Angiogenesis is a complex process involving extensive interplay between cells, soluble factors and extracellular matrix (ECM) components. The construction of a vascular network requires different sequential steps including:
 

(i) the release of proteases from "activated" endothelial cells (ii) degradation of the basement membrane surrounding the existing vessel (iii) migration of the endothelial cells into the interstitial space (iv) endothelial cell proliferation (v) lumen formation (vi) generation of new basement membrane with the recruitment of pericytes (vii) fusion of the newly formed vessels (viii) initiation of blood flow.

A) Basement membrane breakdown: proteolytic enzymes
 

Interaction between the uPA and MMP systems

To initiate the formation of new capillaries, endothelial cells of the existing blood vessels must degrade the underlying basement membrane and invade into the stroma of the neighbouring tissue (153). These processes of endothelial cell invasion and migration require the cooperative activity of the urokinase-plasminogen activator (uPA)  and the matrix metalloproteinases (MMPs) systems.

The urokinase-type (uPA) and tissue-type (tPA) plasminogen activators are serine proteases which convert plasminogen into plasmin. The fibrinolytic activity in blood is mainly regulated by tPA, whereas the activation of plasminogen in tissues is regulated by uPA (17, 153).

uPA is secreted as an inactive single-chain proenzyme. Secreted pro-uPA binds to the uPA receptor (uPAR) present on many different cell types. Cleavage of pro-uPA by plasmin, Factor XIIa or cathepsin B yields the active enzyme consisting of two disulfide-linked chains (17). The interaction of uPA with its receptor concentrates the enzyme activity to the so-called "focal attachment sites" on the cell surface and stimulates signal transduction through the uPAR, leading to induction of cell migration and invasion (19). Plasmin has broad substrate specificity and degrades several ECM components, including fibrin, fibronectin, laminin and the protein core of proteoglycans (153). In addition, plasmin may activate several matrix metalloproteinases such as MMP-1, MMP-3 and MMP-9 (163).

PA activity is controlled at three levels: (i) the expression of uPA and uPAR is upregulated by angiogenic growth factors (88, 147) and cytokines (245). (ii) Pro-uPA needs to be activated proteolytically and (iii) the activity of plasmin and uPA is regulated by a 2-antiplasmin and plasminogen activator inhibitors (PAIs), respectively (11, 19).

The metalloproteinase family consists of at least 16 members, which are expressed as latent enzymes with a similar domain structure (255). They all contain a pre-domain, which is a signal peptide for secretion, a pro-domain, which is removed when the enzyme is proteolytically activated, a catalytic domain containing a Zinc-ion, and besides matrilysin, a "hemopexin" domain, which contains a binding site for tissue inhibitors of metalloproteinases (TIMPs). One specific class of MMPs, the gelatinases, also contain a "fibronectin" domain, that is inserted in the catalytic domain. MMPs are soluble, secreted enzymes with the exception of the recently discovered membrane-type MMP group (MT-MMPs) that contain a transmembrane domain at the carboxy-terminal end and are located at the cell surface (255). MMPs have been classified according to their domain structure (Table 1) and substrate specificity.

Interstitial collagenases (MMP-1, MMP-8 and MMP-13) are capable of degrading fibrillar collagens, whereas type IV collagenases or gelatinases (MMP-2 and MMP-9) are specific for the degradation of type IV collagen. Stromelysins (MMP-3 and MMP-10 and MMP-11) have a wider range of substrate specificity, including proteoglycans, laminin, fibronectin and other MMPs like MMP-1 and MMP-9. Other members of this group include matrilysin (MMP-7) and metalloelastase (MMP-12). The fourth group contains the membrane-associated MMPs MT-MMPs (MT1-MMP, MT2-MMP, MT3-MMP and MT4-MMP), which activate proMMP-2. Recently, some new MMPs (MMP-19 and MMP-20) have been identified, but little is known about their substrate specificity and functional role (255).

Like PAs, MMP gene expression is induced by cytokines and the angiogenic factors basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) (22, 244, 249), whereas the enzymatic activity is regulated by proteolysis of pro-MMPs (163) and by TIMPs (20). PAs and MMPs are secreted together with their inhibitors, ensuring a stringent control of local proteolytic activity, in order to preserve normal tissue structure.

B) Endothelial cell migration and proliferation: angiogenic factors
 

Following proteolytic degradation of the ECM, "leader" endothelial cells start to migrate through the degraded matrix. They are followed by proliferating endothelial cells, which are stimulated by a variety of growth factors, some of which are released from the degraded ECM. Other ECM products, such as peptide fragments of fibrin or hyaluronic acid (221) also stimulate the angiogenic process. Therefore, a local collapse of the ECM results in an increased extracellular concentration of soluble mediators of endothelial cell migration and proliferation.

A variety of angiogenesis inducers have been described (Table 2), which can be divided in three classes (129). The first class consists of the VEGF family and the angiopoietins, which specifically act on endothelial cells. The second class contains most direct-acting molecules, including several cytokines, chemokines (157) and angiogenic enzymes (30, 39), which activate a broad range of target cells besides endothelial cells. The prototype member of this group, FGF-2 was one of the first angiogenic peptides to be characterized. The third group of angiogenic molecules includes the indirect-acting factors, whose effect on angiogenesis results from the release of direct-acting factors from macrophages, endothelial or tumor cells. The most extensively studied are tumor necrosis factor-a (TNF-a) and transforming growth factor-b (TGF-b), which inhibit endothelial cell proliferation in vitro. In vivo, TGF-b induces angiogenesis and stimulates the expression of TNF-a, FGF-2, platelet derived growth factor (PDGF) and VEGF by attracted inflammatory cells (63, 185). TNF-a has been shown to increase the expression of VEGF and its receptors, interleukin-8 (IL-8) and FGF-2 by endothelial cells, thus explaining its angiogenic properties in vivo (87, 261).
 

Only the characteristics of the most prominent angiogenic factors, such as VEGF and FGF-2, and the recently described angiopoietins will be addressed here.

Vascular endothelial growth factor belongs to the VEGF family which currently consists of six members: VEGF-A (or VEGF), placenta growth factor (PlGF), VEGF-B, VEGF-C, VEGF-D and VEGF-E (from orf virus) (246).

VEGF is expressed in different tissues, including brain, kidney, liver and spleen, and by many cell types (246). In vitro, VEGF stimulates ECM degradation, proliferation, migration and tube formation of endothelial cells and induces in these cells the expression of uPA, PAI-1, uPAR and MMP-1 (148, 182, 183, 243). In vivo, VEGF has been shown to regulate vascular permeability, which is considered important for the initiation of angiogenesis (57). The finding that the loss of only a single VEGF allele leads to embryonic lethality implies that this factor plays an irreplaceable role in the development of the vascular system (68).

Transcription of VEGF mRNA is induced by a variety of growth factors and cytokines, including PDGF, epidermal growth factor (EGF), TNF-a, TGF-b and interleukin 1b (IL-1b) (2, 59, 246). VEGF may thus function as a mediator for indirect-acting angiogenic factors such as TGF-b. VEGF levels are also regulated by tissue oxygen tension. Exposure to hypoxia induces VEGF expression rapidly and reversibly, through both increased transcription and stabilization of the mRNA (108, 162). Hypoxic upregulation of VEGF thus provides a compensatory mechanism by which tissues (or tumors) can increase their oxygenation through induction of blood vessel growth. In contrast, normoxia downregulates VEGF production and even causes regression of some newly formed blood vessels. By these opposing processes, the vasculature exactly meets the metabolic demands of the tissue (197).

Alternative exon splicing of the VEGF gene results in different VEGF isoforms containing 121, 145, 165, 189 or 206 amino acid residues, VEGF165 being the predominant form. While VEGF121 does not bind heparin and is freely diffusible, the larger isoforms contain increasingly basic and heparin-binding residues and are bound to the cell surface or sequestered in the ECM, from where they can be released by plasmin cleavage (67).

Two high affinity tyrosine-kinase receptors for VEGF have been identified on vascular endothelium: VEGFR-1 (Flt-1) and VEGFR-2 (Flk-1). Similarly to VEGF, regulation of VEGF receptor gene expression is regulated by hypoxia (248). An additional member of this family VEGFR-3 (Flt-4) is not a receptor for VEGF, but binds VEGF-C and VEGF-D (246) (Fig. 3).

During embryogenesis, expression of VEGFR-1 and VEGFR-2 is initiated at the time of blood island formation. Homozygous mutants inactivating VEGFR-1 or VEGFR-2 are lethal, implying that both receptors are essential for normal development of the embryonic vasculature. Embryos from VEGFR-2 knockout mice were found to lack vasculogenesis and failed to develop blood islands (216). Thus, VEGFR-2 is crucial for differentiation and proliferation of endothelial and hematopoetic cells. Inactivation of the VEGFR-1 gene does not prevent the development of endothelial cells, but impairs their organization in normal vascular channels, due to an increase in the number of endothelial progenitors (79). In adult tissues, VEGFR-1 and VEGFR-2 are predominantly found on vascular endothelial cells, whereas VEGFR-3 is localized mainly on the lymphatic endothelium. Ligand binding induces receptor dimerization and subsequent auto/transphosphorylation. Several studies have indicated that VEGFR-1 and VEGFR-2 have different signal transduction properties (67): interaction of VEGF with VEGFR-2 is critical to induce VEGF-induced biological responses, whereas the function of VEGFR-1 in VEGF-mediated angiogenesis is still unclear.

Recently, neuropilin-1 (NP-1), a cell surface glycoprotein that binds semaphorin/collapsins, mediators of neuronal guidance, has been identified as VEGF165 receptor. NP-1 is expressed in endothelial cells and enhances the mitogenic effects of Flk-1 upon VEGF165stimulation (222).

Besides its function during embryogenesis, VEGF also plays a crucial role in angiogenesis in the adult. VEGF was detected in the ovary during corpus luteum formation (69) and in the uterus during growth of endometrial vessels and at the site of embryo implantation. Also, high VEGF levels were detected during the proliferative phase of wound healing (170). VEGF is equally detectable in areas where endothelial cells are quiescent, such as heart, lung and brain, pointing to the role of VEGF as a endothelial cell-survival factor. Finally, VEGF is thought to play a role in several human cancers, diabetic retinopathy, rheumatoid arthritis and atherosclerosis (67, 114).

Two other endothelial cell-specific receptors, called Tie-1 and Tie-2 (for "tyrosine kinase with immunoglobulin- and EGF-like domains") have been identified several years ago. Knock-out experiments in mice have suggested a role for these receptors in blood vessel maturation. The ligands for Tie-2 have only recently been discovered: angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) both bind Tie-2, but only the binding of Ang-1 results in signal transduction and regulation of blood vessel maturation (227). Therefore, Ang-2 is a natural antagonist of Ang-1 (146).

Recently, a model for the complementary roles of VEGF and angiopoietins in vascular development and angiogenesis has been proposed (104). During embryogenesis, VEGF promotes differentiation and proliferation of endothelial cells and the formation of immature vessels. Ang-1, acting through the Tie-2 receptor, induces blood vessel remodeling and stabilization. In a normal adult vessel, Ang-1 is associated with Tie-2 to keep the vessels in a stable state. Upregulation of Ang-2, by hypoxia or VEGF (171), e.g. in the ovary during corpus luteum formation or by tumor cells, disrupts the interaction between Ang-1 and Tie-2 resulting in destabilization of the vessels. Endothelial cells, which are no longer attached to the perivascular cells and the ECM become responsive to angiogenic signals and, in the presence of VEGF, angiogenesis is promoted. The absence of stimulatory signals will cause regression of the vessels (104).

So far, the FGF family consists of at least 18 members, which share 55% sequence identity at the amino acid level. All FGFs are 18-30 kDa proteins with high affinity for heparin. The prototype member FGF-2 was one of the first angiogenic factors to be characterized and has been extensively studied (188). The single-copy human FGF-2 gene encodes multiple FGF-2 isoforms with molecular weight (MW) ranging from 18,000 to 24,000 (72). The high MW isoforms are colinear NH2-terminal extensions of the better characterized MW 18,000 protein. Both low and high MW FGF-2 isoforms show angiogenic activity in vivo and induce cell proliferation, chemotaxis, and uPA production in cultured endothelial cells (188). Also, FGF-2 was found to induce tube formation in collagen gels and to modulate integrin expression, gap junction intercellular communication and VEGF, Flk-1 and uPAR upregulation in vitro (31, 100).

FGF-2 is expressed at low levels in almost all organs and tissues examined, with high concentrations reached in the brain and pituitary. It is found in many cultured cell types, including fibroblasts, endothelial, smooth muscle and glial cells. Although FGF-2 lacks a leader sequence for secretion, data suggest that FGF-2 is secreted from FGF-2-producing cells by an alternative secretion pathway (73) and accumulates in the ECM, from where it can be released by ECM-degrading enzymes.

Two types of receptors have been identified for FGF-2: high affinity tyrosine-kinase FGF receptors (FGFRs) (123) and low affinity heparan sulfate proteoglycans (HSPGs). Thus far, at least four members of FGFRs have been identified. They represent low number (approx. 10,000 per cell), but high affinity sites (Kd of 20-600 pM). Low affinity binding sites (Kd of 2-20 nM) with high capacity (approx. 1 million sites per cell) were identified as proteoglycans, including syndecan and perlecan, containing heparan sulfate side chains (202). These HSPGs are found in the ECM, the basement membrane and the cell surface. It has been suggested that binding of FGF-2 to HSPGs result in protection of FGF-2 from inactivation in the extracellular environment and in storage of FGF-2 in the ECM and basement membrane. Stored FGF-2 can be released by heparitinase and soluble heparin or after ECM breakdown (202).

A dual receptor model has been proposed for FGF-2 in which interaction of the growth factor with non-signaling HSPGs is required for its binding to the FGFR. Heparin would induce oligomerization of FGF-2, which might be important for receptor dimerization and activation. FGFR activation will then trigger an intracellular signal cascade leading to multiple biological responses, including endothelial cell proliferation and migration, differentiation, protease production, and angiogenesis (211).

FGF-2-deficient mice develop normally without any evident phenotype, i.e. organogenesis, animal growth, life span and female reproductive cycle are unaffected by the absence of FGF-2 (177). Nevertheless, different reports have implicated FGF-2 in both physiological and pathological angiogenesis. Mice lacking FGF-2 showed neuronal defects and delayed wound healing (177). Furthermore, FGF-2 is produced by many tumor cell lines in vitro and is thought to play a role in the growth and neovascularization of solid tumors (23). High levels of FGF-2 are present in endothelial cells of Kaposi’s sarcoma (205) and in proliferating hemangiomas (38), and elevated amounts of FGF-2 have been detected in the serum and urine (166) of patients with advanced colorectal, breast, ovarian and renal carcinomas (53) and soft tissue sarcoma (92).

C) Cell-cell and cell-matrix interactions: adhesion molecules

The processes of cell invasion, migration and proliferation do not only depend on angiogenic enzymes, growth factors and their receptors, but are also mediated by cell adhesion molecules (18). To initiate the angiogenic process, endothelial cells have to dissociate from neighbouring cells before they can invade the underlying tissue. During invasion and migration, the interaction of the endothelial cells with the ECM is mediated by integrins. Also, the final phases of the angiogenic process, including the construction of capillary loops and the determination of the polarity of the endothelial cells, which is required for lumen formation, involve cell-cell contact and cell-ECM interactions (18).

Cell adhesion molecules can be classified into four families depending on their biochemical and structural characteristics. These families include the selectins, the immunoglobulin supergene family, the cadherins and the integrins. Members of each family are implicated in neovascularization (18).

Integrins are a group of cell adhesion receptors, consisting of non-covalently associated a and b subunits, which can heterodimerize in more than 20 combinations. Endothelial cells thus express several distinct integrins, allowing attachment to a wide variety of ECM proteins (58). Integrin avb3 was found to be particularly important during angiogenesis. avb3 is a receptor for a number of proteins with an exposed Arg-Gly-Asp (RGD) sequence, including fibronectin, vitronectin, laminin, von Willebrand factor (vWF), fibrinogen and denatured collagen. In addition, avb3 has been shown to bind MMP-2, in an RGD-independent way, thereby localizing MMP-2-mediated matrix degradation to the endothelial cell surface (28, 58). avb3 is nearly undetectable on quiescent endothelium, but is highly upregulated during cytokine or tumor-induced angiogenesis. In activated endothelium avb3 suppresses the activity of both p53 and the p53-inducible cell-cycle inhibitor p21^WAF1/CIP1, while increasing the Bcl2:Bax ratio, resulting in an anti-apoptotic effect (226). Consequently, avb3 was found to promote melanoma growth by regulating tumor cell survival (184). Another receptor, which has recently been implicated in angiogenesis is integrin avb5. Antibodies directed against avb3  were found to specifically block FGF-2 or TNF-a -induced angiogenesis, whereas antagonists of avb5 blocked VEGF-induced angiogenesis (82). This implies that specific cytokines may stimulate angiogenesis by distinct signaling pathways that may be mediated by specific integrins.

Besides integrins, a number of other cell adhesion molecules are involved in angiogenesis. Vascular endothelial cadherin or VE-cadherin mediates calcium dependent homophilic interactions between endothelial cells. Recently, knockout studies in mice demonstrated that a deficiency or truncation of VE-cadherin induces endothelial apoptosis and inhibits transmission of the endothelial survival signal by VEGF, leading to lethality at 9.5 days of gestation (36). Members of the immunoglobulin superfamily mediate heterophilic cell-cell adhesion. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) are expressed on quiescent endothelium, but are upregulated after stimulation with TNF-a, IL-1 or interferon-g (IFN-g) (24). Furthermore, VCAM-1 can induce chemotaxis in endothelial cells in vitro and angiogenesis in vivo (134). Also members of the selectin family, in particular P-selectin and E-selectin, which promotes adhesion of leukocytes to cytokine-activated vascular endothelium, have been shown to play a role in angiogenesis (134). E–selectin was found to induce endothelial migration and tube formation in vitro and angiogenesis in vivo (167). However, little is known about the mechanism of action of these molecules and mice deficient in both E-selectin and P-selectin are viable and fertile (139).

D) Capillary formation and vessel maturation

After proteolytic degradation of the basement membrane and endothelial cell migration, the newly-forming capillaries synthesize a new basement membrane. During this process extracellular proteolysis must be locally inhibited to permit the deposition and assembly of ECM components. Once a capillary sprout is formed, degradation of the newly formed ECM again occurs at the tip of the sprout, which then allows further invasion. Thus, capillary formation results from alternate cycles of activation and inhibition of extracellular proteolysis. The endothelial cells also form branches, which connect with other branches to form capillary loops. In order to form a lumen, the polarity of the endothelial cells, luminal versus abluminal, has to be established by cell adhesion molecules. Further stabilization of the new capillaries requires the recruitment of pericytes and smooth muscle cells, which is regulated by PDGF. Finally, when sufficient neovascularization has occurred, angiogenic factors are downregulated or the local concentration of inhibitors increases. As a result, the endothelial cells become quiescent and the vessels remain or regress if no longer needed. Thus, angiogenesis requires many interactions that must be tightly regulated in a spatially and temporally manner.