A. SPECIFIC AIMS: Diabetes is widespread in the United States, and its complications have devastating effects on health and quality of life. Impaired wound healing is a serious complication that leads to the development of chronic wounds in the lower extremities. Patients with such wounds display decreased mobility, and overall health, and are the most frequently subjected to limb amputation. Five year mortality following major amputation is over 80%. Wound care practices can be effective, but a large fraction of diabetic ulcers still persist or lead to amputation. Novel therapeutic strategies must focus on the pathological mechanisms underlying impaired healing in order to improve patient outcomes. Wounds become chronic due to diabetic hyperglycemia, including reduced blood flow, hypoxia, and aberrant behavior of immune cells, fibroblasts, and keratinocytes that are critical to healing. Recent unbiased analyses, such as single RNA seq, have revealed the critical involvement of fibroblasts and extracellular matrix (ECM) production. Surprisingly, little attention has been paid to the role of ECM proteins as negative modulators of healing. Work in our laboratory suggests the contribution of the matricellular protein thrombospondin-2 (TSP-2) to diabetic wounds. Normally, TSP-2 is expressed in wounds and contributes to ECM remodeling and vascular regression, in part, by increasing and decreasing the levels of matrix metalloproteinase (MMP)-2/9 and lysyl oxidase (LOX) and ECM crosslinking, respectively. Our recent work has shown that TSP-2 is elevated in the wounds of diabetic patients and mice suggesting a correlation between compromised wound healing and high TSP-2 expression. These findings allowed us to put forward the overarching premise that abnormal ECM production and assembly contribute to prevention of wound closure. We obtained partial confirmation for this hypothesis by generating diabetic mice lacking TSP2 (dbdb-DKO mice) and observed normalization of healing. In this application, we propose studies to explore and exploit ECM phenotypes in diabetes. Specifically, we plan to characterize ECM abnormalities and their contribution to cell dysfunction in vitro and in vivo. In addition, the newly generated dbdb-DKO mouse will be used to probe a novel ECM phenotype. We also aim to translate our findings into a bioengineering approach that will provide proof-of-principle for the utilization of novel ECMs as part of a combinatorial therapeutic strategy. These studies will include ECM obtained from a TSP2 KO pig that we have created for the provision of clinical grade materials. Accordingly, the specific aims of this application are: Specific Aim 1. To test the hypothesis that fibroblast dysfunction and altered ECM production and assembly in diabetes contribute to delayed wound closure. We will perform wound healing in these mice and monitor recovery of biomechanical integrity, a critical aspect of healing. We will also perform in vitro studies to...