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

ObjectiveThe 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-1α subunit is upregulated. However, little is known about the endogenous angiogenic response and the role of HIF-1α in human critical limb ischemia (CLI). We aimed to investigate the extent of the angiogenic response and the expression of HIF-1α in the lower limbs of CLI patients.MethodSkeletal 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-1α expressions were determined by immunohistochemistry. MVD was measured as the median number of microvessels in ×200 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-1α 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.ResultsThe CLI group have significantly higher MVD, with an increase of 2.7-fold compared with the controls (P < 0.001). HIF-1α expression was significantly increased in CLI muscles (P < 0.001) and was localized to vascular endothelial cells.ConclusionsOur findings suggest that the endogenous angiogenic response occurs in CLI. The increased HIF-1α level and colocalization to vascular endothelial cells suggest that HIF-1α plays a role in the physiologic endogenous angiogenic response in CLI. Therefore, augmentation of the HIF-1α pathway may be an important aspect in therapeutic angiogenesis.

Clinical RelevanceThe results of clinical trials that have used therapy with a single proangiogenic factor have been inconsistent and inconclusive. Because many of the angiogenic factors are regulated by hypoxia-inducible factor-1α (HIF-1α), induction of this transcription factor should theoretically provide a more physiologic mechanism of angiogenesis. This study has demonstrated the physiologic angiogenic role of HIF-1α in human critical limb ischemia (CLI). With the encouraging results of a gene therapy method using HIF-1α in preclinical models, therapeutic angiogenesis to augment HIF-1α pathway in human CLI may represent a future promising treatment option for CLI.