Interleukin-10 Improves Diabetic Wound Neovascularization Via Endothelial Progenitor Cell (epc) Recruitment
Emily Steen1, Swathi Balaji1, Kenneth Liechty2, Timothy Crombleholme2, Paul Bollyky3, Sundeep Keswani1.
1BCM, Houston, TX, USA, 2U Colorado SOM, Aurora, CO, USA, 3Stanford University SOM, Stanford, CA, USA.
Introduction: Deficiencies in EPC recruitment and function are well-documented in chronic diabetic wounds. The anti-inflammatory cytokine IL-10 was shown to contribute to neovascularization in a murine myocardial infarction model. We report a novel role for IL-10 in promoting neo-angiogenesis in normal and diabetic wound healing by testing the hypothesis that IL-10 promotes EPC recruitment from bone marrow to dermal wounds by regulating stromal-cell derived factor 1(SDF1-alpha) and vascular endothelial growth factor(VEGF) via a STAT3-dependent mechanism. Methods: We evaluated the impact of IL-10 overexpression(lentiviral IL-10 1x106 TU) in WT and db/db-diabetic wounds(n=4/group): 4mm stented wounds were analyzed at day7 for EPCs(CD133+/Flk1+cells/HPF; confocal microscopy), neovascularization(Meca32+vessels/HPF), and wound healing/inflammation (H&E;F4/80+cells/HPF). Skin-specific, tamoxifen-inducible STAT3 knockout mice modelled the mechanism of EPC mobilization and angiogenesis: circulating EPCs(CD34+/CD133+/Flk1+ cells;FLOW-cytometry) and VEGF and SDF1-alpha in bone marrow, serum and wounds(ELISA;qPCR;IHC) were quantified at day3. In vitro, WT dermal fibroblasts(FB) treated with IL-10(200 ng/ml) were analyzed for VEGF and SDF1-alpha. The effect of IL-10 and IL-10-treated FB-conditioned media on angiogenesis were tested in aortic ring assay. Data presented as mean±SD;p-values by ANOVA. Results: At day7, lenti-IL-10-overexpression significantly improved wound healing, EPC expression(p<0.01), and neovascularization(p<0.001) while significantly reducing macrophage abundance(p<0.001) in normal and diabetic wounds. In WT-mice, lenti-IL-10-overexpression significantly increased circulating EPC levels at day3 post-wounding(2.7±0.5fold;p<0.001), which was associated with increased SDF-1alpha expression in serum/wounds and decreased in marrow(p<0.01). Similarly, lenti-IL-10-overexpression at day3 demonstrated increased serum, wound, and marrow VEGF expression(p<0.05). STAT3-knockdown abrogated the aforementioned day3 and day7 effects of IL-10(p<0.01). In vitro, IL-10 significantly increased FB production of VEGF(p<0.05) and SDF1-a(p<0.05). Relative sprouting area on aortic ring assay increased in IL-10-treated FB-conditioned media as compared to FB-conditioned media alone or media+IL-10 (p<0.01), suggesting FB as a potential target cell for IL-10-induced angiogenic effects. Conclusion: IL-10-driven EPC recruitment via STAT3, VEGF, and SDF-1alpha may be an innovative strategy to induce therapeutic angiogenesis and tissue repair in normal and diabetic wounds.
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