**Author details**

Ioana Berindan‐Neagoe1,2,3\*, Cornelia Braicu<sup>1</sup> , Diana Gulei<sup>2</sup> , Ciprian Tomuleasa1,4 and George Adrian Calin5

\*Address all correspondence to: ioana.neagoe@umfcluj.ro

1 Research Center for Functional Genomics, Biomedicine and Translational Medicine, "Iuliu Hatieganu", University of Medicine and Pharmacy Iuliu‐Hatieganu, Cluj‐Napoca, Romania

2 Medfuture—Research Center for Advanced Medicine, University of Medicine and Pharmacy Iuliu‐Hatieganu, Cluj‐Napoca, Romania

3 Department of Functional Genomics and Experimental Pathology, The Oncological Institute "Prof. Dr. Ion Chiricuta", Cluj‐Napoca, Romania

4 Department of Hematology, The Oncological Institute "Prof. Dr. Ion Chiricuta", Cluj‐Napoca, Romania

5 Department of Experimental Therapeutics, The University of Texas M.D. Anderson Cancer Center, Houston, TX, USA

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**Novel Methods in Angiogenesis Research**

#### **Recent Advances in Angiogenesis Assessment Methods and their Clinical Applications Recent Advances in Angiogenesis Assessment Methods and Their Clinical Applications**

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

http://dx.doi.org/10.5772/66504

#### **Abstract**

Angiogenesis, a natural phenomenon of developing new blood vessels, is an integral part of normal developmental processes as well as numerous pathological states in humans. The angiogenic assays are reliable predictors of certain pathologies in particular tumor growth, metastasis, inflammation, wound healing, tissue regeneration, ischemia, cardiovascular, and ocular diseases. The angiogenic inducer and inhibitor studies rely on both in vivo and in vitro angiogenesis methods, and various animal models are also standardized to assess qualitative and quantitative angiogenesis. Analogously, the discovery and development of anti-angiogenic agents are also based on the choice of suitable angiogenic assays and potential drug targeted sites within the angiogenic process. Similarly, the selection of cell types and compatible experimental conditions resembling the angiogenic disease being studied are also potential challenging tasks in recent angiogenesis studies. The imaging analysis systems for data acquisition from in vivo, in vitro, and in ova angiogenesis assay to preclinic, and clinical research also requires novel but easy-to-use tools and well-established protocols. The proposition of this pragmatic book chapter overviews the recent advances in angiogenesis assessment methods and discusses their applications in numerous disease pathogenesis.

**Keywords:** angiogenesis techniques, *in vitro* angiogenesis, angiogenic mouse models, quantitative angiogenesis, transgenic animal models, angiogenesis in clinical practice, angiogenic inhibitors

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

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2017 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.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Imran Shahid, Waleed H. AlMalki, Mohammed W. AlRabia, Muhammad Ahmed, Mohammad T. Imam, Muhammed K. Saifullah and Muhammad H. Hafeez Imran Shahid, Waleed H. AlMalki, Mohammed W. AlRabia, Muhammad Ahmed, Mohammad T. Imam, Muhammed K. Saifullah and Muhammad H. Hafeez

## **1. Introduction**

The growth of new microvessels from the parent ones is an integral part of new tissue growth in growing organisms. It plays an essential part in human health while playing key roles in wound healing and tissue development [1]. Similarly, the phenomenon is regularly triggered in certain pathological conditions including rheumatoid arthritis, endometriosis, diabetic retinopathy, macular degeneration, tumor growth, and inflammatory conditions in response to certain antigens and toxins [2]. However, almost every normal tissues lack this phenomenon in adulthood, except cyclical events in the female reproductive organs [3]. Physiological angiogenesis in tissues contains a natural balance between endogenous proand anti-angiogenic factors [3]. When this balance gets disturbed and shifts more toward the pro-angiogenic side in certain pathological states (inflammation, ischemia, hypoxia, and cancer), microvascular endothelial cells (ECs) initiate a cascade of angiogenic reactions which may be retracted or progressive and turn microvessels to an angiogenic phenotype [4]. A considerable diversity exists among microvascular endothelial cells in different tissues and organs, and species heterogeneity cannot be ignored in this scenario [4].

Where angiogenesis is useful for tissue growth and development, excessive vessel growth is really problematic and a hallmark to propagate many diseases while contributing to turning tumor cells into cancer, tumor metastasis, psoriasis, arthritis, diabetic retinopathy, and predominantly metabolic disease such as obesity, atherosclerosis, and certain infectious diseases [5]. Conversely, insufficient angiogenesis or neovascularization may cause ischemic tissue states in heart, brain, and peripheral muscles which may lead to high blood pressure, preeclampsia, neurodegeneration, and osteoporosis [5]. In such pathological states, proangiogenic therapies which promote compensatory angiogenesis show promise to treat such pathologies [6]. In parallel to that, angiogenic inhibitors found highly effective in clinical trials as successful strategic treatment approaches with or without conventional chemotherapy for the treatment of solid tumors and metastasis [7]. The potential beneficiary of such novel treatment strategies are patients with aberrant ocular angiogenesis and cancer patients, where defective sight and cancer progression are entirely angiogenesis-dependent [7]. Such treatment paradigms are also heralding a new era of the treatment for other commonly occurring angiogenesis-related diseases.

The formation of new vessels involves many different cell types, and an intricate interplay of various endogenous vascular growth factors, receptors, extracellular matrix (ECM) proteins and the humoral factors [8]. To design and develop potentially effective pro- and anti-angiogenic treatments and to understand molecular mechanisms involved in angiogenesis and neovascularization, numerous in vivo and in vitro assays and animal models of angiogenesis have been developed [9]. Similarly, preclinical angiogenesis assays have also used for drug screening, molecular structure activities, and dosage effects of certain approved anti-angiogenic compounds although such assays are not equivalent and relevant to human disease regarding efficacy [10]. The prime objective of this book chapter is to overview current major and newly introduced angiogenic assays with regard to major advantages and limitations from biological, technical, ethical, and economic perspectives. The major assays which we discuss here include corneal micropocket assay, CAM (chick chorioallantoic membrane) assay, rodent mesentery, Matrigel plug assays, whole-animal assays (zebrafish), and animal models of angiogenesis in the context of cardiovascular, ocular, and adipose tissue diseases. A precise note on genetically engineered animal models for vascular endogenous genes and their spatial, temporal, and conditional expression is also included [9]. It is beyond the scope of this chapter to cover every angiogenesis assays in details, so we briefly overview quantitative techniques and/or methods to assess/evaluate neovascularization in tissues. We also briefly discuss molecular mechanisms and cell signaling pathways involved in angiogenesis and potential anti-angiogenic therapies, their clinical impact, limitations, and future prospects.

## **2. Prerequisite for good angiogenesis assays**

**1. Introduction**

angiogenesis-related diseases.

The growth of new microvessels from the parent ones is an integral part of new tissue growth in growing organisms. It plays an essential part in human health while playing key roles in wound healing and tissue development [1]. Similarly, the phenomenon is regularly triggered in certain pathological conditions including rheumatoid arthritis, endometriosis, diabetic retinopathy, macular degeneration, tumor growth, and inflammatory conditions in response to certain antigens and toxins [2]. However, almost every normal tissues lack this phenomenon in adulthood, except cyclical events in the female reproductive organs [3]. Physiological angiogenesis in tissues contains a natural balance between endogenous proand anti-angiogenic factors [3]. When this balance gets disturbed and shifts more toward the pro-angiogenic side in certain pathological states (inflammation, ischemia, hypoxia, and cancer), microvascular endothelial cells (ECs) initiate a cascade of angiogenic reactions which may be retracted or progressive and turn microvessels to an angiogenic phenotype [4]. A considerable diversity exists among microvascular endothelial cells in different tissues and

Where angiogenesis is useful for tissue growth and development, excessive vessel growth is really problematic and a hallmark to propagate many diseases while contributing to turning tumor cells into cancer, tumor metastasis, psoriasis, arthritis, diabetic retinopathy, and predominantly metabolic disease such as obesity, atherosclerosis, and certain infectious diseases [5]. Conversely, insufficient angiogenesis or neovascularization may cause ischemic tissue states in heart, brain, and peripheral muscles which may lead to high blood pressure, preeclampsia, neurodegeneration, and osteoporosis [5]. In such pathological states, proangiogenic therapies which promote compensatory angiogenesis show promise to treat such pathologies [6]. In parallel to that, angiogenic inhibitors found highly effective in clinical trials as successful strategic treatment approaches with or without conventional chemotherapy for the treatment of solid tumors and metastasis [7]. The potential beneficiary of such novel treatment strategies are patients with aberrant ocular angiogenesis and cancer patients, where defective sight and cancer progression are entirely angiogenesis-dependent [7]. Such treatment paradigms are also heralding a new era of the treatment for other commonly occurring

The formation of new vessels involves many different cell types, and an intricate interplay of various endogenous vascular growth factors, receptors, extracellular matrix (ECM) proteins and the humoral factors [8]. To design and develop potentially effective pro- and anti-angiogenic treatments and to understand molecular mechanisms involved in angiogenesis and neovascularization, numerous in vivo and in vitro assays and animal models of angiogenesis have been developed [9]. Similarly, preclinical angiogenesis assays have also used for drug screening, molecular structure activities, and dosage effects of certain approved anti-angiogenic compounds although such assays are not equivalent and relevant to human disease regarding efficacy [10]. The prime objective of this book chapter is to overview current major and newly introduced angiogenic assays with regard to major advantages and limitations from biological, technical, ethical, and economic perspectives. The major assays which we discuss here include

organs, and species heterogeneity cannot be ignored in this scenario [4].

294 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Before to choose an ideal assay for angiogenesis studies, the investigators and researchers must know the assay kinetics in terms of operating procedures, handling the environment, ethical justification, and assay economy [8]. In vivo angiogenic studies are more informative than in vitro due to complex cellular and molecular activities of angiogenic reactions while providing biology of the assay and showing experimental design are relevant [9]. Similarly, in trauma-based assays (either physical or chemical), where cell damage triggers inflammatory reactions which mimic the release of several pro-angiogenic cytokines, the sensitivity and specificity of the assay are reduced [10]. For such assays, specific precautions must be taken to avoid any inflammatory reaction or to minimize the traumatic tissue state. In parallel to that, the test substance/compound should be designed as being angiogenic in a noninflammatory state. A near to physiological dose of the test compound should be administered for inducing an angiogenic response while to modulate angiogenic assay conditions and dosage response, a dose range of the clinical use must be chosen [10].

Vehicles carrying the test compound in many assays may also affect the pharmacokinetics of the tested drug and alter the dose-response curves among different experimental animals within one group. For such circumstances, the best solution is to compare test animals/samples with vehicle-exposed counterparts [9]. However, for data interpretation, one must be fully acquainted with the fact that how the vehicle-administered tested animals differ from the untreated controls [10]. Spatial and temporal distributions of the tested compounds are also necessary and vital because failure to do so may produce or hinder to generate reliable and rigorous dose-response curves [10]. As in different pathological states newly formed, vessels are delicate in quality and poorly functional, the selection of angiogenic assessment methods (either qualitative or quantitative) also matters to evaluate the morphology and physiology of the neovascularization in diseased tissues [9]. For in vivo angiogenesis assays, histological microscopy provides the detailed information precisely. Mammalian systems adopted for in vivo angiogenesis assays and mouse models for certain cardiovascular, ocular, and cerebral diseases are comparatively more close to relating human pathophysiology than the embryonic CAM assay, embryonic zebrafish (*Xenopus laevis*), and invertebrate (*Hirudo medicinalis*) angiogenic assays [9, 10].

## **3. Key components of an ideal angiogenesis assay**

It would be interesting to describe that despite the much progress in the field of angiogenesis research, there is no single angiogenic model available which may fully elucidate the entire process and molecular mechanisms of the angiogenic and neovascularization process. Some exogenous and endogenous factors hinder the efforts to develop such an ideal system. Due to cell diversity among different tissues where angiogenesis takes place and intricate interplay among different cell signaling pathways of angiogenic reactions, it is an uphill task to develop and validate a unique assay that is optimal for all situations. However, different modalities and ingenious ways with the passage of time in a particular assay facilitate and provide optimisms for better measurements of angiogenesis than the past. In this context, Vallee et al. [11] conclude that "The design and verification of [new] specific, reliable, repeatable, and precise methodology to measure angiogenesis are considered an imperative of high priority in the field of angiogenesis research." Similarly, Auerbach et al. [10] state "Perhaps the most consistent limitation in all these studies and approaches has been the availability of simple, reliable, reproducible, quantitative assays of the angiogenic response." Moreover, it is challenging although not impossible in several angiogenic assays that the quantification of newly formed vessels regarding numbers and lengths. Similarly, the spatial and temporal distribution of tested compound is also necessary to get strong doseresponse curves. Performing an assay in a blinded manner may helpful in this prospect and also to alleviate the influence of any preconceived notions. Analogously, the technical skills to perform any angiogenesis assay are of utmost importance to ensure maximum success.

Despite all these qualms as described above, an ideal angiogenesis assay for quantification of newly formed vessels must feature the following characteristics; first [12], "the release rate [R] and the spatial and temporal concentration distribution [C] of tested compounds should be known to evaluate dose-response curves; second, if tumor cells are used as a source of angiogenic factors, oncogene expression and production of growth factors (either stimulants or inhibitors) must be genetically well defined before the assay proceeding; third, the assay must be designed in a way ensure to provide quantitative measuring parameters of the newly formed vessels (e.g., vascular length [L], surface area [A], volume [V], number of vessels in the network [N], fractal dimensions of the network [Df], and extent of basement membrane [BM]); fourth, the assay should be designed in a way to weigh quantitative measure of morphological characteristics of new vessels (e.g., endothelial cell migration [MR], proliferation rate [PR], canalization rate [CR], blood flow rate [F], and vascular permeability [P]); fifth, a clear demarcation must exist between new and parent vessels; sixth, tissue trauma must be minimized to prevent the formation of new vessels; seventh, in vitro assessment should be verified by in vivo procedures; eighth, angiogenesis assay for long term and with noninvasive monitoring should be preferred; last, the selected assay should be economical, ethically justifiable, robust, and reliable." [12].

## **4. Process of angiogenesis**

Endothelial cell activation, proliferation, and directed migration to form new microvasculature (capillaries) from the parent ones should be a complex process involving many molecular and cell signaling pathway events [2]. Some key regulators to switch on or off gene expression are also participating and influence by positive and negative feedbacks of cellular processes. The normal physiological angiogenesis initiates by sprouting of capillaries under the effect of vascular endothelial growth factors (VEGFs) from parent vessels [13]. It continuous during embryonic development and transiently during female reproductive cycle but almost stops in adult tissues except for some wound healing states [13]. Pathologic angiogenesis remains persistent with the continuous proliferation of ECs in different tissue pathologies and particularly in cancer [3]. Many tumor cells are capable of attracting adjacent blood vasculature from nearby tissues [2]. It was evident by the fact that for solid tumors to grow a certain size, neovascularization is necessary otherwise such tumors rarely metastasize as found in thin melanomas which reside on the avascular basement membrane [2, 13]. Also, for tumor growth, the nutrient supply, oxygen, and waste removal are also essential. The new vasculature fulfills this task while providing immune cells, macrophages, and humoral factors to the vicinity of the tumor cells [2].

**3. Key components of an ideal angiogenesis assay**

296 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

any angiogenesis assay are of utmost importance to ensure maximum success.

**4. Process of angiogenesis**

Despite all these qualms as described above, an ideal angiogenesis assay for quantification of newly formed vessels must feature the following characteristics; first [12], "the release rate [R] and the spatial and temporal concentration distribution [C] of tested compounds should be known to evaluate dose-response curves; second, if tumor cells are used as a source of angiogenic factors, oncogene expression and production of growth factors (either stimulants or inhibitors) must be genetically well defined before the assay proceeding; third, the assay must be designed in a way ensure to provide quantitative measuring parameters of the newly formed vessels (e.g., vascular length [L], surface area [A], volume [V], number of vessels in the network [N], fractal dimensions of the network [Df], and extent of basement membrane [BM]); fourth, the assay should be designed in a way to weigh quantitative measure of morphological characteristics of new vessels (e.g., endothelial cell migration [MR], proliferation rate [PR], canalization rate [CR], blood flow rate [F], and vascular permeability [P]); fifth, a clear demarcation must exist between new and parent vessels; sixth, tissue trauma must be minimized to prevent the formation of new vessels; seventh, in vitro assessment should be verified by in vivo procedures; eighth, angiogenesis assay for long term and with noninvasive monitoring should be preferred; last, the selected assay should be economical, ethically justifiable, robust, and reliable." [12].

Endothelial cell activation, proliferation, and directed migration to form new microvasculature (capillaries) from the parent ones should be a complex process involving many molecular and

It would be interesting to describe that despite the much progress in the field of angiogenesis research, there is no single angiogenic model available which may fully elucidate the entire process and molecular mechanisms of the angiogenic and neovascularization process. Some exogenous and endogenous factors hinder the efforts to develop such an ideal system. Due to cell diversity among different tissues where angiogenesis takes place and intricate interplay among different cell signaling pathways of angiogenic reactions, it is an uphill task to develop and validate a unique assay that is optimal for all situations. However, different modalities and ingenious ways with the passage of time in a particular assay facilitate and provide optimisms for better measurements of angiogenesis than the past. In this context, Vallee et al. [11] conclude that "The design and verification of [new] specific, reliable, repeatable, and precise methodology to measure angiogenesis are considered an imperative of high priority in the field of angiogenesis research." Similarly, Auerbach et al. [10] state "Perhaps the most consistent limitation in all these studies and approaches has been the availability of simple, reliable, reproducible, quantitative assays of the angiogenic response." Moreover, it is challenging although not impossible in several angiogenic assays that the quantification of newly formed vessels regarding numbers and lengths. Similarly, the spatial and temporal distribution of tested compound is also necessary to get strong doseresponse curves. Performing an assay in a blinded manner may helpful in this prospect and also to alleviate the influence of any preconceived notions. Analogously, the technical skills to perform

The parent vessel wall comprises endothelial cell lining, basement membrane, and pericytic cells. Pro-angiogenic growth factors (VEGF, TGF-α, TNF-α) from tumor cells bind to the receptors of ECs and initiate a cascade of cell signaling pathways and angiogenic reactions [2]. Activation and resolution of ECs are two key steps of the angiogenic cascade reactions. When ECs activate and stimulate to grow, the cells secrete proteases, heparanase, and other digestive enzymes that degrade the extracellular matrix (ECM) [13]. ECM degradation allows the secretion of many pro-angiogenic factors from the endothelial cell matrix, and the junctions between ECs become leaky; new microvessel sprouts grow in the direct toward the stimulus [2] (**Figure 1**). For further ECs to grow, proliferate, and migrate, hematopoietic-endothelial progenitor cells (HEPC) also play an essential role [13]. In resolution phase, the new microvasculature tends to mature with the help of pericytic cell adhesion, reconstitution of basement membrane, and

**Figure 1.** Process of angiogenesis from parent vessels: Angiogenesis sprouting initiates when vascular endothelial growth factors (e.g., VEGF-A) bind to VEGFR-2 receptors located on endothelial cells (ECs). ECs release matrix metalloproteinase (MMP) which degrades extracellular matrix (ECM) from which endothelial tip cells migrate. Vascular endothelial growth factors also regulate Notch cell signaling to inhibit proliferation of endothelial stalk cells. Platelet-derived growth factor (PDGF) released from ECs recruits smooth muscle cells (e.g., pericyte) to stabilize the neovasculature. TGF-α = transforming growth factor, TNF-α = tumor necrosis factor.

formation of cell junctions [14]. Interestingly, the resolution phase in tumor surrounding capillary network remains incomplete which results in irregular and tortuous microvasculature with partial ECs, increased cell permeability, and fragmentary basement membrane [15]. Tumor vasculature is disorganized with poor microcirculation, and vessel diameter changes without any differentiation into arterioles, capillaries, and venules [13]. Similarly, tumor vasculature is sprouting type, so assays which quantify sprouting angiogenesis are very useful to study the kinetics of tumor angiogenesis [15].

In the following section, we present the major and currently used preclinical angiogenesis assays in approximate chronological order of their first publication like chick chorioallantoic membrane (CAM), Matrigel plug, and corneal micropocket assays, while the others described in brief. [10]. CAM, Matrigel plug, and zebrafish assays are very useful for new angiogenic inhibitors screening. For a particular research focus, we provide advantages and disadvantages between different assays in a tabular form feasible for the readers (**Table 1**).



formation of cell junctions [14]. Interestingly, the resolution phase in tumor surrounding capillary network remains incomplete which results in irregular and tortuous microvasculature with partial ECs, increased cell permeability, and fragmentary basement membrane [15]. Tumor vasculature is disorganized with poor microcirculation, and vessel diameter changes without any differentiation into arterioles, capillaries, and venules [13]. Similarly, tumor vasculature is sprouting type, so assays which quantify sprouting angiogenesis are very useful to study the

In the following section, we present the major and currently used preclinical angiogenesis assays in approximate chronological order of their first publication like chick chorioallantoic membrane (CAM), Matrigel plug, and corneal micropocket assays, while the others described in brief. [10]. CAM, Matrigel plug, and zebrafish assays are very useful for new angiogenic inhibitors screening. For a particular research focus, we provide advantages and disadvantages

(a) Atypical assay due to avascular tissue

(c) Inaccessible to endogenous blood-

(d) Ethical problems as using a major sensory organ for angiogenic assay

(f) Not a suitable site for tumor growth

(a) Inflammation-mediated angiogenic

tension

(e) Oxygen exposure may affect

(g) Tested compounds are few

(b) Very sensitive to change in O2

(d) Embryonic nonmammalian

angiogenesis in mice than rats

(e) Newly formed vessels are difficult to

activated compounds

(b) Induction of nonspecific inflammation to test substance

borne angiogenic factors

angiogenesis

reactions

procedure

identify

(c) Tumor angiogenesis may assess (c) Not suitable for metabolically

(b) Lacks physiological angiogenesis (b) Real-time observation is limited

nature

between different assays in a tabular form feasible for the readers (**Table 1**).

298 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

**Assay name Advantages Disadvantages**

mice, rat, and rabbit)

is minimized

sprouting

cost

and long-term monitoring

(e) New vessel formation by

(a) Simple to perform and low in

(b) Suitable to study pro- and anti-

**Rodent mesentery assays** (a) Natively sparsely vascularized (a) Less significant for quantitative

angiogenic compounds

(b) Easy to perform in animals (e.g.,

(c) Qualitatively permits noninvasive

(d) Immunologically, cross reaction

**Corneal micropocket assay** (a) Easy-to-identify newly formed vessels

kinetics of tumor angiogenesis [15].

*In vivo* **angiogenesis assays**

**Chick chorioallantoic membrane** 

**assay**


**Table 1.** The advantages and disadvantages of major *in vivo, in vitro* and animal models angiogenesis assays.
