Increased endogenous angiogenic response and hypoxia-inducible factor-1alpha in human critical limb ischemia

The potent physiologic endogenous angiogenic response to ischemic stimuli is often suboptimal, and therefore, a better understanding of the basic mechanisms is essential for the use in therapeutic angiogenesis. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor that promotes ischemia-driven angiogenesis and is induced when the HIF-1alpha subunit is upregulated. However, little is known about the endogenous angiogenic response and the role of HIF-1alpha in human critical limb ischemia (CLI). We aimed to investigate the extent of the angiogenic response and the expression of HIF-1alpha in the lower limbs of CLI patients.

Skeletal muscle biopsy specimens were obtained from the lower limbs of 12 patients with CLI and 12 patients without limb ischemia (controls), with ethical committee approval. Microvessel density (MVD) was determined by using endothelial marker anti-CD31, and HIF-1alpha expressions were determined by immunohistochemistry. MVD was measured as the median number of microvessels in x200 magnification fields. Five random fields per section and three sections per biopsy specimen were analyzed. Enzyme-linked immunoabsorbent assay and Western blotting were used to quantify the HIF-1alpha levels. Colocalization between cell-specific antigens was investigated by double immunofluorescence labelling by using confocal microscopy. Statistical analyses were performed with the Mann-Whitney U test.

The CLI group have significantly higher MVD, with an increase of 2.7-fold compared with the controls (P < 0.001). HIF-1alpha expression was significantly increased in CLI muscles (P < 0.001) and was localized to vascular endothelial cells.

Our findings suggest that the endogenous angiogenic response occurs in CLI. The increased HIF-1alpha level and colocalization to vascular endothelial cells suggest that HIF-1alpha plays a role in the physiologic endogenous angiogenic response in CLI. Therefore, augmentation of the HIF-1alpha pathway may be an important aspect in therapeutic angiogenesis.