Preface

Welcome to the fascinating world of angiogenesis! Back by popular demand, this is the second edition of the *Vasculogenesis and Angiogenesis* Open Access book published in 2011 by InTech. The previous book comprised ten chapters of outstanding scientific content spanning the field of angiogenesis, from developmental biology to regenerative medicine. The original book eli‐ cited substantial enthusiasm, and as of January 2017, its chapters were downloaded more than 23,400 times from the InTech website.

We are now returning to the field, stronger than ever, with more than double the number of chapters and with an expanded content and focus. In order to gain an overview of the impor‐ tant elements in the field, we generated a "word cloud" or an objective weighted list of singleword terms gathered from all the abstracts of the current chapters. This graphical representation shown below highlights the most prominent terms, the frequency of each one being proportional to the font size. Our word cloud highlights a group of essential terms of significance to angiogenesis: factors, signaling, tumors, stem cells, blood and vascular, VEGF, endothelial cells, and therapeutic.

We therefore grouped the chapters into five parts. The first two parts cover cellular and molec‐ ular signaling mechanisms in physiologic and pathologic angiogenesis with application to de‐ velopment, the nervous system, skeletal formation, corneal angiogenesis, and the cardiovascular system. During embryonic development blood vessels form very early and aid in generation of most organs. In healthy adults, new blood vessel formation is not well repre‐ sented; thus most organs appear to be "reticent" to de novo angiogenesis. Of particular note is the role of VEGF, TGF-beta, Notch, SDF-1, CXCR4, MMPs, TIMPs, histone-modifying en‐ zymes, and other signals in angiogenesis as well as the association of imbalances in homeosta‐ sis of these factors with early loss of pregnancy, Alzheimer's disease, cardiovascular diseases, and others. Notable is also the intricate relationship between angiogenesis and endothelial cells, endothelial progenitor cells and hypoxia, and atherosclerosis and growth of coronary collaterals.

The third part grouped chapters on angiogenesis research in cancer. Starting with publication of the original hypothesis by Judah Folkman in 1971 and discovery of VEGF by Napoleone Ferrara in 1989, this topic continues to attract abundant attention and has generated many promising therapies. In addition, cancer stem cells, which can differentiate into cancer endo‐ thelial cells and influence angiogenesis, have become important targets in the fight against cancer. Since numerous experimental approaches in vitro and in vivo are being utilized in an‐ giogenesis research, the fourth chapter was dedicated to presentation and discussion of ad‐ vanced methods and models for the study of angiogenesis. Special emphasis was placed on critical analysis of pros and cons of each method, so that the reader can select adequate models for research and validation of selected targets.

Finally, in the fifth part, utilizing information from basic studies, significant focus has been placed on developing targeted therapies for either promoting or inhibiting angiogenesis. This dichotomy is quite unique to angiogenesis research and has generated a wealth of information that feeds and cross-pollinates both aspects. In scenarios where angiogenesis is desired such as ischemia relief after occlusion of blood vessels, therapies have been developed to induce new blood vessel formation using growth factor delivery and stem cell-mediated gene therapy to overexpress VEGF. Conversely, to limit tumor development, which in most cases relies on de novo vascularization, numerous treatments targeting the intentional stoppage of angiogenesis have been tested. Evidently, one major target is VEGF and its specific receptors. While initial clinical trials with anti-VEGF treatments have proven successful, we had learned throughout the years that these medications may cause significant side effects; in addition, some tumors were found to be resistant to anti-VEGF therapies. Meanwhile, additional potential targets have been identified. These include receptor tyrosine kinases, angiotensin receptors, angioten‐ sin-converting enzymes, and cell surface receptors such as CD146, among others. Clearly there is a need for sustained research and testing of additional targets that would set the basis for effective treatment and benefit an increasing number of patients worldwide.

The purpose of this book is to highlight novel advances in the field and to incentivize scien‐ tists from a variety of fields to pursue angiogenesis as a research avenue. Blood vessel forma‐ tion and maturation to capillaries, arteries, or veins is a fascinating area which can appeal to multiple scientists, students, and professors alike. Angiogenesis is relevant to medicine, engi‐ neering, pharmacology, pathology, genetics, and veterinary sciences and to the many patients suffering from blood vessel diseases and cancer, among others. We are hoping that this book will become a source of inspiration and novel ideas for all and offer this quote as a last word from Paracelsus:

"Medicine is not only a science; it is also an art. It does not consist of compounding pills and plasters; it deals with the very processes of life, which must be understood before they may be guided."

#### **Acknowledgments**

The editors would like to thank Dr. Adriana S. Bankston for helping with the angiogenesis word cloud and to acknowledge financial support from the Department of Bioengineering at the Clemson University, the Harriet and Jerry Dempsey Professorship Award, NIH grants 5P20GM103444-07 and R56HL130950-01, and grants from the Competitiveness Operational Programme 2014–2020; Tissue engineering technologies for cardiac valve regeneration, VALVE-REGEN, ID P\_37\_673, MySMIS code, 103431, contract 50/05.09.2016; and Targeted therapies for heart valve disease in diabetes, THERAVALDIS, ID P\_37\_298, MySMIS code: 104362, contract 115/13.09.2016.

**Signaling Mechanisms in Physiologic and Pathologic Angiogenesis**

giogenesis research, the fourth chapter was dedicated to presentation and discussion of ad‐ vanced methods and models for the study of angiogenesis. Special emphasis was placed on critical analysis of pros and cons of each method, so that the reader can select adequate models

Finally, in the fifth part, utilizing information from basic studies, significant focus has been placed on developing targeted therapies for either promoting or inhibiting angiogenesis. This dichotomy is quite unique to angiogenesis research and has generated a wealth of information that feeds and cross-pollinates both aspects. In scenarios where angiogenesis is desired such as ischemia relief after occlusion of blood vessels, therapies have been developed to induce new blood vessel formation using growth factor delivery and stem cell-mediated gene therapy to overexpress VEGF. Conversely, to limit tumor development, which in most cases relies on de novo vascularization, numerous treatments targeting the intentional stoppage of angiogenesis have been tested. Evidently, one major target is VEGF and its specific receptors. While initial clinical trials with anti-VEGF treatments have proven successful, we had learned throughout the years that these medications may cause significant side effects; in addition, some tumors were found to be resistant to anti-VEGF therapies. Meanwhile, additional potential targets have been identified. These include receptor tyrosine kinases, angiotensin receptors, angioten‐ sin-converting enzymes, and cell surface receptors such as CD146, among others. Clearly there is a need for sustained research and testing of additional targets that would set the basis for

The purpose of this book is to highlight novel advances in the field and to incentivize scien‐ tists from a variety of fields to pursue angiogenesis as a research avenue. Blood vessel forma‐ tion and maturation to capillaries, arteries, or veins is a fascinating area which can appeal to multiple scientists, students, and professors alike. Angiogenesis is relevant to medicine, engi‐ neering, pharmacology, pathology, genetics, and veterinary sciences and to the many patients suffering from blood vessel diseases and cancer, among others. We are hoping that this book will become a source of inspiration and novel ideas for all and offer this quote as a last word

"Medicine is not only a science; it is also an art. It does not consist of compounding pills and plasters; it deals with the very processes of life, which must be understood before they may be

The editors would like to thank Dr. Adriana S. Bankston for helping with the angiogenesis word cloud and to acknowledge financial support from the Department of Bioengineering at the Clemson University, the Harriet and Jerry Dempsey Professorship Award, NIH grants 5P20GM103444-07 and R56HL130950-01, and grants from the Competitiveness Operational Programme 2014–2020; Tissue engineering technologies for cardiac valve regeneration, VALVE-REGEN, ID P\_37\_673, MySMIS code, 103431, contract 50/05.09.2016; and Targeted therapies for heart valve disease in diabetes, THERAVALDIS, ID P\_37\_298, MySMIS code:

**Dr. Dan Simionescu and Dr. Agneta Simionescu**

Clemson University, Clemson, SC, USA

effective treatment and benefit an increasing number of patients worldwide.

for research and validation of selected targets.

from Paracelsus:

**Acknowledgments**

104362, contract 115/13.09.2016.

guided."

X Preface

## **TGF-β Activation and Signaling in Angiogenesis TGF-**β **Activation and Signaling in Angiogenesis**

Paola A. Guerrero and Joseph H. McCarty Paola A. Guerrero and Joseph H. McCarty

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/66405

#### **Abstract**

The transforming growth factor-β (TGF-β) signaling pathway regulates various cellular processes during tissue and organ development and homeostasis. Deregulation of the expression and/or functions of TGF-β ligands, receptors or their intracellular signaling components leads to multiple diseases including vascular pathologies, autoimmune disorders, fibrosis and cancer. In vascular development, physiology and disease TGF-β signaling can have angiogenic and angiostatic properties, depending on expression levels and the tissue context. The objective of this chapter is to analyze the mechanisms that contribute to the activation and signaling of TGF-β in developmental, physiological and pathological angiogenesis, with a particular emphasis on the importance of TGF-β signaling in the mammalian central nervous system (CNS).

**Keywords:** TGF-β, vasculogenesis, angiogenesis, VEGF

## **1. Introduction**

Discovery of TGF-βs was the result of independent efforts by several laboratories [1–4] during characterization of a secreted factor from fibroblasts transformed by the Moloney sarcoma virus (MSV). The TGF-β superfamily is now known to be composed of more than 30 chemokines such as TGF-β1-β3, activins, anti-Müllerian hormone (AMH), bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and NODAL that can signal via canonical and noncanonical receptors and intracellular effector proteins [5].

The best characterized member of the TGF-β family, TGF-β1, is initially produced from a single gene as a large precursor known as pre- and pro-TGF-βs which undergo two proteolytic cleavage events. The first signal peptide is cleaved in the rough endoplasmic reticulum. Furin, a proprotein convertase, subsequently cleaves the protein into two fragments [6]. The carboxy terminus corresponds to the functionally active cytokine and the large amino

terminus is latency-associated protein (LAP), also referred to as the prodomain. Regardless of this processing by furin, the mature and LAP domains remain associated by noncovalent bonds to form the small latent complex (SLC). This complex subsequently covalently interacts with a second gene product, the latent TGF-β binding protein (LTBP), and is incorporated into a larger latent complex (LLC) that associates with the extracellular matrix (ECM) [6]. Threedimensional crystal structure of porcine latent TGF-β1 shows a conformation that resembles a ring-like shape [7]. Two domains were defined in the structure: (i) an arm domain that contains an integrin-binding Arg-Gly-Asp (RGD) peptide motif and (ii) a "straitjacket" domain where the mature TGF-β is encased. At the opposite end of the arm domain, LTBP binds the prodomain forming the "ring head" [7] (**Figure 1**).

After secretion, the LLC complex interacts with various ECM proteins, such as fibronectin and fibrillin, and is maintained in an inactivated form [8]. TGF-β is activated by different mechanisms, including interactions with integrins, alterations in pH and extracellular proteases. αν integrin, which forms heterodimers with five different β integrin subunits (β1, β3, β5, β6 and β8), that bind to LAP-TGF-β1 and LAP-TGFβ-3 [9, 10]. However, only ανβ6 and ανβ8 have been shown to activate the latent TGF-β complex [11]. Activation by both ανβ6 and ανβ8 integrins requires the RGD motif in LAP. Activation by ανB6 requires an intact cytoplasmic domain [12, 13] and the presence of other ECM proteins [14]. Activation by ανβ8, however,

**Figure 1.** TGF-β processing and activation. **(a)**TGF-β precursor undergoes proteolysis at its N-terminus (black arrow head) which results in the removal of its signal peptide. **(b)**In a second proteolytic cleavage event by furin (blue arrow head), the precursor is separated into a large LAP or prodomain (gray) and the mature TGF-β (red) and **(c)**Schematic view of the closed ring structure (left) and unfastened straitjacket (right) conformation corresponding to the inactive LAP-TGF-β and mature TGF-β, respectively.

does not require the integrin cytoplasmic domain, but it is reported to require the presence of metalloproteinases (MMPs) on the cell surface or in the ECM [9]. Additionally, in T cells, ανβ6 and ανβ8 can activate LAP-TGF-β in cooperation with the glycoprotein-A repetitions predominant protein (GARP) [15, 16].

TGF-β is also activated by proteases. Aspartyl (e.g., cathepsin D) [17], cysteine (e.g., calpain) [18] and serine proteases (e.g., plasmin and kallikreins) and metalloproteases have shown to stimulate the release of chemokine from the latent complex, although most of these studies have been performed *in vitro* [19]. Moreover, TGF-β has been reported to be activated by other nonprotease mechanisms such as neuropilin1 (Nrp1), thrombospondin (TSP-1), F-spondin, pregnancy-specific beta-1-glycoprotein 1 (PSG1) and deglycosylation. Likewise, there are chemical and physical settings that activate TGF-β, for example, heat, ultraviolet radiation, physical shear, detergents and reactive oxygen species [8].

Three-dimensional structural studies of the LLC reveal that the RGD motifs are readily available for integrin engagement. Hydrophobic side chains, which have been identified near the RGD motif, likely enhance integrin binding [7]. In the presence of ανβ6, LLC can bind one or two integrin monomers. However, this binding does not induce required conformational changes to promote the complete activation of TGF-β, which is in agreement with prior mutational studies [12, 13, 20]. Furthermore, in accord with previous studies, the crystal structure of latent TGF-β predicts that pulling forces, emanating from the integrin C-terminal cytoplasmic tail that interacts with the cytoskeleton and binding RGD via the N-terminal extracellular region, are counteracted by associations with the ECM. Therefore, in the latent TGF-β, the straitjacket domain is maintained in a closed conformation until tensile forces are applied from both ends of the structure, resulting in loosening of the straitjacket domain and the release of the mature TGF-β. This study also showed that an additional feature of the prodomain is to prevent access to activating receptors [7].
