Print this pageHypoxia Attenuates Insulin-Induced Proliferation and Migration Of Human Diabetic Infrapopliteal Vascular Smooth Muscle Cells
1Veterans Affairs Medical Center Washington, D.C. and Walter Reed Army Medical Center, Washington, DC;2Veterans Affairs Medical Center Washington, D.C., Washington, DC
Objective: The proliferative effects of insulin on infrapopliteal vascular smooth muscle cells (VSMC) have been established, but there is little to explain the paucity of disease in infrapopliteal and pedal arteries beyond an occlusion. We hypothesize that hypoxia attenuates the proliferative and pro-migratory effects of insulin on human diabetic infrapopliteal VSMC in vitro.
Methods: Human VSMC isolated from the infrapopliteal arteries of male diabetics undergoing lower extremity amputation were harvested and grown to sub-confluence in standard conditions. Cells were then exposed to 1% FBS with and without 100ng/ml insulin in oxygen concentrations of 17% (normoxia), 5% and 1%. Cellular proliferation was assayed at 24 hours using [methyl-3H]-thymidine incorporation. Migration assays were performed at 20 hours using the Corning Costar Transwell system and cells were counted at 200X. LDH was assayed and compared among groups as a marker for cytotoxicity. Statistical analysis was accomplished using ANOVA with Tukey post-hoc test or Student t-test as appropriate, with significance set at P<.05.
Results: VSMC in normoxic conditions (17%) had a significant increase in both proliferation and migration when exposed to insulin. A significant increase was not present at both 5% and 1% hypoxia (Figure 1). Of cells exposed to insulin, those at both 5% and 1% oxygen concentrations proliferated at a significantly decreased rate when compared with cells at normoxia. Migration of these insulin-exposed cells was significantly decreased at 1% hypoxia compared to cells at normoxia, but was not significantly different at 5% hypoxia (Figure 2). Hypoxia and insulin exerted no significant effect on cytotoxicity.
Conclusions: The proliferative and pro-migratory effects of insulin on diabetic VSMC are attenuated in hypoxic conditions, in a manner unrelated to cytotoxicity. This observation may explain the relative sparing of distal tibial and pedal arteries from disease in the presence of a more proximal tibial occlusion.
