Abstract 325: Temporally Separated Administration of Proangiogenic Growth Factor Combination Promotes Angiogenesis in Both Diabetic and Nondiabetic Microvascular Endothelial Cells

Background: Diabetes mellitus is a chronic metabolic disease associated with various vascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue including delivery of pro-angiogenic growth factors have shown disappointing results. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We assessed the hypothesis that temporally separated delivery of multiple pro-angiogenic growth factors will improve the efficacy of the angiogenic response in the setting of both diabetes and non-diabetes in vitro. Methods: 96-well tissue culture plates were coated with 60 μL growth factor reduced Matrigel. After gelation, non-diabetic human dermal microvascular endothelial cells (HMVECs) and type-II diabetic cells (D-HMVECs) were plated at 1x104 cells/well with 100 μL basal media EBM-2 with 0.1% bovine serum, and incubated at 37°C. Combinations of growth factors including recombinant human VEGF (50 ng/mL), angiopoietin (Ang)-1 and -2 (250 ng/mL) were administrated at start of the assay and/or at 6 hours into the assay. Images were taken at 3, 6, 9, 12, 18 and 24 hours and used for tube formation quantification by Inverted Microscope at 10x magnification. Results: In untreated control, D-HVECs showed reduced tube formation in compared to HMVECs (n=12; p<0.001). Growth factors combination ‘VEGF and Ang-2 at start of the assay and Ang-1 at 6 hours showed the most significantly increased of tube formation when compared to single growth factor (to VEGF, Ang-1, 2; p<0.001) or growth factors combination without temporally separated delivery (to VEGF/Ang-1, VEGF/Ang-2; p<0.001) in both HMVEC and D-HMVEC. Conclusion: Diabetic endothelial cells showed impaired angiogenesis compared to non-diabetic endothelial cells. Combinations of temporally separated pro-angiogenic growth factors were able to optimize the angiogenic effects and yield the most significant efficacy in the setting of both diabetes and non-diabetes. Future studies will focus on in vivo temporally separated combinations of multiple growth factors.