**6. Animal models of angiogenesis**

that when Matrigel with reduced growth factors is implanted, few cells invade the plug or gel. However, with known angiogenic growth factors (e.g., bFGF), mixed with Matrigel and injected subcutaneously, endothelial cells migrate into the gel and constitute vessel-like structures. A fine network of endothelial cell tubes enlarged by micro- and macro-vessel endothe-

For the quantitative assessment of angiogenesis, Matrigel and surrounded granulation tissue are removed after 1–3 weeks, and immunohistochemistry and histological sections are measured [27]. However, determining the profiles of capillary-like vessels is difficult. Similarly, the hemoglobin (Hb) test does not differentiate the blood flow in newly formed blood vessels and large parent vessels. Fluorochrome-labeled high molecular weight dextran and quantitative vascular specific indicators are alternative methods to assess neovas-

The assay is suitable for tissue regeneration experiment model where neovascularization is coupled with organogenesis, fibrosis, and monocytes/macrophages play a pivotal structural role. A possible drawback of the assay is that Matrigel plug contains only capillary network rather than no tissue without any pro- and anti-angiogenic factors to influence angiogenic

A variation of the Matrigel plug assay is the combination of Matrigel and sponge techniques. Five-hundred microliters of Matrigel is injected subcutaneously into mice and solidify for 20–30 min [27]. After that, the mice are anesthetized, skin overlying Matrigel is shaved, and a small nick is made. A similar nick is made to Matrigel plug, and a sterile polyvinyl sponge with the test compound is introduced into the center of the Matrigel plug with the help of tweezers. The same procedure may use for angiogenic growth factors or test tissue to be implanted in the Matrigel plug. By this modification, neovascularization is directional, and assay sensitivity is increased to measure direct angiogenesis as compared to standard Matrigel plug assay. However, the sponge/Matrigel combined assay is time-consuming, and the total number of

Zebrafish was introduced in 1999 as a whole small angiogenesis model for the screening of pro-angiogenic compounds which directly influence the newly formed vessels [29]. The choice of the whole animal as a tested tissue was based on the remarkable similarity of zebrafish organs to those of a human at the physiological, anatomical, and molecular levels [30]. Moreover, the short generation time (approx. 3 months) and easy to house in small space and relatively large numbers also facilitate to evaluate many tested animals in one assay [31]. The external development of zebrafish embryos and optical transparency during embryonic stage assists continuous microscopic evaluations of different developmental processes from gastrulation to organogenesis [30]. Furthermore, external mode of fertilization also permits easy access to experiment design and assessment. Small tested compounds dissolve to water diffuse directly to fish embryo and induce distinct and dose-dependent angiogenic effects. Both pro- and anti-angiogenic compounds exhibit similar effects in zebrafish as exerted in

lial cells slowly progress to capillary networks in vivo [26].

304 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

cularization [27].

reactions [28].

mammals [31].

assayed animals become limited [27].

**5.7. Whole-animal angiogenesis model**

In biomedical research, mouse models are of utmost importance for a wide variety of medical tests including gene expression, gene knockout, and medical genetic analysis [32]. For this purpose, SCID, transgenic, and genetically engineered mouse models are of particular interests which allow sophisticated investigations for genetically induced pathological states and molecular pathogenesis of certain genetic disorders. Furthermore, such mouse models are useful to study genes essential for angiogenesis and vascular biology [32]. In parallel to that mice with conditional, global knockouts, over-expressing angiogenic factors are also considerable in this prospect [33]. As remarkable similarity exists between human and murine vasculatures, such tools are valuable to search possible molecular interactions among distinct angiogenic factors in the onset and progression of various human diseases [32, 33]. In the following section, we shed light on some practically used mouse models in the context of pathological angiogenesis which directly plays a part in human diseases.

## **6.1. Mouse model of angiogenesis in adipose tissue**

Genetically engineered mouse model for adipose tissue angiogenesis is highly reproducible and produces robust results because the mice are inbred and share a highly similar genetic background. This approach is irrelevant to humans because high caloric intake and little physical exercise are the predisposing factors for developing obesity instead a little genetics involved. Thus, mice fed on high-fat diet present an ideal animal model to study non-genetically related obesity [34].

## *6.1.1. Ob/ob mice*

The mouse carrying the obese mutation (ob) was first described in 1950, and later on, it was shown that the mutation located in the gene coding for a hormone leptin, which regulates appetite and food intake [35]. The hormone binds to leptin receptor (Ob-R) in the hypothalamus and subsequent cell signaling regulates food uptake, energy expenditure as well as fat and glucose metabolism. Ob/ob mice are deficient in leptin exhibit uncontrolled and continuous food intake which results in a gain of body weight. Consequently, mutated mice weight is three times higher, and body fat content elevates up to fivefold as compared to wild-type species. The mutated mice also show decreased physical activity and energy expenditure, infertility, and immune deficiencies. The mutation is recessive, so the heterozygotes do not display such phenotype [35]. Ob/ob mice can be used as an outstanding model to explore the role of angiogenesis in adipose tissue expansion, and with specific angiogenic inhibitors, obesity may be prevented in such mice [36]. As the leptin kinetics in mice to regulate food intake and obesity are homologous to human, such angiogenic model can be used to search novel therapeutic targets to treat obesity and metabolic disorders [37].

#### *6.1.2. Db/db mice*

The mouse strain C57BL/KsJ was first described with an autosomal recessive mutation diabetes (db) in 1966 [38]. Homozygous mice with such mutation are deficient for the leptin receptor and exhibit a phenotype that resembles human diabetes mellitus. The mice with such mutations are also characterized as an obese phenotype. Furthermore, such mice exhibit infertility and hyperglycemia while heterozygotes are typically lived as wild type. Db/db mice can be used to study molecular mechanisms involved in obesity-related diabetes and insulin insensitivity, and the role of angiogenesis and neovascularization can be elucidated in this regard [38].

#### **6.2. Hindlimb ischemic model of angiogenesis**

Most of the angiogenic models described above are very useful to study pathological angiogenesis and search for novel anti-angiogenic treatment in the form of angiogenic inhibitors. However, certain pathological states (e.g., myocardial infarction, stroke, and wound healing/regeneration) in human body require accelerated blood vessel growth to reinstate the proper function of such vital organs [39]. In myocardial infarction, an occluded coronary artery obstructs blood flow to a part of the cardiac muscle tissue which leads to severe tissue hypoxia (ischemia). The cardiac muscle requires a regular supply of oxygen and glucose levels for normal function. To overcome tissue hypoxia, the growth of highly functional arteries is eagerly awaited in such situations. Hind limb ischemia in rat or mice presents an excellent model to study and manipulate newly formed vessels in particular arteries in response to tissue hypoxia [39].

In this assay, the arteries supply blood to one back limb of the mice is ligated to stop the blood circulation in the entire limb [40]. The occlusion of arteries leads to tissue ischemia and the initiation of arteriogenesis from collateral arteries. Pro-angiogenic factors and even antiangiogenic compounds under investigation can be administered to the limb musculature to modulate the arteriogenic response. Doppler angiography is used to evaluate the blood circulation in the hind limb, and the procedure can be repeated in the same animal to know that how the blood flow improves over time. To study newly formed microvessels, the tissue can be excised and stained, and morphology of the blood vessels is elucidated [40].

The assay is the first in class to present therapeutic angiogenesis and widely used in fundamental discoveries to demonstrate that how to generate highly functional and stable arteries therapeutically [39]. On the other side, the potential disadvantage of the assay is very complicated hind limb surgery and requires highly skilled professionals and experienced surgeons. Similarly, the proportion of blood flow in a hind limb may vary after surgery, and it may affect the degrees of tissue hypoxia which ultimately influence on the therapeutic activity of pro- and anti-angiogenic compounds under investigation [39].

#### **6.3. Wound healing assays**

The wound healing assay allows to study and evaluate both angiogenesis and vascular maturation/remodeling in injured or damaged tissues [41]. The assay is usually performed on the skin of mice because other accessible tissues (e.g., tail and ears) do not regenerate well. Two circular holes (approx. 5mm in diameter) are punched through the dorsal skin of anesthetized wild-type C57B16 mice. One hole would serve as control while drugs under investigation can be administered on the other. No bandages or sponge is required as no major blood vessels exist in this region of the skin and wound formation allows very little bleeding in the surrounding area. Wound sealing starts within two weeks, and complete wound healing occurs within a month [41].

Photography and measuring of wound area with calipers provide information about wound size, scar formation, and re-epithelization of the wound [41]. The drugs under investigation (either pro- or anti-angiogenic compounds) in this model may be administered either systemically by oral administration, injection, or topically. The drug effects can be determined by excision of the skin tissue, fixed, and stained with specific dyes. The tested compounds may influence regenerative angiogenesis, vessel morphology, and function. The assay is easy to setup, and surgery is very simple. The wound size remains uniform and homogenous for all animals used in one experiment [41].

The potential disadvantage of the assay is that angiogenesis is inflammation-dependent involving blood clotting phenomenon and other complex biological processes and occurs only in the skin [41]. Similarly, skin tissue regeneration is entirely different as compared to other highly vascular tissues such as the heart and nervous system and does not provide an adequate understanding of the role of angiogenesis in tissue regeneration. Furthermore, tissue regeneration is mainly due to reconstitute new tissue rather than repairing or replacing, which is hard to replace in ischemic insults which produce large patches of dead tissue [41].

#### **6.4. Genetically engineered animal model for angiogenesis**

receptor and exhibit a phenotype that resembles human diabetes mellitus. The mice with such mutations are also characterized as an obese phenotype. Furthermore, such mice exhibit infertility and hyperglycemia while heterozygotes are typically lived as wild type. Db/db mice can be used to study molecular mechanisms involved in obesity-related diabetes and insulin insensitivity, and the role of angiogenesis and neovascularization can be elucidated

Most of the angiogenic models described above are very useful to study pathological angiogenesis and search for novel anti-angiogenic treatment in the form of angiogenic inhibitors. However, certain pathological states (e.g., myocardial infarction, stroke, and wound healing/regeneration) in human body require accelerated blood vessel growth to reinstate the proper function of such vital organs [39]. In myocardial infarction, an occluded coronary artery obstructs blood flow to a part of the cardiac muscle tissue which leads to severe tissue hypoxia (ischemia). The cardiac muscle requires a regular supply of oxygen and glucose levels for normal function. To overcome tissue hypoxia, the growth of highly functional arteries is eagerly awaited in such situations. Hind limb ischemia in rat or mice presents an excellent model to study and manipulate newly formed vessels in particular arteries in response to tis-

In this assay, the arteries supply blood to one back limb of the mice is ligated to stop the blood circulation in the entire limb [40]. The occlusion of arteries leads to tissue ischemia and the initiation of arteriogenesis from collateral arteries. Pro-angiogenic factors and even antiangiogenic compounds under investigation can be administered to the limb musculature to modulate the arteriogenic response. Doppler angiography is used to evaluate the blood circulation in the hind limb, and the procedure can be repeated in the same animal to know that how the blood flow improves over time. To study newly formed microvessels, the tissue

The assay is the first in class to present therapeutic angiogenesis and widely used in fundamental discoveries to demonstrate that how to generate highly functional and stable arteries therapeutically [39]. On the other side, the potential disadvantage of the assay is very complicated hind limb surgery and requires highly skilled professionals and experienced surgeons. Similarly, the proportion of blood flow in a hind limb may vary after surgery, and it may affect the degrees of tissue hypoxia which ultimately influence on the therapeutic activity of

The wound healing assay allows to study and evaluate both angiogenesis and vascular maturation/remodeling in injured or damaged tissues [41]. The assay is usually performed on the skin of mice because other accessible tissues (e.g., tail and ears) do not regenerate well. Two circular holes (approx. 5mm in diameter) are punched through the dorsal skin of anesthetized wild-type C57B16 mice. One hole would serve as control while drugs under investigation can be administered on the other. No bandages or sponge is required as no major blood vessels

can be excised and stained, and morphology of the blood vessels is elucidated [40].

pro- and anti-angiogenic compounds under investigation [39].

in this regard [38].

sue hypoxia [39].

**6.3. Wound healing assays**

**6.2. Hindlimb ischemic model of angiogenesis**

306 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

Gene expression analysis and gene function studies are contributing widely to almost every research in life sciences, biomedical research, biotechnology, molecular pathology, and human health [42]. In vivo applied and functional genomic studies are particularly considered by overexpressing a candidate gene or suppressing the gene expression for the purpose of gene knockout [43]. Such approaches are achieved and applicable by genome manipulation of wild-type animals [43]. Similarly, the generation of transgenic animals by injecting desired DNA constructs to fertilized eggs also presents some standard technology in gene expression studies [44]. Transgenic animal models and DNA constructs (e.g., gene expression plasmids and vectors) with desired gene expression are widely used to study gene function and molecular pathogenesis of diseases, and it create models to demonstrate the complex, intricate interplay between gene overexpression or suppression for the molecular epidemiology of human diseases [44]. In the following section, we briefly overview such novel approaches to be involved in the context of vascular angiogenesis.

The control of target gene expression in vascular cells of transgenic animals by cell or tissue expression plasmid with specific promoters is very helpful to study developmental and pathological gene function in the vasculature [42]. It was found that the promoters derived from the sequences of VEGFR-1, ICAM-2, vWF, or endoglin efficiently work in mouse endothelial cells both in vivo and in vitro with specific intensity and specificity [42]. Similarly, lacZ selective transgene expression was seen in ECs cells under the control of promoters derived from Tie 2 (angiopoietin receptor), ICAM-2, or VE-Cadherin [42]. However, the expression of a transgene in smooth muscle cells (SMCs) is difficult to achieve because most SMC markers are expressed differentially, and SMC growth and cell differentiation are an exclusive process [42]. In contrast, transgenic mice may obtain using selective promoters expressing smooth muscle myosin heavy chain (SM-MHC), smooth muscle α-actin, and SM22α [42]. Such models provide valuable information about the function of a specific gene in a particular tissue, and controlling the expression of such genes may use as a therapeutic approach to certain disease and also for angiogenesis and cancer [42]. However, the transgene expression depends on promoter's characteristics to be used while constructing cell or tissue expression plasmids with the gene of interests including; promoters is constitutively active, capable to express and replicate gene of interest or to express in embryonic or in the adulthood stage [45]. Failure to do so may limit studies in molecular pathogenesis, leads to nonviable transgenic animals or compensatory responses. More powerful tools are being developed based on conditional transgene expression systems [45].

The inhibition of endogenous gene expression is also a potential method to suppress the gene function involved in the molecular pathogenesis of many genetic disorders and infectious diseases [46]. Many studies show that sequence-specific mRNA degradation by double-strand RNA strongly inhibits the function of that gene involved in pathogenesis or propagation of a particular disease [47]. The technique is known as RNA interference (RNAi) and may be used in certain genetic disorders, in cancer, HIV, and other harder to treat infections. Recent publish data indicate that mammalian expression vectors expressing short hairpin RNA (shRNA) under the control of specific vascular promoters inhibit gene expression through an RNAi effect [47].

## **7. Angiogenesis and cancer**

The phenomenon of angiogenesis is fundamental in tumor growth, progression, and metastasis [48]. Angiogenesis itself is the result of a highly orchestrated series of molecular and cellular events including a plethora of genes, signal cascades, and transcription factors which are highly organized and work in a systematic way to generate microvessels in normal physiological angiogenesis [49]. However, the tumor angiogenesis is disorganized, irregular, and not systematic at the level of molecular and cellular events and ultimately propagates many tumors into cancers [48]. The cancer cells contain the ability to stimulate angiogenesis by producing a lot of angiogenic factors including cell growth factors, cytokines, and numerous other molecules [48, 49].

Many pro- and anti-angiogenic molecules involved in the induction of angiogenesis and neovascularization, their receptor ligands, and intracellular signaling pathways have been identified within last 30 years [50]. Much work has been done to develop anti-angiogenic treatment strategies for cancer patients [51]. However, numerous preclinical trials show no promise regarding high efficacy and tolerability with classical anti-angiogenic drugs as monotherapy [51]. It spurred the researchers and investigators to design and develop novel anti-angiogenic compounds to be used in combination with classical cytotoxic agents and radiotherapy [52]. FDA-approved angiogenesis inhibitors in combination with chemotherapy have proven their clinical worth regarding improved patient survival time and patient tolerability in certain cancers [52].

In the coming section, we briefly overview molecular mechanisms of major cell signaling pathways involved in the induction of angiogenesis, and at the end, some brief glimpse about the clinical impacts of newly developed angiogenic inhibitors will be described. The cellular events in the regeneration and propagation of tumor angiogenesis are already explained briefly at page 5 and depicted in **Figure 1**.

## **7.1. VEGF intracellular signaling**

process [42]. In contrast, transgenic mice may obtain using selective promoters expressing smooth muscle myosin heavy chain (SM-MHC), smooth muscle α-actin, and SM22α [42]. Such models provide valuable information about the function of a specific gene in a particular tissue, and controlling the expression of such genes may use as a therapeutic approach to certain disease and also for angiogenesis and cancer [42]. However, the transgene expression depends on promoter's characteristics to be used while constructing cell or tissue expression plasmids with the gene of interests including; promoters is constitutively active, capable to express and replicate gene of interest or to express in embryonic or in the adulthood stage [45]. Failure to do so may limit studies in molecular pathogenesis, leads to nonviable transgenic animals or compensatory responses. More powerful tools are being developed based

The inhibition of endogenous gene expression is also a potential method to suppress the gene function involved in the molecular pathogenesis of many genetic disorders and infectious diseases [46]. Many studies show that sequence-specific mRNA degradation by double-strand RNA strongly inhibits the function of that gene involved in pathogenesis or propagation of a particular disease [47]. The technique is known as RNA interference (RNAi) and may be used in certain genetic disorders, in cancer, HIV, and other harder to treat infections. Recent publish data indicate that mammalian expression vectors expressing short hairpin RNA (shRNA) under the control of specific vascular promoters inhibit gene expression through an RNAi

The phenomenon of angiogenesis is fundamental in tumor growth, progression, and metastasis [48]. Angiogenesis itself is the result of a highly orchestrated series of molecular and cellular events including a plethora of genes, signal cascades, and transcription factors which are highly organized and work in a systematic way to generate microvessels in normal physiological angiogenesis [49]. However, the tumor angiogenesis is disorganized, irregular, and not systematic at the level of molecular and cellular events and ultimately propagates many tumors into cancers [48]. The cancer cells contain the ability to stimulate angiogenesis by producing a lot of angiogenic factors including cell growth factors, cytokines, and numerous

Many pro- and anti-angiogenic molecules involved in the induction of angiogenesis and neovascularization, their receptor ligands, and intracellular signaling pathways have been identified within last 30 years [50]. Much work has been done to develop anti-angiogenic treatment strategies for cancer patients [51]. However, numerous preclinical trials show no promise regarding high efficacy and tolerability with classical anti-angiogenic drugs as monotherapy [51]. It spurred the researchers and investigators to design and develop novel anti-angiogenic compounds to be used in combination with classical cytotoxic agents and radiotherapy [52]. FDA-approved angiogenesis inhibitors in combination with chemotherapy have proven their clinical worth regarding improved patient survival time and patient tolerability in certain

on conditional transgene expression systems [45].

308 Physiologic and Pathologic Angiogenesis - Signaling Mechanisms and Targeted Therapy

effect [47].

**7. Angiogenesis and cancer**

other molecules [48, 49].

cancers [52].

Vascular endothelial-derived growth factor (VEGF) is one of the most important and potent angiogenic molecules which play an integral role in tumor angiogenesis [50]. It presents the first in a class of cytokines which induce vascular leakage and therefore also known as vascular permeability factor. Until now, six members (VEGF-A to VEGF-F) of this unique family of cytokines have been discovered [53]. VEGF-A is mainly involved in angiogenesis and vasculogenesis whereas VEGF-B is a survival factor for ECs, SMCs, and pericytes [54]. VEGF-C and VEGF-D are essential for lymphangiogenesis, and PGF also acts as a survival factor for ECs and modulates VEGF cell signaling [55].

Vascular endothelial growth factors activate ECs by binding to a family of class III transmembrane receptor tyrosine kinases (RTKs) expressing at high levels in endothelial cell lineage [53]. VEGF-R1 and VEGF-R2 are located on ECs and activate during angiogenesis while VEGF-R3 induces intracellular signaling in lymphatic cells. VEGF-R1 acts as a decoy receptor as it is RTK defective and acts as a negative regulator of angiogenesis (**Figure 2**) [54]. The angiogenic multiple cell signaling pathways are initiated as VEGF-A binds to VEGF-R2, and the receptor dimerizes and intracellular receptor domains are phosphorylated in ECs and induce overexpression of growth factors, cell proliferation, mitogenesis, chemotaxis, and prosurvival signaling (**Figure 2**) [55]. VEGF-C binds to VEGFR-3 and initiates mitogenesis in lymphatic cells and stimulates hyperplasia in parent lymphatic vessels [53–55]. The production of VEGF is regulated by several growth factors produced by the tumor cells including, endothelial growth factor (EGF), transforming growth factor (TGF-α & β), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) [54]. Some hormones (e.g., estrogen, thyroid-stimulating hormone (TSH)) and interleukins (e.g., IL-1 & 6) also stimulate VEGFinduced intracellular events in other types of cells [56].

## **7.2. Notch signaling pathways**

The Notch receptors are located on stromal cells and expressed as a heterodimeric complex of two domains, that is, the Notch extracellular domain (NECD) and Notch intracellular domain (NICD) which are associated with each other via noncovalent interactions (**Figure 3a**) [57]. The Notch cell signaling may mimic direct tumor angiogenesis however actively involved to trigger dormant tumors [1]. Notch ligand Delta-like 4 (DLL4) induces cell signaling pathways to improve vascular functions by endocytosis and nonenzymatic dissociation of Notch heterodimer in host stromal cells (**Figure 3a**) [1]. DLL4 inhibition may promote cell proliferation response in ECs which ultimately increase angiogenic sprouting and vessel branching [58]. Despite increased endothelial cell vascularity, the tumor cells perfuse poorly, which reduces cell oxygen concentrations (i.e., increased hypoxia), and consequently, tumor growth is inhibited [59].

**Figure 2.** VEGF-induced intracellular signaling in tumor angiogenesis and angiogenic inhibitors with targeted active sites: The binding of vascular endothelial growth factors (VEGF) to respective transmembrane receptors stimulates a plethora of intracellular signaling pathways which regulate nuclear transcription factors for altered gene expressions of normal cell responses including loss of tumor suppression, activation of hypoxia inducible factor (HIF-α), increased receptor tyrosine kinase activity, increased tumor cell growth, and repression of oncogene mutations. Angiogenic inhibitors to their targeted active sites are also shown with numerical circles in the figure. Only anti-angiogenic compounds approved by the US Food and Drug Administration (FDA) for the treatment of numerous solid tumors and carcinomas are depicted where circle 1 represents growth factor inhibitors (bevacizumab, aflibercept); circle 2, growth factor receptor inhibitors (sunitinib, sorafenib); circle 3, RAS inhibitors (tipifarnib, lonafarnib); circle 4, RAF inhibitors (sorafenib); circle 5, HIF-1α inhibitors (geldanamycin, chetomin, echinomycin, 2ME2); circle 6, PI3K inhibitors (wortmannin, LY294002); circle 7, AKT inhibitors (FARA-A); and circle 8, mTOR inhibitors (rapamycin and analogues). JNK = JUN N-terminal kinase; MAPK = mitogen-activated protein serine/threonine kinase, MAPKK = MAPK kinase, PDK1 = phosphoinositidedependent protein kinase-1; PLC = phospholipase C; PtdInP2 = phosphatidylinositol 4,5-bisphosphate, Ins (1,4,5) = inositol 1,4,5-triphosphates.

In contrast, DLL4 expressed in ECs stimulates Notch 3 receptors located on adjacent cells (e.g., colorectal cancer or T-cell acute lymphoblastic leukemia cells) to activate tumor progression from dormant to active phase [60]. Such findings consider Notch pathways a potential therapeutic target for the design and development of novel anti-angiogenic compounds, although the Notch cell signaling shows a mixed behavior of tumor progression and inhibition in clinical assays [58, 59].

## **7.3. Transforming growth factor-β (TGF-β)**

Transforming growth factor is a ubiquitously expressed paracrine polypeptide of approximately 25 kDa molecular weight [61]. TGF 1 to TGF 3 are three highly homologous isoforms of the polypeptide and discovered in humans and mammals [62]. TGF-β is initially synthesized as a zymogen, and after secretion, an associated peptide is proteolytically sliced to release active form of the growth factor [63].

Active TGF-β binds to constitutively active serine/threonine kinase TGFBR2 receptors to activate TGFBR1 in a heterodimer complex which controls transcription via activation of canonical signal pathways mediated by a family of SMAD proteins (SMAD1-5) (**Figure 3b**) [64]. The Recent Advances in Angiogenesis Assessment Methods and their Clinical Applications http://dx.doi.org/10.5772/66504 311

**Figure 3.** A schematic diagram of Notch and TGF-β induced cell signaling pathways in tumor angiogenesis: (a) Notch cell signaling pathways: The ligand DLL4 dissociates Notch heterodimers by nonenzymatic degradation and cell endocytosis. Notch extracellular domain exposes Notch to ADAM metalloproteases and γ-secretase in sending cells (tip cells) for proteolytic cleavage and the release of Notch intracellular domain which translocates to the nucleus of receiving cells (stalk cells) for the transcriptional activation of Notch target genes (shown in the nucleus of the stalk cells). The DLL4 ligand and Notch inhibitors are also depicted in the red rectangular boxes. (b) Transforming growth factor (TGF-β) induced intracellular signaling pathway: In normal cells, the binding of TGF-β to transmembrane TGFBR2 receptors activates TGFBR1 receptors which upregulate the expression of a series of SMAD proteins (SMAD 2, 3, and 4) and cause cell cycle arrest and apoptosis. However, TGF-β stimulates other molecular pathways in transformed cells to inhibit cell apoptosis and accelerates cell migration and metastasis. In contrast, a second type 1 receptor (ALK1) is expressed in ECs which stimulates cell proliferation and migration via activating SMADs 1 and 5 genes.

activation of SMAD 1 and 5 proteins in transformed cells inhibits apoptosis and mediates cell proliferation and migration via the activation of other cell signaling pathways [65]. However, in normal cells, the stimulation of SMAD 2, 3, and 4 exhibits cell cycle arrests and apoptosis [66]. Similarly, the SMAD 2, 3, and 4 proteins increase the expression of PAI-1which is essential for vessel maturation in angiogenesis (**Figure 3b**) [66].
