Vascular Function in Diabetic Wound Healing
Okonkwo, Uzoagu Alexis
MetadataShow full item record
Vascular deficits are recognized as a fundamental contributing factor of diabetes-associated diseases. In diabetic wound healing, changes in capillary growth and function are thought to greatly affect wound healing outcomes. Although multiple previous studies have demonstrated that the proangiogenic phase of wound healing is greatly blunted in the context of diabetes, a comprehensive understanding of the mechanisms that regulate skin revascularization and capillary stabilization in diabetic wounds is lacking. The work presented here uses imaging modalities and molecular analysis to carefully examine how vascular maturation and functionality differs in diabetic wounds compared to normal wild type wounds. To begin to examine vascular deficits in the context of diabetes, we first sought data in human subjects to show that alterations exist in human diabetic ulcers. For these experiments, we analyzed existing genomic databases to determine if human diabetic foot ulcers show differential expression of vascular maturation markers. We found that in human diabetic foot skin there is significant downregulation in many maturation factors involved in capillary pruning and stability. Following these experiments, we employed an experimental model of diabetic wound healing, that of the genetically diabetic db/db mouse, to examine vascular repair in diabetic wounds in detail. In the db/db mouse experiments, we first we confirmed that genetically diabetic mice show a slower closure rate and delayed onset of angiogenesis during wound healing with closure achieved between days 17-19 compared to days 11-13 in wild type control. With this knowledge, we went on to examine the expression of a large group of known factors that influence capillary recruitment, maturation, and stability, and along with markers that are expressed in dermal pericytes. These studies demonstrated that diabetic wounds have significant perturbations in almost all aspects of vascular regrowth and maturation. Specifically, we saw significant down regulation in the following factors: VEGF-A, SPRY2, PEDF, LRP6, TSP1, IP10, CXCR3, PDGFR-β, HB-EGF, EGFR, TGFβ-1, Sema3a, Nrp1, Ang2, NG2, and RGS5. Next, we investigated angiogenesis by staining for CD31+ ECs. We found that at D10 post wounding that diabetic mice have significantly decreased levels of capillaries in the wound bed. Pericyte coverage, a marker of capillary maturation, was then quantified in both wild type and diabetic wounds. We found a trend of diabetic wound capillaries having an increased number of capillaries without pericyte association. Turning our attention to functional attributes of wound capillaries, the permeability of capillaries was compared in diabetic wounds and normal wild type wounds at D10 post wounding using FITC-conjugated, high molecular weight dextran. Here, we concluded that diabetic vessels exhibit significantly increased amounts of extravascular leakage of FITC-dextran reagent compared to normal wounds. Lastly, we used microCT to explore the dynamics of vessel growth and regression during wound healing. This approach allowed us to compare capillary architecture in detail in diabetic and normal wild type wounds. Our data suggests that vessels initially exhibit a significantly increased number of vessel volume, vessel surface area, vessel length, and vessel branches at D7 compared to D14 and 21. Together, these studies provide novel information about the complexity of the perturbation in angiogenesis that is seen in wounds of diabetic individuals.
Subjectwound healing, diabetes, angiogenesis