Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.

The biology of VEGF and its receptors.

The establishment of a vascular supply is required for organ development and differentiation as well as for tissue repair and reproductive functions in the adult1 Neovascularization (angiogenesis) is also implicated in the pathogenesis of a number of disorders These include: proliferative retinopathies, age-related macular degeneration, tumors, rheumatoid arthritis, and psoriasis1,2 A strong correlation has been noted between density of microvessels in primary breast cancers and their nodal metastases and patient survival3 Similarly, a correlation has been reported between vascularity and invasive behavior in several other tumors4–6

The biology of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. Hypoxia has been shown to be a major inducer of VEGF gene transcription. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high-affinity VEGF receptors. The role of VEGF in developmental angiogenesis is emphasized by the finding that loss of a single VEGF allele results in defective vascularization and early embryonic lethality. VEGF is critical also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with various VEGF inhibitors in a variety of malignancies are ongoing. Very recently, an anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the Food and Drug Administration as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.

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Vascular endothelial growth factor: basic science and clinical progress.

Shaker A. Mousa and Thomas M. Reilly The DuPont Merck Pharmaceutical Company Cardiovascular Diseases Div., Exp. Station, Wilmington, DE 19880-0400 The present investigation was undertaken to compare the effects of different direct thrombin inhibitors including hirudin, DUP 714 (Ac-D-Phe-Pro-boro Arg) and PPACK (Phe-Pro-Arg-chloromethyl ketone) with different platelet GPIIblIIIa antagonists including linear Arginine-Glycine-Aspartate (RGD), cyclic RGD, peptidomimetic and non-peptide inhibitors on various platelet functions including: (a) platelet aggregation, (b) fibrinogen binding, (c) platelet plasminogen activator inhibitor type-l (PAl -1) release, (d) platelet serotonin release, and (e) platelet intracellular Ca++ mobilization. Hirudin, DUP 714 and PPACK were effective in inhibiting all of the responses (a-e) mediated by thrombin, with ICsos ranging from 0.002 to 0.9 uM depending upon the concentration of thrombin used, but not those mediated by other platelet agonists including collagen, ADP, and arachidonic acid. In contrast, linear RGD, constrained cyclic RGD (XM648), an RGD peptidomimetic (YY751), a non-peptide GPIIb/IIIa antagonist were effective in blocking fibrinogen binding and platelet aggregation mediated by any of the agonists. The potency of XL 111, XM648 and YY751 in inhibiting thrombin-mediated platelet aggregation (a) and fibrinogen binding (b) ranged from ICso of 0.05 to 1.0 uM in (a) and IC50 of 0.01 to 0.005 uM in (b). However, all platelet GPIIb/IIIa antagonists tested were ineffective in blocking either (c), (d) or (e). Flow cytometric analysis using dual fluorescence markers for the platelet GPIIblIIIa membrane receptors (FIrC-Iabeled cyclic RGD analog, XL086) and for the alpha granule (PE-monoclonal antibody for P-selectin) demonstrated a dissociation between the platelet GPIIb/IIIa receptors by inhibitors and platelet granular secretion. These data suggest that GPIIb/IIIa receptor blockade as compared with direct inhibition of thrombin, does not inhibit intracellular CaH mobilization signal transduction. These data also show that GPIIb/IIIa antagonists, in contrast to thrombin inhibitors, inhibit platelet aggregation mediated by different agonists. The universal anti-aggregatory efficacy of the GPIIb/IIIa antagonists and efficacy of the thrombin inhibitors against Cardiovascular Disease 2 Edited by L.L. Gallo, Plenum Press, New York, 1995 255 thrombin-induced platelet secretion suggest potential benefits of their combinations in various thrombotic disorders.

Cardiovascular Disease 2

Vascular endothelial growth factor (VEGF) is a fundamental regulator of normal and abnormal angiogenesis. Recent evidence indicates that VEGF is essential for embryonic vasculogenesis and angiogenesis. Furthermore, VEGF is required for the cyclical blood vessel proliferation in the female reproductive tract and for longitudinal bone growth and endochondral bone formation. Substantial experimental evidence also implicates VEGF in pathological angiogenesis. Anti-VEGF monoclonal antibodies or other VEGF inhibitors block the growth of many tumor cell lines in nude mice. Furthermore, the concentrations of VEGF are elevated in the aqueous and vitreous humors of patients with proliferative retinopathies such as the diabetic retinopathy. In addition, VEGF-induced angiogenesis results in a therapeutic benefit in several animal models of myocardial or limb ischemia. Currently, both therapeutic angiogenesis using recombinant VEGF or VEGF gene transfer and inhibition of VEGF-mediated pathological angiogenesis are being pursued.

Molecular and biological properties of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has recently been shown to increase cytosolic free calcium in endothelial cells. In the present study, we investigated the coronary vascular effects of recombinant human and native guinea pig VEGF/VPF in isolated canine coronary arteries in the presence and absence of intimal endothelium, indomethacin, and NG-monomethyl-L-arginine, a competitive nitric oxide synthase inhibitor. Addition of recombinant VEGF/VPF (1-660 pM) in coronary arteries that had been previously contracted with prostaglandin F2 alpha induced a slow, dose-dependent relaxation, reaching a maximum of -59.1 +/- 6.7% (mean +/- SE, n = 19). Mechanical disruption of the intimal endothelium completely abolished the observed relaxation. No direct vascular effect of recombinant VEGF/VPF on the endothelium-disrupted coronary arteries was noted. Pretreatment of endothelium-intact coronary arteries with 5 microM of indomethacin did not alter the observed relaxation (-57.3 +/- 7.0%, n = 18), whereas pretreatment with either NG-monomethyl-L-arginine or 10 microM of genistein, a known inhibitor of tyrosine kinase, significantly inhibited the relaxation. Addition of native VEGF/VPF (1-100 pM) also induced an endothelium-dependent relaxation in the isolated coronary arteries. Heating of recombinant VEGF/VPF (70 degrees C, 25 min) or prior incubation with a specific antibody raised against a VEGF/VPF peptide completely abolished the relaxation. Finally, recombinant VEGF/VPF stimulated a slow rise in cytosolic free calcium in cultured human endothelial cells that was qualitatively similar to that of native VEGF/VPF.(ABSTRACT TRUNCATED AT 250 WORDS)

J. K. Zaleski

Papers

Vascular endothelial growth factor induces EDRF-dependent relaxation in coronary arteries.