Regulation of tumor angiogenesis by the local environment

Kelly Hall, Sophia Ran

Department of Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine and SimmonsCooper Cancer Institute, Springfield, IL, 62794-9678, USA

TABLE OF CONTENTS

1. Abstract

2. Introduction

3. Mechanisms of tumor blood vessel formation

3.1. Step 1: tipping the angiogenic balance

3.1.1. Role of VEGF-A in induction of tumor angiogenesis

3.1.2. Regulation of VEGF-A expression and activity

3.2. Step 2: destabilization of pre-existing blood vessels

3.2.1. Role of angiopoietins in stabilization and destabilization of blood vessels

3.3. Step 3: blood vessel sprouting

3.3.1. Role of Notch and its ligand Delta-like 4 (Dll4) in vascular sprouting

3.4. Step 4: induction of endothelial cell (EC) migration, proliferation, and survival

3.5. Step 5: stabilization of new vessels

3.5.1. Role of PDGF factors in maturation and stabilization of new vessels

3.5.2. Role of Ang-1in regulation of vessel integrity 4. Abnormalities of tumor vessels and consequences of their malfunction for tumor growth and anti-cancer therapy 5. Anti-angiogenic therapies for the treatment of solid tumors

6. Concluding remarks

7. Acknowledgement

8. References

1. ABSTRACT

Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays an essential role in embryogenesis, inflammation, wound healing, tumor growth and metastasis. The tumor microenvironment contains excessive amounts of pro-angiogenic factors derived from neoplastic, stromal, and infiltrating immune cells. The imbalance of pro-angiogenic and anti-angiogenic factors promotes abnormal angiogenesis, creating numerous blood vessels with structural abnormalities and functional defects. These defective vessels often create an inflammatory environment within the tumor that promotes coagulation, thrombosis, and impairs blood supply, causing further complications to the cancer patient. The structural and functional abnormalities of the tumor vessels promote hematogenous metastasis, which is strongly associated with shorter patient survival. Furthermore, tumor blood vessels are poorly perfused, which impedes drug delivery to the tumor, thus reducing the efficacy of anti-cancer agents. Tumor angiogenesis is widely studied as an important target for suppressing tumor growth and metastasis. This review will briefly summarize the current findings related to regulation of angiogenesis by the tumor microenvironment, while highlighting potential targets for inhibiting this process.

2. INTRODUCTION

Blood vasculature is essential for tissue health serving as a highway for the delivery of oxygen and nutrients. Diffusion of oxygen through the tissues is limited to approximately 200 �m (1), thus cells must be located within this distance from a blood capillary to prevent death from hypoxia and nutrient deprivation. The blood vasculature also transports growth factors, cytokines, and hormones throughout the body. Additionally, blood vessels regulate vascular tone essential for controlling both systemic and local blood flow. Blood vasculature also contributes to regulation of the immune response by controlling access of circulating immune cells to the sites of inflammation and immune stress. Blood vessels control the fibrinolytic status of the normal endothelium permitting coagulation upon vascular injury to prevent excessive blood loss. Wound healing relies heavily on all functions of blood vessels, including control of the coagulation cascade, immune cell trafficking, and angiogenesis.

Angiogenesis is defined as the formation of new blood vessels from pre-existing vessels with the possible aid of endothelial cell progenitors. Normal physiological angiogenesis plays an essential role in embryogenesis, inflammation, wound healing and female reproductive functions. The formation, maturation and regression of new vessels induced by normal physiological needs are regulated by the environmental cues that coordinate blood delivery and tissue expansion or remodeling. In contrast, abnormal angiogenesis induced by tumors that typically contain disproportionately high levels of pro-angiogenic factors creates structurally deficient and functionally impaired blood vessels (2). These incorrectly constructed vessels often create an inflammatory environment within the tumor that promotes coagulation and thrombosis, causing further complications to the patient (3,4). The structural and functional abnormalities of the tumor vessels promote hematogenous metastasis, which is linked to decreased patient survival (5). Importantly, tumor blood vessels are poorly perfused, which impedes drug delivery to the tumor, thus reducing the efficacy of anti-cancer agents. It is, therefore, widely accepted that tumor angiogenesis is an important target for suppressing tumor growth and metastasis. This review will briefly summarize the current concepts of angiogenesis in the context of the tumor microenvironment, while highlighting potential targets for inhibiting this process.

3. MECHANISMS OF TUMOR BLOOD VESSEL FORMATION

The process of angiogenesis can be tentatively separated into five main steps regulated by the microenvironment. In reality, no step is clearly separated from another and some steps might occur simultaneously. For the purpose of this review, we will describe five discrete steps in the tumor-inducing angiogenic cascade that are typically required to form a new vessel. As illustrated in Figure 1, these steps include: 1) induction of angiogenesis by tipping the balance of pro- and anti-angiogenic factors; 2) vessel destabilization; 3) vascular sprouting; 4) induction of endothelial cell migration, proliferation, and survival; and 5) vessel maturation and stabilization.

3.1. Step 1: tipping the angiogenic balance

In adulthood, blood vessels generally remain in a state of quiescence in which they are neither growing nor regressing. Maintenance of the quiescent state requires a balance of pro- and anti-angiogenic factors whose expression is regulated by the local environment of the vessel, endothelium-supporting mural cells and the endothelial cells themselves. However, conditions created by solid tumors turn on an angiogenic switch that tips the balance in favor of angiogenesis, resulting in transformation of quiescent vessels to actively sprouting vessels (6). The angiogenic switch is a complex mechanism involving a wide array of factors. Of these, vascular endothelial growth factor (VEGF-A) has been shown to play a particularly important role (7).

3.1.1. Role of VEGF-A in induction of tumor angiogenesis

The vascular endothelial growth factor (VEGF) family includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). Each of these proteins participates in angiogenesis, lymphangiogenesis, or both to varying degrees. VEGF-A is a homodimeric glycoprotein that is generated through several alternatively spliced isoforms (8). VEGF-A₁₆₅ is the most abundant of the three main isoforms (i.e., VEGF₁₂₁, VEGF₁₆₅, and VEGF₁₈₉) (8). VEGF-A can be produced by a variety of cell types present in the tumor microenvironment, including infiltrating macrophages (9,10,11), mast cells (12), neutrophils (13), platelets (14), stromal fibroblasts (15), endothelial cells (16) and neoplastic cells (17) (Table 1). Thus, both the local environment and the tumor contribute to high intratumoral concentrations of VEGF-A.

Members of the VEGF family bind two types of receptors: 1) VEGF receptors (VEGFR) that contain a tyrosine kinase domain; and 2) neuropilins that lack a cytosolic domain. The tyrosine kinase VEGFR family consists of VEGFR-1, -2, and -3. They are primarily expressed on blood and lymphatic endothelium, but have also been detected on monocytes, a subset of hematopoietic cells, and on neoplastic cells of different tumor types (18,19). VEGF-A binds VEGFR-1 and VEGFR-2, but not VEGFR-3 (8). VEGF-A has a higher affinity to VEGFR-1 but the signaling transduction of VEGFR-2 has increased functional impact on endothelial cells (20). One study concluded that VEGFR-2 is the primary signaling receptor, whereas VEGFR-1 is a decoy receptor (21). However, other studies demonstrated a functional role for both receptors on tumor (22,23) and endothelial cells (24). Expression of functional VEGFR-1 and VEGFR-2 receptors was also shown in several human tumors on both endothelial and malignant epithelial cells (Table 1). Based on these studies, both receptors can contribute to tumor progression through autocrine (25) and paracrine (26) loops.

VEGF ligands also have affinity for members of the Neuropilin (NRP) family, including neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2). Originally, NRP-1 and NRP-2 were identified as neuronal receptors involved in axon guidance (8). Subsequently, both neuropilins were found to be expressed on vascular smooth muscle and endothelial cells, and to participate in angiogenesis (27) and lymphangiogenesis (28). Blood vascular endothelial cells primarily express NRP-1 (27), where lymphatic endothelial cells mainly express NRP-2 (29). NRP-1 binds an exon of VEGF₁₆₅ encoding for a heparin-binding domain, which contrasts the binding site known to interact with its cognate tyrosine kinase receptor, VEGFR-2 (28). NRP-1 presumably plays an ancillary role in VEGFR-dependent activation of endothelial cells by cooperating with tyrosine kinase receptors and enhancing their signaling (28,30). Co-expression of NRP-1 with VEGFR-2 on porcine aortic cells mediated a four-fold increase in VEGF-A binding to VEGFR-2 that subsequent increase in chemotaxis response of these cells for VEGF-A (31). Both NRP-1 (32,33) and NRP-2 (32) also form functional complexes with VEGFR-1 that enhance its activity. In addition, VEGF-A can signal through neuropilins in the absence of VEGFRs (34,35). Neuropilins are abundantly expressed on both endothelial (36,37) and malignant epithelial cells (38,37) in a variety of tumors, and contribute to tumor progression through paracrine and autocrine mechanisms.

3.1.2. Regulation of VEGF-A expression and activity

Expression of VEGF-A is elevated in response to hypoxia and inflammation, conditions characteristic of most epithelial malignancies (39). All solid tumors contain hypoxic pockets primarily caused by the lack of coordination between growth of the tumor mass and generation of new blood vessels to support tumor cell proliferation (8). Tumor hypoxia promotes expression of VEGF-A through stabilization of a transcription factor, hypoxia inducible factor-1 (HIF-1) alpha that binds the VEGF-A promoter and activates its transcription (40,41). Upregulation of VEGF-A by hypoxia has been demonstrated in many human tumors, including breast (42), colorectal (43), brain (42,44) and gallbladder cancers (45) (see Table 1).

Another parameter that plays a significant role in regulation of VEGF-A expression is tumor-associated inflammation. Many inflammatory cytokines that are present in the tumor environment up-regulate VEGF-A expression (Table 2). For instance, IL-1 beta induces VEGF-A expression in gastric carcinoma cells via extracellular signal regulated kinases 1 and 2 (ERK1/2) and p38-dependent pathways (46). IL-6 dependent upregulation of VEGF-A has been shown in non-small cell lung (47) and gastric cancers (48). VEGF-A transcription is potently enhanced by the binding of the transcription factor nuclear factor kappa B (NF-κB) to the VEGF-A promoter (49,50). Increased production of VEGF-A exacerbates tumor inflammation by recruiting macrophages and neutrophils (51,52,53) that secrete additional angiogenic factors and further promote vascular formation (54).

VEGF-A signal transduction activates pathways that mediate survival, proliferation, migration and invasion of endothelial cells, all of which are essential for induction of angiogenesis. VEGF-A binding to VEGFR-1 induces the activation of phospholipase C gamma and the mitogen-activated protein kinase (MAPK) cascade, promoting migration and proliferation of endothelial cells (55,56). VEGFR-2 signaling induces endothelial cell proliferation, survival and changes in gene expression via activation of the MAPK and phosphatidylinositol 3-kinase (PI3K)/AKT pathways (57,58). Additional downstream gene targets of VEGF-A signaling are nitric oxide (NO) (59,60) and angiopoietin-2 (Ang-2) (61), both of which regulate steps of angiogenesis that immediately follow the induction of the angiogenic switch.

3.2. Step 2: destabilization of pre-existing vessels

New vessels sprout and branch from pre-existing vasculature (62). Normally, tight endothelial cell-to-cell interactions and contacts between endothelial and peri-endothelial mural cells keep the vasculature stable (62). These contacts must destabilize to permit sprouting, division and migration of the endothelial cells in order to initiate the formation of new blood vessels (63). Destabilization of blood vasculature decreases the number and intensity of endothelial cell interactions with mural cells, creating leaky and dilated vessels that are primed for the action of VEGF-A (63). Ang-2 is thought to be primarily responsible for this critical event (64,65).

3.2.1. Role of angiopoietins in stabilization and destabilization of blood vessels

The angiopoietin (Ang) family consists of four secreted glycoproteins: Ang-1, Ang-2, Ang-3, and Ang-4 (64,63). Ang-1 and Ang-2 were the first discovered and are the most well-studied members. Ang-1 is secreted by endothelial cells, pericytes and vascular smooth muscle cells, whereas Ang-2 is primarily produced by endothelial cells, stored in Weibel-Palade bodies, and released upon activation (66). While Ang-1 is expressed in many tissues, Ang-2 expression is localized to areas of vascular remodeling (65). These secreted proteins are the ligands for a tyrosine kinase receptor, Tie-2 (63). A structurally related receptor, Tie-1, does not bind known angiopoietins but might facilitate binding and signal transduction mediated by Tie-2▪angiopoietin interactions (63). Tie receptors are expressed on endothelial cells (67), leukemia cells (63,68) and a sub-set of hematopoietic cells (69,27). Tie-2 is also expressed on smooth muscle cells, neuronal cells and stem cells (70,66). In blood vascular endothelial cells, both Ang-1 and Ang-2 bind Tie-2, but only Ang-1 causes Tie-2 phosphorylation under normal conditions. Based on these observations, Ang-1 has been proposed to stimulate Tie-2 signal transduction in blood vascular endothelial cells, whereas Ang-2 is suggested to antagonize activation of the Tie-2 pathway (65).

The signaling of Tie-2 induced by Ang-1 resulting in stabilization of blood endothelium is mediated through the PI3K pathway (71,72) that induces NOS expression and promotes endothelial cell survival (71,73). Endothelial cell motility is also induced by the PI3K pathway through interaction with signaling components, such as growth factor receptor-bound protein 7 (GRB-7) and src homology 2-containing tyrosine phosphatase protein (SHP2), leading to activation of focal adhesion kinase (FAK) (71). Association of the docking protein, Dok-R, with phosphorylated Tie-2 leads to the phosphorylation of Nck/Pak kinases that are involved in cell migration (71). Induction of Tie-2 signaling by Ang-1 binding mainly results in activation of endothelial cells. However, in some contexts, angiopoietin-dependent Tie-2 activation can result in an inhibitory effect through activation of A20-binding inhibitor of NF-kappaB 2 (ABIN2) (72,74).

The destabilizing effect on vasculature is mediated by overexpression of Ang-2 in a variety of human tumors including liver (75,76), gastric (77), renal cell (78) non-small cell lung (79), ovarian (80), prostate (81) and breast (82) cancers (Table 3). In several cancers, overexpression of Ang-2 is associated with increased lymph node metastasis, venous invasion, and high microvascular density (MVD) (83,84). In normal tissues, expression of Ang-2 is restricted to the endothelium (65). In tumors, however, Ang-2 is expressed in both neoplastic epithelial (85,83) and endothelial cells (86). Similarly to VEGF-A, tumor expression of Ang-2 is regulated by hypoxia and inflammatory cytokines (86).

Expression of Ang-2 is regulated by both VEGF-A (61) and fibroblast growth factor-2 (basic FGF or bFGF) (7). Basic FGF has been shown to increase Ang-2 expression up to 2-fold in bovine microvascular endothelial cells (7). The same study reported that hypoxic conditions also promoted Ang-2 expression by 3- to 5-fold (61,87). Hypoxia has been shown to significantly increase Ang-2 (88) and Tie-2 (89) expression in bovine microvascular (88) and human umbilical cord vein endothelial cells (HUVEC) (89). However, it is currently unclear whether hypoxia induces Ang-2 directly, through binding of HIF-1 alpha to the Ang-2 promoter, or indirectly, through upregulation of VEGF-A (41) or bFGF (90,91) that subsequently increases Ang-2 expression.

Ang-2 competes with Ang-1 for the Tie-2 receptor, and can prevent Ang-1-mediated stabilizing effects (92). The destabilization effect of Ang-2 prevents maturation of angiogenic vasculature, which can lead to regression of blood vessels. In a rat glioma model, overexpression of Ang-2 in tumor endothelial cells in the absence of VEGF-A led to vessel regression (93). However, in the presence of both VEGF-A and Ang-2, vasculature growth is renewed (93). The results of this study suggested that in the absence of VEGF-A, Ang-2 destabilizes vasculature leading to vessel regression, but in the presence of VEGF-A, Ang-2 promotes angiogenesis by priming the vessels (93). This showed, for the first time, that Ang-2 plays an important role in vessel dismantling that is mediated, in part, by increased expression of matrix metalloproteinases (MMPs). Another study demonstrated that overexpression of Ang-2 in gastric carcinoma cells up-regulated MMP-1 and MMP-9 expression (94). In human glioma cells, Ang-2 was shown to induce expression of another member of this family, MMP-2 (95). The MMPs promote angiogenesis by degrading the extracellular matrix (ECM) (reviewed in (96)) that allows tumor expansion and dissemination of metastatic cells. Thus, Ang-2 can promote tumor growth through both endothelial-dependent and -independent mechanisms.

3.3. Step 3: blood vessel sprouting

3.3.1. Role of Notch and its ligand Delta-like 4 (Dll4) in regulation of tumor vessel sprouting

The destabilization of the pre-existing capillaries by VEGF-A, Ang-2 and MMPs prepares for endothelial cell sprouting. Tip cells, endothelial cells that guide the developing vessel, develop filopodia-like extensions and sprout from the primary vessel. The quantity and activity of tip cells are regulated by two cell surface proteins, Notch and its ligand, Delta-like 4 (Dll4) (97). Activation of Notch signaling occurs when Notch binds to Dll4 on a neighboring cell, leading to proteolytic cleavage of the Notch intracellular domain followed by downstream signaling (100). An in vitro model of angiogenesis demonstrated that suppression of Notch signaling in HUVEC increased sprouting by 2- to 4-fold (101). Additionally, inhibition of Notch/Dll4 signaling by the inactivation of Dll4 allele, Notch cleavage inhibition, and deletion of Notch all enhance the number of tip cells in the mouse retina (99). Based on these observations, it is hypothesized that Notch signaling induction by Dll4 regulates tip cell numbers and plays a major role in vascular sprouting (99).

Dll4/Notch signaling between tip cells and supporting stalk cells acts as a negative feedback loop limiting endothelial response to VEGF-A. VEGF-A stimulates the activation of tip cells (97) that includes an increase in Dll4 expression (98). Overexpression of Dll4 in HUVEC was shown to increase expression of the angiogenic factors VEGF-A, basic FGF, hepatocyte growth factor (HGF) and VEGFR-1 (102). At the same time, VEGFR-2 and NRP-1 expression decreased and soluble VEGFR-1 increased concomitant with a reduction of proliferation and migratory response to VEGF-A, which was reversed by inhibition of the Notch pathway (98,103).

Tumor angiogenesis is modulated by both increased expression of Dll4 and Notch proteins, and activation of Notch signaling. Upregulation of Dll4 expression has been reported in human bladder and renal cell cancers (98,104). Forced overexpression of Dll4 in murine tumor lines decreased blood vessel density, accompanied by a decrease in tumor size and an increase in hypoxia (105). The blockade of Dll4 in murine lung carcinoma and glioma models initially promoted vascular sprouting and branching, thus increasing vessel density. However, the blockade ultimately increased hypoxia due to incomplete vascular remodeling and poor perfusion, which led to inhibition of tumor growth (106). These studies suggest that the Dll4/Notch signaling pathway is a finely balanced regulatory system designed to control vessel sprouting and maturation. As such, it is a potential target for inhibition of new tumor vasculature.

3.4. Step 4: induction of endothelial cell migration, proliferation and survival

Tip cells lead the migration of endothelial cells, following signals from malignant and other cells within the tumor microenvironment. The main chemotactic signal inducing this step is VEGF-A, which stimulates migration of endothelial cells through binding to VEGFR-1 and activation of PLC gamma and MAPK pathways (55,56). VEGFR-2 can also mediate migration of microvascular endothelial cells via activation of Src homology 2 domain containing adaptor protein B (Shb) and the subsequent activation of the PI3K pathway (107). In addition to recruiting endothelial cells, VEGF-A also attracts hematopoietic stem cells and endothelial progenitor cells (EPC) (108). An increase in endothelial cells at angiogenic regions has two causes: VEGF-A-dependent recruitment of EPC from the blood circulation and emergence of differentiated endothelial cells from pre-existing vessels.

Both Ang-1 and Ang-2 are also implicated in induction of migratory responses in blood vascular endothelial cells. Ang-1 has been shown to induce HUVEC migration, whereas Ang-2 was reported to block this response (109). However, another study showed that Ang-2 stimulates migration of a murine brain capillary endothelial cell line via activation of Tie-2 signaling (110). The question of whether Ang-2 causes phosphorylation of Tie-2 and downstream signaling is still debatable. It appears that a low concentration of Ang-2 does not induce Tie-2 signaling; however, a high dose of Ang-2 (more than 800 ng/ml) was shown to cause Tie-2 phosphorylation in HUVEC (111). A significantly lower concentration of Ang-2 (200 ng/ml) was sufficient for activation of Tie-2 signaling in human cord blood-derived EPCs. This suggests that Ang-2 might contribute to tumor angiogenesis by promoting EPC recruitment (112), in addition to its known role in pre-sensitizing tumor vasculature to VEGF-A's effect.

Migration of endothelial cells can also be induced by HGF and bFGF, both of which are up-regulated in a variety of solid tumors. HGF induces endothelial cell migration through binding and activation of its tyrosine kinase receptor, C-mesenchymal-epithelial transition factor (c-Met), which leads to activation of several pathways (113). c-Met signaling increases cell motility by either direct activation of PI3K or through the indirect mechanism of phosphorylating the effector Gab1 (114). The PI3K pathway is also implicated in chemotaxis of endothelial cells induced by bFGF through activation of fibroblast growth factor receptor 1 (FGFR1) signaling (115). Other prominent stimulators of tumor endothelial cell migration are inflammatory cytokines such as IL-8 and IL-6, which play a role in tumor progression and angiogenesis (116,117).

Some of the factors involved in endothelial cell migration can also signal for proliferation and survival. Preventing apoptosis of migrating endothelial cells is extremely important because, after vessel destabilization, endothelial cells no longer receive pro-survival signals from interactions with the ECM or cell-to-cell contacts with peri-endothelial or adjacent stromal cells (118). Therefore, the survival of migrating endothelial cells intimately depends on the intracellular activation of pro-survival pathways by the tumor's environmental cues. Numerous studies have shown that VEGF-A is one of the most potent pro-survival factors for endothelial cells under adverse conditions (58,119,120). For instance, the survival of serum-starved HUVEC is significantly increased when exogenous VEGF-A activates the pro-survival PI3K/AKT pathway (58). VEGF-A binding to VEGFR-2 also induces proliferation of endothelial cells via activation of PLC gamma pathway (121). Both proliferation and survival traits promoted by VEGF-A signaling are essential for endothelial cells to complete the angiogenic cascade, culminating in creation of new vessels.

Other factors that contribute to endothelial cell survival and proliferation are Ang-1 and bFGF. Ang-1 increases endothelial cell survival by stimulating the AKT pathway and by up-regulating the expression of an apoptotic inhibitor, survivin (71). Blocking or neutralizing Ang-1 diminishes the ability of endothelial cells to survive under stressful conditions (122,123). For instance, Ang-1 has been shown to suppress endothelial cell death induced by the anticancer drug doxorubicin, demonstrating the potential chemoprotective ability of Ang-1 in the tumor microenvironment (124). Basic FGF also protects HUVEC from cell death, as demonstrated by the reduction of apoptosis induced by the cytotoxic HIV protein, gp120 (125). All known endothelial pro-survival factors activate similar intracellular pathways involving ERK1/2, PI3K, and AKT signaling (125).

3.5. Step 5: vessel stabilization

Stabilization of the new vessels is essential for proper vessel function and is promoted by endothelial cell-to-cell contact or by interactions between endothelial cells and pericytes. Two main factors that promote these interactions are platelet-derived growth factor (PDGF) and Ang-1.

3.5.1. Role of PDGF factors in maturation and stabilization of new vessels

The PDGF family consists of four factors: PDGF-A, -B, -C, and -D. These proteins form either homodimers or a heterodimer PDGF-AB factor (126). PDGF receptors (PDGFR) are dimers consisting of either PDGFR-alpha or -beta chains. PDGF ligands have different affinities to the receptor dimers. PDGF-AA binds only PDGFR-alpha homodimers, PDGF-AB can bind PDGFR-alpha/alpha and PDGFR-alpha/beta, whereas PDGF-BB binds all three receptor dimers (i.e. PDGFR-alpha/alpha, -alpha/beta, and -beta/beta). Differential expression of PDGF ligands and receptors creates a complex interplay. For example, PDGF-BB ligand and PDGFR-beta/beta receptor are expressed on endothelial cells and pericytes, respectively (127). PDGF-BB expressed by endothelial cells stabilizes newly-formed blood vessels by recruiting pericytes. This role of PDGF-B has been shown in retention motif-deficient (pdgf-beta^ret/ret) mice that had significantly fewer pericytes overall with remaining pericytes detached from the endothelium (128). Exogenous PDGF-B improved recruitment of pericytes but not the abnormal attachment of the pericytes to the endothelium. The role of PDGFR-beta in pericyte recruitment was also shown in a mouse wound-healing model, where inhibition of this receptor correlated with a reduction in pericytes (127). In an in vivo angiogenic model, PDGF-AB co-expression with bFGF promoted stabilization of newly-formed blood vessels by pericyte recruitment (129). This effect was significantly blocked by inhibition of PDGFR-beta (129).

Several studies showed that expression of PDGF ligands and receptors is regulated by VEGF-A, FGF-2 and other angiogenic factors commonly found in the tumor environment. For instance, VEGF-A was shown to enhance endothelial PDGF-B expression, whereas FGF-2 enhanced expression of PDGF receptor beta (PDGFR beta) on peri-endothelial mural cells (130). FGF-2 increased expression of PDGF receptors on blood vascular endothelial cells (129). Both VEGF-A and bFGF induced migration of mural cells in vitro (130). PDGF expression is up-regulated by hypoxia in a HIF-1 alpha-dependent manner, as demonstrated in studies of glioblastoma cells (131) and human breast cancer (132). The induction of PDGF ligands and receptor expression by mediators of angiogenesis suggests that PDGF plays an important role during formation of new vessels, mainly through recruitment of pericytes and stabilization of the endothelial monolayer.

3.5.2. Role of Ang-1 in regulation of vessel integrity

Ang-1 is another regulator of vessel integrity and a potent inhibitor of vascular permeability (133). Both of these functions stem from the ability of Ang-1 to promote pericyte migration and survival (133,134). This is exemplified by the in vitro wound-healing study in which Ang-1 promoted migration and survival of pericytes stressed by TNF alpha or high glucose concentration (134). Antagonistic roles of Ang-1 and Ang-2 in regulation of blood vasculature were also shown in another study, demonstrating that Ang-1 increases expression of endothelial cell-activating HGF, whereas Ang-2 inhibits this effect (135). Neutralization of HGF blocked Ang-1-induced migration of smooth muscle cells, suggesting that the effects of Ang-1 on perivascular cell recruitment might be mediated, in part, by HGF (135). Based on this study, it has been proposed that Ang-1 can stabilize blood vessels directly, on the endothelium via binding to the Tie-2 receptor, and through indirect mechanisms such as upregulation of HGF.

In the tumor microenvironment, Ang-1 and PDGF are expressed by endothelial, stromal, and tumor-infiltrating immune cells. Overexpression of Ang-1 was detected in brain, breast, and lung tumors (64), whereas high expression of PDGF was found in colorectal (136), pancreatic (137) and small cell lung cancers (138). Although both proteins are thought to stabilize vasculature, tumor vessels are notoriously leaky and characterized by abnormal pericyte coverage. This suggests that, in the tumor microenvironment, destabilizing vascular factors are more prevalent or more potent than Ang-1 and PDGF. Overexpression of Ang-1 and PDGF in tumors not only fails to promote maturation of blood vessels but also contributes to resistance to anti-angiogenic therapy and chemotherapy (139). Nevertheless, the presence of these factors ensures survival and functionality of a sufficient number of vessels to sustain the needs of a continuously expanding tumor mass.

4. ABNORMALITIES OF TUMOR VESSELS AND CONSEQUENCES OF THEIR MALFUNCTION FOR TUMOR GROWTH AND ANTI-CANCER THERAPY

Although tumors typically contain increased blood vessel density compared with normal tissues (140), tumor vessels are characterized by diverse abnormalities induced by the aberrant microenvironment. Tumor blood vasculature is chaotically arranged, disorganized, and has atypical branching patterns that lack a normal distinct vessel hierarchy (141,142). The morphological features of tumor vessels are blurred, which prevents their categorization to a specific vascular type. The following structural abnormalities of tumor vessels cause leakiness and dilation, resulting in irregular blood flow (143). First, a defective monolayer of disorganized, overlapping, and loosely connected endothelial cells contributes to leaking (144). Second, the basement membrane includes redundant layers and extensions that project away from the vessel (2). Third, the plasma membrane of perivascular cells displays cytoplasmic processes extending away from endothelial cells rather than toward blood vessels (5). Collectively, these morphologic abnormalities prevent tight association of endothelial cells with the basement membrane and pericytes, which significantly contributes to hyperpermeability of the tumor vessels (2,5).

Structural abnormalities associated with tumor vessels are attributed, in part, to an imbalance of pro- and anti-angiogenic factors found in the tumor microenvironment. In general, production of pro-angiogenic factors vastly exceeds expression of negative vascular regulators (145). The most prominent angiogenic factors in all solid tumors include VEGF-A (146), Ang-2 (82), basic FGF (136), and TGF beta (147). Notch/Dll4 feedback that controls branching and endothelial tip cell number is also drastically increased in tumors (141). Other angiogenic factors produced in tumors include inflammatory mediators and chemokines that contribute to the lack of directional migration of the growing vessels, leading to heterogeneous and disorganized arrangement of the tumor vasculature.

The prevalence of pro-angiogenic VEGF-A and Ang-2 over anti-angiogenic factors leads to overactive angiogenesis and a reduced number of stabilized and mature blood vessels. The abnormalities in the perivascular cells (5) and basement membrane (2) suggest that the local concentrations of pro-angiogenic factors are overwhelmingly higher than the stabilizing signals. As a result, the newly-formed vessels are unable to mature due to continuous stimulation by promoters of angiogenesis. One example of how pro-angiogenic stimuli may overpower stabilizing factors is in the fine balance between Ang-1 and Ang-2 proteins. Ang-2 is stored in Weibel-Palade bodies within the endothelial cells and, when angiogenic signals are received, it is quickly released in the interstitial space between the endothelial and perivascular cells (66). Ang-2 concentration quickly exceeds Ang-1 levels secreted by the endothelial and perivascular cells. Ang-2 then transiently replaces Ang-1 in the Tie-2 binding site, leading to destabilization of the vasculature. However, unlike Ang-1, Ang-2 does not bind to the extracellular matrix (148) and quickly dissipates, causing Ang-1 local concentration to increase. Once the balance is tilted toward the Ang-1 factor, interactions within the endothelial monolayer are tightened and the vessel becomes less permeable and more stable. This normal interplay maintained by a proper ratio between Ang-1 and Ang-2 is severely skewed in the tumor environment, where both factors are overexpressed (Table 3), thus off-setting the natural balance. Continuous destabilization of the new tumor blood vessels plays a major role in critical events underlying tumor pathology, including vascular hyperpermeability, chronic inflammation and poor tissue perfusion. Additionally, Ang-2 overexpression is likely to promote both hematogenous and lymphatic metastasis because of its potent ability to dismantle vessels, making them accessible to tumor cell penetration.

Structural abnormalities of the tumor vessels result in malfunction of tumor blood vasculature. Insufficient blood perfusion prevents attainment of the necessary amount of oxygen and nutrients to match the continuous expansion of the tumor mass. This leads to perpetual hypoxia and nutrient deprivation within the tumor microenvironment that, in turn, increases the expression of angiogenic factors. Consequently, tumor angiogenesis: a) promotes growth of the primary tumor by increasing the vessel density and blood supply; b) promotes metastasis by increasing the number of easy-to-penetrate vascular channels; and c) generates a chronically inflamed site by creating dilated and leaky vessels that expose numerous adhesion sites for circulating immune cells. Additionally, poor perfusion of the tumor tissue creates a major problem for delivery of anti-cancer drugs to avascular tumor regions. Shielding these regions from cytotoxic drug activity creates reservoirs of tumor cells ready to repopulate necrotic areas accessible to chemotherapy. Thus, the pathology of tumor angiogenesis contributes to tumor progression by two independent mechanisms: functional blood vessels promote tumor expansion, while malfunctioning vessels protect tumor cells from cytotoxic drugs.

5. ANTI-ANGIOGENIC THERAPIES FOR THE TREATMENT OF SOLID TUMORS

Angiogenesis became a major target in cancer treatment because of its important role in tumor growth and metastasis. It was initially hypothesized that preventing or reducing tumor angiogenesis would inhibit tumor growth by decreasing delivery of nutrients and oxygen, which would ultimately lead to tumor death. VEGF-A has become the primary target for such a strategy, as a key mediator of tumor angiogenesis that is almost ubiquitously up-regulated in solid tumors (149). In the past decade, many drugs have been developed that target VEGF-A, including antibodies that neutralize this factor or block its receptors, and small organic compounds that inhibit VEGF-A receptor tyrosine kinase activity. In experimental human cancer xenograft models, the anti-VEGF-A strategy demonstrated a remarkable potency to suppress tumor growth, resulting in as much as a 90% tumor inhibition compared with untreated or saline-treated tumors (150,151). However, this success did not translate in human clinical trials. For instance, in a phase I/II trial of 75 metastatic breast cancer patients, only 17% of patients responded to a humanized anti-VEGF-A monoclonal antibody, bevacizumab, and only four patients remained progression-free for more than one year (152). Many other trials showed no objective responses in the majority of patients and no sustainable benefits for patients with partial response. One of the main differences between experimental tumor models and human patients is the status of angiogenic growth: in animal models, treatment typically begins when the tumors are barely established and angiogenesis is at a vigorous stage, while human patients often have tumors with well-established vasculature that is no longer sensitive to VEGF-A. The attempt to translate anti-VEGF-A therapy as a single treatment into clinics has proven to be largely ineffective, underscoring the extreme complexity of human cancers as compared to animal models. Recently, however, studies that combine anti-VEGF-A therapy and chemotherapy show more promising results. For instance, in a phase III trial with 462 metastatic breast cancer patients, use of bevacizumab in combination with the chemotherapeutic drug capecitabine had a 19.8% response, compared to 9.1% for capecitabine alone (153).

The relative success of combination therapy may lend support to the theory of vessel normalization (154). The inability to form properly functioning vessels within the tumor and the low perfusion of abnormal tumor blood vessels is believed to decrease the delivery of chemotherapeutic drugs and, therefore, decrease the effectiveness of cancer treatment (154). It has been hypothesized that stabilizing tumor vessels by balancing angiogenic factors might improve cancer treatment by normalizing the vessels and increasing perfusion, thus increasing drug delivery (155). Anti-VEGF-A therapy has shown a vessel-normalization effect, both in experimental models (156) and in human clinical trials (157).

Several other angiogenic factors have been proposed as targets for anti-angiogenic therapy, including the Notch/Dll4 pathway, PDGF receptors, and a combination of either target with anti-VEGF-A drugs. In rodent tumor models, blocking Dll4 increased hypoxia and retarded tumor growth (106,158). In mouse tumor models, combination therapy against VEGF-A and PDGFR reduced tumor vessel density, the number of perivascular cells, and tumor growth (159). In clinical trials for renal cell carcinoma, the addition of anti-PDGFR to anti-VEGF-A increased the response from less than 10% to greater than 40% (160). Undoubtedly, insights gained in the rapidly developing angiogenesis field will continue to uncover new targets for attacking tumor vasculature. Additionally, the recent success of combinations of VEGF-A targeting reagents and chemotherapeutic drugs calls for inclusion of anti-angiogenic drugs as a standard modality for anti-cancer treatment.

6. CONCLUDING REMARKS

Tumor angiogenesis is a multi-step process involving interactions among epithelial, stromal, tumor-infiltrating, endothelial, and endothelium-supporting cells. All components of the tumor microenvironment secrete factors that influence vascular formation. Tumors contain excessive amounts of angiogenic factors unbalanced by negative regulators, which disrupts the regulatory mechanisms of physiological angiogenesis. Consequently, tumor vessels develop abnormally, leading to increased tumor growth and metastasis, and resistance to therapy. Studying the similarities and differences of physiological and tumor angiogenesis will aid in a better understanding of tumor biology, and give rise to potential new anti-cancer targets.

7. ACKNOWLEDGMENTS

Sophia Ran is supported by NIH (#1R15CA125682-01) and Illinois William E. McElroy Foundation.

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Key Words: Angiogenesis, Tumor Environment, VEGF-A, Angiopoietins, Anti-Angiogenic Targets, Review