Recently , vascular endothelial growth factor-C (VEGF-C or VEGF-2) was described as a specific ligand for the endothelial receptor tyrosine kinases VEGFR-2 and VEGFR-3. In vivo data , limited to constitutive overexpression in transgenic mice , have been interpreted as evidence that the growth-promoting effects of VEGF-C are restricted to development of the lymphatic vasculature. The current studies were designed to test the hypothesis that constitutive expression of VEGF-C in adult animals promotes angiogenesis. In vitro, VEGF-C exhibited a dose-dependent mitogenic and chemotactic effect on endothelial cells , particularly for microvascular endothelial cells (72% and 95% potency , respectively , compared with VEGF-A/ VEGF-1). VEGF-C stimulated release of nitric oxide from endothelial cells and increased vascular permeability in the Miles assay; the latter effect was attenuated by pretreatment with the nitric oxide synthase inhibitor N -nitro-L-arginine methyl ester. Both VEGFR-2 and VEGFR-3 receptors were shown to be expressed in human saphenous vein and internal mammary artery. The potential for VEGF-C to promote angiogenesis in vivo was then tested in a rabbit ischemic hindlimb model. Ten days after ligation of the external iliac artery , VEGF-C was administered as naked plasmid DNA (pcVEGF-C; 500 g) from the polymer coating of an angioplasty balloon (n ‫؍‬ 8 each) or as recombinant human protein (rhVEGF-C; 500 g) by direct intra-arterial infusion. Physiological and anatomical assessments of angiogenesis 30 days later showed evidence of therapeutic angiogenesis for both pcVEGF-C and rhVEGF-C. Hindlimb blood pressure ratio (ischemic/normal) after pcVEGF-C increased to 0.83 ؎ 0.03 after pcVEGF-C versus 0.59 ؎ 0.04 (P < 0.005) in pGSVLacZ controls and to 0.76 ؎

Previous studies have indicated that advanced age is associated with impaired angiogenesis in part because of reduced levels of vascular endothelial growth factor (VEGF) expression. To investigate potential mechanisms responsible for this age-dependent defect in VEGF expression, aortic smooth muscle cells isolated from young rabbits (ages 6 -8 months) or old rabbits (ages 4 -5 years) were exposed to normoxic (21% oxygen) or hypoxic (0.1% oxygen) conditions. Hypoxia-induced VEGF expression was significantly lower in old versus young cells. VEGF mRNA stability in hypoxic conditions was similar in both young and old cells. However, transient transfection with a luciferase reporter gene that was transcriptionally regulated by the VEGF promoter revealed a significant defect in VEGF up-regulation following hypoxia in old versus young cells (a 43 versus 117% increase in luciferase activity, p < 0.05); this difference was not seen when a deletion construct lacking the hypoxia-inducible 1 (HIF-1) binding site was used. Moreover, although HIF-1␣-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1␣ protein and DNA binding activity were significantly reduced in old versus young smooth muscle cells that were exposed to hypoxia. We propose that age-dependent reduction in hypoxia-induced VEGF expression results from reduced HIF-1 activity and may explain the previously described age-dependent impairment of angiogenesis in response to ischemia.

Background-Normally, quiescent endothelial cells (EC) line the inner surface of arteries and protect against thrombosis and neointimal growth. A variety of noxious stimuli, including balloon angioplasty, may compromise EC integrity, thereby initiating proliferation and triggering the local release of cytokines, including tumor necrosis factor-␣ (TNF-␣). Methods and Results-In vivo blockade of TNF-␣ using a soluble receptor molecule results in accelerated reendothelialization at sites of balloon angioplasty, suggesting an important physiological role of TNF-␣ in attenuating regrowth of endothelium after balloon angioplasty. Our studies reveal that TNF-␣, an apoptosis-inducing cytokine, induces G1 cell-cycle arrest in proliferating EC. Quiescent EC are relatively immune to TNF-induced apoptosis versus proliferating EC, which display repression of the E2F transcription factor coincident with TNF-induced apoptosis and cell-cycle arrest. We also show that in this setting, E2F overexpression exerts a survival effect in proliferating EC and restores cell-cycle progression, in direct contrast to results of prior reports, which revealed that deregulated expression of E2F in normally cycling cells induces apoptosis. Conclusions-These data demonstrate that TNF-induced apoptosis is highly dependent on cell-cycle activity and that E2F can function as survival factor under certain conditions. (Circulation. 1998;98:2883-2890.)