Abstract 17495: Thrombospondin-1/CD36 Pathway Critically Contributes to SHH Signaling Defect and EPC Dysfunction on Wound Healing in Type 1 Diabetes

Abstract

Introduction: Refractory wounds in diabetic patients is a significant clinical problem. We reported previously that sonic hedgehog (SHH), a morphogenic protein central to wound repair, is deficient in diabetes, but the underlying mechanisms are poorly understood. We hypothesized that thrombospondin-1 (TSP-1) through its receptor CD36 contributes to SHH signaling defect in cutaneous tissue and endothelial progenitor cells (EPCs), resulting in impaired wound healing in type 1 diabetic mice. Methods and Results: Cutaneous expression of SHH and its signaling molecules including Patched (Ptc), Smoothened (Smo), and Gli were significantly decreased in streptozotocin (STZ)-induced type 1 diabetic mice (male, C57BL/6J, 12 weeks) vs. their age-matched control littermates (Western blot analysis, n=5, p<0.05 vs. control), which were reversed in STZ-treated TSP-1 -/- mice (homozygote, C57BL/6J background, n=5, p<0.05 vs. STZ-treated WT mice). Excisional wound (6-mm punch biopsy) closure was significantly delayed in STZ mice (n=6, p<0.05 vs. control) with reduced wound blood flow (Laser Doppler), which was not observed in STZ-treated TSP-1 -/- mice (n=5, p<0.05 vs. STZ-treated WT mice). The protective effect of TSP-1 depletion on wound repair was abolished following topical treatment with the selective SHH inhibitor cyclopamine (5 μ M, 30 min/day, n=5, p<0.05 vs. STZ-treated TSP-1 -/- mice). Isolated EPCs from bone marrow of STZ mice showed significantly impaired angiogenesis (Matrigel tube formation, n=4, p<0.05 vs. control), which was rescued by exogenous SHH (1 mg/L, 24 hours, n=4, p<0.05 vs. STZ-control). Increased EPC angiogenesis in STZ-treated TSP-1 -/- mice (n=5, p<0.05 vs. STZ-WT) was inhibited by the selective SHH inhibitor cyclopamine (5 μ M, 24 hours, n=5, p<0.05). SHH, Ptc, Smo and Gli levels were significantly increased in EPCs from TSP-1 -/- mice and CD36 -/- mice, but not CD47 -/- mice (n=6, p<0.05 vs. WT). Normal EPCs treated with recombinant human TSP-1 protein (2.2 nM, 24 hour) exhibited impaired angiogenesis, an effect that was not observed in EPCs from CD36 -/- mice (n=5, p<0.05). Conclusion: TSP-1/CD36 pathway critically contributes to SHH signaling defect, resulting in EPC dysfunction and impaired wound healing in type 1 diabetic mice.