Endothelial Cells and the Initiation of Angiogenesis

Angiogenesis may be better regarded as a cascade rather than a linear process, with any of a variety of factors able to push the vessel through any single stage of its growth. Divergent consequences may result from stimulation by any one factor. Effects on blood vessel growth depend on what other angiogenic or antiangiogenic factors are available, on the precise expression of receptors and second messenger systems by the vessel itself, and on whether its matrix environment is either permissive or hostile to vascular invasion.

The initial stages of angiogenesis conveniently can be investigated using cultured endothelial cells, although differences are apparent between endo-thelial cells derived from different sources. The expression of receptors for angiogenic factors may differ between arterioles, capillaries, and venules such that angiogenic activity may be observed in some endothelial cell types but not in others [18]. Furthermore, culture conditions may themselves change endothelial cell gene expression, enhancing angiogenic responses where the resting endothelium may show none. Indeed, demonstrating angiogenic activity on endothelial cells in vitro is only one step in determining whether a factor may and does stimulate vessel growth in vivo.

4.1. VEGF as a Prototypical Direct-Acting Angiogenic Factor

The upregulation of VEGF by resident fibroblasts and its expression by migrating macrophages, and the upregulation of VEGF and its receptors by endothelial cells in the growing vessel, each indicate the importance of this factor in maintaining pathological vascular growth. This view is now strongly supported by interventional studies in both man and animals, which points to an almost ubiquitous role for VEGF in both physiological and pathological angiogenesis [19].

VEGF is sufficient to induce angiogenesis in a variety of animal models, although it is unlikely that it ever does so alone in physiological or pathological conditions. The complexity of cellular events that follow exposure of isolated endothelial cells to VEGF illustrates the processes that can initiate angiogenesis (Fig. 1), many of which are shared by other angiogenic factors. The interested reader is directed to other reviews of the molecular biology of VEGF for more detail [19, 20]. This section attempts to illustrate how the initial responses of endothelial cells to angiogenic factors indicate diverse molecular targets through which this process could be pharmacologically manipulated.

4.2. VEGF and Its Receptors

VEGF-A is a member of a family of growth factors that also includes VEGF-B through F, placental growth factor, and, more distantly, platelet-derived growth factor. VEGF-A exists as at least seven different homodi-meric isoforms that arise from alternative splicing of mRNA from a single gene. VEGF-A121, VEGF-A145, and VEGF-A165 have clearly demonstrated biological effects on blood vessel endothelial cells.

VEGF acts through receptors on the cell surface of which three with diverse functions and activities have been most clearly defined on blood vessel endothelial cells. VEGFR1 (vascular endothelial growth factor receptor-1) (Flt-1) and VEGFR2 (KDR/Flk-1) are receptor tyrosine kinases related to the platelet-derived growth factor family of receptors, whereas neuropilin-1 lacks a cytoplasmic kinase domain and may function to facilitate VEGF interaction with other VEGFRs. Angiogenesis can be inhibited either by inactivating VEGF (e.g., using anti-VEGF antibodies) or by blocking its receptor tyrosine kinases. Both these approaches have led to encouraging clinical trials in man, particularly in renal tumors where mutations in the von Hippel-Lindau tumor suppressor gene lead to overproduction of VEGF [21, 22].

VEGFR2 appears to mediate most of the biological activities of VEGF on endothelial cells. VEGFR1 may contribute to the angiogenic activity of VEGF by enhancing the production of proteases and growth factors.

Fig. 1. Angiogenic signaling by vascular endothelial growth factor. VEGFR activation follows binding of VEGF and is facilitated by neuropilin and av/33-integrin. Tyrosine kinase activity of active receptors phosphorylates at several sites, activating PKC/Erk, PI3K/Akt/PKB, and FAK/ paxillin pathways. These in turn stimulate endothelial cell proliferation, survival, and migration. In addition, the production of both polypeptide and low molecular weight growth factors cascades the angiogenic response. VEGFRs may be cleaved by 7-secretase to yield inhibitory, soluble forms. For more detailed explanations, see text.

Activated VEGFR1 alone, however, has relatively weak tyrosine kinase activity and may be insufficient to stimulate endothelial cell proliferation [20]. On the other hand, inhibition of angiogenesis by factors such as pigment epithelium-derived factor (PEDF) is associated with reduced VEGF-induced phosphorylation of VEGFR1 [23].

VEGFR1 is also produced in a soluble form, overexpression of which inhibits VEGF effects on endothelial cells, suggesting that VEGFR1 may function primarily as a negative regulator of VEGF activity at VEGFR2 [24]. PEDF increases endothelial cell 7-secretase activity, increases cleavage and intracellular translocation of the transmembrane domain of VEGFR1, and inhibits angiogenesis [23]. The precise balance between pro- and antiangio-genic actions of VEGFR1 may depend on pathophysiological context and the balance between the production of membrane-bound and soluble forms.

In mature tissues, VEGFR3 is primarily localized to lymphatic endothelial cells, where binding of VEGF-C stimulates lymphangiogenesis [25]. Some inflamed tissues, such as from arthritic joints, display VEGFR3 colocalized with other blood vessel markers on endothelial cells, although a role in the accelerated blood vessel angiogenesis in this tissue has not been clearly demonstrated [26].

4.3. VEGF Signal Transduction During Angiogenesis

Activation of VEGFR2 results in several parallel intracellular signaling events. A protein kinase C (PKC)/extracellular receptor kinase (Erk) pathway appears to predominantly mediate proliferative actions of VEGF on endothelial cells. A separate phosphatidylinositol 3'-kinase (PI3K)/protein kinase B (Akt/PKB) pathway predominantly enhances endothelial cell survival. Several pathways may mediate endothelial cell migration, involving PIP3, the p38 mitogen-activated protein kinase (MAPK), and focal adhesion kinase.

4.3.1. The PKC/Erk Pathway and Endothelial Cell Proliferation

Receptor tyrosine kinase activation leads to autophosphorylation of specific tyrosine residues in the intracellular domain of VEGFR2. A proliferative signaling cascade is initiated, inter alia, by autophosphorylation of Tyr1175, creating a binding site for phospholipase C-71 which, in turn, activates PKC [27]. Erk translocates to the nucleus where it phosphorylates transcription factors such as c-Jun, inducing transcription of c-fos and leading to cell proliferation. PKC-dependent activation of Erk leads to downstream activation of phospholipase A2 and consequent production of prosta-cyclin by endothelial cells, and also induces cyclooxygenase-2 (COX-2) expression [28, 29]. Decreasing COX-2 expression or the administration of

COX-2-specific inhibitors can decrease VEGF-induced endothelial cell proliferation, indicating that COX-2 mediates some of VEGF's angiogenic actions.

The Erk pathway may be subject to negative feedback through increased production of protein tyrosine phosphatase (SH-PTP1). Anti-SH-PTP1 antibodies increase VEGF-induced endothelial cell proliferation, suggesting that upregulation of SH-PTP1 damps down the angiogenic effects of VEGF [28].

4.3.2. The PI3K/Akt/PKB Pathway and Endothelial Cell Survival

Survival signaling pathways include VEGFR2-dependent PI3K activation, leading to accumulation of phosphatidylinositol (3,4,5)P3 (PIP3) and activation of Akt/PKB. Akt/PKB enhances endothelial cell survival by inhibiting caspase activity, in part through survivin; by upregulating the antiapoptotic proteins A1 and Bcl-2; and by inhibiting B-cell lymphoma 2 (Bcl-2)-associated death promoter homologue. Endothelial cell survival may also be facilitated by Erk and MAPK pathways, for example, through downregulation of Rho-kinase [30].

4.3.3. Pathways Mediating Endothelial Cell Migration

Endothelial cell migration is mediated by two additional pathways that stimulate cytoskeletal reorganization. These involve the p38 MAPK and focal adhesion kinase with paxillin phosphorylation, respectively [31]. PIP3 also activates the small GTP-binding protein Rac which facilitates endothelial cell migration. The PI3K pathway may also contribute to VEGF-enhanced endothelial cell proliferation [32].

4.3.4. Other Modulators and Mediators of VEGF-Enhanced Angiogenesis

Increased nitric oxide generation mediates some angiogenic effects of VEGF, including endothelial cell proliferation and migration. Endothelial nitric oxide synthase (eNOS) activation results from several events downstream of VEGFR2 activation. Increased intracellular Ca2+ mobilization results in rapid nitric oxide production. Akt/PKB-dependent phosphory-lation of eNOS in response to VEGF also rapidly increases eNOS activity by removing the need for Ca2+ to stimulate nitric oxide generation [33]. In addition, VEGFR2 stimulation enhances eNOS expression via PKC/Erk, resulting in more sustained endothelial activation [34].

VEGF enhancement of VEGFR2 activity may be facilitated by the association of VEGFR2 with other endothelial cell surface molecules such as avß3-integrin and neuropilin-1 [19, 35]. Integrin facilitation of VEGFR2 activation depends on its association with ligands such as vitronectin in the pericellular matrix. Coexpression of neuropilin-1 with VEGFR2 more than doubles the

VEGFR2 binding to VEGF-A165 and endothelial cell migration. VEGFR1 and VEGFR2 activities may also be enhanced by their upregulated expression in neovascular endothelial cells [19].

4.4. Autocrine Production of Angiogenic Factors by Endothelial Cells

VEGF stimulation of endothelial cells also induces their autocrine production of a cascade of other growth factors, including platelet-activating factor [36]. Increased platelet-activating factor expression stimulates endothelial cell migration and further promotes the expression of fibroblast growth factor (FGF)-1, FGF-2, and macrophage inflammatory protein 2 [19].

Autocrine expression of VEGF by endothelial cells has also been demonstrated in response, for example, to hypoxia, growth factors, and oxidative stress [37, 38]. Induced endothelial cell expression of VEGF itself may be dependent on a number of second messenger systems, and can be inhibited by blocking MAPK, PI3K, and PKC [37].

other angiogenic factors may also induce the production of autocrine growth factors by endothelial cells, including FGF-2, ccL2 and ccL3 chemokines, and connective tissue growth factor (CTGF/CCN2) [39-42]. induction of FGF-2 expression by endothelial cells has been found to be necessary for the angiogenic activity of a number of angiogenic molecules such as substance P and eicosanoids [40, 43]. For example, blocking antibodies to FGF-2 will inhibit the apparently direct, neurokinin-1 (NK1) receptor-mediated stimulation of endothelial cell proliferation by substance P.

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