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Laboratory of Experimental Oncology, University Hospital Gasthuisberg, Catholic University of Leuven, Leuven, Belgium (A.H., B.L., A.T.V.O., E.A.D.B.); and Department of Cancer Medicine, Ninewells Hospital, Dundee, United Kingdom (M.S.H.)
Abstract
Abstract I. Introduction II. Vascular Endothelial Growth Factor A. The Locations and Structures of Vascular Endothelial Growth Factor B. Vascular Endothelial Growth Factor Receptors C. Signal Transduction D. Regulation of Gene Expression E. Physiological Regulation of Vascular Endothelial Growth Factor in Normal Adults III. Vascular Endothelial Growth Factor in Tumor Growth A. Vascular Endothelial Growth Factor and Tumorigenesis B. Lymphangiogenesis and Tumor Growth C. The Role of Vascular Endothelial Growth Factor in the Impaired Host Antitumor Immune Response D. Vascular Endothelial Growth Factor as a Prognostic Factor or an Indicator of Angiogenesis E. Potential Therapeutic Applications Related to Vascular Endothelial Growth Factor IV. Vascular Endothelial Growth Factor in Nonmalignant Disease A. Rheumatoid Arthritis and Osteoarthritis B. Cardiovascular Ischemia C. Peripheral Vascular Disease D. Diabetes Mellitus E. Endometriosis, Preeclampsia, and Ovarian Hyperstimulation Syndrome F. Psoriatic Skin Disease V. Conclusions and Future Directions
Angiogenesis is a hallmark of wound healing, the menstrual cycle, cancer, and various ischemic and inflammatory diseases. A rich variety of pro- and antiangiogenic molecules have already been discovered. Vascular endothelial growth factor (VEGF) is an interesting inducer of angiogenesis and lymphangiogenesis, because it is a highly specific mitogen for endothelial cells. Signal transduction involves binding to tyrosine kinase receptors and results in endothelial cell proliferation, migration, and new vessel formation. In this article, the role of VEGF in physiological and pathological processes is reviewed. We also discuss how modulation of VEGF expression creates new therapeutic possibilities and describe recent developments in this field.
Humans are complex multicellular organisms, and all cells require a dependable, finely controlled supply of oxygen. The diffusion of oxygen through tissues is limited to 100 to 200 µm; therefore, a highly developed vascular system has evolved to ensure that all cells are within this distance of a supply of oxygen. The system needs to be maintained through angiogenesis, the process of new blood vessel development from pre-existing vasculature. This involves endothelial cell division, selective degradation of the basement membrane and the surrounding extracellular matrix, endothelial cell migration, and the formation of a tubular structure. Once blood vessels have been established, the endothelial cells undergo tissue-specific changes to generate functionally distinct vessels. During embryogenesis, blood vessels form by the differentiation of endothelial cell precursors (angioblasts), which associate to form primitive vessels. This process is called vasculogenesis.
Angiogenesis is subject to a complex control system with proangiogenic and antiangiogenic factors. In adults, angiogenesis is tightly controlled by this "angiogenic balance", i.e., a physiological balance between the stimulatory and inhibitory signals for blood vessel growth. In normal circumstances, the formation of new blood vessels occurs during wound healing, organ regeneration, and in the female reproductive system during ovulation, menstruation, and the formation of the placenta. It is also an important factor in several pathological processes such as tumor growth, rheumatoid arthritis, diabetic retinopathy, and psoriasis. A switch to the angiogenic phenotype depends on a local change in the balance between angiogenic stimulators and inhibitors.
One of the most important proangiogenic factors is vascular endothelial growth factor (VEGF1). VEGF also potentiates microvascular hyperpermeability, which can both precede and accompany angiogenesis. In this review, we summarize the properties and functions of VEGF and its place in the process of angiogenesis in malignancy and other conditions.
II. Vascular Endothelial Growth Factor
A. The Locations and Structures of Vascular Endothelial Growth Factor
The VEGF family currently comprises seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. All members have a common VEGF homology domain. This core region is composed of a cystine knot motif, with eight invariant cysteine residues involved in inter- and intramolecular disulfide bonds at one end of a conserved central four-stranded 
-sheet within each monomer, which dimerize in an antiparallel, side-by-side orientation (Neufeld et al., 1999
; Ortega et al., 1999). Figure 1 represents the three-dimensional structure of VEGF.
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VEGF-A is a 34- to 42-kDa, dimeric, disulfide-bound glycoprotein. In normal tissues, the highest levels of VEGF-A mRNA are found in adult lung, kidney, heart, and adrenal gland. Lower, but still readily detectable, quantities of VEGF-A transcript levels occur in liver, spleen, and gastric mucosa. VEGF-A exists in at least seven homodimeric isoforms. The monomers consist of 121, 145, 148, 165, 183, 189, or 206 amino acids (Fig. 2). The primary VEGF-A transcript derives from a single VEGF-A gene, coding for eight exons (Poltorak et al., 1997; Neufeld et al., 1999). The amino acids encoded by exons 1 to 5 and 8 are conserved in all isoforms except VEGF-A148, whereas variable alternative splicing occurs in exons 6 and 7, which encode two distinct heparin-binding domains. The presence or absence of these domains influences solubility and receptor binding. The heparin-binding domain encoded by exon 6 determines binding to the extracellular matrix, and therefore isoforms containing this domain (VEGF-A145, VEGF-A189, and VEGF-A206) are bound tightly to cell surface heparin-containing proteoglycans in the extracellular matrix, whereas those lacking the domain are diffusible. VEGF-A165, which contains only one heparin-binding region encoded by exon 7, is moderately diffusible, and VEGF-A121, which lacks the domains encoded by both exons 6 and 7, is highly diffusible (Ortega et al., 1999).
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Plasmin is involved in the degradation of the extracellular matrix, both directly through digestion of components of the basement membrane and indirectly by activating collagenases from zymogens, and it liberates VEGF-A165, VEGF-A189, and VEGF-A206. Cleavage of VEGF-A165 and VEGF-A189 by plasmin results in fragments containing the 110-amino terminal amino acids (VEGF-A110) that are highly diffusible (Houck et al., 1992). Plasmin is generated from plasminogen by plasminogen activators, and it has been postulated that endothelial cells secrete tissue and urokinase type plasminogen activators (tPA and uPA) in response to VEGF and basic fibroblast growth factor (bFGF) (Houck et al., 1992; Cohen et al., 1995; Neufeld et al., 1999). VEGF-A189 is also cleaved directly by uPA into 38-kDa homodimers, independently from the activation of plasmin, probably within the sequence encoded by exon 6 (Plouet et al., 1997). These homodimers have a weaker ability to circulate, in comparison to the 34-kDa homodimers generated by plasmin, but they have a higher mitogenic activity. It is postulated that VEGF-A206 can undergo a similar maturation process following exposure to uPA. In contrast, VEGF-A165 is not cleaved by uPA.
VEGF-B was discovered in 1995. It is abundantly expressed in the adult myocardium, skeletal muscle, and pancreas. In mouse embryonal tissues, high expression is seen in the developing heart, brown fat, muscle (including the smooth muscle layer in embryonic arteries), and in the spinal cord. The VEGF-B gene is composed of seven exons. Exons 3 and 4 encode for invariant cysteine residues that are responsible for a cysteine knot motif with two disulfide bridges. Alternative splicing of exon 6 generates two isoforms of VEGF-B, VEGF-B167 (21 kDa) and VEGF-B186 (32 kDa). The C-terminal domain of VEGF-B167 is hydrophilic, structurally related to the heparin-binding domain of some VEGF-A isoforms, and interacts easily with a coreceptor neuropilin-1. Conversely, the C-terminal domain of VEGF-B186 is hydrophobic, modified by O-linked glycosylation, and requires limited proteolysis to bind to neuropilin-1. VEGF-B167 is bound to the surface of cells or pericellular heparan sulfate proteoglycans, whereas VEGF-B186 is secreted freely. This difference in behavior is explained by the presence of a basic region at the C terminus of the VEGF-B167 isoform, which is not present in the VEGF-B186 isoform. Both VEGF-B167 and VEGF-B186 are produced as disulfide-linked homodimers and can generate disulfide-linked heterodimers with VEGF-A when coexpressed. In the absence of heparin, the VEGF-B167–VEGF-A165 heterodimer remains associated with the cell. As the homodimers of VEGF-A165 diffuse efficiently into the extracellular space, VEGF-B167 appears to determine the release of the heterodimers from the cell surface and may therefore control the bioavailability of VEGF-A (Waltenberger et al., 1994; Olofsson et al., 1996, 1998; Neufeld et al., 1999; Ortega et al., 1999; Scrofani et al., 2000). The two VEGF-B isoforms are differentially expressed, with VEGF167 being predominant, suggesting that splicing events are strictly controlled (Olofsson et al., 1996, 1998, 1999; Joukov et al., 1997; Neufeld et al., 1999; Scrofani et al., 2000).
In adult tissues, VEGF-C is expressed most prominently in heart, placenta, ovary, small intestine, and the thyroid gland, whereas in embryonal tissue, expression occurs where lymphatic vessels undergo sprouting from embryonic veins, such as the perimetanephric, axillary, and jugular areas. The gene for VEGF-C spans more than 40 kb of DNA and consists of seven exons. The VEGF homology domain of VEGF-C is encoded by exons 3 and 4, and exons 5 and 7 encode cysteine-rich motifs. In comparison to other ligands of the VEGF family, there is a spacing of the cysteine residues at the C terminus, reminiscent of the BR3P sequence (C-terminal silk domain). Two 2.4-kb and 2.0-kb VEGF-C mRNAs can be detected by Northern blotting of RNA from many embryonal and adult tissues. Tumor cells almost exclusively express the 2.4-kb mRNA form. The identity of the 2.0-kb VEGF-C mRNA remains to be determined. Newly synthesized VEGF-C is a preproprotein, with a predicted molecular mass of 46.9 kD, consisting of an N-terminal signal sequence followed by an N-terminal propeptide, the VEGF homology domain, and a cysteine-rich C-terminal propeptide. The VEGF homology domain contains three putative glycosylation sites. VEGF-C is secreted as a disulfide-bonded homodimer that is proteolytically processed from the precursor polypeptide, and the secreted form contains the C-terminal silk domain. The fully processed VEGF-C is a noncovalent dimer (Joukov et al., 1996, 1997; Witzenbichler et al., 1998; Ortega et al., 1999; Li and Eriksson, 2001).
VEGF-D is found in adult tissues, particularly lung, heart, skeletal muscle, colon, and small intestine. In embryonal tissues, it is abundant in the developing lung (Yamada et al., 1997; Ortega et al., 1999; Li and Eriksson, 2001). The human gene of VEGF-D is located on chromosome Xp22.31. The human cDNA encodes a protein of 354 amino acids. Together with VEGF-C, VEGF-D defines a subfamily of the VEGFs, with close similarities in VEGF homology domains and long N- and C-terminal domains. VEGF-D is synthesized as a preproprotein, which requires proteolytic processing in both the N- and C-terminal regions for activity, and the fully processed growth factor is a noncovalent dimer.
VEGF-E is an Orf virus-encoded VEGF (NZ-strains). The gene products have only 19 to 25% amino acid identity with VEGF, have no apparent basic domain, and seem to be involved in the process of pathological angiogenesis in virus-infected lesions. Its cDNA structure is exonless in the viral genome, suggesting a phylogenetic origin in the vertebrate genome. It has a potent endothelial cell growth stimulatory activity and vascular permeability activity similar to those of VEGF-A165, without the heparin-binding region. Histologically, the lesions caused by the Orf virus are highly vascular and edematous, with an increased number of vessels produced by proliferation of endothelial cells, and contain extensive inflammatory infiltrates of a mixed character. The extensive dermal vascular response seems to be a direct effect of VEGF (Ogawa et al., 1998; Ortega et al., 1999).
B. Vascular Endothelial Growth Factor Receptors
Three VEGF tyrosine kinase receptors have been identified: The fms-like tyrosine kinase Flt-1 (VEGFR-1/Flt-1), the kinase domain region, also referred to as fetal liver kinase (VEGFR-2/KDR/Flk-1), and Flt-4 (VEGFR-3). Each receptor has seven immunoglobulin-like domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insert domain (Ortega et al., 1999).
VEGFR-2 appears to be the most important receptor in VEGF-induced mitogenesis and permeability (Waltenberger et al., 1994; Zachary, 1998). It has a lower affinity for VEGF-A (Kd = 400–800 pM) than VEGFR-1 (Terman et al., 1994). VEGF-C, VEGF-D, and VEGF-E are also ligands for this receptor. Receptor activation during angiogenesis induces the production of platelet-activating factor (PAF) by endothelial cells, stimulates their mitosis and migration, and increases vascular permeability (Joukov et al., 1996; Achen and Stacker, 1998; Ogawa et al., 1998; Ortega et al., 1999; Andre et al., 2000; Partanen et al., 2000; Bernatchez et al., 2002). PAF has many crucial roles in the induction of angiogenesis (Bernatchez et al., 2002). Not only is it involved in inflammatory cell rolling and adhesion (Prescott et al., 1984), but in vitro studies indicate that PAF promotes the expression of potent angiogenic factors and chemokines, including acid fibroblast factor, basic fibroblast growth factor (bFGF), and macrophage inflammatory protein 2 (Bussolino et al., 1995). It also potentiates the migration of cultured endothelial cells.
The role of VEGFR-1 in endothelial cell function is less clear. VEGFR-1 has a much weaker kinase activity and is unable to generate a mitogenic response in endothelial cells when stimulated by VEGF, although it has the highest affinity for VEGF-A (Kd = 15–100 pM) (Terman et al., 1994). It modulates endothelial cell division at the earliest stages of vascular development from the fourth to fifth day of endothelial cell differentiation, just before the formation of the first primitive blood vessels. Embryos and differentiated endothelial cells lacking VEGFR-1 (VEGFR-1-/-) show increased vascularization and number of endothelial cells, accompanied by an increased endothelial cell mitotic index. Kearney et al. (2002) suggested that VEGFR-1 modulates the endothelial cell cycle by affecting one or more molecular signaling pathways. Potentially, VEGFR-1 could negatively modulate pathological vascularization and in this way dampen the proangiogenic effects of VEGFR-2 (Dvorak, 2002), but further investigation is needed. VEGFR-1 is also important in cell migration. The binding of VEGF-A165 to VEGFR-1 induces directed migration of mononuclear phagocytes across an endothelial cell monolayer as well as their activation, based on the expression of tissue factor procoagulant activity. The interaction between VEGF-A165 and VEGFR-1 additionally induces a chemotactic reponse in polymorphonuclear cells. It is significant that monocytes and polymorphonuclear cells only express the gene for VEGFR-1 and not for VEGFR-2 (Berse et al., 1992; Barleon et al., 1996). The relevance of this finding to tumor growth and wound healing will be discussed later. Plouët et al. (1997) showed that the processing of VEGF-A189 into the 38-kDa homodimer, VEGF-A110, or the 34-kDa homodimer, by uPA and plasmin, respectively, is a prerequisite for VEGF-A189 diffusion toward endothelial cells, activation of the VEGFR-2 signal transduction pathway, and the triggering of endothelial cell proliferation. VEGFR-1 also binds VEGF-B, a process dominated by hydrophobic contacts. However, only a poor mitogenic signal for endothelial cells is induced, and VEGF-B is an inefficient endothelial cell mitogen (Olofsson et al., 1998; Ortega et al., 1999; Andre et al., 2000). Endothelial cells respond to VEGF-B binding to VEGFR-1 by increasing expression and activity of uPA and PAI-1 (plasminogen activator inhibitor 1). The expression of PAI-1 precedes that of uPA and may serve to protect the extracellular matrix from extensive proteolysis (Olofsson et al., 1998).
VEGFR-3 differs from the other two VEGFRs by undergoing proteolytic cleavage in the extracellular domain into two disulfide-linked polypeptides. The 4.5- and 5.8-kb VEGFR-3 mRNAs encode polypeptides, which differ in their C termini, and apparently in their signaling properties, as a result of alternative 3' exon splicing (Joukov et al., 1996; Ortega et al., 1999; Partanen et al., 2000). VEGFR-3 binds only VEGF-C and VEGF-D. Since this receptor is generally restricted to lymphatic endothelial cells, activation stimulates mitosis, migration, differentiation, and survival of these cells (Achen et al., 1998; Partanen et al., 2000).
Neuropilin-1 is a type I transmembrane receptor with a molecular weight of 130 to 135 kDa. It does not function independently but acts as a coreceptor. Although the highly conserved short cytoplasmic domain of neuropilin-1 suggests that this is an important component, no binding partners or obvious protein homology domains have been reported within this portion (Peles et al., 1997; Soker et al., 1998; Latil et al., 2000). However, neuropilin-1 possesses five discrete extracellular domains, and this diversity of protein modules is consistent with the possibility of multiple-binding ligands (Peles et al., 1997; Soker et al., 1998). Neuropilin-1 binds VEGF-A165 (Kd = 200–300 pM), and when coexpressed with VEGFR-2, it enhances the binding of VEGF-A165 to VEGFR-2 4- to 6-fold. The chemotaxis of endothelial cells coexpressing these receptors toward a gradient of VEGF-A165 is about 2.5 times faster than that of endothelial cells expressing VEGFR-2 alone. VEGF-B167 can also bind neuropilin-1 through its heparin-binding domain, encoded by exon 6B. This domain is highly homologous to the neuropilin-1 binding epitopes of VEGF-A165. VEGF-B167 and neuropilin-1 are coexpressed prominently in the smooth muscle cells surrounding larger blood vessels. Furthermore, neuropilin-1 in the endothelial cells of large vessels is located in a juxtacrine relationship with the smooth muscle cell layer producing VEGF-B. Unlike VEGF-A165, VEGF-B167 induces a biological response in cells that express neuropilin-1, but not VEGFRs. The signal transduction pathway is as yet unknown, although Cai and Reed (1999) characterized a neuropilin-1-interacting protein that interacts with the cytoplasmic domain. Neuropilin-1 may also potentiate the effects of VEGF-B in cells where VEGFR-1 is expressed (Makinen et al., 1999; Neufeld et al., 1999). Neuropilin-1 potentiates PAF synthesis; Bernatchez et al. (2002) hypothesized that the pathological role of neuropilin-1, when coexpressed with VEGFR-2, might be a result of increased PAF synthesis by endothelial cells, with sustained vascular permeability, inflammation, and endothelial cell migration.
The second immunoglobulin-like domain in the extracellular domain of VEGFR-1 and VEGFR-3 is responsible for specific ligand recognition. It is incapable of binding any of its ligands in the absence of the other flanking domains 1 and 3. These flanking domains play an important role in maintaining the binding site in a spatial conformation compatible with ligand binding. They do not contribute major binding determinants, since these domains can be switched between VEGFR-1 and 3 with 25 and 32% identity, respectively, without any marked influence on domain 2-mediated ligand binding and subsequent receptor activation. VEGFR-2 also shows specific binding determinants within the second immunoglobulin-like domain, but additional elements are required for full ligand binding. The flanking domain sequences contribute to VEGF binding to this receptor. Immunoglobulin-like domains 4 to 7 are required for maximal transphosphorylation, which initiates the signal transduction cascade (Davis-Smyth et al., 1996).
VEGFR-1 and VEGFR-2 are expressed predominantly by vascular endothelial cells. Their promotors contain a 5' flanking sequence essential for endothelial expression, and the receptors are up-regulated during angiogenesis. There is increasing evidence to support the expression and functional importance of VEGFR-1 and VEGFR-2 in cell types other than endothelial cells. They are present in tumor cells, where they are coexpressed with VEGF, and they are also expressed by smooth muscle cells, pancreatic beta cells, and osteoblasts (Ortega et al., 1999). VEGFR-3 is found mainly in venous endothelium during early embryonic development, together with VEGFR-2, but later in fetal development it becomes mainly confined to lymphatic endothelial cells. Although the lymphatic system seems to develop from large central veins in the embryonic jugular, retroperitoneal, and peri-mesonephric regions, very few adult tissues retain VEGFR-3 expression in their venous endothelia. VEGFR-3 is up-regulated in lymphangiogenic vessels but not in angiogenic vessels (Joukov et al., 1996; Lymboussaki et al., 1998; Ortega et al., 1999; Partanen et al., 2000; Karpanen et al., 2001). Neuropilin-1 is expressed in the tips of actively growing axons of certain classes of neuron. It has an important role in axon growth and guidance in the developing embryo. It is also found in the endothelial cells of blood vessels, in the surrounding mesenchymal cells, and in cardiac, placental, pulmonary, hepatic, skeletal, renal, and pancreatic tissues, as well as in a variety of tumor-derived cells (Peles et al., 1997; Soker et al., 1998). Figure 3 gives an overview of the different receptors of the VEGF family.
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The intracellular signaling pathways governing VEGF expression are still poorly explored.
Binding of VEGF to VEGFR-1 has been implicated in important pleiotropic functions, as discussed above. However, the underlying molecular mechanisms have not yet been resolved. This is at least partly due to the apparent inability of the receptor to respond to VEGF-A by increased kinase activity. It is possible that the low kinase activity is due to a small number of tightly regulated phosphorylation sites. Accordingly, a repressor sequence has been identified in the VEGFR-1 juxtamembrane domain, and replacement of this region by that of VEGFR-2 allows VEGFR-1 to respond to VEGF-A stimulation (Gille et al., 2000). The function of the repressor sequence may be to influence the folding of the receptor, and hence the accessibility of the kinase domain active site. Alternatively, the repressor sequence may regulate interactions with phosphatases (Claesson-Welsh, 2003).
Tyrosine residues known to be phosphorylated are Tyr1213, Tyr1333, Tyr1242, and Tyr1327. Three Src homology 2 domain-containing proteins bind to the phosphorylation site Tyr1213, namely SHP-2, phospholipase C 
1 (PLC1), and growth factor receptor-bound 2. The phosphorylation site Tyr1333 allows binding of PLC1 and the adaptor molecules Crk and Nck. Tyr1242 and Tyr1327 are poorly phosphorylated and have not been shown to participate in the binding of signal transduction molecules (Ito et al., 1998).
Binding of VEGF to VEGFR-2 results in autophosphorylation of the following tyrosine residues: Tyr951 and Tyr996, present in the kinase-insert domain; Tyr1054 and Tyr1059, present in the kinase domain; and Tyr1175 and Tyr1214 in the C-terminal tail.
Autophosphorylation of Tyr951 creates a binding site for the VEGFR-associated protein (Wu et al., 2000) and Tyr1175 creates a binding site for Sck (Warner et al., 2000) and PLC1 (Takahashi et al., 2001). Binding of PLC1 activates protein kinase C, which in turn activates Ras. This pathway induces the activation of the extracellular regulated kinase (Erk) pathway [p42/44 mitogen-activated protein kinase (MAPK)]. Erk can translocate to the nucleus, where it phosphorylates and activates transcription factors, including c-Jun and the ternary complex factor, which in turn induce immediate transcription of the c-fos gene (Mazure et al., 1997; Rak et al., 2000; Arsham et al., 2002).
VEGFR-2 also activates phosphatidylinositol 3'-kinase (PI3K), which results in an increase of the lipid phosphatidylinositol (3,4,5)P3, leading to activation of protein kinase B (Akt/PKB), endothelial nitric oxide synthase, and the small GTP-binding protein Rac. Akt/PKB inhibits B-cell lymphoma 2 (Bcl-2)-associated death promoter homolog and caspase-9, thereby promoting cell survival (Gerber et al., 1998). Endothelial nitric oxide synthase generates nitric oxide, resulting in an increase of vascular permeability and cellular migration (Fulton et al., 1999), which is further promoted by Rac (Cross et al., 2003).
VEGF-induced cytoskeletal reorganization and cell migration is also established through interaction of VEGFR-2 with p38MAPK and focal adhesion kinase together with its substrate paxilin (Rousseau et al., 1997). Figure 4 shows several VEGFR signal transduction pathways that are known thus far.
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Binding of VEGF to VEGFR-3 results in association of the receptor with the adaptor proteins Shc and growth factor receptor-bound 2 via Tyr1337 (Pajusola et al., 1994). Activation of the classical Erk pathway is dependent on PLC/PK-mediated p42/44 MAPK activation and independent of Ras. VEGFR-3 activation leads to induction of PI3K and stimulation of Akt/PKB, which could be important for the survival of blood and lymphatic endothelial cells (Makinen et al., 2001).
D. Regulation of Gene Expression
1. Hypoxia. Under normal physiological conditions, each of the approximately 1014 cells in the adult human body is provided with an adequate supply of oxygen to meet its metabolic demands through the concerted function of the pulmonary, hematopoietic, and cardiovascular systems. Oxygen is transported by circulating erythrocytes, the production of which is controlled by the glycoprotein hormone erythropoietin (EPO). EPO-producing cells in the liver and kidneys can sense oxygen concentration and respond to systemic hypoxia by increasing EPO gene transcription. Hypoxia can also be restricted to cells within a localized region of a specific organ, usually as a result of insufficient perfusion. VEGF-A plays a central role in angiogenesis and neovascularization, increasing delivery of both oxygen and energy substrates. VEGF-A expression can be induced when cells are subjected to hypoxia or hypoglycemia. This response seems to depend on Hypoxia Regulated/Responsive Element/Enhancer sequences in the 5' and 3' regions of the VEGF-A gene (Dachs and Tozer, 2000; Ryan et al., 2000; Tsuzuki et al., 2000).
The hypoxia-inducible protein complex HIF-1 binds to the enhancer sequences of the VEGF-A gene, EPO gene, and other critical genes, such as those for glycolytical enzymes and glucose transporters. Both transcription and RNA stability can be enhanced (Jones et al., 2001). HIF-1 is a heterodimer, consisting of the HIF-1
and HIF-1 subunits, or aryl hydrocarbon receptor nuclear translocator, and both are basic-helix-loop-helix per-aryl hydrocarbon receptor nuclear translocator-sim proteins. HIF-1 is constitutively expressed. However, HIF-1 is degraded under normoxic conditions by ubiquitination (Iyer et al., 1998), which is enhanced by binding on von Hippel-Lindau protein and p53 through the recruitment of ubiquitin ligases (Fig. 5). Hypoxic conditions inhibit the protein ubiquitination and stabilize the HIF-1 protein. The exact mechanism by which oxygen tension is sensed is unknown. Some authors have postulated that HIF-1 itself, as an iron-containing protein, can perform this function, whereas others have suggested that a NAPDH-linked oxidase is involved (Forsythe et al., 1996; Ryan et al., 1998; Srinivas et al., 1998; Dachs and Tozer, 2000; Richard et al., 2000; Tsuzuki et al., 2000). In addition to hypoxia, growth factors such as insulin-like growth factors (IGF) 1 and 2 and bFGF have been shown to increase HIF-1 expression. The modulation of HIF-1 by growth factors or hypoxia is apparently a parallel-independent pathway (Bermont et al., 2000). Dimerization of HIF-1 and HIF-1 is required in the cytosol for a stable association with the nuclear compartment. Steroid receptor coactivator and transcription intermediary factor, both transcriptional coactivators, interact with HIF-1 to potentiate its activation (Dachs and Tozer, 2000).
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The expression of VEGF in hypoxic tumor cells is influenced by the circadian organization of molecular clockwork. Clock genes govern physiologic and behavioral circadian rhythms via an autoregulatory transcription-translation feedback loop. The basic helix-loop-helix per-aryl hydrocarbon receptor nuclear translocator-sim protein domain proteins CLOCK and BMAL 1 form a heterodimer and activate transcription of the Per and Cry genes. At a critical concentration of the PER and CRY proteins, CLOCK/BMAL-mediated activation of the Per and Cry genes is attenuated in a negative feedback loop. VEGF, CLOCK, and BMAL 1 mRNA levels in implanted sarcoma 180 tumors in mice show a circadian oscillation, with high and low levels of VEGF during the light and dark phases, respectively (Koyanagi et al., 2003). The negative component of the molecular loop governs the rhythmic change in hypoxia-induced VEGF gene transcription.
The genes for VEGFR-1 and VEGFR-2 seem to be differentially regulated by hypoxia in cultured endothelial cells. Hypoxia induces an up-regulation of VEGFR-1 mRNA, and an HIF binding site has been described in the promotor region of VEGFR-1, but it is not certain if posttranscriptional mechanisms participate in the regulation of VEGFR-1 expression. VEGFR-2 expression remains unchanged, or moderately down-regulated, in hypoxic conditions in vitro. These findings are inconsistent with the hypoxia-induced up-regulation of both receptors observed in several in vivo models. Since the VEGFR-2 gene does not possess a HIF binding site in its promotor region, the in vivo response to hypoxia could possibly be explained by a post-transcriptional increase in mRNA stability and the stimulatory effects of an as yet unidentified paracrine mediator released by ischemic tissues, which is absent in the supernatant of endothelial cells. One can conclude that both VEGFR-1 and VEGFR-2 are up-regulated in hypoxic conditions (Dachs and Tozer, 2000; Fang et al., 2001). VEGF-B and VEGF-C mRNA are apparently not regulated by hypoxia. It is unclear whether VEGF-D and VEGF-E expression is induced by hypoxia (Enholm et al., 1997).
Reactive oxygen species (ROS) (superoxide, hydrogen peroxide, and their metabolites) are regarded as cytotoxic and mutagenic. Reactive oxygen may play a role in neoplastic growth, because a variety of cell lines derived from human cancers demonstrate significantly elevated hydrogen peroxide. The mechanism of ROS generation in malignant cells is not fully understood but may involve de novo synthesis through defective respiration, byproducts of oxidative metabolism, or the induction of ROS-generating enzymes, such as Nox-1. Nox-1 is a recently identified homolog of gp91phox, the catalytic subunit of the phagocyte superoxide generating NADPH-oxidase. Arbiser et al. (2002) showed that Nox-1 expression strongly enhances the tumorigenic potential of DU-145 prostate epithelial cells, with a corresponding increase in tumor vascularity, implying that Nox-1 is angiogenic. Nox-1 signals angiogenic and tumorigenic effects partly through hydrogen peroxide, resulting in an increase of both the synthesis of VEGF mRNA and the bioactivity of matrix metalloproteinase-9 (MMP-9). It activates the extracellular signal-regulated kinase pathway and NF-
B-dependent transcription, both of which have been implicated in growth and angiogenesis.
2. Growth Factors and Cytokines. Tumor necrosis factor-alpha (TNF-) is an inflammatory cytokine with a wide spectrum of biological activity, including angiogenesis. It influences the formation of new vessels indirectly, rather than by directly promoting the sprout of endothelial cells and their growth. The release of angiogenic molecules (e.g., bFGF, PAF, VEGF-A, and VEGF-C), and the up-regulation of proteolytic systems (e.g., uPA) are biological events seemingly triggered by TNF-. Moreover, it has been proven that TNF- also increases the transcription of the VEGFR-2 gene in vascular endothelial cells and has been postulated that the up-regulation of Sp-1 transcription by TNF- leads to increased binding of this factor to the Sp-1 binding site of the VEGFR-2 promotor region. This would explain the increase in VEGFR-2 expression. TNF- probably also increases the transcription of neuropilin-1. The augmented expression of neuropilin-1 and VEGFR-2, after TNF- stimulation, accounts for the increased migration of endothelial cells and the stimulation of wound repair by VEGF-A165 (Giraudo et al., 1998; Ristimaki et al., 1998).
Several growth factors, such as tissue growth factor- (TGF-), epidermal growth factor (EGF), and platelet-derived growth factor BB (PDGF-BB) induce VEGF-A mRNA expression. It has been shown that VEGF-A mRNA is induced in vivo in wounds by PDGF in fibroblasts and by keratinocyte growth factor in epidermal keratinocytes (Enholm et al., 1997). Serum, which contains various growth stimulatory agents, induces an approximately 5-fold increase in VEGF-C mRNA in starved human fibroblasts in vitro. VEGF-C mRNA levels also increased in response to PDGF and to a lesser extent, by EGF and TGF-. Knowing that PDGF is released from platelets following tissue injury, it is possible that VEGF-C is induced in wounds, contributing to the repair of tissue injury. None of these growth factors or serum induces changes in VEGF-B mRNA levels (Enholm et al., 1997). Cytokines such as IL-1 in human synovial fibroblasts, IL-1 in aortic smooth muscle cells, and IL-6 in tumor cell lines have been shown to stimulate VEGF-A expression. VEGF-C and VEGFR-2 mRNA levels are increased in human umbilical vein endothelial cells (HUVEC) treated with IL-1 (Ristimaki et al., 1998).
The expression of the VEGF-A isoforms 121 and 165 can also be regulated by IGF-1. The intrinsic activity of this growth factor has been investigated in endometrial adenocarcinoma cells devoid of estrogen receptors. IGF-1 fails to increase the transcription of the VEGF-A gene. There is a delayed increase of VEGF-A mRNA, so the regulation of mRNA occurs at a posttranscriptional level. In colorectal carcinoma cell lines, IGF-1 appears to operate at both transcriptional and posttranscriptional levels (Akagi et al., 1998). Therefore, the regulation level of VEGF-A expression appears to be tissue specific, or it may depend on the profile of oncogene and antioncogene mutations (Bermont et al., 2000). It is not known whether this growth factor plays a significant role in the VEGF regulation of nontumoral angiogenic processes.
The biological activity of many cytokines and growth factors is mediated by activation of a signal transducer and activator of transcription (Stat). Niu et al. (2002) showed that constitutively activated Stat3 is capable of activating VEGF expression. Moreover, Stat3-induced VEGF-A up-regulation requires the Stat3 binding site at position -848 in the VEGF-A promotor region, providing evidence that VEGF-A is a direct target gene of Stat3. They demonstrated that the interruption of Stat3 signaling inhibits VEGF-A expression, identifying Stat3 as a promising molecular target for antiangiogenic therapy. Since Stat3 is downstream of several important angiogenic tyrosine kinases, such as Src and EGFR, blocking Stat3 may inhibit the neovascularization mediated by multiple angiogenic signaling pathways. For example, targeting Stat3 in tumors may potentially inhibit VEGF-A expression induced by hypoxia and oncogenic tyrosine kinases, both of which are dependent on Src activation. The constitutive activation of Stat3 may also stimulate tumor angiogenesis by down-regulating the expression of angiostatic mediators, as disrupting Stat3 signaling in tumor cells activates the expression of IP-10 and IFN-, inhibitors of angiogenesis.
Cyclooxygenase (COX)-1 and -2 enzymes convert arachidonic acid to prostaglandins and thromboxanes. COX-1 is constitutively expressed and is responsible for normal kidney and platelet function, as well as the maintenance of the gastrointestinal mucosa. The COX-2 enzyme can be induced by a variety of proinflammatory cytokines (e.g., IL-1), growth factors (e.g., EGF), TGF-, inducible nitric oxide synthetase, and ultraviolet B radiation (Gately, 2000). The role of COX-2 in cancer promotion has been demonstrated experimentally in a model of human familial adenomatous polyposis. Oshima et al. (1996) showed that the tumor burden was significantly reduced by the genetic knockout of COX-2. COX-2 expression has been reported in 80% of gastrointestinal adenocarcinomas (Eberhart et al., 1994). It is localized to the neoplastic epithelium, stromal cells, and new angiogenic endothelial cells, whereas COX-1 is distributed widely within mature blood vessels (Masferrer et al., 1999). COX-2 overexpression results in dedifferentiation, adhesion to extracellular matrices, and inhibition of programmed cell death in intestinal cells (Tsujii et al., 1997). Although the exact mechanisms by which COX-2 promotes tumor cell growth are unclear, it stimulates endothelial motility and tube formation in Caco-2 and HCA-7 cells by increasing the production of proangiogenic factors such as VEGF-A. Using an MKN45 xenograft model, Sawaoka et al. (1999) demonstrated that COX-2 inhibition by NS-398, a selective COX-2 inhibitor, suppresses protein levels of VEGF-A and bFGF, and that the angiogenic and apoptotic indices are significantly associated (Sawaoka et al., 1999). They postulated that apoptosis was induced by suppressing angiogenesis. Celecoxib, a potent and selective COX-2 inhibitor, induces apoptosis by blocking Akt activation (Hsu et al., 2000). Nonsteroidal anti-inflammatory drugs (NSAID) inhibit the activity of mitogen-activated protein kinase, an important intermediate in several signaling pathways involved in the activation of transcription factors and early response genes leading to cell proliferation and differentiation and essential for the induction of angiogenesis. These nonselective COX inhibitors also inhibit mitogen-activated protein kinase translocation into the nucleus (Jones et al., 1999). Furthermore, NSAIDs increase the expression of the von Hippel Lindau tumor suppressor, which leads to increased ubiquination of total protein. This results in decreased HIF-1 accumulation, hypoxia-induced VEGF/VEGFR-1 expression, and the inhibition of hypoxia induced angiogenesis (Jones et al., 2002).
Prostaglandin E2 (PGE2) production has been shown to be selectively linked to COX-2 activity through the inducible PGE2 synthase pathway in both inflammatory and aberrant cell growth processes (Murakami et al., 2000). Experimental studies have shown that PGE2 production, or the addition of PGE2 to cell cultures, can mediate important carcinogenic mechanisms. It has been suggested that PGE2 stimulates angiogenesis by increasing Bcl-2 levels and inhibiting apoptosis. However, the precise link between prostaglandin production and Bcl-2 synthesis has not been elucidated. PGE2 also seems to inhibit the immune response against cancer (Kambayashi et al., 1995) and perhaps enhances the invasiveness of neoplastic cells by increasing matrix metalloproteinase-2 (MMP-2) activation (Tsujii et al., 1997). PGE2, a potent stimulator of osteogenesis in vivo, causes rapid induction of VEGF-A mRNA and VEGF-A production in osteoblastic cells in vitro (Harada et al., 1994). The mechanisms of PGE2 up-regulation of VEGF-A still have to be elucidated. Since angiogenesis is associated with the early phase of bone formation, the induction of VEGF-A may play a role in PGE2 stimulation of bone formation in vivo. Further studies on how the products of COX-2 mediate the transcriptional regulation of the VEGF-A gene are needed.
3. Hormonal Regulation. Estrogens stimulate VEGF-A gene transcription and stabilize VEGF-A mRNA, prolonging the half-life of the transcripts (Shweiki et al., 1993; Hyder et al., 1996; Ruohola et al., 1999). This interaction has been studied in human breast cancer (MCF-7) and uterine cell lines. MCF-7 cells express both estrogen receptors and , which normally cause transactivation by binding an estrogen response element in the promotor region of certain genes. The 5' regulatory regions of VEGF-A have not been found to contain any classical estrogen response element. However, the regulatory regions contain several AP-1 and Sp1 sites, which are known to mediate estrogen action. The exact mechanisms remain to be determined (Ruohola et al., 1999). Progestins have been reported to increase VEGF-A expression in the human uterus and in human breast cancer cells (T47-D), associated with transcriptional activation of the VEGF gene (Hyder et al., 1996). The antiestrogens tamoxifen and toremifene, both of which are used in the treatment of breast cancer, do not inhibit the estrogen-induced increase of mRNA expression. On the contrary, they increase VEGF-A mRNA. It has been postulated that the increase in VEGF-A expression is also mediated by an AP-1-related mechanism, but this also requires further study (Gagliardi and Collins, 1993; Hyder et al., 1996; Adams et al., 2000). The clinical impact of these findings will be discussed later.
The effect of testosterone on VEGF-A expression has been studied in the androgen-dependent S115 mouse breast cancer cell line and human prostatic tissue. Transcriptional activation causes an increase in VEGF-A expression, as well as stabilization of the mRNA. The upstream regulatory region of the VEGF gene does not seem to contain any androgen or gonadotrophin response elements. However, the ligand-bound androgen receptor has been found to modulate transcription indirectly via other transcription factors, such as the AP-1 complex (Ruohola et al., 1999).
VEGF-B and VEGF-C are also expressed in MCF7 and S115 breast cancer cells, but they are not affected by hormonal treatment to the same extent as VEGF-A. VEGF-D mRNA is expressed only in S115 cells and is not regulated by testosterone (Ruohola et al., 1999).
E. Physiological Regulation of Vascular Endothelial Growth Factor in Normal Adults
Physiological angiogenesis is mainly restricted to wound healing and the female reproductive cycle.
1. Wound Healing. Wound healing can be subdivided into four phases: acute inflammation, re-epithelialization, granulation tissue formation, and tissue remodelling.
Following cutaneous injury, hemorrhage is stopped by platelet activation and the initiation of the clotting cascade. This results in the formation of a clot, consisting of platelets, embedded in a mesh of cross-linked fibrin fibers, together with small amounts of plasma fibronectin, vitronectin, and thrombospondin. The activated platelets release several cytokines and growth factors stored in their granules. One of these growth factors is VEGF-A, which attracts circulating neutrophils and monocytes. This chemotactic response is mediated by VEGFR-1, expressed on the inflammatory cells (Schaffer and Nanney, 1996; Martin, 1997). Wound healing experiments in VEGF-A transgenic mice, described by Detmar et al. (1998), showed increased leukocyte rolling and adhesion in the postcapillary venules at the wound site. This can be explained by the molecular changes in the surface of the endothelial cells, such as the expression of E- and P-selectin. The exact function of VEGF-A in this process is not clear. As well as phagocytosing particles and releasing toxic metabolites and enzymes, the recruited neutrophils and monocytes also produce a series of proinflammatory cytokines, including IL-1 and TNF-. The leukocyte-derived cytokines, together with TGF- released from platelets, and serum derived factors, are able to induce VEGF-A gene expression in the keratinocytes at the wound margins. VEGF-A expression in these cells is also regulated by HIF-1. This heterodimer is formed in hypoxic regions in the healing wound or after stimulation by IGF-1 and IGF-2, as described above (Frank et al., 1995; Senger and van de Water, 2000). It is notable that only keratinocytes express VEGF-A during this process, with the exception of a few mononuclear cells.
During re-epithelialization, the activated keratinocytes migrate along the interface between the clot and the underlying healthy dermis. This movement is only possible when the fibrin barrier of the clot is dissolved. The most important fibrinolytic enzyme is plasmin, which is derived from plasminogen within the clot itself, and can be activated either by tPA or uPA. Both of these activators are up-regulated in the migrating keratinocytes. VEGF-A regulates the production of plasminogen activators and PAI-1 in endothelial cells. It is unclear whether the increased VEGF-A expression during re-epithelialization also stimulates the production of uPA and tPA by keratinocytes in an autocrine fashion (Schaffer and Nanney, 1996; Martin, 1997). Several matrix metalloproteinases, each of which cleaves a specific subset of matrix proteins, are also up-regulated by wound-edge keratinocytes (Lohi et al., 2001; Mirastschijski et al., 2002; Saarialho-Kere et al., 2002).
An ingrowth of new blood vessels and fibroblasts, together with the synthesis of the components of connective tissue matrix (collagen type I and III), transform the fibrin-fibronectin stroma into the highly cellular and vascular granulation tissue of healing wounds. Endothelial cell migration requires freedom from the constraints of the basement membrane. VEGF-A, released by keratinocytes, monocytes, and endothelial cells, acts in a paracrine and autocrine manner on the capillaries at the wound edge. VEGFR-1 and VEGFR-2 are up-regulated in these endothelial cells, and their activation by VEGF-A results in the generation of proteases such as uPA, tPA, plasmin, and collagenase, which are capable of digesting basal lamina components. VEGF-A also induces an increase in permeability (Dvorak et al., 1995b) by disorganizing the endothelial junction proteins (VE-cadherin and occludin). This process results in gap formation and decreased barrier properties between adjacent endothelial cells. It is proposed that this enhances the supply of proteins and cells needed to form granulation tissue. Furthermore, receptor activation leads to the up-regulation of v3 integrins, which are expressed transiently at the tips of sprouting capillaries and enhance endothelial cell migration. Finally, receptor activation results in endothelial cell migration (VEGFR-1) and proliferation (VEGFR-2), as mentioned earlier.
In addition to endothelial migration from adjacent pre-existing blood vessels, another source of endothelialization is the recruitment of bone marrow-derived endothelial progenitor cells (EPC) from the peripheral circulation (Takahashi et al., 1999). EPCs can be considered to be embryonic angioblasts, which are VEGFR-2+/AC133+ (a hematopoietic stem cell marker, with an as yet unrecognized function), and migrate, proliferate, and capable of differentiating into mature endothelial cells, which are VEGFR-2+/AC133- (Caprioli et al., 1998). Hattori et al. (2001) showed that high plasma levels of VEGF promoted the mobilization of EPCs through activation of metalloproteinases, adhesion molecules, and remodeling of the extracellular matrix within the bone marrow. EPC mobilization promotes an up-regulation of Id1 and Id3, induced by the raised plasma VEGF-A (Benezra et al., 2001). Id proteins (Id1, 2, 3, and 4) contain a highly conserved dimerization motif known as the helix-loop-helix (HLH) domain, which mediates its interaction with other proteins. Their primary targets are the basic HLH transcription factors controlling cell type-specific gene expression and the expression of cell cycle regulatory genes. Heterodimerization between Id and bHLH proteins prevents transcription factors from binding DNA (Lassar et al., 1994). The exact pathways activated by VEGF-induced Id up-regulation need further elucidation. There is comobilization of VEGFR-1+ myeloid cells and EPCs, suggesting that these hematopoietic precursor cells are associated with the newly formed vessels and may be essential for the incorporation of EPCs (Benezra et al., 2001).
Endothelial cells assemble as solid cords, which subsequently acquire a lumen. VEGF-A121, VEGF-A165, and their receptors on endothelial cells (VEGFR-1 and VEGFR-2) enhance and increase lumen formation, in addition to increasing vessel length. Other molecules affecting lumen formation are the integrins v3. Intercalation or thinning of endothelial cells and fusion of pre-existing vessels is a third mechanism which allows newly formed vessels to increase their diameter and length (Schaffer and Nanney, 1996; Martin, 1997).
VEGF-A also acts on pericytes in an autocrine and paracrine manner to stimulate their proliferation and migration. In the normal retina, the recruitment of pericytes is significantly delayed and lags behind the formation of the endothelial plexus. The time interval during which the vasculature is maintained without a pericyte coating determines a window of plasticity for the vasculature to be remodelled and adjusted to the physiological needs of the tissue. It is proposed, but not yet proven, that this mechanism is also important during wound healing (Benjamin et al., 1998; Yonekura et al., 1999). In a study of wound healing in pig skin, Paavonen et al. (2000) observed that VEGFR-3-positive lymphatic vessels appear in the wound simultaneously with blood vessels but regress sooner. Angiogenenic blood vessels are negative for VEGFR-3 during normal blood vessel regeneration in wounds. Lymphangiogenesis may be an important phenomenon in wound healing, but it needs further investigation.
Granulation tissue is eventually remodeled, as blood vessels are resorbed and fibroblasts disappear. The end result is a scar composed largely of dense type I collagen, with occasional widely dispersed fibrocytes and blood vessels.
2. The Female Reproductive Cycle. The ovarian vasculature is not distributed equally; primordial and preantral follicles do not have their own vascular supply but are supported by vessels in the surrounding stroma. As the antrum develops in the follicle, the thecal layer acquires a vascular sheath, consisting of two capillary networks, one in each of the theca interna and externa. The capillary networks are connected, and all capillary blood passes from the theca interna into small vessels, which become continuous with the ovarian stromal veins. All capillaries remain outside the basement membrane of the follicle, so that the granulosa layer remains avascular until the time of ovulation. The vascular density increases during follicle development from the preantral to the antral stage (Gordon et al., 1995). One of the earliest signs of follicular atresia is the reduced vascularity of the follicle. This limits the access to nutrients, substrates, and trophic hormones (Hazzard and Stouffer, 2000). At ovulation, the follicle is converted into the corpus luteum. During ovulation in the rat, the inner capillary plexus adjacent to the basement membrane expands by sprouting into the avascular granulosa layer to form a dense network of sinusoidal capillaries. The outer capillary plexus delays sprouting but eventually becomes interconnected in the corpus luteum. This sinusoidal meshwork differentiates into arterial and venous systems. A decrease in the density of blood vessels during early luteolysis has been observed, and this decline may be related to morphological changes in endothelial cells, including apoptosis. During luteolysis, the larger microvessels are maintained, probably to assist in resorption of the luteal mass (Hazzard and Stouffer, 2000).
VEGF-A is important in luteal angiogenesis. VEGF-A mRNA or protein is detectable in the granulosa cells of primordial and primary follicles, as they progressively become localized to the granulosa surrounding the oocyte and theca cells of the preovulatory follicle. After ovulation, VEGF-A mRNA and protein expression are observed in granulosa-derived luteal cells. VEGF-A expression in the corpus luteum appears highest early in the luteal phase and declines after the mid-luteal phase, with little or no expression in the late corpus luteum (Fraser and Lunn, 2000). Neutralization of VEGF-A during the early luteal phase inhibits the development of the normally extensive capillary bed. Luteal function, reflected by the secretion of progesterone, is reduced by 60%. Whether oxygen tension is a primary regulator of VEGF-A expression in ovarian tissues is unknown. Follicles with a high content of dissolved oxygen also contain the highest follicular fluid concentration of VEGF-A. These follicles display better fertilization rates and embryo development than do oocytes from severely hypoxic follicles (Hazzard and Stouffer, 2000).
Gonadotrophic hormones, particularly luteinizing hormone (LH), appear to be major regulators of angiogenesis in the ovary (Hyder and Stancel, 1999). The LH-stimulated luteinization of granulosa cells at the time of ovulation is associated with enhanced VEGF-A expression. Further studies are needed to determine whether the action of LH is critical for vascularization of the theca in the developing antral follicle, and whether the rescue of the corpus luteum by human chorionic gonadotrophin (hCG) in early pregnancy involves modification of the luteal vasculature. It is speculative whether the decline in VEGF-A expression in the late luteal phase of the menstrual cycle is related to reduced sensitivity of the corpus luteum to LH. Laitinen et al. (1997) showed that LH/hCG decreased the expression of VEGF-C in luteinized granulosa cells. VEGF-B expression is not affected. This implies different roles for the VEGF species in the developing corpus luteum (Laitinen et al., 1997; Hazzard and Stouffer, 2000).
Shifren et al. (1996) described the cellular localization and abundance of both VEGF-A mRNA and protein in the cycling human endometrium. VEGF-A mRNA was observed in the cytoplasm of glandular epithelial cells, accompanied by diffuse stromal staining. VEGF-A expression increased in the secretory phase throughout the endometrium. Both estradiol and progesterone induced the expression of VEGF-A (Shifren et al., 1996).
III. Vascular Endothelial Growth Factor in Tumor Growth
A. Vascular Endothelial Growth Factor and Tumorigenesis
All solid tumors are composed of two compartments: the malignant cells and the stroma. The latter is composed of many elements, which can be grouped into four categories: new blood vessels, inflammatory cells, connective tissues, and a fibrin-gel matrix. Tumor fibrin originates from the extravasation and extravascular clotting of plasma fibrinogen by tumor cell prothrombinase and is dependent on increased vascular permeability. Hyperpermeability and macromolecular transvascular transport are facilitated by the existence of interendothelial junctions, caveolae (vesiculo-vacuolar organelles), and fenestrations, all of which are induced by VEGF-A produced by tumor cells (Hobbs et al., 1998; Monsky et al., 1999). The increased permeability and decreased selectivity of neovascular fenestrated endothelium, compared with the naturally occurring fenestrated endothelium, results from the absence of the basement membrane and the decreased anionic glycocalix on the luminal surface of the fenestral diaphragms. The mechanism by which VEGF-A modulates the changes leading to an increase in capillary permeability is unknown.
The transformation from provisional matrix (fibrin-fibronectin gel) to the mature collageneous tumoral stroma requires the following events: an influx of monocytes that differentiate into macrophages, the replication of fibroblasts and endothelial cells, the migration of macrophages, endothelial cells, and fibroblasts into the fibrin-fibronectin gel, and finally, the degradation of this provisional matrix and progressive replacement by matrix proteins, proteoglycans, and glycosaminoglycans produced by fibroblasts (Dvorak et al., 1995a). The mature tumoral stroma resembles the granulation tissue of healing wounds. There are only small differences between the formation of tumoral stroma and the process of wound healing. First, wound healing is preceded by tissue injury. Such an insult is generally not associated with tumoral stroma generation, which is initiated by the increased permeability of local blood vessels. Second, platelets, which are important in wound healing, have not been found outside blood vessels in solid tumors. However, tumor cells themselves express clotting factors and secrete growth factors, which function as fibroblast mitogens and chemoattractants. Third, fibrin and fibronectin persist in tumoral stroma, whereas both appear only transiently in wounds that heal normally. Fourth, the abundant fibrin deposited around some tumors may serve as a barrier to lymphocytes, macrophages, and other inflammatory cells (Dvorak, 1986). The persistence of fibrin and fibronectin in tumoral stroma is probably due to the fact that tumors produce VEGF-A constitutively. This explains the expression: "Tumors are wounds that do not heal".
Blood vessels in the core and periphery of developing tumors differ. Irrespective of the host or tumor type, core vessels have certain common features: open interendothelial junctions, fenestrated endothelium, a discontinuous or absent basement membrane, and abnormally convoluted lumina. In contrast, peripheral vessels are large and venular and are formed by endothelial cells characterized by fenestrations (formed by fusion of intracellular caveolae) and attenuation. Open interendothelial junctions are not as prevalent as in core vessels (Roberts and Palade, 1997). It is notable that vessels in the tumor periphery represent a true transition from normal nonproliferating host vessels to tumor vessels generated in response to VEGF-A, without the further complicating influences such as tumor secreted proteases, necrosis, hypoxia, and increased interstitial pressure, present in the tumor core (Dvorak, 1986; Nagy et al., 1989). Tsuji et al. (2002) performed morphometric studies on resected specimens of human colorectal carcinoma. They described a significant increase in the mean microvessel diameter as the pathological process progressed from normal (7.03 µm), to localized carcinoma (7.99 µm), and then to metastatic carcinoma (9.75 µm). The risk of regional or distant metastasis increased as the microvessel diameter increased. Giant capillaries (>60 µm) have been identified as a site for cancer cell intravasation. Whether VEGF is involved in the genesis of enlarged microvessels remains to be determined. The number of VEGF-positive cells failed to correlate with the microvessel diameter (Tsuji et al., 2002).
During tumorigenesis, neoplastic lesions initially undergo an avascular growth phase to a size not much greater than 2 to 3 mm3. This phase is followed by a second event that distinguishes a growing tumor from one that is dormant, the switch from the avascular to vascular phenotype, or "the angiogenic switch". This initiates a cascade of events that results in the expansion of tumor volume and subsequent metastasis. The formation of new blood vessels provides a mechanism by which an in situ tumor lesion can circumvent the critical limitations of the oxygen diffusion distance and restrictions on nutrient exchange (Folkman, 1995; Carmeliet, 2000; Carmeliet and Jain, 2000; Kerbel, 2000). The angiogenic switch is regulated by the net balance between positive and negative regulators of new capillary growth (Folkman, 1986; Hanahan, 2002). Figure 6 gives an overview of the stages of tumor development, growth, and metastasis, in which angiogenesis plays an important role.
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An important inhibitor of angiogenesis is the p53 tumor suppressor gene. p53 arrests cell cycle progression under nonviable conditions and mediates hypoxia-induced apoptosis. The p53 gene product promotes the expression of inhibitors of angiogenesis, such as thrombospondin, and inhibits the expression of VEGF-A, but not VEGF-B or VEGF-C (Fontanini et al., 1998; Ravi et al., 2000). p53 is mutated in 50% of all cancers and is also inactivated by viral oncoproteins (Yuan et al., 2002). HIF-1 has been found to be overexpressed in most human cancers, since active tumor growth is accompanied by hypoxia (Graeber et al., 1996; Elson et al., 2000). Hypoxic conditions lead to the up-regulation of both p53 and HIF-1 (Carmeliet et al., 1998; Wenger et al., 1998). HIF-1 binds to and stabilizes p53, provided the latter is not mutated (Zhong et al., 1999). This association initiates apoptosis: The angiogenic switch is maintained in the off position, since positive factors are overruled by the angiogenesis inhibitors. The loss of p53 expression enhances the heterodimerization of HIF-1 with HIF-1 and up-regulates the expression of VEGF-A in tumor cells (Blagosklonny et al., 1998; Zhong et al., 1999; Blancher et al., 2000). In this situation, there is a net balance of activators over inhibitors, and new blood vessels will be formed (Dachs and Tozer, 2000; Ryan et al., 2000). In human tumors, the p63/p51/p73L/p40/KET gene, a new p53 family member (p63 gene), is not mutated as frequently as p53, but certain isoforms are occasionally lacking or overexpressed. TAp63 and dNp63, the two major isoforms of p63, bind to the VEGF-promotor region, and respectively, down- and up-regulate VEGF gene expression. The modulation of VEGF expression is closely correlated with the distinct roles of both isoforms in the regulation of HIF-1 stability. Senoo et al. (2002) showed that the degradation of HIF-1 is stimulated by TAp63. The other isoform, dNp63
