S. Balaji1, R. Ranjan1, C. Moles1, N. Hann1, T. M. Crombleholme1,2, S. G. Keswani1 1Cincinnati Children’s Hospital Medical Center,Pediatric General Thoracic And Fetal Surgery,Cincinnati, OH, USA 2Children’s Hospital Of Colorado,Center For Children’s Surgery,Aurora, CO, USA
Introduction: The diabetic wound healing phenotype is in part characterized by impaired neovascularization and deficient endothelial progenitor cell (EPC) recruitment. Over expression of angiogenic growth factors to improve EPC recruitment and wound healing is a novel strategy to treat diabetic wounds. Angiopoetin-1 (Ang1) is a potent mobilizer of EPCs from the bone marrow (BM) and we have shown that Ang1 has direct effects on reepithelialization. A suggested mechanism for EPC mobilization from the BM is mediated through MMP9 and stem cell factor (SCF). Taken together we hypothesize that Ang1 overexpression in diabetic wounds will recruit EPCs and improve neovascularization and wound healing.
Methods: To track BM derived EPCs we used an endothelial lineage BM labelled murine model with diabetes. FVBN mice were lethally irradiated and subsequently reconstituted with BM from syngeneic TIE-2/LacZ donor mice. Diabetes was induced with streptozotocin (STZ). 8 mm wounds were created in BM transplanted mice (BMT) (n=12) and treated with 10 8 PFU of AdAng1, AdGFP or PBS. Wounds were harvested at day 7 post wounding and analyzed for epithelial gap, vessel density and Tie-2/LacZ positive EPCs recruitment. Serum levels of VEGF, proMMP9 and SCF were assessed by ELISA. To determine if Ang1 effects were EPC dependent a simiar experiment was conducted in EPC mobilization deficient mice (MMP9 KO). Data presented as mean+/-SEM, p-values by ANOVA.
Results: All mice survived at 4 weeks post BMT. Mice with STZ-induced diabetes had blood glucose >350mg/dl. Ang1 resulted in significantly improved reepithelialization (Ang1 2.3+/-.2mm; GFP 3.9+/-.2; PBS 4.0+/-.1 p<.0001), neovascularization (Ang1 6.8+/-.3Caps/Hpf; GFP 3.0+/-.4; PBS 2.9+/-.3 p<.0001) and EPC recruitment (Ang1 5.3+/-.4 EPCs/Hpf; GFP 2.1+/-.3; PBS 2.2+/-.3 p<.0001) in diabetic BMT wounds at day 7. Ang1 treatment resulted in increased levels of proMMP9 (Ang1 9.7+/-.8 ng/ml; GFP 6.3+/-.9; PBS 6.4+/-.4 p<.01) and SCF (Ang1 265+/-28 pg/ml; GFP 119+/-16; PBS159+/-12 p<.001) but had no effect on VEGF. Peripheral blood EPC (CD34+/Cd133+/Flk1+) counts at day 3 post wounding using FACS demonstrated impaired EPC mobilization in MMP9 KO mice compared to wild type controls (MMP9 KO 0.66+/-0.02; WT 1.1+/-0.05 p<0.001), which was rescued by SCF administration (MMP9 KO+SCF 1.23+/-0.09; WT 1.1+/-0.05 p=ns), validating this model for EPC loss-of-function mechanistic studies. In MMP9 KO mice, AdAng1 similarly accelerated reepithelialization (Ang1 3.2+/-.1mm; GFP 4.1+/-.2; PBS 3.8+/-.2; p<.05), but had no effect on neovascularization (Ang1 4.1+/-.5 Caps/HPF; GFP 3.9+/-.4; PBS 3.9+/-.6).
Conclusion: Our results demonstrate that Ang1 results in improved neovascularization which is dependent on EPC recruitment and that it has direct effects on wound reepithelialization. These data may represent a novel strategy to correct the impaired diabetic neovascularization phenotype and improve diabetic wound healing.