Vascular endothelial growth factor (VEGF) is a hypoxia-inducible endothelial cell mitogen and survival factor. Its receptor VEGFR-2 (KDR/Flk-1) mediates these effects. We studied the expression of VEGF and VEGFR-2 in ischemic human and rabbit skeletal muscle by immunohistochemistry and in situ hybridization. Human samples were obtained from eight lower limb amputations because of acute or chronic critical ischemia. In chronically ischemic human skeletal muscle VEGF and VEGFR-2 expression was restricted to atrophic and regenerating skeletal myocytes, whereas in acutely ischemic limbs VEGF and VEGFR-2 were expressed diffusely in the affected muscle. Hypoxia-inducible factor-1alpha was associated with VEGF and VEGFR-2 expression both in acute and chronic ischemia but not in regeneration. Hindlimb ischemia was induced in 20 New Zealand White rabbits by excising the femoral artery. Magnetic resonance imaging and histological sections revealed extensive ischemic damage in the thigh and leg muscles of ischemic rabbit hindlimbs with VEGF expression similar to acute human lower limb ischemia. After 1 and 3 weeks of ischemia VEGF expression was restricted to regenerating myotubes and by 6 weeks regeneration and expression of VEGF was diminished. VEGFR-2 expression was co-localized with VEGF expression in regenerating myotubes. Macrophages and an increased number of capillaries were associated with areas of ischemic muscle expressing VEGF and VEGFR-2. In conclusion, two patterns of VEGF and VEGFR-2 expression in human and rabbit ischemic skeletal muscle are demonstrated. In acute skeletal muscle ischemia VEGF and VEGFR-2 are expressed diffusely in the affected muscle. In chronic skeletal muscle ischemia and in skeletal muscle recovering from ischemia VEGF and VEGFR-2 expression are restricted to atrophic and regenerating muscle cells suggesting the operation of an autocrine pathway that may promote survival and regeneration of myocytes.

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A signi cant reduction in genomic 5-methylcytosine content was detected in advanced human atherosclerotic lesions and in lesions of ApoE knock-out mice. SMC were shown to develop hypomethylation in vitro during transformation from a contractile to synthetic phenotype. Balloon denudation of New Zealand White rabbit aorta caused proliferation of intimal SMC with concomitant genomic hypomethylation in the thickened intima. By using in situ hybridization the overall transcriptional activity was found to be increased in clusters of lesion SMC. Marked heterogeneity was seen in MTase mRNA expression in various types of atherosclerotic lesions among intimal and medial SMC.These ndings show that (1) genomic hypomethylation occurs during atherogenesis in human, mouse and rabbit lesions and that it correlates with increased transcriptional activity; (2) MTase is expressed in atherosclerotic lesions; and (3) hypomethylation is present in advanced lesions at the same level as in malignant tumors and may affect cellular proliferation and gene expression in atherosclerotic lesions.

Rationale: We studied a possibility that shRNAs can lead to transcriptional gene activation at the promoter level via epigenetic mechanism. Objective: The purpose of this study was to test the effects on vascular endothelial growth factor (VEGF-A) expression by promoter targeted small hairpin RNAs (shRNAs) in vitro and in experimental animals in vivo using stable local lentiviral gene transfer. Methods and Results