Age-dependent defect in vascular endothelial growth factor expression is associated with reduced hypoxia-inducible factor 1 activity.

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 alpha-mRNA expression was shown to be similar in young and old smooth muscle cells, HIF-1 alpha 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.

Age-dependent increase in c-fos activity and cyclin A expression in vascular smooth muscle cells. A potential link between aging, smooth muscle cell proliferation and atherosclerosis.

OBJECTIVE: Aging can be defined as a progressive deterioration of biological functions after the organism has attained its maximal reproductive competence, which is usually associated with a decrease in proliferative ability in most cell types. However, in certain pathological situations such as atherosclerosis and restenosis, aging has been shown to be associated with a higher level of vascular smooth muscle cell (VSMC) proliferation and neointimal lesion formation after angioplasty. In the present study, we investigated potential mechanisms involved in the age-dependent increase in VSMC proliferation. METHODS AND RESULTS: Primary cultures of VSMCs were isolated from young (6-8-month-old) and old (4-5-year-old) New Zealand rabbits. Results from cell counting assays and FACS analysis were consistent with a shortening of the cell cycle in old VSMCs. Western blot analysis in serum stimulated cells showed a significant increase in the level of cyclin A and cyclin-dependent kinase 2 proteins in the old vs. young VSMCs. In marked contrast, expression of cyclin E in VSMCs was not influenced by aging. Transient transfection assays showed an age-dependent increase in transcription from the human cyclin A promoter. Parallel studies demonstrated that the expression of the AP1 transcription factor c-fos, which interacts with the cyclin A promoter and stimulates VSMC proliferation, was also increased in old VSMCs. Consistent with this notion, electrophoretic mobility shift assays demonstrated an increase in AP1 DNA-binding activity in old VSMCs. CONCLUSIONS: These studies suggest that age-associated increase in c-fos activity contributes to augmented cyclin A expression and VSMC proliferation in old animals. These mechanisms might contribute to the higher prevalence and severity of atherosclerosis in the elderly.

Restoration of E2F expression rescues vascular endothelial cells from tumor necrosis factor-alpha-induced apoptosis.

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-alpha (TNF-alpha). METHODS AND RESULTS: In vivo blockade of TNF-alpha using a soluble receptor molecule results in accelerated reendothelialization at sites of balloon angioplasty, suggesting an important physiological role of TNF-alpha in attenuating regrowth of endothelium after balloon angioplasty. Our studies reveal that TNF-alpha, 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.

Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotes angiogenesis in the setting of tissue ischemia.

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(omega)-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 microg) from the polymer coating of an angioplasty balloon (n = 8 each) or as recombinant human protein (rhVEGF-C; 500 microg) 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 +/- 0.04 after rhVEGF-C versus 0.58 +/- 0.03 (P < 0.01) in control rabbits receiving rabbit serum albumin. Doppler-derived iliac flow reserve was 2.7 +/- 0.1 versus 2.0 +/- 0.2 (P < 0.05) for pcVEGF-C versus LacZ controls and 2.9 +/- 0.3 versus 2.1 +/- 0.2 (P < 0.05) for rhVEGF-C versus albumin controls. Neovascularity was documented by angiography in vivo (angiographic scores: 0.85 +/- 0.05 versus 0.51 +/- 0.02 (P < 0.001) for plasmid DNA and 0.74 +/- 0.08 versus 0.53 +/- 0.03 (P < 0.05) for protein), and capillary density (per mm2) was measured at necropsy (252 +/- 12 versus 183 +/- 10 (P < 0.005) for plasmid DNA and 229 +/- 20 versus 164 +/- 20 (P < 0.05) for protein). In contrast to the results of gene targeting experiments, constitutive expression of VEGF-C in adult animals promotes angiogenesis in the setting of limb ischemia. VEGF-C and its receptors thus constitute an apparently redundant pathway for postnatal angiogenesis and may represent an alternative to VEGF-A for strategies of therapeutic angiogenesis in patients with limb and/or myocardial ischemia.