Introduction: Diabetic patients suffer poor clinical outcomes after ischemic insult, as demonstrated by increased morbidity and mortality following cerebrovascular insult, myocardial infarction, and wounding. The pro-angiogenic protein vascular endothelial growth factor (VEGF) is thought to play an important role in this diabetic dysfunction. While the literature supports this concept, the mechanism by which diabetes affects VEGF production remains unknown. VEGF production is regulated by the transcriptional regulator HIF-1α, which in turn is controlled by oxygen tension. Under normoxic conditions HIF-1α is degraded by the activity of prolyl hydroxylases (PHDs) which mark the protein for degradation. Here we demonstrate that diabetes actually increases HIF-1α RNA transcription, as well as HIF-1α protein stability. We further demonstrate that high glucose increases HIF-1α stability by decreasing PHD transcription. This further demonstrates that VEGF downregulation in diabetics is secondary to interference with HIF-1α's ability to act as a transcriptional regulator, and not by direct interference with HIF-1α production or stability.

Methods: A murine model of soft tissue ischemia was used to specifically isolate and quantify the amount of VEGF production in diabetic mice by ELISA. Diabetes was induced by intraperitoneal injection of streptozotocin. Cell culture experiments were done on mouse embryonic fibroblasts (MEF) in 5 mM (low glucose) and 25 mM (high glucose) under normoxic (21% O2) and hypoxic (0.5% O2) conditions. Protein was extracted from these cells for use in western blot analysis for HIF-1α. RNA from these cells was used for analysis by Taqman RT-PCR.

Results: VEGF protein expression was reduced 6-fold in diabetic mice when compared to age matched controls. Western blot analysis demonstrated a qualitative increase in HIF-1α protein in the MEF cell line under high glucose, hypoxic conditions when compared to low glucose, hypoxic cultures. RT-PCR analysis of HIF-1α demonstrated a 10-fold increase in HIF-1α RNA production under diabetic hypoxic conditions while PHD RNA production was decreased 7-fold.

Conclusion: Diabetes results in impaired neovascularization, leading to functional deficits after ischemic injury. High glucose conditions interfere with the ability of HIF-1α to act as a transcriptional regulator, causing the downregulation of downstream proangiogenic genes such as VEGF. Our data indicate that HIF-1α transcription is increased by high glucose conditions. On the protein level HIF-1α stability is also increased by diabetes, supported by the fact that PHD transcription is decreased by high glucose. Finally we demonstrate that the downstream affect of this HIF-1α dysregulation is the downregulation of VEGF protein in response to ischemia, thus leading to a defect in neovascularization.