Introduction: Our lab and others have previously shown that bone marrow progenitor cells contribute to ischemic neovascularization and that this process is impaired in diabetes. However, characterization of these cells and their specific dysfunction in diabetes remains unclear. In this study, we identify MSCs as the progenitor subset which mobilizes from the bone marrow to migrate and engraft in peripheral tissue in response to ischemia and show that this process is significantly impaired at the mobilization step in diabetes.
Methods: A previously described in-vivo model of soft tissue ischemia was utilized in streptazotocin-induced (STZ) diabetic mice (C57/BL6). Bone marrow, whole blood and ischemic skin was harvested for FACS analysis at various timepoints from D0 through D14. In bone marrow, MSCs were identified as lin-/sca1+/CD45-. Hematopoietic cells (HSCs) were identified as lin-/sca1+/ckit+/CD45+. In circulation, EPCs were identified as lin-/flk1+/CXCR4+. To identify progenitor cells once engrafted in peripheral tissue, a murine parabiotic model was employed, pairing GFP and wild-type mice. Two weeks after ischemic surgery on the wild-type mouse, tissue was harvested and analyzed by FACS for endothelial (CD31+/flk1+) and bone marrow (GFP+/CD45) markers. Vascular density in ischemic skin and bone marrow progenitor engraftment was confirmed by CD31 and GFP immunohistochemistry.
Results: FACS analysis of wild-type and diabetic bone marrow revealed a significant decrease in MSCs immediately following ischemic surgery. This population was restored by D7 in wild-type but not diabetic animals. Circulating levels of EPCs were depleted within 12hrs after surgery in diabetics while cells continued to mobilize and circulate in wild-type animals. While a similar change was observed in bone marrow HSCs, engrafted bone marrow-derived cells were CD45-, indicating an MSC origin. Vascular density was significantly reduced in diabetic animals, as evidenced by fewer CD31+/flk1+ cells by FACS analysis in peripheral tissue and lower vessel counts by CD31 immunostaining.
Conclusions: This study represents a significant step in identifying the origin of EPCs and further describing their vasculogenic impairment in diabetes. For the first time, we are able to confirm a mesenchymal, as opposed to hematopoietic precursor cell gives rise to circulating EPCs. Furthermore, this process was found to be arrested at the mobilization step in diabetes. Based on these results, augmentation of the MSC population by cell-based therapies or mobilizing agents represent powerful alternatives to current therapies for the ischemic vascular complications of diabetes.
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