa‐ ry to follow patients for enough time to document a clinical cure. In assessing drug efficacy in high-transmission settings, temporary suppression of infection for 14 days has not been considered sufficient. The public health goal of treatment is to reduce transmission of the in‐ fection to others, i.e. to reduce the infectious reservoir. A secondary but equally important objective of treatment is to prevent the emergence and spread of resistance to antimalarials. Tolerability, the adverse effect profile and the speed of therapeutic response are also impor‐ tant considerations. A brief summary of the WHO policies (WHO, 2010) for treatment of un‐ complicated *falciparum* malaria is listed below:

*2.2.2. ACT is a "policy standard" for first line malaria treatment*

species (WHO 2006; 2007).

also for public health control of malaria.

pies for treatment of uncomplicated malaria (WHO 2006; 2007).

Antimalarial combination therapies can improve treatment efficacies of failing individual components and provide some protection for individual components against the develop‐ ment of higher levels of resistance. ACTs have been advocated as the best available op‐ tion, and are the most commonly adopted regimen in countries changing antimalarial policy in the last decade. ACTs are most preferred for their enhancement of efficacy (Price 2000; White and Olliaro, 1998; White 1999a), lower malaria incidence and their po‐ tential to lower the rate at which resistance emerges and spreads (Nosten et al., 2000; White 1999b). Five ACTs recommended by a WHO Expert Consultative Group in 2010 include AM-lumefantrine (Coartem), AS-mefloquine (Artequin), AS-amodiaquine, and AS-sulfadoxine/pyrimethamine. Recently, WHO has endorsed ACTs as the "policy stand‐ ard" for all malaria infections in areas where *P. falciparum* is the predominant infecting

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181

ARTs rapidly reduce parasitemia, but have poor efficacy as short course monotherapy. When used in combination with another agent, the rapid reduction in parasite numbers results in relatively few parasites being exposed to the second drug (to which significant resistance may already exist), theoretically preventing emergence of additional resistance mutations (White 2004). Furthermore, since ARTs themselves are not required to mediate final cure, there should also be little opportunity for ART resistance to develop. In addi‐ tion, rapid reduction of the parasite burden in vivo by ACT drug combinations reduces the frequency of gametocyte generation, increases the rates of cure and may also reduce transmission of resistant parasites (Price, 2000). Most currently recommended drug com‐ binations for *falciparum* malaria are variants of ACT where a rapidly acting ART com‐ pound is combined with a longer half-life drug of a different class. ARTs used include DHA, AS, AM and companion drugs include mefloquine, amodiaquine, sulfadoxine/pyri‐ methamine, lumefantrine, piperaquine, pyronaridine, and chlorproguanil/dapsone. The standard of care must be to cure malaria by killing the last parasite. Combination anti‐ malarial treatment is vital not only to the successful treatment of individual patients but

ACTs continue to be the mainstay treatment of uncomplicated falciparum malaria. For the next 8–10 years, no alternative medicines to the ART derivatives able to offer similar high levels of therapeutic efficacy are expected to enter the market. For this reason, WHO has fo‐ cused its efforts not only to increase access to quality ACTs, but also to contain the risk of development of *falciparum* resistance, associated with the large-scale use of oral monothera‐

In January 2006, WHO appealed to manufacturers to stop marketing oral ART mono‐ therapies and instead to promote quality ACTs in line with WHO policy. This position has been widely disseminated via WHO Offices, WHO briefings to hospital staff and in regional and inter-country briefings to representatives of national health. Major procure‐ ment and funding agencies and international suppliers have accepted the WHO recom‐ mendation and agreed not to fund or procure oral ART monotherapies. In April 2006, the Global Malaria Programme of WHO provided a technical briefing to 25 pharmaceuti‐

Artemisinin-based combination therapies (ACTs) are the treatment recommended by WHO in 2010 for all cases of uncomplicated *falciparum* malaria as first-line treatment including:


Second-line treatment:


Note: The ART derivatives (oral, rectal, or parenteral formulations) and partner medicines of ACTs are not recommended as monotherapy for uncomplicated malaria due to high rates of recrudescence associated with ART monotherapy.

To treat severe malaria, the primary objective of antimalarial treatment is to prevent death. Prevention of recrudescence and avoidance of minor adverse effects are secondary. In treat‐ ing cerebral malaria, prevention of neurological deficit is also an important objective. In the treatment of severe malaria in pregnancy, saving the life of the mother is the primary objec‐ tive. The following WHO policies are recommended for treatment of severe and complicat‐ ed *falciparum* malaria as first-line treatment (WHO 2010):

Any of the following antimalarial medicines have been recommended by the WHO in 2010 for initial treatment.


Follow-on treatment: once the patient recovers enough and can tolerate oral treatment, the following options can be used to complete treatment:


Consistent with WHO recommendations (2006; 2010), malaria endemic countries which are experiencing resistance to currently used antimalarial drug monotherapies (chloroquine, sulphadoxine/pyrimethamine or amodiaquine) should change treatment policies to the highly effective ART-based combination treatments (ACTs).

#### *2.2.2. ACT is a "policy standard" for first line malaria treatment*

tant considerations. A brief summary of the WHO policies (WHO, 2010) for treatment of un‐

Artemisinin-based combination therapies (ACTs) are the treatment recommended by WHO in 2010 for all cases of uncomplicated *falciparum* malaria as first-line treatment including:

**•** an effective alternative ACT (efficacy of ACTs depend on efficacy of the partner medicine,

Note: The ART derivatives (oral, rectal, or parenteral formulations) and partner medicines of ACTs are not recommended as monotherapy for uncomplicated malaria due to high rates

To treat severe malaria, the primary objective of antimalarial treatment is to prevent death. Prevention of recrudescence and avoidance of minor adverse effects are secondary. In treat‐ ing cerebral malaria, prevention of neurological deficit is also an important objective. In the treatment of severe malaria in pregnancy, saving the life of the mother is the primary objec‐ tive. The following WHO policies are recommended for treatment of severe and complicat‐

Any of the following antimalarial medicines have been recommended by the WHO in 2010

Follow-on treatment: once the patient recovers enough and can tolerate oral treatment, the

Consistent with WHO recommendations (2006; 2010), malaria endemic countries which are experiencing resistance to currently used antimalarial drug monotherapies (chloroquine, sulphadoxine/pyrimethamine or amodiaquine) should change treatment policies to the

therefore it is possible to use two different ACTs as 1st and 2nd line options)

complicated *falciparum* malaria is listed below:

**•** artesunate plus sulfadoxine-pyrimethamine,

**•** quinine + tetracycline or doxycycline or clindamycin

of recrudescence associated with ART monotherapy.

ed *falciparum* malaria as first-line treatment (WHO 2010):

following options can be used to complete treatment:

highly effective ART-based combination treatments (ACTs).

**•** dihydroartemisinin plus piperaquine.

**•** artemether plus lumefantrine, **•** artesunate plus amodiaquine, **•** artesunate plus mefloquine,

180 Research Directions in Tumor Angiogenesis

Second-line treatment:

for initial treatment.

**•** artemether (i.m.)

**•** artesunate (i.v. or i.m.)

**•** full course of an ACT or

**•** quinine (i.v. infusion or i.m. injection).

**•** quinine + clindamycin or doxycycline

Antimalarial combination therapies can improve treatment efficacies of failing individual components and provide some protection for individual components against the develop‐ ment of higher levels of resistance. ACTs have been advocated as the best available op‐ tion, and are the most commonly adopted regimen in countries changing antimalarial policy in the last decade. ACTs are most preferred for their enhancement of efficacy (Price 2000; White and Olliaro, 1998; White 1999a), lower malaria incidence and their po‐ tential to lower the rate at which resistance emerges and spreads (Nosten et al., 2000; White 1999b). Five ACTs recommended by a WHO Expert Consultative Group in 2010 include AM-lumefantrine (Coartem), AS-mefloquine (Artequin), AS-amodiaquine, and AS-sulfadoxine/pyrimethamine. Recently, WHO has endorsed ACTs as the "policy stand‐ ard" for all malaria infections in areas where *P. falciparum* is the predominant infecting species (WHO 2006; 2007).

ARTs rapidly reduce parasitemia, but have poor efficacy as short course monotherapy. When used in combination with another agent, the rapid reduction in parasite numbers results in relatively few parasites being exposed to the second drug (to which significant resistance may already exist), theoretically preventing emergence of additional resistance mutations (White 2004). Furthermore, since ARTs themselves are not required to mediate final cure, there should also be little opportunity for ART resistance to develop. In addi‐ tion, rapid reduction of the parasite burden in vivo by ACT drug combinations reduces the frequency of gametocyte generation, increases the rates of cure and may also reduce transmission of resistant parasites (Price, 2000). Most currently recommended drug com‐ binations for *falciparum* malaria are variants of ACT where a rapidly acting ART com‐ pound is combined with a longer half-life drug of a different class. ARTs used include DHA, AS, AM and companion drugs include mefloquine, amodiaquine, sulfadoxine/pyri‐ methamine, lumefantrine, piperaquine, pyronaridine, and chlorproguanil/dapsone. The standard of care must be to cure malaria by killing the last parasite. Combination anti‐ malarial treatment is vital not only to the successful treatment of individual patients but also for public health control of malaria.

ACTs continue to be the mainstay treatment of uncomplicated falciparum malaria. For the next 8–10 years, no alternative medicines to the ART derivatives able to offer similar high levels of therapeutic efficacy are expected to enter the market. For this reason, WHO has fo‐ cused its efforts not only to increase access to quality ACTs, but also to contain the risk of development of *falciparum* resistance, associated with the large-scale use of oral monothera‐ pies for treatment of uncomplicated malaria (WHO 2006; 2007).

In January 2006, WHO appealed to manufacturers to stop marketing oral ART mono‐ therapies and instead to promote quality ACTs in line with WHO policy. This position has been widely disseminated via WHO Offices, WHO briefings to hospital staff and in regional and inter-country briefings to representatives of national health. Major procure‐ ment and funding agencies and international suppliers have accepted the WHO recom‐ mendation and agreed not to fund or procure oral ART monotherapies. In April 2006, the Global Malaria Programme of WHO provided a technical briefing to 25 pharmaceuti‐ cal companies involved in the production and marketing of ART monotherapies. Out of these, 15 declared their willingness to stop marketing ART monotherapies over a short period of time, but 10 companies did not disclose their marketing plans for the future (meeting report available at: www.who.int/malaria/docs/ Meeting\_briefing19April.pdf). In addition, some countries, like China and Pakistan, have been visited by WHO delega‐ tions to address multiple domestic manufacturers involved in this sector. The evolving position of manufacturers and of National Drug Regulatory Authorities (NDRA) in ma‐ laria endemic countries is monitored and displayed on the WHO Global Malaria Pro‐ gramme website front-page: http://malaria.who.int/.

ma, and lung cancer cells (Lu et al., 2009). Moreover, artemisone (second generation ART compound) has shown better activity than ART and considerable synergistic interactions

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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183

ART has been found to act either directly by inducing DNA damage (genotoxicity) or in‐ directly by interfering with a range of signaling pathways involved in several hallmarks of malignancy. Direct DNA damage is only described in specific systems, however, while indirect effects are more commonly noted in the literature. In pancreatic cells (Panc-1), artesunate was shown to cause DNA fragmentation and membrane damage. Interesting‐ ly, low doses of artesunate were associated with oncosis-like cell death, whereas higher concentrations were shown to induce apoptosis (Du et al., 2010). The extent and type of cellular damage seems to depend on the phenotype and the origin of cell line, and it may also vary in a time- and dose-dependent manner (Crespo-Ortiz and Wei, 2012). No‐ tably, higher sensitivity to AS was observed in rapidly growing cell lines when com‐

Moreover, the highly stable ARTs and ART-derived trioxane dimers were shown to in‐ hibit growth and selectively kill several human cancer cell lines without inducing cyto‐ toxic effects on normal neighboring cells. One proposed mechanism by which ART targets cancer cells involves cleavage of the endoperoxide bridge by the relatively high concentrations of iron in cancer cells, resulting in iron depletion in those cells coupled with generation of free radicals such as reactive oxygen species (ROS) capable of induc‐ ing subsequent oxidative damage. This mechanism resembles the known mechanism of action of ART in malarial parasites. In addition to possessing higher iron influx via transferrin receptors, cancer cells are also sensitive to oxygen radicals because of a rela‐ tive deficiency in antioxidant enzymes. A significant positive correlation can be made be‐ tween AS sensitivity and transferrin receptor levels as well as between AS sensitivity

Expression profiling of several classes of tumor cells has shown that ART treatment caused selective expression changes of many oncogenes and tumor suppressor genes than genes responsible for iron metabolism, which suggests that the anti-cancer proper‐ ties of ARTs cannot be explained simply by the global toxic effects of oxidative damage. Alternatively, DHA, AS, and AM may well be to modulating genes and proteins coordi‐ nating growth signals, apoptosis, proliferation capacity, angiogenesis and tissue invasion, and metastasis. A complex network of interactions through different pathways may en‐ hance the anti-cancer effect of these endoperoxide drugs leading to cancer control and

ARTs have also been observed to attenuate multidrug resistance in cancer patients, an effect due in part to the inhibition of glutathione S-transferase activity. ART and its bioactive de‐ rivatives elicit their anti-cancer effects by concurrently activating, inhibiting and/or attenuat‐ ing multiple complementary cell signaling pathways, which have been described in a variety of human cancer cell systems as well as in athymic mouse xenograft models. The ART compounds exert common as well as distinct cellular effects depending on the pheno‐

type and tissue origin of the human cancer cells tested. (Firestone and Sundar 2009)

with other anti-cancer agents (Gravett et al., 2010).

pared with slow growing cancer cells (Efferth et al., 2003).

and expression of ATP binding cassette transporters (Efferth, 2006).

cell death (Crespo-Ortiz and Wei, 2012).

In May 2007, the 60th World Health Assembly resolved to take strong action against oral monotherapies and approved the resolution WHA60.18, which:


The above-mentioned benefits of ACTs make them an important tool for malaria treatment and control that has led to their increased use by 2010, most countries (89 countries), adopt‐ ed ACTs as their first-line treatment of uncomplicated *falciparum* malaria. Only two coun‐ tries adopted ACTs exclusively as second-line treatment (Bosman and Mendis, 2007).

#### **2.3. Anti-cancer activities of ARTs**

ART and its bioactive derivatives (AS, DHA, and AM) exhibit potent anti-cancer effects in a variety of human cancer cell model systems. The pleiotropic response in cancer cells to ART includes: 1) growth inhibition by cell cycle arrest, 2) apoptosis, 3) inhibition of angiogenesis, 4) disruption of cell migration, and 5) modulation of nuclear receptor responsiveness. These effects of ARTs result from perturbations of many cellular signaling pathways *in vitro* and in animal models. Considerable research has been focused on the most active ART com‐ pounds, namely, DHA and AS.

Molecular, cellular and physiological studies have demonstrated that, depending on the tis‐ sue type and experimental system, ART and its derivatives arrest cell growth, induce an apoptotic response, alter hormone responsive properties and/or inhibit angiogenesis of hu‐ man cancer cells. The Developmental Therapeutics Program of the National Cancer Institute (NCI), USA, which analyzed the activity of AS on 55 human cancer cell lines (IC50 values shown between nano- to micro-molar range, depending on the cancer cell line), showed that AS displays inhibitory activity against leukemia, colon, melanoma, breast, ovarian, prostate, central nervous system (CNS), and renal cancer cells (Efferth et al., 2001; 2003; Efferth, 2006). DHA also has remarkable anti-neoplastic activity against pancreatic, leukemic, osteosarco‐ ma, and lung cancer cells (Lu et al., 2009). Moreover, artemisone (second generation ART compound) has shown better activity than ART and considerable synergistic interactions with other anti-cancer agents (Gravett et al., 2010).

cal companies involved in the production and marketing of ART monotherapies. Out of these, 15 declared their willingness to stop marketing ART monotherapies over a short period of time, but 10 companies did not disclose their marketing plans for the future (meeting report available at: www.who.int/malaria/docs/ Meeting\_briefing19April.pdf). In addition, some countries, like China and Pakistan, have been visited by WHO delega‐ tions to address multiple domestic manufacturers involved in this sector. The evolving position of manufacturers and of National Drug Regulatory Authorities (NDRA) in ma‐ laria endemic countries is monitored and displayed on the WHO Global Malaria Pro‐

In May 2007, the 60th World Health Assembly resolved to take strong action against oral

**1.** urges Member States to progressively cease the provision, in both the public and pri‐ vate sectors, of oral ART monotherapies, to promote the use of ART-combination thera‐ pies, and to implement policies that prohibit the production, marketing, distribution

**2.** requests international organizations and financing bodies to adjust their policies so as progressively cease to fund the provision and distribution of oral ART monotherapies, and to join in campaigns to prohibit the production, marketing, distribution and use of

The above-mentioned benefits of ACTs make them an important tool for malaria treatment and control that has led to their increased use by 2010, most countries (89 countries), adopt‐ ed ACTs as their first-line treatment of uncomplicated *falciparum* malaria. Only two coun‐

ART and its bioactive derivatives (AS, DHA, and AM) exhibit potent anti-cancer effects in a variety of human cancer cell model systems. The pleiotropic response in cancer cells to ART includes: 1) growth inhibition by cell cycle arrest, 2) apoptosis, 3) inhibition of angiogenesis, 4) disruption of cell migration, and 5) modulation of nuclear receptor responsiveness. These effects of ARTs result from perturbations of many cellular signaling pathways *in vitro* and in animal models. Considerable research has been focused on the most active ART com‐

Molecular, cellular and physiological studies have demonstrated that, depending on the tis‐ sue type and experimental system, ART and its derivatives arrest cell growth, induce an apoptotic response, alter hormone responsive properties and/or inhibit angiogenesis of hu‐ man cancer cells. The Developmental Therapeutics Program of the National Cancer Institute (NCI), USA, which analyzed the activity of AS on 55 human cancer cell lines (IC50 values shown between nano- to micro-molar range, depending on the cancer cell line), showed that AS displays inhibitory activity against leukemia, colon, melanoma, breast, ovarian, prostate, central nervous system (CNS), and renal cancer cells (Efferth et al., 2001; 2003; Efferth, 2006). DHA also has remarkable anti-neoplastic activity against pancreatic, leukemic, osteosarco‐

tries adopted ACTs exclusively as second-line treatment (Bosman and Mendis, 2007).

gramme website front-page: http://malaria.who.int/.

182 Research Directions in Tumor Angiogenesis

monotherapies and approved the resolution WHA60.18, which:

and the use of counterfeit antimalarial medicines;

counterfeit antimalarial medicines;

**2.3. Anti-cancer activities of ARTs**

pounds, namely, DHA and AS.

ART has been found to act either directly by inducing DNA damage (genotoxicity) or in‐ directly by interfering with a range of signaling pathways involved in several hallmarks of malignancy. Direct DNA damage is only described in specific systems, however, while indirect effects are more commonly noted in the literature. In pancreatic cells (Panc-1), artesunate was shown to cause DNA fragmentation and membrane damage. Interesting‐ ly, low doses of artesunate were associated with oncosis-like cell death, whereas higher concentrations were shown to induce apoptosis (Du et al., 2010). The extent and type of cellular damage seems to depend on the phenotype and the origin of cell line, and it may also vary in a time- and dose-dependent manner (Crespo-Ortiz and Wei, 2012). No‐ tably, higher sensitivity to AS was observed in rapidly growing cell lines when com‐ pared with slow growing cancer cells (Efferth et al., 2003).

Moreover, the highly stable ARTs and ART-derived trioxane dimers were shown to in‐ hibit growth and selectively kill several human cancer cell lines without inducing cyto‐ toxic effects on normal neighboring cells. One proposed mechanism by which ART targets cancer cells involves cleavage of the endoperoxide bridge by the relatively high concentrations of iron in cancer cells, resulting in iron depletion in those cells coupled with generation of free radicals such as reactive oxygen species (ROS) capable of induc‐ ing subsequent oxidative damage. This mechanism resembles the known mechanism of action of ART in malarial parasites. In addition to possessing higher iron influx via transferrin receptors, cancer cells are also sensitive to oxygen radicals because of a rela‐ tive deficiency in antioxidant enzymes. A significant positive correlation can be made be‐ tween AS sensitivity and transferrin receptor levels as well as between AS sensitivity and expression of ATP binding cassette transporters (Efferth, 2006).

Expression profiling of several classes of tumor cells has shown that ART treatment caused selective expression changes of many oncogenes and tumor suppressor genes than genes responsible for iron metabolism, which suggests that the anti-cancer proper‐ ties of ARTs cannot be explained simply by the global toxic effects of oxidative damage. Alternatively, DHA, AS, and AM may well be to modulating genes and proteins coordi‐ nating growth signals, apoptosis, proliferation capacity, angiogenesis and tissue invasion, and metastasis. A complex network of interactions through different pathways may en‐ hance the anti-cancer effect of these endoperoxide drugs leading to cancer control and cell death (Crespo-Ortiz and Wei, 2012).

ARTs have also been observed to attenuate multidrug resistance in cancer patients, an effect due in part to the inhibition of glutathione S-transferase activity. ART and its bioactive de‐ rivatives elicit their anti-cancer effects by concurrently activating, inhibiting and/or attenuat‐ ing multiple complementary cell signaling pathways, which have been described in a variety of human cancer cell systems as well as in athymic mouse xenograft models. The ART compounds exert common as well as distinct cellular effects depending on the pheno‐ type and tissue origin of the human cancer cells tested. (Firestone and Sundar 2009)

#### **2.4. Anti-cancer mechanism of ART and its derivatives**

The anti-cancer potential of ARTs has been demonstrated in various cancer cells including those of leukemia and other cancer cells of breast, ovary, liver, lung, pancreas and colon (Tan et al., 2011).The mechanisms of action of ARTs in cancer cells are associated with: 1) anti-angiogenic effects, 2) induction of apoptosis, 3) oxidative stress response, 4) oncogenes and tumor suppressor genes, and 5) multidrug resistance (Figure 2) (Efferth 2006; 2007).

age by release of highly alkylating carbon-centered radicals and ROS. Radicals may play a role in the cell alterations reported in ARTs-treated cancer cells such as enhanced apoptosis, arrest of growth, inhibition of angiogenesis, and DNA damage. Microarray analyses found that the action of ARTs seems to be modulated by the expression of oxi‐ dative stress enzymes including catalase, thioredoxin reductase, superoxide dismutase and the glutathione S-transferase family. ARTs-sensitive cells demonstrate down-regulat‐ ed oxidation enzymes whereas over-expression of these enzymes renders cancer cells less sensitive to chemotherapeutic agents. The antineoplastic toxicity of ARTs appears to be also modulated by calcium metabolism, endoplasmic reticulum (ER) stress, and the ex‐ pression of the translationally controlled tumor protein, TCTP, a calcium binding protein which has been also postulated as a parasite target. Although the expression of the TCTP gen, *tctp,* was initially correlated with cancer cell response to ARTs, a functional role for TCTP in the action of ARTs has yet to be found. As for malaria parasites, the

Expression profiling of several classes of tumor cells has shown that ART treatment caus‐ es selective expression changes of many more oncogenes and tumor suppressor genes than genes responsible for iron metabolism, which suggests that the anti-cancer proper‐ ties of ART cannot be explained simply by the global toxic effects of oxidative damage. ART has also been observed to attenuate multidrug resistance in cancer patients, an ef‐ fect due in part to the inhibition of glutathione S-transferase activity. ART and its bioac‐ tive derivatives elicit their anti-cancer effects by concurrently activating, inhibiting and/or attenuating multiple complementary cell signaling pathways, which have been described in a variety of human cancer cell systems as well as in athymic mouse xenograft models. The ART compounds exert common as well as distinct cellular effects depending on the phenotype and tissue origin of the human cancer cells tested. (Firestone and Sundar,

Studies have identified potential general anti-cancer mechanisms of anti-cancer ARTs such as normalization of the upregulated Wnt/β-catenin pathway in colorectal cancer. Other pathways for anti-cancer activity include inhibition of enhanced angiogenesis associated with tumors. ARTs have been shown to inhibit proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC), inhibit VEGF binding to surface recep‐ tors on HUVEC and reduce expression of VEGF receptors Flt-1 and KDR/flk-1 on HUVECs. In cancer cells, artemisinins reduce expression of the VEGF receptor KDR/flk-1 in tumor and endothelial cells and slow the growth of human ovarian cancer HO-8910 xenografts in nude mice. HUVEC apoptosis by artesunate is associated with downregulation of Bcl-2 (B-cell leukemia/lymphoma 2) and upregulation of BAX (Bcl-2-associated X protein). In addition, mRNA expression of 30 out of 90 angiogenesis-related genes correlated significantly with the cellular response to ARTs, supporting the hypothesis that ARTs exert their anti-tumor

+ ATPase (SERCA) as a target of ARTs in cancer cells has

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185

role of sarcoendoplasmic Ca2

2009).

also been explored (Crespo-Ortiz and Wei, 2012).

*2.4.2. Potential general mechanisms of ART and its derivatives*

effects by inhibition of tumor angiogenesis (Krishna et al., 2008).

**Figure 2.** Schema of tumor angiogenesis induced by hypoxia and the inhibitions of tumor growth by antiangiogenic artemisinins (ART) and its derivatives of dihydroartemisinin (DHA), artesunate (AS) and artemether (AM) follows three directions, including the inhibition of tumor cell synthesis of angiogenic proteins, the neutralization of angiogenic proteins by antibodies or traps, and the inhibition of endothelial cell binding to angiogenic proteins or direct induc‐ tion of endothelial cell apoptosis.

#### *2.4.1. Anti-cancer mechanism of ARTs based on antimalarial actions*

The endoperoxide moiety of ART has been shown to be pharmacologically important and responsible for antimalarial activity against the malaria parasites. The potent anticancer action of ARTs can be also attributed to the endoperoxide bond. In most of the *in vitro* cancer cell lines tested, preloading of cancer cells with iron or iron-saturated holo‐ transferrin triggers ART cytotoxicity with an increase in ARTs activity up to 100-fold against some cell lines. It has been hypothesized that iron-activated ARTs induce dam‐ age by release of highly alkylating carbon-centered radicals and ROS. Radicals may play a role in the cell alterations reported in ARTs-treated cancer cells such as enhanced apoptosis, arrest of growth, inhibition of angiogenesis, and DNA damage. Microarray analyses found that the action of ARTs seems to be modulated by the expression of oxi‐ dative stress enzymes including catalase, thioredoxin reductase, superoxide dismutase and the glutathione S-transferase family. ARTs-sensitive cells demonstrate down-regulat‐ ed oxidation enzymes whereas over-expression of these enzymes renders cancer cells less sensitive to chemotherapeutic agents. The antineoplastic toxicity of ARTs appears to be also modulated by calcium metabolism, endoplasmic reticulum (ER) stress, and the ex‐ pression of the translationally controlled tumor protein, TCTP, a calcium binding protein which has been also postulated as a parasite target. Although the expression of the TCTP gen, *tctp,* was initially correlated with cancer cell response to ARTs, a functional role for TCTP in the action of ARTs has yet to be found. As for malaria parasites, the role of sarcoendoplasmic Ca2 + ATPase (SERCA) as a target of ARTs in cancer cells has also been explored (Crespo-Ortiz and Wei, 2012).

Expression profiling of several classes of tumor cells has shown that ART treatment caus‐ es selective expression changes of many more oncogenes and tumor suppressor genes than genes responsible for iron metabolism, which suggests that the anti-cancer proper‐ ties of ART cannot be explained simply by the global toxic effects of oxidative damage. ART has also been observed to attenuate multidrug resistance in cancer patients, an ef‐ fect due in part to the inhibition of glutathione S-transferase activity. ART and its bioac‐ tive derivatives elicit their anti-cancer effects by concurrently activating, inhibiting and/or attenuating multiple complementary cell signaling pathways, which have been described in a variety of human cancer cell systems as well as in athymic mouse xenograft models. The ART compounds exert common as well as distinct cellular effects depending on the phenotype and tissue origin of the human cancer cells tested. (Firestone and Sundar, 2009).

#### *2.4.2. Potential general mechanisms of ART and its derivatives*

**2.4. Anti-cancer mechanism of ART and its derivatives**

184 Research Directions in Tumor Angiogenesis

The anti-cancer potential of ARTs has been demonstrated in various cancer cells including those of leukemia and other cancer cells of breast, ovary, liver, lung, pancreas and colon (Tan et al., 2011).The mechanisms of action of ARTs in cancer cells are associated with: 1) anti-angiogenic effects, 2) induction of apoptosis, 3) oxidative stress response, 4) oncogenes and tumor suppressor genes, and 5) multidrug resistance (Figure 2) (Efferth 2006; 2007).

**Figure 2.** Schema of tumor angiogenesis induced by hypoxia and the inhibitions of tumor growth by antiangiogenic artemisinins (ART) and its derivatives of dihydroartemisinin (DHA), artesunate (AS) and artemether (AM) follows three directions, including the inhibition of tumor cell synthesis of angiogenic proteins, the neutralization of angiogenic proteins by antibodies or traps, and the inhibition of endothelial cell binding to angiogenic proteins or direct induc‐

The endoperoxide moiety of ART has been shown to be pharmacologically important and responsible for antimalarial activity against the malaria parasites. The potent anticancer action of ARTs can be also attributed to the endoperoxide bond. In most of the *in vitro* cancer cell lines tested, preloading of cancer cells with iron or iron-saturated holo‐ transferrin triggers ART cytotoxicity with an increase in ARTs activity up to 100-fold against some cell lines. It has been hypothesized that iron-activated ARTs induce dam‐

*2.4.1. Anti-cancer mechanism of ARTs based on antimalarial actions*

tion of endothelial cell apoptosis.

Studies have identified potential general anti-cancer mechanisms of anti-cancer ARTs such as normalization of the upregulated Wnt/β-catenin pathway in colorectal cancer. Other pathways for anti-cancer activity include inhibition of enhanced angiogenesis associated with tumors. ARTs have been shown to inhibit proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVEC), inhibit VEGF binding to surface recep‐ tors on HUVEC and reduce expression of VEGF receptors Flt-1 and KDR/flk-1 on HUVECs. In cancer cells, artemisinins reduce expression of the VEGF receptor KDR/flk-1 in tumor and endothelial cells and slow the growth of human ovarian cancer HO-8910 xenografts in nude mice. HUVEC apoptosis by artesunate is associated with downregulation of Bcl-2 (B-cell leukemia/lymphoma 2) and upregulation of BAX (Bcl-2-associated X protein). In addition, mRNA expression of 30 out of 90 angiogenesis-related genes correlated significantly with the cellular response to ARTs, supporting the hypothesis that ARTs exert their anti-tumor effects by inhibition of tumor angiogenesis (Krishna et al., 2008).

#### *2.4.3. Anti-angiogenesis of ARTs including Anti-proliferation*

In the process of angiogenesis, the formation of new blood vessels from pre-existing ones is essential for the supply of tumors with oxygen and nutrients. If cancers reach a size for which diffusion alone cannot supply enough oxygen and nutrients angiogenesis is promot‐ ed by numerous pro-angiogenic or anti-angiogenic factors. The anti-angiogenic activities of ARTs were shown using various models of angiogenesis, namely, proliferation, migration and tube formation of endothelial cells. As a consequence, inhibitors of angiogenesis were considered as interesting possibilities for cancer therapy. As shown by several groups around the world, ART and its derivatives inhibit angiogenesis, and a detailed description of the ART-induced anti-angiogenic mechanisms will be described in Section 3.

sites into food vacuoles, where enzymatic degradation takes place (Semenov et al., 1998; Shenai et al., 2000). The release of heme-iron during hemoglobin digestion facilitates the cleavage of the endoperoxide moiety by a Fe (II) Fenton reaction. Breaking the endoperoxide bridge of ART results in the generation of reactive oxygen species, such as hydroxyl radicals and superoxide anions, which damage the food vacuole membranes and leads to subse‐ quent auto-digestion (Krishna et al., 2004; O'Neill and Posner, 2004). In addition, the heme iron (II)-mediated decomposition of ART leads to the generation of carbon-centered radical species (Butler et al., 1998). The cleavage of the endoperoxide bond of ART and its deriva‐ tives also leads to the alkylation of heme and some *Plasmodium*-specific proteins, including the *Plasmodium falciparum* translationally controlled tumor protein (TCTP) and the sarco/ endoplasmic reticulum Ca2+ ATPase (SERCA) ortholog of *Plasmodium falciparum* (Eckstein-Ludwig et al., 2003). Recent observations indicate, however, that heme iron (II) and oxida‐ tive stress are not the only mechanisms of ART's anti-malarial activity (Parapini et al., 2004).

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By comparing the baseline antioxidant mRNA gene expression in the NCI cell line panel with the IC50 values for AS, oxidative stress was found to play a role in the anti-tumor activi‐ ty of AS (Efferth, 2006). The expression of thioredoxin reductase and catalase correlated sig‐ nificantly with the IC50 values for AS against the tumor cell lines in the NCI panel. As tumor cells contain much less iron than erythrocytes, but more than other normal tissues (Shter‐ man et al., 1991), the question arises as to whether iron may be critical for ART's activity against tumor cells (Payne, 2003). The growth of tumors in rats was significantly retarded by daily oral administration of ferrous sulfate followed by dihydroartemisinin, while treatment with each drug applied alone had no effect (Moore et al., 1995). Cellular iron uptake and in‐ ternalization are mediated by binding of transferrin-iron complexes to the transferrin recep‐ tor (CD71) expressed on the cell surface membrane which leads to subsequent iron endocytosis. CD71 is normally expressed in the basal epidermis, endocrine pancreas, hepa‐ tocytes, Kupfer cells, testis, and pituitary, while most other tissues are CD71-negative. In contrast, CD71 is highly expressed in proliferating and malignant cells (Sutherland et al.,

1981) and it is widely distributed among clinical tumors (Gatter et al., 1983).

mia cells than to normal lymphocytes (Lai et al., 2005).

*2.4.6. Oncogenes and tumor suppressor genes*

tomatosis viral oncogene homolog

Interestingly, exposure of ART and its derivatives produces no or only marginal cytotoxicity to non-tumor cells. Human breast cells do not respond to treatment with transferrin plus DHA, while the growth of breast cancer cells is significantly inhibited (Singh and Lai, 2001). Similarly, ART tagged to transferrin has been shown to be more cytotoxic to MOLT-4 leuke‐

Oncogenes and tumor suppressor genes frequently affect downstream processes in tumor cells. The expression of several oncogenes and tumor suppressor genes has been shown to correlate with response to artesunate, including the epidermal growth factor receptor (*EGFR*), the tumor growth factor ß (*TGFB*), FBJ murine osteosarcoma viral oncogene homo‐ logue B (*FOSB*), *FOS*-like antigen-2 (*FOSL2*), the multiple endocrine neoplasia 1 gene (*MEN1*), *v-myb* avian myeloblastosis viral oncogene homolog (*MYB*), *v-myc* avian myelocy‐

#### *2.4.4. Induction of apoptosis*

ARTs induce cell cycle arrest in various cell types (Efferth, 2006). For example, DHA and AS effectively mediate G1 phase arrest in HepG2 and Hep3B cells (Hou et al., 2008), and DHA treatment has been shown to reduce cell numbers of HCT116 colon cancer cells in S phase (Lu et al., 2011). Interestingly, DHA treatment has also been shown to trigger G2 phase ar‐ rest in OVCA-420 ovarian cancer cells (Jiao et al., 2007). Thus, ART-mediated cell cycle arrest is possibly cell type dependent. ARTs have also been shown to induce apoptotic cell death in a number of cell types, in which the mitochondrial-mediated apoptotic pathway plays a decisive role (Lu et al., 2011). For instance, DHA has been shown to enhance Bax and re‐ duces Bcl-2 expression in cancer cells (Hou et al., 2008; Chen et al., 2009). DHA-induced apoptosis is abrogated by the loss of Bak and is largely reduced in cells with siRNA-mediat‐ ed down-regulation of Bak or NOXA (Handrick et al., 2010). DHA has been shown to acti‐ vate caspase-8, however, which is related to the death receptor-mediated apoptotic pathway in HL-60 cells (Liu et al., 2008). DHA has also been shown to enhance Fas expression and activates caspase-8 in ovarian cancer cells (Chen et al., 2009). In addition, DHA enhances death receptor 5 and activates both mitochondrial- and death receptor-mediated apoptotic pathways in prostate cancer cells (He et al., 2010). ARTs-induced apoptosis in cancer cells may involve p38 MAPK, however, rather than p53 (Hou et al., 2008; Lu et al., 2008).

Since most anti-cancer drugs kill tumor cells by the induction of apoptosis, the same may be true for ART and its derivatives. AS was first shown to promote apoptosis in tumor cells (Efferth *et al*., 1996). This has been subsequently confirmed by other groups (Li et al., 2001; Sadava et al., 2002; Singh and Lai, 2004; Wang et al., 2002; Yamachika et al., 2004). By micro‐ array and hierarchical cluster analyses, several apoptosis-regulating genes were identified, whose mRNA expression correlated significantly with the IC50 values for AS in the NCI can‐ cer cell lines (Efferth et al., 2003).

#### *2.4.5. Oxidative stress response*

ART is first activated in malaria parasites by intra-parasitic heme-iron, which catalyzes the cleavage of the endoperoxide bond. The *Plasmodium* trophozoites and schizonts live within red blood cells, where hemoglobin serves as an amino acid source. It is taken up by the para‐ sites into food vacuoles, where enzymatic degradation takes place (Semenov et al., 1998; Shenai et al., 2000). The release of heme-iron during hemoglobin digestion facilitates the cleavage of the endoperoxide moiety by a Fe (II) Fenton reaction. Breaking the endoperoxide bridge of ART results in the generation of reactive oxygen species, such as hydroxyl radicals and superoxide anions, which damage the food vacuole membranes and leads to subse‐ quent auto-digestion (Krishna et al., 2004; O'Neill and Posner, 2004). In addition, the heme iron (II)-mediated decomposition of ART leads to the generation of carbon-centered radical species (Butler et al., 1998). The cleavage of the endoperoxide bond of ART and its deriva‐ tives also leads to the alkylation of heme and some *Plasmodium*-specific proteins, including the *Plasmodium falciparum* translationally controlled tumor protein (TCTP) and the sarco/ endoplasmic reticulum Ca2+ ATPase (SERCA) ortholog of *Plasmodium falciparum* (Eckstein-Ludwig et al., 2003). Recent observations indicate, however, that heme iron (II) and oxida‐ tive stress are not the only mechanisms of ART's anti-malarial activity (Parapini et al., 2004).

By comparing the baseline antioxidant mRNA gene expression in the NCI cell line panel with the IC50 values for AS, oxidative stress was found to play a role in the anti-tumor activi‐ ty of AS (Efferth, 2006). The expression of thioredoxin reductase and catalase correlated sig‐ nificantly with the IC50 values for AS against the tumor cell lines in the NCI panel. As tumor cells contain much less iron than erythrocytes, but more than other normal tissues (Shter‐ man et al., 1991), the question arises as to whether iron may be critical for ART's activity against tumor cells (Payne, 2003). The growth of tumors in rats was significantly retarded by daily oral administration of ferrous sulfate followed by dihydroartemisinin, while treatment with each drug applied alone had no effect (Moore et al., 1995). Cellular iron uptake and in‐ ternalization are mediated by binding of transferrin-iron complexes to the transferrin recep‐ tor (CD71) expressed on the cell surface membrane which leads to subsequent iron endocytosis. CD71 is normally expressed in the basal epidermis, endocrine pancreas, hepa‐ tocytes, Kupfer cells, testis, and pituitary, while most other tissues are CD71-negative. In contrast, CD71 is highly expressed in proliferating and malignant cells (Sutherland et al., 1981) and it is widely distributed among clinical tumors (Gatter et al., 1983).

Interestingly, exposure of ART and its derivatives produces no or only marginal cytotoxicity to non-tumor cells. Human breast cells do not respond to treatment with transferrin plus DHA, while the growth of breast cancer cells is significantly inhibited (Singh and Lai, 2001). Similarly, ART tagged to transferrin has been shown to be more cytotoxic to MOLT-4 leuke‐ mia cells than to normal lymphocytes (Lai et al., 2005).

#### *2.4.6. Oncogenes and tumor suppressor genes*

*2.4.3. Anti-angiogenesis of ARTs including Anti-proliferation*

*2.4.4. Induction of apoptosis*

186 Research Directions in Tumor Angiogenesis

cer cell lines (Efferth et al., 2003).

*2.4.5. Oxidative stress response*

In the process of angiogenesis, the formation of new blood vessels from pre-existing ones is essential for the supply of tumors with oxygen and nutrients. If cancers reach a size for which diffusion alone cannot supply enough oxygen and nutrients angiogenesis is promot‐ ed by numerous pro-angiogenic or anti-angiogenic factors. The anti-angiogenic activities of ARTs were shown using various models of angiogenesis, namely, proliferation, migration and tube formation of endothelial cells. As a consequence, inhibitors of angiogenesis were considered as interesting possibilities for cancer therapy. As shown by several groups around the world, ART and its derivatives inhibit angiogenesis, and a detailed description

ARTs induce cell cycle arrest in various cell types (Efferth, 2006). For example, DHA and AS effectively mediate G1 phase arrest in HepG2 and Hep3B cells (Hou et al., 2008), and DHA treatment has been shown to reduce cell numbers of HCT116 colon cancer cells in S phase (Lu et al., 2011). Interestingly, DHA treatment has also been shown to trigger G2 phase ar‐ rest in OVCA-420 ovarian cancer cells (Jiao et al., 2007). Thus, ART-mediated cell cycle arrest is possibly cell type dependent. ARTs have also been shown to induce apoptotic cell death in a number of cell types, in which the mitochondrial-mediated apoptotic pathway plays a decisive role (Lu et al., 2011). For instance, DHA has been shown to enhance Bax and re‐ duces Bcl-2 expression in cancer cells (Hou et al., 2008; Chen et al., 2009). DHA-induced apoptosis is abrogated by the loss of Bak and is largely reduced in cells with siRNA-mediat‐ ed down-regulation of Bak or NOXA (Handrick et al., 2010). DHA has been shown to acti‐ vate caspase-8, however, which is related to the death receptor-mediated apoptotic pathway in HL-60 cells (Liu et al., 2008). DHA has also been shown to enhance Fas expression and activates caspase-8 in ovarian cancer cells (Chen et al., 2009). In addition, DHA enhances death receptor 5 and activates both mitochondrial- and death receptor-mediated apoptotic pathways in prostate cancer cells (He et al., 2010). ARTs-induced apoptosis in cancer cells

of the ART-induced anti-angiogenic mechanisms will be described in Section 3.

may involve p38 MAPK, however, rather than p53 (Hou et al., 2008; Lu et al., 2008).

Since most anti-cancer drugs kill tumor cells by the induction of apoptosis, the same may be true for ART and its derivatives. AS was first shown to promote apoptosis in tumor cells (Efferth *et al*., 1996). This has been subsequently confirmed by other groups (Li et al., 2001; Sadava et al., 2002; Singh and Lai, 2004; Wang et al., 2002; Yamachika et al., 2004). By micro‐ array and hierarchical cluster analyses, several apoptosis-regulating genes were identified, whose mRNA expression correlated significantly with the IC50 values for AS in the NCI can‐

ART is first activated in malaria parasites by intra-parasitic heme-iron, which catalyzes the cleavage of the endoperoxide bond. The *Plasmodium* trophozoites and schizonts live within red blood cells, where hemoglobin serves as an amino acid source. It is taken up by the para‐

Oncogenes and tumor suppressor genes frequently affect downstream processes in tumor cells. The expression of several oncogenes and tumor suppressor genes has been shown to correlate with response to artesunate, including the epidermal growth factor receptor (*EGFR*), the tumor growth factor ß (*TGFB*), FBJ murine osteosarcoma viral oncogene homo‐ logue B (*FOSB*), *FOS*-like antigen-2 (*FOSL2*), the multiple endocrine neoplasia 1 gene (*MEN1*), *v-myb* avian myeloblastosis viral oncogene homolog (*MYB*), *v-myc* avian myelocy‐ tomatosis viral oncogene homolog

(*MYC*), *c-src* tyrosine kinase (*CSK*), *v-raf* murine sarcoma viral oncogene homolog B1 (*BRAF*), the *RAS* oncogene family members *ARHC, ARHE*, *RAB2 and RAN*, the breast cancer susceptibility gene 2 (*BRCA2*), and others (Efferth et al., 2003).

damage and several other stresses, including drug stress, the amount of p53 is increased due to disruption of its degradation. Artesunate could inhibit HSCs proliferation in vitro through increase the expression of p53 (Efferth et al., 2006; Hou et al., 2008; Lu et al., 2008).

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A prominent feature of ART and its derivatives in malaria treatment shows early signs of cross-resistance to other antimalarial drugs. ARTs are therefore very valuable for the treat‐ ment of otherwise unresponsive, multidrug-resistant malaria parasites (Li and Weina 2011). Therefore, it is reasonable to ask whether ARTs are involved in the multidrug-resistance phenotypes observed in tumor cells. A comparison of the microarray-based mRNA expres‐ sion of the multidrug resistance-conferring *ABCB1* gene (*MDR1*; P-glycoprotein) was con‐ ducted with the IC50 values determined for tumor cells treated with AS and dihydroartemisinyl ester stereoisomer 1, but no significant relationships were observed.

Similarly, the flow cytometric measurement of the fluorescent probe rhodamine 123, which represents a functional assay for P-glycoprotein, did not reveal significant correlations, and similar results were obtained with other ARTs. As a control, we used the established antitumor drug docetaxel (taxotere), which is a known substrate of *MDR1* (Shirakawa et al., 1999). The IC50 values determined for cells treated with docetaxel correlated both with rhod‐ amine 123 efflux and *MDR1* mRNA expression. To validate these results obtained by corre‐ lation analyses, cell lines over-expressing *MDR1*/P-glycoprotein as well as other drug resistance-conferring genes were used. AS was shown similarly active towards drug-sensi‐ tive and multidrug resistant cell lines (Efferth et al., 2002; 2003). Likewise, methotrexate-re‐ sistant CEM/MTX1500LV cells with an amplification of the dihydrofolate reductase (*DHFR*) gene and hydroxyurea-resistant CEM/HUR90 cells with over-expression of ribonucleotide reductase (RRPM2) were not cross-resistant to AS. In addition, other research has shown that ART increased the tissue permeability for standard cytostatic drugs. i.e. doxorubicin in

mouse embryonic stem cell-derived embryoid bodies (Wartenberg et al., 2003).

**3. Anti-cancer effect of ARTs** *via* **an anti-angiogenic activity**

In the process of angiogenesis, the formation of new blood vessels from pre-existing ones is essential for the supply of tumors with oxygen and nutrients and for the spread of metastat‐ ic cells throughout the body. Normal angiogenesis is strictly controlled by some transient, typical physiological processes such as reproduction, development, wound healing; contin‐ ued angiogenesis is also a characteristic of pathological alteration such as neoplasia. Neopla‐ sia is an angiogenesis-dependent disease, and the growth of tumors, intravasation and metastases require angiogenesis. In human and experimental cancers, new vessels are re‐ quired for increased delivery of nutrients and are a target for invading tumor cells, and there is a large body of evidence to support a key role for angiogenesis in disease progres‐ sion. The growth, invasion and metastasis of tumors have been shown to be dependent on

angiogenesis. A summary of the anti-angiogenic effects of ARTs is shown in Table 1.

*2.4.7. Multidrug resistance*

The epidermal growth factor receptor (*EGFR*) represents an exquisite target for therapeu‐ tic interventions, and molecular approaches to study the expression of the EGFR gene have yielded some very interesting findings. Glioblastoma cells transfected with a dele‐ tion-activated *EGFR* cDNA were more resistant to AS than the control cells which agrees well with microarray gene expression data (Efferth et al., 2003). In addition to playing a role in drug resistance, the activation of *EGFR*-coupled signaling routes drives mitogenic and other cancer-promoting processes, e.g. proliferation, angiogenesis, and inhibition of apoptosis (Efferth 2006). In addition, combination treatment of the EGFR tyrosine kinase inhibitor, OSI-774, plus AS was investigated and synergistic effects were found in glio‐ blastoma cells transfected with a deletion-activated *EGFR* cDNA, and additive effects were shown to occur in cells transfected with wild-type *EGFR* (Efferth et al., 2004a). A profile of chromosomal gains and losses was determined by comparative genomic hy‐ bridization in nine non-transfected glioblastoma cell lines, and this profile correlated well with the IC50 values determined after treatment of the same glioblastoma cell lines with the combination treatment of AS and OSI-774. Genes located at genomic loci correlating to cellular response to AS and OSI-774 may serve as candidate genes to determine drug sensitivity and resistance (Efferth 2007).

By screening a panel of isogenic *Saccaromyces cerevisiae* strains with defined genetic muta‐ tions in DNA repair, DNA checkpoint, and cell proliferation genes, one yeast strain with a defective mitosis-regulating *BUB3* gene showed increased sensitivity to AS treatment. An‐ other strain with a defective proliferation-regulating *CLN2* gene showed increased AS resist‐ ance over the wild-type strain. None of the other DNA repair or DNA check-point deficient isogenic strains were different from wild-type yeast (Efferth et al., 2001). The conditional ex‐ pression of the *CDC25A* gene by a tetracycline repressor expression vector (tet-off system) has been shown to increase cellular sensitivity to AS treatment (Efferth et al., 2003). CDC25A is a key regulator of the cell cycle, which drives cells from the G1 phase into S phase. AS has been shown to down-regulate the expression of the CDC25A protein which supports the hy‐ pothesis that AS interferes with cell cycle regulation (Efferth et al., 2003).

The IC50 values for artesunate were correlated with the constitutive mRNA expression levels measured by microarray hybridization. Scientists selected expression data of 559 genes de‐ posited in the NCI's database (http://dtp.nci.nih.gov). The mRNA expression has been deter‐ mined as reported. These genes belong to different categories of biological functions (63 apoptosis-regulating genes, 113 proliferation associated genes, 140 anti-oxidative stress re‐ sponse genes, 90 angiogenesis-regulating genes, 123 oncogenes and tumor suppressor genes). For example, p53, the ''guardian of the genome'', is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apopto‐ sis, senescence and DNA repair. In normal cells, p53 is frequently undetectable due to fast ubiquitination by mdm-2 and subsequent proteasomal degradation. However, upon DNA damage and several other stresses, including drug stress, the amount of p53 is increased due to disruption of its degradation. Artesunate could inhibit HSCs proliferation in vitro through increase the expression of p53 (Efferth et al., 2006; Hou et al., 2008; Lu et al., 2008).

#### *2.4.7. Multidrug resistance*

(*MYC*), *c-src* tyrosine kinase (*CSK*), *v-raf* murine sarcoma viral oncogene homolog B1 (*BRAF*), the *RAS* oncogene family members *ARHC, ARHE*, *RAB2 and RAN*, the breast cancer

The epidermal growth factor receptor (*EGFR*) represents an exquisite target for therapeu‐ tic interventions, and molecular approaches to study the expression of the EGFR gene have yielded some very interesting findings. Glioblastoma cells transfected with a dele‐ tion-activated *EGFR* cDNA were more resistant to AS than the control cells which agrees well with microarray gene expression data (Efferth et al., 2003). In addition to playing a role in drug resistance, the activation of *EGFR*-coupled signaling routes drives mitogenic and other cancer-promoting processes, e.g. proliferation, angiogenesis, and inhibition of apoptosis (Efferth 2006). In addition, combination treatment of the EGFR tyrosine kinase inhibitor, OSI-774, plus AS was investigated and synergistic effects were found in glio‐ blastoma cells transfected with a deletion-activated *EGFR* cDNA, and additive effects were shown to occur in cells transfected with wild-type *EGFR* (Efferth et al., 2004a). A profile of chromosomal gains and losses was determined by comparative genomic hy‐ bridization in nine non-transfected glioblastoma cell lines, and this profile correlated well with the IC50 values determined after treatment of the same glioblastoma cell lines with the combination treatment of AS and OSI-774. Genes located at genomic loci correlating to cellular response to AS and OSI-774 may serve as candidate genes to determine drug

By screening a panel of isogenic *Saccaromyces cerevisiae* strains with defined genetic muta‐ tions in DNA repair, DNA checkpoint, and cell proliferation genes, one yeast strain with a defective mitosis-regulating *BUB3* gene showed increased sensitivity to AS treatment. An‐ other strain with a defective proliferation-regulating *CLN2* gene showed increased AS resist‐ ance over the wild-type strain. None of the other DNA repair or DNA check-point deficient isogenic strains were different from wild-type yeast (Efferth et al., 2001). The conditional ex‐ pression of the *CDC25A* gene by a tetracycline repressor expression vector (tet-off system) has been shown to increase cellular sensitivity to AS treatment (Efferth et al., 2003). CDC25A is a key regulator of the cell cycle, which drives cells from the G1 phase into S phase. AS has been shown to down-regulate the expression of the CDC25A protein which supports the hy‐

The IC50 values for artesunate were correlated with the constitutive mRNA expression levels measured by microarray hybridization. Scientists selected expression data of 559 genes de‐ posited in the NCI's database (http://dtp.nci.nih.gov). The mRNA expression has been deter‐ mined as reported. These genes belong to different categories of biological functions (63 apoptosis-regulating genes, 113 proliferation associated genes, 140 anti-oxidative stress re‐ sponse genes, 90 angiogenesis-regulating genes, 123 oncogenes and tumor suppressor genes). For example, p53, the ''guardian of the genome'', is a transcription factor that can bind to promoter regions of hundreds of genes where it either activates or suppresses gene expression. Thereby, p53 serves as a tumor suppressor by inducing cell cycle arrest, apopto‐ sis, senescence and DNA repair. In normal cells, p53 is frequently undetectable due to fast ubiquitination by mdm-2 and subsequent proteasomal degradation. However, upon DNA

pothesis that AS interferes with cell cycle regulation (Efferth et al., 2003).

susceptibility gene 2 (*BRCA2*), and others (Efferth et al., 2003).

188 Research Directions in Tumor Angiogenesis

sensitivity and resistance (Efferth 2007).

A prominent feature of ART and its derivatives in malaria treatment shows early signs of cross-resistance to other antimalarial drugs. ARTs are therefore very valuable for the treat‐ ment of otherwise unresponsive, multidrug-resistant malaria parasites (Li and Weina 2011). Therefore, it is reasonable to ask whether ARTs are involved in the multidrug-resistance phenotypes observed in tumor cells. A comparison of the microarray-based mRNA expres‐ sion of the multidrug resistance-conferring *ABCB1* gene (*MDR1*; P-glycoprotein) was con‐ ducted with the IC50 values determined for tumor cells treated with AS and dihydroartemisinyl ester stereoisomer 1, but no significant relationships were observed.

Similarly, the flow cytometric measurement of the fluorescent probe rhodamine 123, which represents a functional assay for P-glycoprotein, did not reveal significant correlations, and similar results were obtained with other ARTs. As a control, we used the established antitumor drug docetaxel (taxotere), which is a known substrate of *MDR1* (Shirakawa et al., 1999). The IC50 values determined for cells treated with docetaxel correlated both with rhod‐ amine 123 efflux and *MDR1* mRNA expression. To validate these results obtained by corre‐ lation analyses, cell lines over-expressing *MDR1*/P-glycoprotein as well as other drug resistance-conferring genes were used. AS was shown similarly active towards drug-sensi‐ tive and multidrug resistant cell lines (Efferth et al., 2002; 2003). Likewise, methotrexate-re‐ sistant CEM/MTX1500LV cells with an amplification of the dihydrofolate reductase (*DHFR*) gene and hydroxyurea-resistant CEM/HUR90 cells with over-expression of ribonucleotide reductase (RRPM2) were not cross-resistant to AS. In addition, other research has shown that ART increased the tissue permeability for standard cytostatic drugs. i.e. doxorubicin in mouse embryonic stem cell-derived embryoid bodies (Wartenberg et al., 2003).

#### **3. Anti-cancer effect of ARTs** *via* **an anti-angiogenic activity**

In the process of angiogenesis, the formation of new blood vessels from pre-existing ones is essential for the supply of tumors with oxygen and nutrients and for the spread of metastat‐ ic cells throughout the body. Normal angiogenesis is strictly controlled by some transient, typical physiological processes such as reproduction, development, wound healing; contin‐ ued angiogenesis is also a characteristic of pathological alteration such as neoplasia. Neopla‐ sia is an angiogenesis-dependent disease, and the growth of tumors, intravasation and metastases require angiogenesis. In human and experimental cancers, new vessels are re‐ quired for increased delivery of nutrients and are a target for invading tumor cells, and there is a large body of evidence to support a key role for angiogenesis in disease progres‐ sion. The growth, invasion and metastasis of tumors have been shown to be dependent on angiogenesis. A summary of the anti-angiogenic effects of ARTs is shown in Table 1.


**3.1. Anti-angiogenic effects of ARTs**

**Artemisinin (ART)**

**Dihydroartemisinin (DHA)**

*3.1.1. In vitro anti-angiogenic effects of ART and its derivatives*

been shown to increase the potency of ARTs in killing cancer cells.

addition to their cytotoxic effects (Anfosso et al., 2006).

VEGF-C and reducing tumor lymphangiogenesis (Wang et al., 2008).

While most of the research on the anti-cancer activities of ARTs has been performed with cell lines *in vitro*, there are a few reports in the literature showing activity *in vivo* against xen‐ ograft tumors, e.g., breast tumors, ovarian cancer, Kaposi sarcoma, fibrosarcoma, or liver cancer. The *in vitro* data in the literature supports the hypothesis that ART and its deriva‐ tives kill or inhibit the growth of many types of cancer cell lines, including drug-resistant cell lines, suggesting that ART could become the basis of a new class of anti-cancer drugs. In addition, the co-administration of holotransferrin and other iron sources with ARTs has

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ARTs are antimalarial agents, but also reveal profound antitumor activity *in vitro* and *in vivo*. Ina microarray study of cancer cells treated at the 50% inhibition concentration with eight ARTs, (ART, AS, arteether, artemisetene, arteanuine B, dihydroartemisinylester stereo‐ isomers 1 and 2) the mRNA expression data of 89 known angiogenesis-related genes was obtained and correlated against the sensitivity of these tumor cells to ARTs treatment. The constitutive expression of 30 genes correlated significantly with the cellular response to ARTs. The finding cell sensitivity and resistance of tumor cells could be predicted by the mRNA expression of angiogenesis related genes supports the hypothesis that ARTs reveal their antitumor effects at least, in part, by inhibition of tumor angiogenesis. As many chemopreventive drugs exert anti-angiogenic features, ARTs might also be chemo-preventive in

A recent study demonstrated that ART-induced cell growth arrest in A375M malignant melanoma tumor cells also affected the viability of A375P cutaneous melanoma tumor cells with both cytotoxic and growth inhibitory effects, while ART was not effective in inhibiting the growth of other tumor cell lines (MCF7 and MKN). In addition, ART treat‐ ment affected the migratory ability of A375M cells by reducing metalloproteinase 2 (MMP-2) productions and down-regulating αvβ3 integrin expression. These findings sup‐ port the hypothesis that ART may serve as a chemotherapeutic agent for melanoma treatment (Buommino et al., 2009). Furthermore, IL-1beta-induced p38 mitogen-activated protein kinase (MAPK) activation and upregulation of VEGF-C mRNA, and VEGF-C re‐ ceptor protein levels in LLC cells were also suppressed by ART or by the p38 MAPK in‐ hibitor SB-203580, suggesting that p38 MAPK could serve as a mediator of proinflammatory cytokine-induced VEGF-C expression. These data support the hypothesis that ART may be useful for the prevention of lymph node metastasis by downregulating

DHA and AS have been shown to be remarkable inhibitors of tumor cell growth and sup‐ pression of angiogenesis *in vitro*. The anti-cancer activity of ARTs has been demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) growth inhibition

VEGF = vascular endothelial growth factor; HIF = hypoxia-inducible factor; NF-kB = nuclear factor of kappa light poly‐ peptide gene enhancer in B cells 1; KDR = kinase insert domain protein recepto; MMP = matrix metalloproteinase; BMP = bone morphogenic protein; αvβ3 = Transmembrane heterodimeric protein expressed on sprouting endothelial cells; HUVEC = human umbilical vein endothelial cells. CAM = chorioallantoic membrane

**Table 1.** Anti-angiogenic effects of ART and its derivatives

#### **3.1. Anti-angiogenic effects of ARTs**

#### *3.1.1. In vitro anti-angiogenic effects of ART and its derivatives*

While most of the research on the anti-cancer activities of ARTs has been performed with cell lines *in vitro*, there are a few reports in the literature showing activity *in vivo* against xen‐ ograft tumors, e.g., breast tumors, ovarian cancer, Kaposi sarcoma, fibrosarcoma, or liver cancer. The *in vitro* data in the literature supports the hypothesis that ART and its deriva‐ tives kill or inhibit the growth of many types of cancer cell lines, including drug-resistant cell lines, suggesting that ART could become the basis of a new class of anti-cancer drugs. In addition, the co-administration of holotransferrin and other iron sources with ARTs has been shown to increase the potency of ARTs in killing cancer cells.

#### **Artemisinin (ART)**

**Artemisinins Effects/Mechanism References**

Dell'Eva et al., 2004 Huan-huan et al., 2004 Chen et al., 2004a Chen et al., 2004b Chen et al., 2004c Zhou et al., 2007 Zhou et al., 2007 Chen et al., 2010a He et al., 2011 Liu et al., 2011

Chen et al., 2003 Chen et al., 2004a Lee et al., 2006 Wu et al., 2006 Wang et al., 2007 Chen et al., 2010b Hwang et al., 2010 Zhou et al., 2010 Rasheed et al., 2010 Wang et al., 2010 Aung et al., 2011 Lu et al., 2011 Wang et al., 2011

Anfosso et al., 2006 Anfosso et al., 2006 Wang et al., 2008 Buommino et al., 2009

D'Alessandro et al., 2007

2011

Galal et al., 2009 Soomro et al.,

1) Induction of apoptosis in KS-IMM cells 2) Reduced F1t-1 and KDR/flk-1 expressions 3) Lowered VEGF and KDR/flk-1 expression 4) inhibited the proliferation of HUVEC 5) Inhibited HUVEC and VEGF expression 6) Suppress angiogenic ability & Decreased VEGF

8) Decreased VEGF and Ang-1 secretion 9) Decreased the secretion of VEGF and IL-8 10) Either increased cytotoxicity or cytostasis

1) DHA was more effective than AS 2) Reduced VEGF binding to its receptors 3) Induced K562 cells apoptosis, inhibited VEGF 4) Reduced VEGF secretion by RPMI8226 cells 5) Attenuated the levels of VEGFR-3/Flt-4. 6) Decreased KDR levels and NF-kB DNA binding

7) Inhibition of PKCalpha/Raf/MAPKs 8) Decreased VEGF receptor KDR/flk-1 9) Inhibited the expression of several MMPs 10) DHA inactivates NF-kappaB and potentiates

12) Inducted iron-dependent endoplasmic reticulum stress

less anti-angiogenic effect than DHA in all the experimental

1) Active against solid tumor-derived cell lines and good

2) More active in vitro and in vivo than the commonly used

VEGF = vascular endothelial growth factor; HIF = hypoxia-inducible factor; NF-kB = nuclear factor of kappa light poly‐ peptide gene enhancer in B cells 1; KDR = kinase insert domain protein recepto; MMP = matrix metalloproteinase; BMP = bone morphogenic protein; αvβ3 = Transmembrane heterodimeric protein expressed on sprouting endothelial

Thioacetal ARTs inhibitiory activity upon HUVEC Oh et al., 2003 ART-glycolipid hybrids Showed potent in vivo anti-angiogenic activity on CAM Ricci et al., 2010

cells; HUVEC = human umbilical vein endothelial cells. CAM = chorioallantoic membrane

13) DHA inhibits formation of HUVECs, MMP9

2) Decreased MMP2, MMP9 and BMP1 levels 3) Decreased VEGF-C, IL-1 β-induced p38 4) Decreased αvβ3 transcription

11) Down-regulated VEGF

correlation with other ARTs

models

AS

**Table 1.** Anti-angiogenic effects of ART and its derivatives

1) Decreased VEGF-A transcription

7) Decreased HIF-1α levels

Artesunate (AS)

190 Research Directions in Tumor Angiogenesis

Dihydro-artemisinin

Artemisinin (ART)

2nd Artemisinin artemisone

Artemisinin-like compounds (ART-like)

(DHA)

ARTs are antimalarial agents, but also reveal profound antitumor activity *in vitro* and *in vivo*. Ina microarray study of cancer cells treated at the 50% inhibition concentration with eight ARTs, (ART, AS, arteether, artemisetene, arteanuine B, dihydroartemisinylester stereo‐ isomers 1 and 2) the mRNA expression data of 89 known angiogenesis-related genes was obtained and correlated against the sensitivity of these tumor cells to ARTs treatment. The constitutive expression of 30 genes correlated significantly with the cellular response to ARTs. The finding cell sensitivity and resistance of tumor cells could be predicted by the mRNA expression of angiogenesis related genes supports the hypothesis that ARTs reveal their antitumor effects at least, in part, by inhibition of tumor angiogenesis. As many chemopreventive drugs exert anti-angiogenic features, ARTs might also be chemo-preventive in addition to their cytotoxic effects (Anfosso et al., 2006).

A recent study demonstrated that ART-induced cell growth arrest in A375M malignant melanoma tumor cells also affected the viability of A375P cutaneous melanoma tumor cells with both cytotoxic and growth inhibitory effects, while ART was not effective in inhibiting the growth of other tumor cell lines (MCF7 and MKN). In addition, ART treat‐ ment affected the migratory ability of A375M cells by reducing metalloproteinase 2 (MMP-2) productions and down-regulating αvβ3 integrin expression. These findings sup‐ port the hypothesis that ART may serve as a chemotherapeutic agent for melanoma treatment (Buommino et al., 2009). Furthermore, IL-1beta-induced p38 mitogen-activated protein kinase (MAPK) activation and upregulation of VEGF-C mRNA, and VEGF-C re‐ ceptor protein levels in LLC cells were also suppressed by ART or by the p38 MAPK in‐ hibitor SB-203580, suggesting that p38 MAPK could serve as a mediator of proinflammatory cytokine-induced VEGF-C expression. These data support the hypothesis that ART may be useful for the prevention of lymph node metastasis by downregulating VEGF-C and reducing tumor lymphangiogenesis (Wang et al., 2008).

#### **Dihydroartemisinin (DHA)**

DHA and AS have been shown to be remarkable inhibitors of tumor cell growth and sup‐ pression of angiogenesis *in vitro*. The anti-cancer activity of ARTs has been demonstrated by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) growth inhibition assay of four human cancer cell lines, cervical cancer HeLa, uterus chorion cancer JAR, em‐ bryo transversal cancer RD and ovarian cancer HO-8910 treated with DHA and AS. IC50 val‐ ues obtained through this MTT growth inhibition assay demonstrated that DHA was more effective at inhibiting cancer cell lines than AS. The anti-angiogenic activities of DHA and AS were tested on *in vitro* models of angiogenesis by assessing the proliferation, migration and tube formation of human umbilical vein endothelial (HUVE) cells. The results showed that DHA and AS significantly inhibited angiogenesis in a dose-dependent manner. These results also showed that DHA was more effective than ART in inhibiting angiogenesis (Chen et al., 2003).

tor under induction of cell apoptosis, inhibition of the migration, and formation of tubelike structures in LECs (Wang et al., 2007). In addition, to investigate the effects of DHA on cell cycle progression and NF-kappaB activity in pancreatic cancer cells, the cell cycle progression was determined. The translocation and DNA-binding activity of NF-kappaB were inhibited in DHA-treated cells in a dose-dependent manner, indicated the inactiva‐ tion effects of DHA in pancreatic cancer cells. Study shows that DHA induces cell cycle arrest and apoptosis in pancreatic cancer cells, and this effect might be due to inhibition

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One study showed that DHA is an effective anti-metastatic agent that functions by downregulating the MMP-9 gene which is associated with metastasis. 1) DHA was shown to re‐ duce phorbol myristate acetate (PMA)-induced activation of MMP-9 and MMP-2 and further inhibited cell invasion and migration. 2) DHA was also shown to suppress the PMAenhanced expression of the levels of MMP-9 protein and mRNA, and enhanced transcrip‐ tional activity of the MM-9 gene through suppression of NF-kappaB and activation of AP-1 without changing the level of tissue inhibition of metalloproteinase (TIMP)-1. 3) DHA has been shown to reduce PMA-enhanced MMP-2 expression by suppressing membrane-type 1 MMP (MT1-MMP), but was not shown to t alter TIMP-2 levels. 4) DHA was shown to inhib‐ it PMA-induced NF-kappaB and c-Jun nuclear translocation, which are upstream of PMAinduced MMP-9 expression which enhances metastasis. 5) DHA strongly repressed the PMA-induced phosphorylation of Raf/ERK and JNK, which are dependent on the PKC al‐ pha pathway. In summary, this study demonstrated that the anti-invasive effects of DHA may occur through inhibition of PKC alpha/Raf/ERK and JNK phosphorylation and reduc‐ tion of NF-kappaB and AP-1 activation, leading to down-regulation of MMP-9 expression.

Wang et al. demonstrated that DHA enhances gemcitabine-induced growth inhibition and apoptosis in both BxPC-3 and PANC-1 cell lines *in vitro*. The effect is at least partially due to the DHA-driven deactivation of gemcitabine-induced NF-kappaB activation, which in turn leads to a tremendous decrease in the expression of NF-kappaB target gene products, such as c-myc, cyclin D1, Bcl-2, Bcl-xL (Wang et al., 2010). DHA was also shown to exhibit signifi‐ cant anti-cancer activity against the renal epithelial LLC cell line. In addition, DHA was shown to induce apoptosis of LLC cells and influenced the expression of the vascular endo‐ thelial growth factor (VEGF) receptor KDR/flk-1. Furthermore, in both tumor xenografts, a greater degree of growth inhibition was achieved when DHA and chemotherapeutic drugs were used in combination. The combined effect of DHA administered with chemotherapy

The effect of DHA was investigated using *in vitro*/*in vivo* optical imaging combined with cell/tumor growth assays of the pancreatic cancer cell line BxPc3-RFP which stably expresses red fluorescence protein. DHA inhibited the proliferation and viability of pancreatic cancer cells in a dose-dependent manner and induced apoptosis. The results of this experiment demonstrated DHA-induced down-regulation of PCNA and Bcl-2, and up-regulation of Bax. VEGF expression was down-regulated by DHA in cells under normoxic, but not hypox‐ ic, conditions. The anti-angiogenic effect of DHA appears to be a complicated process (Aung

drugs on LLC tumor metastasis was shown to be significant (Zhou et al., 2010).

of NF-kappaB signaling (Chen et al., 2010b).

(Hwang et al., 2010)

The effect of DHA on human multiple myeloma-induced angiogenesis under hypoxia and elucidated its mechanism of action has been performed. An *in vivo* chicken chorioallantoic membrane model was used to examine the effect of DHA on multiple myeloma-induced an‐ giogenesis. Compared with conditioned medium of control, conditioned medium from hu‐ man multiple myeloma RPMI8226 cells pretreated with 3 µM DHA in hypoxia was observed to reduce microvessel growth on chicken chorioallantoic membranes by approxi‐ mately 28.6% (P < 0.05). The level of VEGF in conditioned medium was determined by en‐ zyme-linked immunosorbent assay. The results confirmed that 3 µM DHA could significantly decrease VEGF secretion by RPMI8226 cells (P < 0.05), which correlated well with the reduction of multiple myeloma-induced angiogenesis on chicken chorioallantoic membranes. Western blot and reverse transcription-PCR results revealed that DHA downre‐ gulated the expression of VEGF in RPMI8226 cells in hypoxia. Therefore, DHA possesses potential as an antiangiogenic drug in multiple myeloma therapy and thereby may improve patient outcome (Wu et al., 2006).

The effect of DHA on VEGF expression and apoptosis in chronic myeloid leukemia (CML) K562 cells was assessed. The results demonstrated that in addition to its anti-proliferation effect on CML cells, DHA was also found to induce K562 cells apoptosis. The percentage of apoptotic cells was increased to 6.9 and 15.8% after being treated with 5 and 10 µM DHA for 48 h, respectively (P < 0.001). All these experiments suggested that DHA could inhibit the VEGF expression and secretion effectively in K562 cells, even at a lower concentration (2 µM, P < 0.05). Moreover, we further assessed the stimulating angiogenic activity of CM from K562 cells on CAM model. Also, the angiogenic activity was decreased in response to the CM from K562 cells pretreated with DHA in a dose-dependent manner. Taken together, these results from our study together with its known low toxicity make it possible that DHA might present potential anti-leukemia effect as a treatment for CML therapy, or as an ad‐ junct to standard chemotherapeutic regimens (Lee et al., 2006)

DHA was found to have a potent ability in influencing lymphatic endothelial cells (LECs) behavior. DHA also exerted a significant inhibitory effect on migration and tubelike formation of LECs in a dose-dependent manner. Quantitative RT-PCR further showed that DHA remarkably downregulated the expression of antiapoptotic bcl-2 mRNA, but upregulated that of the proapoptotic gene bax mRNA. In addition, DHA could strongly attenuate the mRNA and protein levels of VEGFR-3/Flt-4. In summary, these findings indicate that DHA may be useful as a potential lymphangiogenesis inhibi‐ tor under induction of cell apoptosis, inhibition of the migration, and formation of tubelike structures in LECs (Wang et al., 2007). In addition, to investigate the effects of DHA on cell cycle progression and NF-kappaB activity in pancreatic cancer cells, the cell cycle progression was determined. The translocation and DNA-binding activity of NF-kappaB were inhibited in DHA-treated cells in a dose-dependent manner, indicated the inactiva‐ tion effects of DHA in pancreatic cancer cells. Study shows that DHA induces cell cycle arrest and apoptosis in pancreatic cancer cells, and this effect might be due to inhibition of NF-kappaB signaling (Chen et al., 2010b).

assay of four human cancer cell lines, cervical cancer HeLa, uterus chorion cancer JAR, em‐ bryo transversal cancer RD and ovarian cancer HO-8910 treated with DHA and AS. IC50 val‐ ues obtained through this MTT growth inhibition assay demonstrated that DHA was more effective at inhibiting cancer cell lines than AS. The anti-angiogenic activities of DHA and AS were tested on *in vitro* models of angiogenesis by assessing the proliferation, migration and tube formation of human umbilical vein endothelial (HUVE) cells. The results showed that DHA and AS significantly inhibited angiogenesis in a dose-dependent manner. These results also showed that DHA was more effective than ART in inhibiting angiogenesis

The effect of DHA on human multiple myeloma-induced angiogenesis under hypoxia and elucidated its mechanism of action has been performed. An *in vivo* chicken chorioallantoic membrane model was used to examine the effect of DHA on multiple myeloma-induced an‐ giogenesis. Compared with conditioned medium of control, conditioned medium from hu‐ man multiple myeloma RPMI8226 cells pretreated with 3 µM DHA in hypoxia was observed to reduce microvessel growth on chicken chorioallantoic membranes by approxi‐ mately 28.6% (P < 0.05). The level of VEGF in conditioned medium was determined by en‐ zyme-linked immunosorbent assay. The results confirmed that 3 µM DHA could significantly decrease VEGF secretion by RPMI8226 cells (P < 0.05), which correlated well with the reduction of multiple myeloma-induced angiogenesis on chicken chorioallantoic membranes. Western blot and reverse transcription-PCR results revealed that DHA downre‐ gulated the expression of VEGF in RPMI8226 cells in hypoxia. Therefore, DHA possesses potential as an antiangiogenic drug in multiple myeloma therapy and thereby may improve

The effect of DHA on VEGF expression and apoptosis in chronic myeloid leukemia (CML) K562 cells was assessed. The results demonstrated that in addition to its anti-proliferation effect on CML cells, DHA was also found to induce K562 cells apoptosis. The percentage of apoptotic cells was increased to 6.9 and 15.8% after being treated with 5 and 10 µM DHA for 48 h, respectively (P < 0.001). All these experiments suggested that DHA could inhibit the VEGF expression and secretion effectively in K562 cells, even at a lower concentration (2 µM, P < 0.05). Moreover, we further assessed the stimulating angiogenic activity of CM from K562 cells on CAM model. Also, the angiogenic activity was decreased in response to the CM from K562 cells pretreated with DHA in a dose-dependent manner. Taken together, these results from our study together with its known low toxicity make it possible that DHA might present potential anti-leukemia effect as a treatment for CML therapy, or as an ad‐

DHA was found to have a potent ability in influencing lymphatic endothelial cells (LECs) behavior. DHA also exerted a significant inhibitory effect on migration and tubelike formation of LECs in a dose-dependent manner. Quantitative RT-PCR further showed that DHA remarkably downregulated the expression of antiapoptotic bcl-2 mRNA, but upregulated that of the proapoptotic gene bax mRNA. In addition, DHA could strongly attenuate the mRNA and protein levels of VEGFR-3/Flt-4. In summary, these findings indicate that DHA may be useful as a potential lymphangiogenesis inhibi‐

(Chen et al., 2003).

192 Research Directions in Tumor Angiogenesis

patient outcome (Wu et al., 2006).

junct to standard chemotherapeutic regimens (Lee et al., 2006)

One study showed that DHA is an effective anti-metastatic agent that functions by downregulating the MMP-9 gene which is associated with metastasis. 1) DHA was shown to re‐ duce phorbol myristate acetate (PMA)-induced activation of MMP-9 and MMP-2 and further inhibited cell invasion and migration. 2) DHA was also shown to suppress the PMAenhanced expression of the levels of MMP-9 protein and mRNA, and enhanced transcrip‐ tional activity of the MM-9 gene through suppression of NF-kappaB and activation of AP-1 without changing the level of tissue inhibition of metalloproteinase (TIMP)-1. 3) DHA has been shown to reduce PMA-enhanced MMP-2 expression by suppressing membrane-type 1 MMP (MT1-MMP), but was not shown to t alter TIMP-2 levels. 4) DHA was shown to inhib‐ it PMA-induced NF-kappaB and c-Jun nuclear translocation, which are upstream of PMAinduced MMP-9 expression which enhances metastasis. 5) DHA strongly repressed the PMA-induced phosphorylation of Raf/ERK and JNK, which are dependent on the PKC al‐ pha pathway. In summary, this study demonstrated that the anti-invasive effects of DHA may occur through inhibition of PKC alpha/Raf/ERK and JNK phosphorylation and reduc‐ tion of NF-kappaB and AP-1 activation, leading to down-regulation of MMP-9 expression. (Hwang et al., 2010)

Wang et al. demonstrated that DHA enhances gemcitabine-induced growth inhibition and apoptosis in both BxPC-3 and PANC-1 cell lines *in vitro*. The effect is at least partially due to the DHA-driven deactivation of gemcitabine-induced NF-kappaB activation, which in turn leads to a tremendous decrease in the expression of NF-kappaB target gene products, such as c-myc, cyclin D1, Bcl-2, Bcl-xL (Wang et al., 2010). DHA was also shown to exhibit signifi‐ cant anti-cancer activity against the renal epithelial LLC cell line. In addition, DHA was shown to induce apoptosis of LLC cells and influenced the expression of the vascular endo‐ thelial growth factor (VEGF) receptor KDR/flk-1. Furthermore, in both tumor xenografts, a greater degree of growth inhibition was achieved when DHA and chemotherapeutic drugs were used in combination. The combined effect of DHA administered with chemotherapy drugs on LLC tumor metastasis was shown to be significant (Zhou et al., 2010).

The effect of DHA was investigated using *in vitro*/*in vivo* optical imaging combined with cell/tumor growth assays of the pancreatic cancer cell line BxPc3-RFP which stably expresses red fluorescence protein. DHA inhibited the proliferation and viability of pancreatic cancer cells in a dose-dependent manner and induced apoptosis. The results of this experiment demonstrated DHA-induced down-regulation of PCNA and Bcl-2, and up-regulation of Bax. VEGF expression was down-regulated by DHA in cells under normoxic, but not hypox‐ ic, conditions. The anti-angiogenic effect of DHA appears to be a complicated process (Aung et al., 2011). DHA was shown to significantly inhibit NF-κB DNA-binding activity, which in turn results in a tremendous decrease in the expression of NF-κB-targeted pro-angiogenic gene products such as VEGF, IL-8, COX-2, and MMP-9 in *vitro*: These findings suggest that DHA could be developed as a novel agent against pancreatic cancer (Wang et al., 2011). Ad‐ ditional supporting evidence of the potential of DHA to be used as an anti-pancreatic cancer agent were shown through a DHA driven up-regulation of glucose-regulated protein 78 (GRP78), which is known to be involved in endoplasmic reticulum stress (ER stress),. Fur‐ ther study demonstrated that DHA could enhance expression of GRP78 as well as the growth arrest and DNA-damage-inducible gene 153 at both the mRNA and protein levels. These studies suggest that redox imbalance may result in DHA-induced ER stress, which may contribute, at least in part, to its anti-cancer activity (Lu et al., 2011).

AS at low concentration was shown to significantly decrease VEGF and Ang-1 secretion by human multiple myeloma cells (line RPMI8226, P < 0.05), which correlated well with the re‐ duction of angiogenesis induced by the myeloma RPMI8226 cells. This study also showed that AS down-regulated the expression of VEGF and Ang-1 in RPMI8226 cells and reduced the activation of extracellular signal regulated kinase 1 (ERK1) as well. Therefore, AS has been shown to block ERK1/2 activation, downregulate VEGF and Ang-1 expression and in‐ hibit angiogenesis induced by human multiple myeloma RPMI8226 cells. Combined with previous published data, the results from this study supports the hypothesis that AS pos‐

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195

AS has also been shown to decrease the secretion of VEGF and IL-8 from TNFα- or hy‐ poxia-stimulated rheumatoid arthritis fibroblast-like synoviocyte (line RA FLS) in a dosedependent manner. In addition, AS treatment resulted in the inhibition of TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. AS treatment was shown to prevent Akt phosphorylation, but there was no evidence that phosphorylation of p38 and ERK was averted. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF, IL-8 secretion, and HIF-1α expression induced by TNFα or hypoxia. Therefore, AS has been shown to inhibit angiogenic factor expression in the RA FLS cell line, and this lat‐ est study provides new evidence that, as a low-cost agent, AS may have therapeutic po‐

Using a polyploid cell line, research on the role of AS in impacting cell cycle arrest was as‐ sessed. The results of this study show that AS treatment of polyploid cells resulted in a dose-dependent decreases in cell number, which was associated with either increased cyto‐ toxicity or cytostasis. Of the two possibilities, cytostasis, a simultaneous arrest at all phases of the cell cycle, appeared to be a more likely possibility. This deduction was supported by molecular profiling, which showed reductions in cell cycle transit proteins. AS appeared to maintain cells in this arrested state, however, reculturing these treated cells in drug-free me‐ dium resulted in significant reductions in cell viability. Taken together, these observations indicate AS and its related compounds may be effective for the treatment of polyploid tu‐ mors, and that activity is related to the cell cycle schedule. Therefore, it is important to care‐ fully select the most appropriate schedule to maximize AS efficacy when using AS as a

There are many reports discussing the *in vivo* anti-cancer activity of ARTs which may pro‐

The effect of ART on tumor growth, lymphangiogenesis, metastasis and survival in mouse Lewis lung carcinoma (LLC) models was examined. The results of this study showed that orally administered artemisinin inhibited lymph node and lung metastasis and prolonged

sesses potential anti-myeloma activity (Chen et al., 2010a).

tential for rheumatoid arthritis (He et al., 2011).

primary or adjuvant anti-tumor therapy (Liu et al., 2011)

*3.1.2. In vivo anti-angiogenic effects of ART and its derivatives*

**Artemisinin (ART)**

vide insight into the potential activity of ARTs as anti-cancer agents.

#### **Artesunate (AS)**

AS has been shown to inhibit the growth of Kaposi's sarcoma cells, a highly angiogenic mul‐ tifocal tumor, and the degree of cell growth inhibition correlated with the induction of apop‐ tosis. AS was also shown to inhibit the growth of normal human umbilical endothelial cells and of KS-IMM cells that were established from a Kaposi's sarcoma lesion obtained from a renal transplant patient. The inhibition of cell growth correlated with the induction of apop‐ tosis in KS-IMM cells. Apoptosis was not observed in normal endothelial cells, which showed drastically increased cell doubling times upon AS treatment (Dell'Eva et al., 2004).

AS has been shown to greatly inhibit cell proliferation and differentiation of endothelial cells in a dose-dependent manner in the range of 12.5-100 µM. AS was also shown to re‐ duce Flt-1 and KDR/flk-1 expression of endothelial cells when dosed *in vitro* in a range of 0.1-0.5 µM. In subsequent studies by the same author, the AS-driven apoptosis of a human microvascular dermal endothelial cell line was studied. The apoptosis was detected utiliz‐ ing a morphological dual staining assay composed of ethidium bromide and acridine or‐ ange as well as a DNA fragmentation TUNEL assay quantified by a flow cytometric propidium iodide (PI) assay. The results suggest that the anti-angiogenic effect induced by AS treatment might occur by the induction of cellular apoptosis (Huan-huan et al., 2004). In addition, the inhibitory effect of AS on *in vitro* angiogenesis was tested using aortic cells cultured in a fibrin gel. AS was shown to effectively suppress the stimulating angiogenic ability of chronic myeloid leukemia cells (line K562) when the K562 cells were pretreated for 48 h with AS in a time-dependent manner (days 3-14). AS treatment was also found to decrease the VEGF level in chronic myeloma K562 cells, even at a lower concentration (2 µmol/l, P < 0.01). (Zhou et al., 2007).

The addition of Fe(II)-glycine sulfate and transferrin has been shown to enhance the cytotoxici‐ ty (10.3-fold) of free AS *in vitro*. AS microencapsulated in maltosyl-ß- cyclodextrin, and ARTs were tested against CCRF-CEM leukemia and U373 astrocytoma cells *in vitro* (Efferth et al., 2004). Treatment with AS at more than 2.5 µM for 48 h inhibited the proliferation of human vein endothelial cells (HUVEC) in a concentration dependent manner using an MTT (3-(4,5-dime‐ thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) based growth proliferation assay (p < 0.05). The IC50 value of this growth inhibition assay was 20.7 µM, and HUVEC cells were also shown to be growth inhibited by 88.7% after treatment with 80 µM AS (Chen et al., 2004b).

AS at low concentration was shown to significantly decrease VEGF and Ang-1 secretion by human multiple myeloma cells (line RPMI8226, P < 0.05), which correlated well with the re‐ duction of angiogenesis induced by the myeloma RPMI8226 cells. This study also showed that AS down-regulated the expression of VEGF and Ang-1 in RPMI8226 cells and reduced the activation of extracellular signal regulated kinase 1 (ERK1) as well. Therefore, AS has been shown to block ERK1/2 activation, downregulate VEGF and Ang-1 expression and in‐ hibit angiogenesis induced by human multiple myeloma RPMI8226 cells. Combined with previous published data, the results from this study supports the hypothesis that AS pos‐ sesses potential anti-myeloma activity (Chen et al., 2010a).

AS has also been shown to decrease the secretion of VEGF and IL-8 from TNFα- or hy‐ poxia-stimulated rheumatoid arthritis fibroblast-like synoviocyte (line RA FLS) in a dosedependent manner. In addition, AS treatment resulted in the inhibition of TNFα- or hypoxia-induced nuclear expression and translocation of HIF-1α. AS treatment was shown to prevent Akt phosphorylation, but there was no evidence that phosphorylation of p38 and ERK was averted. TNFα- or hypoxia-induced secretion of VEGF and IL-8 and expression of HIF-1α were hampered by treatment with the PI3 kinase inhibitor LY294002, suggesting that inhibition of PI3 kinase/Akt activation might inhibit VEGF, IL-8 secretion, and HIF-1α expression induced by TNFα or hypoxia. Therefore, AS has been shown to inhibit angiogenic factor expression in the RA FLS cell line, and this lat‐ est study provides new evidence that, as a low-cost agent, AS may have therapeutic po‐ tential for rheumatoid arthritis (He et al., 2011).

Using a polyploid cell line, research on the role of AS in impacting cell cycle arrest was as‐ sessed. The results of this study show that AS treatment of polyploid cells resulted in a dose-dependent decreases in cell number, which was associated with either increased cyto‐ toxicity or cytostasis. Of the two possibilities, cytostasis, a simultaneous arrest at all phases of the cell cycle, appeared to be a more likely possibility. This deduction was supported by molecular profiling, which showed reductions in cell cycle transit proteins. AS appeared to maintain cells in this arrested state, however, reculturing these treated cells in drug-free me‐ dium resulted in significant reductions in cell viability. Taken together, these observations indicate AS and its related compounds may be effective for the treatment of polyploid tu‐ mors, and that activity is related to the cell cycle schedule. Therefore, it is important to care‐ fully select the most appropriate schedule to maximize AS efficacy when using AS as a primary or adjuvant anti-tumor therapy (Liu et al., 2011)

#### *3.1.2. In vivo anti-angiogenic effects of ART and its derivatives*

There are many reports discussing the *in vivo* anti-cancer activity of ARTs which may pro‐ vide insight into the potential activity of ARTs as anti-cancer agents.

#### **Artemisinin (ART)**

et al., 2011). DHA was shown to significantly inhibit NF-κB DNA-binding activity, which in turn results in a tremendous decrease in the expression of NF-κB-targeted pro-angiogenic gene products such as VEGF, IL-8, COX-2, and MMP-9 in *vitro*: These findings suggest that DHA could be developed as a novel agent against pancreatic cancer (Wang et al., 2011). Ad‐ ditional supporting evidence of the potential of DHA to be used as an anti-pancreatic cancer agent were shown through a DHA driven up-regulation of glucose-regulated protein 78 (GRP78), which is known to be involved in endoplasmic reticulum stress (ER stress),. Fur‐ ther study demonstrated that DHA could enhance expression of GRP78 as well as the growth arrest and DNA-damage-inducible gene 153 at both the mRNA and protein levels. These studies suggest that redox imbalance may result in DHA-induced ER stress, which

AS has been shown to inhibit the growth of Kaposi's sarcoma cells, a highly angiogenic mul‐ tifocal tumor, and the degree of cell growth inhibition correlated with the induction of apop‐ tosis. AS was also shown to inhibit the growth of normal human umbilical endothelial cells and of KS-IMM cells that were established from a Kaposi's sarcoma lesion obtained from a renal transplant patient. The inhibition of cell growth correlated with the induction of apop‐ tosis in KS-IMM cells. Apoptosis was not observed in normal endothelial cells, which showed drastically increased cell doubling times upon AS treatment (Dell'Eva et al., 2004). AS has been shown to greatly inhibit cell proliferation and differentiation of endothelial cells in a dose-dependent manner in the range of 12.5-100 µM. AS was also shown to re‐ duce Flt-1 and KDR/flk-1 expression of endothelial cells when dosed *in vitro* in a range of 0.1-0.5 µM. In subsequent studies by the same author, the AS-driven apoptosis of a human microvascular dermal endothelial cell line was studied. The apoptosis was detected utiliz‐ ing a morphological dual staining assay composed of ethidium bromide and acridine or‐ ange as well as a DNA fragmentation TUNEL assay quantified by a flow cytometric propidium iodide (PI) assay. The results suggest that the anti-angiogenic effect induced by AS treatment might occur by the induction of cellular apoptosis (Huan-huan et al., 2004). In addition, the inhibitory effect of AS on *in vitro* angiogenesis was tested using aortic cells cultured in a fibrin gel. AS was shown to effectively suppress the stimulating angiogenic ability of chronic myeloid leukemia cells (line K562) when the K562 cells were pretreated for 48 h with AS in a time-dependent manner (days 3-14). AS treatment was also found to decrease the VEGF level in chronic myeloma K562 cells, even at a lower concentration (2

The addition of Fe(II)-glycine sulfate and transferrin has been shown to enhance the cytotoxici‐ ty (10.3-fold) of free AS *in vitro*. AS microencapsulated in maltosyl-ß- cyclodextrin, and ARTs were tested against CCRF-CEM leukemia and U373 astrocytoma cells *in vitro* (Efferth et al., 2004). Treatment with AS at more than 2.5 µM for 48 h inhibited the proliferation of human vein endothelial cells (HUVEC) in a concentration dependent manner using an MTT (3-(4,5-dime‐ thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) based growth proliferation assay (p < 0.05). The IC50 value of this growth inhibition assay was 20.7 µM, and HUVEC cells were also shown to be growth inhibited by 88.7% after treatment with 80 µM AS (Chen et al., 2004b).

may contribute, at least in part, to its anti-cancer activity (Lu et al., 2011).

**Artesunate (AS)**

194 Research Directions in Tumor Angiogenesis

µmol/l, P < 0.01). (Zhou et al., 2007).

The effect of ART on tumor growth, lymphangiogenesis, metastasis and survival in mouse Lewis lung carcinoma (LLC) models was examined. The results of this study showed that orally administered artemisinin inhibited lymph node and lung metastasis and prolonged survival without retarding tumor growth. ART-treated mice showed significant decreases in lymph node metastasis, tumor lymphangiogenesis and expression of VEGF-C as compared to control mice. (Wang et al., 2008).

reduced following drug-treatment with no apparent toxic effects on the nude mice. AS ad‐ ministration was shown to dramatically reduce VEGF expression on tumor cells and KDR/ flk-1 expression on endothelial cells as well as tumor cells. Accordingly, these results sup‐ port the hypothesis that AS is capable of inhibiting angiogenesis in *vitro* and *in vivo*. These findings together with the known low toxicity of AS are clues that AS may be a promising

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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197

Further studies on the anti-angiogenic effects of AS have been conducted *in vivo* and *in vitro*. The anti-angiogenic effect of AS *in vivo* was evaluated utilizing the chicken chorioallantoic membrane (CAM) neovascularization model. At low concentrations of 10 nM/100 µl/egg, AS was shown to significantly inhibit CAM angiogenesis, and completely inhibited angiogene‐ sis at concentrations of 80 nM/100 µl/egg. The results of this study suggest that the anti-an‐ giogenic effect induced by AS might occur by the induction of cellular apoptosis. These findings and the known low toxicity of AS support the hypothesis that AS might be a prom‐ ising candidate as an angiogenesis inhibitor (Huan-huan et al., 2004). Similarly, AS was shown to significantly impair primary tumor growth and metastasis in the chicken embryo metastasis (CAM) model where AS was shown to suppress invasion and metastasis of nonsmall cell lung cancer (NSCLC) cells. The transcriptional findings of these experiments showed AS treatment reduced transcription of u-PA, MMP-2 and MMP-7, supporting the hypothesis that AS has promise as a novel therapeutic for NSCLC (Rasheed et al., 2010).

Also, AS has been studied in a variety of tumor models as a potential antitumor drug. In one study of vascularization, a critical element of tumor metastasis, AS was shown to strongly reduce angiogenesis of Kaposi's sarcoma cells *in vivo* by inhibiting vascularization in Matri‐ gel plugs injected subcutaneously into syngenic mice. This data suggests that AS represents a promising candidate drug for the treatment of the highly angiogenic Kaposi's sarcoma. As a low-cost drug, it might be of particular interest for use in areas of the world where Kapo‐

The efficacy of AS, as an anti-cancer agent, to reduce tumor growth was studied in rats giv‐ en AS subcutaneously at a dose of 50 mg/kg/day and at a dose of 100 mg/kg/day for 15 days. The results of this experiment showed animals with AS treated tumors showed a reduction in tumor growth by 41%, in the 50 mg/kg treatment group and 62% in the 100 mg/kg treat‐ ment group. The density of micro-vessels which was used as a measure of angiogenic activi‐ ty in the tumors of animals treated with 100 mg/kg of AS daily was at least four times lower than in the control group (Chen et al., 2004b). The anti-angiogenic activity of AS *in vivo* was also evaluated in nude mice implanted with a human ovarian cancer cell line (HO-8910). Evaluation of angiogenesis in the AS treated and control animals with an ovarian cancer xenograft were determined through immunohistochemical staining for microvessel forma‐ tion (CD31), VEGF and the VEGF receptor KDR/flk-1. Tumor growth was noted to be de‐ creased, and the density of the tumor microvessels was reduced following AS treatment

The anti-angiogenic effect of AS was further evaluated *in vivo* in the chicken chorioallantoic membrane (CAM) neovascularization model. The results showed that stimulating angiogen‐ ic activity was decreased in response to the treatment of myeloblastic K562 cells with ART,

angiogenesis inhibitor (Chen et al., 2004c).

si's sarcoma is highly prevalent. (Dell'Eva et al., 2004).

with no apparent toxicity to the animals (Chen et al., 2004a, 2004b).

#### **Dihydroartemisinin (DHA)**

The anti-angiogenic activity of DHA *in vitro* and *in vivo*, and investigated DHA-induced apoptosis in human umbilical vein endothelial cells (HUVEC). DHA markedly reduced VEGF binding to its receptors on the surface of HUVEC. The expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on HUVEC were lower following DHA treat‐ ment as shown by an immunocytochemical staining assay. The *in vivo* anti-angiogenic ac‐ tivity was evaluated in the chicken chorioallantoic membrane (CAM) neovascularization model. DHA significantly inhibited CAM angiogenesis at low concentrations (5-30 nmol/100 microl per egg). This group also investigated both qualitatively and quantita‐ tively the induction of HUVEC apoptosis by DHA. A dose-related (5-80 µM) and timedependent (6-36 h) increase in DHA-induced HUVEC apoptosis was observed by flow cytometry. These results suggest that the anti-angiogenic effect induced by DHA might occur by induction of cellular apoptosis and inhibition of expression of VEGF receptors. These findings and the known low toxicity of DHA indicate that it might be a promising candidate angiogenesis inhibitor (Chen et al., 2004a).

The anti-angiogenic effect of DHA on pancreatic cancer was assessed using BxPC-3 xeno‐ grafts subcutaneously established in BALB/c nude mice. DHA demonstrated remarkable activity against pancreatic cancer studies concuted *in vivo.* DHA treatment resulted in re‐ duced tumor volume and decreased microvessel density, and there were additional tran‐ scriptional effects demonstrated in these studies as well regarding the expression of NFκB-related pro-angiogenic gene products which were down-regulated. This finding of relating to the inhibition of NF-κB activation is likely one of the mechanisms involved in DHA anti-angiogenic activity against human pancreatic cancer. This suggests that DHA could be developed as a novel agent against pancreatic cancer (Wang et al., 2011). In a further study, the co-administration of the chemotherapeutic agent gemcitabine with DHA was shown to result in remarkably enhanced anti-tumor effects, as demonstrated by significantly increased apoptosis, as well as a decreased Ki-67 index, reduced NF-kap‐ paB activity, reduced downstream angiogenic gene products, and predictably, significant‐ ly reduced tumor volume. The authors conclude that inhibition of gemcitabine-induced NF-kappaB activation is one of the mechanisms by which DHA could promote its antitumor effect on pancreatic cancer (Wang et al., 2010).

#### **Artesunate (AS)**

The anti-angiogenic effect *in vivo* of artesunate was evaluated in nude mice f implanted with human ovarian cancer cells (HO-8910). The effects of artesunate on angiogenesis in this *in vivo* study were evaluated by immune-histochemical staining for microvessel associated an‐ tigens (CD31), VEGF and the VEGF receptor KDR/flk-1. AS significantly inhibited angiogen‐ esis in a concentration-dependent form in the range of 0.5-50 µM. The IC50 of AS for HUVE cells was 21 µM. Growth of the xenograft tumor was decreased and microvessel density was reduced following drug-treatment with no apparent toxic effects on the nude mice. AS ad‐ ministration was shown to dramatically reduce VEGF expression on tumor cells and KDR/ flk-1 expression on endothelial cells as well as tumor cells. Accordingly, these results sup‐ port the hypothesis that AS is capable of inhibiting angiogenesis in *vitro* and *in vivo*. These findings together with the known low toxicity of AS are clues that AS may be a promising angiogenesis inhibitor (Chen et al., 2004c).

survival without retarding tumor growth. ART-treated mice showed significant decreases in lymph node metastasis, tumor lymphangiogenesis and expression of VEGF-C as compared

The anti-angiogenic activity of DHA *in vitro* and *in vivo*, and investigated DHA-induced apoptosis in human umbilical vein endothelial cells (HUVEC). DHA markedly reduced VEGF binding to its receptors on the surface of HUVEC. The expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on HUVEC were lower following DHA treat‐ ment as shown by an immunocytochemical staining assay. The *in vivo* anti-angiogenic ac‐ tivity was evaluated in the chicken chorioallantoic membrane (CAM) neovascularization model. DHA significantly inhibited CAM angiogenesis at low concentrations (5-30 nmol/100 microl per egg). This group also investigated both qualitatively and quantita‐ tively the induction of HUVEC apoptosis by DHA. A dose-related (5-80 µM) and timedependent (6-36 h) increase in DHA-induced HUVEC apoptosis was observed by flow cytometry. These results suggest that the anti-angiogenic effect induced by DHA might occur by induction of cellular apoptosis and inhibition of expression of VEGF receptors. These findings and the known low toxicity of DHA indicate that it might be a promising

The anti-angiogenic effect of DHA on pancreatic cancer was assessed using BxPC-3 xeno‐ grafts subcutaneously established in BALB/c nude mice. DHA demonstrated remarkable activity against pancreatic cancer studies concuted *in vivo.* DHA treatment resulted in re‐ duced tumor volume and decreased microvessel density, and there were additional tran‐ scriptional effects demonstrated in these studies as well regarding the expression of NFκB-related pro-angiogenic gene products which were down-regulated. This finding of relating to the inhibition of NF-κB activation is likely one of the mechanisms involved in DHA anti-angiogenic activity against human pancreatic cancer. This suggests that DHA could be developed as a novel agent against pancreatic cancer (Wang et al., 2011). In a further study, the co-administration of the chemotherapeutic agent gemcitabine with DHA was shown to result in remarkably enhanced anti-tumor effects, as demonstrated by significantly increased apoptosis, as well as a decreased Ki-67 index, reduced NF-kap‐ paB activity, reduced downstream angiogenic gene products, and predictably, significant‐ ly reduced tumor volume. The authors conclude that inhibition of gemcitabine-induced NF-kappaB activation is one of the mechanisms by which DHA could promote its anti-

The anti-angiogenic effect *in vivo* of artesunate was evaluated in nude mice f implanted with human ovarian cancer cells (HO-8910). The effects of artesunate on angiogenesis in this *in vivo* study were evaluated by immune-histochemical staining for microvessel associated an‐ tigens (CD31), VEGF and the VEGF receptor KDR/flk-1. AS significantly inhibited angiogen‐ esis in a concentration-dependent form in the range of 0.5-50 µM. The IC50 of AS for HUVE cells was 21 µM. Growth of the xenograft tumor was decreased and microvessel density was

to control mice. (Wang et al., 2008).

candidate angiogenesis inhibitor (Chen et al., 2004a).

tumor effect on pancreatic cancer (Wang et al., 2010).

**Artesunate (AS)**

**Dihydroartemisinin (DHA)**

196 Research Directions in Tumor Angiogenesis

Further studies on the anti-angiogenic effects of AS have been conducted *in vivo* and *in vitro*. The anti-angiogenic effect of AS *in vivo* was evaluated utilizing the chicken chorioallantoic membrane (CAM) neovascularization model. At low concentrations of 10 nM/100 µl/egg, AS was shown to significantly inhibit CAM angiogenesis, and completely inhibited angiogene‐ sis at concentrations of 80 nM/100 µl/egg. The results of this study suggest that the anti-an‐ giogenic effect induced by AS might occur by the induction of cellular apoptosis. These findings and the known low toxicity of AS support the hypothesis that AS might be a prom‐ ising candidate as an angiogenesis inhibitor (Huan-huan et al., 2004). Similarly, AS was shown to significantly impair primary tumor growth and metastasis in the chicken embryo metastasis (CAM) model where AS was shown to suppress invasion and metastasis of nonsmall cell lung cancer (NSCLC) cells. The transcriptional findings of these experiments showed AS treatment reduced transcription of u-PA, MMP-2 and MMP-7, supporting the hypothesis that AS has promise as a novel therapeutic for NSCLC (Rasheed et al., 2010).

Also, AS has been studied in a variety of tumor models as a potential antitumor drug. In one study of vascularization, a critical element of tumor metastasis, AS was shown to strongly reduce angiogenesis of Kaposi's sarcoma cells *in vivo* by inhibiting vascularization in Matri‐ gel plugs injected subcutaneously into syngenic mice. This data suggests that AS represents a promising candidate drug for the treatment of the highly angiogenic Kaposi's sarcoma. As a low-cost drug, it might be of particular interest for use in areas of the world where Kapo‐ si's sarcoma is highly prevalent. (Dell'Eva et al., 2004).

The efficacy of AS, as an anti-cancer agent, to reduce tumor growth was studied in rats giv‐ en AS subcutaneously at a dose of 50 mg/kg/day and at a dose of 100 mg/kg/day for 15 days. The results of this experiment showed animals with AS treated tumors showed a reduction in tumor growth by 41%, in the 50 mg/kg treatment group and 62% in the 100 mg/kg treat‐ ment group. The density of micro-vessels which was used as a measure of angiogenic activi‐ ty in the tumors of animals treated with 100 mg/kg of AS daily was at least four times lower than in the control group (Chen et al., 2004b). The anti-angiogenic activity of AS *in vivo* was also evaluated in nude mice implanted with a human ovarian cancer cell line (HO-8910). Evaluation of angiogenesis in the AS treated and control animals with an ovarian cancer xenograft were determined through immunohistochemical staining for microvessel forma‐ tion (CD31), VEGF and the VEGF receptor KDR/flk-1. Tumor growth was noted to be de‐ creased, and the density of the tumor microvessels was reduced following AS treatment with no apparent toxicity to the animals (Chen et al., 2004a, 2004b).

The anti-angiogenic effect of AS was further evaluated *in vivo* in the chicken chorioallantoic membrane (CAM) neovascularization model. The results showed that stimulating angiogen‐ ic activity was decreased in response to the treatment of myeloblastic K562 cells with ART, and tumor growth was inhibited when K562 cells were pretreated with ART in a dose-de‐ pendent manner (3-12 µmol/l). Further analyses of the level of VEGF expression by Western blot and also assays of VEGF mRNA by RT-PCR in K562 cells showed that ART could inhib‐ it VEGF expression, and the inhibition correlated well with the level of VEGF secreted in the culture medium. These findings suggest that AS may have potential as a treatment for chronic myelogenous leukemia (CML) or as an adjunct to standard chemotherapeutic regi‐ mens (Zhou et al., 2007).

thelial growth factor (VEGF) and interleukin-8 (CXCL-8). The data showed that artemisone is significantly less anti-angiogenic than DHA in all the experimental models tested, sug‐ gesting that artemisone will be safer to use than the current clinical artemisinins during pregnancy for an antimalarial indication but perhaps less efficacious for an anti-angiogenic

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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199

Angiogenesis and vasculogenesis refer to the growth of blood vessels. Angiogenesis is the growth most often associated with repair of damaged vessels or the growth of smaller blood vessels, while vasculogenesis is the process by which the primary blood system is being cre‐ ated or changed. Vasculogenesis occurs during the very early developmental stages of an or‐ ganism when the blood vessel pathways are created. Angiogenesis, while a similar process, does not depend on the same set of genes as vasculogenesis, and this process is activated instead in the presence of an injury to a blood vessel. In the last three decades, considerable research has been reported that supports the hypothesis that tumor growth and metastasis require angiogenesis. Angiogenesis, the proliferation and migration of endothelial cells re‐ sulting in the formation of new blood vessels, is an important process for the progression of tumors (Figure 3). ARTs have been shown in a number of published reports to have anti-

**Figure 3.** The modes of vasculogenesis and angiogenesis. Vasculogenesis occurs during the very early developmental stages of an organism when the blood vessel pathways are created. Angiogenesis, while a similar process, does not depend on the same set of genes as vasculogenesis, and this process is activated instead in the presence of an injury to a blood vessel. Angiogenesis finishes the circulatory connections begun by vasculogenesis and builds arteries and

veins from the capillaries (Modified from Hanahan, 1997

indication as part of a anti-cancer regimen (D'Alessandro et al., 2007).

angiogenic effects.

**3.2. Mechanistic perspectives for the anti-angiogenic activities of ARTs**

#### *3.1.3. Anti-angiogenic effects of novel ARTs and ART-like compounds*

Artesunate has been shown to exhibit anti-angiogenic, anti-tumorigenic and anti-viral prop‐ erties in addition to its known antimalarial properties. The array of activities of the ARTs, and the recent emergence of malaria resistance to AS, prompted one group to synthesize and evaluate several novel ART-like derivatives. Sixteen distinct derivatives were therefore synthesized, and the *in vitro* cytotoxic effects of each were tested with different cell lines. The *in vivo* anti-angiogenic properties were evaluated using a zebrafish embryo model. This groupreported the identification of several novel ART-like compounds that are easily syn‐ thesized, stable at room temperature, may overcome drug-resistance pathways and are more active *in vitro* and *in vivo* than the commonly used AS. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and cancer (Soomro et al., 2011).

Twelve ART acetal dimers were synthesized and tested for antitumor activity against 60 *in vitro* tumor cell lines compiled by the National Cancer Institute (NCI), producing a mean GI50 concentration between 8.7 (least active) and 0.019 µM (most active). The significant ac‐ tivity of the compounds in this preliminary screen led to additional *in vitro* antitumor and anti-angiogenesis studies. Several active dimers were also evaluated in the *in vivo* NCI hol‐ low fiber assay followed by a preliminary xenograft study. The title compounds were found to be active against solid tumor-derived cell lines and showed good correlation with other artemisinin-based molecules in the NCI database (Galal et al., 2009).

In addition, various thioacetal ART derivatives can inhibit the angiogenesis and might be angiogenesis inhibitors. In particular, 10 alpha-phenylthiodihydroartemisinins, 10 beta-ben‐ zenesulfonyl-9-epi-dihydroartemisinin and 10 alpha-mercaptodihydroartemisinin exhibit strong growth inhibition activity against HUVEC proliferation. Compound 11 have a good inhibitory activity upon HUVEC tube formation, and 5 and 11 show a strong inhibitory ef‐ fect on angiogenesis using CAM assay at 5 µg/egg by 90% (Oh et al., 2004).

Artemisone is a novel 10-alkylamino derivative which is not metabolized to DHA. It was se‐ lected as a clinical drug candidate on the basis of its potency *in vitro* against *Plasmodium falci‐ parum* and its lack of detectable neurotoxicity in both *in vitro* and *in vivo* screens. Artemisone was tested *in vitro* and *in vivo* for anti-angiogenic effects which may support its use as an anti-angiogenic agent as an adjunct to standard tumor chemotherapy. The various studies of artemisone's anti-angiogenic activity include proliferation of human endothelial cells and their migration on a fibronectin matrix, the sprouting of new vessels from rat aorta sections grown in collagen, and the production of pro-angiogenic cytokines such as vascular endo‐ thelial growth factor (VEGF) and interleukin-8 (CXCL-8). The data showed that artemisone is significantly less anti-angiogenic than DHA in all the experimental models tested, sug‐ gesting that artemisone will be safer to use than the current clinical artemisinins during pregnancy for an antimalarial indication but perhaps less efficacious for an anti-angiogenic indication as part of a anti-cancer regimen (D'Alessandro et al., 2007).

#### **3.2. Mechanistic perspectives for the anti-angiogenic activities of ARTs**

and tumor growth was inhibited when K562 cells were pretreated with ART in a dose-de‐ pendent manner (3-12 µmol/l). Further analyses of the level of VEGF expression by Western blot and also assays of VEGF mRNA by RT-PCR in K562 cells showed that ART could inhib‐ it VEGF expression, and the inhibition correlated well with the level of VEGF secreted in the culture medium. These findings suggest that AS may have potential as a treatment for chronic myelogenous leukemia (CML) or as an adjunct to standard chemotherapeutic regi‐

Artesunate has been shown to exhibit anti-angiogenic, anti-tumorigenic and anti-viral prop‐ erties in addition to its known antimalarial properties. The array of activities of the ARTs, and the recent emergence of malaria resistance to AS, prompted one group to synthesize and evaluate several novel ART-like derivatives. Sixteen distinct derivatives were therefore synthesized, and the *in vitro* cytotoxic effects of each were tested with different cell lines. The *in vivo* anti-angiogenic properties were evaluated using a zebrafish embryo model. This groupreported the identification of several novel ART-like compounds that are easily syn‐ thesized, stable at room temperature, may overcome drug-resistance pathways and are more active *in vitro* and *in vivo* than the commonly used AS. These promising findings raise the hopes of identifying safer and more effective strategies to treat a range of infections and

Twelve ART acetal dimers were synthesized and tested for antitumor activity against 60 *in vitro* tumor cell lines compiled by the National Cancer Institute (NCI), producing a mean GI50 concentration between 8.7 (least active) and 0.019 µM (most active). The significant ac‐ tivity of the compounds in this preliminary screen led to additional *in vitro* antitumor and anti-angiogenesis studies. Several active dimers were also evaluated in the *in vivo* NCI hol‐ low fiber assay followed by a preliminary xenograft study. The title compounds were found to be active against solid tumor-derived cell lines and showed good correlation with other

In addition, various thioacetal ART derivatives can inhibit the angiogenesis and might be angiogenesis inhibitors. In particular, 10 alpha-phenylthiodihydroartemisinins, 10 beta-ben‐ zenesulfonyl-9-epi-dihydroartemisinin and 10 alpha-mercaptodihydroartemisinin exhibit strong growth inhibition activity against HUVEC proliferation. Compound 11 have a good inhibitory activity upon HUVEC tube formation, and 5 and 11 show a strong inhibitory ef‐

Artemisone is a novel 10-alkylamino derivative which is not metabolized to DHA. It was se‐ lected as a clinical drug candidate on the basis of its potency *in vitro* against *Plasmodium falci‐ parum* and its lack of detectable neurotoxicity in both *in vitro* and *in vivo* screens. Artemisone was tested *in vitro* and *in vivo* for anti-angiogenic effects which may support its use as an anti-angiogenic agent as an adjunct to standard tumor chemotherapy. The various studies of artemisone's anti-angiogenic activity include proliferation of human endothelial cells and their migration on a fibronectin matrix, the sprouting of new vessels from rat aorta sections grown in collagen, and the production of pro-angiogenic cytokines such as vascular endo‐

*3.1.3. Anti-angiogenic effects of novel ARTs and ART-like compounds*

artemisinin-based molecules in the NCI database (Galal et al., 2009).

fect on angiogenesis using CAM assay at 5 µg/egg by 90% (Oh et al., 2004).

mens (Zhou et al., 2007).

198 Research Directions in Tumor Angiogenesis

cancer (Soomro et al., 2011).

Angiogenesis and vasculogenesis refer to the growth of blood vessels. Angiogenesis is the growth most often associated with repair of damaged vessels or the growth of smaller blood vessels, while vasculogenesis is the process by which the primary blood system is being cre‐ ated or changed. Vasculogenesis occurs during the very early developmental stages of an or‐ ganism when the blood vessel pathways are created. Angiogenesis, while a similar process, does not depend on the same set of genes as vasculogenesis, and this process is activated instead in the presence of an injury to a blood vessel. In the last three decades, considerable research has been reported that supports the hypothesis that tumor growth and metastasis require angiogenesis. Angiogenesis, the proliferation and migration of endothelial cells re‐ sulting in the formation of new blood vessels, is an important process for the progression of tumors (Figure 3). ARTs have been shown in a number of published reports to have antiangiogenic effects.

**Figure 3.** The modes of vasculogenesis and angiogenesis. Vasculogenesis occurs during the very early developmental stages of an organism when the blood vessel pathways are created. Angiogenesis, while a similar process, does not depend on the same set of genes as vasculogenesis, and this process is activated instead in the presence of an injury to a blood vessel. Angiogenesis finishes the circulatory connections begun by vasculogenesis and builds arteries and veins from the capillaries (Modified from Hanahan, 1997

As malignant tissues grow, metastases and solid tumors require extra blood supply for thriving and survival. Thus, cancer cells induce neovascularization by regulating proteins and pathways involved in the generation and restructure of new vasculature. Angiogenesis process leads to enhanced proliferation of endothelial cells through induction of VEGF, fi‐ broblast growth factor (FGF), its receptors, and cytokines. This event occurs via multiple ef‐ fects including hypoxia-driven activation of expression of HIF-1*α* and the aryl hydrocarbon receptor nuclear translocator (ARNT). Angiogenesis control is mediated by angiostatin, en‐ dostatin, thrombospondin, TIMPs, PAI-1, and others. Due to their role in tumor survival, the pro-angiogenic factors and the molecules involved in their regulatory networks are relevant drug targets (Crespo-Ortiz and Wei, 2012)

sion of genes implicated in angiogenesis. The mRNA expression data of angiogenesis-relat‐ ed genes correlated well with the 50% growth inhibition concentration values for eight ARTs (ART, AS, arteether, artemisetene, arteanuine B, dihydroartemisinylester stereoisom‐ ers 1 and 2). The constitutive expression of 30 different genes correlated significantly with the cellular response to ARTs. The finding that drug sensitivity and resistance of tumor cells could be predicted by the mRNA expression of angiogenesis related genes supports the hypothesis that the antitumor activity of ARTs may be due, at least in part, by inhibi‐ tion of tumor angiogenesis. As many chemo-preventive drugs exert anti-angiogenic fea‐ tures, ARTs might also have a chemo-preventive effect in addition to their cytotoxic effects

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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201

These findings are consistent with previous published work (Wartenberg et al., 2003) show‐ ing an artemisinin-dependent decrease in expression levels of hypoxia-inducible factor 1a (HIF-1a; H1F1A), which is known to be a transcriptional activator of VEGFA and is critical in neovasculogenesis in hypoxic tissues. The inhibition of angiogenesis by ART (at a concen‐ tration of 12 mM) involving VEGF and HIF-1a was also demonstrated in leukemic and glio‐ ma cells (Huang et al., 2008; Zhou et al., 2007). Loss of HIF-1α and VEGF expression by artemisinin appears to depend on ROS as co-treatment with free-radical scavengers such as vitamin E and mannitol reversed the effects of artemisinin (Wartenberg et al., 2003). The sensitivity and resistance of these tumor cells has been shown to correlate with mRNA ex‐ pression of angiogenesis-related genes. This suggests that the anti-tumor effects of ARTs are potentially due to their role in inhibiting tumor angiogenesis (Anfosso et al., 2006). The find‐ ing that tumor cell drug sensitivity and resistance could be predicted by mRNA expression of angiogenesis-related genes supports the hypothesis that artemisinins their anti-tumor ef‐

In addition, an investigation to determine the sensitivity and resistance of cancer cells to‐ wards AS was conducted. The gene-hunting approach applied by us delivered several novel candidate genes that may regulate the response of cancer cells to AS. These results merit fur‐ ther investigations to prove the contribution of these genes for AS resistance. Study demon‐ strated that AS was no inhibitor of ABC transporters ABCB1 and ABCG2. Although AS may exhibit specific inhibitory functions towards particular ABC transporters, but not towards a wide spectrum of several different ABC transporters. This approach showed that response of tumor cells towards AS is multi-factorial in nature and is determined by gene expression associated with AS sensitivity on the one hand and with gene expression associated with AS

The anti-cancer mechanism of ARTs is likely to be related to the cleavage of the iron- or heme-mediated peroxide bridge, followed by the generation of ROS (Mercer et al., 2011; Zhang et al., 2010). The anti-cancer potential of ARTs is possibly connected to the expression of TfR. The synergism of AS and iron (II)-glycine sulfate co-treatment is unsuitable for all types of tumor cells. Endoplasmic reticulum stress is partially involved in some cases of

ARTs-mediated anti-proliferation (Lu et al., 2010; Stockwin et al., 2009).

fects at least in part by inhibition of tumor angiogenesis.

resistance on the other hand (Sertel et al., 2010).

*3.2.2. Anti-proliferative mechanisms of ARTs*

(Anfosso et al., 2006).

Cancers are capable of spreading through the body by two mechanisms: invasion and meta‐ stasis. Invasion is the direct migration and penetration by cancer cells into neighboring tis‐ sues. Metastasis is the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then grow in a new focus (metastasize) in normal tis‐ sues elsewhere in the body. Without a connection to a network of blood vessels, a tumor can only grow to about the size of a pinhead (1-2 mm), that is to say a tumor is in a vascular, quiescent status. When a subgroup of cells within the tumor switches to an angiogenic phe‐ notype by changing the local equilibrium between positive and negative regulators of angio‐ genesis, tumor starts to grow rapidly and becomes clinically detectable. Anti-angiogenesis therapy is a novel approach in cancer treatment and prevention of tumor metastasis. It is therefore expected that angiogenesis inhibitors may be clinically useful for the treatment of tumors.

#### *3.2.1. Anti-cancer mechanism of ARTs on angiogenesis-related genes*

Angiogenesis involves tissue restructuring, and genes that regulate angiogenesis, such as che‐ mokine receptors, can also affect tumor metastasis. A vital requirement of neovasculogenesis is endothelial mitosis, which occurs in response to activation by pro-angiogenic signaling from VEGF and its receptors. Three human genes encode for VEGF (VEGFA, VEGFB and VEGFC) and splice variants add more heterogeneity to the biological actions of the VEGF gene family (Tischer et al., 1991). Analysis of VEGF transcripts in cultured vascular smooth muscle cells by PCR and cDNA cloning revealed three different forms of the VEGF coding region, which has also been previously reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indi‐ cates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 factor bind‐ ing sites. Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured vascular smooth muscle cells after treatment with the phorbol ester 12-O-tetradeca‐ noyl-phorbol-13-acetate (Tischer et al., 1991).

In a study using an US National Cancer Institute (NCI) panel of 60 tumor cell lines, ART and related compounds displayed anti-angiogenic activities based on the altered expres‐ sion of genes implicated in angiogenesis. The mRNA expression data of angiogenesis-relat‐ ed genes correlated well with the 50% growth inhibition concentration values for eight ARTs (ART, AS, arteether, artemisetene, arteanuine B, dihydroartemisinylester stereoisom‐ ers 1 and 2). The constitutive expression of 30 different genes correlated significantly with the cellular response to ARTs. The finding that drug sensitivity and resistance of tumor cells could be predicted by the mRNA expression of angiogenesis related genes supports the hypothesis that the antitumor activity of ARTs may be due, at least in part, by inhibi‐ tion of tumor angiogenesis. As many chemo-preventive drugs exert anti-angiogenic fea‐ tures, ARTs might also have a chemo-preventive effect in addition to their cytotoxic effects (Anfosso et al., 2006).

These findings are consistent with previous published work (Wartenberg et al., 2003) show‐ ing an artemisinin-dependent decrease in expression levels of hypoxia-inducible factor 1a (HIF-1a; H1F1A), which is known to be a transcriptional activator of VEGFA and is critical in neovasculogenesis in hypoxic tissues. The inhibition of angiogenesis by ART (at a concen‐ tration of 12 mM) involving VEGF and HIF-1a was also demonstrated in leukemic and glio‐ ma cells (Huang et al., 2008; Zhou et al., 2007). Loss of HIF-1α and VEGF expression by artemisinin appears to depend on ROS as co-treatment with free-radical scavengers such as vitamin E and mannitol reversed the effects of artemisinin (Wartenberg et al., 2003). The sensitivity and resistance of these tumor cells has been shown to correlate with mRNA ex‐ pression of angiogenesis-related genes. This suggests that the anti-tumor effects of ARTs are potentially due to their role in inhibiting tumor angiogenesis (Anfosso et al., 2006). The find‐ ing that tumor cell drug sensitivity and resistance could be predicted by mRNA expression of angiogenesis-related genes supports the hypothesis that artemisinins their anti-tumor ef‐ fects at least in part by inhibition of tumor angiogenesis.

In addition, an investigation to determine the sensitivity and resistance of cancer cells to‐ wards AS was conducted. The gene-hunting approach applied by us delivered several novel candidate genes that may regulate the response of cancer cells to AS. These results merit fur‐ ther investigations to prove the contribution of these genes for AS resistance. Study demon‐ strated that AS was no inhibitor of ABC transporters ABCB1 and ABCG2. Although AS may exhibit specific inhibitory functions towards particular ABC transporters, but not towards a wide spectrum of several different ABC transporters. This approach showed that response of tumor cells towards AS is multi-factorial in nature and is determined by gene expression associated with AS sensitivity on the one hand and with gene expression associated with AS resistance on the other hand (Sertel et al., 2010).

#### *3.2.2. Anti-proliferative mechanisms of ARTs*

As malignant tissues grow, metastases and solid tumors require extra blood supply for thriving and survival. Thus, cancer cells induce neovascularization by regulating proteins and pathways involved in the generation and restructure of new vasculature. Angiogenesis process leads to enhanced proliferation of endothelial cells through induction of VEGF, fi‐ broblast growth factor (FGF), its receptors, and cytokines. This event occurs via multiple ef‐ fects including hypoxia-driven activation of expression of HIF-1*α* and the aryl hydrocarbon receptor nuclear translocator (ARNT). Angiogenesis control is mediated by angiostatin, en‐ dostatin, thrombospondin, TIMPs, PAI-1, and others. Due to their role in tumor survival, the pro-angiogenic factors and the molecules involved in their regulatory networks are relevant

Cancers are capable of spreading through the body by two mechanisms: invasion and meta‐ stasis. Invasion is the direct migration and penetration by cancer cells into neighboring tis‐ sues. Metastasis is the ability of cancer cells to penetrate into lymphatic and blood vessels, circulate through the bloodstream, and then grow in a new focus (metastasize) in normal tis‐ sues elsewhere in the body. Without a connection to a network of blood vessels, a tumor can only grow to about the size of a pinhead (1-2 mm), that is to say a tumor is in a vascular, quiescent status. When a subgroup of cells within the tumor switches to an angiogenic phe‐ notype by changing the local equilibrium between positive and negative regulators of angio‐ genesis, tumor starts to grow rapidly and becomes clinically detectable. Anti-angiogenesis therapy is a novel approach in cancer treatment and prevention of tumor metastasis. It is therefore expected that angiogenesis inhibitors may be clinically useful for the treatment of

Angiogenesis involves tissue restructuring, and genes that regulate angiogenesis, such as che‐ mokine receptors, can also affect tumor metastasis. A vital requirement of neovasculogenesis is endothelial mitosis, which occurs in response to activation by pro-angiogenic signaling from VEGF and its receptors. Three human genes encode for VEGF (VEGFA, VEGFB and VEGFC) and splice variants add more heterogeneity to the biological actions of the VEGF gene family (Tischer et al., 1991). Analysis of VEGF transcripts in cultured vascular smooth muscle cells by PCR and cDNA cloning revealed three different forms of the VEGF coding region, which has also been previously reported in HL60 cells. The three forms of the human VEGF protein chain predicted from these coding regions are 189, 165, and 121 amino acids in length. Comparison of cDNA nucleotide sequences with sequences derived from human VEGF genomic clones indi‐ cates that the VEGF gene is split among eight exons and that the various VEGF coding region forms arise from this gene by alternative splicing. Analysis of the VEGF gene promoter region revealed a single major transcription start, which lies near a cluster of potential Sp1 factor bind‐ ing sites. Northern blot analysis demonstrated that the level of VEGF transcripts is elevated in cultured vascular smooth muscle cells after treatment with the phorbol ester 12-O-tetradeca‐

In a study using an US National Cancer Institute (NCI) panel of 60 tumor cell lines, ART and related compounds displayed anti-angiogenic activities based on the altered expres‐

drug targets (Crespo-Ortiz and Wei, 2012)

200 Research Directions in Tumor Angiogenesis

*3.2.1. Anti-cancer mechanism of ARTs on angiogenesis-related genes*

noyl-phorbol-13-acetate (Tischer et al., 1991).

tumors.

The anti-cancer mechanism of ARTs is likely to be related to the cleavage of the iron- or heme-mediated peroxide bridge, followed by the generation of ROS (Mercer et al., 2011; Zhang et al., 2010). The anti-cancer potential of ARTs is possibly connected to the expression of TfR. The synergism of AS and iron (II)-glycine sulfate co-treatment is unsuitable for all types of tumor cells. Endoplasmic reticulum stress is partially involved in some cases of ARTs-mediated anti-proliferation (Lu et al., 2010; Stockwin et al., 2009).

In normal cells, cyclin-dependent kinases (CDK) are the proteins translating signals in order to guide cells through the cell-division cycle. Normal growth relies on the ability to translate signals in order to replicate and divide in an effective manner (McDonald and El-Deiry, 2000). Uncontrolled proliferation in cancer cells is known to result from mutations inducing amplification of growth signals, deregulation of checkpoints, and loss of sensitivity to growth inhibitors. Abnormal cell growth is also triggered by deregulation of programmed cell death or apoptosis (Vogelstein and Kinzler, 2004). ARTs have been shown to effectively induce cell growth arrest in cancer lines either by disrupting the cell cycle kinetics or by in‐ terfering with proliferation-interacting pathways.

al., 2009).Overall, a wide body of research supports the hypothesis that ARTs are capable of

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203

Angiogenesis is promoted by numerous factors including cytokines such as VEGF, bFGF, PDGF and others. It is negatively regulated by angiostatin, endostatin, thrombospondin, TIMPs and other factors. The factors that are produced in tumor cells as well as in surround‐ ing stromal cells act in a balance to promote either pro-angiogenic or anti-angiogenic proc‐ esses. Among the cytokines for regulating angiogenesis, VEGF and angiopoietin-1 (Ang-1) have specific modulating effects on the growth of vascular endothelial cells, and they play a key role in the process of angiogenesis (Thurston 2002). VEGF is a homodimeric 34-42 kDa, a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeabilityenhancing activities specific for endothelial cells. Two receptor tyrosine kinases have been described as putative VEGF receptors, Flt-1 and KDR. Flt-1 (fms-like tyrosine kinase), and KDR (kinase-insert-domain-containing receptor) proteins have been shown to bind VEGF

ART and DHA have been shown to significantly inhibit angiogenesis in a dose-dependent manner as demonstrated by measurement of the proliferation, migration and tube formation of human umbilical vein endothelial (HUVE) cells (Chen et al., 2003). DHA was shown to markedly reduce VEGF binding to its receptors on the surface of HUVE cells and reduced the expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on HUVE cells. ART derivatives also inhibited HUVE cell tube formation and exhibited anti-angiogenic ef‐ fects (Oh et al., 2004). By utilizing the chicken chorioallantoic membrane (CAM) culture technique, it is possible to detect the microangium-like structures formed by *in vitro* cultivat‐ ed arterial rings associated with angiogenesis. By using this method, AS has been shown to also have anti-angiogenic effects. Treatment with AS significantly inhibited chicken cho‐ rioallantoic membrane (CAM) angiogenesis, proliferation, and differentiation of human mi‐ crovascular dermal endothelial cells in a dose-dependent manner and reduced Flt-1 and

Tumor hypoxia activates the transcription factor hypoxia inducible factor-1α (HIF-1α). This adaptation increases tumor angiogenesis to support the survival of poorly nourished cancer cells. Hypoxic tumors are resistant to radiation and many anti-cancer agents. HIF-1α is acti‐ vated during angiostatic therapy, and HIF-1α has also been shown to up-regulate the ex‐ pression of transferrin receptors. Since ART is selectively toxic to iron-loaded cells, radio and drug-resistant tumors might be selectively susceptible to attack by a treatment strategy

These findings are consistent with previous findings (Wartenberg et al., 2003) that noted ARTdependent decreases in expression levels of HIF-1α. HIF-1α is known to be a transcriptional activator of VEGF, and it plays a crucial role in neo-vasculogenesis in hypoxic tissues. ART treatment of leukemic and glioma cells in vitro at a concentration of 12 mM was shown in an‐ other study to inhibit angiogenesis. This ART driven angiogenesis inhibition was shown to in‐ volve suppression of VEGF and HIF-1α expression at the transcriptional level. (Huang et al.,

consisting of iron-loading and ART treatment (Li et al., 2008; Zhou et al., 2007).

interfering with several pathways known to be involved in neoplasia.

*3.2.3. Anti-VEGF mechanisms of ARTs*

KDR/flk-1 expression (Huan-Huan et al., 2004).

with high affinity.

DHA and AS are very potent growth inhibitors, and multiple studies have demonstrated that DHA is the most potent anti-cancer artemisinin-like compound (DHA *>* AS *>* AM) (Ef‐ ferth et al., 2003; Woerdenbag et al., 1993). Recently, artemisone has shown impressive anti‐ tumor efficacy in 7 cells lines including melanoma and breast cancer cells (Gravett et al., 2010). ART compounds have been shown to exert cytostatic and cytotoxic action on cancer cells (Efferth et al., 2003; Hou et al., 2008). ART-induced growth arrest has been reported at all cell cycle phases; however, arrest at the G0/G1 to S transition seems to be more common‐ ly affected (Efferth et al., 2003). Arrest at all cell cycle phases at the same time has been inter‐ preted as a cytostatic effect. Disruption of the cell cycle at G2/M was observed after DHA treatment in osteosarcoma, pancreas, leukemia (Yao et al., 2008) and ovarian cancer cells (Jiao et al., 2007). Similarly, AS interferes with G2 in osteosarcoma, ovarian, and other differ‐ ent cancer lines (Ji et al., 2011).

Several ART derivatives displayed higher cytotoxicity to murine bone marrow cells than to murine Ehrlich ascites tumor cells in a clonogenic assay (Beekman et al., 1998). The IC50 val‐ ues for HeLa cervical cancer cells, uterine chorion cancer JAR cells, embryo transversal can‐ cer RD and ovarian cancer HO-8910 cell lines after 48-h treatment with ART and DHA ranged from 15 to 50 µM and from 8 to 33 µM, respectively (Chen et al., 2003). ART potenti‐ ated the differentiation of 1α, 25-dihydroxyvitamin-D3-induced HL-60 leukemia cell pre‐ dominantly into monocytes and all-*trans* RA-induced cell differentiation into granulocytes, respectively (Kim et al., 2003). Signal transducers involved in the differentiation process, such as extracellular-signal regulated kinase (ERK) and protein kinase C ß1 (PKCB1) were affected by ART.

Inhibition of proliferation may also be attributed to down-regulation of interacting proteins targeting multiple pathways (Firestone and Sundar, 2009). It has been shown that DHA treatment of pancreatic cells (BxPC3, AsPC-1) inhibited cell viability by decreasing the levels of proliferating cell nuclear antigen (PCNA) and cyclin D with parallel increase in p21 (Chen et al., 2009). Another study in the same system showed that DHA counters NF-*κ*B factor ac‐ tivation leading to inhibition of its targets in the proliferation (c-myc, cyclin D) and apoptot‐ ic pathways (Bcl2, Bcl-xl) (Wang et al., 2010). In prostate cancer, DHA has been shown to induce cell cycle arrest by disrupting the interaction of Sp1 (specificity protein 1) and the CDK4 promoter (Willoughby et al., 2009). Dissociation of the Sp1-CDK4 complex promotes caspase activation and cell death. In addition, another study has identified AS as a topoiso‐ merase II inhibitor which inhibits growth by interaction with multiple pathways (Youns et al., 2009).Overall, a wide body of research supports the hypothesis that ARTs are capable of interfering with several pathways known to be involved in neoplasia.

#### *3.2.3. Anti-VEGF mechanisms of ARTs*

In normal cells, cyclin-dependent kinases (CDK) are the proteins translating signals in order to guide cells through the cell-division cycle. Normal growth relies on the ability to translate signals in order to replicate and divide in an effective manner (McDonald and El-Deiry, 2000). Uncontrolled proliferation in cancer cells is known to result from mutations inducing amplification of growth signals, deregulation of checkpoints, and loss of sensitivity to growth inhibitors. Abnormal cell growth is also triggered by deregulation of programmed cell death or apoptosis (Vogelstein and Kinzler, 2004). ARTs have been shown to effectively induce cell growth arrest in cancer lines either by disrupting the cell cycle kinetics or by in‐

DHA and AS are very potent growth inhibitors, and multiple studies have demonstrated that DHA is the most potent anti-cancer artemisinin-like compound (DHA *>* AS *>* AM) (Ef‐ ferth et al., 2003; Woerdenbag et al., 1993). Recently, artemisone has shown impressive anti‐ tumor efficacy in 7 cells lines including melanoma and breast cancer cells (Gravett et al., 2010). ART compounds have been shown to exert cytostatic and cytotoxic action on cancer cells (Efferth et al., 2003; Hou et al., 2008). ART-induced growth arrest has been reported at all cell cycle phases; however, arrest at the G0/G1 to S transition seems to be more common‐ ly affected (Efferth et al., 2003). Arrest at all cell cycle phases at the same time has been inter‐ preted as a cytostatic effect. Disruption of the cell cycle at G2/M was observed after DHA treatment in osteosarcoma, pancreas, leukemia (Yao et al., 2008) and ovarian cancer cells (Jiao et al., 2007). Similarly, AS interferes with G2 in osteosarcoma, ovarian, and other differ‐

Several ART derivatives displayed higher cytotoxicity to murine bone marrow cells than to murine Ehrlich ascites tumor cells in a clonogenic assay (Beekman et al., 1998). The IC50 val‐ ues for HeLa cervical cancer cells, uterine chorion cancer JAR cells, embryo transversal can‐ cer RD and ovarian cancer HO-8910 cell lines after 48-h treatment with ART and DHA ranged from 15 to 50 µM and from 8 to 33 µM, respectively (Chen et al., 2003). ART potenti‐ ated the differentiation of 1α, 25-dihydroxyvitamin-D3-induced HL-60 leukemia cell pre‐ dominantly into monocytes and all-*trans* RA-induced cell differentiation into granulocytes, respectively (Kim et al., 2003). Signal transducers involved in the differentiation process, such as extracellular-signal regulated kinase (ERK) and protein kinase C ß1 (PKCB1) were

Inhibition of proliferation may also be attributed to down-regulation of interacting proteins targeting multiple pathways (Firestone and Sundar, 2009). It has been shown that DHA treatment of pancreatic cells (BxPC3, AsPC-1) inhibited cell viability by decreasing the levels of proliferating cell nuclear antigen (PCNA) and cyclin D with parallel increase in p21 (Chen et al., 2009). Another study in the same system showed that DHA counters NF-*κ*B factor ac‐ tivation leading to inhibition of its targets in the proliferation (c-myc, cyclin D) and apoptot‐ ic pathways (Bcl2, Bcl-xl) (Wang et al., 2010). In prostate cancer, DHA has been shown to induce cell cycle arrest by disrupting the interaction of Sp1 (specificity protein 1) and the CDK4 promoter (Willoughby et al., 2009). Dissociation of the Sp1-CDK4 complex promotes caspase activation and cell death. In addition, another study has identified AS as a topoiso‐ merase II inhibitor which inhibits growth by interaction with multiple pathways (Youns et

terfering with proliferation-interacting pathways.

ent cancer lines (Ji et al., 2011).

202 Research Directions in Tumor Angiogenesis

affected by ART.

Angiogenesis is promoted by numerous factors including cytokines such as VEGF, bFGF, PDGF and others. It is negatively regulated by angiostatin, endostatin, thrombospondin, TIMPs and other factors. The factors that are produced in tumor cells as well as in surround‐ ing stromal cells act in a balance to promote either pro-angiogenic or anti-angiogenic proc‐ esses. Among the cytokines for regulating angiogenesis, VEGF and angiopoietin-1 (Ang-1) have specific modulating effects on the growth of vascular endothelial cells, and they play a key role in the process of angiogenesis (Thurston 2002). VEGF is a homodimeric 34-42 kDa, a heparin-binding glycoprotein with potent angiogenic, mitogenic and vascular permeabilityenhancing activities specific for endothelial cells. Two receptor tyrosine kinases have been described as putative VEGF receptors, Flt-1 and KDR. Flt-1 (fms-like tyrosine kinase), and KDR (kinase-insert-domain-containing receptor) proteins have been shown to bind VEGF with high affinity.

ART and DHA have been shown to significantly inhibit angiogenesis in a dose-dependent manner as demonstrated by measurement of the proliferation, migration and tube formation of human umbilical vein endothelial (HUVE) cells (Chen et al., 2003). DHA was shown to markedly reduce VEGF binding to its receptors on the surface of HUVE cells and reduced the expression levels of two major VEGF receptors, Flt-1 and KDR/flk-1, on HUVE cells. ART derivatives also inhibited HUVE cell tube formation and exhibited anti-angiogenic ef‐ fects (Oh et al., 2004). By utilizing the chicken chorioallantoic membrane (CAM) culture technique, it is possible to detect the microangium-like structures formed by *in vitro* cultivat‐ ed arterial rings associated with angiogenesis. By using this method, AS has been shown to also have anti-angiogenic effects. Treatment with AS significantly inhibited chicken cho‐ rioallantoic membrane (CAM) angiogenesis, proliferation, and differentiation of human mi‐ crovascular dermal endothelial cells in a dose-dependent manner and reduced Flt-1 and KDR/flk-1 expression (Huan-Huan et al., 2004).

Tumor hypoxia activates the transcription factor hypoxia inducible factor-1α (HIF-1α). This adaptation increases tumor angiogenesis to support the survival of poorly nourished cancer cells. Hypoxic tumors are resistant to radiation and many anti-cancer agents. HIF-1α is acti‐ vated during angiostatic therapy, and HIF-1α has also been shown to up-regulate the ex‐ pression of transferrin receptors. Since ART is selectively toxic to iron-loaded cells, radio and drug-resistant tumors might be selectively susceptible to attack by a treatment strategy consisting of iron-loading and ART treatment (Li et al., 2008; Zhou et al., 2007).

These findings are consistent with previous findings (Wartenberg et al., 2003) that noted ARTdependent decreases in expression levels of HIF-1α. HIF-1α is known to be a transcriptional activator of VEGF, and it plays a crucial role in neo-vasculogenesis in hypoxic tissues. ART treatment of leukemic and glioma cells in vitro at a concentration of 12 mM was shown in an‐ other study to inhibit angiogenesis. This ART driven angiogenesis inhibition was shown to in‐ volve suppression of VEGF and HIF-1α expression at the transcriptional level. (Huang et al., 2008; Zhou et al., 2007). Loss of HIF-1α and VEGF expression after ART treatment appears to be dependent on production of ROS because co-treatment with free-radical scavengers such as vitamin E and mannitol reversed the effects of ART (Wartenberg et al., 2003).

lyzed, and the results demonstrated that ART induces cell growth arrest in the A375M cell line, and affects the viability of A375P melanoma cells through both cytotoxic and growth inhibitory effects. In addition, ART was shown to affect the migratory ability of A375M mel‐ anoma cells by reducing metalloproteinase 2 (MMP-2) production and down-regulating al‐ pha v beta 3 integrin expression (Buommino et al., 2009). Other studies, however, showed ART was not effective in inhibiting proliferation of other tumor cell lines such as MCF7, a

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

205

Similarly, ARTs have been shown to inhibit Matrigel invasion of 6 non-small cell cancer (NSCLC) cell lines and inhibited urokinase-type plasminogen activator (u-PA) activity, -pro‐ tein and -mRNA expression. Furthermore, in a PCR-metastasis array, ARTs were shown to inhibit the expression of several matrix metalloproteinases (MMPs), especially MMP-2 and MMP-7 mRNA/protein. In luciferase reporter assays, ARTs were shown to down-regulate MMP-2-, MMP-7- and u-PA-promoter/-enhancer activity, in parallel to AP-1- and NF-kBtransactivation. Si-RNA knockdown of u-PA, MMP-2 and MMP-7 abolished ART's ability to inhibit invasion, further supporting hypotheses of the anti-cancer activity of ARTs In conclu‐ sion, this study showed that ART treatment suppresses invasion and metastasis in NSCLC, specifically targeting transcription of u-PA, MMP-2 and MMP-7. These studies all support the utility of ART compounds as novel therapeutic agents or adjunct therapies for NSCLC

DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimen‐ sional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA en‐ hanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. Accordingly, it is possible that a redox imbalance may be the mecha‐ nism behind DHA-induced ER stress, which may contribute, at least in part, to its anti-can‐

DHA has been shown to enhance gemcitabine-induced growth inhibition and apoptosis in both BxPC-3 and PANC-1 cell lines. The mechanism is at least partially due to DHA's role in deactivating gemcitabine-induced NF-kappaB activation, which, in turn, so as to dramatical‐ ly decreases the expression of its target gene products, such as c-myc, cyclin D1, Bcl-2, BclxL. *In vivo* studies have shown that, gemcitabine also manifested remarkably enhanced antitumor effects when combined with DHA, as demonstrated by significantly increased apoptosis, as well as decreased Ki-67 index, NF-kappaB activity and its related gene prod‐

breast adenocarcinoma cell line, and MKN, a gastric carcinoma line.

(Rasheed et al., 2010).

cer activity (Lu et al., 2011).

*In vitro*, VEGF is a potent endothelial cell mitogen. In cultured endothelial cells, VEGF has been shown to activate phospholipase C and induce rapid increases of free cytosolic Ca2+. VEGF has also been shown to stimulate the release of von Willebrand factor from endothe‐ lial cells and induce expression of tissue factor activity in endothelial cells as well as in mon‐ ocytes, and. VEGF has been shown to be involved in the chemotaxis of monocytes and osteoblasts. *In vivo*, VEGF can induce angiogenesis as well as increase microvascular perme‐ ability. As a vascular permeability factor, VEGF acts directly on the endothelium and does not degranulate mast cells. It promotes extravasation of plasma fibrinogen, leading to fibrin deposition which alters the tumor extracellular matrix. The modified extracellular matrix subsequently promotes the migration of macrophages, fibroblasts and endothelial cells. Based on its *in vitro* and *in vivo* properties, VEGF is believed to play important roles in in‐ flammation and also in normal and pathological aspects of angiogenesis, a process that is associated with wound healing, embryonic development, growth, and metastasis of solid tu‐ mors. Elevated levels of VEGF have been reported in synovial fluids of rheumatoid arthritis patients and in sera from cancer patients.

In the last three decades, there is a growing body of evidence on the role of angiogenesis in tumor growth and metastases of tumors (Firestone and Sundar, 2009). Angiogenesis can be divided into a series of temporally regulated responses, including induction of proteases, migration of endothelial cells, cell proliferation and differentiation. This is a highly complex process, in which a number of cytokines and growth factors released by endothelial cells, tumor cells and matrix cells are involved. The expression of VEGF has been suggested to be related to some fundamental features of solid tumors, such as the growth rate, the density of tumor microvessels, and the development of tumor metastases.

It is interesting to note that torilin, another sesquiterpene (derived from the fruits of *Torilis japonica)*, has also been shown to be a potent anti-angiogenic factor which also inhibits blood vessel formation by disrupting VEGFA expression. A similar finding was also shown by us‐ ing DHA (Kim et al., 2007). Hence, the ability of ART to inhibit angiogenesis may be due to its chemical nature as a sesquiterpene. Another compelling finding is that other phytoses‐ quiterpene lactones, such as costunolide from *Saussurea lappa*, can inhibit KDR signaling (Jeong et al., 2002). Comparisons with other sesquiterpenes may shed more light on the unique features of the anti-cancer actions of ART, and potentially lead to better angiostatic drug design. Taken together, ART and its derivatives, and other sesquiterpene lactones, have been shown to have potent anti-angiogenic effects in tumor cells. These observations have many implications in terms of cancer therapy as well as cancer prevention since angio‐ genesis is a promotional event.

#### *3.2.4. Other anti-angiogenic mechanisms of ARTs*

Anti-cancer activity of ARTs has been reported both *in vitro* and *in vivo*. The inhibitory ef‐ fects of ART on the migratory ability of melanoma cell lines (A375P and A375M) were ana‐ lyzed, and the results demonstrated that ART induces cell growth arrest in the A375M cell line, and affects the viability of A375P melanoma cells through both cytotoxic and growth inhibitory effects. In addition, ART was shown to affect the migratory ability of A375M mel‐ anoma cells by reducing metalloproteinase 2 (MMP-2) production and down-regulating al‐ pha v beta 3 integrin expression (Buommino et al., 2009). Other studies, however, showed ART was not effective in inhibiting proliferation of other tumor cell lines such as MCF7, a breast adenocarcinoma cell line, and MKN, a gastric carcinoma line.

2008; Zhou et al., 2007). Loss of HIF-1α and VEGF expression after ART treatment appears to be dependent on production of ROS because co-treatment with free-radical scavengers such as

*In vitro*, VEGF is a potent endothelial cell mitogen. In cultured endothelial cells, VEGF has been shown to activate phospholipase C and induce rapid increases of free cytosolic Ca2+. VEGF has also been shown to stimulate the release of von Willebrand factor from endothe‐ lial cells and induce expression of tissue factor activity in endothelial cells as well as in mon‐ ocytes, and. VEGF has been shown to be involved in the chemotaxis of monocytes and osteoblasts. *In vivo*, VEGF can induce angiogenesis as well as increase microvascular perme‐ ability. As a vascular permeability factor, VEGF acts directly on the endothelium and does not degranulate mast cells. It promotes extravasation of plasma fibrinogen, leading to fibrin deposition which alters the tumor extracellular matrix. The modified extracellular matrix subsequently promotes the migration of macrophages, fibroblasts and endothelial cells. Based on its *in vitro* and *in vivo* properties, VEGF is believed to play important roles in in‐ flammation and also in normal and pathological aspects of angiogenesis, a process that is associated with wound healing, embryonic development, growth, and metastasis of solid tu‐ mors. Elevated levels of VEGF have been reported in synovial fluids of rheumatoid arthritis

In the last three decades, there is a growing body of evidence on the role of angiogenesis in tumor growth and metastases of tumors (Firestone and Sundar, 2009). Angiogenesis can be divided into a series of temporally regulated responses, including induction of proteases, migration of endothelial cells, cell proliferation and differentiation. This is a highly complex process, in which a number of cytokines and growth factors released by endothelial cells, tumor cells and matrix cells are involved. The expression of VEGF has been suggested to be related to some fundamental features of solid tumors, such as the growth rate, the density of

It is interesting to note that torilin, another sesquiterpene (derived from the fruits of *Torilis japonica)*, has also been shown to be a potent anti-angiogenic factor which also inhibits blood vessel formation by disrupting VEGFA expression. A similar finding was also shown by us‐ ing DHA (Kim et al., 2007). Hence, the ability of ART to inhibit angiogenesis may be due to its chemical nature as a sesquiterpene. Another compelling finding is that other phytoses‐ quiterpene lactones, such as costunolide from *Saussurea lappa*, can inhibit KDR signaling (Jeong et al., 2002). Comparisons with other sesquiterpenes may shed more light on the unique features of the anti-cancer actions of ART, and potentially lead to better angiostatic drug design. Taken together, ART and its derivatives, and other sesquiterpene lactones, have been shown to have potent anti-angiogenic effects in tumor cells. These observations have many implications in terms of cancer therapy as well as cancer prevention since angio‐

Anti-cancer activity of ARTs has been reported both *in vitro* and *in vivo*. The inhibitory ef‐ fects of ART on the migratory ability of melanoma cell lines (A375P and A375M) were ana‐

vitamin E and mannitol reversed the effects of ART (Wartenberg et al., 2003).

patients and in sera from cancer patients.

204 Research Directions in Tumor Angiogenesis

genesis is a promotional event.

*3.2.4. Other anti-angiogenic mechanisms of ARTs*

tumor microvessels, and the development of tumor metastases.

Similarly, ARTs have been shown to inhibit Matrigel invasion of 6 non-small cell cancer (NSCLC) cell lines and inhibited urokinase-type plasminogen activator (u-PA) activity, -pro‐ tein and -mRNA expression. Furthermore, in a PCR-metastasis array, ARTs were shown to inhibit the expression of several matrix metalloproteinases (MMPs), especially MMP-2 and MMP-7 mRNA/protein. In luciferase reporter assays, ARTs were shown to down-regulate MMP-2-, MMP-7- and u-PA-promoter/-enhancer activity, in parallel to AP-1- and NF-kBtransactivation. Si-RNA knockdown of u-PA, MMP-2 and MMP-7 abolished ART's ability to inhibit invasion, further supporting hypotheses of the anti-cancer activity of ARTs In conclu‐ sion, this study showed that ART treatment suppresses invasion and metastasis in NSCLC, specifically targeting transcription of u-PA, MMP-2 and MMP-7. These studies all support the utility of ART compounds as novel therapeutic agents or adjunct therapies for NSCLC (Rasheed et al., 2010).

DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimen‐ sional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA en‐ hanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. Accordingly, it is possible that a redox imbalance may be the mecha‐ nism behind DHA-induced ER stress, which may contribute, at least in part, to its anti-can‐ cer activity (Lu et al., 2011).

DHA has been shown to enhance gemcitabine-induced growth inhibition and apoptosis in both BxPC-3 and PANC-1 cell lines. The mechanism is at least partially due to DHA's role in deactivating gemcitabine-induced NF-kappaB activation, which, in turn, so as to dramatical‐ ly decreases the expression of its target gene products, such as c-myc, cyclin D1, Bcl-2, BclxL. *In vivo* studies have shown that, gemcitabine also manifested remarkably enhanced antitumor effects when combined with DHA, as demonstrated by significantly increased apoptosis, as well as decreased Ki-67 index, NF-kappaB activity and its related gene prod‐ ucts, and predictably, significantly reduced tumor volume. The inhibition of gemcitabine-in‐ duced NF-kappaB activation is one of the mechanisms that is known by which DHA dramatically promotes its anti-tumor effect on pancreatic cancer (Wang et al., 2010).

issued (Protocol Number: ISRCTN05203252, 2008. http://www.controlled-trials.com/

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

207

**2.** Phase II study of AS treatment as an adjunct to treat non-small cell lung cancer

This study was designed to compare the efficacy and toxicity of AS treatment combined with NP (a chemotherapy regimen of vinorelbine and cisplatin) and NP alone in the treat‐ ment of advanced non-small cell lung cancer (NSCLC). One hundred and twenty cases of advanced NSCLC were randomly divided into an NP chemotherapy group and a combined AS with NP therapy group. Patients in the control group were treated with the NP regimen of vinorelbine and cisplatin. Patients in the trial group were treated with the NP regimen supplemented with intravenous AS injections (120 mg, once-a-day intravenous injection, from the 1st day to 8th day, for 8 days). At least two 21-day-cycles of treatment were per‐ formed. There were no significant differences in the short-term survival rates, mean survival times and the 1-year survival rates between the trial group and the control group, which were 44 weeks and 45 weeks, respectively. The disease controlled rate of the trial group (88.2%) was significantly higher than that of the control group (72.7%) (P < 0.05), and the tri‐ al group's time to disease progression (24 weeks) was significantly longer than that of the control group (20 weeks). No significant difference was found in toxicity between the two treatment groups. Therefore. AS combined with NP can increase the disease controlled rate and prolong the time to progression of patients with advanced NSCLC without significant

The purpose of this study was to evaluate the tolerability of an adjunctive therapy with AS for a period of 4 weeks in patients over the age of 18 years with advanced metastatic breast cancer, which was defined as a histologically or cytologically confirmed. Women of child‐ bearing potential were tested to rule out pregnancy prior to their treatment. Relevant neuro‐ logical symptoms, adverse events, and the relation between adverse events and the use of AS, as an adjunct, saliva cortisol profile, overall response rate, clinical benefit, and assess‐ ment of patients' expectations will be monitored as study endpoints. No result of this study has yet been publicly issued (Protocol Number: NCT00764036, 2011, http://www.cancer.gov/

AS was successfully used in the treatment of laryngeal squamous cell carcinoma where treated patients showed a substantial reduction in tumor size (by 70%) after two months of treatment (Singh and Verma, 2002). Furthermore, AS used in combination with standard chemotherapy increased survival and substantially reduced metastasis in patients with ma‐ lignant skin cancer (Berger et al., 2005). Another report describes a beneficial improvement in a patient with pituitary macroadenoma who was treated with artemether for 12 months (Singh and Panwar, 2006). Other cases describing the use of ARTs for treatment of cancer

ISRCTN05203252/).

(Completed)

side effects (Zhang et al., 2008).

**3.** Phase I study with metastatic breast cancer (Completed)

*3.3.2. Treatment reports of ARTs used to treat cancers*

clinicaltrials/search/view/print?cdrid=616937&version=HealthProfessional).

have been reported in the Cancer Smart Bomb Part I and II study (White, 2002)

Embryotoxicity appears to be connected with defective angiogenesis and vasculogenesis in certain stages of embryo development. This may prevent the use of ART derivatives in ma‐ laria during pregnancy, when both mother and fetus are at high risk of death. Artemisone is a novel 10-alkylamino derivative which is not metabolized to DHA. It was selected as a clin‐ ical drug candidate on the basis of its high efficacy against *Plasmodium falciparum in vitro* and its lack of detectable neurotoxicity in both *in vitro* and *in vivo* screens. A comparative study of the anti-angiogenic properties of both artemisone and dihydroartemisinin in different model systems was conducted. In this study, the effects of both artemisone and DHA were evaluated by measuring the proliferation of human endothelial cells and their migration on a fibronectin matrix, the sprouting of new vessels from rat aorta sections grown in collagen, and the production of pro-angiogenic cytokines such as VEGF and interleukin-8 (CXCL-8). The data show that artemisone is significantly less anti-angiogenic than DHA in all the ex‐ perimental models, suggesting that it will be safer to use than the current clinical ARTs dur‐ ing pregnancy (D'Alessandro et al., 2007).

#### **3.3. Anti-cancer clinical trials and case treatments of ARTs**

Antitumor activity of ARTs has also been documented in human trials and individual clini‐ cal cases. ART, AM and AS have been used in cancer therapy, and they have been shown to be well tolerated without significant side effects (Table 3).

#### *3.3.1. Clinical trials of ARTs as anti-cancer agents*

Clinical evidence has accumulated showing that ART-derived drugs have promise for treat‐ ment of laryngeal carcinomas, uveal melanomas and pituitary macroadenomas. AS is also in phase I-II trials for treatment of breast, colorectal and non-small cell lung cancers. Similarly, a clinical trial in 120 patients with advanced non-small cell lung cancer has shown that arte‐ sunate in combination with a chemotherapy regimen of vinorelbine and cisplatin elevated 1 year survival rate by 13% with a significant improvement in disease control and time to progression. No additional AS-related side effects were reported (Zhang et al., 2008).

**1.** Phase I study of oral AS to treat colorectal cancer (Completed)

The primary objective of this study was to determine the effects of oral AS in inducing apop‐ tosis in patients awaiting surgical treatment of colorectal adenocarcinoma. The secondary objective of this study was to establish the tolerability of oral AS for the treatment of colorec‐ tal cancer. Subjects were randomized to receive either 200 mg AS or placebo orally once dai‐ ly for 14 days while awaiting surgery for definitive surgical treatment of colorectal adenocarcinoma. A significant difference in the proportion of colorectal adenocarcinoma cells exhibiting apoptosis was noted between the two treatment groups (placebo and AS), assessed at the time of surgery after two weeks of drug treatment. No result was publicly issued (Protocol Number: ISRCTN05203252, 2008. http://www.controlled-trials.com/ ISRCTN05203252/).

**2.** Phase II study of AS treatment as an adjunct to treat non-small cell lung cancer (Completed)

This study was designed to compare the efficacy and toxicity of AS treatment combined with NP (a chemotherapy regimen of vinorelbine and cisplatin) and NP alone in the treat‐ ment of advanced non-small cell lung cancer (NSCLC). One hundred and twenty cases of advanced NSCLC were randomly divided into an NP chemotherapy group and a combined AS with NP therapy group. Patients in the control group were treated with the NP regimen of vinorelbine and cisplatin. Patients in the trial group were treated with the NP regimen supplemented with intravenous AS injections (120 mg, once-a-day intravenous injection, from the 1st day to 8th day, for 8 days). At least two 21-day-cycles of treatment were per‐ formed. There were no significant differences in the short-term survival rates, mean survival times and the 1-year survival rates between the trial group and the control group, which were 44 weeks and 45 weeks, respectively. The disease controlled rate of the trial group (88.2%) was significantly higher than that of the control group (72.7%) (P < 0.05), and the tri‐ al group's time to disease progression (24 weeks) was significantly longer than that of the control group (20 weeks). No significant difference was found in toxicity between the two treatment groups. Therefore. AS combined with NP can increase the disease controlled rate and prolong the time to progression of patients with advanced NSCLC without significant side effects (Zhang et al., 2008).

**3.** Phase I study with metastatic breast cancer (Completed)

ucts, and predictably, significantly reduced tumor volume. The inhibition of gemcitabine-in‐ duced NF-kappaB activation is one of the mechanisms that is known by which DHA

Embryotoxicity appears to be connected with defective angiogenesis and vasculogenesis in certain stages of embryo development. This may prevent the use of ART derivatives in ma‐ laria during pregnancy, when both mother and fetus are at high risk of death. Artemisone is a novel 10-alkylamino derivative which is not metabolized to DHA. It was selected as a clin‐ ical drug candidate on the basis of its high efficacy against *Plasmodium falciparum in vitro* and its lack of detectable neurotoxicity in both *in vitro* and *in vivo* screens. A comparative study of the anti-angiogenic properties of both artemisone and dihydroartemisinin in different model systems was conducted. In this study, the effects of both artemisone and DHA were evaluated by measuring the proliferation of human endothelial cells and their migration on a fibronectin matrix, the sprouting of new vessels from rat aorta sections grown in collagen, and the production of pro-angiogenic cytokines such as VEGF and interleukin-8 (CXCL-8). The data show that artemisone is significantly less anti-angiogenic than DHA in all the ex‐ perimental models, suggesting that it will be safer to use than the current clinical ARTs dur‐

Antitumor activity of ARTs has also been documented in human trials and individual clini‐ cal cases. ART, AM and AS have been used in cancer therapy, and they have been shown to

Clinical evidence has accumulated showing that ART-derived drugs have promise for treat‐ ment of laryngeal carcinomas, uveal melanomas and pituitary macroadenomas. AS is also in phase I-II trials for treatment of breast, colorectal and non-small cell lung cancers. Similarly, a clinical trial in 120 patients with advanced non-small cell lung cancer has shown that arte‐ sunate in combination with a chemotherapy regimen of vinorelbine and cisplatin elevated 1 year survival rate by 13% with a significant improvement in disease control and time to

The primary objective of this study was to determine the effects of oral AS in inducing apop‐ tosis in patients awaiting surgical treatment of colorectal adenocarcinoma. The secondary objective of this study was to establish the tolerability of oral AS for the treatment of colorec‐ tal cancer. Subjects were randomized to receive either 200 mg AS or placebo orally once dai‐ ly for 14 days while awaiting surgery for definitive surgical treatment of colorectal adenocarcinoma. A significant difference in the proportion of colorectal adenocarcinoma cells exhibiting apoptosis was noted between the two treatment groups (placebo and AS), assessed at the time of surgery after two weeks of drug treatment. No result was publicly

progression. No additional AS-related side effects were reported (Zhang et al., 2008).

**1.** Phase I study of oral AS to treat colorectal cancer (Completed)

dramatically promotes its anti-tumor effect on pancreatic cancer (Wang et al., 2010).

ing pregnancy (D'Alessandro et al., 2007).

206 Research Directions in Tumor Angiogenesis

**3.3. Anti-cancer clinical trials and case treatments of ARTs**

be well tolerated without significant side effects (Table 3).

*3.3.1. Clinical trials of ARTs as anti-cancer agents*

The purpose of this study was to evaluate the tolerability of an adjunctive therapy with AS for a period of 4 weeks in patients over the age of 18 years with advanced metastatic breast cancer, which was defined as a histologically or cytologically confirmed. Women of child‐ bearing potential were tested to rule out pregnancy prior to their treatment. Relevant neuro‐ logical symptoms, adverse events, and the relation between adverse events and the use of AS, as an adjunct, saliva cortisol profile, overall response rate, clinical benefit, and assess‐ ment of patients' expectations will be monitored as study endpoints. No result of this study has yet been publicly issued (Protocol Number: NCT00764036, 2011, http://www.cancer.gov/ clinicaltrials/search/view/print?cdrid=616937&version=HealthProfessional).

#### *3.3.2. Treatment reports of ARTs used to treat cancers*

AS was successfully used in the treatment of laryngeal squamous cell carcinoma where treated patients showed a substantial reduction in tumor size (by 70%) after two months of treatment (Singh and Verma, 2002). Furthermore, AS used in combination with standard chemotherapy increased survival and substantially reduced metastasis in patients with ma‐ lignant skin cancer (Berger et al., 2005). Another report describes a beneficial improvement in a patient with pituitary macroadenoma who was treated with artemether for 12 months (Singh and Panwar, 2006). Other cases describing the use of ARTs for treatment of cancer have been reported in the Cancer Smart Bomb Part I and II study (White, 2002)

#### **1.** Metastatic uveal melanomas treated with AS

Berger et al. reported on the first long-term treatment of two cancer patients with AS in com‐ bination with standard chemotherapy. These patients with metastatic uveal melanoma were treated on a compassionate-use basis, after standard chemotherapy alone was ineffective in stopping tumor growth. The therapy regimen was well tolerated with no additional side ef‐ fects other than those caused by standard chemotherapy alone. One patient experienced a temporary response after the addition of AS to Fotemustine while the disease was progress‐ ing under therapy with Fotemustine alone. The second patient first experienced a stabiliza‐ tion of the disease after the addition of AS to Dacarbazine, followed by objective regressions of splenic and lung metastases. This patient is still alive 47 months after first diagnosis with stage IV uveal melanoma, a diagnosis with a median survival of 2-5 months, without addi‐ tional side effects. One patient experienced a temporary response after the addition of AS while the disease was progressing under standard therapy with Fotemustine alone. This pa‐ tient died after 24 months.

consequence of his disease. AM was administered orally to the patient over a period of 12 months. Although the tumor remained consistent in size, CT scans showed a reduction in tumor density, and clinically, the related symptoms and signs improved significantly as therapy progressed. Overall, the AM treatment was beneficial in improving the patient's quality of life. AM and other ART analogs appear to have promise for treatment of this type

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

209

ARTs are largely non-toxic, with related compounds having been administered to over 2 million patients; both children and adult, world-wide without reports of significant serious side effects, and ARTs are very inexpensive when compared to conventional cancer drugs. The final results of current clinical trials utilizing ARTs as therapy or adjunct against a wide variety of cancers have not yet been published although initial findings released suggest positive results. How positive or efficacious these results are remains to be seen until full and final results are published. One question, however, remains - why are there not more

ARTs as angiogenesis inhibitors are unique cancer-fighting agents because they tend to in‐ hibit the growth of blood vessels rather than tumor cells. Therefore, the angiogenesis inhibi‐ tor therapy does not necessarily kill tumors but instead may prevent tumors from growing.

Following oral administration, however, AS and DHA have short mean residence times (MRT) of 1.95 and 2.71 hr, respectively, and ART has a longer MRT of 7.4 hr. Intramuscular AM and arteether have longer MRTs ranging from 13.9 - 42.9 hrs in humans s due to pro‐ longed absorption and accumulation at the injection sites. The shortest MRT (0.90 hr) was found in humans following intravenous injection of AS (Table 2). It is obvious that the dif‐ ferent ARTs administered in different regimens have significant differences in pharmacoki‐ netic (PK) characteristics in humans, and only intramuscular ARTs can provide a long period of therapy. Therefore, injectable AM has been recommended as a longer acting com‐

Pharmacokinetics (PK) studies of ARTs show three phases (absorption, distribution, and elimination) of ART drugs in blood following oral, intravenous, or intramuscular adminis‐ tration. After multiple daily administrations, four ARTs showed declining daily drug con‐ centrations (ART, DHA, AS, and AM) which has been shown is believed to be due to an auto-induced metabolism pathway during multiple oral treatments in patients and health subjects (van Agtmael et al., 1999; Ashton et al., 1996; 1998; Khanh et al., 1999; Park et al., 1998). The Cmax and AUC values of ARTs are markedly reduced from one-third to one-sev‐ enth on the last dose day compared with the first day. The decrease in drug exposure levels during treatment is not disease-related, since the PK profile of ART drugs on the last day shows a similar decrease to that reported in healthy subjects. Similar time-dependent de‐

**3.4. Why are not there more trials or more wide-spread use of ARTs?**

trials or more wide-spread use of ARTs as an off-label cancer treatment?

This type of therapy may therefore need to be administered over a long period.

pound that may be suitable for cancer treatment (White, 2002).

of cancer (Singh and Panwar, 2006).

*3.4.1. PK mismatch of ARTs in cancer therapy*

Despite the small number of treated patients, AS may be a promising adjuvant drug for the treatment of melanoma and possibly other tumors in combination with standard chemothera‐ py. AS is well tolerated, and the lack of serious side effects will facilitate prospective random‐ ized trials in the near future. From *in vitro* studies already conducted (Efferth et al., 2004b), it is further conceivable that loading tumor cells with bivalent iron, by simply providing Fe2+ in tab‐ let form, might increase the susceptibility of cancer cells to AS treatment. It is tempting to spec‐ ulate that, in the case of the second patient previously discussed, the addition of Fe2+ had an actual clinical impact and resulted in an improved response to therapy (Berger et al., 2005).

**2.** Laryngeal carcinoma treated with AS

AS injections and tablets were used in one study to treat a laryngeal squamous cell carcino‐ ma patient over a period of nine months. The tumor was significantly reduced in size by 70% after two months of treatment. Overall, AS treatment of the patient was beneficial in prolong‐ ing and improving quality of life. Without treatment, laryngeal cancer patients die within an average of 12 months. The patient lived for nearly one year and eight months until his death due to pneumonia.

The observations that the patient regained his voice, appetite, and weight after a short term treatment with AS, and the fact that the tumor was significantly reduced in size without any apparent adverse side effects suggests that AS treatment could be an effective and economi‐ cal alternative treatment for cancer, especially in cases of late cancer detection where availa‐ ble treatments are limited. Since this case report was published, several patients with different types of cancers have begun treatment with ART and its analogs with promising re‐ sults. AS therapy has potential to prevent and treat a wide range of cancers given its efficacy, low cost, and due to the common mechanisms of action demonstrated against various cancer cells (Singh and Verma, 2002).

**3.** Pituitary macroadenoma treated with AM

AM, an ART analogue, was used to treat a 75-year old male patient with pituitary macroa‐ denoma. This patient presented with vision, hearing, and locomotion-related problems as a consequence of his disease. AM was administered orally to the patient over a period of 12 months. Although the tumor remained consistent in size, CT scans showed a reduction in tumor density, and clinically, the related symptoms and signs improved significantly as therapy progressed. Overall, the AM treatment was beneficial in improving the patient's quality of life. AM and other ART analogs appear to have promise for treatment of this type of cancer (Singh and Panwar, 2006).

#### **3.4. Why are not there more trials or more wide-spread use of ARTs?**

ARTs are largely non-toxic, with related compounds having been administered to over 2 million patients; both children and adult, world-wide without reports of significant serious side effects, and ARTs are very inexpensive when compared to conventional cancer drugs. The final results of current clinical trials utilizing ARTs as therapy or adjunct against a wide variety of cancers have not yet been published although initial findings released suggest positive results. How positive or efficacious these results are remains to be seen until full and final results are published. One question, however, remains - why are there not more trials or more wide-spread use of ARTs as an off-label cancer treatment?

#### *3.4.1. PK mismatch of ARTs in cancer therapy*

**1.** Metastatic uveal melanomas treated with AS

tient died after 24 months.

208 Research Directions in Tumor Angiogenesis

due to pneumonia.

cells (Singh and Verma, 2002).

**3.** Pituitary macroadenoma treated with AM

**2.** Laryngeal carcinoma treated with AS

Berger et al. reported on the first long-term treatment of two cancer patients with AS in com‐ bination with standard chemotherapy. These patients with metastatic uveal melanoma were treated on a compassionate-use basis, after standard chemotherapy alone was ineffective in stopping tumor growth. The therapy regimen was well tolerated with no additional side ef‐ fects other than those caused by standard chemotherapy alone. One patient experienced a temporary response after the addition of AS to Fotemustine while the disease was progress‐ ing under therapy with Fotemustine alone. The second patient first experienced a stabiliza‐ tion of the disease after the addition of AS to Dacarbazine, followed by objective regressions of splenic and lung metastases. This patient is still alive 47 months after first diagnosis with stage IV uveal melanoma, a diagnosis with a median survival of 2-5 months, without addi‐ tional side effects. One patient experienced a temporary response after the addition of AS while the disease was progressing under standard therapy with Fotemustine alone. This pa‐

Despite the small number of treated patients, AS may be a promising adjuvant drug for the treatment of melanoma and possibly other tumors in combination with standard chemothera‐ py. AS is well tolerated, and the lack of serious side effects will facilitate prospective random‐ ized trials in the near future. From *in vitro* studies already conducted (Efferth et al., 2004b), it is further conceivable that loading tumor cells with bivalent iron, by simply providing Fe2+ in tab‐ let form, might increase the susceptibility of cancer cells to AS treatment. It is tempting to spec‐ ulate that, in the case of the second patient previously discussed, the addition of Fe2+ had an actual clinical impact and resulted in an improved response to therapy (Berger et al., 2005).

AS injections and tablets were used in one study to treat a laryngeal squamous cell carcino‐ ma patient over a period of nine months. The tumor was significantly reduced in size by 70% after two months of treatment. Overall, AS treatment of the patient was beneficial in prolong‐ ing and improving quality of life. Without treatment, laryngeal cancer patients die within an average of 12 months. The patient lived for nearly one year and eight months until his death

The observations that the patient regained his voice, appetite, and weight after a short term treatment with AS, and the fact that the tumor was significantly reduced in size without any apparent adverse side effects suggests that AS treatment could be an effective and economi‐ cal alternative treatment for cancer, especially in cases of late cancer detection where availa‐ ble treatments are limited. Since this case report was published, several patients with different types of cancers have begun treatment with ART and its analogs with promising re‐ sults. AS therapy has potential to prevent and treat a wide range of cancers given its efficacy, low cost, and due to the common mechanisms of action demonstrated against various cancer

AM, an ART analogue, was used to treat a 75-year old male patient with pituitary macroa‐ denoma. This patient presented with vision, hearing, and locomotion-related problems as a ARTs as angiogenesis inhibitors are unique cancer-fighting agents because they tend to in‐ hibit the growth of blood vessels rather than tumor cells. Therefore, the angiogenesis inhibi‐ tor therapy does not necessarily kill tumors but instead may prevent tumors from growing. This type of therapy may therefore need to be administered over a long period.

Following oral administration, however, AS and DHA have short mean residence times (MRT) of 1.95 and 2.71 hr, respectively, and ART has a longer MRT of 7.4 hr. Intramuscular AM and arteether have longer MRTs ranging from 13.9 - 42.9 hrs in humans s due to pro‐ longed absorption and accumulation at the injection sites. The shortest MRT (0.90 hr) was found in humans following intravenous injection of AS (Table 2). It is obvious that the dif‐ ferent ARTs administered in different regimens have significant differences in pharmacoki‐ netic (PK) characteristics in humans, and only intramuscular ARTs can provide a long period of therapy. Therefore, injectable AM has been recommended as a longer acting com‐ pound that may be suitable for cancer treatment (White, 2002).

Pharmacokinetics (PK) studies of ARTs show three phases (absorption, distribution, and elimination) of ART drugs in blood following oral, intravenous, or intramuscular adminis‐ tration. After multiple daily administrations, four ARTs showed declining daily drug con‐ centrations (ART, DHA, AS, and AM) which has been shown is believed to be due to an auto-induced metabolism pathway during multiple oral treatments in patients and health subjects (van Agtmael et al., 1999; Ashton et al., 1996; 1998; Khanh et al., 1999; Park et al., 1998). The Cmax and AUC values of ARTs are markedly reduced from one-third to one-sev‐ enth on the last dose day compared with the first day. The decrease in drug exposure levels during treatment is not disease-related, since the PK profile of ART drugs on the last day shows a similar decrease to that reported in healthy subjects. Similar time-dependent de‐


that many patients reach after treatment with these compounds. After an initial quick re‐

**Artemisinins Cancer targets Clinical studies Protocols & References** Artesunate Colorectal cancer Clinical trial, Phase I ISRCTN05203252, 2011 UK

> Case report Case report

Artemether Pituitary macroadenoma Case report Singh& Panwar 2006 INDIA

**Table 3.** Anti-cancer effects of artemisinin (ART), artesunate (AS), and artemether (AM) in case reports of treatments

ARTs are anti-angiogenic agents with a variety of targets that inhibit tumor angiogenesis in two ways: 1) blockade of angiogenic pathways and 2) inhibition of endogenous angiogene‐ sis (Efferth, 2006; 2007; Crespo-Ortiz and Wei, 2012). Cancers produce a variety of angiogen‐ ic factors or cytokines to stimulate angiogenesis, which is essential for tumor growth and metastasis (Cao and Liu, 2007). These cancer-derived angiogenic factors include VEGF, fi‐ broblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGFs), angiopoietins (Angs), hepatocyte growth factor (HGF), and insulin-like growth factors (IGFs). The angio‐ genic signals triggered by these angiogenic factors are mediated by their specific tyrosine kinase receptors (TKRs) expressed in endothelial cells (Nissen *et al*., 2007; Xue *et al*., 2008).

ARTs responses seem to be mediated by those angiogenic factors with strong multi-tar‐ geted anti-angiogenic potency. However, ART targets cancer cells is cleavage of the en‐ doperoxide bridge by the relatively high concentrations of iron in cancer cells, resulting in generation of free radicals such as reactive oxygen species (ROS) and subsequent oxi‐ dative damage as well as iron depletion in the cells. Studies demonstrated that co-admin‐ istration of holotransferrin and other iron sources with ARTs have been shown to increase the potency of ARTs in killing cancer cells (Lai et al., 2009; Mercer et al., 2011; Zhang et al., 2010). Also, DHA in combination with butyric acid acts synergistically at

Current combinations with chemotherapy for the treatment of patients with cancer have produced only modest beneficial effects (Cao *et al*., 2009). Optimization of anti-angiogenic therapy is urgently needed in order to maximize therapeutic efficacy of these drugs. Obvi‐ ously, defining novel therapeutic targets other than VEGF would be an important approach

Clinical trial Phase I-II

Zhang et al., 2008 CHINA Berger et al., 2005 GERMANY Singh & Verma, 2002 INDIA NCT00764036 2008

http://dx.doi.org/10.5772/54109

211

GERMANY Verified Feb. 2009

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

Clinical Trial, Phase I-II

sponse, many patients seem to stabilize without a complete remission (White, 2002).

Non-small cell lung cancer Metastatic uveal melanoma Laryngeal carcinoma Metastatic breast cancer

*3.4.2. Possible optimization of clinical trials with ARTs*

All clinical trials listed here are completed

and clinical trials (Ghantous et al., 2010)

low dose (Singh and Lai, 2005).

Artesunate

\*The data was fitted with WinNonlin (V5.0) by author. \*\*Oral 750 mg mefloquine at 24 hr after IV injection. PK = phar‐ macokinetics; PD = pharmacodynamics; MRT = Mean residence time; PC50 = Mean time for parasitemia to fall by half; AUIC = area under inhibitory curve; QHS = Artemisinin; DHA = Dihydroartemisinin; AM = Artemether; AE = Arteether; AS = Artesunic acid; MPC = minimum parasiticidal concentration; IM = intramuscular.

**Table 2.** Pharmacokinetics (PK) parameters of intravenous artesunate (AS), oral AS, oral dihydroartemisinin (DHA), oral artemisinin (ART), intramuscular artemether (AM) and arteether (AE) in human treatments with uncomplicated and severe/complicated malaria on day 1\*.

clines have been reported in animals dosed with oral AM (Classen et al., 1999). One possible explanation for the decrease in plasma concentration-time during treatment is an increase in metabolic capacity due to auto-induction of hepatic drug-metabolizing enzymes.

Similar observations have shown that decreasing absorption of ART derivatives may be a problem for the longer-term use required for treatment of cancer. In treating malaria, the ART derivatives are given for a short four or five day course. In these short treatments, no absorption resistance has been observed to occur. Recent information has come to light that indicates that the intestine builds up resistance to absorbing oral ART compounds very quickly, within several days. Resistance is demonstrated by a >30% drop of the original rate of absorption. Research indicates that this resistance can be overcome very quickly by dis‐ continuing use of the ART compounds for several days to a week; when resumed, their ab‐ sorption will be at the previous higher level. One study author, Dr. Lai, pointed out that this intestinal resistance and subsequent lowered absorption rate may be the basis of the plateau that many patients reach after treatment with these compounds. After an initial quick re‐ sponse, many patients seem to stabilize without a complete remission (White, 2002).


**Table 3.** Anti-cancer effects of artemisinin (ART), artesunate (AS), and artemether (AM) in case reports of treatments and clinical trials (Ghantous et al., 2010)

#### *3.4.2. Possible optimization of clinical trials with ARTs*

clines have been reported in animals dosed with oral AM (Classen et al., 1999). One possible explanation for the decrease in plasma concentration-time during treatment is an increase in

**Table 2.** Pharmacokinetics (PK) parameters of intravenous artesunate (AS), oral AS, oral dihydroartemisinin (DHA), oral artemisinin (ART), intramuscular artemether (AM) and arteether (AE) in human treatments with uncomplicated and

\*The data was fitted with WinNonlin (V5.0) by author. \*\*Oral 750 mg mefloquine at 24 hr after IV injection. PK = phar‐ macokinetics; PD = pharmacodynamics; MRT = Mean residence time; PC50 = Mean time for parasitemia to fall by half; AUIC = area under inhibitory curve; QHS = Artemisinin; DHA = Dihydroartemisinin; AM = Artemether; AE = Arteether;

Similar observations have shown that decreasing absorption of ART derivatives may be a problem for the longer-term use required for treatment of cancer. In treating malaria, the ART derivatives are given for a short four or five day course. In these short treatments, no absorption resistance has been observed to occur. Recent information has come to light that indicates that the intestine builds up resistance to absorbing oral ART compounds very quickly, within several days. Resistance is demonstrated by a >30% drop of the original rate of absorption. Research indicates that this resistance can be overcome very quickly by dis‐ continuing use of the ART compounds for several days to a week; when resumed, their ab‐ sorption will be at the previous higher level. One study author, Dr. Lai, pointed out that this intestinal resistance and subsequent lowered absorption rate may be the basis of the plateau

metabolic capacity due to auto-induction of hepatic drug-metabolizing enzymes.

**PK Parameters AS AS DHA ART AM AE**

1052 (DHA); 198

Tmax (hr) 0.13 0.75 1.4 2.4 6.0 8.2

1334 (DHA); 210

t1/2 (absorption, hr) 0.36 (DHA) 0.67 1.21 1.88 3.2 t1/2 (elimination, hr) 0.67 (DHA); 0.05 (AS)0.70 (DHA) 0.85 2.3 7.83 22.7 MRT (hr) 0.90 (DHA) 1.95 (DHA) 2.71 7.41 13.94 42.9

(AS)

(AS)

Tlag (hr) 0.2 0.45

AS = Artesunic acid; MPC = minimum parasiticidal concentration; IM = intramuscular.

First loading dosage 120 mg 100 mg 200 mg 500 mg 3.2 mg/kg 4.8 mg/kg

Intravenous Oral Oral Oral Intramuscula

r

50 mg b.i.d. x 4 100 mg x 4 250 x 2 x 5 1.6mg/kg x 41.6mg/kg

500 mg 600 mg 3000 mg 9.6 mg/kg 12.8 mg/kg

437.5 588.0 74.9 110.1

1329 2601 1230 4702

Intramuscular

x 5

Route of administration

Maintaining dosage Oral 100 mg at

210 Research Directions in Tumor Angiogenesis

Total dose 220 mg and

Cmax (ng/ml) 2646 (DHA);

AUC0-24 hr (ng·h/ml) 2378 (DHA); 1146 (AS)

severe/complicated malaria on day 1\*.

8 hr

mefloquine\*\*

11343(AS)

ARTs are anti-angiogenic agents with a variety of targets that inhibit tumor angiogenesis in two ways: 1) blockade of angiogenic pathways and 2) inhibition of endogenous angiogene‐ sis (Efferth, 2006; 2007; Crespo-Ortiz and Wei, 2012). Cancers produce a variety of angiogen‐ ic factors or cytokines to stimulate angiogenesis, which is essential for tumor growth and metastasis (Cao and Liu, 2007). These cancer-derived angiogenic factors include VEGF, fi‐ broblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGFs), angiopoietins (Angs), hepatocyte growth factor (HGF), and insulin-like growth factors (IGFs). The angio‐ genic signals triggered by these angiogenic factors are mediated by their specific tyrosine kinase receptors (TKRs) expressed in endothelial cells (Nissen *et al*., 2007; Xue *et al*., 2008).

ARTs responses seem to be mediated by those angiogenic factors with strong multi-tar‐ geted anti-angiogenic potency. However, ART targets cancer cells is cleavage of the en‐ doperoxide bridge by the relatively high concentrations of iron in cancer cells, resulting in generation of free radicals such as reactive oxygen species (ROS) and subsequent oxi‐ dative damage as well as iron depletion in the cells. Studies demonstrated that co-admin‐ istration of holotransferrin and other iron sources with ARTs have been shown to increase the potency of ARTs in killing cancer cells (Lai et al., 2009; Mercer et al., 2011; Zhang et al., 2010). Also, DHA in combination with butyric acid acts synergistically at low dose (Singh and Lai, 2005).

Current combinations with chemotherapy for the treatment of patients with cancer have produced only modest beneficial effects (Cao *et al*., 2009). Optimization of anti-angiogenic therapy is urgently needed in order to maximize therapeutic efficacy of these drugs. Obvi‐ ously, defining novel therapeutic targets other than VEGF would be an important approach to increase clinical responses as a majority of cancer patients have been shown to demon‐ strate intrinsic resistance to anti-VEGF therapy. Given the fact that most tumors produce a broad spectrum of angiogenic factors to stimulate angiogenesis and to sustain the establish‐ ed vasculature, it is not surprising that blockade of a single angiogenic pathway would be insufficient to suppress tumor growth and multitargeted "dirty drugs" would be more effec‐ tive. In support of this view, anti-angiogenic monotherapy with tyrosine kinase inhibitors such as sunitinib and sorafenib targeting multiple signaling pathways has been shown to re‐ sult in increased survival in patients treated for metastatic renal cell carcinoma (Escudier *et al*., 2007; Motzer *et al*., 2006)

mors may create completely different environments, leading to dissimilar angiogenic pro‐ files and drug responses. Young human or animal subjects are susceptible to angiogenic stimuli by triggering relatively robust angiogenic responses under physiological and patho‐ logical settings. In contrast, older human or animal subjects often show delayed or impaired

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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213

In animal tumor models, the endpoint of any drug study is the effect of the drug on tu‐ mor size, whereas in human patients, survival improvements by ARTs are often the clin‐ ical endpoints measure. Therapeutic efficacy of anti-angiogenic agents is often assessed as monotherapy in animals whereas the same agents are delivered to cancer patients as combinatorial therapy with chemotoxic drugs. In animal tumor models, delivery of che‐ motherapeutic drugs alone at the conventional dose levels often produces overwhelming anti-tumor effects and addition of anti-angiogenic agents as an extra component would be difficult to enhance the chemotherapeutic effect. Thus, the anti-angiogenic monothera‐ py with most available drugs has not demonstrated clinical benefits in cancer patients

Unlike humans, inbred experimental homogenous mice represent the same genetic back‐ ground and tumors are artificially manipulated to grow at the same or at least a similar pace. Unsurprisingly, these genetically identical animals would produce a similar response to the same drug. Indeed, anti-angiogenic monotherapy in mice regardless of whether the tumor implanted is a xenograft or derived from a genetically prone mouse tumor model shows the predicted power of drug tumor suppression. Thus, this type of animal model would not be appropriate for assessment of the therapeutic efficacy of ARTs in human can‐ cer patients. Therefore, the difference in anti-angiogenic profiles between human and mouse cancers in relation to the therapeutic efficacy of drug treatment may well explain the varia‐

There have been a variety of reasons to believe that anti-angiogenic drugs for clinical use as a cancer treatment may have a number of side effects. First, the generation of new blood ves‐ sels is a very complicated, multi-step biological process, and VEGF plays an important role in a variety of biological processes such as hematopoiesis, myelopoiesis and endothelial cell survival. Therefore, anti-angiogenic therapy could cause several toxicities due to these pleio‐ tropic biologic effects. Furthermore, many of the angiogenic inhibitors tested target multiple tyrosine kinases in several different pathways, and thus toxicities may not only arise from the inhibition of one pathway but also possibly from the concomitant inhibition of several pathways. Moreover, many of these biological agents are used or will be used in combina‐ tion with other cytotoxic agents as a treatment strategy. It is not surprising that there is more toxicity in some studies using combination therapies involving angiogenesis inhibitors than those using single agents. The toxicities associated with administration of angiogenesis in‐ hibitors have been shown to include bleeding, disturbed wound healing, thrombosis, hyper‐ tension, hypothyroidism and fatigue, proteinuria and edema, skin toxicity, leukopenia,

angiogenic responses under the same conditions.

(Cao, 2009; 2011; Hurwitz et al., 2004).

tion in human cancer patient responses to ARTs therapy.

lymphopenia, and immunomodulation (Wu et al., 2008).

*3.4.4. Potential toxicities of ARTs in the cancer therapy*

ARTs are delivered to cancer patients by systemic administration, which may lead to a uni‐ versal impact on healthy vasculatures distributed in multiple tissues and organs (Cao, 2010). In the conventional view of anti-cancer drugs, off-tumor targets would be associated with unwanted adverse effects of drugs. Interestingly, clinical benefits of ARTs have been posi‐ tively associated with neurotoxicity and embryotoxicity, which have been shown to result from the systemic effects of these drugs (Li et al., 2009). In preclinical tumor models, it has been demonstrated that anti-angiogenic agents administered at a low dose normalize vascu‐ latures in healthy tissues including those fenestrated vasculatures in endocrine organs such as bone marrow, liver and adrenal gland without affecting the tumor vasculature (Xue *et al*., 2008). Normalization of tumor VEGF-induced vascular tortuosity in non-tumor tissues has been shown to significantly prolong the survival of tumor-bearing mice by improving the cancer associated systemic syndrome. These findings suggest that the off-tumor targets of anti-angiogenic agents such as ARTs may provide clinical benefits to cancer patients. Un‐ fortunately, clinical trials based on improvement of paraneoplastic syndrome and cancer ca‐ chexia by ARTs have neither been designed nor reported (Cao, 2011).

#### *3.4.3. Animal model results differ from human cancer patients*

Preclinical models for assessment of anti-angiogenic and antitumor activities are xenograft tumor models in mice that carry implanted mouse or human tumors. Although this is a commonly used animal tumor model for studying anti-angiogenic and antitumor effects of different molecules, the relevance of this xenograft model to the clinical setting is far from reality. The subcutaneous implantation site does not usually represent physiologically or‐ thotropic sites where human tumors arise. The tissue site is probably one of the most impor‐ tant issues related to response of tumors to drugs because angiogenic vessels in various tissues may express different receptors that are activated by specific ligands. Selective ex‐ pression of different subsets of the same ligand receptors exists in different tissues. Differen‐ tial expression of angiogenic factor receptors in various tissues and organs may lead to distinctive ARTs specific responses.

It is known that angiogenesis occurs at different rates in various aged populations (Rivard *et al*., 1999). Thus, a difference between human cancers and mouse tumor models is the speed of cancer development. In human patients, spontaneous development of a clinical detectable cancer may take years whereas development of a similar sized mouse tumor may only take weeks (O'Reilly *et al*., 1994). The differential growth rates between human and mouse tu‐ mors may create completely different environments, leading to dissimilar angiogenic pro‐ files and drug responses. Young human or animal subjects are susceptible to angiogenic stimuli by triggering relatively robust angiogenic responses under physiological and patho‐ logical settings. In contrast, older human or animal subjects often show delayed or impaired angiogenic responses under the same conditions.

In animal tumor models, the endpoint of any drug study is the effect of the drug on tu‐ mor size, whereas in human patients, survival improvements by ARTs are often the clin‐ ical endpoints measure. Therapeutic efficacy of anti-angiogenic agents is often assessed as monotherapy in animals whereas the same agents are delivered to cancer patients as combinatorial therapy with chemotoxic drugs. In animal tumor models, delivery of che‐ motherapeutic drugs alone at the conventional dose levels often produces overwhelming anti-tumor effects and addition of anti-angiogenic agents as an extra component would be difficult to enhance the chemotherapeutic effect. Thus, the anti-angiogenic monothera‐ py with most available drugs has not demonstrated clinical benefits in cancer patients (Cao, 2009; 2011; Hurwitz et al., 2004).

Unlike humans, inbred experimental homogenous mice represent the same genetic back‐ ground and tumors are artificially manipulated to grow at the same or at least a similar pace. Unsurprisingly, these genetically identical animals would produce a similar response to the same drug. Indeed, anti-angiogenic monotherapy in mice regardless of whether the tumor implanted is a xenograft or derived from a genetically prone mouse tumor model shows the predicted power of drug tumor suppression. Thus, this type of animal model would not be appropriate for assessment of the therapeutic efficacy of ARTs in human can‐ cer patients. Therefore, the difference in anti-angiogenic profiles between human and mouse cancers in relation to the therapeutic efficacy of drug treatment may well explain the varia‐ tion in human cancer patient responses to ARTs therapy.

#### *3.4.4. Potential toxicities of ARTs in the cancer therapy*

to increase clinical responses as a majority of cancer patients have been shown to demon‐ strate intrinsic resistance to anti-VEGF therapy. Given the fact that most tumors produce a broad spectrum of angiogenic factors to stimulate angiogenesis and to sustain the establish‐ ed vasculature, it is not surprising that blockade of a single angiogenic pathway would be insufficient to suppress tumor growth and multitargeted "dirty drugs" would be more effec‐ tive. In support of this view, anti-angiogenic monotherapy with tyrosine kinase inhibitors such as sunitinib and sorafenib targeting multiple signaling pathways has been shown to re‐ sult in increased survival in patients treated for metastatic renal cell carcinoma (Escudier *et*

ARTs are delivered to cancer patients by systemic administration, which may lead to a uni‐ versal impact on healthy vasculatures distributed in multiple tissues and organs (Cao, 2010). In the conventional view of anti-cancer drugs, off-tumor targets would be associated with unwanted adverse effects of drugs. Interestingly, clinical benefits of ARTs have been posi‐ tively associated with neurotoxicity and embryotoxicity, which have been shown to result from the systemic effects of these drugs (Li et al., 2009). In preclinical tumor models, it has been demonstrated that anti-angiogenic agents administered at a low dose normalize vascu‐ latures in healthy tissues including those fenestrated vasculatures in endocrine organs such as bone marrow, liver and adrenal gland without affecting the tumor vasculature (Xue *et al*., 2008). Normalization of tumor VEGF-induced vascular tortuosity in non-tumor tissues has been shown to significantly prolong the survival of tumor-bearing mice by improving the cancer associated systemic syndrome. These findings suggest that the off-tumor targets of anti-angiogenic agents such as ARTs may provide clinical benefits to cancer patients. Un‐ fortunately, clinical trials based on improvement of paraneoplastic syndrome and cancer ca‐

Preclinical models for assessment of anti-angiogenic and antitumor activities are xenograft tumor models in mice that carry implanted mouse or human tumors. Although this is a commonly used animal tumor model for studying anti-angiogenic and antitumor effects of different molecules, the relevance of this xenograft model to the clinical setting is far from reality. The subcutaneous implantation site does not usually represent physiologically or‐ thotropic sites where human tumors arise. The tissue site is probably one of the most impor‐ tant issues related to response of tumors to drugs because angiogenic vessels in various tissues may express different receptors that are activated by specific ligands. Selective ex‐ pression of different subsets of the same ligand receptors exists in different tissues. Differen‐ tial expression of angiogenic factor receptors in various tissues and organs may lead to

It is known that angiogenesis occurs at different rates in various aged populations (Rivard *et al*., 1999). Thus, a difference between human cancers and mouse tumor models is the speed of cancer development. In human patients, spontaneous development of a clinical detectable cancer may take years whereas development of a similar sized mouse tumor may only take weeks (O'Reilly *et al*., 1994). The differential growth rates between human and mouse tu‐

chexia by ARTs have neither been designed nor reported (Cao, 2011).

*3.4.3. Animal model results differ from human cancer patients*

distinctive ARTs specific responses.

*al*., 2007; Motzer *et al*., 2006)

212 Research Directions in Tumor Angiogenesis

There have been a variety of reasons to believe that anti-angiogenic drugs for clinical use as a cancer treatment may have a number of side effects. First, the generation of new blood ves‐ sels is a very complicated, multi-step biological process, and VEGF plays an important role in a variety of biological processes such as hematopoiesis, myelopoiesis and endothelial cell survival. Therefore, anti-angiogenic therapy could cause several toxicities due to these pleio‐ tropic biologic effects. Furthermore, many of the angiogenic inhibitors tested target multiple tyrosine kinases in several different pathways, and thus toxicities may not only arise from the inhibition of one pathway but also possibly from the concomitant inhibition of several pathways. Moreover, many of these biological agents are used or will be used in combina‐ tion with other cytotoxic agents as a treatment strategy. It is not surprising that there is more toxicity in some studies using combination therapies involving angiogenesis inhibitors than those using single agents. The toxicities associated with administration of angiogenesis in‐ hibitors have been shown to include bleeding, disturbed wound healing, thrombosis, hyper‐ tension, hypothyroidism and fatigue, proteinuria and edema, skin toxicity, leukopenia, lymphopenia, and immunomodulation (Wu et al., 2008).

In addition, there are published studies showing potential toxicities associated with the use of ARTs as anti-angiogenic agents. Various animal studies have documented neurotoxicity and embryotoxicity associated with ARTs administration, which has raised the question of whether those toxicities might occur in humans, particularly, in anti-cancer therapy and pre‐ vention of metastasis.

na, 2010b). The mechanisms and the pharmacokinetic profiles that affect reproductive toxicity in animal species are currently understood. These animal studies have shown that only injectable AS (intramuscular, intravenous, or subcutaneous) induces reprotoxici‐ ty at a lower dose (0.6-1.0 mg/kg) than the therapeutic dose (2-4 mg/kg) in humans. Oth‐ er doses in different regimens (oral artemisinins or intramuscular AM) are safe at higher levels (6.1-51.0 mg/kg) than the therapeutic doses used. Orally dosing, the most common‐ ly used route of administration in pregnant women with Artemisinin-based combination therapies (ACTs), has been shown to result in lower peak drug concentrations and short‐

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215

Toxicokinetic and tissue distribution data has shown that the severe embryotoxicity in‐ duced by injectable AS is associated with six risk factors: 1) Injectable AS can provide much higher peak concentrations (3–25 fold) than oral ARTs or intramuscular AM when administered to animals. *In vitro* results have shown that the drug exposure level and time are important factors required for induction of embryotoxicity (Longo et al., 2006a; 2006b; 2008). *In vivo* studies have shown that the drug exposure level, however, is more important than the drug exposure time as AS and DHA both have been shown to have very short half–lives (< 1 h) when administered to various animal species. 2) AS is com‐ pletely converted to DHA, and therefore, AS serves as a prodrug of DHA. DHA has been shown to be more effective than AS in inhibition of angiogenesis and vasculogene‐ sis *in vitro* (Chen et al., 2004a; White et al., 2006). 3) Among the ARTs, AS has been shown to have the highest conversion rate to DHA. The conversion rate of AS to DHA was shown to range from 38.2–72.7% while of the conversion rate of AM and AE to DHA ranges from 12.4–14.2%. 4) The conversion rate of AS to DHA was significantly in‐ creased in pregnant animals than in non–pregnant rats following multiple injections. 5) The buildup of high peak concentrations of AS and DHA in the plasma of pregnant rats was significantly higher than those of non–pregnant animals after repeated dosing. 6) In‐ jectable AS administration results in a higher distribution of AS and DHA in the tissues of feto–placental units in pregnant animals after multiple administrations (Li et al., 2008).

It is not clear how these findings from animals translate to human patients treated for ma‐ laria in with a 3-5 day treatment regimen (WHO, 2006b; Wang, 1989). Data from limited clinical trials in pregnant women (1837 cases) exposed to ART compounds and ACTs, in‐ cluding a small number (176 cases) in the first trimester, have not shown an increase in the rates of abortion or stillbirth; they have also not shown evidence of abnormalities. Since more than 99% of pregnant patients have been treated with oral ARTs or intramuscular AM in the previously referenced trials, the lack of sensitivity and enhanced repair capabilities of humans to respond to ARTs induced embryotoxicity may explain the lack of embryotoxici‐

The possible embryotoxicity associated with ARTs therapy should be avoided by limiting ex‐ posure of pregnant women in the first trimester which is the critical period for induction of em‐ bryo damage and resorption. In addition, to protect pregnant women from embryotoxicity associated with ARTs treatment, injectable AS should be used very cautiously. There is agree‐ ment that ART derivatives should not be withheld at any stage of pregnancy, in cases of severe

er exposure times, which is less likely to induce embryotoxicity.

ty observed.

#### **Neurotoxicity of ARTs**

Studies with laboratory animals have demonstrated neurotoxicity associated with a number of adverse effects including movement disturbances, spasticity, balance deficits, brainstem tissue damage, and even death following administration of some intramus‐ cular doses of oil-soluble AM and arteether, or intragastric water-soluble artelinate. There are significant differences in neurotoxicity observed between rats, dogs and rhe‐ sus monkeys after treatment with different ARTs suggesting that the exposure time re‐ quired to induce neurotoxicity after dosing with ARTs is likely to be longer in humans. Since toxicity is dependent on chemical/drug exposure levels and time (Ran‐ gan et al., 1997; Rozman and Doull 2000), the neurotoxicity of ARTs has been demon‐ strated to occur through continued drug exposure over a longer period of time rather than through an elevated drug exposure level over a shorter period of time (Jorgensen 1980; Li et al., 2002 and 2006; Rozman 1998). Accordingly, the 3-5 days dosing dura‐ tion currently used in ART antimalarial therapy should be quite safe. Neurotoxicity may be caused in humans, however, with inappropriate dose regimens, and therefore, sustained drug exposure times appear to be the critical factor to assess and prevent neurotoxicity (Li and Hickman, 2011).

The current clinical dose regimens of three-day ART combined therapies (ACTs) for un‐ complicated cases of malaria, and the dose regimens recommended for intravenous AS treatments for severe malaria which include a few days of a loading doses may be too short of a drug exposure time to induce neurotoxicity in humans. Also, with regard to acute toxicity, humans appear to be less sensitive than animals (Geyer et al., 1990; Kim‐ brough 1990), and humans appear to have much better repair capabilities than animals to respond to such toxicity (Culotta and Koshland 1994). TK/TD analysis of neurotoxicity after ART treatment has provided a wealth of data to provide a means of predicting the neurotoxic exposure time of ARTs in humans (Li and Hickman, 2011). Based on this da‐ ta, we predict the safe dosing duration of ARTs in the neurotoxic exposure time should be longer than 7 days (168 hr). Advances in our knowledge of ART-induced neurotoxici‐ ty can help refine the treatment regimens used to treat malaria with ACTs as well as in‐ jectable AS products to avoid the risk of neurotoxicity. If the drug exposure time of ARTs administered for anti-cancer therapy occurs over 14 days or even longer anti-can‐ cer, ARTs-induced neurotoxicity may well occur (Li and Hickman, 2011).

#### **Embryotoxicity of ARTs**

In animal work, there is clear evidence of ARTs-induced embryo death and some evi‐ dence of morphological abnormalities in mice, rats, hamsters, guinea pig, rabbits and monkeys in early pregnancy especially after administration of injectable AS (Li and Wei‐ na, 2010b). The mechanisms and the pharmacokinetic profiles that affect reproductive toxicity in animal species are currently understood. These animal studies have shown that only injectable AS (intramuscular, intravenous, or subcutaneous) induces reprotoxici‐ ty at a lower dose (0.6-1.0 mg/kg) than the therapeutic dose (2-4 mg/kg) in humans. Oth‐ er doses in different regimens (oral artemisinins or intramuscular AM) are safe at higher levels (6.1-51.0 mg/kg) than the therapeutic doses used. Orally dosing, the most common‐ ly used route of administration in pregnant women with Artemisinin-based combination therapies (ACTs), has been shown to result in lower peak drug concentrations and short‐ er exposure times, which is less likely to induce embryotoxicity.

In addition, there are published studies showing potential toxicities associated with the use of ARTs as anti-angiogenic agents. Various animal studies have documented neurotoxicity and embryotoxicity associated with ARTs administration, which has raised the question of whether those toxicities might occur in humans, particularly, in anti-cancer therapy and pre‐

Studies with laboratory animals have demonstrated neurotoxicity associated with a number of adverse effects including movement disturbances, spasticity, balance deficits, brainstem tissue damage, and even death following administration of some intramus‐ cular doses of oil-soluble AM and arteether, or intragastric water-soluble artelinate. There are significant differences in neurotoxicity observed between rats, dogs and rhe‐ sus monkeys after treatment with different ARTs suggesting that the exposure time re‐ quired to induce neurotoxicity after dosing with ARTs is likely to be longer in humans. Since toxicity is dependent on chemical/drug exposure levels and time (Ran‐ gan et al., 1997; Rozman and Doull 2000), the neurotoxicity of ARTs has been demon‐ strated to occur through continued drug exposure over a longer period of time rather than through an elevated drug exposure level over a shorter period of time (Jorgensen 1980; Li et al., 2002 and 2006; Rozman 1998). Accordingly, the 3-5 days dosing dura‐ tion currently used in ART antimalarial therapy should be quite safe. Neurotoxicity may be caused in humans, however, with inappropriate dose regimens, and therefore, sustained drug exposure times appear to be the critical factor to assess and prevent

The current clinical dose regimens of three-day ART combined therapies (ACTs) for un‐ complicated cases of malaria, and the dose regimens recommended for intravenous AS treatments for severe malaria which include a few days of a loading doses may be too short of a drug exposure time to induce neurotoxicity in humans. Also, with regard to acute toxicity, humans appear to be less sensitive than animals (Geyer et al., 1990; Kim‐ brough 1990), and humans appear to have much better repair capabilities than animals to respond to such toxicity (Culotta and Koshland 1994). TK/TD analysis of neurotoxicity after ART treatment has provided a wealth of data to provide a means of predicting the neurotoxic exposure time of ARTs in humans (Li and Hickman, 2011). Based on this da‐ ta, we predict the safe dosing duration of ARTs in the neurotoxic exposure time should be longer than 7 days (168 hr). Advances in our knowledge of ART-induced neurotoxici‐ ty can help refine the treatment regimens used to treat malaria with ACTs as well as in‐ jectable AS products to avoid the risk of neurotoxicity. If the drug exposure time of ARTs administered for anti-cancer therapy occurs over 14 days or even longer anti-can‐

cer, ARTs-induced neurotoxicity may well occur (Li and Hickman, 2011).

In animal work, there is clear evidence of ARTs-induced embryo death and some evi‐ dence of morphological abnormalities in mice, rats, hamsters, guinea pig, rabbits and monkeys in early pregnancy especially after administration of injectable AS (Li and Wei‐

vention of metastasis.

214 Research Directions in Tumor Angiogenesis

**Neurotoxicity of ARTs**

neurotoxicity (Li and Hickman, 2011).

**Embryotoxicity of ARTs**

Toxicokinetic and tissue distribution data has shown that the severe embryotoxicity in‐ duced by injectable AS is associated with six risk factors: 1) Injectable AS can provide much higher peak concentrations (3–25 fold) than oral ARTs or intramuscular AM when administered to animals. *In vitro* results have shown that the drug exposure level and time are important factors required for induction of embryotoxicity (Longo et al., 2006a; 2006b; 2008). *In vivo* studies have shown that the drug exposure level, however, is more important than the drug exposure time as AS and DHA both have been shown to have very short half–lives (< 1 h) when administered to various animal species. 2) AS is com‐ pletely converted to DHA, and therefore, AS serves as a prodrug of DHA. DHA has been shown to be more effective than AS in inhibition of angiogenesis and vasculogene‐ sis *in vitro* (Chen et al., 2004a; White et al., 2006). 3) Among the ARTs, AS has been shown to have the highest conversion rate to DHA. The conversion rate of AS to DHA was shown to range from 38.2–72.7% while of the conversion rate of AM and AE to DHA ranges from 12.4–14.2%. 4) The conversion rate of AS to DHA was significantly in‐ creased in pregnant animals than in non–pregnant rats following multiple injections. 5) The buildup of high peak concentrations of AS and DHA in the plasma of pregnant rats was significantly higher than those of non–pregnant animals after repeated dosing. 6) In‐ jectable AS administration results in a higher distribution of AS and DHA in the tissues of feto–placental units in pregnant animals after multiple administrations (Li et al., 2008).

It is not clear how these findings from animals translate to human patients treated for ma‐ laria in with a 3-5 day treatment regimen (WHO, 2006b; Wang, 1989). Data from limited clinical trials in pregnant women (1837 cases) exposed to ART compounds and ACTs, in‐ cluding a small number (176 cases) in the first trimester, have not shown an increase in the rates of abortion or stillbirth; they have also not shown evidence of abnormalities. Since more than 99% of pregnant patients have been treated with oral ARTs or intramuscular AM in the previously referenced trials, the lack of sensitivity and enhanced repair capabilities of humans to respond to ARTs induced embryotoxicity may explain the lack of embryotoxici‐ ty observed.

The possible embryotoxicity associated with ARTs therapy should be avoided by limiting ex‐ posure of pregnant women in the first trimester which is the critical period for induction of em‐ bryo damage and resorption. In addition, to protect pregnant women from embryotoxicity associated with ARTs treatment, injectable AS should be used very cautiously. There is agree‐ ment that ART derivatives should not be withheld at any stage of pregnancy, in cases of severe and complicated malaria, if the life of the mother is at risk. It is believed that oral ARTs regi‐ mens are much safer than parenteral administrations in pregnant patients. When relating the animal and human toxicity associated with ARTs administration, there are differences in sensi‐ tivity, the timing of the most vulnerable period of the embryo to ARTs administration, and the different pharmacokinetic profiles between animals and humans which may possibly provide a greater margin of safety for the use of ARTs by pregnant women.

**1.** Over-expression of VEGF results in increased angiogenesis in normal and pathological conditions. The existence of an alternative splicing site at the 3'untranslated region of VEGF mRNA results in the expression of isoforms with a C-terminal region which are down-regulated in tumors and may have differential inhibitory effects. This suggests that control of splicing can be an important regulatory mechanism of angiogenesis in

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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217

**2.** The VEGF family includes VEGF-A, -B, -C, -D, -E factors and the placenta growth factor (PIGF). The most studied and best characterized member of the VEGF family is VEGF-A or VEGF, which is secreted by tumors- and plays an important role in both normal and tumor-associated angiogenesis. The biologic effect of VEGF-A is exerted through interaction with cell surface receptors that include VEGF receptor-1 (VEGFR-1, flt-1) and VEGF receptor-2 (VEGFR-2, KDR/flk-1), which are selectively located on vascular endothelium and are up-regulated during angiogenesis, and VEGFR-3, a lymphatic growth factor. The role of VEGFR-1 seems to be complex, and studies indicate that VEGFR-1 may negatively regulate angiogenesis, although it has also been shown that it contributes to vascular sprouting and metastasis. The VEGF-A–VEGFR-2 interaction al‐ so plays a crucial role in angiogenesis, through the coordinated signaling of endothelial cell proliferation, migration and recruitment of endothelial cell progenitor cells. VEGF-B has recently been found to be largely necessary for vascular survival rather than an‐

**3.** Placental growth factor (PlGF), a member of the VEGF family of growth factors, is in‐ duced as tumors lose responsiveness to VEGF-directed therapies (Van de Veire et al., 2010). PlGF was first described, crystallized and identified as a ligand for VEGFR1 in the early 1990s (Ribatti, 2008). The functional biology of PlGF is still being explored. PlGF appears to have direct effects on some malignant cells and has been shown to in‐

**4.** Angiopoietins (Angs) are another family of endothelial cell-specific molecules that bind Tie receptors, and they play an important role in vessel maintenance, growth and stabilization. There are four types of angiopoietins known: Ang-1, -2, -3 and -4. Tie1 mRNA is highly expressed in embryonic vascular endothelium, angioblasts, en‐ docardium, and lung capillaries while it is weekly expressed in the endocardium of adults The Tie2 receptor takes part in vessel maturation by transducing survival sig‐ nals for endothelial cells. Ang-1 acts as an agonist promoting vessel stabilization in a paracrinal manner, whileAng-2 is an autocrine antagonist inducing vascular destabi‐ lization at high concentrations. Ang-2 has been found to be dramatically increased during vascular remodeling, and it has been implicated in tumor-associated angio‐ genesis and tumor progression. It has been found that VEGF also activates the Tie2

**5.** The Notch signaling pathway is critical for many developmental processes including physiologic angiogenesis. The Notch pathway has also been shown to have a key role in tumor angiogenesis. Preclinical and clinical studies of various anti-angiogenic combina‐ tions suggests that the mechanism associated with poor efficacy may involve tumor re‐

cancer cells (Biselli-Chicote et al., 2012).

giogenesis (Ferrara, 2009; 2010; Zhang et al., 2009).

receptor (Makrilia et al., 2009).

crease cell proliferation and migration (Chen et al., 2009d).

In accordance with WHO recommendations and the new research described above, the two major issues for considering ART drug use in a program for prevention or manage‐ ment of malaria in pregnant women are safety and efficacy (WHO, 2006a). First, the ex‐ posure to injectable AS should be very limited, during the early sensitive period (GD 15 to week 6 in humans), which is the likely critical phase for induction of embryo damage. This is essentially the same recommendation that the WHO has provided where ARTs should not be used in the first trimester of pregnancy in women. Secondly, in uncompli‐ cated malaria WHO recommends that the oral ARTs, including ACTs, should only be used in the second and third trimester when other treatments are considered unsuitable? We feel that oral regimens could be used to treat pregnant women in all trimesters, how‐ ever, when other treatments are considered unavailable, because the common oral ARTs regimens utilized provide a lower peak concentration and short exposure time, and that can make these ARTs combination drugs safer for use in pregnant women than intrave‐ nous or intramuscular injection of AS. Therefore, this policy should also suitable in anticancer therapy and prevention (Li and Weina, 2011).

### **4. Therapeutic implications of new and alternative mechanisms of antiangiogenesis**

Until recently, normal and abnormal processes of angiogenesis were considered to be based on a limited number of known mechanisms. Recent advances have been made in identifying a number of novel alternate processes involved in angiogenesis. If these new findings of al‐ ternate mechanisms are confirmed, cancer therapy strategies may also be affected

#### **4.1. New signaling molecules and pathways that influence the angiogenic response**

The first generation of clinically useful anti-angiogenic agents including ARTs focused on VEGF and targets in the VEGF pathway. VEGF and its receptors represent one of the bestvalidated signaling pathways in angiogenesis (Ferrara et al., 2003), and the current FDA ap‐ proved anti-angiogenic agents inhibit the VEGF pathway (Ferrara, 2010). The strengths and limitations of this therapeutics are now clear. Some tumors do not respond to VEGF-direct‐ ed therapies *de novo*, and others become non-responsive or resistant over time by switching to other angiogenic pathways. The next generation of angiogenesis-directed therapeutics will expand the field beyond the VEGF pathway and become more disease selective. New signaling molecules and pathways, including new VEGF-independent cancer angiogenesis pathways, have been recently reported (Teicher, 2011):

**1.** Over-expression of VEGF results in increased angiogenesis in normal and pathological conditions. The existence of an alternative splicing site at the 3'untranslated region of VEGF mRNA results in the expression of isoforms with a C-terminal region which are down-regulated in tumors and may have differential inhibitory effects. This suggests that control of splicing can be an important regulatory mechanism of angiogenesis in cancer cells (Biselli-Chicote et al., 2012).

and complicated malaria, if the life of the mother is at risk. It is believed that oral ARTs regi‐ mens are much safer than parenteral administrations in pregnant patients. When relating the animal and human toxicity associated with ARTs administration, there are differences in sensi‐ tivity, the timing of the most vulnerable period of the embryo to ARTs administration, and the different pharmacokinetic profiles between animals and humans which may possibly provide

In accordance with WHO recommendations and the new research described above, the two major issues for considering ART drug use in a program for prevention or manage‐ ment of malaria in pregnant women are safety and efficacy (WHO, 2006a). First, the ex‐ posure to injectable AS should be very limited, during the early sensitive period (GD 15 to week 6 in humans), which is the likely critical phase for induction of embryo damage. This is essentially the same recommendation that the WHO has provided where ARTs should not be used in the first trimester of pregnancy in women. Secondly, in uncompli‐ cated malaria WHO recommends that the oral ARTs, including ACTs, should only be used in the second and third trimester when other treatments are considered unsuitable? We feel that oral regimens could be used to treat pregnant women in all trimesters, how‐ ever, when other treatments are considered unavailable, because the common oral ARTs regimens utilized provide a lower peak concentration and short exposure time, and that can make these ARTs combination drugs safer for use in pregnant women than intrave‐ nous or intramuscular injection of AS. Therefore, this policy should also suitable in anti-

**4. Therapeutic implications of new and alternative mechanisms of anti-**

Until recently, normal and abnormal processes of angiogenesis were considered to be based on a limited number of known mechanisms. Recent advances have been made in identifying a number of novel alternate processes involved in angiogenesis. If these new findings of al‐

The first generation of clinically useful anti-angiogenic agents including ARTs focused on VEGF and targets in the VEGF pathway. VEGF and its receptors represent one of the bestvalidated signaling pathways in angiogenesis (Ferrara et al., 2003), and the current FDA ap‐ proved anti-angiogenic agents inhibit the VEGF pathway (Ferrara, 2010). The strengths and limitations of this therapeutics are now clear. Some tumors do not respond to VEGF-direct‐ ed therapies *de novo*, and others become non-responsive or resistant over time by switching to other angiogenic pathways. The next generation of angiogenesis-directed therapeutics will expand the field beyond the VEGF pathway and become more disease selective. New signaling molecules and pathways, including new VEGF-independent cancer angiogenesis

ternate mechanisms are confirmed, cancer therapy strategies may also be affected

**4.1. New signaling molecules and pathways that influence the angiogenic response**

a greater margin of safety for the use of ARTs by pregnant women.

216 Research Directions in Tumor Angiogenesis

cancer therapy and prevention (Li and Weina, 2011).

pathways, have been recently reported (Teicher, 2011):

**angiogenesis**


sistance and recurrence, which has led to the search for alternative angiogenic treatment strategies. Significant progress has been made in shedding light on the complex mecha‐ nisms by which Notch signaling can influence tumor growth by disrupting vasculature in an array of tumor models (Ridgway et al., 2006). The Notch pathway is being investi‐ gated as a target for anti-angiogenesis treatment. The VEGF and Notch pathways inter‐ act and intersect such that the VEGF pathway stimulates angiogenesis while the Notch pathway helps to guide cell fate decisions that appropriately shape activation (Li and Harris, 2009; Garcia and Kandel, 2012).

inhibitory potency are in clinical trials with a focus on anti-angiogenic activity. Matrix metalloproteinases (MMPs) are a family of enzymes that cleave the extracellular matrix, a process which is considered important for the formation of new blood vessels. Inhibi‐ tion of MMPs activity seems to be a crucial step in the process of vessel stabilization during the resolution phase of angiogenesis, since uncontrolled proteolysis results in re‐

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

219

Angiogenesis is an essential process in tumor growth, and new basic science research find‐ ings in angiogenesis have had considerable impact on cancer therapy research, as the sur‐ vival and proliferation of cancer is fundamentally dependent on angiogenesis,. In past years, numerous anti-angiogenic agents were developed, and some of them have been applied clinically. Angiogenesis is a complex and multistep process, however, and the intertwining of interrelated angiogenesis pathways is still not completely understood. Discoveries of new and alternative angiogenesis signaling molecules and pathways, combined with studies on the major signaling proteins and pathways related to tumor angiogenesis, have led to new

Similarly, angiopreventive strategies may involve various targets including angiogenic mol‐ ecules from tumors cells, inflammatory system and their respective receptors on endothelial cells such as VEGF, PDGF, FGF and their receptors, angiopoietin (Ang) family, endothelial cells, matrix metalloproteinases (MMPs), cyclooxygenases (COXs), lipoxygenases (LOXs) etc. Inflammation, for example, has been shown to be one of the most important processes in mediating angiogenesis, and may be a valid target for mediating anti-angiogenic therapies. Accordingly, long-term angiostasis treatments will likely be an important element in pre‐ venting metastasis of tumors that have been treated and are in remission. Emphasis should be placed on screening and identification of non-toxic anti-angiogenic molecules or com‐ pounds and their further evaluation in clinical trials to discover the most efficacious anti-

VEGF has a number of different gene family members including VEGF-A, -B, -C, -D, and -E and placental growth factor (PlGF). Among them, VEGF-A (or VEGF) has been the most well-characterized and is considered a key angiogenic factor with various splicing variants such as VEGF-A125, -A145, -A165, -A183, -A189, and -A206. VEGF-A is indispensable during em‐ bryonic vascular development, and even the loss of a single VEGF-A allele in mice has been shown to result in embryonic lethality due to defective vasculature. Hypoxia, often seen in the center of tumors, strongly up-regulates VEGF-A expression via increased production of hypoxia inducible factor (HIF). Under normal conditions, HIF is ubiquitinated and degener‐ ated by binding to von Hippel-Lindau (VHL) proteins, but, in under hypoxic conditions, HIF cannot bind to VHL, resulting in increased active HIF. HIF acts as a transcriptional acti‐ vator by mediating transcription at the HIF-1 binding site, the hypoxia response element (HRE), and by enhancing transcription of many pro-angiogenic genes including VEGF-A

gression of newly formed vessels (Makrilia et al., 2009).

**4.2. Potential targets in angiogenesis and angioprevention**

drug development research to target tumor angiogenesis.

angiogenic treatments for cancer therapy.

**VEGF-A (VEGF)**

gene (Ichihara et al., 2011).

Delta-like ligand 4 (Dll4) is a key endothelial Notch ligand. The Notch pathway and the VEFG pathway interrelate via the interaction between Dll4 and VEGF. This cross-talk occurs through VEGF-induced upregulation of Dll4 and Dll4 downregula‐ tion of the VEGFR signaling. Both pathways are essential for normal angiogenesis, and blockade of one may produce compensatory changes in the other. Dll4–Notch signaling has sparked high interest in exploring molecular targets in these intercon‐ nected pathways for cancer therapy (Oon and Harris, 2011)


inhibitory potency are in clinical trials with a focus on anti-angiogenic activity. Matrix metalloproteinases (MMPs) are a family of enzymes that cleave the extracellular matrix, a process which is considered important for the formation of new blood vessels. Inhibi‐ tion of MMPs activity seems to be a crucial step in the process of vessel stabilization during the resolution phase of angiogenesis, since uncontrolled proteolysis results in re‐ gression of newly formed vessels (Makrilia et al., 2009).

#### **4.2. Potential targets in angiogenesis and angioprevention**

Angiogenesis is an essential process in tumor growth, and new basic science research find‐ ings in angiogenesis have had considerable impact on cancer therapy research, as the sur‐ vival and proliferation of cancer is fundamentally dependent on angiogenesis,. In past years, numerous anti-angiogenic agents were developed, and some of them have been applied clinically. Angiogenesis is a complex and multistep process, however, and the intertwining of interrelated angiogenesis pathways is still not completely understood. Discoveries of new and alternative angiogenesis signaling molecules and pathways, combined with studies on the major signaling proteins and pathways related to tumor angiogenesis, have led to new drug development research to target tumor angiogenesis.

Similarly, angiopreventive strategies may involve various targets including angiogenic mol‐ ecules from tumors cells, inflammatory system and their respective receptors on endothelial cells such as VEGF, PDGF, FGF and their receptors, angiopoietin (Ang) family, endothelial cells, matrix metalloproteinases (MMPs), cyclooxygenases (COXs), lipoxygenases (LOXs) etc. Inflammation, for example, has been shown to be one of the most important processes in mediating angiogenesis, and may be a valid target for mediating anti-angiogenic therapies. Accordingly, long-term angiostasis treatments will likely be an important element in pre‐ venting metastasis of tumors that have been treated and are in remission. Emphasis should be placed on screening and identification of non-toxic anti-angiogenic molecules or com‐ pounds and their further evaluation in clinical trials to discover the most efficacious antiangiogenic treatments for cancer therapy.

#### **VEGF-A (VEGF)**

sistance and recurrence, which has led to the search for alternative angiogenic treatment strategies. Significant progress has been made in shedding light on the complex mecha‐ nisms by which Notch signaling can influence tumor growth by disrupting vasculature in an array of tumor models (Ridgway et al., 2006). The Notch pathway is being investi‐ gated as a target for anti-angiogenesis treatment. The VEGF and Notch pathways inter‐ act and intersect such that the VEGF pathway stimulates angiogenesis while the Notch pathway helps to guide cell fate decisions that appropriately shape activation (Li and

Delta-like ligand 4 (Dll4) is a key endothelial Notch ligand. The Notch pathway and the VEFG pathway interrelate via the interaction between Dll4 and VEGF. This cross-talk occurs through VEGF-induced upregulation of Dll4 and Dll4 downregula‐ tion of the VEGFR signaling. Both pathways are essential for normal angiogenesis, and blockade of one may produce compensatory changes in the other. Dll4–Notch signaling has sparked high interest in exploring molecular targets in these intercon‐

**6.** Fibroblast growth factors (FGFs) in signaling pathways are a family of heparin-binding proteins required for the development and differentiation of various organs from the early stages of embryogenesis. Acidic and basic fibroblast growth factors (aFGF or FGF1 and bFGF or FGF2 respectively) are described as inducers of angiogenesis. FGFs stimu‐ late endothelial cell proliferation and migration, as well as production of collagenase and plasminogen activator. FGFs induce sprouting of blood vessels *in vivo* in the chick chorioallantoic membrane and cornea, thus supporting their role in angiogenesis (Mak‐ rilia et al., 2009). In addition, the HGF/c-Met pathway is upregulated in some tumors as an alternate angiogenic pathway. The HGF/c-Met tyrosine kinase signaling pathway is upregulated in many cancers resulting in invasive growth consisting of physiological

processes including proliferation, invasion and angiogenesis (Eder et al., 2009).

angiogenesis, metastasis and survival (Teicher and Fricker, 2010).

gation as an anti-angiogenic agent. (Hait et al., 2009).

**7.** The CXCL12 (SDF-1)/CXCR4 pathway represents a stromal chemokine axis involved in tumor angiogenesis. CXCR2 is a G-protein coupled receptor with several ligands in‐ cluding interleukin-8 and other angiogenic cytokines and may represent a useful target for anti-angiogenic agents. The CXCL12/CXCR4 axis is involved in tumor progression,

**8.** Sphingosine-1-phosphate (S-1-P) is a bioactive lipid that regulates many cellular and physiological processes including cell proliferation, survival, motility, angiogenesis, vascular maturation, immunity and lymphocyte trafficking. Sphingosine-1-phosphate can be neutralized with a monoclonal antibody. Anti-S-1-P antibodies are under investi‐

**9.** Several small molecules and antibodies targeting additional pro-angiogenic cell surface molecules are under investigation as anti-angiogenic agents. Tumor necrosis factor-a (TNF-α), transforming growth factor-α (TGF-α), epidermal growth factor (EGF), colo‐ ny-stimulating factors (CSFs) and others have been implicated in the process of angio‐ genesis. Several multi-targeted kinase inhibitors each with a unique pattern of

Harris, 2009; Garcia and Kandel, 2012).

218 Research Directions in Tumor Angiogenesis

nected pathways for cancer therapy (Oon and Harris, 2011)

VEGF has a number of different gene family members including VEGF-A, -B, -C, -D, and -E and placental growth factor (PlGF). Among them, VEGF-A (or VEGF) has been the most well-characterized and is considered a key angiogenic factor with various splicing variants such as VEGF-A125, -A145, -A165, -A183, -A189, and -A206. VEGF-A is indispensable during em‐ bryonic vascular development, and even the loss of a single VEGF-A allele in mice has been shown to result in embryonic lethality due to defective vasculature. Hypoxia, often seen in the center of tumors, strongly up-regulates VEGF-A expression via increased production of hypoxia inducible factor (HIF). Under normal conditions, HIF is ubiquitinated and degener‐ ated by binding to von Hippel-Lindau (VHL) proteins, but, in under hypoxic conditions, HIF cannot bind to VHL, resulting in increased active HIF. HIF acts as a transcriptional acti‐ vator by mediating transcription at the HIF-1 binding site, the hypoxia response element (HRE), and by enhancing transcription of many pro-angiogenic genes including VEGF-A gene (Ichihara et al., 2011).

#### **VEGF Receptors (VEGFR)**

VEGF family members bind to VEGFR (VEGFR-1, VEGFR-2, and VEGFR-3), and VEGF-A binds to VEGFR-1 and VEGFR-2. Although the affinity of VEGF-A to VEGFR-1 is 10-fold higher than it's binding to VEGFR-2, VEGF-A signaling is mainly mediated by VEGFR-2 be‐ cause of its intense kinase activity (Olsson et al., 2006). VEGFR-2 signaling in endothelial cells is mediated through downstream cascades such as PI3K/AKT, p38/MAPK, and PLCγ/ MAPK, triggering proliferation and migration of endothelial cells, production of proteases, and hyperpermeability of vessels. Currently, researchers agree that VEGF-A/VEGFR-2 sig‐ naling is the key pathway for tumor angiogenesis.

**PDGF**

**PDGF Receptors (PDGFR)**

**Delta-like ligand 4 (DLL4)**

The role of platelet-derived growth factor (PDGF) in angiogenesis is not yet fully under‐ stood. More recently, PDGF has been found to stimulate angiogenesis *in vivo,* and experi‐ ments with knockout mice have suggested a role for PDGF in the recruitment of pericytes that are needed for the development of capillaries in tumors. PDGF has also been implicat‐ ed in the vascular aging process. It has been shown that some tumors overcome inhibition of VEGF-mediated angiogenesis by upregulating members of the PDGF family. Epithelial cancers are characterized by paracrine PDGF signaling, whereas autocrine PDGF signaling is implicated in neoplasms such as leukemias, gliomas and sarcomas (Yang et al., 2009). Thus far, four PDGF family members have been identified, PDGF-A, -B, -C, and -D. They form 5 different forms of homodimers and heterodimers, PDGF-AA, -AB, -BB, -CC, and - DD. PDGFs generally act in a paracrine manner in epithelial cancers, while they have been shown to act in an autocrine manner in gliomas, sarcomas, and leukemia. PDGFs are secret‐ ed from various cells, and PDGF-A and -C are mainly secreted from epithelial cells, muscle, and neuronal progenitors while PDGF-B is secreted from vascular endothelial cells. PDGF-

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PDGFs transmit their signal via PDGFRs. When PDGFRs bind PDGFs, PDGFRs dimerize, are autophosphorylated at tyrosine residues in the PDGFR intracellular domain, and the phosphoyrlated PDFGR dimer has been shown to activate downstream pathways, includ‐ ing PI3K, Ras-MAPK, and PLCγ. There are 2 types of PDGFRs, PDGFR-α and PDGFR-β. PDGFRs can form 3 kinds of homodimers and heterodimers, PDGFR-αα, -ββ, and αβ. Con‐ sidering the five PDGF dimers described above, there could be multiple and complex PDGF/PDGFR pairings. To date, however, there are only three PDGF/PDGFR pairs proven to be functional *in vivo*, PDGF-AA/PDGFR-αα, PDGF-CC/PDGFR-αα, and PDGF-BB/ PDGFR-ββ. PDGFR-α has been shown to be involved in embryonic development, while

The PDGFR-α-induced pathway is involved in organogenesis such as in alveogenesis, villus morphogenesis, hair morphogenesis, and oligodendrogenesis. In addition, PDGFR-α may indirectly promote angiogenesis by recruiting stromal fibroblast-producing VEGF-A and other pro-angiogenic factors. PDGFR-β is expressed in pericytes but not in endothelial cells, and PCGFR-β signaling is believed to play a role in angiogenesis. Due to PDGFR-β's expres‐ sion in pericytes as opposed to endothelial cells, the PDGFR-β signaling pathway does not increase the number of tumor vessels but acts to form mature tumor vessels by recruiting PDGFR-β-expressing pericytes, and, in turn, acting to accelerate tumor growth. Blocking the PDGFR-β pathway inhibits the maturation of blood vessels, eliciting detachment of peri‐ cytes and disruption of tumor vessels, while blocking the VEGFR pathway impairs forma‐ tion of early-stage immature vessels lacking pericyte coverage but does not affect existing

Delta-like ligand 4 (DLL4) belongs to the Delta/Jagged family of transmembrane ligands that binds to Notch receptors. Delta–Notch signaling has been shown to mediate cell–cell

D secretion is, unfortunately, not well understood (Andrae et al., 2008).

PDGFR-β has been shown to be involved in angiogenesis (Cao et al., 2008).

mature, large blood vessels well-covered with pericytes (Ichihara et al., 2011).

VEGF-B and PlGF bind only to VEGFR-1, in contrast to VEGF-A, which binds to both VEGFR-1 and -2. VEGFR-1 signaling has more complex roles in angiogenesis compared with that of VEGFR-2. VEGFR-1 exists as a decoy receptor with high affinity for VEGF-A, and its low kinase activity prevents VEGF-A from binding to VEGFR-2, so VEGFR-1 actually functions as a negative regulator of angiogenesis. In fact, VEGFR-1 tyrosine kin‐ ase-deficient mice, with normal ligand binding ability and deficient signal transduction, have been shown to develop normally, which means VEGFR-1 tyrosine kinase activity is not indispensable, at least during development. On the other hand, there is growing evi‐ dence that VEGFR-1 can mediate signaling to downstream cascades. VEGFR-1 signaling in bone marrow cells such as macrophage lineage cells has been shown in a subcutane‐ ous injected tumor model to mobilize them to tumor tissues, contributing to angiogene‐ sis and tumor progression (Muramatsu et al., 2010). It has also been reported that VEGFR-1 signaling might be associated with metastasis. Lymphangiogenesis plays an im‐ portant role in the tumor microenvironment and the formation of new lymphatic blood vessels is considered the first step of tumor metastasis. VEGFR-3 has been shown to in‐ duce lymphangiogenesis after binding VEGF-C or –D (Ichihara et al., 2011).

#### **Angiopoietin/Tie2**

Ang/Tie2 signaling is an endothelial cell-specific pathway, like VEGF/VEGFR signaling, but it is difficult to target for cancer therapy because of the complex nature of this sig‐ naling pathway which will be reviewed in depth later in this chapter. Angiopoietins play an important role in vessel stabilization and maturation, although they cannot directly induce tumor angiogenesis. There are four types of angiopoietins that are known: Ang-1, -2, -3 and -4. Tie1 mRNA is highly expressed in embryonic vascular endothelium, angio‐ blasts and in the endocardium; however, in adult tissues it is expressed strongly in lung capillaries but weakly in the endocardium. The Tie2 receptor takes part in vessel matura‐ tion by mediating survival signals for endothelial cells. Ang-1 acts as an agonist promot‐ ing vessel stabilization in a paracrinal manner, whereas Ang-2 is an autocrine antagonist inducing vascular destabilization at high concentrations. Ang-2 has been found to be dra‐ matically increased during vascular remodeling and is implicated in tumor-associated an‐ giogenesis and tumor progression. As a further demonstration of the interrelated nature of angiogenic pathways, it has been shown that VEGF also activates the Tie2 receptor (Singh and Milner, 2009; Thomas and Augustin, 2009).

#### **PDGF**

**VEGF Receptors (VEGFR)**

220 Research Directions in Tumor Angiogenesis

**Angiopoietin/Tie2**

naling is the key pathway for tumor angiogenesis.

VEGF family members bind to VEGFR (VEGFR-1, VEGFR-2, and VEGFR-3), and VEGF-A binds to VEGFR-1 and VEGFR-2. Although the affinity of VEGF-A to VEGFR-1 is 10-fold higher than it's binding to VEGFR-2, VEGF-A signaling is mainly mediated by VEGFR-2 be‐ cause of its intense kinase activity (Olsson et al., 2006). VEGFR-2 signaling in endothelial cells is mediated through downstream cascades such as PI3K/AKT, p38/MAPK, and PLCγ/ MAPK, triggering proliferation and migration of endothelial cells, production of proteases, and hyperpermeability of vessels. Currently, researchers agree that VEGF-A/VEGFR-2 sig‐

VEGF-B and PlGF bind only to VEGFR-1, in contrast to VEGF-A, which binds to both VEGFR-1 and -2. VEGFR-1 signaling has more complex roles in angiogenesis compared with that of VEGFR-2. VEGFR-1 exists as a decoy receptor with high affinity for VEGF-A, and its low kinase activity prevents VEGF-A from binding to VEGFR-2, so VEGFR-1 actually functions as a negative regulator of angiogenesis. In fact, VEGFR-1 tyrosine kin‐ ase-deficient mice, with normal ligand binding ability and deficient signal transduction, have been shown to develop normally, which means VEGFR-1 tyrosine kinase activity is not indispensable, at least during development. On the other hand, there is growing evi‐ dence that VEGFR-1 can mediate signaling to downstream cascades. VEGFR-1 signaling in bone marrow cells such as macrophage lineage cells has been shown in a subcutane‐ ous injected tumor model to mobilize them to tumor tissues, contributing to angiogene‐ sis and tumor progression (Muramatsu et al., 2010). It has also been reported that VEGFR-1 signaling might be associated with metastasis. Lymphangiogenesis plays an im‐ portant role in the tumor microenvironment and the formation of new lymphatic blood vessels is considered the first step of tumor metastasis. VEGFR-3 has been shown to in‐

duce lymphangiogenesis after binding VEGF-C or –D (Ichihara et al., 2011).

(Singh and Milner, 2009; Thomas and Augustin, 2009).

Ang/Tie2 signaling is an endothelial cell-specific pathway, like VEGF/VEGFR signaling, but it is difficult to target for cancer therapy because of the complex nature of this sig‐ naling pathway which will be reviewed in depth later in this chapter. Angiopoietins play an important role in vessel stabilization and maturation, although they cannot directly induce tumor angiogenesis. There are four types of angiopoietins that are known: Ang-1, -2, -3 and -4. Tie1 mRNA is highly expressed in embryonic vascular endothelium, angio‐ blasts and in the endocardium; however, in adult tissues it is expressed strongly in lung capillaries but weakly in the endocardium. The Tie2 receptor takes part in vessel matura‐ tion by mediating survival signals for endothelial cells. Ang-1 acts as an agonist promot‐ ing vessel stabilization in a paracrinal manner, whereas Ang-2 is an autocrine antagonist inducing vascular destabilization at high concentrations. Ang-2 has been found to be dra‐ matically increased during vascular remodeling and is implicated in tumor-associated an‐ giogenesis and tumor progression. As a further demonstration of the interrelated nature of angiogenic pathways, it has been shown that VEGF also activates the Tie2 receptor The role of platelet-derived growth factor (PDGF) in angiogenesis is not yet fully under‐ stood. More recently, PDGF has been found to stimulate angiogenesis *in vivo,* and experi‐ ments with knockout mice have suggested a role for PDGF in the recruitment of pericytes that are needed for the development of capillaries in tumors. PDGF has also been implicat‐ ed in the vascular aging process. It has been shown that some tumors overcome inhibition of VEGF-mediated angiogenesis by upregulating members of the PDGF family. Epithelial cancers are characterized by paracrine PDGF signaling, whereas autocrine PDGF signaling is implicated in neoplasms such as leukemias, gliomas and sarcomas (Yang et al., 2009). Thus far, four PDGF family members have been identified, PDGF-A, -B, -C, and -D. They form 5 different forms of homodimers and heterodimers, PDGF-AA, -AB, -BB, -CC, and - DD. PDGFs generally act in a paracrine manner in epithelial cancers, while they have been shown to act in an autocrine manner in gliomas, sarcomas, and leukemia. PDGFs are secret‐ ed from various cells, and PDGF-A and -C are mainly secreted from epithelial cells, muscle, and neuronal progenitors while PDGF-B is secreted from vascular endothelial cells. PDGF-D secretion is, unfortunately, not well understood (Andrae et al., 2008).

#### **PDGF Receptors (PDGFR)**

PDGFs transmit their signal via PDGFRs. When PDGFRs bind PDGFs, PDGFRs dimerize, are autophosphorylated at tyrosine residues in the PDGFR intracellular domain, and the phosphoyrlated PDFGR dimer has been shown to activate downstream pathways, includ‐ ing PI3K, Ras-MAPK, and PLCγ. There are 2 types of PDGFRs, PDGFR-α and PDGFR-β. PDGFRs can form 3 kinds of homodimers and heterodimers, PDGFR-αα, -ββ, and αβ. Con‐ sidering the five PDGF dimers described above, there could be multiple and complex PDGF/PDGFR pairings. To date, however, there are only three PDGF/PDGFR pairs proven to be functional *in vivo*, PDGF-AA/PDGFR-αα, PDGF-CC/PDGFR-αα, and PDGF-BB/ PDGFR-ββ. PDGFR-α has been shown to be involved in embryonic development, while PDGFR-β has been shown to be involved in angiogenesis (Cao et al., 2008).

The PDGFR-α-induced pathway is involved in organogenesis such as in alveogenesis, villus morphogenesis, hair morphogenesis, and oligodendrogenesis. In addition, PDGFR-α may indirectly promote angiogenesis by recruiting stromal fibroblast-producing VEGF-A and other pro-angiogenic factors. PDGFR-β is expressed in pericytes but not in endothelial cells, and PCGFR-β signaling is believed to play a role in angiogenesis. Due to PDGFR-β's expres‐ sion in pericytes as opposed to endothelial cells, the PDGFR-β signaling pathway does not increase the number of tumor vessels but acts to form mature tumor vessels by recruiting PDGFR-β-expressing pericytes, and, in turn, acting to accelerate tumor growth. Blocking the PDGFR-β pathway inhibits the maturation of blood vessels, eliciting detachment of peri‐ cytes and disruption of tumor vessels, while blocking the VEGFR pathway impairs forma‐ tion of early-stage immature vessels lacking pericyte coverage but does not affect existing mature, large blood vessels well-covered with pericytes (Ichihara et al., 2011).

#### **Delta-like ligand 4 (DLL4)**

Delta-like ligand 4 (DLL4) belongs to the Delta/Jagged family of transmembrane ligands that binds to Notch receptors. Delta–Notch signaling has been shown to mediate cell–cell communication and regulates cell fate determination. Delta/Notch signaling is also criti‐ cally important for proper vascular development. One particular endothelial cell Notch ligand, DLL4, has been shown to be required for regulation of tip cell formation during angiogenesis. Activation of the Delta/Notch signaling pathway has been shown to de‐ crease endothelial tip cell numbers. Conversely, decreased DLL4 signaling increases tip cell formation. Upregulation of DLL4 was also found in tumor vessels. Two groups have demonstrated independently that inhibiting DLL4 leads to tumor growth suppression by deregulating angiogenesis, resulting in increased, but non-functional vessels. Importantly, this strategy is also effective in slowing the growth of tumors that are relatively resistant to anti-VEGF therapy, and DLL4 inhibition also exhibits an additive effect when com‐ bined with anti-VEGF therapy to slow the growth of anti-VEGF resistant tumors (Du‐ fraine et al., 2008, Ferrara, 2010).

FGF2 respectively) have been shown to be inducers of angiogenesis. FGFs stimulate en‐ dothelial cell proliferation and migration, as well as production of collagenase and plas‐ minogen activator. FGFs have also been shown to induce sprouting of blood vessels *in vivo* in the chick chorioallantoic membrane and cornea, thus supporting their role in an‐

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The FGF signaling pathway plays an important role in embryonic organogenesis, and dis‐ turbance of this pathway leads to various kinds of developmental defects. In the adult or‐ ganism, FGF/FGFR signaling is involved in important physiological processes such as the regulation of wound healing and angiogenesis. FGFs are heparin-binding growth factors that are part of a family that includes 23 members, FGF1-23 (Turner and Grose, 2010). Only 18 FGF members work as FGF ligands, because FGF11, 12, 13, and 14 are not functional li‐ gands for FGFR, and the FGF15 gene does not exist in humans. Among these family mem‐ bers, FGF1 and FGF2 have been shown to possess a potent pro-angiogenic effect and they play a role in inducing proliferation and migration of endothelial cells (Daniele et al., 2012).

FGFRs belong to a receptor family consisting of FGFR-1, -2, -3, and -4 (Turner and Grose, 2010). FGFRs are expressed in most cells and have various functions, including normal cell growth, differentiation, and angiogenesis., FGFR over expression or mutation has been shown to be associated with a variety of different neoplasms FGFR activation has been shown to induce angiogenesis in both cell cultures and in animal models (Cao

The transforming growth factor-β (TGF-β) is thought to have both pro- and anti-angiogenic properties. Low TGF-β levels contribute to a switch in angiogenesis, by up-regulating angio‐ genic factors and proteinases. On the other hand, high TGF-β levels have been shown to in‐ hibit endothelial cell growth, stimulate smooth muscle cells differentiation and recruitment and promote basement membrane reformation. In cancer cells, multiple mutations in the TGF-β signaling pathway have been described. Elevated TGF-β levels have been shown to induce proliferation of cancer cells, the surrounding stromal cells, immune cells, endothelial cells and smooth muscle cells. High levels of endoglin, which is part of the TGF-β receptor complex, have been detected in cancer patients and are directly correlated with tumor meta‐ stasis. More specifically, during the initial stages of tumorigenesis, TGF-β inhibits tumor growth and development by inhibiting cell proliferation and by inducing apoptosis. In later stages, tumor stages become resistant to the tumor suppressioni activity of TGF-β, TGF-β

**1.** The role of integrin ανβ3 in mediating angiogenesis has been shown through its bind‐ ing of extracellular matrix components and matrix metalloproteinase-2, thus helping to connect new vessels with pre-existing ones, to produce the intra-tumoral vascular net‐ work. Ephrin ligands and ephrin receptors play a critical role in blood vessel assembly.

giogenesis (Makrilia et al., 2009).

**FGF Receptors (FGFR)**

**TGF-β**

et al., 2008; Korc and Friesel, 2009).

takes on a pro-oncogenic role (Pardali and Dijke, 2009).

Other important tumor angiogenesis targets include:

In fact, not all of the endothelial cells are stimulated, due to a mechanism deciding which endothelial cells should react to angiogenic stimulus and which should not. The DLL4/Notch pathway plays a key role in this mechanism. DLL1, DLL3, DLL4, Jagged1, and Jagged2 bind to the Notch receptor as ligands. Among these ligands related to tu‐ mor angiogenesis, DLL4 has been the most intensely investigated, because DLL4 is strongly expressed in tumor vascular endothelial cells but more weakly in normal vascu‐ lar endothelial cells. DLL4 is a transmembrane ligand, and its expression in tumor ves‐ sels is regulated by VEGF-A. VEGF-A up-regulates DLL4 in sprouting endothelial cells (tip cells), and up-regulated DLL4 interacts with Notch in the adjacent endothelial cells (stalk cells). In reverse, the DLL4/Notch pathway down-regulates VEGFR-2 expression in Notch-expressing endothelial cells, resulting in the reduction of VEGF-A-induced sprout‐ ing and branching (Lobov et al., 2007). Thus the DLL4/Notch pathway can be considered a negative feedback VEGFR pathway (Ichihara et al., 2011).

#### **Notch**

Notch receptors are single-pass transmembrane proteins in a family consisting of Notch1, Notch2, Notch3, and Notch4. The Notch receptor signaling pathway has a characteristic mechanism for signal transduction. After ligand binding, the Notch receptor is cleaved at an extracellular domain by proteases such as ADAM10 or TACE, followed by cleavage at a transmembrane domain by γ-secretase. As a consequence, the Notch intracellular domain translocates to the nucleus and activates the transcription of target genes. Blocking the DLL4/Notch signaling pathway leads to increased angiogenesis, such as the enhancement of tip-cell formation, branching, and vessel density. Paradoxically, blockade of the DLL4/Notch signaling also leads to the inhibition of tumor growth in a variety of tumor models. This is possibly due to an increase in the number of non-functional tumor vessels induced by the DLL4/Notch blockade which in turn results in tumor hypoxia (Scehnet et al., 2007).

#### **FGF1 and FGF2**

The fibroblast growth factor (FGF) family has been implicated in neurogenesis, organ de‐ velopment, branching morphogenesis, angiogenesis and various pathologic processes in‐ cluding cancer. Acidic and basic fibroblast growth factors (aFGF or FGF1 and bFGF or FGF2 respectively) have been shown to be inducers of angiogenesis. FGFs stimulate en‐ dothelial cell proliferation and migration, as well as production of collagenase and plas‐ minogen activator. FGFs have also been shown to induce sprouting of blood vessels *in vivo* in the chick chorioallantoic membrane and cornea, thus supporting their role in an‐ giogenesis (Makrilia et al., 2009).

The FGF signaling pathway plays an important role in embryonic organogenesis, and dis‐ turbance of this pathway leads to various kinds of developmental defects. In the adult or‐ ganism, FGF/FGFR signaling is involved in important physiological processes such as the regulation of wound healing and angiogenesis. FGFs are heparin-binding growth factors that are part of a family that includes 23 members, FGF1-23 (Turner and Grose, 2010). Only 18 FGF members work as FGF ligands, because FGF11, 12, 13, and 14 are not functional li‐ gands for FGFR, and the FGF15 gene does not exist in humans. Among these family mem‐ bers, FGF1 and FGF2 have been shown to possess a potent pro-angiogenic effect and they play a role in inducing proliferation and migration of endothelial cells (Daniele et al., 2012).

#### **FGF Receptors (FGFR)**

FGFRs belong to a receptor family consisting of FGFR-1, -2, -3, and -4 (Turner and Grose, 2010). FGFRs are expressed in most cells and have various functions, including normal cell growth, differentiation, and angiogenesis., FGFR over expression or mutation has been shown to be associated with a variety of different neoplasms FGFR activation has been shown to induce angiogenesis in both cell cultures and in animal models (Cao et al., 2008; Korc and Friesel, 2009).

#### **TGF-β**

communication and regulates cell fate determination. Delta/Notch signaling is also criti‐ cally important for proper vascular development. One particular endothelial cell Notch ligand, DLL4, has been shown to be required for regulation of tip cell formation during angiogenesis. Activation of the Delta/Notch signaling pathway has been shown to de‐ crease endothelial tip cell numbers. Conversely, decreased DLL4 signaling increases tip cell formation. Upregulation of DLL4 was also found in tumor vessels. Two groups have demonstrated independently that inhibiting DLL4 leads to tumor growth suppression by deregulating angiogenesis, resulting in increased, but non-functional vessels. Importantly, this strategy is also effective in slowing the growth of tumors that are relatively resistant to anti-VEGF therapy, and DLL4 inhibition also exhibits an additive effect when com‐ bined with anti-VEGF therapy to slow the growth of anti-VEGF resistant tumors (Du‐

In fact, not all of the endothelial cells are stimulated, due to a mechanism deciding which endothelial cells should react to angiogenic stimulus and which should not. The DLL4/Notch pathway plays a key role in this mechanism. DLL1, DLL3, DLL4, Jagged1, and Jagged2 bind to the Notch receptor as ligands. Among these ligands related to tu‐ mor angiogenesis, DLL4 has been the most intensely investigated, because DLL4 is strongly expressed in tumor vascular endothelial cells but more weakly in normal vascu‐ lar endothelial cells. DLL4 is a transmembrane ligand, and its expression in tumor ves‐ sels is regulated by VEGF-A. VEGF-A up-regulates DLL4 in sprouting endothelial cells (tip cells), and up-regulated DLL4 interacts with Notch in the adjacent endothelial cells (stalk cells). In reverse, the DLL4/Notch pathway down-regulates VEGFR-2 expression in Notch-expressing endothelial cells, resulting in the reduction of VEGF-A-induced sprout‐ ing and branching (Lobov et al., 2007). Thus the DLL4/Notch pathway can be considered

Notch receptors are single-pass transmembrane proteins in a family consisting of Notch1, Notch2, Notch3, and Notch4. The Notch receptor signaling pathway has a characteristic mechanism for signal transduction. After ligand binding, the Notch receptor is cleaved at an extracellular domain by proteases such as ADAM10 or TACE, followed by cleavage at a transmembrane domain by γ-secretase. As a consequence, the Notch intracellular domain translocates to the nucleus and activates the transcription of target genes. Blocking the DLL4/Notch signaling pathway leads to increased angiogenesis, such as the enhancement of tip-cell formation, branching, and vessel density. Paradoxically, blockade of the DLL4/Notch signaling also leads to the inhibition of tumor growth in a variety of tumor models. This is possibly due to an increase in the number of non-functional tumor vessels induced by the

DLL4/Notch blockade which in turn results in tumor hypoxia (Scehnet et al., 2007).

The fibroblast growth factor (FGF) family has been implicated in neurogenesis, organ de‐ velopment, branching morphogenesis, angiogenesis and various pathologic processes in‐ cluding cancer. Acidic and basic fibroblast growth factors (aFGF or FGF1 and bFGF or

fraine et al., 2008, Ferrara, 2010).

222 Research Directions in Tumor Angiogenesis

**Notch**

**FGF1 and FGF2**

a negative feedback VEGFR pathway (Ichihara et al., 2011).

The transforming growth factor-β (TGF-β) is thought to have both pro- and anti-angiogenic properties. Low TGF-β levels contribute to a switch in angiogenesis, by up-regulating angio‐ genic factors and proteinases. On the other hand, high TGF-β levels have been shown to in‐ hibit endothelial cell growth, stimulate smooth muscle cells differentiation and recruitment and promote basement membrane reformation. In cancer cells, multiple mutations in the TGF-β signaling pathway have been described. Elevated TGF-β levels have been shown to induce proliferation of cancer cells, the surrounding stromal cells, immune cells, endothelial cells and smooth muscle cells. High levels of endoglin, which is part of the TGF-β receptor complex, have been detected in cancer patients and are directly correlated with tumor meta‐ stasis. More specifically, during the initial stages of tumorigenesis, TGF-β inhibits tumor growth and development by inhibiting cell proliferation and by inducing apoptosis. In later stages, tumor stages become resistant to the tumor suppressioni activity of TGF-β, TGF-β takes on a pro-oncogenic role (Pardali and Dijke, 2009).

Other important tumor angiogenesis targets include:

**1.** The role of integrin ανβ3 in mediating angiogenesis has been shown through its bind‐ ing of extracellular matrix components and matrix metalloproteinase-2, thus helping to connect new vessels with pre-existing ones, to produce the intra-tumoral vascular net‐ work. Ephrin ligands and ephrin receptors play a critical role in blood vessel assembly.

**2.** The role of VE-cadherin in neovascularization has been shown in a number of studies.

multiple chromosomes, and multiple centrosomes, raising the possibility that such insta‐ bility may contribute to resistance to anti-angiogenic therapies. Tumor-associated blood vessels are excessively branched and hemorrhagic, and blood flow through these mal‐ formed vessels is often chaotic and may impede delivery of chemotherapy to the tumor

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A new therapeutic strategy targeting tumor vasculature has gained increased attention in the scientific community. This method involves targeting abnormal tumor vessel function by inducing vessel normalization. It is well known that tumor blood vessels are highly abnormal in structure and function, characterized by a tortuous, chaotic, and irregular branching network. In the tumor vasculature, ECs are highly activated, lose their polari‐ ty and alignment, and detach from the basement membrane, all resulting in a leaky, fe‐ nestrated network that facilitates bleeding and increases interstitial fluid pressure. Apart from the ECs, the entire vessel wall, including the basement membrane and the covering pericytes, becomes abnormal in most tumors. Tumor ECs are typically covered with few‐ er and more abnormal pericytes, and their associated basement membrane is only loose‐ ly associated and inhomogeneous in structure. It is suspected that this abnormal vasculature impedes the distribution of chemotherapy and oxygen. Traditional anti-an‐ giogenic therapy aims to maximally inhibit angiogenesis and to prune existing tumor vessels, however, this strategy can also increase the risk of aggravating hypoxia and en‐

Recent genetic and pharmacological studies have revealed that targeting abnormal tumor vessel function by the induction of vessel normalization can offer alternative options for an‐ ti-angiogenic therapy. Vessel normalization can be achieved by several different approaches, including blockade of VEGF, genetic modulation of the oxygen sensors prolyl hydroxylase domain containing protein 2 (PHD2), targeting of mechanisms that affect pericyte coverage and vessel maturation, and targeting myeloid cells via blockade or genetic loss of PlGF. Ves‐ sel normalization could provide a means to increase the responsiveness to chemotherapy, immunotherapy, or radiation, and may contribute to restricting tumor dissemination (Rolny

One recent study demonstrated that boron targeting of the largest possible proportion of tu‐ mor cells contributes to the success of boron neutron capture therapy (BNCT), and tumor blood vessel normalization improves the delivery of boron to the tumor. In this study, blood vessel normalization was induced by administering two doses of thalidomide (Th) in tumorbearing hamsters on two consecutive days. The effect of blood vessel normalization to en‐ hance the efficacy of boronophenylalanine (BPA) administration was assessed through *in vivo* BNCT studies at the RA-3 Nuclear Reactor utilizing tumor-bearing hamsters. Overall tumor control at 28 days post-treatment was significantly higher for Th+ BPA-BNCT than for Th- BPA-BNCT with a tumor volume reduction of 84 ± 3% in the Th+ BPA-BNCT group compared to 67 ± 5% in the Th- BPA-BNCT group. Pretreatment with thalidomide enhanced the therapeutic efficacy of BNCT and reduced precancerous tissue toxicity (Molinari et al., 2012). Some studies confirmed, however, that antibodies to VEGF in combination with che‐ motherapeutic agents produce synergistic cytotoxicity in a range of cancers. Research data

itself (Ferrara, 2010; Gordon et al., 2010).

hancing tumor cell invasiveness (Carmeliet and Jain, 2011).

et al., 2011; Schmidt and Carmeliet, 2011).


#### **4.3. Vascular normalization in anti-angiogenic cancer therapy**

Normal vasculature comprises organized layers of endothelial cells (ECs) and pericytes. There is evidence for paracrine signaling between ECs and specialized organ-specific cells; hence, there is some variation in the structure and function of blood vessels depending on their anatomic location. Pericyte-EC crosstalk facilitates vascular growth and homeostasis, and once vessels mature, these cells become dormant. Blood vessel proliferation is an essen‐ tial physiological process, and vessel sprouting is one of the major mechanisms of expansion in the network of vessels in growing tumors through filopodia and endothelial stalk cells.

Unlike blood vessels in normal tissue, the tumor-associated vasculature is irregular and unstable, probably due to the over-production of pro-angiogenic proteins such as VEGF. Tumor vessels are distinct in several respects relative to normal vasculature as they are disorganized and tortuous and their spatial distribution is significantly heterogeneous, resulting in uneven drug distribution in tumors. Tumor vessels do not follow the hierar‐ chy of arterioles, capillaries and venules, and tumor vessels are leakier than normal ves‐ sels since tumor-associated endothelial cells are widened and loosely connected. Recent studies suggest that tumor ECs have cytogenetic abnormalities including aneuploidy, multiple chromosomes, and multiple centrosomes, raising the possibility that such insta‐ bility may contribute to resistance to anti-angiogenic therapies. Tumor-associated blood vessels are excessively branched and hemorrhagic, and blood flow through these mal‐ formed vessels is often chaotic and may impede delivery of chemotherapy to the tumor itself (Ferrara, 2010; Gordon et al., 2010).

**2.** The role of VE-cadherin in neovascularization has been shown in a number of studies.

apoptotic signal of VEGFs.

224 Research Directions in Tumor Angiogenesis

(Makrilia et al., 2009)

sults in regression of newly formed vessels.

**4.3. Vascular normalization in anti-angiogenic cancer therapy**

**3.** Cadherins have been shown to establish endothelial cell junctional stability in the vessel wall and enhance endothelial cell survival by promoting the transmission of the anti-

**4.** Cyclooxygenase-2, an enzyme known to regulate cellular processes such as apoptosis,

**5.** The fibrinolytic system is another angiogenesis target, and the activation of this system depends on the conversion of plasminogen to plasmin by the tissue-type plasminogen

**6.** Matrix metalloproteinases (MMPs) are a family of enzymes that cleave the extracellular matrix, a process which is considered important for the formation of new blood vessels. Inhibition of the activity of MMPs seems to be a crucial step in the process of vessel sta‐ bilization during the resolution phase of angiogenesis, as uncontrolled proteolysis re‐

**7.** The hypoxia-inducible factors (HIFs) mediate transcriptional responses to localized hy‐ poxia in normal tissues and in cancers, and HIFs have been shown to promote tumor progression by altering cellular metabolism and stimulating angiogenesis. Under condi‐ tions of abundant oxygen (N8–10%), HIF-α proteins are translated, but the proteins are rapidly degraded. Stabilization of HIF proteins in hypoxic cancer cells is thought to pro‐ mote tumor progression, largely by inducing the localized expression of specific target genes encoding VEGF, glycolytic enzymes (PGK, ALDA), glucose transporters (GLUT1) and proteins regulating motility (lysl oxidase) and metastasis (CXCR4, E-cadherin).

Normal vasculature comprises organized layers of endothelial cells (ECs) and pericytes. There is evidence for paracrine signaling between ECs and specialized organ-specific cells; hence, there is some variation in the structure and function of blood vessels depending on their anatomic location. Pericyte-EC crosstalk facilitates vascular growth and homeostasis, and once vessels mature, these cells become dormant. Blood vessel proliferation is an essen‐ tial physiological process, and vessel sprouting is one of the major mechanisms of expansion in the network of vessels in growing tumors through filopodia and endothelial stalk cells.

Unlike blood vessels in normal tissue, the tumor-associated vasculature is irregular and unstable, probably due to the over-production of pro-angiogenic proteins such as VEGF. Tumor vessels are distinct in several respects relative to normal vasculature as they are disorganized and tortuous and their spatial distribution is significantly heterogeneous, resulting in uneven drug distribution in tumors. Tumor vessels do not follow the hierar‐ chy of arterioles, capillaries and venules, and tumor vessels are leakier than normal ves‐ sels since tumor-associated endothelial cells are widened and loosely connected. Recent studies suggest that tumor ECs have cytogenetic abnormalities including aneuploidy,

also has been shown to have an angiogenic effect via thromboxane-A2.

activator (tPA) and the urokinase-type plasminogen activator (uPA).

A new therapeutic strategy targeting tumor vasculature has gained increased attention in the scientific community. This method involves targeting abnormal tumor vessel function by inducing vessel normalization. It is well known that tumor blood vessels are highly abnormal in structure and function, characterized by a tortuous, chaotic, and irregular branching network. In the tumor vasculature, ECs are highly activated, lose their polari‐ ty and alignment, and detach from the basement membrane, all resulting in a leaky, fe‐ nestrated network that facilitates bleeding and increases interstitial fluid pressure. Apart from the ECs, the entire vessel wall, including the basement membrane and the covering pericytes, becomes abnormal in most tumors. Tumor ECs are typically covered with few‐ er and more abnormal pericytes, and their associated basement membrane is only loose‐ ly associated and inhomogeneous in structure. It is suspected that this abnormal vasculature impedes the distribution of chemotherapy and oxygen. Traditional anti-an‐ giogenic therapy aims to maximally inhibit angiogenesis and to prune existing tumor vessels, however, this strategy can also increase the risk of aggravating hypoxia and en‐ hancing tumor cell invasiveness (Carmeliet and Jain, 2011).

Recent genetic and pharmacological studies have revealed that targeting abnormal tumor vessel function by the induction of vessel normalization can offer alternative options for an‐ ti-angiogenic therapy. Vessel normalization can be achieved by several different approaches, including blockade of VEGF, genetic modulation of the oxygen sensors prolyl hydroxylase domain containing protein 2 (PHD2), targeting of mechanisms that affect pericyte coverage and vessel maturation, and targeting myeloid cells via blockade or genetic loss of PlGF. Ves‐ sel normalization could provide a means to increase the responsiveness to chemotherapy, immunotherapy, or radiation, and may contribute to restricting tumor dissemination (Rolny et al., 2011; Schmidt and Carmeliet, 2011).

One recent study demonstrated that boron targeting of the largest possible proportion of tu‐ mor cells contributes to the success of boron neutron capture therapy (BNCT), and tumor blood vessel normalization improves the delivery of boron to the tumor. In this study, blood vessel normalization was induced by administering two doses of thalidomide (Th) in tumorbearing hamsters on two consecutive days. The effect of blood vessel normalization to en‐ hance the efficacy of boronophenylalanine (BPA) administration was assessed through *in vivo* BNCT studies at the RA-3 Nuclear Reactor utilizing tumor-bearing hamsters. Overall tumor control at 28 days post-treatment was significantly higher for Th+ BPA-BNCT than for Th- BPA-BNCT with a tumor volume reduction of 84 ± 3% in the Th+ BPA-BNCT group compared to 67 ± 5% in the Th- BPA-BNCT group. Pretreatment with thalidomide enhanced the therapeutic efficacy of BNCT and reduced precancerous tissue toxicity (Molinari et al., 2012). Some studies confirmed, however, that antibodies to VEGF in combination with che‐ motherapeutic agents produce synergistic cytotoxicity in a range of cancers. Research data shows that the process of normalization of tumor blood vessel structure is not always bene‐ ficial. In the case of cerebral tumors, for example, the process of tumor vessel normalization may induce a re-establishment of the low permeability characteristics of normal brain micro‐ vasculature, preventing the delivery of chemotherapeutics (Ribatti, 2011).

mor vessels were thought to be lined exclusively by endothelial cells (ECs). Therapeutic benefits from promising anti-angiogenic strategies targeting genetically stable ECs, how‐ ever, are frequently limited by the development of resistance, implying an oversimplified view of tumor vasculature. Recently, great advances in our understanding of cancer vas‐ cularization have emerged with several novel mechanisms proposed. In fact, the latest studies of the most lethal ovarian cancers characterized by widespread metastases within the peritoneal cavity have revealed that in addition to ECs, other cells, including bone marrow-derived and plastic tumor cells, contribute to tumor vascularization There are two proposed mechanisms by which tumor-infiltrating bone marrow-derived cells might participate in tumor angiogenesis: (1) direct incorporation in the tumor vasculature and (2) as a source of angiogenic factors such as VEGF-A and MMP-9, which may in turn in‐

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Current anti-angiogenic therapies have been designed on the assumption that endothelial cells forming the tumor vasculature exhibit genetic stability. Recent studies demonstrate that this is not the case. Tumor endothelial cells possess a distinct phenotype, differing from normal endothelial cells at both the molecular and functional levels. This finding challenges the concept that tumor angiogenesis exclusively depends on normal endothelial cell recruit‐ ment from the surrounding vascular network. Indeed, recent data suggest alternative strat‐ egies for tumor vascularization, and it has been reported that tumor vessels may be derived from an intratumor embryonic-like vasculogenesis. This condition might be due to differen‐ tiation of normal stem and progenitor cells of either hematopoietic origin or cells resident in tissues. Cancer stem cells may also participate in tumor vasculogenesis by virtue of their

During cancer progression, tumors require a blood supply for growth and use the blood supply for metastatic dissemination. It is logical that a stronger ability to form *de novo* net‐ works and channels providing a stable blood supply may confer a survival advantage for tumors. Ovarian cancers, as discussed previously, can generate tumor vasculature from di‐ verse origins, including EC, EPC, and tumor cells, reflecting a vast capacity for neovasculari‐ zation, which may help to explain its high malignancy. Thus, anti-angiogenic and vascular targeting strategies against alternative tumor vascularization mechanisms are clearly prom‐

The existence of multiple signal pathways and complex regulatory systems in vascular for‐ mation means that inhibition of just a single pathway will presumably trigger alternative vascularization mechanisms and additional signal pathways. Therefore, exploring other novel signals in neovascularization is essential for further studies to efficiently target blood vessels in cancer therapy. On the other hand, with the emergence of the concept of normali‐ zation of tumor vasculature as a novel form of anti-angiogenic therapy, new vascular signals involved in vascular remodeling are becoming appealing therapeutic strategies to research‐ ers, as a better understanding of these normalization mechanisms may ultimately lead to more effective therapies. Indeed, several novel ligand/receptor pathways are emerging to in‐ clude: Slit/Robo, semaphoring/plexins, Netrin-1/UNC5B, Delta-like 4/Notch, and others. In‐ terestingly, the first three ligand/receptor pairs are all formerly known to be involved with

crease the bioavailability of angiogenic factors.

stem and progenitor cell properties (Bussolati et al., 2011).

ising as improved, more efficacious cancer therapies.

Despite having an abundant number of vessels, tumors are usually hypoxic and nutrientdeprived because their vessels malfunction. Such abnormal milieu can fuel disease progres‐ sion and resistance to treatment. Traditional anti-angiogenesis strategies attempt to reduce the tumor vascular supply, but their success is restricted by insufficient efficacy or develop‐ ment of resistance. Preclinical and initial clinical evidence have shown that normalization of tumor vascular abnormalities is emerging as a complementary therapeutic paradigm for cancer therapy and other vascular disorders, which affect more than half a billion people worldwide. Clearly, additional randomized prospective multi-centered trials should be con‐ ducted in larger patient populations to confirm these initial clinical data. In addition, critical questions regarding whether vessel normalizing agents can improve tumor oxygenation and drug delivery in human cancers remain to be answered (Carmeliet and Jain, 2011).

#### **4.4. New vascularization/angiogenesis mechanisms in cancer therapy**

Before discussing the different ways a tumor is vascularized, we should emphasize that these mechanisms are not mutually exclusive. In fact, in most cases, angiogenesis and neo‐ vascularization mechanisms are interlinked, being involved concurrently in physiological as well as in pathological angiogenesis. Although the molecular regulation of endothelial sprouting has been extensively studied and reviewed in the literature, the morphogenic and molecular events associated with alternative cancer vascularization mechanisms are not nearly as well understood. Cancer cells are not generally controlled by normal regulatory mechanisms, but tumor growth is highly dependent on the supply of oxygen, nutrients, and host-derived regulators. It is now established that tumor vasculature is not necessarily de‐ rived from endothelial cell sprouting. Cancer tissue can acquire vasculature by a variety of mechanisms to include co-opting pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis, or vasculogenic mimicry. The best-known molecular pathway driving tumor vascularization is the hypoxia-adaptation mechanism. Other pathways involving a broad and diverse spectrum of genetic aberrations, however, are associated with the development of the "angiogenic phenotype." Based on this knowledge, novel forms of antivascular modalities have been developed in the past decade.

When applying these targeted therapies, the stage of tumor progression, the type of vascula‐ rization of the given cancer tissue, and the molecular machinery behind the vascularization process all need to be considered. A further challenge is finding the most appropriate com‐ binations of antivascular therapies and standard radio- and chemotherapies. The most promising therapeutic plan of action will involve the integration of recent discoveries in this field into a rational strategy to for developing effective clinical modalities using antivascular therapy for cancer (Döme et al., 2007).

Neovascularization is essential for tumor growth and metastasis. An adequate vascula‐ ture feeds tumor growth and enhances the potential of metastasis. For many years, tu‐ mor vessels were thought to be lined exclusively by endothelial cells (ECs). Therapeutic benefits from promising anti-angiogenic strategies targeting genetically stable ECs, how‐ ever, are frequently limited by the development of resistance, implying an oversimplified view of tumor vasculature. Recently, great advances in our understanding of cancer vas‐ cularization have emerged with several novel mechanisms proposed. In fact, the latest studies of the most lethal ovarian cancers characterized by widespread metastases within the peritoneal cavity have revealed that in addition to ECs, other cells, including bone marrow-derived and plastic tumor cells, contribute to tumor vascularization There are two proposed mechanisms by which tumor-infiltrating bone marrow-derived cells might participate in tumor angiogenesis: (1) direct incorporation in the tumor vasculature and (2) as a source of angiogenic factors such as VEGF-A and MMP-9, which may in turn in‐ crease the bioavailability of angiogenic factors.

shows that the process of normalization of tumor blood vessel structure is not always bene‐ ficial. In the case of cerebral tumors, for example, the process of tumor vessel normalization may induce a re-establishment of the low permeability characteristics of normal brain micro‐

Despite having an abundant number of vessels, tumors are usually hypoxic and nutrientdeprived because their vessels malfunction. Such abnormal milieu can fuel disease progres‐ sion and resistance to treatment. Traditional anti-angiogenesis strategies attempt to reduce the tumor vascular supply, but their success is restricted by insufficient efficacy or develop‐ ment of resistance. Preclinical and initial clinical evidence have shown that normalization of tumor vascular abnormalities is emerging as a complementary therapeutic paradigm for cancer therapy and other vascular disorders, which affect more than half a billion people worldwide. Clearly, additional randomized prospective multi-centered trials should be con‐ ducted in larger patient populations to confirm these initial clinical data. In addition, critical questions regarding whether vessel normalizing agents can improve tumor oxygenation and

drug delivery in human cancers remain to be answered (Carmeliet and Jain, 2011).

Before discussing the different ways a tumor is vascularized, we should emphasize that these mechanisms are not mutually exclusive. In fact, in most cases, angiogenesis and neo‐ vascularization mechanisms are interlinked, being involved concurrently in physiological as well as in pathological angiogenesis. Although the molecular regulation of endothelial sprouting has been extensively studied and reviewed in the literature, the morphogenic and molecular events associated with alternative cancer vascularization mechanisms are not nearly as well understood. Cancer cells are not generally controlled by normal regulatory mechanisms, but tumor growth is highly dependent on the supply of oxygen, nutrients, and host-derived regulators. It is now established that tumor vasculature is not necessarily de‐ rived from endothelial cell sprouting. Cancer tissue can acquire vasculature by a variety of mechanisms to include co-opting pre-existing vessels, intussusceptive microvascular growth, postnatal vasculogenesis, glomeruloid angiogenesis, or vasculogenic mimicry. The best-known molecular pathway driving tumor vascularization is the hypoxia-adaptation mechanism. Other pathways involving a broad and diverse spectrum of genetic aberrations, however, are associated with the development of the "angiogenic phenotype." Based on this knowledge, novel forms of antivascular modalities have been developed in the past decade. When applying these targeted therapies, the stage of tumor progression, the type of vascula‐ rization of the given cancer tissue, and the molecular machinery behind the vascularization process all need to be considered. A further challenge is finding the most appropriate com‐ binations of antivascular therapies and standard radio- and chemotherapies. The most promising therapeutic plan of action will involve the integration of recent discoveries in this field into a rational strategy to for developing effective clinical modalities using antivascular

Neovascularization is essential for tumor growth and metastasis. An adequate vascula‐ ture feeds tumor growth and enhances the potential of metastasis. For many years, tu‐

**4.4. New vascularization/angiogenesis mechanisms in cancer therapy**

therapy for cancer (Döme et al., 2007).

vasculature, preventing the delivery of chemotherapeutics (Ribatti, 2011).

226 Research Directions in Tumor Angiogenesis

Current anti-angiogenic therapies have been designed on the assumption that endothelial cells forming the tumor vasculature exhibit genetic stability. Recent studies demonstrate that this is not the case. Tumor endothelial cells possess a distinct phenotype, differing from normal endothelial cells at both the molecular and functional levels. This finding challenges the concept that tumor angiogenesis exclusively depends on normal endothelial cell recruit‐ ment from the surrounding vascular network. Indeed, recent data suggest alternative strat‐ egies for tumor vascularization, and it has been reported that tumor vessels may be derived from an intratumor embryonic-like vasculogenesis. This condition might be due to differen‐ tiation of normal stem and progenitor cells of either hematopoietic origin or cells resident in tissues. Cancer stem cells may also participate in tumor vasculogenesis by virtue of their stem and progenitor cell properties (Bussolati et al., 2011).

During cancer progression, tumors require a blood supply for growth and use the blood supply for metastatic dissemination. It is logical that a stronger ability to form *de novo* net‐ works and channels providing a stable blood supply may confer a survival advantage for tumors. Ovarian cancers, as discussed previously, can generate tumor vasculature from di‐ verse origins, including EC, EPC, and tumor cells, reflecting a vast capacity for neovasculari‐ zation, which may help to explain its high malignancy. Thus, anti-angiogenic and vascular targeting strategies against alternative tumor vascularization mechanisms are clearly prom‐ ising as improved, more efficacious cancer therapies.

The existence of multiple signal pathways and complex regulatory systems in vascular for‐ mation means that inhibition of just a single pathway will presumably trigger alternative vascularization mechanisms and additional signal pathways. Therefore, exploring other novel signals in neovascularization is essential for further studies to efficiently target blood vessels in cancer therapy. On the other hand, with the emergence of the concept of normali‐ zation of tumor vasculature as a novel form of anti-angiogenic therapy, new vascular signals involved in vascular remodeling are becoming appealing therapeutic strategies to research‐ ers, as a better understanding of these normalization mechanisms may ultimately lead to more effective therapies. Indeed, several novel ligand/receptor pathways are emerging to in‐ clude: Slit/Robo, semaphoring/plexins, Netrin-1/UNC5B, Delta-like 4/Notch, and others. In‐ terestingly, the first three ligand/receptor pairs are all formerly known to be involved with neuronal axon guidance, implying a possibility that other neural guidance cues may also function as vascular signals. Agents developed from these pathways that control the mor‐ phology of the vascular system can induce tumor vascular normalization and, thus, alleviate hypoxia and increase the efficacy of conventional therapies if both are carefully scheduled. Alternatively, blockade of these pathways may result in increased amounts of immature, nonfunctioning vessels, which results in reduced tumor growth, as is the case with blockade of Delta-like 4 (Tang et al., 2009)

gen to the tumor, increasing its growth. Thus, angiogenesis plays a key role in cancer pro‐ gression and development of metastases. Anti-angiogenic therapies have demonstrated significant efficacy in some patients, however, several side effects of anti-angiogenic therapy have been noted in the literature. In addition, the cost of several of these therapies is very

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

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VEGF is an important growth factor that promotes angiogenesis and participates in a variety of physiological and pathological processes. Over-expression of VEGF results in increased angiogenesis in normal and pathological conditions. There is significant evidence that alter‐ native splicing of VEGF gene and other genes involved in angiogenesis can regulate the an‐ giogenic process in tumors. Alternative therapies might replace or improve existing ones. In particular, there is a place for pharmaceutical modulation of angiogenic factors affecting pre-mRNA splicing. This can be brought about not only by alteration in the splicing of hepa‐ rin-binding isoforms of VEGF but also by the relative balance of pro- and anti-angiogenic isoforms. The concept of an angiogenic splicing phenotype, which controls a number of dif‐ ferent proteins that can be activated or deactivated by over-expression or activation of key splicing control factors, is an opportunity for intervention that should be explored in greater

In eukaryotes genes consist of coding sequences (exons) interspersed with non-coding se‐ quences (introns). The regulation of alternative inclusion/exclusion of exons, or parts of exons, during RNA processing of pre-mRNA into mRNA (alternative splicing) allows a dra‐ matic increase of the protein repertoire versus the gene repertoire. In a number of cases, al‐ ternative splicing of mRNA has been shown to generate proteins with distinct, sometimes opposite, functions from a given gene. Angiogenesis is the process of vascularization in physiological conditions, and there are a number of pathologies, including cancer, where an‐ giogenesis favors tumor progression and dissemination of metastasis. In this chapter, we discuss some key examples showing how alternative splicing may induce a switch from an‐ ti-angiogenic to pro-angiogenic functions reciprocally. For some of these splicing events, the molecular mechanisms that trigger alternative splicing toward one or the other direction are now becoming known. The emergence of strategies enabling the regulation of alternative

In tumors of the nervous system, tumor derived endothelial cells (TECs) may present the same genetic amplification or chromosomal aberrations of the tumor of origin. In human xenografts of renal carcinoma, melanoma, and liposarcoma, murine TECs are aneuploid, bearing alterations similar to those observed in human TECs. This observation remains un‐ explained. It cannot be ascribed to cell fusion among tumor and endothelial cells, as no hu‐ man DNA was present in murine TECs. The researchers who conducted this research speculated that the tumor microenvironment may produce factors capable of inducing ge‐ netic instability, or loss of tumor suppressors and/or check point activity, resulting in aneu‐ ploidy. Altogether, these data suggest two different explanations for the origin of TECs. The first is that they originate from a common progenitor of tumor and endothelial cells targeted by neoplastic transformation; the second is that the effect of the tumor microenvironment

splicing opens new routes for anti-angiogenic therapies (Munaut et al., 2010).

high and may not affordable for many patients worldwide

detail in the near future (Biselli-Chicote et al., 2012).

leads to genetic instability (Gardlik et al., 2011a).

In addition, newly published findings suggest that vessels in many non-malignant dis‐ eases are also abnormal. Pharmacological approaches used to normalize vessels in cancer can also induce vessel normalization in other angiogenic disorders in animal models and in patients. Moreover, vascular normalization with bevacizumab has provided the first medical treatment to improve hearing in patients with type II neurofibromatosis. Despite treatment advances for coronary and peripheral arterial disease, the burden of these ill‐ nesses remains high. To this end, normalization of abnormal vessels has been proposed as a novel strategy to stabilize vulnerable atherosclerotic plaques. One of the challenges for this therapeutic approach is that these strategies stimulate the formation of immature, leaky and disorganized vessels that are poorly perfused, exhibit signs of vessel disorgani‐ zation and are prone to regression once therapy is halted. Therapeutic normalization of such neovessels would offer the advantage of creating more mature vessels that could deliver oxygen and nutrients more rapidly and efficiently to the ischemic tissue and thereby restore tissue performance (Carmeliet and Jain, 2011).

In contrast with the anti-angiogenic therapy, vascular targeting therapy aims at destroy‐ ing the existing vasculature of a tumor. Three different classes of vascular targeting ther‐ apeutics have been proposed, cytoskeletal disruption, targeted gene delivery, and drug targeting of tumor endothelial cells. The first class, cytoskeletal disruptors, utilizes a combination of combretastatin derivatives which stops blood flow and inhibits tumor growth through the disruption of the tubulin cytoskeleton of endothelial cells which, in turn, leads to vasculature thrombosis. The second class of vascular targeted therapeutics is targeted gene delivery to the neovasculature. This is achieved by using cationic nano‐ particles bound to an integrin ανβ3 directed ligand that delivers a mutant gene to tumor vessels. The third class of vascular targeting therapeutics is cationic liposome-based vas‐ cular targeting therapy, which relies on a selective propensity for drug delivery to acti‐ vated tumor endothelial cells. The mechanism on which this targeted drug delivery is based relies on the negative charge associated with angiogenic endothelial cells which in turn attracts cationic liposomes which can actively bind negatively charged angiogenic endothelial cells and deliver cytotoxic drugs (Makrilia et al., 2009).

#### **4.5. Anti-angiogenic gene therapy for cancer**

Tumor growth and progression depends on angiogenesis, a process of new blood vessel for‐ mation from preexisting vascular endothelial cells. Tumors promote angiogenesis by secret‐ ing or activating angiogenic factors that stimulate endothelial proliferation and migration and capillary morphogenesis. The newly formed blood vessels provide nutrients and oxy‐ gen to the tumor, increasing its growth. Thus, angiogenesis plays a key role in cancer pro‐ gression and development of metastases. Anti-angiogenic therapies have demonstrated significant efficacy in some patients, however, several side effects of anti-angiogenic therapy have been noted in the literature. In addition, the cost of several of these therapies is very high and may not affordable for many patients worldwide

neuronal axon guidance, implying a possibility that other neural guidance cues may also function as vascular signals. Agents developed from these pathways that control the mor‐ phology of the vascular system can induce tumor vascular normalization and, thus, alleviate hypoxia and increase the efficacy of conventional therapies if both are carefully scheduled. Alternatively, blockade of these pathways may result in increased amounts of immature, nonfunctioning vessels, which results in reduced tumor growth, as is the case with blockade

In addition, newly published findings suggest that vessels in many non-malignant dis‐ eases are also abnormal. Pharmacological approaches used to normalize vessels in cancer can also induce vessel normalization in other angiogenic disorders in animal models and in patients. Moreover, vascular normalization with bevacizumab has provided the first medical treatment to improve hearing in patients with type II neurofibromatosis. Despite treatment advances for coronary and peripheral arterial disease, the burden of these ill‐ nesses remains high. To this end, normalization of abnormal vessels has been proposed as a novel strategy to stabilize vulnerable atherosclerotic plaques. One of the challenges for this therapeutic approach is that these strategies stimulate the formation of immature, leaky and disorganized vessels that are poorly perfused, exhibit signs of vessel disorgani‐ zation and are prone to regression once therapy is halted. Therapeutic normalization of such neovessels would offer the advantage of creating more mature vessels that could deliver oxygen and nutrients more rapidly and efficiently to the ischemic tissue and

In contrast with the anti-angiogenic therapy, vascular targeting therapy aims at destroy‐ ing the existing vasculature of a tumor. Three different classes of vascular targeting ther‐ apeutics have been proposed, cytoskeletal disruption, targeted gene delivery, and drug targeting of tumor endothelial cells. The first class, cytoskeletal disruptors, utilizes a combination of combretastatin derivatives which stops blood flow and inhibits tumor growth through the disruption of the tubulin cytoskeleton of endothelial cells which, in turn, leads to vasculature thrombosis. The second class of vascular targeted therapeutics is targeted gene delivery to the neovasculature. This is achieved by using cationic nano‐ particles bound to an integrin ανβ3 directed ligand that delivers a mutant gene to tumor vessels. The third class of vascular targeting therapeutics is cationic liposome-based vas‐ cular targeting therapy, which relies on a selective propensity for drug delivery to acti‐ vated tumor endothelial cells. The mechanism on which this targeted drug delivery is based relies on the negative charge associated with angiogenic endothelial cells which in turn attracts cationic liposomes which can actively bind negatively charged angiogenic

Tumor growth and progression depends on angiogenesis, a process of new blood vessel for‐ mation from preexisting vascular endothelial cells. Tumors promote angiogenesis by secret‐ ing or activating angiogenic factors that stimulate endothelial proliferation and migration and capillary morphogenesis. The newly formed blood vessels provide nutrients and oxy‐

thereby restore tissue performance (Carmeliet and Jain, 2011).

endothelial cells and deliver cytotoxic drugs (Makrilia et al., 2009).

**4.5. Anti-angiogenic gene therapy for cancer**

of Delta-like 4 (Tang et al., 2009)

228 Research Directions in Tumor Angiogenesis

VEGF is an important growth factor that promotes angiogenesis and participates in a variety of physiological and pathological processes. Over-expression of VEGF results in increased angiogenesis in normal and pathological conditions. There is significant evidence that alter‐ native splicing of VEGF gene and other genes involved in angiogenesis can regulate the an‐ giogenic process in tumors. Alternative therapies might replace or improve existing ones. In particular, there is a place for pharmaceutical modulation of angiogenic factors affecting pre-mRNA splicing. This can be brought about not only by alteration in the splicing of hepa‐ rin-binding isoforms of VEGF but also by the relative balance of pro- and anti-angiogenic isoforms. The concept of an angiogenic splicing phenotype, which controls a number of dif‐ ferent proteins that can be activated or deactivated by over-expression or activation of key splicing control factors, is an opportunity for intervention that should be explored in greater detail in the near future (Biselli-Chicote et al., 2012).

In eukaryotes genes consist of coding sequences (exons) interspersed with non-coding se‐ quences (introns). The regulation of alternative inclusion/exclusion of exons, or parts of exons, during RNA processing of pre-mRNA into mRNA (alternative splicing) allows a dra‐ matic increase of the protein repertoire versus the gene repertoire. In a number of cases, al‐ ternative splicing of mRNA has been shown to generate proteins with distinct, sometimes opposite, functions from a given gene. Angiogenesis is the process of vascularization in physiological conditions, and there are a number of pathologies, including cancer, where an‐ giogenesis favors tumor progression and dissemination of metastasis. In this chapter, we discuss some key examples showing how alternative splicing may induce a switch from an‐ ti-angiogenic to pro-angiogenic functions reciprocally. For some of these splicing events, the molecular mechanisms that trigger alternative splicing toward one or the other direction are now becoming known. The emergence of strategies enabling the regulation of alternative splicing opens new routes for anti-angiogenic therapies (Munaut et al., 2010).

In tumors of the nervous system, tumor derived endothelial cells (TECs) may present the same genetic amplification or chromosomal aberrations of the tumor of origin. In human xenografts of renal carcinoma, melanoma, and liposarcoma, murine TECs are aneuploid, bearing alterations similar to those observed in human TECs. This observation remains un‐ explained. It cannot be ascribed to cell fusion among tumor and endothelial cells, as no hu‐ man DNA was present in murine TECs. The researchers who conducted this research speculated that the tumor microenvironment may produce factors capable of inducing ge‐ netic instability, or loss of tumor suppressors and/or check point activity, resulting in aneu‐ ploidy. Altogether, these data suggest two different explanations for the origin of TECs. The first is that they originate from a common progenitor of tumor and endothelial cells targeted by neoplastic transformation; the second is that the effect of the tumor microenvironment leads to genetic instability (Gardlik et al., 2011a).

In addition, the differentiation of cancer stem cells into endothelial cells and the consequent involvement of these cells in tumor vascularization have been recently described in different tumors. The definitive proof that tumor stem cells are bipotent relies on the ability of clones of tumor stem cells to differentiate *in vitro* and *in vivo* into both tumor epithelial and endo‐ thelial cells (Bussolati et al., 2008; 2009). More recently, the ability of tumor cells to differen‐ tiate into endothelial cells has also been reported for cancer stem cells present in neuroblastomas (Alvero et al., 2009). In particular, only a fraction of stem cells, characterized by CD133 and CD144 co-expression (Wang et al., 2010), or in a recent report, by co-expres‐ sion of Oct4 and tenascin C, shows vasculogenic potential and is selectively localized in the proximity of tumor vessels (Pezzolo et al., 2011). An alternative mechanism of tumor blood perfusion implies the possibility that tumor cells form channels connected to the tumor vas‐ culature, a process defined as "vasculogenic mimicry". Alternatively, the process of tumor vasculogenic mimicry could be interpreted as being dependent on tumor stem cells (El Hal‐ lanti et al., 2010; Yao et al., 2011), as a transitional step in stem cell differentiation toward endothelial cells (Bussolati et al., 2011).

oping highly tumor-specific anti-angiogenic applications which utilize drugs such as the

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

Our current knowledge of the anti-cancer mechanism of ARTs is derived from our knowledge of the antimalarial activity of ARTs. The potent anti-cancer activity of ARTs can be attributed to the endoperoxide bond of the ARTs compounds which is shared with the antiparasitic activity of ARTs. In most of the cancers studied, preloading of can‐ cer cells with iron or iron-saturated holotransferrin triggers ART cytotoxicity with an in‐ crease in the activity of ARTs. It has been hypothesized that iron-activated ARTs induce damage by release of highly alkylating carbon-centered radicals and radical oxygen spe‐ cies (ROS). Generation of free radicals may play a role in the cell alterations reported in ARTs-treated cancer cells such as enhanced apoptosis, arrest of growth, inhibition of an‐ giogenesis, and DNA damage. In addition, ARTs-sensitive cancer cells have been shown to have down-regulated expression of oxidation enzymes while cancer cells with over-ex‐ pression of these molecules are more resistant to ARTs therapy. The antineoplastic toxici‐ ty of ARTs appears to be also modulated by calcium metabolism, endoplasmic reticulum (ER) stress, and the expression of the translationally controlled tumor protein, TCTP, a binding calcium protein which has been also postulated as a parasite target. Although the expression of the TCTP gen, *tctp,* was initially correlated with cancer cell responses to ARTs, a functional role for TCTP in the anti-cancer activity of ARTs has yet to be

+ ATPase (SERCA) as a

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231

ARTs which have demonstrated anti-angiogenic efficacy with little toxicity.

**5.1. Further targeting anti-angiogenesis of ART and its derivatives**

found. As for malaria parasites, the role of sarcoendoplasmic Ca2

amined. (Firestone and Sundar, 2009).

target of ARTs in cancer cells has also been explored (Crespo-Ortiz and Wei, 2012).

ART and its bioactive derivatives elicit their anti-cancer effects by concurrently activating, inhibiting and/or attenuating multiple complementary cell signaling pathways, especially those associated with the VEGF family, based on published data. The precise mechanism of new and alternative actions and other primary targets of ARTs, however, will require fur‐ ther study. In anti-cancer therapy, it has been postulated that ARTs may target organelles such as pathways involving PlGF growth factors (a VEGF subfamily),, angiopoietins, such as the Angs proteins, the Notch signaling pathway, signaling pathways involving fibroblast growth factors (FGFs), and the matrix metalloproteinase (MMPs) family of enzymes (Cres‐ po-Ortiz and Wei, 2012). In a recent study, investigators discovered a panel of genes con‐ taining many fundamental regulators of angiogenic regulators, such as VEGF, was found that correlate with the cellular response to AS. These genes govern the stimulation, prolifer‐ ation and migration of endothelial cells, a fundamental step in vessel formation. The investi‐ gators decided to further limit their cluster analysis by including in the cluster analysis only those genes whose mRNA expression correlated with GI50 values of at least four ARTs (An‐ fosso et al., 2006). Three human genes coding for VEGF (VEGFA, VEGFB, and VEGFC) were discovered in this cluster of ARTs-affected angiogenic regulating genes.. Despite the contin‐ uous investigations on new targets, the ART compounds exert common as well as distinct cellular effects depending on the phenotype and tissue origin of the human cancer cells ex‐

In cancer therapy, recent investigations have focused on using genetically modified bacteria to actually block tumor angiogenesis. Despite recent progress, only a few studies on bacteri‐ al tumor therapy have focused on anti-angiogenesis. Bacteria-mediated anti-angiogenic ther‐ apy for cancer, however, is an attractive approach given that solid tumors are often characterized by increased vascularization.

The first modern attempts at using bacteria for therapeutic purposes were made more than 40 years ago by showing that bacteria could predominantly replicate in solid tu‐ mors. The first indications of this phenomenon, however, date back to the 19th century. These findings remained largely unexplored until the turn of the 20th century, when on‐ colytic bacteria capable of lysing host cells were first studied by various research groups. The utilization of bacterial systems for therapeutic anti-cancer purposes is further en‐ hanced by genetic modifications, which make them a very promising tool for targeted delivery of genes and their products. Specific advantages of using bacteria for anti-can‐ cer gene therapy include the natural oncolytic potential of some strains/species, direct targeting of tumor tissues and the ease of positive regulation/eradication. The anti-cancer effect of tumor-targeting bacteria can also be achieved after oral administration, which may circumvent the use of intravenous routes of delivery and associated adverse events of intravenous therapy (Chen et al., 2009; Gardlik et al., 2011b).

#### **5. Further development of ARTs as anti-angiogenic cancer agents**

Cancer angiogenesis has been confirmed by measurement of high proliferation indices for endothelial cells, not only in rapidly growing animal tumors, but also in human tumors. The rationale for developing anti-angiogenic strategies for cancer therapy was based on the fact that physiological angiogenesis only occurs in a limited number of situations, such as in wound healing and during menstrual cycle. This suggests there is an opportunity for devel‐ oping highly tumor-specific anti-angiogenic applications which utilize drugs such as the ARTs which have demonstrated anti-angiogenic efficacy with little toxicity.

#### **5.1. Further targeting anti-angiogenesis of ART and its derivatives**

In addition, the differentiation of cancer stem cells into endothelial cells and the consequent involvement of these cells in tumor vascularization have been recently described in different tumors. The definitive proof that tumor stem cells are bipotent relies on the ability of clones of tumor stem cells to differentiate *in vitro* and *in vivo* into both tumor epithelial and endo‐ thelial cells (Bussolati et al., 2008; 2009). More recently, the ability of tumor cells to differen‐ tiate into endothelial cells has also been reported for cancer stem cells present in neuroblastomas (Alvero et al., 2009). In particular, only a fraction of stem cells, characterized by CD133 and CD144 co-expression (Wang et al., 2010), or in a recent report, by co-expres‐ sion of Oct4 and tenascin C, shows vasculogenic potential and is selectively localized in the proximity of tumor vessels (Pezzolo et al., 2011). An alternative mechanism of tumor blood perfusion implies the possibility that tumor cells form channels connected to the tumor vas‐ culature, a process defined as "vasculogenic mimicry". Alternatively, the process of tumor vasculogenic mimicry could be interpreted as being dependent on tumor stem cells (El Hal‐ lanti et al., 2010; Yao et al., 2011), as a transitional step in stem cell differentiation toward

In cancer therapy, recent investigations have focused on using genetically modified bacteria to actually block tumor angiogenesis. Despite recent progress, only a few studies on bacteri‐ al tumor therapy have focused on anti-angiogenesis. Bacteria-mediated anti-angiogenic ther‐ apy for cancer, however, is an attractive approach given that solid tumors are often

The first modern attempts at using bacteria for therapeutic purposes were made more than 40 years ago by showing that bacteria could predominantly replicate in solid tu‐ mors. The first indications of this phenomenon, however, date back to the 19th century. These findings remained largely unexplored until the turn of the 20th century, when on‐ colytic bacteria capable of lysing host cells were first studied by various research groups. The utilization of bacterial systems for therapeutic anti-cancer purposes is further en‐ hanced by genetic modifications, which make them a very promising tool for targeted delivery of genes and their products. Specific advantages of using bacteria for anti-can‐ cer gene therapy include the natural oncolytic potential of some strains/species, direct targeting of tumor tissues and the ease of positive regulation/eradication. The anti-cancer effect of tumor-targeting bacteria can also be achieved after oral administration, which may circumvent the use of intravenous routes of delivery and associated adverse events

endothelial cells (Bussolati et al., 2011).

230 Research Directions in Tumor Angiogenesis

characterized by increased vascularization.

of intravenous therapy (Chen et al., 2009; Gardlik et al., 2011b).

**5. Further development of ARTs as anti-angiogenic cancer agents**

Cancer angiogenesis has been confirmed by measurement of high proliferation indices for endothelial cells, not only in rapidly growing animal tumors, but also in human tumors. The rationale for developing anti-angiogenic strategies for cancer therapy was based on the fact that physiological angiogenesis only occurs in a limited number of situations, such as in wound healing and during menstrual cycle. This suggests there is an opportunity for devel‐ Our current knowledge of the anti-cancer mechanism of ARTs is derived from our knowledge of the antimalarial activity of ARTs. The potent anti-cancer activity of ARTs can be attributed to the endoperoxide bond of the ARTs compounds which is shared with the antiparasitic activity of ARTs. In most of the cancers studied, preloading of can‐ cer cells with iron or iron-saturated holotransferrin triggers ART cytotoxicity with an in‐ crease in the activity of ARTs. It has been hypothesized that iron-activated ARTs induce damage by release of highly alkylating carbon-centered radicals and radical oxygen spe‐ cies (ROS). Generation of free radicals may play a role in the cell alterations reported in ARTs-treated cancer cells such as enhanced apoptosis, arrest of growth, inhibition of an‐ giogenesis, and DNA damage. In addition, ARTs-sensitive cancer cells have been shown to have down-regulated expression of oxidation enzymes while cancer cells with over-ex‐ pression of these molecules are more resistant to ARTs therapy. The antineoplastic toxici‐ ty of ARTs appears to be also modulated by calcium metabolism, endoplasmic reticulum (ER) stress, and the expression of the translationally controlled tumor protein, TCTP, a binding calcium protein which has been also postulated as a parasite target. Although the expression of the TCTP gen, *tctp,* was initially correlated with cancer cell responses to ARTs, a functional role for TCTP in the anti-cancer activity of ARTs has yet to be found. As for malaria parasites, the role of sarcoendoplasmic Ca2 + ATPase (SERCA) as a target of ARTs in cancer cells has also been explored (Crespo-Ortiz and Wei, 2012).

ART and its bioactive derivatives elicit their anti-cancer effects by concurrently activating, inhibiting and/or attenuating multiple complementary cell signaling pathways, especially those associated with the VEGF family, based on published data. The precise mechanism of new and alternative actions and other primary targets of ARTs, however, will require fur‐ ther study. In anti-cancer therapy, it has been postulated that ARTs may target organelles such as pathways involving PlGF growth factors (a VEGF subfamily),, angiopoietins, such as the Angs proteins, the Notch signaling pathway, signaling pathways involving fibroblast growth factors (FGFs), and the matrix metalloproteinase (MMPs) family of enzymes (Cres‐ po-Ortiz and Wei, 2012). In a recent study, investigators discovered a panel of genes con‐ taining many fundamental regulators of angiogenic regulators, such as VEGF, was found that correlate with the cellular response to AS. These genes govern the stimulation, prolifer‐ ation and migration of endothelial cells, a fundamental step in vessel formation. The investi‐ gators decided to further limit their cluster analysis by including in the cluster analysis only those genes whose mRNA expression correlated with GI50 values of at least four ARTs (An‐ fosso et al., 2006). Three human genes coding for VEGF (VEGFA, VEGFB, and VEGFC) were discovered in this cluster of ARTs-affected angiogenic regulating genes.. Despite the contin‐ uous investigations on new targets, the ART compounds exert common as well as distinct cellular effects depending on the phenotype and tissue origin of the human cancer cells ex‐ amined. (Firestone and Sundar, 2009).

In addition, most of these studies were based on the consideration that an ideal ARTs as an anti-angiogenic drugs may target different types of tumor, assuming endothelial cells to be similar in different tumor types and genetically stable. The therapeutic efficacy of ARTs, however, was not as successful as expected and endothelial cells acquired drug re‐ sistance. This setback is possibly due to the fact that most anti-angiogenic drugs were tested on normal endothelial cells. In light of the involvement of angiogenesis and vascu‐ logenesis in tumor vascularization, it can be speculated that tumor cytotoxic therapies, radiotherapy, and anti-angiogenic drugs, may stimulate vasculogenesis by inducing tu‐ mor hypoxia and/or an epithelial mesenchymal transition. Therefore, targeting both an‐ giogenesis and vasculogenesis in tumors may be required to inhibit tumor vascularization, growth, and invasion. In particular, an improved knowledge of the rela‐ tive contribution of vasculogenesis to tumor vascularization is likely to be critical for de‐ velopment of specific therapeutic strategies (Li et al., 2011).

mally occurs only in pathological conditions such as injury or endometrial development. Large number of angiogenic factors, such as VEGF and bFGF. etc., is secreted by tumor cells are required to cause endothelial cell recruitment and proliferation (Ferrara et al., 2003).

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

233

These stimuli are constantly present so the differentiation of the tumor endothelium into a mature vessel network is rarely complete, and tumor vessels show an abnormal mor‐ phology. The immature vessel network of tumors is a promising anti-angiogenic target for ARTs compounds. The body of knowledge of endothelial cell physiology and tumor angiogenesis obtained through recent research has been crucial to actually understand some of the mechanisms of how ARTs actually exert their anti-angiogenic effects (Ef‐ ferth, 2005; 2007).Endothelial cells are non-transformed cells, and they should be quite accessible to treat with physiologically achievable concentrations of ARTs (Efferth, 2006; Crespo-Ortiz and Wei, 2012). This therapeutic strategy may involve various targets in‐ cluding angiogenic molecules from tumors cells and inflammatory system (such as neu‐ trophils and macrophages) such as VEGF, bFGF, TNFa, IL-8, etc. and their respective receptors on endothelial cells, endothelial cells itself, matrix metalloproteinases (MMPs), cyclooxygenases (COXs), lipoxygenases (LOXs) etc (see Section 3). Therefore, long-term angiostasis treatment will likely be necessary for cancer prevention and control. This multitargeted anti-angiogenic strategy suggests drug discovery and development should be focused on finding small, non-toxic anti-angiogenic molecules or compounds to be

used in a multifaceted cancer control regimen (Firestone and Sundar, 2009).

*vivo* model systems (Chen and Cleck, 2009a; Cao et al., 2009; 2011).

Crespo-Ortiz and Wei, 2012).

Large numbers of chemopreventive agents, such as ARTs, have been shown to possess anticancer activities in many studies. These agents achieve anti-cancer activities through various mechanisms by targeting different aspects of cancer progression and development. Since an‐ giogenesis is a pre-requisite for the growth of solid tumors, vascular targeting has been ex‐ plored as a potential strategy to suppress tumor growth and metastasis. In this regard, many phytochemicals or ARTs have been shown to target tumor angiogenesis using *in vitro* and *in*

Since, angiogenesis is critically important for physiological process such as wound-heal‐ ing, acute injury healing, and healing of chronic ulceration of the gastrointestinal muco‐ sa, administration of ARTs compounds that inhibit tumor angiogenesis might also suppress physiological angiogenesis and produce critical side effects when dosed over a long period of time. Therefore, anti-angiogenic chemopreventive ARTs and phytochemi‐ cals should be studied and analyzed first for their selective targeting of tumor specific angiogenesis to find the most effective anti-tumor combinations (Bhat and Singh, 2008;

**5.3. Combination strategies to enhance efficacy and to prevent resistance of ARTs**

Anti-angiogenesis is a cytostatic therapy that is likely to have greatest effect when combined with cytotoxic therapy. It has recently been suggested that anti-angiogenic drugs represent the universal chemosensitizing agents for cancer treatment. There are a number of mechanisms for the observed synergism between anti-angiogenic agents and anti-cancer chemotherapeutic agents that have been proposed 1) the normalization of tumor microvessels by anti-angiogen‐

#### **5.2. Prevention and therapy strategies of ARTs for cancer treatments**

Angiogenesis inhibition therapy does not necessarily kill tumors but instead may prevent tumors from growing. Therefore, this type of therapy may need to be administered over a long period of time. Some common components of human diets also act as mild angio‐ genesis inhibitors and have therefore been proposed for angioprevention, the prevention of metastasis through the inhibition of angiogenesis. Phytochemicals and ART-mediated anti-angiogenic intervention is a growing area of research that may provide an effective cancer prevention strategy. Suppression of pathological angiogenesis by phytochemicals and ARTs could have potential applications in cancer prevention and therapy as well as in other diseases with similar etiology. Chemopreventive phytochemicals are generally non-toxic and hence will produce minimal side effects. In addition, endothelial cells lack induced drug resistance and, therefore, angioprevention could be a preferred strategy for cancer control in comparison to other therapies such as radiotherapy and chemotherapy.

Several anti-angiogenic strategies have been developed to inhibit tumor growth by targeting different components of tumor angiogenesis. Non-toxic natural chemopreventive agents that could be part of the daily human diet have been shown to safely target and inhibit dif‐ ferent aspects and components of the process of angiogenesis. ART and its derivatives, and other sesquiterpene lactones, have been shown to have potent anti-angiogenic effects in tu‐ mor cells as well as in healthy rat embryos in culture. These observations have many impli‐ cations in terms of cancer therapy as well as cancer prevention since angiogenesis is a promotional event (Firestone and Sundar, 2009).

Studies have shown that, the upper limit of a tumor mass in the absence of angiogenesis is 1-2 mm, and this size limit is related to the maximum size possible for simple diffusion of nutrients and gases like CO2 and O2. This 1-2 mm tumor size can be maintained by the bal‐ ance of cell proliferation and apoptosis leading to dormancy of small tumors for many years. Therefore, for tumor growth and the development of vasculature is critical to proceed towards tumor progression and metastasis., Endothelial cells are a preferential target for therapy because they are common to all solid tumors, and endothelial cell proliferation nor‐ mally occurs only in pathological conditions such as injury or endometrial development. Large number of angiogenic factors, such as VEGF and bFGF. etc., is secreted by tumor cells are required to cause endothelial cell recruitment and proliferation (Ferrara et al., 2003).

In addition, most of these studies were based on the consideration that an ideal ARTs as an anti-angiogenic drugs may target different types of tumor, assuming endothelial cells to be similar in different tumor types and genetically stable. The therapeutic efficacy of ARTs, however, was not as successful as expected and endothelial cells acquired drug re‐ sistance. This setback is possibly due to the fact that most anti-angiogenic drugs were tested on normal endothelial cells. In light of the involvement of angiogenesis and vascu‐ logenesis in tumor vascularization, it can be speculated that tumor cytotoxic therapies, radiotherapy, and anti-angiogenic drugs, may stimulate vasculogenesis by inducing tu‐ mor hypoxia and/or an epithelial mesenchymal transition. Therefore, targeting both an‐ giogenesis and vasculogenesis in tumors may be required to inhibit tumor vascularization, growth, and invasion. In particular, an improved knowledge of the rela‐ tive contribution of vasculogenesis to tumor vascularization is likely to be critical for de‐

Angiogenesis inhibition therapy does not necessarily kill tumors but instead may prevent tumors from growing. Therefore, this type of therapy may need to be administered over a long period of time. Some common components of human diets also act as mild angio‐ genesis inhibitors and have therefore been proposed for angioprevention, the prevention of metastasis through the inhibition of angiogenesis. Phytochemicals and ART-mediated anti-angiogenic intervention is a growing area of research that may provide an effective cancer prevention strategy. Suppression of pathological angiogenesis by phytochemicals and ARTs could have potential applications in cancer prevention and therapy as well as in other diseases with similar etiology. Chemopreventive phytochemicals are generally non-toxic and hence will produce minimal side effects. In addition, endothelial cells lack induced drug resistance and, therefore, angioprevention could be a preferred strategy for cancer control in comparison to other therapies such as radiotherapy and chemotherapy.

Several anti-angiogenic strategies have been developed to inhibit tumor growth by targeting different components of tumor angiogenesis. Non-toxic natural chemopreventive agents that could be part of the daily human diet have been shown to safely target and inhibit dif‐ ferent aspects and components of the process of angiogenesis. ART and its derivatives, and other sesquiterpene lactones, have been shown to have potent anti-angiogenic effects in tu‐ mor cells as well as in healthy rat embryos in culture. These observations have many impli‐ cations in terms of cancer therapy as well as cancer prevention since angiogenesis is a

Studies have shown that, the upper limit of a tumor mass in the absence of angiogenesis is 1-2 mm, and this size limit is related to the maximum size possible for simple diffusion of nutrients and gases like CO2 and O2. This 1-2 mm tumor size can be maintained by the bal‐ ance of cell proliferation and apoptosis leading to dormancy of small tumors for many years. Therefore, for tumor growth and the development of vasculature is critical to proceed towards tumor progression and metastasis., Endothelial cells are a preferential target for therapy because they are common to all solid tumors, and endothelial cell proliferation nor‐

velopment of specific therapeutic strategies (Li et al., 2011).

232 Research Directions in Tumor Angiogenesis

promotional event (Firestone and Sundar, 2009).

**5.2. Prevention and therapy strategies of ARTs for cancer treatments**

These stimuli are constantly present so the differentiation of the tumor endothelium into a mature vessel network is rarely complete, and tumor vessels show an abnormal mor‐ phology. The immature vessel network of tumors is a promising anti-angiogenic target for ARTs compounds. The body of knowledge of endothelial cell physiology and tumor angiogenesis obtained through recent research has been crucial to actually understand some of the mechanisms of how ARTs actually exert their anti-angiogenic effects (Ef‐ ferth, 2005; 2007).Endothelial cells are non-transformed cells, and they should be quite accessible to treat with physiologically achievable concentrations of ARTs (Efferth, 2006; Crespo-Ortiz and Wei, 2012). This therapeutic strategy may involve various targets in‐ cluding angiogenic molecules from tumors cells and inflammatory system (such as neu‐ trophils and macrophages) such as VEGF, bFGF, TNFa, IL-8, etc. and their respective receptors on endothelial cells, endothelial cells itself, matrix metalloproteinases (MMPs), cyclooxygenases (COXs), lipoxygenases (LOXs) etc (see Section 3). Therefore, long-term angiostasis treatment will likely be necessary for cancer prevention and control. This multitargeted anti-angiogenic strategy suggests drug discovery and development should be focused on finding small, non-toxic anti-angiogenic molecules or compounds to be used in a multifaceted cancer control regimen (Firestone and Sundar, 2009).

Large numbers of chemopreventive agents, such as ARTs, have been shown to possess anticancer activities in many studies. These agents achieve anti-cancer activities through various mechanisms by targeting different aspects of cancer progression and development. Since an‐ giogenesis is a pre-requisite for the growth of solid tumors, vascular targeting has been ex‐ plored as a potential strategy to suppress tumor growth and metastasis. In this regard, many phytochemicals or ARTs have been shown to target tumor angiogenesis using *in vitro* and *in vivo* model systems (Chen and Cleck, 2009a; Cao et al., 2009; 2011).

Since, angiogenesis is critically important for physiological process such as wound-heal‐ ing, acute injury healing, and healing of chronic ulceration of the gastrointestinal muco‐ sa, administration of ARTs compounds that inhibit tumor angiogenesis might also suppress physiological angiogenesis and produce critical side effects when dosed over a long period of time. Therefore, anti-angiogenic chemopreventive ARTs and phytochemi‐ cals should be studied and analyzed first for their selective targeting of tumor specific angiogenesis to find the most effective anti-tumor combinations (Bhat and Singh, 2008; Crespo-Ortiz and Wei, 2012).

#### **5.3. Combination strategies to enhance efficacy and to prevent resistance of ARTs**

Anti-angiogenesis is a cytostatic therapy that is likely to have greatest effect when combined with cytotoxic therapy. It has recently been suggested that anti-angiogenic drugs represent the universal chemosensitizing agents for cancer treatment. There are a number of mechanisms for the observed synergism between anti-angiogenic agents and anti-cancer chemotherapeutic agents that have been proposed 1) the normalization of tumor microvessels by anti-angiogen‐ ic therapy which enhances chemotherapeutic drug delivery, 2) prevention of tumor cell repo‐ pulation by anti-angiogenic drugs during the break periods after maximum tolerated dose chemotherapy and 3) augmentation of the antivascular effects of chemotherapeutics by antiangiogenic drugs (Makrilia et al., 2009). There is growing evidence supporting the use of ART and its derivatives in cancer therapy given their potent antiproliferative, antimetastatic and anti-angiogenic activity, which makes them potential anti-cancer drugs. In a combination ther‐ apy for cancer, the antineoplastic action of ART may contribute to an independent antitumor activity with no additional side effects. The benefits of combining ARTs with other anti-cancer agents have been investigated showing that the multifactorial activity of ARTs in different pathways may provide synergism and improve overall activity (Liu et al., 2011).

with cisplatin in non-small cell lung cancer A549 in mice (Zhou et al., 2010). In rat C6 glioma cells, addition of 1 *μ*M DHA increased the cytotoxic effect of temozolomide, a DNA alkylat‐ ing agent used in the treatment of brain cancer, by 177%,. Further investigation showed that DHA promotes apoptotic and necrotic activity of temozolomide through ROS generation

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

235

Recently, an enhancement of the anti-cancer activity of AS was shown in different combi‐ nation regimens. A striking synergy was achieved in combinations of AS and the immu‐ nomodulator drug, lenalidomide (Liu et al., 2011). Overall, this evidence suggests that DHA and AS have remarkable ability to potentiate antitumor agents and to counter tu‐ mor resistance. ARTs also have been shown to improve ionizing-based therapies. In the glioma cell line U373MG, DHA treatment was shown to inhibit the radiation- induced expression of GST with concomitant ROS generation. A combination treatment with DHA has been shown to be more effective than radiation or DHA alone (Kim et al., 2006). The adjuvant effect of ARTs in other cancer treatments including hyperbaric oxy‐

Current available ARTs in combination with chemotherapy for the treatment of cancer pa‐ tients have produced only modest beneficial effects (Cao *et al*., 2009). Optimization of antiangiogenic therapy is urgently needed in order to maximize therapeutic efficacy of these drugs. Thus, development of a new generation of drugs targeting diverse angiogenic path‐ ways is expected to improve the anticancer benefits of ARTs therapy. In preclinical tumor models, it has been shown that a combination of anti-angiogenic agents with different mech‐ anistic principles yielded a synergistic effect on tumor suppression. Translation of this pre‐ clinical finding to patient therapy would suggest acombination of different of antiangiogenic agents combined with a chemotherapeutic agent will enhance cancer therapy,

The mechanism(s) underlying the interaction of combinations of anti-angiogenic agents such as the ARTs plus chemotherapeutic treatment, and, indeed, the mechanism of ART action against cancer is still not fully understood, however, many studies in this field of research are ongoing and will guide the basis of further studies and clinical trials (Liu et al., 2011). Scientists investigating the cancer-fighting properties of ARTs have found early evidence that combining it with an existing cancer drug has the potential to make each drug more ef‐ fective in combination versus when these drugs are used alone. There is currently limited published data exploring the value of ARTs as a combination partner in treatment regimens. These studies have used simple approaches to studying drug–drug interactions, and as a

At high concentrations, ARTs appear to be active against cancer *in vivo.* The use of ARTs at high concentrations or for long drug exposure times, however, has substantial risk of severe toxicities, including embryotoxicity and neurotoxicity. Animal studies have shown that high peak concentrations of AS and DHA can induce embryotoxicity, and the longer exposure times associated with therapy using oil-soluble ARTs, such as AM, will produce fatal neuro‐

and such a combination should be considered in future clinical trials (Cao, 2011).

consequence, their conclusions are still open to debate (Liu, 2008).

**5.4. Strategies to avoid potential drug toxicities of ARTs**

(Huang et al., 2008).

gen has also been reported (Ohgami et al., 2010).

Drug combinations that involve ARTs have been reported *in vitro*, which show value in this approach, both as a sensitizing agent to chemotherapy in solid tumors (Sieber et al., 2009), and as a synergistic partner with doxorubicin in leukemia (Efferth et al., 2007). Incubation of cancer cells with DHA alone was found to be less effective than in combination with holo‐ transferrin, indicating that intracellular iron plays a role in the cytotoxic effects of DHA (Lai and Singh, 1995). In addition to conventional chemotherapies, ART was also shown to be ef‐ fective when combined with the immune modulatory drug LEN (Galustian and Dalgleish, 2009). These *in vitro* studies demonstrated the effects of ARTs on the cell cycle, and these studiesalso demonstrated restoration of cytotoxicity in an ART-resistant cell by adopting a pulsed-schedule of combination treatment.

Many anti-angiogenic and antivascular agents are now in clinical trials for the treatment of cancer. It is conceivable that loading tumor cells with bivalent iron by simply providing Fe2+ in tablet form might increase the susceptibility of cancer cells to the action of AS. In a clinical study of humans with uveal melanoma, one of the patients enrolled was treated with biva‐ lent iron and artesunate, and it is tempting to speculate the addition of Fe2+ had an actual clinical impact and resulted in an improved response to therapy (Berger et al., 2005). Contin‐ ued research in this area is encouraged by the recent success of a Phase II clinical trial of AS combined with NP chemotherapy in treatment of advanced non-small cell lung cancer. The disease controlled rate of the trial group of AS plus NP chemotherapy (88.2%) was signifi‐ cantly higher than that of the NP chemotherapy alone group (72.7%), and the trial group's time to progression (24 weeks) was significantly longer than that of the NP chemotherapy alone group (20 weeks). AS combined with NP chemotherapy can increase the short-term survival rate of patients with advanced non-small cell lung cancer and prolong the time to progression without extra side effects (Zhang et al., 2008). The diversity in the targets of ART supports the possibility that it could be used in combination with other agents.

In addition, it has been reported that resistant cancer cell lines become sensitive by adding ART to the conventional treatment (chemosensitization). Interestingly, DHA and AS have ex‐ hibited the strongest chemosensitizing/synergistic effects, while other ARTs shows only ad‐ ditive and antagonistic interactions (Singh and Lai, 2005). DHA was shown to synergistically enhance tumor growth inhibition by 45% when administered in combination with gemcita‐ bine, while other ARTs showed only additive effects (Wang et al., 2010). Consistent with this observation, a greater antitumor activity was observed when DHA was used in a combina‐ tion with cyclosphosphamide in murine Lewis lung carcinoma cell line or in combination with cisplatin in non-small cell lung cancer A549 in mice (Zhou et al., 2010). In rat C6 glioma cells, addition of 1 *μ*M DHA increased the cytotoxic effect of temozolomide, a DNA alkylat‐ ing agent used in the treatment of brain cancer, by 177%,. Further investigation showed that DHA promotes apoptotic and necrotic activity of temozolomide through ROS generation (Huang et al., 2008).

ic therapy which enhances chemotherapeutic drug delivery, 2) prevention of tumor cell repo‐ pulation by anti-angiogenic drugs during the break periods after maximum tolerated dose chemotherapy and 3) augmentation of the antivascular effects of chemotherapeutics by antiangiogenic drugs (Makrilia et al., 2009). There is growing evidence supporting the use of ART and its derivatives in cancer therapy given their potent antiproliferative, antimetastatic and anti-angiogenic activity, which makes them potential anti-cancer drugs. In a combination ther‐ apy for cancer, the antineoplastic action of ART may contribute to an independent antitumor activity with no additional side effects. The benefits of combining ARTs with other anti-cancer agents have been investigated showing that the multifactorial activity of ARTs in different

Drug combinations that involve ARTs have been reported *in vitro*, which show value in this approach, both as a sensitizing agent to chemotherapy in solid tumors (Sieber et al., 2009), and as a synergistic partner with doxorubicin in leukemia (Efferth et al., 2007). Incubation of cancer cells with DHA alone was found to be less effective than in combination with holo‐ transferrin, indicating that intracellular iron plays a role in the cytotoxic effects of DHA (Lai and Singh, 1995). In addition to conventional chemotherapies, ART was also shown to be ef‐ fective when combined with the immune modulatory drug LEN (Galustian and Dalgleish, 2009). These *in vitro* studies demonstrated the effects of ARTs on the cell cycle, and these studiesalso demonstrated restoration of cytotoxicity in an ART-resistant cell by adopting a

Many anti-angiogenic and antivascular agents are now in clinical trials for the treatment of cancer. It is conceivable that loading tumor cells with bivalent iron by simply providing Fe2+ in tablet form might increase the susceptibility of cancer cells to the action of AS. In a clinical study of humans with uveal melanoma, one of the patients enrolled was treated with biva‐ lent iron and artesunate, and it is tempting to speculate the addition of Fe2+ had an actual clinical impact and resulted in an improved response to therapy (Berger et al., 2005). Contin‐ ued research in this area is encouraged by the recent success of a Phase II clinical trial of AS combined with NP chemotherapy in treatment of advanced non-small cell lung cancer. The disease controlled rate of the trial group of AS plus NP chemotherapy (88.2%) was signifi‐ cantly higher than that of the NP chemotherapy alone group (72.7%), and the trial group's time to progression (24 weeks) was significantly longer than that of the NP chemotherapy alone group (20 weeks). AS combined with NP chemotherapy can increase the short-term survival rate of patients with advanced non-small cell lung cancer and prolong the time to progression without extra side effects (Zhang et al., 2008). The diversity in the targets of ART

supports the possibility that it could be used in combination with other agents.

In addition, it has been reported that resistant cancer cell lines become sensitive by adding ART to the conventional treatment (chemosensitization). Interestingly, DHA and AS have ex‐ hibited the strongest chemosensitizing/synergistic effects, while other ARTs shows only ad‐ ditive and antagonistic interactions (Singh and Lai, 2005). DHA was shown to synergistically enhance tumor growth inhibition by 45% when administered in combination with gemcita‐ bine, while other ARTs showed only additive effects (Wang et al., 2010). Consistent with this observation, a greater antitumor activity was observed when DHA was used in a combina‐ tion with cyclosphosphamide in murine Lewis lung carcinoma cell line or in combination

pathways may provide synergism and improve overall activity (Liu et al., 2011).

pulsed-schedule of combination treatment.

234 Research Directions in Tumor Angiogenesis

Recently, an enhancement of the anti-cancer activity of AS was shown in different combi‐ nation regimens. A striking synergy was achieved in combinations of AS and the immu‐ nomodulator drug, lenalidomide (Liu et al., 2011). Overall, this evidence suggests that DHA and AS have remarkable ability to potentiate antitumor agents and to counter tu‐ mor resistance. ARTs also have been shown to improve ionizing-based therapies. In the glioma cell line U373MG, DHA treatment was shown to inhibit the radiation- induced expression of GST with concomitant ROS generation. A combination treatment with DHA has been shown to be more effective than radiation or DHA alone (Kim et al., 2006). The adjuvant effect of ARTs in other cancer treatments including hyperbaric oxy‐ gen has also been reported (Ohgami et al., 2010).

Current available ARTs in combination with chemotherapy for the treatment of cancer pa‐ tients have produced only modest beneficial effects (Cao *et al*., 2009). Optimization of antiangiogenic therapy is urgently needed in order to maximize therapeutic efficacy of these drugs. Thus, development of a new generation of drugs targeting diverse angiogenic path‐ ways is expected to improve the anticancer benefits of ARTs therapy. In preclinical tumor models, it has been shown that a combination of anti-angiogenic agents with different mech‐ anistic principles yielded a synergistic effect on tumor suppression. Translation of this pre‐ clinical finding to patient therapy would suggest acombination of different of antiangiogenic agents combined with a chemotherapeutic agent will enhance cancer therapy, and such a combination should be considered in future clinical trials (Cao, 2011).

The mechanism(s) underlying the interaction of combinations of anti-angiogenic agents such as the ARTs plus chemotherapeutic treatment, and, indeed, the mechanism of ART action against cancer is still not fully understood, however, many studies in this field of research are ongoing and will guide the basis of further studies and clinical trials (Liu et al., 2011). Scientists investigating the cancer-fighting properties of ARTs have found early evidence that combining it with an existing cancer drug has the potential to make each drug more ef‐ fective in combination versus when these drugs are used alone. There is currently limited published data exploring the value of ARTs as a combination partner in treatment regimens. These studies have used simple approaches to studying drug–drug interactions, and as a consequence, their conclusions are still open to debate (Liu, 2008).

#### **5.4. Strategies to avoid potential drug toxicities of ARTs**

At high concentrations, ARTs appear to be active against cancer *in vivo.* The use of ARTs at high concentrations or for long drug exposure times, however, has substantial risk of severe toxicities, including embryotoxicity and neurotoxicity. Animal studies have shown that high peak concentrations of AS and DHA can induce embryotoxicity, and the longer exposure times associated with therapy using oil-soluble ARTs, such as AM, will produce fatal neuro‐ toxicity (Li et al., 2007a). To prevent embryotoxicity in pregnant women with malaria, cur‐ rent WHO policy recommendations on the use of ARTs in uncomplicated malaria state that ARTs should be used only in the second and third trimester, limiting the use of ARTS in the first trimester to cases where it is the only effective treatment available (WHO 2006b).

signed to improve the anti-cancer activities of ART. This second generation ART derivatives have shown remarkable anti-angiogenic effects and cytotoxicity towards tumor cells (D'Alessandro et al., 2007; Krishna et al., 2008). Ether-linked dimers of DHA, for example, have been shown to cause accumulation of tumor cells in the G1 phase of the cell cycle (Morrissey et al., 2010). New growth-inhibitory ART derivatives containing cyan and aryl groups have been shown to cause accumulation of P388 and A549 cells in the G1 phase (Li et al., 2001). Finally, deoxoartemisinyl cyanoarylmethyl ART derivatives with cytotoxic activi‐ ty have been shown to induce a significant accumulation of L1210 and P388 cells in the G1 phase (Wu et al., 2001)., ART-like endoperoxides have also been synthesized chemically which show greater cytotoxicity towards tumor cells than native ART, which aids in pre‐

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

237

As mentioned above, AS is completely converted to DHA and is best described as a prodrug of DHA. DHA has been shown to be more effective than AS in inhibition of angiogenesis and vasculogenesis *in vitro* (Longo et al., 2006a; 2006b; Chen et al., 2004b; White et al., 2006). In addition, the embryotoxicity and neurotoxicity of ARTs can be reduced by using artemi‐ sone, which is a novel derivative of ART that is not metabolized to DHA (D'Alessandro et

Artemisone is a novel amino alkyl ART that has recently entered Phase II clinical trials (D'Alessandro et al., 2007). The compound was rationally designed to have reduced lipophi‐ licity in order to impede transport to the brain and embryo. In addition, the inclusion of a thiomorpholine 1,1-dioxide group at the C10 position blocks the conversion of artemisone to the more lipophilic DHA. This structural modification does not affect anti-parasitic activity but reduces neurotoxicity and embryotoxicity, as assessed *in vitro* against primary neuronal brain stem cell cultures from fetal rats and *in vivo* in female rats (Schmuck et al., 2009). The retention of artemisone antimalarial activity infers that chemical activation of the peroxide bridge to a toxic parasiticidal chemical species remains unchanged, but recent literature also suggests that artemisone has a direct cytotoxic activity without activation of the endoperox‐ ide bridge. In fact, two subsequent studies have provided conflicting results concerning the dependence of the pharmacological activity of artemisone on iron-activation of the endoper‐

Interestingly, an *in vitro* study by D'Alessandro et al, showed that the anti-angiogenic effects of artemisone were reduced compared with DHA, and it was suggested that this reduction may limit the potential of artemisone to cause embryotoxicity mediated by defective angio‐ genesis and vasculogenesis during embryo development (D'Alessandro et al., 2007). Togeth‐ er these studies suggest that, while artemisone was designed to optimize safety by physicochemical means, the structural changes induced to create artemisone may also affect the intracellular chemical and molecular pathways which underlie toxicity, perhaps via re‐ duced or alternative mechanisms of bio-activation and/or reduced cellular accumulation, when compared with the traditional ARTs. Therefore, artemisone represents an exciting novel compound in which increased anti-parasitic activity is combined with a reduced po‐

tential to cause both embryotoxicity and neurotoxicity.

serving the natural resources of the *A. annua* plants (Soomro et al., 2011).

al., 2007; Schmuck et al., 2009).

oxide group.

Studies with laboratory animals have demonstrated fatal neurotoxicity associated with intramuscular administration of AM and AE or oral administration of artelinic acid. These effects suggest that the exposure time of ARTs was extended in these studies due to the accumulation of drug in the bloodstream, and this accumulation, in turn, resulted in neurotoxicity. In one study, the drug exposure time with a neurotoxic outcome (neu‐ rotoxic exposure time) was evaluated as a predictor of neurotoxicity *in vivo* (Li and Hick‐ man, 2011). The neurotoxic exposure time represents a total time spent above the lowest observed neurotoxic effect levels (LONEL) in plasma. The dose of AE required to induce minimal neurotoxicity required a 2-3 fold longer exposure time in rhesus monkeys (179.5 hr) than in rats (67.1 hr) and dogs (113.2 hr) when using a daily dose of 6-12.5 mg/kg for 7-28 days, indicating that the safe dosing duration in monkeys should be longer than 7 days under this exposure. Oral artelinic acid treatment required much longer LONEL levels (8-fold longer) than intramuscular AE to induce neurotoxicity, suggesting that wa‐ ter-soluble ARTs appear to be much safer than oil-soluble ARTs. Due to the lower doses (2-4 mg/kg) used with current ARTs and the more rare use of AE in treating humans, the exposure time is much shorter in humans. Therefore, the current regimen of 3-5 days dosing duration should be quite safe. Advances in our knowledge of ART-induced neu‐ rotoxicity can help refine the treatment regimens used to treat malaria with oral ARTs as well as injectable AS products to avoid the risk of neurotoxicity. Although the watersoluble ARTs, like AS, appear to be much safer, further study is needed in when em‐ ploying ARTs as anti-cancer agents (Li and Hickman, 2011).

Thus, rapid elimination of ARTs in oral formulations is safer than slow-release or oilbased intramuscular formulations (Efferth and Kaina, 2010). Remarkably, although ARTs derivatives have been widely used as antimalarials, their toxicity in humans have been shown to be negligible. In cancer therapy, ARTs may have multiple benefits as it can be used in combination with no additional side effects, but also it enhances potency and re‐ duces doses of more toxic anti-cancer partners. Clinical doses used in malaria treatment after ART administration of 2 mg/kg in patients raise plasma concentrations to 2640 ± 1800 *μ*g/L (approximately 6*.*88 *±* 4*.*69 mM) which can be considered up to 3 orders of magnitude higher than those ART concentrations with antitumor activity (Efferth et al., 2003). It becomes relevant to closely monitor the safety of long-term ARTs-based thera‐ pies as severe side effects may be highly unusual but significant. So far, ARTs treat‐ ments for as long as 12 months have been reported with no relevant side effects (Berger et al., 2005; Singh and Verma, 2002; Singh and Panwar 2006).

#### **5.5. Strategies to utiliize current and novel ARTs as anti-cancer agents**

A number of first generation derivatives of ART have been created (DHA, AS, AM and AE), and other novel compounds have been synthesized as second generation derivatives de‐ signed to improve the anti-cancer activities of ART. This second generation ART derivatives have shown remarkable anti-angiogenic effects and cytotoxicity towards tumor cells (D'Alessandro et al., 2007; Krishna et al., 2008). Ether-linked dimers of DHA, for example, have been shown to cause accumulation of tumor cells in the G1 phase of the cell cycle (Morrissey et al., 2010). New growth-inhibitory ART derivatives containing cyan and aryl groups have been shown to cause accumulation of P388 and A549 cells in the G1 phase (Li et al., 2001). Finally, deoxoartemisinyl cyanoarylmethyl ART derivatives with cytotoxic activi‐ ty have been shown to induce a significant accumulation of L1210 and P388 cells in the G1 phase (Wu et al., 2001)., ART-like endoperoxides have also been synthesized chemically which show greater cytotoxicity towards tumor cells than native ART, which aids in pre‐ serving the natural resources of the *A. annua* plants (Soomro et al., 2011).

toxicity (Li et al., 2007a). To prevent embryotoxicity in pregnant women with malaria, cur‐ rent WHO policy recommendations on the use of ARTs in uncomplicated malaria state that ARTs should be used only in the second and third trimester, limiting the use of ARTS in the

Studies with laboratory animals have demonstrated fatal neurotoxicity associated with intramuscular administration of AM and AE or oral administration of artelinic acid. These effects suggest that the exposure time of ARTs was extended in these studies due to the accumulation of drug in the bloodstream, and this accumulation, in turn, resulted in neurotoxicity. In one study, the drug exposure time with a neurotoxic outcome (neu‐ rotoxic exposure time) was evaluated as a predictor of neurotoxicity *in vivo* (Li and Hick‐ man, 2011). The neurotoxic exposure time represents a total time spent above the lowest observed neurotoxic effect levels (LONEL) in plasma. The dose of AE required to induce minimal neurotoxicity required a 2-3 fold longer exposure time in rhesus monkeys (179.5 hr) than in rats (67.1 hr) and dogs (113.2 hr) when using a daily dose of 6-12.5 mg/kg for 7-28 days, indicating that the safe dosing duration in monkeys should be longer than 7 days under this exposure. Oral artelinic acid treatment required much longer LONEL levels (8-fold longer) than intramuscular AE to induce neurotoxicity, suggesting that wa‐ ter-soluble ARTs appear to be much safer than oil-soluble ARTs. Due to the lower doses (2-4 mg/kg) used with current ARTs and the more rare use of AE in treating humans, the exposure time is much shorter in humans. Therefore, the current regimen of 3-5 days dosing duration should be quite safe. Advances in our knowledge of ART-induced neu‐ rotoxicity can help refine the treatment regimens used to treat malaria with oral ARTs as well as injectable AS products to avoid the risk of neurotoxicity. Although the watersoluble ARTs, like AS, appear to be much safer, further study is needed in when em‐

Thus, rapid elimination of ARTs in oral formulations is safer than slow-release or oilbased intramuscular formulations (Efferth and Kaina, 2010). Remarkably, although ARTs derivatives have been widely used as antimalarials, their toxicity in humans have been shown to be negligible. In cancer therapy, ARTs may have multiple benefits as it can be used in combination with no additional side effects, but also it enhances potency and re‐ duces doses of more toxic anti-cancer partners. Clinical doses used in malaria treatment after ART administration of 2 mg/kg in patients raise plasma concentrations to 2640 ± 1800 *μ*g/L (approximately 6*.*88 *±* 4*.*69 mM) which can be considered up to 3 orders of magnitude higher than those ART concentrations with antitumor activity (Efferth et al., 2003). It becomes relevant to closely monitor the safety of long-term ARTs-based thera‐ pies as severe side effects may be highly unusual but significant. So far, ARTs treat‐ ments for as long as 12 months have been reported with no relevant side effects (Berger

A number of first generation derivatives of ART have been created (DHA, AS, AM and AE), and other novel compounds have been synthesized as second generation derivatives de‐

first trimester to cases where it is the only effective treatment available (WHO 2006b).

236 Research Directions in Tumor Angiogenesis

ploying ARTs as anti-cancer agents (Li and Hickman, 2011).

et al., 2005; Singh and Verma, 2002; Singh and Panwar 2006).

**5.5. Strategies to utiliize current and novel ARTs as anti-cancer agents**

As mentioned above, AS is completely converted to DHA and is best described as a prodrug of DHA. DHA has been shown to be more effective than AS in inhibition of angiogenesis and vasculogenesis *in vitro* (Longo et al., 2006a; 2006b; Chen et al., 2004b; White et al., 2006). In addition, the embryotoxicity and neurotoxicity of ARTs can be reduced by using artemi‐ sone, which is a novel derivative of ART that is not metabolized to DHA (D'Alessandro et al., 2007; Schmuck et al., 2009).

Artemisone is a novel amino alkyl ART that has recently entered Phase II clinical trials (D'Alessandro et al., 2007). The compound was rationally designed to have reduced lipophi‐ licity in order to impede transport to the brain and embryo. In addition, the inclusion of a thiomorpholine 1,1-dioxide group at the C10 position blocks the conversion of artemisone to the more lipophilic DHA. This structural modification does not affect anti-parasitic activity but reduces neurotoxicity and embryotoxicity, as assessed *in vitro* against primary neuronal brain stem cell cultures from fetal rats and *in vivo* in female rats (Schmuck et al., 2009). The retention of artemisone antimalarial activity infers that chemical activation of the peroxide bridge to a toxic parasiticidal chemical species remains unchanged, but recent literature also suggests that artemisone has a direct cytotoxic activity without activation of the endoperox‐ ide bridge. In fact, two subsequent studies have provided conflicting results concerning the dependence of the pharmacological activity of artemisone on iron-activation of the endoper‐ oxide group.

Interestingly, an *in vitro* study by D'Alessandro et al, showed that the anti-angiogenic effects of artemisone were reduced compared with DHA, and it was suggested that this reduction may limit the potential of artemisone to cause embryotoxicity mediated by defective angio‐ genesis and vasculogenesis during embryo development (D'Alessandro et al., 2007). Togeth‐ er these studies suggest that, while artemisone was designed to optimize safety by physicochemical means, the structural changes induced to create artemisone may also affect the intracellular chemical and molecular pathways which underlie toxicity, perhaps via re‐ duced or alternative mechanisms of bio-activation and/or reduced cellular accumulation, when compared with the traditional ARTs. Therefore, artemisone represents an exciting novel compound in which increased anti-parasitic activity is combined with a reduced po‐ tential to cause both embryotoxicity and neurotoxicity.

Increased knowledge of the molecular mechanisms of ART-derived drugs and recent synthe‐ sis of novel ART derivatives demonstrates that further pharmacokinetic and pharmacody‐ namic analyses of novel ART derivatives are needed to understand why these compounds differ in efficacy and toxicity. This information will prove useful for the rationale design of more-effective ART-based molecules for use as anti-cancer agents. New derivatives of ARTs may act not only as treatment drugs, but also may have potential as potent cancer preventa‐ tive agents due to their inhibition of tumor promotion and progression.

**6. Perspectives and conclusion**

of p53 status in genotoxicity need to be further analyzed.

highly recurrent and aggressive tumors or advanced stage cancers.

ART and its bioactive derivatives are potent anti-cancer phytochemicals that pose minimal risks to human patients. ART has been shown to arrest cancer cell growth, induce apoptosis, disrupt angiogenic pathways and has other anti-cancer properties through pleiotropic effects as shown against a variety of human cancer cell lines. In addition, ART-related compounds have been shown to inhibit tumor promotion and progression, suggesting these molecules is not only effective as treatment therapeutics, but also as potential anti-cancer preventive agents. ARTs have been recommended and widely used as antimalarials for six years, and they have saved the lives of many patients infected with malaria (WHO, 2006a). Supporting evidence indicates that ART-like compounds may be a therapeutic alternative or adjunct for use in treating highly metastatic and aggressive cancers that have no other long term effec‐ tive therapy (Morrissey et al., 2010) particularly against cancer cells that have developed drug resistance (Wang et al., 2010). Furthermore, antimalarial endoperoxides may act syn‐ ergistically with other anti-cancer drugs with no additional side effects. The many antitumor activities, both direct and indirect, of ARTs compounds, however are not entirely explained. So far, the precise molecular events involved in how, when, and where radical oxygen spe‐ cies (ROS) production is initially triggered in cancer cells remain to be defined. In addition, the relevance of any ROS-independent mechanism should be also addressed; these might not be obvious but possibly important for ART-mediated cytotoxicity in some cancer cells. Some other aspects such as the direct DNA damage induced by ART-like compounds and the role

The Use of Artemisinin Compounds as Angiogenesis Inhibitors to Treat Cancer

http://dx.doi.org/10.5772/54109

239

Characterizing the anti-cancer effects of existing and novel ARTs derivatives remains an im‐ portant research goal, and research also needs to be focused on unveiling the mechanisms of cancer cell cytotoxicity by identifying their relation to particular cancer biomarkers and mol‐ ecules. ARTs seem to regulate key players participating in multiple pathways such as VEGF, NF-*κ*B, survivin, NOXA, HIF-1*α*, and BMI-1. These molecules and others are to be revealed, which in turn may be involved in drug response, drug interactions, mechanisms of resist‐ ance, and collateral effects in normal cells. A better understanding of common mechanisms under similar conditions in different cell systems will greatly aid the development of target‐ ed ART derivatives. This will improve ARTs cytotoxicity by lowering IC50, emerging of re‐ sistance, drug associated toxicity, and potentiating drug interactions. Furthermore, novel endoperoxide compounds and combinational therapies can be addressed to target or co-tar‐ get markers of carcinoma progression and prevent invasiveness and metastatic properties in

Even though the utility of ARTs in the clinical setting have already been assessed, specific in‐ teractions with established chemotherapy regimens need to be further dissected in different cancer cell lines and their associated phenotypes. This will be crucial to implement clinical trials and treatment of individual cases. Due to the toxicity of ARTs, long-term therapy also requires close monitoring. It is important to note that the prototype drug, ART, seems to modulate responses leading to antagonistic interactions with other anti-cancer drugs. While it may be useful to have the prototype drug as a control *in vitro*, however, its pharmacokinet‐ ic properties may differ from the semisynthetic ARTs. Therefore, ART antagonistic reactions and resistance must be cautiously validated using different semisynthetic derivatives. DHA,

Recently, a series of DHA derivatives were synthesized via an aza-Michael addition reaction, and these novel compounds showed a high selectivity index and an IC50 in the nanomolar range against HeLa cells (0.37 *μ*M) (Feng et al., 2000). In another study, a series of deoxoarte‐ misinins and carboxypropyldeoxoartemisinin compounds were synthesized, and the antitu‐ mor effects of these compounds were not associated with lipophilicity, as has commonly been assumed, but instead was associated with distinctive boat/chair molecular conforma‐ tions which facilitated the interaction of these novel compounds with receptors (Lee et al., 2000). In many studies, there has been an emphasis on the nature and stereochemistry of the dimer linker which may influence anti-cancer activity. It has also been shown, however, that the linker by its own is inactive. Morrisey et al. have described that an ARTs dimer exhibits up to 30-fold more activity than ARTs against prostate cancer lines (Morrissey et al., 2010). This dimer selectively exerted both higher cytostatic activity and apoptosis in C4-2 (a cell line derived from LNCaP) and LNCaP cells compared to ARTs (Morrissey et al., 2010). The ster‐ eoisomery of the linker may be associated with enhanced anti-cancer activity (Alagbala et al., 2006). In another study, C12 non-acetal dimers and one trimer of deoxoartemisinin showed similar potency to that of the conventional anti-cancer drugs against many cell lines. The linker with one amide or one sulfur-centered 2 ethylene group was essential for potent anticancer activity (Jung et al., 2003). The mechanism underlying the antiproliferative action of the ARTs-derived dimers is not clear and requires further study.

Recently, a series of easily synthesized, potent ARTs-like derivatives with anti-cancer activi‐ ty were created. These endoperoxides exhibit high chemical stability and greater cytotoxicity than AS against cancer cell lines. These compounds also exhibit relevant anti-angiogenic properties as judged by studies in a zebrafish model (Soomro et al., 2011). To overcome the short half-lives of ARTs, novel, longer lasting derivatives will be required. One such example is synthetic trioxolanes, endoperoxide drugs which were created to provide long lasting effi‐ cacy against *Schistosoma* species. ARTs compounds share the endoperoxide bridge structural feature of the trioxolanes, and they have been shown to have prophylactic activity towards the younger developmental stages of *Schistosoma* but are ineffective as curative agents. The synthetic trioxalane compounds incorporate the endoperoxide "warhead" with enhanced pharmacokinetic properties and exhibit greater efficacy as curative agents against establish‐ ed Schistosoma infections (Xiao et al., 2007). Given that ARTs may be potentially used as an‐ ti-cancer drugs and possibly in other parasitic and viral infections, the development of novel endoperoxide compounds with enhanced pharmacokinetic properties and targeted anti-can‐ cer activity is essential. These promising research findings suggest it is possible to identify safer and more effective strategies to treat a range of infections and cancer (Crespo-Ortiz and Wei, 2012).

#### **6. Perspectives and conclusion**

Increased knowledge of the molecular mechanisms of ART-derived drugs and recent synthe‐ sis of novel ART derivatives demonstrates that further pharmacokinetic and pharmacody‐ namic analyses of novel ART derivatives are needed to understand why these compounds differ in efficacy and toxicity. This information will prove useful for the rationale design of more-effective ART-based molecules for use as anti-cancer agents. New derivatives of ARTs may act not only as treatment drugs, but also may have potential as potent cancer preventa‐

Recently, a series of DHA derivatives were synthesized via an aza-Michael addition reaction, and these novel compounds showed a high selectivity index and an IC50 in the nanomolar range against HeLa cells (0.37 *μ*M) (Feng et al., 2000). In another study, a series of deoxoarte‐ misinins and carboxypropyldeoxoartemisinin compounds were synthesized, and the antitu‐ mor effects of these compounds were not associated with lipophilicity, as has commonly been assumed, but instead was associated with distinctive boat/chair molecular conforma‐ tions which facilitated the interaction of these novel compounds with receptors (Lee et al., 2000). In many studies, there has been an emphasis on the nature and stereochemistry of the dimer linker which may influence anti-cancer activity. It has also been shown, however, that the linker by its own is inactive. Morrisey et al. have described that an ARTs dimer exhibits up to 30-fold more activity than ARTs against prostate cancer lines (Morrissey et al., 2010). This dimer selectively exerted both higher cytostatic activity and apoptosis in C4-2 (a cell line derived from LNCaP) and LNCaP cells compared to ARTs (Morrissey et al., 2010). The ster‐ eoisomery of the linker may be associated with enhanced anti-cancer activity (Alagbala et al., 2006). In another study, C12 non-acetal dimers and one trimer of deoxoartemisinin showed similar potency to that of the conventional anti-cancer drugs against many cell lines. The linker with one amide or one sulfur-centered 2 ethylene group was essential for potent anticancer activity (Jung et al., 2003). The mechanism underlying the antiproliferative action of

Recently, a series of easily synthesized, potent ARTs-like derivatives with anti-cancer activi‐ ty were created. These endoperoxides exhibit high chemical stability and greater cytotoxicity than AS against cancer cell lines. These compounds also exhibit relevant anti-angiogenic properties as judged by studies in a zebrafish model (Soomro et al., 2011). To overcome the short half-lives of ARTs, novel, longer lasting derivatives will be required. One such example is synthetic trioxolanes, endoperoxide drugs which were created to provide long lasting effi‐ cacy against *Schistosoma* species. ARTs compounds share the endoperoxide bridge structural feature of the trioxolanes, and they have been shown to have prophylactic activity towards the younger developmental stages of *Schistosoma* but are ineffective as curative agents. The synthetic trioxalane compounds incorporate the endoperoxide "warhead" with enhanced pharmacokinetic properties and exhibit greater efficacy as curative agents against establish‐ ed Schistosoma infections (Xiao et al., 2007). Given that ARTs may be potentially used as an‐ ti-cancer drugs and possibly in other parasitic and viral infections, the development of novel endoperoxide compounds with enhanced pharmacokinetic properties and targeted anti-can‐ cer activity is essential. These promising research findings suggest it is possible to identify safer and more effective strategies to treat a range of infections and cancer (Crespo-Ortiz and

tive agents due to their inhibition of tumor promotion and progression.

238 Research Directions in Tumor Angiogenesis

the ARTs-derived dimers is not clear and requires further study.

Wei, 2012).

ART and its bioactive derivatives are potent anti-cancer phytochemicals that pose minimal risks to human patients. ART has been shown to arrest cancer cell growth, induce apoptosis, disrupt angiogenic pathways and has other anti-cancer properties through pleiotropic effects as shown against a variety of human cancer cell lines. In addition, ART-related compounds have been shown to inhibit tumor promotion and progression, suggesting these molecules is not only effective as treatment therapeutics, but also as potential anti-cancer preventive agents. ARTs have been recommended and widely used as antimalarials for six years, and they have saved the lives of many patients infected with malaria (WHO, 2006a). Supporting evidence indicates that ART-like compounds may be a therapeutic alternative or adjunct for use in treating highly metastatic and aggressive cancers that have no other long term effec‐ tive therapy (Morrissey et al., 2010) particularly against cancer cells that have developed drug resistance (Wang et al., 2010). Furthermore, antimalarial endoperoxides may act syn‐ ergistically with other anti-cancer drugs with no additional side effects. The many antitumor activities, both direct and indirect, of ARTs compounds, however are not entirely explained. So far, the precise molecular events involved in how, when, and where radical oxygen spe‐ cies (ROS) production is initially triggered in cancer cells remain to be defined. In addition, the relevance of any ROS-independent mechanism should be also addressed; these might not be obvious but possibly important for ART-mediated cytotoxicity in some cancer cells. Some other aspects such as the direct DNA damage induced by ART-like compounds and the role of p53 status in genotoxicity need to be further analyzed.

Characterizing the anti-cancer effects of existing and novel ARTs derivatives remains an im‐ portant research goal, and research also needs to be focused on unveiling the mechanisms of cancer cell cytotoxicity by identifying their relation to particular cancer biomarkers and mol‐ ecules. ARTs seem to regulate key players participating in multiple pathways such as VEGF, NF-*κ*B, survivin, NOXA, HIF-1*α*, and BMI-1. These molecules and others are to be revealed, which in turn may be involved in drug response, drug interactions, mechanisms of resist‐ ance, and collateral effects in normal cells. A better understanding of common mechanisms under similar conditions in different cell systems will greatly aid the development of target‐ ed ART derivatives. This will improve ARTs cytotoxicity by lowering IC50, emerging of re‐ sistance, drug associated toxicity, and potentiating drug interactions. Furthermore, novel endoperoxide compounds and combinational therapies can be addressed to target or co-tar‐ get markers of carcinoma progression and prevent invasiveness and metastatic properties in highly recurrent and aggressive tumors or advanced stage cancers.

Even though the utility of ARTs in the clinical setting have already been assessed, specific in‐ teractions with established chemotherapy regimens need to be further dissected in different cancer cell lines and their associated phenotypes. This will be crucial to implement clinical trials and treatment of individual cases. Due to the toxicity of ARTs, long-term therapy also requires close monitoring. It is important to note that the prototype drug, ART, seems to modulate responses leading to antagonistic interactions with other anti-cancer drugs. While it may be useful to have the prototype drug as a control *in vitro*, however, its pharmacokinet‐ ic properties may differ from the semisynthetic ARTs. Therefore, ART antagonistic reactions and resistance must be cautiously validated using different semisynthetic derivatives. DHA, AS, and AM are the only endoperoxides currently licensed for therapeutic use. So far, AM has been shown to share similar anti-cancer properties as DHA and AS (Wu et al., 2009).

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[3] D'Alessandro, S., Gelati, M., Basilico, N., Parati, EA., Haynes, RK., Taramelli, D. (2007) Differential effects on angiogenesis of two antimalarial compounds, dihy‐ droartemisinin and artemisone: implications for embryotoxicity. Toxicology 241, 66–

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Cancer research is a permanent discovery of new genes and pleiotropic interactions. The study of the antitumor activity of ARTs compounds may become even more complex as immunologi‐ cal hallmarks are also involved in the generation of tumors. Immunological hallmarks in can‐ cer cells include the ability to induce chronic inflammatory response, evasion of tumor recognition, and ability to induce tolerance (Cavallo et al., 2011). Whether ART may participate in the mechanisms involved in these events has yet to be determined. Overall, the real potential and benefits of the ART drug class remain yet to be uncovered. The imminent possibility of ARTs being included in the arsenal of anti-cancer drugs has opened the door for challenging re‐ search in this area, one that seems to fulfill many expectations (Crespo-Ortiz and Wei, 2012).

In conclusion, the inhibition of angiogenesis induced by ART-derived drugs has been shown to be a mechanism of anti-cancer activity *in vitro* and *in vivo*. In particular, cancer angiogene‐ sis plays a key role in the growth, invasion, and metastasis of tumors. ARTs-induced inhibi‐ tion of angiogenesis could be a promising therapeutic strategy for treatment and prevention of cancer. Other anti-cancer mechanisms induced by ARTs have been recognized recently that have guided various clinical trials in anti-cancer therapy. Since new and alternative an‐ giogenesis mechanisms have been found, further research on the mechanism of efficacy and toxicity could lead us to understand more deeply the possibilities inherent in therapeutic de‐ velopment of ARTs for malaria, cancer, and other indications. The new therapeutic strategies for use of ARTs as anti-angiogenic agents should be considered to avoid problems associated with reproductive toxicity and neurotoxicity. Taken together, ART and its derivatives have been shown to have potent anti-angiogenic and antivasculogenic effects in tumor cells. These observations have many implications in terms of cancer therapy and prevention as well as avoidance of drug toxicity associated with inhibition of angiogenesis.

#### **Acknowledgements**

This material has been reviewed by Walter Reed Army Institute of Research. There is no ob‐ jection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting the views of the Department of the Army or the Department of Defense.

#### **Author details**

Qigui Li\* , Peter Weina and Mark Hickman

\*Address all correspondence to: qigui.li@us.army.mil

Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, USA

#### **References**

AS, and AM are the only endoperoxides currently licensed for therapeutic use. So far, AM has been shown to share similar anti-cancer properties as DHA and AS (Wu et al., 2009).

Cancer research is a permanent discovery of new genes and pleiotropic interactions. The study of the antitumor activity of ARTs compounds may become even more complex as immunologi‐ cal hallmarks are also involved in the generation of tumors. Immunological hallmarks in can‐ cer cells include the ability to induce chronic inflammatory response, evasion of tumor recognition, and ability to induce tolerance (Cavallo et al., 2011). Whether ART may participate in the mechanisms involved in these events has yet to be determined. Overall, the real potential and benefits of the ART drug class remain yet to be uncovered. The imminent possibility of ARTs being included in the arsenal of anti-cancer drugs has opened the door for challenging re‐ search in this area, one that seems to fulfill many expectations (Crespo-Ortiz and Wei, 2012).

In conclusion, the inhibition of angiogenesis induced by ART-derived drugs has been shown to be a mechanism of anti-cancer activity *in vitro* and *in vivo*. In particular, cancer angiogene‐ sis plays a key role in the growth, invasion, and metastasis of tumors. ARTs-induced inhibi‐ tion of angiogenesis could be a promising therapeutic strategy for treatment and prevention of cancer. Other anti-cancer mechanisms induced by ARTs have been recognized recently that have guided various clinical trials in anti-cancer therapy. Since new and alternative an‐ giogenesis mechanisms have been found, further research on the mechanism of efficacy and toxicity could lead us to understand more deeply the possibilities inherent in therapeutic de‐ velopment of ARTs for malaria, cancer, and other indications. The new therapeutic strategies for use of ARTs as anti-angiogenic agents should be considered to avoid problems associated with reproductive toxicity and neurotoxicity. Taken together, ART and its derivatives have been shown to have potent anti-angiogenic and antivasculogenic effects in tumor cells. These observations have many implications in terms of cancer therapy and prevention as well as

This material has been reviewed by Walter Reed Army Institute of Research. There is no ob‐ jection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official, or as reflecting

Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver

avoidance of drug toxicity associated with inhibition of angiogenesis.

the views of the Department of the Army or the Department of Defense.

, Peter Weina and Mark Hickman

\*Address all correspondence to: qigui.li@us.army.mil

**Acknowledgements**

240 Research Directions in Tumor Angiogenesis

**Author details**

Qigui Li\*

Spring, USA


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**Chapter 8**

**3-D Microvascular Tissue Constructs for**

Mani T. Valarmathi, Stefanie V. Biechler and

Additional information is available at the end of the chapter

John W. Fuseler

**1. Introduction**

http://dx.doi.org/10.5772/53118

**Exploring Concurrent Temporal and Spatial**

**Regulation of Postnatal Neovasculogenesis**

Development of postnatal new blood vessels occurs essentially by two temporally distinct but interrelated processes, vasculogenesis and angiogenesis. Vasculogenesis is the process of blood vessel formation occurring by a de novo production of endothelial cells in an embryo (primitive vascular network) or a formerly avascular area when endothelial precursor cells (angioblasts, hemangioblasts or stem cells) migrate and differentiate in response to local cues (such as growth factors and extracellular matrix) to form new intact blood vessels (Ri‐ sau and Flamme, 1995). Angiogenesis refers principally to the sprouting of new blood ves‐ sels from the differentiated endothelium of pre-existing vessels. These vascular trees or plexuses are then pruned, remodeled and extended through angiogenesis to become larger caliber vessels (Carmeliet, 2000). In addition, there exists yet another unique mechanism of neovascularization, the postnatal vasculogenesis, where new blood vessels are formed by the process of fusion and differentiation of endothelial progenitors of bone marrow origin (Valarmathi et al., 2009). This indicates a potential role for bone marrow-derived progenitor cells in postnatal neovasculogenesis and/or neoangiogenesis. This implies that additional mechanisms besides angiogenesis can occur in the adult, and has opened up the possibility to investigate the embryonic origin and development of these putative progenitor cells.

The adult bone marrow contains two subsets of multipotential stem cells, hematopoietic stem cells (HSCs) and bone marrow stromal cells or mesenchymal stem cells (BMSCs/ MSCs). BMSCs are a readily available heterogeneous population of cells that can be directed to differentiate into multiple mesenchymal and non-mesenchymal cells either in vitro or in

and reproduction in any medium, provided the original work is properly cited.

© 2013 T. Valarmathi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of Postnatal Neovasculogenesis**

Mani T. Valarmathi, Stefanie V. Biechler and John W. Fuseler

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53118

#### **1. Introduction**

Development of postnatal new blood vessels occurs essentially by two temporally distinct but interrelated processes, vasculogenesis and angiogenesis. Vasculogenesis is the process of blood vessel formation occurring by a de novo production of endothelial cells in an embryo (primitive vascular network) or a formerly avascular area when endothelial precursor cells (angioblasts, hemangioblasts or stem cells) migrate and differentiate in response to local cues (such as growth factors and extracellular matrix) to form new intact blood vessels (Ri‐ sau and Flamme, 1995). Angiogenesis refers principally to the sprouting of new blood ves‐ sels from the differentiated endothelium of pre-existing vessels. These vascular trees or plexuses are then pruned, remodeled and extended through angiogenesis to become larger caliber vessels (Carmeliet, 2000). In addition, there exists yet another unique mechanism of neovascularization, the postnatal vasculogenesis, where new blood vessels are formed by the process of fusion and differentiation of endothelial progenitors of bone marrow origin (Valarmathi et al., 2009). This indicates a potential role for bone marrow-derived progenitor cells in postnatal neovasculogenesis and/or neoangiogenesis. This implies that additional mechanisms besides angiogenesis can occur in the adult, and has opened up the possibility to investigate the embryonic origin and development of these putative progenitor cells.

The adult bone marrow contains two subsets of multipotential stem cells, hematopoietic stem cells (HSCs) and bone marrow stromal cells or mesenchymal stem cells (BMSCs/ MSCs). BMSCs are a readily available heterogeneous population of cells that can be directed to differentiate into multiple mesenchymal and non-mesenchymal cells either in vitro or in

© 2013 T. Valarmathi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

vivo (Wakitani et al., 1995; Pittenger et al., 1999; Makino et al., 1999; Fukuda et al., 2001; Bianco et al., 2001; Valarmathi et al., 2009; 2010). Most noticeably, BMSCs have been induced to undergo maturation and differentiation towards vascular endothelial and smooth muscle cell lineages. Previous reports indicate that BMSCs and bone marrow-derived multipotent adult progenitor cells (MAPCs) can be differentiated into endothelial-like cells in vitro and contribute to neoangiogenesis in vivo (Oswald et al., 2004; Reyes et al., 2002; Al-Khaldi et al., 2003). Additionally, it has been shown that BMSCs can augment collateral remodeling and perfusion in ischemic models through paracrine mechanisms rather than by cellular incor‐ poration upon local delivery (Kinnaird et al., 2004). Therefore, the identification of bonemarrow-derived (hematopoietic and non-hematopoietic stem cells) and non-bone-marrowderived (tissue-resident stem/progenitor cells – adipose, neural, heart, skeletal muscle; peripheral and cord blood-derived stem cells) endothelial progenitors cells (EPCs) has led to the realization of potential postnatal vasculogenesis (Urbich and Dimmeler, 2004).

fashion. Moreover, unlike ESCs derivation, obtaining autologous or allogeneic BMSCs is fea‐ sible and can potentially be exploited to develop tissue-engineered blood vessel constructs for therapeutic purposes. Similarly, when compared to bone marrow-derived BMSCs, re‐ peated isolation and rapid expansion of sufficient yield of autologous and/or allogeneic nonbone-marrow-derived resident stem cells/progenitors, especially from vital organs for routine therapeutic purposes are highly constrained. On the contrary, to a certain extent, au‐ tologous and/or allogeneic bone marrow-derived BMSCs are amenable for repeated isola‐ tion and reasonable in vitro expansion from the patients. In addition, the significant advantage of using these BMSCs is their low immunogenicity. And these autologous or allo‐ geneic BMSCs have been reported to be immunomodulatory and immunotolerogenic both in vitro as well as in vivo. (Aggarwal and Pittenger, 2005). Taken together, these data strong‐ ly indicate that BMSCs can represent the potential cell of choice for adult autologous and/or

3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of…

http://dx.doi.org/10.5772/53118

263

Extracellular molecules initiate biological signals and play a critical role in the control of cellu‐ lar proliferation, differentiation, and morphogenesis. Many parameters, such as the presence and amount of soluble factors such as hormones, growth factors, and cytokines or the insolu‐ ble factors such as the physical configuration of the matrix which mediates the cell-cell interac‐ tions and cell-matrix interactions, exert a strong influence on the success of angiogenic processes in vitro and presumably in vivo (Even-Ram and Yamada, 2005; Carlson, 2007). The likelihood and ultimate success of in vitro cellular differentiation depends on how closer the cell-matrix interactions and relationships' mimic to those found during normal development or regeneration. In vascular tissue engineering, the application of these principles in vivo will be important to ensure that the matrix/scaffold to be implanted can support endothelial cell proliferation and migration resulting in endothelial tube formation (Ingber and Folkman, 1989). The vital issue for realistic clinical application is whether these scaffolds with pre‐ formed network of endothelial capillaries/microvessels can survive implantation into tissue

We therefore hypothesized that under appropriate in vitro physicochemical microenviron‐ mental cues (combination of growth factors and extra cellular matrix, ECM) multipotent adult BMSCs could be differentiated into vascular endothelial and smooth muscle cell line‐ ages. To test this hypothesis, we characterized the intrinsic vasculogenic differentiation po‐ tential of adult BMSCs when seeded onto a three-dimensional (3-D) tubular scaffold engineered from aligned type I collagen strands and cultured in both vasculogenic and nonvasculogenic growth media. In these culture conditions, BMSCs differentiated and matured into both endothelial and smooth muscle/pericyte cell lineages and showed microvascular morphogenesis. We also explored the potential of the 3-D model system to undergo postna‐

The differentiation of rat BMSCs was carried out on a 3-D tubular scaffold made up of aligned type I collagen-gel fibers. Rat BMSCs were isolated from the tibial and femoral bone

allogeneic stem cell based vascularized tissue regeneration.

defects and subsequently be able to anastomose to the host vasculature.

tal de novo vasculogenesis.

**2. Experimental approach**

A variety of cellular types can be mobilized from the bone marrow reservoir and can home to sites of neovascularization, where they enhance the angiogenic process (Bertolini et al., 2006). While the concept that vascular progenitors are delivered and recruited was initially conceived and based on the circulating endothelial stem/progenitors (EPCs) paradigm, cur‐ rently, it has become obvious that other classes of vasculogenic cells can also be derived from the in situ de novo differentiation of precursor cells. Among these, a population of Tie2 expressing mesenchymal precursor cells was recently identified, which are capable of in vi‐ tro expansion and of generating Tie2 negative but α-SMA positive cells when re-introduced into the tumors (De Palma et al., 2005). On the one hand, the mechanism by which these cells (EPCs) are recruited from the bone marrow to sites of new blood vessel formation re‐ mains an area of active study, but on the other hand, the mechanisms of by which EPCs egress from the bone marrow and subsequent recruitment to sites of neovascularization/ neoangiogenesis warrants further investigation.

Even though there exists ample evidence for the existence of EPCs, proof of a functional re‐ quirement for EPCs mobilization and vascular recruitment in human cancer necessitates fur‐ ther experimentation. In addition, to address the temporal as well as spatial contribution of not only these EPCs but also other marrow-derived stem/progenitors to a specific tumor en‐ vironment require the generation of novel tumor model systems. And these model systems can be utilized to evaluate the subtle contribution of marrow-derived stem/progenitors dur‐ ing the early phases of the neo-angiogenic switch besides oncogenic transformation.

For the above mentioned reasons, embryonic, fetal and postnatal stem cells as well as vari‐ ous types of progenitor cells, can be a potential cellular source for vascular tissue engineer‐ ing (Levenberg, 2005). However, the source for the early-stage developmental cells is restricted. The utility of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in facilitating vascularized tissue/organ regeneration is still in its incipient stages. A number of issues, including a propensity for some implanted ESCs/iPSCs to form benign teratomas and/or malignant teratocarcinomas in the regenerating tissue/organ, remain to be addressed. In contrast to both ESCs/iPSCs, it has been well established that the adult stem cell, BMSCs exhibit multilineage differentiation potential in a well-controlled, predictable fashion. Moreover, unlike ESCs derivation, obtaining autologous or allogeneic BMSCs is fea‐ sible and can potentially be exploited to develop tissue-engineered blood vessel constructs for therapeutic purposes. Similarly, when compared to bone marrow-derived BMSCs, re‐ peated isolation and rapid expansion of sufficient yield of autologous and/or allogeneic nonbone-marrow-derived resident stem cells/progenitors, especially from vital organs for routine therapeutic purposes are highly constrained. On the contrary, to a certain extent, au‐ tologous and/or allogeneic bone marrow-derived BMSCs are amenable for repeated isola‐ tion and reasonable in vitro expansion from the patients. In addition, the significant advantage of using these BMSCs is their low immunogenicity. And these autologous or allo‐ geneic BMSCs have been reported to be immunomodulatory and immunotolerogenic both in vitro as well as in vivo. (Aggarwal and Pittenger, 2005). Taken together, these data strong‐ ly indicate that BMSCs can represent the potential cell of choice for adult autologous and/or allogeneic stem cell based vascularized tissue regeneration.

Extracellular molecules initiate biological signals and play a critical role in the control of cellu‐ lar proliferation, differentiation, and morphogenesis. Many parameters, such as the presence and amount of soluble factors such as hormones, growth factors, and cytokines or the insolu‐ ble factors such as the physical configuration of the matrix which mediates the cell-cell interac‐ tions and cell-matrix interactions, exert a strong influence on the success of angiogenic processes in vitro and presumably in vivo (Even-Ram and Yamada, 2005; Carlson, 2007). The likelihood and ultimate success of in vitro cellular differentiation depends on how closer the cell-matrix interactions and relationships' mimic to those found during normal development or regeneration. In vascular tissue engineering, the application of these principles in vivo will be important to ensure that the matrix/scaffold to be implanted can support endothelial cell proliferation and migration resulting in endothelial tube formation (Ingber and Folkman, 1989). The vital issue for realistic clinical application is whether these scaffolds with pre‐ formed network of endothelial capillaries/microvessels can survive implantation into tissue defects and subsequently be able to anastomose to the host vasculature.

We therefore hypothesized that under appropriate in vitro physicochemical microenviron‐ mental cues (combination of growth factors and extra cellular matrix, ECM) multipotent adult BMSCs could be differentiated into vascular endothelial and smooth muscle cell line‐ ages. To test this hypothesis, we characterized the intrinsic vasculogenic differentiation po‐ tential of adult BMSCs when seeded onto a three-dimensional (3-D) tubular scaffold engineered from aligned type I collagen strands and cultured in both vasculogenic and nonvasculogenic growth media. In these culture conditions, BMSCs differentiated and matured into both endothelial and smooth muscle/pericyte cell lineages and showed microvascular morphogenesis. We also explored the potential of the 3-D model system to undergo postna‐ tal de novo vasculogenesis.

#### **2. Experimental approach**

vivo (Wakitani et al., 1995; Pittenger et al., 1999; Makino et al., 1999; Fukuda et al., 2001; Bianco et al., 2001; Valarmathi et al., 2009; 2010). Most noticeably, BMSCs have been induced to undergo maturation and differentiation towards vascular endothelial and smooth muscle cell lineages. Previous reports indicate that BMSCs and bone marrow-derived multipotent adult progenitor cells (MAPCs) can be differentiated into endothelial-like cells in vitro and contribute to neoangiogenesis in vivo (Oswald et al., 2004; Reyes et al., 2002; Al-Khaldi et al., 2003). Additionally, it has been shown that BMSCs can augment collateral remodeling and perfusion in ischemic models through paracrine mechanisms rather than by cellular incor‐ poration upon local delivery (Kinnaird et al., 2004). Therefore, the identification of bonemarrow-derived (hematopoietic and non-hematopoietic stem cells) and non-bone-marrowderived (tissue-resident stem/progenitor cells – adipose, neural, heart, skeletal muscle; peripheral and cord blood-derived stem cells) endothelial progenitors cells (EPCs) has led to

the realization of potential postnatal vasculogenesis (Urbich and Dimmeler, 2004).

neoangiogenesis warrants further investigation.

262 Research Directions in Tumor Angiogenesis

A variety of cellular types can be mobilized from the bone marrow reservoir and can home to sites of neovascularization, where they enhance the angiogenic process (Bertolini et al., 2006). While the concept that vascular progenitors are delivered and recruited was initially conceived and based on the circulating endothelial stem/progenitors (EPCs) paradigm, cur‐ rently, it has become obvious that other classes of vasculogenic cells can also be derived from the in situ de novo differentiation of precursor cells. Among these, a population of Tie2 expressing mesenchymal precursor cells was recently identified, which are capable of in vi‐ tro expansion and of generating Tie2 negative but α-SMA positive cells when re-introduced into the tumors (De Palma et al., 2005). On the one hand, the mechanism by which these cells (EPCs) are recruited from the bone marrow to sites of new blood vessel formation re‐ mains an area of active study, but on the other hand, the mechanisms of by which EPCs egress from the bone marrow and subsequent recruitment to sites of neovascularization/

Even though there exists ample evidence for the existence of EPCs, proof of a functional re‐ quirement for EPCs mobilization and vascular recruitment in human cancer necessitates fur‐ ther experimentation. In addition, to address the temporal as well as spatial contribution of not only these EPCs but also other marrow-derived stem/progenitors to a specific tumor en‐ vironment require the generation of novel tumor model systems. And these model systems can be utilized to evaluate the subtle contribution of marrow-derived stem/progenitors dur‐

For the above mentioned reasons, embryonic, fetal and postnatal stem cells as well as vari‐ ous types of progenitor cells, can be a potential cellular source for vascular tissue engineer‐ ing (Levenberg, 2005). However, the source for the early-stage developmental cells is restricted. The utility of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in facilitating vascularized tissue/organ regeneration is still in its incipient stages. A number of issues, including a propensity for some implanted ESCs/iPSCs to form benign teratomas and/or malignant teratocarcinomas in the regenerating tissue/organ, remain to be addressed. In contrast to both ESCs/iPSCs, it has been well established that the adult stem cell, BMSCs exhibit multilineage differentiation potential in a well-controlled, predictable

ing the early phases of the neo-angiogenic switch besides oncogenic transformation.

The differentiation of rat BMSCs was carried out on a 3-D tubular scaffold made up of aligned type I collagen-gel fibers. Rat BMSCs were isolated from the tibial and femoral bone marrow of adult rats. The BMSCs isolated from the bone marrow were expanded, main‐ tained and passaged to make sure that the attached marrow stromal cells were devoid of any non-adhering populations of cells. Phenotypic characterization of the BMSCs for cell surface markers was performed by confocal microscopy (qualitative evaluation) and singlecolor flow cytometry (quantitative analysis). This adherent population of cells was further purified and enriched by indirect magnetic cell sorting. The cells were subjected to CD90 positive selection. The resultant enriched CD90+ /CD34- /CD45 fractions were expanded by subculturing and subjected to flow cytometric analysis to validate the proper phenotype. This population of purified BMSCs was used in all experiments. For vasculogenic differen‐ tiation of BMSCs, the expanded and purified population of CD90+ BMSCs were seeded into the collagen-gel tubular scaffold and cultured either in vasculogenic or non-vasculogenic culture medium for 28 days. At regular intervals of 7, 14, 21 and 28 days the tube cultures were assayed by RT-qPCR, immunofluorescence, ultrastructural and biochemical analyses for various endothelial and smooth muscle differentiation markers as shown in table 1, 2 and 3. The time points at which these analyses were carried out cover the optimal range of both vasculogenesis and angiogenic processes seen in vivo and/or in vitro and mimic the progression of microvascular development.

**Figure 1.** Fabrication of 3-D collagen gel based tubular scaffolds engineered from aligned type I collagen fibers. Type I collagen was extracted from bovine calf hides (A). Bovine collagen type I was extruded with a device that contained two counter-rotating cones to generate the 3-D collagen gel tubular scaffolds. The dimensions of tubes produced for this set of experiments had a length of 30 mm with a luminal diameter of 4 mm and an external diameter of 5 mm, leaving a wall thickness of 0.5 mm. The collagen tubes had a defined fiber angle of 18° relative to the central axis of

3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of…

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265

The initial step is to isolate the mononuclear cells from the bone marrow by aspiration and centrifugation followed by plating and isolation of the cells based on differential ad‐ herence capacity to tissue culture dishes (passage 0 cells). Rat BMSCs were isolated from the bone marrow of adult 300g Sprague Dawley®™ SD®™ rats (Harlan Sprague Daw‐ ley, Inc.). Briefly, after deep anesthesia, the femoral and tibial bones were removed asep‐ tically and cleaned extensively to remove associated soft connective tissues. The marrow cavities of these bones were flushed with Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) and combined. The isolated marrow plugs were triturated, and passed through needles of decreasing gauge (from 18 gauge to 22 gauge) to break up clumps and cellular aggregates. The resulting single-cell suspensions were centrifuged at 200g for 5 minutes. Nucleated cells were counted using a Neubauer chamber. Cells were plat‐ ed at a density of 5 X 106 – 2 X 107 cells per T75 cm2 flasks in basal media composed of DMEM supplemented with 10% fetal bovine serum (FBS, lot-selected; Hyclone), gentami‐ cin (50 µg/ml) and amphotericin B (250 ng/ml) and incubated in a humidified atmos‐ phere of 5% CO2 at 37°C for 7 days. The medium was replaced, and changed three times per week until the cultures become ~70% confluent (between 12 and 14 days). Cells were trypsinized using 0.05% trypsin-0.1% EDTA and re-plated at a density of 1 x 106 cells per T75 cm2 flasks. After three passages, attached marrow stromal cells were devoid of any non-adhering population of cells. These passaged BMSCs were cryopreserved and stored

the tube and had pores ranging from 1 to 10 μm (B). (A and B, courtesy of M.J. Yost)

in liquid nitrogen until further use (Valarmathi et al., 2011).

**3.2. BMSCs isolation, expansion and maintenance**

#### **3. Research methods**

#### **3.1. Fabrication of tubular scaffold**

The 3-D collagen type I tube served as a scaffold on which rat BMSCs differentiation cul‐ tures were carried out (Figure 1). The details of the production and properties of the col‐ lagen tubular scaffolds have previously been described (Yost et al., 2004). Briefly, a 25 mg/ml solution of bovine collagen type I was extruded with a device that contained two counter-rotating cones. The liquid collagen was fed between the two cones and forced through a circular annulus in the presence of an NH3-air (50-50 vol/vol) chamber. This process results in a hollow cylindrical tube of aligned collagen fibrils with an inner cen‐ tral lumen. The dimensions of tubes produced for this set of experiments had a length of 30 mm with a luminal diameter of 4 mm and an external diameter of 5 mm, leaving a wall thickness of 0.5 mm. The collagen tubes had a defined fiber angle of 18° relative to the central axis of the tube and had pores ranging from 1 to 10 µm. The rationale for the particular orientation of collagen fibers was based on our previous work on cardiovascu‐ lar tissue engineering (Yost et al., 2004). When proepicardial organ (PE) cells were seed‐ ed onto this scaffold, they underwent maturation and differentiation and produced elongated vessel-like structures reminiscent of in vivo-like phenotype (Valarmathi et al., 2008). The tubes were sterilized using gamma radiation 1200 Gy followed by Stratalinker UV crosslinker 1800 (Stratagene) and then placed in Mosconas's solution (in mM: 136.8 NaCl, 28.6 KCl, 11.9 NaHCO3, 9.4 glucose, 0.08 NaH2PO4, pH 7.4) (Sigma-Aldrich) con‐ taining 1 µl/ml gentamicin (Sigma-Aldrich) and incubated in 5% CO2 at 37°C until cellu‐ lar seeding (Valarmathi et al., 2010).

**Figure 1.** Fabrication of 3-D collagen gel based tubular scaffolds engineered from aligned type I collagen fibers. Type I collagen was extracted from bovine calf hides (A). Bovine collagen type I was extruded with a device that contained two counter-rotating cones to generate the 3-D collagen gel tubular scaffolds. The dimensions of tubes produced for this set of experiments had a length of 30 mm with a luminal diameter of 4 mm and an external diameter of 5 mm, leaving a wall thickness of 0.5 mm. The collagen tubes had a defined fiber angle of 18° relative to the central axis of the tube and had pores ranging from 1 to 10 μm (B). (A and B, courtesy of M.J. Yost)

#### **3.2. BMSCs isolation, expansion and maintenance**

marrow of adult rats. The BMSCs isolated from the bone marrow were expanded, main‐ tained and passaged to make sure that the attached marrow stromal cells were devoid of any non-adhering populations of cells. Phenotypic characterization of the BMSCs for cell surface markers was performed by confocal microscopy (qualitative evaluation) and singlecolor flow cytometry (quantitative analysis). This adherent population of cells was further purified and enriched by indirect magnetic cell sorting. The cells were subjected to CD90

subculturing and subjected to flow cytometric analysis to validate the proper phenotype. This population of purified BMSCs was used in all experiments. For vasculogenic differen‐

the collagen-gel tubular scaffold and cultured either in vasculogenic or non-vasculogenic culture medium for 28 days. At regular intervals of 7, 14, 21 and 28 days the tube cultures were assayed by RT-qPCR, immunofluorescence, ultrastructural and biochemical analyses for various endothelial and smooth muscle differentiation markers as shown in table 1, 2 and 3. The time points at which these analyses were carried out cover the optimal range of both vasculogenesis and angiogenic processes seen in vivo and/or in vitro and mimic the

The 3-D collagen type I tube served as a scaffold on which rat BMSCs differentiation cul‐ tures were carried out (Figure 1). The details of the production and properties of the col‐ lagen tubular scaffolds have previously been described (Yost et al., 2004). Briefly, a 25 mg/ml solution of bovine collagen type I was extruded with a device that contained two counter-rotating cones. The liquid collagen was fed between the two cones and forced through a circular annulus in the presence of an NH3-air (50-50 vol/vol) chamber. This process results in a hollow cylindrical tube of aligned collagen fibrils with an inner cen‐ tral lumen. The dimensions of tubes produced for this set of experiments had a length of 30 mm with a luminal diameter of 4 mm and an external diameter of 5 mm, leaving a wall thickness of 0.5 mm. The collagen tubes had a defined fiber angle of 18° relative to the central axis of the tube and had pores ranging from 1 to 10 µm. The rationale for the particular orientation of collagen fibers was based on our previous work on cardiovascu‐ lar tissue engineering (Yost et al., 2004). When proepicardial organ (PE) cells were seed‐ ed onto this scaffold, they underwent maturation and differentiation and produced elongated vessel-like structures reminiscent of in vivo-like phenotype (Valarmathi et al., 2008). The tubes were sterilized using gamma radiation 1200 Gy followed by Stratalinker UV crosslinker 1800 (Stratagene) and then placed in Mosconas's solution (in mM: 136.8 NaCl, 28.6 KCl, 11.9 NaHCO3, 9.4 glucose, 0.08 NaH2PO4, pH 7.4) (Sigma-Aldrich) con‐ taining 1 µl/ml gentamicin (Sigma-Aldrich) and incubated in 5% CO2 at 37°C until cellu‐

/CD34-

/CD45-

fractions were expanded by

BMSCs were seeded into

positive selection. The resultant enriched CD90+

progression of microvascular development.

**3. Research methods**

264 Research Directions in Tumor Angiogenesis

**3.1. Fabrication of tubular scaffold**

lar seeding (Valarmathi et al., 2010).

tiation of BMSCs, the expanded and purified population of CD90+

The initial step is to isolate the mononuclear cells from the bone marrow by aspiration and centrifugation followed by plating and isolation of the cells based on differential ad‐ herence capacity to tissue culture dishes (passage 0 cells). Rat BMSCs were isolated from the bone marrow of adult 300g Sprague Dawley®™ SD®™ rats (Harlan Sprague Daw‐ ley, Inc.). Briefly, after deep anesthesia, the femoral and tibial bones were removed asep‐ tically and cleaned extensively to remove associated soft connective tissues. The marrow cavities of these bones were flushed with Dulbecco's Modified Eagle Medium (DMEM; Invitrogen) and combined. The isolated marrow plugs were triturated, and passed through needles of decreasing gauge (from 18 gauge to 22 gauge) to break up clumps and cellular aggregates. The resulting single-cell suspensions were centrifuged at 200g for 5 minutes. Nucleated cells were counted using a Neubauer chamber. Cells were plat‐ ed at a density of 5 X 106 – 2 X 107 cells per T75 cm2 flasks in basal media composed of DMEM supplemented with 10% fetal bovine serum (FBS, lot-selected; Hyclone), gentami‐ cin (50 µg/ml) and amphotericin B (250 ng/ml) and incubated in a humidified atmos‐ phere of 5% CO2 at 37°C for 7 days. The medium was replaced, and changed three times per week until the cultures become ~70% confluent (between 12 and 14 days). Cells were trypsinized using 0.05% trypsin-0.1% EDTA and re-plated at a density of 1 x 106 cells per T75 cm2 flasks. After three passages, attached marrow stromal cells were devoid of any non-adhering population of cells. These passaged BMSCs were cryopreserved and stored in liquid nitrogen until further use (Valarmathi et al., 2011).

#### **3.3. Immunophenotyping of BMSCs by flow cytometry and confocal microscopy**

BMSCs are a heterogeneous population of cells with varying degrees of cell shapes and sizes (Anokhina et al., 2007). Stringent characterization of BMSCs used in experimental proce‐ dures is required for various cell surface markers; this is to ensure that the employed popu‐ lation of cells contains solely stem/progenitor cells. This will obviate the possible contamination of marrow-derived endothelial cells and macrophages that are part of the ad‐ herent population of cultured cells. Therefore, characterization of BMSCs included qualita‐ tive evaluation for various cell surface markers and was performed on cells grown in the Lab-tekTM chamber slide systemTM (Nunc) using a Zeiss LSM 510 Meta confocal scanning la‐ ser microscope (Carl Zeiss, Inc.), and quantitative analysis of the same set of markers was performed by single-color flow cytometry using a Coulter® EPICS® XL™ Flow Cytometer (Beckman Coulter, Inc.) as previously described (Valarmathi et al., 2009).

ports and the minimal criteria for defining multipotent mesenchymal stromal cells, enun‐ ciated by the international society for cellular therapy (ISCT) position statement

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Phenotypic characterization using the same set of markers on passage 3 BMSCs by confocal microscopy also revealed that the cells were negative for CD11b, CD31, CD34, CD44, CD45 and OX43 (Figure 3, A-J, O-P), and strongly positive for CD73 and CD90 (Figure 3, K-N). The permeabilized cells when stained for Vcam1 (CD106), Flt1 (Vegfr1), Flk1 (Vegfr2) and VE-cadherin (Figure 4, A-H) revealed faintly detectable cytoplasmic and/or nuclear signal of these endothelial antigens. While these cells showed abundant cytoplasmic expression of smooth muscle antigen, calponin (Figure 4, I-J). Phenotypic characterization and evaluation of these markers on clonally expanded BMSCs showed similar expression patterns consis‐

**Primary Antibodies Dilutions Source Cell Target**

CD11b 1:50 BD Pharmingen Leukocytes CD31 1:10 Abcam Endothelial CD44 1:10 Gene Tex, Inc Leukocytes CD45 1:50 BD Pharmingen Hematopoietic

CD73 1:50 BD Pharmingen BMSCs CD90 1:50 BD Pharmingen BMSCs CD106 1:50 BD Pharmingen Endothelial OX43 1:10 Gene Tex, Inc Endothelial

CD34 1:100 Santa Cruz Biotechnology Endothelial Flt-1 1:100 Santa Cruz Biotechnology Endothelial Flk-1 1:100 Santa Cruz Biotechnology Endothelial VE- cadherin 1:100 Santa Cruz Biotechnology Endothelial Pecam1 1:100 Santa Cruz Biotechnology Endothelial Vwf 1:100 Santa Cruz Biotechnology Endothelial Tomato lectin 1:50 Vector Laboratories Endothelial Fibronectin 1:200 Abcam Endothelial

α-SMA 1:100 Sigma-Aldrich Smooth Muscle Calponin 1:5000 Sigma-Aldrich Smooth Muscle

(Valarmathi et al., 2009; Reyes et al., 2002; Dominici et al., 2006).

tent with their parent culture.

BMSCs characterization markers

Endothelial cell differentiation markers

Smooth muscle cell differentiation markers

**Table 2.** Primary antibodies used in this study (Valarmathi et al., 2009).


**Table 1.** Criteria to identify BMSCs/MSCs (Dominici et al., 2006)

Immunophenotyping of passage 3 undifferentiated BMSCs for various cell surface mark‐ ers by flow cytometry revealed that the fluorescent intensity and distribution of the cells stained for CD11b, CD31, CD34, CD44, CD45 and CD106 were not significantly different from the intensity and distribution of cells stained with isotype controls (Figure 2 A-E, H). In addition, these cells were negative for the rat endothelial cell surface marker OX43 (Figure 2, I), an antigen expressed on all vascular endothelial cells of rat, indicating that these cultures were devoid of any possible hematopoietic stem and/or progenitor cells as well as differentiated bone-marrow-derived endothelial cells. In contrast, BMSCs exhibit‐ ed high expression of CD73 and CD90 surface antigens (Figure 2, F-G), which is consis‐ tent with their undifferentiated state. Furthermore, flow cytometric analysis of the same passage 3 BMSCs for various other vascular endothelial cell surface antigens and smooth muscle cell intracellular antigens revealed that these cells were negative for Flt1, Flk1 and VE-cadherin (Figure 2, J-L) and, were predominantly positive for calponin (Figure 2, M). The expression profiles of these surface molecules were consistent with previous re‐ ports and the minimal criteria for defining multipotent mesenchymal stromal cells, enun‐ ciated by the international society for cellular therapy (ISCT) position statement (Valarmathi et al., 2009; Reyes et al., 2002; Dominici et al., 2006).

**3.3. Immunophenotyping of BMSCs by flow cytometry and confocal microscopy**

(Beckman Coulter, Inc.) as previously described (Valarmathi et al., 2009).

International Society for Cellular Therapy (ISCT): Mesenchymal and Tissue Stem Cell Committee

**Criteria to Identify BMSCs/MSCs**

266 Research Directions in Tumor Angiogenesis

1. Adherence to plastic in standard culture conditions

2. Phenotype Positive (≥95%+)

3. In vitro differentiation: Osteoblasts, Chondroblasts and Adipocytes.

(Demonstrated by staining of in vitro cell culture)

**Table 1.** Criteria to identify BMSCs/MSCs (Dominici et al., 2006)

BMSCs are a heterogeneous population of cells with varying degrees of cell shapes and sizes (Anokhina et al., 2007). Stringent characterization of BMSCs used in experimental proce‐ dures is required for various cell surface markers; this is to ensure that the employed popu‐ lation of cells contains solely stem/progenitor cells. This will obviate the possible contamination of marrow-derived endothelial cells and macrophages that are part of the ad‐ herent population of cultured cells. Therefore, characterization of BMSCs included qualita‐ tive evaluation for various cell surface markers and was performed on cells grown in the Lab-tekTM chamber slide systemTM (Nunc) using a Zeiss LSM 510 Meta confocal scanning la‐ ser microscope (Carl Zeiss, Inc.), and quantitative analysis of the same set of markers was performed by single-color flow cytometry using a Coulter® EPICS® XL™ Flow Cytometer

> CD73 CD90 CD105

Immunophenotyping of passage 3 undifferentiated BMSCs for various cell surface mark‐ ers by flow cytometry revealed that the fluorescent intensity and distribution of the cells stained for CD11b, CD31, CD34, CD44, CD45 and CD106 were not significantly different from the intensity and distribution of cells stained with isotype controls (Figure 2 A-E, H). In addition, these cells were negative for the rat endothelial cell surface marker OX43 (Figure 2, I), an antigen expressed on all vascular endothelial cells of rat, indicating that these cultures were devoid of any possible hematopoietic stem and/or progenitor cells as well as differentiated bone-marrow-derived endothelial cells. In contrast, BMSCs exhibit‐ ed high expression of CD73 and CD90 surface antigens (Figure 2, F-G), which is consis‐ tent with their undifferentiated state. Furthermore, flow cytometric analysis of the same passage 3 BMSCs for various other vascular endothelial cell surface antigens and smooth muscle cell intracellular antigens revealed that these cells were negative for Flt1, Flk1 and VE-cadherin (Figure 2, J-L) and, were predominantly positive for calponin (Figure 2, M). The expression profiles of these surface molecules were consistent with previous re‐

Negative (≤2%+) CD11b or CD14

CD79α or CD19 HLA-DR

CD34 CD45 Phenotypic characterization using the same set of markers on passage 3 BMSCs by confocal microscopy also revealed that the cells were negative for CD11b, CD31, CD34, CD44, CD45 and OX43 (Figure 3, A-J, O-P), and strongly positive for CD73 and CD90 (Figure 3, K-N). The permeabilized cells when stained for Vcam1 (CD106), Flt1 (Vegfr1), Flk1 (Vegfr2) and VE-cadherin (Figure 4, A-H) revealed faintly detectable cytoplasmic and/or nuclear signal of these endothelial antigens. While these cells showed abundant cytoplasmic expression of smooth muscle antigen, calponin (Figure 4, I-J). Phenotypic characterization and evaluation of these markers on clonally expanded BMSCs showed similar expression patterns consis‐ tent with their parent culture.


**Table 2.** Primary antibodies used in this study (Valarmathi et al., 2009).

**Figure 2.** Immunophenotyping of passage 3 undifferentiated rat BMSCs by flow cytometry. Single parameter histo‐ grams showing the relative fluorescence intensity of staining (abscissa) and the number of cells analyzed, events (ordi‐ nate). Isotype controls were included in each experiment to identify the level of background fluorescence. The intensity and distribution of cells stained for hematopoietic and endothelial markers; CD11b, CD31, CD34, CD44, CD45, CD106, OX43, Flt-1, Flk-1 and VE-cadherin (green, shaded peaks) were not significantly different from those of isotype control (red, open peaks) (Panels A-E, H-L). The fluorescent intensity was greater (shifted to right) when BMSCs were stained with CD73, CD90 and calponin (green) compared to isotype control (red) (Panels F, G, M). The predomi‐ nant population of BMSCs uniformly expressed CD90 surface molecule, consistent with their undifferentiated state. Adapted from Valarmathi et al., 2009.

**Figure 3.** Immunophenotyping of passage 3 undifferentiated rat BMSCs by confocal microscopy. Phenotypic charac‐ terization and evaluation revealed that the permeabilized cells were negative for CD11b, CD31, CD34, CD44, CD45 and OX43 (Figure 3, A-J, O-P), indicating that these cultures were devoid of any potential hematopoietic and/or endo‐ thelial cells of bone marrow origin. However, the cells consistently expressed both CD73 and CD90 (Figure3, K-N) sur‐ face antigens, a property of mesenchymal/stromal stem cells. Isotype controls were included in each experiment to identify the level of background staining. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (red, rhoda‐ mine phalloidin). Merged images (B, D, F, H, J-N, P). (A-P, scale bar 100 μm). Adapted from Valarmathi et al., 2009.

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269

**Figure 3.** Immunophenotyping of passage 3 undifferentiated rat BMSCs by confocal microscopy. Phenotypic charac‐ terization and evaluation revealed that the permeabilized cells were negative for CD11b, CD31, CD34, CD44, CD45 and OX43 (Figure 3, A-J, O-P), indicating that these cultures were devoid of any potential hematopoietic and/or endo‐ thelial cells of bone marrow origin. However, the cells consistently expressed both CD73 and CD90 (Figure3, K-N) sur‐ face antigens, a property of mesenchymal/stromal stem cells. Isotype controls were included in each experiment to identify the level of background staining. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (red, rhoda‐ mine phalloidin). Merged images (B, D, F, H, J-N, P). (A-P, scale bar 100 μm). Adapted from Valarmathi et al., 2009.

**Figure 2.** Immunophenotyping of passage 3 undifferentiated rat BMSCs by flow cytometry. Single parameter histo‐ grams showing the relative fluorescence intensity of staining (abscissa) and the number of cells analyzed, events (ordi‐ nate). Isotype controls were included in each experiment to identify the level of background fluorescence. The intensity and distribution of cells stained for hematopoietic and endothelial markers; CD11b, CD31, CD34, CD44, CD45, CD106, OX43, Flt-1, Flk-1 and VE-cadherin (green, shaded peaks) were not significantly different from those of isotype control (red, open peaks) (Panels A-E, H-L). The fluorescent intensity was greater (shifted to right) when BMSCs were stained with CD73, CD90 and calponin (green) compared to isotype control (red) (Panels F, G, M). The predomi‐ nant population of BMSCs uniformly expressed CD90 surface molecule, consistent with their undifferentiated state.

Adapted from Valarmathi et al., 2009.

268 Research Directions in Tumor Angiogenesis

**3.4. Purification and enrichment of CD90+**

scribed previously (Valarmathi et al., 2010).

seeded into the collagen-gel tubes at a density of 0.5 x 106

**3.5. BMSCs vasculogenic differentiation**

**(MACS)**

cells/cm2

7, 14, 21 and 28 days.

**regeneration**

 **BMSCs by Magnetic-Activated Cell Sorting**

/CD34-

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271

cells/30 mm tube lengths and cul‐

/CD45- frac‐

Purification and enrichment of input BMSCs (such as CD45-, CD34-, CD105) are manda‐ tory either using MACS (magnetic activated cell sorter) or FACS (fluorescent activated cell sorter). Since the unpurified fraction may contain sizable number of contaminating adherent macrophages and bone marrow-derived endothelial progenitors and differenti‐ ated endothelial cells. The adherent populations of BMSCs were further purified by indi‐ rect magnetic cell labeling method using an autoMACS™ Pro Separator (Miltenyi Biotech). The cells were subjected to CD90 positive selection by incubating the cells with FITC- labeled anti-CD90 antibodies (BD Pharmingen), followed by incubation with anti-FITC magnetic microbeads (Miltenyi Biotech), and passed through the magnetic columns

3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of…

tions were expanded by subcultivation and subjected to flow cytometric analysis as de‐

For vasculogenic differentiation of BMSCs, the purified population of CD90+ BMSCs were

tured either in mesenchymal stem cell growth medium supplemented with 10% FBS, peni‐ cillin and streptomycin (Poietics® MSCGM™ BulletKit®; Lonza Ltd.) or microvascular endothelial cell growth medium (Clonetics® EGM®-MV Bullet Kit®; Lonza Ltd.) supplement‐ ed with 5% FBS, bovine brain extract, human epidermal growth factor (hEGF), hydrocorti‐ sone, amphotericin B and gentamicin for 28 days. These BMSCs seeded tubes were cultured either in vasculogenic or non-vasculogenic medium for the defined time periods of 7, 14, 21 and 28 days. In addition, BMSCs were seeded in 65-mm Petri dishes at a density of 3 x 103

**4. BMSCs based postnatal de novo vasculogenesis and in situ vascular**

The 3-D collagen-gel tubular scaffold has previously been used to create models of vascular‐ ized bone development (Valarmathi et al., 2008a; 2008b). Here we report the utility of a 3-D tubular construct for its ability to support the vasculogenic differentiation of BMSCs culmi‐ nating into microvascular structures, which are similar to those structures resulting from

In the developing vertebrate embryo, the initial event of blood vessel formation is the differ‐ entiation of vascular endothelial cells, which subsequently cover the entire interior surface of all blood vessels. Angioblasts are a subpopulation of primitive mesodermal cells that are committed to differentiate into endothelial cells and later on form the primitive vascular lab‐

postnatal de novo vasculogenesis and angiogenesis (Valarmathi et al., 2008a; 2008b).

and cultured in non-vasculogenic (MSCGM) or vasculogenic (EGMMV) media for

as per the manufacturer's instructions. The resultant enriched CD90+

**Figure 4.** Confocal microscopic analysis of various vascular related antigens on passage 3 undifferentiated rat BMSCs. Evaluation of the permeabilized cells for Vcam1 (CD106), Flt1 (Vegfr1), Flk1 (Vegfr2) and VE-cadherin revealed faintly detectable cytoplasmic and/or nuclear signals of these endothelial antigens (Figure 4, A-H). Whereas, these cells showed abundant cytoplasmic expression of smooth muscle antigen, calponin (Figure 4, I-J). Suggesting that BMSCs constitutively express very low-levels of endothelial associated antigens as well as very high-levels of smooth muscle specific antigens. Isotype and/or negative controls were included in each experiment to identify the level of back‐ ground staining. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (red, rhodamine phalloidin). Merged images (B, D-F, H-J). (A-J, scale bar 100 μm). Adapted from Valarmathi et al., 2009.

#### **3.4. Purification and enrichment of CD90+ BMSCs by Magnetic-Activated Cell Sorting (MACS)**

Purification and enrichment of input BMSCs (such as CD45-, CD34-, CD105) are manda‐ tory either using MACS (magnetic activated cell sorter) or FACS (fluorescent activated cell sorter). Since the unpurified fraction may contain sizable number of contaminating adherent macrophages and bone marrow-derived endothelial progenitors and differenti‐ ated endothelial cells. The adherent populations of BMSCs were further purified by indi‐ rect magnetic cell labeling method using an autoMACS™ Pro Separator (Miltenyi Biotech). The cells were subjected to CD90 positive selection by incubating the cells with FITC- labeled anti-CD90 antibodies (BD Pharmingen), followed by incubation with anti-FITC magnetic microbeads (Miltenyi Biotech), and passed through the magnetic columns as per the manufacturer's instructions. The resultant enriched CD90+ /CD34- /CD45- frac‐ tions were expanded by subcultivation and subjected to flow cytometric analysis as de‐ scribed previously (Valarmathi et al., 2010).

#### **3.5. BMSCs vasculogenic differentiation**

**Figure 4.** Confocal microscopic analysis of various vascular related antigens on passage 3 undifferentiated rat BMSCs. Evaluation of the permeabilized cells for Vcam1 (CD106), Flt1 (Vegfr1), Flk1 (Vegfr2) and VE-cadherin revealed faintly detectable cytoplasmic and/or nuclear signals of these endothelial antigens (Figure 4, A-H). Whereas, these cells showed abundant cytoplasmic expression of smooth muscle antigen, calponin (Figure 4, I-J). Suggesting that BMSCs constitutively express very low-levels of endothelial associated antigens as well as very high-levels of smooth muscle specific antigens. Isotype and/or negative controls were included in each experiment to identify the level of back‐ ground staining. Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (red, rhodamine phalloidin). Merged

images (B, D-F, H-J). (A-J, scale bar 100 μm). Adapted from Valarmathi et al., 2009.

270 Research Directions in Tumor Angiogenesis

For vasculogenic differentiation of BMSCs, the purified population of CD90+ BMSCs were seeded into the collagen-gel tubes at a density of 0.5 x 106 cells/30 mm tube lengths and cul‐ tured either in mesenchymal stem cell growth medium supplemented with 10% FBS, peni‐ cillin and streptomycin (Poietics® MSCGM™ BulletKit®; Lonza Ltd.) or microvascular endothelial cell growth medium (Clonetics® EGM®-MV Bullet Kit®; Lonza Ltd.) supplement‐ ed with 5% FBS, bovine brain extract, human epidermal growth factor (hEGF), hydrocorti‐ sone, amphotericin B and gentamicin for 28 days. These BMSCs seeded tubes were cultured either in vasculogenic or non-vasculogenic medium for the defined time periods of 7, 14, 21 and 28 days. In addition, BMSCs were seeded in 65-mm Petri dishes at a density of 3 x 103 cells/cm2 and cultured in non-vasculogenic (MSCGM) or vasculogenic (EGMMV) media for 7, 14, 21 and 28 days.

#### **4. BMSCs based postnatal de novo vasculogenesis and in situ vascular regeneration**

The 3-D collagen-gel tubular scaffold has previously been used to create models of vascular‐ ized bone development (Valarmathi et al., 2008a; 2008b). Here we report the utility of a 3-D tubular construct for its ability to support the vasculogenic differentiation of BMSCs culmi‐ nating into microvascular structures, which are similar to those structures resulting from postnatal de novo vasculogenesis and angiogenesis (Valarmathi et al., 2008a; 2008b).

In the developing vertebrate embryo, the initial event of blood vessel formation is the differ‐ entiation of vascular endothelial cells, which subsequently cover the entire interior surface of all blood vessels. Angioblasts are a subpopulation of primitive mesodermal cells that are committed to differentiate into endothelial cells and later on form the primitive vascular lab‐ yrinth (Risau and Flamme, 2000). In addition, endothelial cells can also arise from heman‐ gioblasts, a common precursor for both hematopoietic and endothelial cells (His et al., 1900).

be induced to undergo microvascular development. Hence, we developed a 3-D culture sys‐

3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of…

aligned, porous, biocompatible collagen-fiber tubular scaffold for differentiation purposes. Here, we utilized two types of growth media for vasculogenic differentiation purpose, MSCGM (non-vasculogenic) as control and EGMMV (vasculogenic) preferentially for micro‐ vascular differentiation. Both of these culture media consistently promoted the vasculogenic differentiation of BMSCs and also supported the formation of endothelium lined vessel-like

A number of early and late stage markers associated with rodent vascular development in vivo were used in this study to characterize the rat BMSCs derived microvascular structures at mRNA and protein levels, which included: CD31/Pecam1, Flt1 (Vegfr1), Flk1 (Vegfr2/ Kdr), VE-cadherin (CD144), CD34, Tie1, Tek (Tie2), and Von Willibrand factor (Vwf). Plate‐ let/endothelial cell adhesion molecule, also known as CD31, is a transmembrane protein ex‐ pressed abundantly early in vascular development that may mediate leukocyte adhesion and migration, angiogenesis, and thrombosis (Albelda et al., 1991). The other early stage dif‐ ferentiation markers Flk1 and Flt1 that are receptors for the vascular endothelial cell growth factor-A (Vegf) essentially play a vital role in embryonic vascular and hematopoietic devel‐ opment (Shalaby et al., 1997). Similarly, VE-cadherin, a member of the cadherin family of adhesion receptors, is a specific and constitutive marker of endothelial cell plays an impor‐ tant role in early vascular assembly. Vascular markers that are expressed at a later stage in‐ clude CD34 and Tie-2 (Bautch et al., 2000). CD34 is a transmembrane surface glycoprotein that is expressed in endothelial cells and hematopoietic stem cells. Tie1 and Tek are receptor kinases on endothelial cells that are essential for vascular development and remodeling in the embryo and may also mediate maintenance and repair of the adult vascular system. In late phases of vasculogenesis, the mature endothelial cells will synthesize and secrete Vwf homolog, a plasma protein that mediates platelet adhesion to damaged blood vessels and

rat BMSCs was seeded and cultured on a highly

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**length (bp)**

**Annealing temperature (°C)**

**GenBank accession**

**No**

tem in which a pure population of CD90+

structures within the constructs.

stabilizes blood coagulation factor VIII.

**Genes Forward primer Reverse primer Product**

Pecam1 5'–CGAAATCTAGGCCTCAGCAC–3' 5'–CTTTTTGTCCACGGTCACCT–3' 227 56 NM\_03159.1 Kdr 5'–TAGCGGGATGAAATCTTTGG–3' 5'–TTGGTGAGGATGACCGTGTA–3' 207 56 NM\_013062.1 Tie1 5'–AAGGTCACACACACGGTGAA–3' 5'–TGGTGGCTGTACATTTTGGA–3' 174 56 XM\_233462.4

Tek 5'–CCGTGCTGCTGAACAACTTA–3' 5'–AATAGCCGTCCACGATTGTC–3' 201 56 NM\_001105737.1

Vwf 5'–GCTCCAGCAAGTTGAAGACC–3' 5'–GCAAGTCACTGTGTGGCACT–3' 163 56 XM\_342759.3 Gapdh 5'–TTCAATGGCACAGTCAAGGC–3' 5'–TCACCCCATTTGATGTTAGCG–3' 101 56 XR\_007416.1

**Table 3.** RT-qPCR primer sequences used in this study (Valarmathi et al., 2009; Rozen and Skaletsky, 2000).

In adults, endothelial precursor cells have been identified in bone marrow, peripheral blood and blood vessels (Prater et al., 2007). Two subsets of multipotential stem cells, HSCs and BMSCs/MSCs are resident in the postnatal bone marrow. Of these cells, BMSCs can be dif‐ ferentiated into osteoblasts, chondrocytes, adipocytes, smooth muscle cells and hemato‐ poietic supportive stroma either in vitro or in vivo (Bianco et al., 2001). Previous studies have provided substantial evidence that bone-marrow-derived stem and/or progenitor cells can be differentiated into either endothelial or smooth muscle cells in vitro and in pathologi‐ cal situations are capable of contributing to neoangiogenesis in vivo by cellular integration (Carmeliet and Luttun, 2001).

Although there are a plethora of studies focused on developing viable scaffolds for osteo‐ genic, chondrogenic, adipogenic and musculogenic differentiation of BMSCs (Lanza et al., 2000), the optimal scaffolds that are capable of inducing and supporting the growth and dif‐ ferentiation of BMSCs into vascular cell lineages are yet to be identified and characterized. Despite the much known vasculogenic potential and transgermal plasticity of BMSCs; none of these studies explicitly demonstrated the postnatal de novo vasculogenic potential of BMSCs in vitro (Reyes et al., 2002; Oswald et al., 2004; Brey et al., 2005).

When compared to 2-D planar cultures, the potential 3-D models of vasculogenesis allow us to understand the role of specific factors under more physiological and spatial conditions with respect to dimensionality, architecture and cell polarity. Nevertheless, the molecular composition and the natural complexity and diversity of in vivo extra cellular matrix (ECM) organization cannot be easily mimicked or reproduced in vitro (Vailhe et al., 2001). In addi‐ tion, even though quite a few in vitro 3-D models of vasculogenesis based on fibrin and col‐ lagen gels are in vogue (Folkman and Haudenschild, 1980); none have explored the behavior of BMSCs and their intrinsic vasculogenic differentiation potential on a topograph‐ ically structured 3-D tubular scaffold made of uniformly aligned type I collagen fibers.

Previous studies demonstrated that the formation of endothelial tubes in vitro was largely influenced by the nature of the substrate (Kleinman et al., 1982). The formation of endotheli‐ um lined tubular structures was enhanced when the substrate was rich in laminin (Madri et al., 1988), whereas a matrix rich in type I collagen would not promote rapid tubulogenesis (Montesano et al., 1983; Ingber and Folkman, 1989). Similarly, Ingber and Folkman (1989) documented that under a given cocktail of growth factors, the local physical nature of the interaction between endothelial cells and the underlying matrix/substrate ultimately deter‐ mined the tubular morphogenesis. Substrates containing abundant fibronectin promoted adhesion, spreading and growth of endothelial cells. In contrast, less adhesive substrate or matrix materials that were arranged three-dimensionally permitted the endothelial cells to retract and form tubes (Ingber and Folkman, 1989).

In general, successful in vitro differentiation of cells depends on cell-cell as well as cell-ma‐ trix interactions. Therefore, we hypothesized that under appropriate in vitro local environ‐ mental cues (combination of growth factors and ECM) multipotent postnatal BMSCs could be induced to undergo microvascular development. Hence, we developed a 3-D culture sys‐ tem in which a pure population of CD90+ rat BMSCs was seeded and cultured on a highly aligned, porous, biocompatible collagen-fiber tubular scaffold for differentiation purposes. Here, we utilized two types of growth media for vasculogenic differentiation purpose, MSCGM (non-vasculogenic) as control and EGMMV (vasculogenic) preferentially for micro‐ vascular differentiation. Both of these culture media consistently promoted the vasculogenic differentiation of BMSCs and also supported the formation of endothelium lined vessel-like structures within the constructs.

yrinth (Risau and Flamme, 2000). In addition, endothelial cells can also arise from heman‐ gioblasts, a common precursor for both hematopoietic and endothelial cells (His et al., 1900).

In adults, endothelial precursor cells have been identified in bone marrow, peripheral blood and blood vessels (Prater et al., 2007). Two subsets of multipotential stem cells, HSCs and BMSCs/MSCs are resident in the postnatal bone marrow. Of these cells, BMSCs can be dif‐ ferentiated into osteoblasts, chondrocytes, adipocytes, smooth muscle cells and hemato‐ poietic supportive stroma either in vitro or in vivo (Bianco et al., 2001). Previous studies have provided substantial evidence that bone-marrow-derived stem and/or progenitor cells can be differentiated into either endothelial or smooth muscle cells in vitro and in pathologi‐ cal situations are capable of contributing to neoangiogenesis in vivo by cellular integration

Although there are a plethora of studies focused on developing viable scaffolds for osteo‐ genic, chondrogenic, adipogenic and musculogenic differentiation of BMSCs (Lanza et al., 2000), the optimal scaffolds that are capable of inducing and supporting the growth and dif‐ ferentiation of BMSCs into vascular cell lineages are yet to be identified and characterized. Despite the much known vasculogenic potential and transgermal plasticity of BMSCs; none of these studies explicitly demonstrated the postnatal de novo vasculogenic potential of

When compared to 2-D planar cultures, the potential 3-D models of vasculogenesis allow us to understand the role of specific factors under more physiological and spatial conditions with respect to dimensionality, architecture and cell polarity. Nevertheless, the molecular composition and the natural complexity and diversity of in vivo extra cellular matrix (ECM) organization cannot be easily mimicked or reproduced in vitro (Vailhe et al., 2001). In addi‐ tion, even though quite a few in vitro 3-D models of vasculogenesis based on fibrin and col‐ lagen gels are in vogue (Folkman and Haudenschild, 1980); none have explored the behavior of BMSCs and their intrinsic vasculogenic differentiation potential on a topograph‐ ically structured 3-D tubular scaffold made of uniformly aligned type I collagen fibers.

Previous studies demonstrated that the formation of endothelial tubes in vitro was largely influenced by the nature of the substrate (Kleinman et al., 1982). The formation of endotheli‐ um lined tubular structures was enhanced when the substrate was rich in laminin (Madri et al., 1988), whereas a matrix rich in type I collagen would not promote rapid tubulogenesis (Montesano et al., 1983; Ingber and Folkman, 1989). Similarly, Ingber and Folkman (1989) documented that under a given cocktail of growth factors, the local physical nature of the interaction between endothelial cells and the underlying matrix/substrate ultimately deter‐ mined the tubular morphogenesis. Substrates containing abundant fibronectin promoted adhesion, spreading and growth of endothelial cells. In contrast, less adhesive substrate or matrix materials that were arranged three-dimensionally permitted the endothelial cells to

In general, successful in vitro differentiation of cells depends on cell-cell as well as cell-ma‐ trix interactions. Therefore, we hypothesized that under appropriate in vitro local environ‐ mental cues (combination of growth factors and ECM) multipotent postnatal BMSCs could

BMSCs in vitro (Reyes et al., 2002; Oswald et al., 2004; Brey et al., 2005).

retract and form tubes (Ingber and Folkman, 1989).

(Carmeliet and Luttun, 2001).

272 Research Directions in Tumor Angiogenesis

A number of early and late stage markers associated with rodent vascular development in vivo were used in this study to characterize the rat BMSCs derived microvascular structures at mRNA and protein levels, which included: CD31/Pecam1, Flt1 (Vegfr1), Flk1 (Vegfr2/ Kdr), VE-cadherin (CD144), CD34, Tie1, Tek (Tie2), and Von Willibrand factor (Vwf). Plate‐ let/endothelial cell adhesion molecule, also known as CD31, is a transmembrane protein ex‐ pressed abundantly early in vascular development that may mediate leukocyte adhesion and migration, angiogenesis, and thrombosis (Albelda et al., 1991). The other early stage dif‐ ferentiation markers Flk1 and Flt1 that are receptors for the vascular endothelial cell growth factor-A (Vegf) essentially play a vital role in embryonic vascular and hematopoietic devel‐ opment (Shalaby et al., 1997). Similarly, VE-cadherin, a member of the cadherin family of adhesion receptors, is a specific and constitutive marker of endothelial cell plays an impor‐ tant role in early vascular assembly. Vascular markers that are expressed at a later stage in‐ clude CD34 and Tie-2 (Bautch et al., 2000). CD34 is a transmembrane surface glycoprotein that is expressed in endothelial cells and hematopoietic stem cells. Tie1 and Tek are receptor kinases on endothelial cells that are essential for vascular development and remodeling in the embryo and may also mediate maintenance and repair of the adult vascular system. In late phases of vasculogenesis, the mature endothelial cells will synthesize and secrete Vwf homolog, a plasma protein that mediates platelet adhesion to damaged blood vessels and stabilizes blood coagulation factor VIII.


**Table 3.** RT-qPCR primer sequences used in this study (Valarmathi et al., 2009; Rozen and Skaletsky, 2000).

In any type of in vitro cellular differentiation, the cytodifferentiated cells need to be critically evaluated for their maturation and differentiation at transcriptional, translational and func‐ tional levels. Therefore, to study the expression pattern of key vasculogenic gene transcripts in the 3-D tube constructs; we examined the time-dependent expression pattern of Pecam1, Kdr, Tie1, Tek and Vwf at mRNA level in the tube constructs by real-time PCR (Figure 5 A-D).

Constitutive expressions of these markers were detected at low to very low levels in undif‐ ferentiated BMSCs. RT-qPCR results showed that differentiation of BMSCs under vasculo‐ genic culture conditions for 28 days resulted in increased expression of transcripts coding for various endothelial cell associated proteins such as Pecam1, Kdr, Tek and Vwf. The peak expression of Vwf, the endothelial specific protein occurred around day 21 (over 400 fold) indicating that the differentiating cells acquired a distinctive phenotype and biosynthetic ac‐ tivity of differentiated and matured endothelial cells. The upregulation of Tek during this period may represent the continual development and remodeling of the developing micro‐ vessels. Whereas differentiation of BMSCs under non-vasculogenic conditions for 14 days showed signs of early and rapid induction of transcripts coding for both early and late stage endothelial cell markers such as Kdr, Tie1, Tek and Vwf. The peak expression of Vwf occur‐

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As revealed by immunostaining for various vasculogenic markers, day 21 vasculogenic and non-vasculogenic tube cultures showed that BMSCs were able to adhere, proliferate, migrate and, undergo complete maturation and differentiation into microvascular structures (Figure 6 A-C). BMSCs derived microvessel formation is a combination of de novo vasculogenesis i.e., in situ endothelial cell differentiation and endothelium-lined tube formation, and angiogenesis, endothelial sprouting from existing endothelial tubes. In addition, these microvessels are stabi‐

**Figure 6.** Localization of BMSCs derived endothelial cells by Texas Red labeled Lycopersicon Esculentum lectin/Toma‐ to Lectin (LEL/TL) staining. BMSCs cultured in collagen-gel tubular scaffolds in both vasculogenic and non-vasculogen‐ ic culture conditions were incubated with tomato lectin (1:50 in 10 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid, pH 7.5; 0.15 M NaCl), and was used to identify endothelial cells. Confocal laser scanning microscopic analysis of sections of day 14 tubular scaffolds in these media conditions demonstrated the typical cob‐ blestone appearance of differentiating endothelial cells (A), fusion and self-assembly (B), and evolving primitive capil‐ lary plexus with attempted lumen formation (B-C, white arrows). Cells were also stained for nuclei (blue, DAPI). Image (A) shows a projection representing 19 sections collected at 5.05 μm intervals (90.90 μm). Image (B) shows a projec‐ tion representing 13 sections collected at 4.05 μm intervals (48.60 μm). Image (C) shows a projection representing 15 sections collected at 6 μm intervals (84.00 μm). Merged images (A-C). (A-B, scale bar 100 μm; C, scale bar 50 μm).

To validate the findings of mRNA expression pattern of important vasculogenic markers in these tube cultures and to determine whether these messages were in fact translated into pro‐ teins, immunostaining of the BMSC tube culture was carried out (Figure 7 A-L; Figure 8 A-L).

lized by association with BMSC-derived smooth muscle cells and/or pericytes.

red during day 14 (over 20 fold).

**Figure 5.** Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of various key vas‐ culogenic markers, platelet/endothelial cell adhesion molecule 1 (Pecam1), kinase insert domain protein receptor (Kdr/Flk1/Vegfr-2), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (Tie1), endothelial-specific re‐ ceptor tyrosine kinase (Tek/Tie2) and Von Willebrand factor homology (Vwf) as a function of time (abscissa). BMSCs cultured in Petri dishes (2-D culture) in mesenchymal stem cell growth medium (A) and, in microvascular growth medi‐ um (B). BMSCs cultured in collagen-gel tubular scaffolds (3-D culture) in mesenchymal stem cell growth medium (C) and, in microvascular growth medium (D). The calibrator control included passage 4 BMSCs day 0 sample and; the target gene expression was normalized by a non-regulated reference gene expression, Gapdh. The expression ratio (ordinate) was calculated using the REST-XL version 2 software (Pfaffl 2001; Pfaffl et al., 2002). The values are means ± standard errors for three cultures (n=3), \*p<0.005; \*\*P<0.001. Adapted from Valarmathi et al., 2009.

Constitutive expressions of these markers were detected at low to very low levels in undif‐ ferentiated BMSCs. RT-qPCR results showed that differentiation of BMSCs under vasculo‐ genic culture conditions for 28 days resulted in increased expression of transcripts coding for various endothelial cell associated proteins such as Pecam1, Kdr, Tek and Vwf. The peak expression of Vwf, the endothelial specific protein occurred around day 21 (over 400 fold) indicating that the differentiating cells acquired a distinctive phenotype and biosynthetic ac‐ tivity of differentiated and matured endothelial cells. The upregulation of Tek during this period may represent the continual development and remodeling of the developing micro‐ vessels. Whereas differentiation of BMSCs under non-vasculogenic conditions for 14 days showed signs of early and rapid induction of transcripts coding for both early and late stage endothelial cell markers such as Kdr, Tie1, Tek and Vwf. The peak expression of Vwf occur‐ red during day 14 (over 20 fold).

In any type of in vitro cellular differentiation, the cytodifferentiated cells need to be critically evaluated for their maturation and differentiation at transcriptional, translational and func‐ tional levels. Therefore, to study the expression pattern of key vasculogenic gene transcripts in the 3-D tube constructs; we examined the time-dependent expression pattern of Pecam1, Kdr, Tie1, Tek and Vwf at mRNA level in the tube constructs by real-time PCR (Figure 5 A-D).

274 Research Directions in Tumor Angiogenesis

**Figure 5.** Real-time reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of various key vas‐ culogenic markers, platelet/endothelial cell adhesion molecule 1 (Pecam1), kinase insert domain protein receptor (Kdr/Flk1/Vegfr-2), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (Tie1), endothelial-specific re‐ ceptor tyrosine kinase (Tek/Tie2) and Von Willebrand factor homology (Vwf) as a function of time (abscissa). BMSCs cultured in Petri dishes (2-D culture) in mesenchymal stem cell growth medium (A) and, in microvascular growth medi‐ um (B). BMSCs cultured in collagen-gel tubular scaffolds (3-D culture) in mesenchymal stem cell growth medium (C) and, in microvascular growth medium (D). The calibrator control included passage 4 BMSCs day 0 sample and; the target gene expression was normalized by a non-regulated reference gene expression, Gapdh. The expression ratio (ordinate) was calculated using the REST-XL version 2 software (Pfaffl 2001; Pfaffl et al., 2002). The values are means ±

standard errors for three cultures (n=3), \*p<0.005; \*\*P<0.001. Adapted from Valarmathi et al., 2009.

As revealed by immunostaining for various vasculogenic markers, day 21 vasculogenic and non-vasculogenic tube cultures showed that BMSCs were able to adhere, proliferate, migrate and, undergo complete maturation and differentiation into microvascular structures (Figure 6 A-C). BMSCs derived microvessel formation is a combination of de novo vasculogenesis i.e., in situ endothelial cell differentiation and endothelium-lined tube formation, and angiogenesis, endothelial sprouting from existing endothelial tubes. In addition, these microvessels are stabi‐ lized by association with BMSC-derived smooth muscle cells and/or pericytes.

**Figure 6.** Localization of BMSCs derived endothelial cells by Texas Red labeled Lycopersicon Esculentum lectin/Toma‐ to Lectin (LEL/TL) staining. BMSCs cultured in collagen-gel tubular scaffolds in both vasculogenic and non-vasculogen‐ ic culture conditions were incubated with tomato lectin (1:50 in 10 mM N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid, pH 7.5; 0.15 M NaCl), and was used to identify endothelial cells. Confocal laser scanning microscopic analysis of sections of day 14 tubular scaffolds in these media conditions demonstrated the typical cob‐ blestone appearance of differentiating endothelial cells (A), fusion and self-assembly (B), and evolving primitive capil‐ lary plexus with attempted lumen formation (B-C, white arrows). Cells were also stained for nuclei (blue, DAPI). Image (A) shows a projection representing 19 sections collected at 5.05 μm intervals (90.90 μm). Image (B) shows a projec‐ tion representing 13 sections collected at 4.05 μm intervals (48.60 μm). Image (C) shows a projection representing 15 sections collected at 6 μm intervals (84.00 μm). Merged images (A-C). (A-B, scale bar 100 μm; C, scale bar 50 μm).

To validate the findings of mRNA expression pattern of important vasculogenic markers in these tube cultures and to determine whether these messages were in fact translated into pro‐ teins, immunostaining of the BMSC tube culture was carried out (Figure 7 A-L; Figure 8 A-L).

**Figure 7.** Expression pattern of various vasculogenic markers in tubular scaffold by confocal microscopy. Localization of key endothelial and smooth muscle cell phenotypic markers of day 21 non-vasculogenic tube cultures demonstrat‐ ed the expression of CD34 (A-C), Pecam1 (D-F), Vwf (G-I), VE-cadherin (J-L) and α-SMA (B-C, E-F, H-I, K-L). Dual immu‐ nostainings of non-vasculogenic tube cultures (mesenchymal stem cell growth media, MSCGM) revealed areas of elongated and flattened cells composed of varying degrees of mature endothelial and smooth muscle cells (A-L). These cells were organized into a loose delicate network of nascent capillary-like structures composed of mature en‐ dothelial and smooth muscle cells and showed evidence of central lumen formation (white arrows, D-I). In addition, tube-like structures were emanating from the mixed population of vasculogenic cells represented by their distinct morphology and phenotypic expression (white arrows, A-C; white arrows, J-L). Cells were also stained for nuclei (blue, DAPI). Images (A-C) show a projection representing 15 sections collected at 5.05 μm intervals (70.70 μm). Images (D-F) show a projection representing 19 sections collected at 5.05 μm intervals (90.90 μm). Images (J-L) show a projection representing 19 sections collected at 4.04 μm intervals (72.90 μm). Merged images (A-L). (A-L, scale bar 50 μm). Adapted from Valarmathi et al., 2009.

**Figure 8.** Expression pattern of various vasculogenic markers in tubular scaffold by confocal microscopy. Localization of key endothelial and smooth muscle cell phenotypic markers of day 21 vasculogenic tube cultures demonstrated the expression of Flk1 (A, C), VE-cadherin (D, F), Vwf (G, I; J, L), tomato lectin (E-F; H-I) and α-SMA (B-C, K-L). Dual immunos‐ tainings of vasculogenic tube cultures (microvascular endothelial growth medium, EGMMV) revealed areas of elongat‐ ed cells composed of both mature endothelial and smooth muscle cells (A-L). These cells formed developing microvessl-like structures (A-F, J-L). The linear nascent capillary-like structures showed a translucent central lumen (white arrows, D-F). In addition, the cells were organized into a loose network of vascular cells and were in a ribbonlike configuration (G-I). These aligned vascular cells transformed into thin tube-like structures reminiscent of in vivo microvessel morphogenesis (J-L). Cells were also stained for nuclei (blue, DAPI). Images (D-F) show a projection repre‐ senting 13 sections collected at 3.05 μm intervals (36.60 μm). Images (G-I) show a projection representing 18 sections collected at 7.05 μm intervals (119.8 μm). Images (J-L) show a projection representing 27 sections collected at 6.05 μm intervals (157.3 μm). Merged images (A-L). (A-C, J-L scale bar 100 μm; D-F, G-I scale bar 50 μm). Adapted from Valar‐

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mathi et al., 2009.

**Figure 8.** Expression pattern of various vasculogenic markers in tubular scaffold by confocal microscopy. Localization of key endothelial and smooth muscle cell phenotypic markers of day 21 vasculogenic tube cultures demonstrated the expression of Flk1 (A, C), VE-cadherin (D, F), Vwf (G, I; J, L), tomato lectin (E-F; H-I) and α-SMA (B-C, K-L). Dual immunos‐ tainings of vasculogenic tube cultures (microvascular endothelial growth medium, EGMMV) revealed areas of elongat‐ ed cells composed of both mature endothelial and smooth muscle cells (A-L). These cells formed developing microvessl-like structures (A-F, J-L). The linear nascent capillary-like structures showed a translucent central lumen (white arrows, D-F). In addition, the cells were organized into a loose network of vascular cells and were in a ribbonlike configuration (G-I). These aligned vascular cells transformed into thin tube-like structures reminiscent of in vivo microvessel morphogenesis (J-L). Cells were also stained for nuclei (blue, DAPI). Images (D-F) show a projection repre‐ senting 13 sections collected at 3.05 μm intervals (36.60 μm). Images (G-I) show a projection representing 18 sections collected at 7.05 μm intervals (119.8 μm). Images (J-L) show a projection representing 27 sections collected at 6.05 μm intervals (157.3 μm). Merged images (A-L). (A-C, J-L scale bar 100 μm; D-F, G-I scale bar 50 μm). Adapted from Valar‐ mathi et al., 2009.

**Figure 7.** Expression pattern of various vasculogenic markers in tubular scaffold by confocal microscopy. Localization of key endothelial and smooth muscle cell phenotypic markers of day 21 non-vasculogenic tube cultures demonstrat‐ ed the expression of CD34 (A-C), Pecam1 (D-F), Vwf (G-I), VE-cadherin (J-L) and α-SMA (B-C, E-F, H-I, K-L). Dual immu‐ nostainings of non-vasculogenic tube cultures (mesenchymal stem cell growth media, MSCGM) revealed areas of elongated and flattened cells composed of varying degrees of mature endothelial and smooth muscle cells (A-L). These cells were organized into a loose delicate network of nascent capillary-like structures composed of mature en‐ dothelial and smooth muscle cells and showed evidence of central lumen formation (white arrows, D-I). In addition, tube-like structures were emanating from the mixed population of vasculogenic cells represented by their distinct morphology and phenotypic expression (white arrows, A-C; white arrows, J-L). Cells were also stained for nuclei (blue, DAPI). Images (A-C) show a projection representing 15 sections collected at 5.05 μm intervals (70.70 μm). Images (D-F) show a projection representing 19 sections collected at 5.05 μm intervals (90.90 μm). Images (J-L) show a projection representing 19 sections collected at 4.04 μm intervals (72.90 μm). Merged images (A-L). (A-L, scale bar 50 μm).

Adapted from Valarmathi et al., 2009.

276 Research Directions in Tumor Angiogenesis

It is well known that endothelial cells share a large majority of their characteristic anti‐ genic markers with other types of hematopoietic and mesenchymal cells (Bertolini et al., 2006). Therefore, antigens such as CD31, CD34, CD144 (VE-cadherin), CD146, Vwf or CD105 are not only expressed by endothelial cells but also expressed by hematopoietic cells (specifically HSCs), platelets and certain subpopulations of fibroblasts. Hence to identify the differentiated and matured endothelial cells in the tubular scaffold a battery of various early and late stage vasculogenic markers such as Pecam1, CD34, Flt1, Flk1, VE-cadherin, fibronectin and Vwf were employed. In addition, tomato lectin, another marker specific for rat vascular endothelial cells, was found closely associated with Flk1 and Vwf staining. These endothelial associated markers localized to endothelial cell clus‐ ters and capillary-like structures that were present throughout the tubular construct. This suggests that BMSC-derived endothelial cells assembled into endothelium-lined tube-like structures and initiated the process of vasculogenesis, consistent with our previous re‐ port (Figure 9 A-H) (Valarmathi et al., 2008a). In addition, the BMSC-derived cells and the microvessel-like structures expressed the smooth muscle antigens, α-SMA and calpo‐ nin. These α-SMA positive cells were recruited in juxtaposition to the tandemly arranged endothelial cells and, were attached and wrapped around in such a way that is reminis‐ cent of in vivo microvessel morphogenesis.

In order to quantitate the degree of vasculogenesis, image morphometric analyses were used to determine the capillary density, vessel breadth and length (integrated morphom‐ etry subroutine of MetaMorph 6.1). In brief, confocal scanning laser microscopic color images were converted to 8-bit monochrome images for both image and fractal analysis (Fuseler et al., 2007; Fernandez et al., 2001; Grizzi et al., 2005; Valarmathi et al., 2012). The fluorescence vasculature in an image was defined as a Region of Interest (ROI) by being thresholded using the "set threshold" subroutine of MetaMorph Image analysis software (v.6.1). The morphological descriptors of fiber breadth were a representative measure of blood vessel diameter. Fiber length was measured using the integrated morphometry algorithm of MetaMorph. Using this technique we were able to reconfirm what our confocal images demonstrated. In microvascular media, the BMSC-derived mi‐ crovessels were longer (63%) and broader (37%) when compared with microvessels gen‐ erated in basal media (Figure 10).

Similarly, it is critically important to characterize the ultrastructural morphology of any stem cells that are directed to differentiate into vascular lineage cells. Scanning electron mi‐ croscopic (SEM) analysis of the tubular constructs depicted the pattern of microvessel mor‐ phogenesis and maturity. These formed nascent capillary-like structures and elongated tube-like structures revealed patent lumen-like structures, elucidating the vessel-maturation (Figure 11). Besides, transmission electron microscopic (TEM) analysis (Hanaichi et al., 1986) revealed elongated capillary-like structures lined by differentiating endothelial cells (Figure 12A). These cells showed electron dense bodies as well as numerous small pinocytotic vesi‐ cles adjacent to the endothelial cell membranes as well as in their cytoplasm (Figure 12B, small black arrows). In addition these cells exhibited variously sized cell-cell junctions, which have the appearance of typical in vivo endothelial tight junctions (Figure 12C-F).

**Figure 9.** Expression pattern of vasculogenic markers α-SMA and tomato lectin (TL). Localization of BMSC-derived vas‐ cular progenitor cells on the collagen tube scaffold by confocal microscopy is seen in panels A, C, E, and G. The merged images showing the actin cytoskeleton are shown in panels B, D,F, and H. Sections of a day 9 osteogenic tube culture demonstrated nascent capillary-like structures positive for α-SMA (red, A). These elongated cord-like structures showed strong colocalization of α-SMA and actin (merged, B). In the tubes nuclei appeared aligned (blue, DAPI). In adjacent fields, abundant sheets of parallel oriented α-SMA positive cells were abutting the nascent vessel-like struc‐ tures (red, C). The merged image (D) shows actin and α-SMA in vessel-like structures that appear to be in different planes with almost a perpendicular alignment with the underlying α-SMA positive cells (D). Similar sections of osteo‐

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It is well known that endothelial cells share a large majority of their characteristic anti‐ genic markers with other types of hematopoietic and mesenchymal cells (Bertolini et al., 2006). Therefore, antigens such as CD31, CD34, CD144 (VE-cadherin), CD146, Vwf or CD105 are not only expressed by endothelial cells but also expressed by hematopoietic cells (specifically HSCs), platelets and certain subpopulations of fibroblasts. Hence to identify the differentiated and matured endothelial cells in the tubular scaffold a battery of various early and late stage vasculogenic markers such as Pecam1, CD34, Flt1, Flk1, VE-cadherin, fibronectin and Vwf were employed. In addition, tomato lectin, another marker specific for rat vascular endothelial cells, was found closely associated with Flk1 and Vwf staining. These endothelial associated markers localized to endothelial cell clus‐ ters and capillary-like structures that were present throughout the tubular construct. This suggests that BMSC-derived endothelial cells assembled into endothelium-lined tube-like structures and initiated the process of vasculogenesis, consistent with our previous re‐ port (Figure 9 A-H) (Valarmathi et al., 2008a). In addition, the BMSC-derived cells and the microvessel-like structures expressed the smooth muscle antigens, α-SMA and calpo‐ nin. These α-SMA positive cells were recruited in juxtaposition to the tandemly arranged endothelial cells and, were attached and wrapped around in such a way that is reminis‐

In order to quantitate the degree of vasculogenesis, image morphometric analyses were used to determine the capillary density, vessel breadth and length (integrated morphom‐ etry subroutine of MetaMorph 6.1). In brief, confocal scanning laser microscopic color images were converted to 8-bit monochrome images for both image and fractal analysis (Fuseler et al., 2007; Fernandez et al., 2001; Grizzi et al., 2005; Valarmathi et al., 2012). The fluorescence vasculature in an image was defined as a Region of Interest (ROI) by being thresholded using the "set threshold" subroutine of MetaMorph Image analysis software (v.6.1). The morphological descriptors of fiber breadth were a representative measure of blood vessel diameter. Fiber length was measured using the integrated morphometry algorithm of MetaMorph. Using this technique we were able to reconfirm what our confocal images demonstrated. In microvascular media, the BMSC-derived mi‐ crovessels were longer (63%) and broader (37%) when compared with microvessels gen‐

Similarly, it is critically important to characterize the ultrastructural morphology of any stem cells that are directed to differentiate into vascular lineage cells. Scanning electron mi‐ croscopic (SEM) analysis of the tubular constructs depicted the pattern of microvessel mor‐ phogenesis and maturity. These formed nascent capillary-like structures and elongated tube-like structures revealed patent lumen-like structures, elucidating the vessel-maturation (Figure 11). Besides, transmission electron microscopic (TEM) analysis (Hanaichi et al., 1986) revealed elongated capillary-like structures lined by differentiating endothelial cells (Figure 12A). These cells showed electron dense bodies as well as numerous small pinocytotic vesi‐ cles adjacent to the endothelial cell membranes as well as in their cytoplasm (Figure 12B, small black arrows). In addition these cells exhibited variously sized cell-cell junctions, which have the appearance of typical in vivo endothelial tight junctions (Figure 12C-F).

cent of in vivo microvessel morphogenesis.

278 Research Directions in Tumor Angiogenesis

erated in basal media (Figure 10).

**Figure 9.** Expression pattern of vasculogenic markers α-SMA and tomato lectin (TL). Localization of BMSC-derived vas‐ cular progenitor cells on the collagen tube scaffold by confocal microscopy is seen in panels A, C, E, and G. The merged images showing the actin cytoskeleton are shown in panels B, D,F, and H. Sections of a day 9 osteogenic tube culture demonstrated nascent capillary-like structures positive for α-SMA (red, A). These elongated cord-like structures showed strong colocalization of α-SMA and actin (merged, B). In the tubes nuclei appeared aligned (blue, DAPI). In adjacent fields, abundant sheets of parallel oriented α-SMA positive cells were abutting the nascent vessel-like struc‐ tures (red, C). The merged image (D) shows actin and α-SMA in vessel-like structures that appear to be in different planes with almost a perpendicular alignment with the underlying α-SMA positive cells (D). Similar sections of osteo‐

genic cultured tubes illustrate the sprouting and branching tubular structures positive for the rat endothelial marker tomato lectin (red, E-H). A plexus of arborizing endothelial cells was observed. These plexuses contained capillary-like vessels lined with endothelial cells (red, G). Similarly in the merged panel, the apparent multilayered nature of the ves‐ sels was observed. However in these fields, the plexuses of endothelial cells were not in a perpendicular arrangement but rather in a web-like orientation (merged, H). Figures C and D were projections representing 19 sections collected at 2 μm intervals. Figures G and H were projections representing 32 sections collected at 9 μm intervals. Merged im‐ ages (A-H) (A-D, Scale bar 20 μm; E-H, Scale bar 50 μm). Adapted from Valarmathi et al., 2008a.

**Figure 10.** Morphometric analysis of microvessel-like structures generated in the walls of collagen-gel tubular con‐ structs in different culture media, viz., basal or microvascular medium.

**Figure 11.** Scanning electron microscopic (SEM) analysis of tubular constructs. SEM analysis of day 28 tubular con‐ structs under both vasculogenic and non-vasculogenic culture conditions showed the typical cobblestone appearance of differentiated endothelial cells (A, F), stratification and networking (B, G), and the presence of smooth-walled tubelike structures with its attached smooth muscle cells and/or pericytes (black arrows, D, E, I, J). Multiple smooth musclelike cells were wrapping around these tube-like structures (black asterisks, Figure C-E, H-J). These cylindrical structures revealed the presence of evolving patent lumens (white asterisks, C-E, H, J). Some of these luminal surfaces showed the regular cobblestone arrangement of endothelial cells (white asterisks, C, D). Adapted from Valarmathi et al., 2009.

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genic cultured tubes illustrate the sprouting and branching tubular structures positive for the rat endothelial marker tomato lectin (red, E-H). A plexus of arborizing endothelial cells was observed. These plexuses contained capillary-like vessels lined with endothelial cells (red, G). Similarly in the merged panel, the apparent multilayered nature of the ves‐ sels was observed. However in these fields, the plexuses of endothelial cells were not in a perpendicular arrangement but rather in a web-like orientation (merged, H). Figures C and D were projections representing 19 sections collected at 2 μm intervals. Figures G and H were projections representing 32 sections collected at 9 μm intervals. Merged im‐

**Figure 10.** Morphometric analysis of microvessel-like structures generated in the walls of collagen-gel tubular con‐

structs in different culture media, viz., basal or microvascular medium.

ages (A-H) (A-D, Scale bar 20 μm; E-H, Scale bar 50 μm). Adapted from Valarmathi et al., 2008a.

280 Research Directions in Tumor Angiogenesis

**Figure 11.** Scanning electron microscopic (SEM) analysis of tubular constructs. SEM analysis of day 28 tubular con‐ structs under both vasculogenic and non-vasculogenic culture conditions showed the typical cobblestone appearance of differentiated endothelial cells (A, F), stratification and networking (B, G), and the presence of smooth-walled tubelike structures with its attached smooth muscle cells and/or pericytes (black arrows, D, E, I, J). Multiple smooth musclelike cells were wrapping around these tube-like structures (black asterisks, Figure C-E, H-J). These cylindrical structures revealed the presence of evolving patent lumens (white asterisks, C-E, H, J). Some of these luminal surfaces showed the regular cobblestone arrangement of endothelial cells (white asterisks, C, D). Adapted from Valarmathi et al., 2009.

Furthermore, the ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL tagged with the fluorescent probe (Dil-Ac-LDL) (Voy‐ ta et al., 1984). BMSC-derived endothelial cells and the nascent capillary-like structures were brilliantly fluorescent whereas the fluorescent intensity of smooth muscle cells/pericytes was barely detectable as reported previously (Valarmathi et al., 2009). This suggests that the formed endothelial cells were not only fully differentiated but also functionally competent

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283

This behavior of BMSCs and their exhibition of vasculogenic differentiation potential can be attributed to the nature of microenvironmental factors in this culture conditions. The pre‐ conditioned factors in the growth microenvironment rendered by the aligned type I collagen fibers of the tubular scaffold and the soluble differentiating factors provided by the vasculo‐ genic and non-vasculogenic media may be behind the BMSC fate determination. Further work is ongoing to determine whether our prevascularised tubular scaffolds can survive im‐ plantation into a tissue defect and is able to anastomose promptly with vascular sprouts em‐ anating from the host. Finally, our morphological, molecular, immunological and biochemical data reveal the intrinsic vasculogenic differentiation potential of BMSCs under

Previously, it has been shown that mature vascular endothelium can give rise to smooth muscle cell (SMC) via endothelial-mesenchymal transdifferentiation, coexpressing both en‐ dothelial and SMC-specific phenotypic markers (Frid et al., 2002). Recently, it has been show that Flk1-expressing blast cells derived from embryonic stem cells can act as precursors that can differentiate into both endothelial and mural cell populations of the vasculature (Yama‐ shita et al., 2000). In this study, clonal analyses revealed the bi-lineage potential of BMSCs, suggesting that both endothelial and smooth muscle/pericytes could be derived from single colonies. However, in general, BMSC-derived colonies are clonal or nearly clonal. The colo‐ nies of BMSCs resultant from a number of cells may represent co-existence of several sub‐ clones, each capable of differentiating into specific lineages. Hence, single cell-derived colonies that are stably transfected with lineage specific markers are needed to gain more

Our results indicate that the 3-D tubular scaffold with its unique characteristics provides a favorable microenvironment that permits the development of in situ microvascular structures. Moreover, this is the first ever documentation that explicitly demonstrates that adult BMSCs under appropriate in vitro environmental cues can be induced to un‐ dergo vasculogenic differentiation culminating in microvessel morphogenesis. Our model recapitulates many aspects of in vivo de novo vasculogenesis. Thus, this unique culture system provides an in vitro model to investigate the maturation and differentiation of BMSC-derived vascular endothelial and smooth muscle cells in the context of postnatal vasculogenesis. In addition, it allows us to elucidate various molecular mechanisms un‐ derlying the origin of both endothelial and smooth muscle cells and especially to gain a deeper insight and validate the emerging concept of 'one cell and two fates' hypothesis

and matured (Figure 13A-J).

appropriate 3-D environmental conditions.

meaningful insights and address the origin of both lineages.

of vascular development (Yamashita et al., 2000).

**Figure 12.** Transmission electron microscopic (TEM) analysis of tubular constructs. TEM analysis of day 28 tubular con‐ structs under both vasculogenic and non-vasculogenic culture conditions showed a vessel-like structure containing many small dense bodies within endothelial cells on either side of the lumen (A). Note the most obvious feature of endothelial cells, the concentration of small vesicles (pinocytotic vesicles) adjacent to the endothelial cell membranes (B, black arrows). The interdigitating endothelial cells showing junctional regions (C, E, inserts, lower magnification). The typical adherent junction could be visualized between two overlapping endothelial cell processes (D, F, inserts, higher magnification).

Furthermore, the ability to identify endothelial cells based on their increased metabolism of Ac-LDL was examined using Ac-LDL tagged with the fluorescent probe (Dil-Ac-LDL) (Voy‐ ta et al., 1984). BMSC-derived endothelial cells and the nascent capillary-like structures were brilliantly fluorescent whereas the fluorescent intensity of smooth muscle cells/pericytes was barely detectable as reported previously (Valarmathi et al., 2009). This suggests that the formed endothelial cells were not only fully differentiated but also functionally competent and matured (Figure 13A-J).

This behavior of BMSCs and their exhibition of vasculogenic differentiation potential can be attributed to the nature of microenvironmental factors in this culture conditions. The pre‐ conditioned factors in the growth microenvironment rendered by the aligned type I collagen fibers of the tubular scaffold and the soluble differentiating factors provided by the vasculo‐ genic and non-vasculogenic media may be behind the BMSC fate determination. Further work is ongoing to determine whether our prevascularised tubular scaffolds can survive im‐ plantation into a tissue defect and is able to anastomose promptly with vascular sprouts em‐ anating from the host. Finally, our morphological, molecular, immunological and biochemical data reveal the intrinsic vasculogenic differentiation potential of BMSCs under appropriate 3-D environmental conditions.

Previously, it has been shown that mature vascular endothelium can give rise to smooth muscle cell (SMC) via endothelial-mesenchymal transdifferentiation, coexpressing both en‐ dothelial and SMC-specific phenotypic markers (Frid et al., 2002). Recently, it has been show that Flk1-expressing blast cells derived from embryonic stem cells can act as precursors that can differentiate into both endothelial and mural cell populations of the vasculature (Yama‐ shita et al., 2000). In this study, clonal analyses revealed the bi-lineage potential of BMSCs, suggesting that both endothelial and smooth muscle/pericytes could be derived from single colonies. However, in general, BMSC-derived colonies are clonal or nearly clonal. The colo‐ nies of BMSCs resultant from a number of cells may represent co-existence of several sub‐ clones, each capable of differentiating into specific lineages. Hence, single cell-derived colonies that are stably transfected with lineage specific markers are needed to gain more meaningful insights and address the origin of both lineages.

Our results indicate that the 3-D tubular scaffold with its unique characteristics provides a favorable microenvironment that permits the development of in situ microvascular structures. Moreover, this is the first ever documentation that explicitly demonstrates that adult BMSCs under appropriate in vitro environmental cues can be induced to un‐ dergo vasculogenic differentiation culminating in microvessel morphogenesis. Our model recapitulates many aspects of in vivo de novo vasculogenesis. Thus, this unique culture system provides an in vitro model to investigate the maturation and differentiation of BMSC-derived vascular endothelial and smooth muscle cells in the context of postnatal vasculogenesis. In addition, it allows us to elucidate various molecular mechanisms un‐ derlying the origin of both endothelial and smooth muscle cells and especially to gain a deeper insight and validate the emerging concept of 'one cell and two fates' hypothesis of vascular development (Yamashita et al., 2000).

**Figure 12.** Transmission electron microscopic (TEM) analysis of tubular constructs. TEM analysis of day 28 tubular con‐ structs under both vasculogenic and non-vasculogenic culture conditions showed a vessel-like structure containing many small dense bodies within endothelial cells on either side of the lumen (A). Note the most obvious feature of endothelial cells, the concentration of small vesicles (pinocytotic vesicles) adjacent to the endothelial cell membranes (B, black arrows). The interdigitating endothelial cells showing junctional regions (C, E, inserts, lower magnification). The typical adherent junction could be visualized between two overlapping endothelial cell processes (D, F, inserts,

higher magnification).

282 Research Directions in Tumor Angiogenesis

scaffolds in microvascular endothelial cell growth medium revealed typical abundant punctate perinuclear bright red fluorescence of the differentiated and matured endothelial cells (G-J). These labeled vascular cells were organized into small discrete clusters (G), self-organized into numerous small capillaries with a central lumen (white arrow, H), assem‐ bled into solid cord of cells (white arrow, I) and, transformed into tube-like structure with attempted lumen formation (white arrows, J). Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (green, Alexa® 488 phalloidin). Im‐ ages (A-B) show a projection representing 13 sections collected at 3.05 μm intervals (36.60 μm). Images (C-D) show a projection representing 20 sections collected at 4.05 μm intervals (76.95 μm). Images (E-F) show a projection repre‐ senting 4 sections collected at 2.05 μm intervals (6.15 μm). Image (H) shows a projection representing 20 sections col‐ lected at 5 μm intervals (95.00 μm). Image (I) shows a projection representing 13 sections collected at 4 μm intervals (48.00 μm). Image (J) shows a projection representing 22 sections collected at 5 μm intervals (105.00 μm). Merged

3-D Microvascular Tissue Constructs for Exploring Concurrent Temporal and Spatial Regulation of…

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285

Here we report a unique 3-D culture system that recapitulates many aspects of postnatal de novo vasculogenesis and angiogenesis. This is the first comprehensive report that evidently demonstrates that BMSCs under appropriate in vitro environmental conditions can be in‐ duced to undergo vasculogenic differentiation culminating in microvessels. Since BMSCs differentiated into both endothelial and smooth muscle cell lineages, this in vitro model sys‐ tem provides a tool for investigating the cellular and molecular origin of both vascular en‐ dothelial cells and smooth muscle cells. In addition, this system can potentially be harnessed to develop in vitro engineering of microvascular trees, especially using autologous bone-

"This work was supported by an award from the American Heart Association." – National Scientist Development Grant (11SDG5280022) as well "This material is based upon work supported by the National Science Foundation/EPSCoR under Grant No. (EPS – 0903795)." –

The South Carolina Project for Organ Biofabrication, for Valarmathi Thiruvanamalai.

, Stefanie V. Biechler and John W. Fuseler

\*Address all correspondence to: valarmathi.thiruvanamalai@uscmed.sc.edu or valarma‐

Laboratory of Stem Cell Biology and Tissue Engineering, Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina, USA

marrow-derived BMSCs for therapeutic purposes in regenerative medicine.

images (A-J). (A-J, scale bar 50 μm). Adapted from Valarmathi et al., 2009.

**5. Conclusions**

**Acknowledgements**

**Author details**

Mani T. Valarmathi\*

thi64@hotmail.com

**Figure 13.** Characterization of BMSC-derived endothelial cells by Dil-Ac-LDL uptake. BMSCs cultured in collagen-gel tubular scaffolds in non-vasculogenic (mesenchymal stem cell growth medium, MSCGM) and vasculogenic (microvas‐ cular endothelial cell growth medium, EGMMV) culture conditions were incubated with 10 μg / ml of Dil-Ac-LDL for 4 to 6 hours. Confocal laser scanning microscopic analysis of sections of day 21 tubular scaffolds in MSC growth medium revealed abundant punctate perinuclear bright red fluorescence of the differentiated and matured endothelial cells (A-F). These labeled vascular cells were organized into a discrete cluster (A, B), assembled into tangled capillary-like structures on top of a cluster (white asterisks, C, D) and, transformed into nascent linear and branching capillary-like structures (white asterisks, E, F). Similarly, confocal laser scanning microscopic analysis of sections of day 21 tubular scaffolds in microvascular endothelial cell growth medium revealed typical abundant punctate perinuclear bright red fluorescence of the differentiated and matured endothelial cells (G-J). These labeled vascular cells were organized into small discrete clusters (G), self-organized into numerous small capillaries with a central lumen (white arrow, H), assem‐ bled into solid cord of cells (white arrow, I) and, transformed into tube-like structure with attempted lumen formation (white arrows, J). Cells were also stained for nuclei (blue, DAPI) and fibrillar actin (green, Alexa® 488 phalloidin). Im‐ ages (A-B) show a projection representing 13 sections collected at 3.05 μm intervals (36.60 μm). Images (C-D) show a projection representing 20 sections collected at 4.05 μm intervals (76.95 μm). Images (E-F) show a projection repre‐ senting 4 sections collected at 2.05 μm intervals (6.15 μm). Image (H) shows a projection representing 20 sections col‐ lected at 5 μm intervals (95.00 μm). Image (I) shows a projection representing 13 sections collected at 4 μm intervals (48.00 μm). Image (J) shows a projection representing 22 sections collected at 5 μm intervals (105.00 μm). Merged images (A-J). (A-J, scale bar 50 μm). Adapted from Valarmathi et al., 2009.

#### **5. Conclusions**

Here we report a unique 3-D culture system that recapitulates many aspects of postnatal de novo vasculogenesis and angiogenesis. This is the first comprehensive report that evidently demonstrates that BMSCs under appropriate in vitro environmental conditions can be in‐ duced to undergo vasculogenic differentiation culminating in microvessels. Since BMSCs differentiated into both endothelial and smooth muscle cell lineages, this in vitro model sys‐ tem provides a tool for investigating the cellular and molecular origin of both vascular en‐ dothelial cells and smooth muscle cells. In addition, this system can potentially be harnessed to develop in vitro engineering of microvascular trees, especially using autologous bonemarrow-derived BMSCs for therapeutic purposes in regenerative medicine.

#### **Acknowledgements**

"This work was supported by an award from the American Heart Association." – National Scientist Development Grant (11SDG5280022) as well "This material is based upon work supported by the National Science Foundation/EPSCoR under Grant No. (EPS – 0903795)." – The South Carolina Project for Organ Biofabrication, for Valarmathi Thiruvanamalai.

#### **Author details**

**Figure 13.** Characterization of BMSC-derived endothelial cells by Dil-Ac-LDL uptake. BMSCs cultured in collagen-gel tubular scaffolds in non-vasculogenic (mesenchymal stem cell growth medium, MSCGM) and vasculogenic (microvas‐ cular endothelial cell growth medium, EGMMV) culture conditions were incubated with 10 μg / ml of Dil-Ac-LDL for 4 to 6 hours. Confocal laser scanning microscopic analysis of sections of day 21 tubular scaffolds in MSC growth medium revealed abundant punctate perinuclear bright red fluorescence of the differentiated and matured endothelial cells (A-F). These labeled vascular cells were organized into a discrete cluster (A, B), assembled into tangled capillary-like structures on top of a cluster (white asterisks, C, D) and, transformed into nascent linear and branching capillary-like structures (white asterisks, E, F). Similarly, confocal laser scanning microscopic analysis of sections of day 21 tubular

284 Research Directions in Tumor Angiogenesis

Mani T. Valarmathi\* , Stefanie V. Biechler and John W. Fuseler

\*Address all correspondence to: valarmathi.thiruvanamalai@uscmed.sc.edu or valarma‐ thi64@hotmail.com

Laboratory of Stem Cell Biology and Tissue Engineering, Department of Cell Biology and Anatomy, School of Medicine, University of South Carolina, Columbia, South Carolina, USA

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### *Edited by Jianyuan Chai*

Angiogenesis is an extension process of the cardiovascular network within human body. It is usually triggered by the demand of oxygen and nutrients from the fast growing tissue and uncontrollably dividing cells, as seen during wound healing and tumor progression. This book focuses on tumor angiogenesis and includes 8 chapters written by highly experienced scholars from five different countries. It is the goal of this book to provide readers with an update on the molecular and cellular mechanisms of this biological process and hopefully to point out some new research directions for future therapeutic adventure.

Research Directions in Tumor Angiogenesis

Research Directions in

Tumor Angiogenesis

*Edited by Jianyuan Chai*

Photo by arenacreative / iStock