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Critical lower limb ischemia is a common cause for amputation. To develop new therapeutic strategies, more information is needed about molecular mechanisms of tissue responses to ischemic stress and factors inducing angiogenesis. Using a DNA array of 8400 genes, gene expression patterns in human skeletal muscle samples collected from lower limbs amputated due to acute-on-chronic or chronic critical lower limb ischemia, were compared with the control samples collected from the same limb. The results were confirmed by RT-PCR and immunohistochemistry. In acute-on-chronic ischemia, 291 genes were significantly upregulated and 174 genes were downregulated (change in 5.5% of all genes) as compared to control samples. Significant induction of the hypoxia-inducible angiogenic pathway involving hypoxia-inducible factor-1alpha (HIF-1alpha), HIF-2alpha, vascular endothelial growth factor (VEGF) and its angiogenic receptor VEGFR-2, as well as tumor necrosis factor-alpha (TNF-alpha) with its downstream signaling machinery promoting inflammation and cell death, were found in acute-on-chronic ischemia. In chronic critical ischemia, gene expression changes were much less striking than in acute-on-chronic ischemia, with 74 genes significantly upregulated and 34 genes downregulated (change in 1.3% of all genes). In the chronic situation, the anabolic and survival factors, insulin-like growth factor-1 (IGF-1) and IGF-2, were upregulated in atrophic and regenerating myocytes together with attenuated HIF, VEGF, and VEGFR-2 expression in the same cells. In conclusion, acute-on-chronic and chronic human skeletal muscle ischemia result in distinct gene expression patterns. These findings may be of importance in the design of novel therapies, such as therapeutic vascular growth, for patients suffering from lower limb ischemia.

A better understanding of atherogenesis at the level of gene expression could lead to the identification of new therapeutic strategies for vascular diseases. With DNA array technology, it is possible to identify multiple, simultaneous changes in gene expression in small tissue samples from atherosclerotic arteries. We analyzed gene expression in normal arteries and in immunohistologically characterized human advanced atherosclerotic lesions using an array of 18376 cDNA fragments. The array method was first validated by detecting a group of genes (n=17) that were already known to be connected to atherogenesis. These genes included e.g. Apolipoprotein E, CD68, TIMP and phospholipase D. Next we detected 75 differentially expressed genes that were previously not connected to atherogenesis. A subgroup of genes involved in cell signaling and proliferation was selected for further analyzes with in situ hybridization and RT-PCR which confirmed array results by showing induction in advanced lesions of Janus kinase 1 (JAK-1) which is an important signaling molecule in activated macrophages; VEGF receptor-2 which mediates angiogenic and vasculoprotective effects of VEGF; and an unknown gene, which mapped on chromosome 19. It is concluded that DNA array technology enables fast screening of gene expression in small samples of atherosclerotic lesions. The technique will be useful for the identification of new factors, such as JAK-1 and VEGF receptor-2, which may play an important role in atherogenesis.

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.