Decreased Numbers of SDF-1A-Expressing Cells in Peripheral Wounds of Diabetic Mice

Katherine Gallagher, MD, Lee Goldstein, MD, April Nedeau, MD, Haiying Chen, MD, Jun Liu, MD, Min Xaio, MD and Omaida Velazquez, MD
University of Pennsylvania, Philadelphia, PA

Introduction: Work from our laboratory and others have reported a crucial role for bone marrow-derived endothelial cells (EPC) in wound healing and a deficiency in circulating EPC in diabetic mice. Stromal derived growth factor 1-alpha (SDF-1A) has been identified as the specific chemokine that mediates recruitment of EPC into ischemic tissue undergoing post-natal vasculogenesis but the effect of the diabetes phenotype on SDF-1A expression at the wound level has not been previously studied. In addition, while it is known that SDF-1A serves as a homing signal for EPC to migrate from circulation to tissue, it remains unknown as to whether SDF-1A levels at the wound influence mobilization of EPC into circulation from central stores, such as the bone marrow. We hypothesized that SDF-1A expression is decreased in wounds with induction of the diabetic phenotype and that augmentation of levels of SDF-1A in wounds of diabetic mice results in increased circulating EPC.
Methods: 20 FVB mice underwent 4 mm punch biopsy wounding of their hindlimb. 15 of these mice were pre-treated with streptozocin (STZ) to induce diabetes (mean glucose = 471mg/dL) one week prior to the wounding. Wounds from STZ diabetic mice (N=5) and non-diabetic controls (N=5) were analyzed for SDF-1A expression by immunohistochemistry. The percentage of cells expressing SDF-1A was quantified relative to the total wound cellularity in 4 serial cross-sections per wound, counting 10 random high power fields (HPF) at 100X magnification. The circulating EPC pool was quantified in the remaining STZ diabetic mice using flow cytometric analysis. Peripheral blood from mice treated with either direct wound injections of 25 ug/Kg purified SDF-1A (N=5) or saline wound injections (N=5), was harvested and EPC were identified as cells labeled with both CXCR4 (immature progenitor cell marker) and VEGFR-2 (mature endothelial cell marker).
Results: Peripheral wounds of STZ-induced diabetic mice had a significantly decreased (by nearly 50%) number of cells expressing SDF-1A compared with non-diabetic controls (P<0.001, Figure 1). The number of EPC in peripheral circulation in wounded STZ-induced diabetic mice treated with SDF-1A demonstrated a 2-fold increase when compared to saline-injected controls (P<0.05, Figure 2).
Conclusions: To our knowledge, this is the first study to demonstrate that in mice with diabetes, there is a pronounced decrease in the number of SDF-1A-expressing cells within peripheral skin wounds. Moreover, the direct injection of SDF-1A purified protein into the wounds enhances mobilization of EPC into circulation. The mechanisms for this latter effect requires further study.
This work has significant clinical implications for the development of potential therapies that address both the diabetes related deficit in the number of circulating EPC and the wound-level expression of the key EPC-homing chemokine, SDF-1A.