Development of elastin-like polypeptide fusion protein containing the binding domain of receptors for advanced glycation end-products for diabetic chronic skin wounds

Chronic and non-healing skin wounds are some of the most significant complications in patients with advanced diabetes. A contributing mechanism to this pathology is the non-enzymatic glycation of proteins due to hyperglycemia, leading to the formation of advanced glycation end products (AGEs). AGEs bind to the receptor for AGEs (RAGE), which triggers pro-inflammatory signals that may inhibit the proliferative phase of wound healing. Soluble forms of RAGE (sRAGE) may be used as a competitive inhibitor of AGE-mediated signaling; however, sRAGE is short-lived in the highly proteolytic wound environment. Previous studies have also found detrimental effects of AGEs on growth factor activities that are critical to the normal wound healing process, including epidermal growth factor (EGF), keratinocyte growth factor (KGF), and stromal cell-derived factor-1 (SDF-1). The aim of this dissertation work was to develop a recombinant fusion protein containing the binding domain of RAGE (vRAGE) linked to elastin-like polypeptides (ELPs) that self-assembles into coacervates to shield from proteolysis and provide a drug depot. This protein could act as a competitive inhibitor of AGEs to restore the AGE-mediated impaired biological mechanisms in the treatment of diabetic chronic wounds. This dissertation aimed to characterize the physical properties of vRAGE-ELP and investigate the biological activity in vitro and in vivo. Furthermore, a synergistic effect of vRAGE-ELP, when used in combination with another ELP fusion protein, SDF1-ELP that was previously found to accelerate wound healing and promote vascularization, was also investigated. We report that vRAGE-ELP self-assembles into coacervates around 30-31C. The coacervate size was concentration and temperature-dependent, ranging between 500 and 1600 nm. vRAGE-ELP reversed several AGE-mediated changes in cultured human umbilical vein endothelial cells (HUVECs), including a decrease in viable cell number, an increase in levels of reactive oxygen species (ROS), and an increased expression of the pro-inflammatory marker, intercellular adhesion molecule-1 (ICAM-1). vRAGE-ELP was stable in elastases (common wound fluid proteases) in vitro for 7 days. When used in a single topical application on full-thickness excisional skin wounds in diabetic mice, wound closure was accelerated, with 90% and 100% wound closure on post-wounding days 28 and 35, respectively, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. Furthermore, the combination of vRAGE-ELP and SDF1-ELP increased viable cell numbers in AGE-stimulated HUVECs, promoted tube formation, and accelerated scratch wound assay in vitro. When used in full-thickness excisional skin wounds of diabetic mice, wound closure in the combination groups reached almost 100% on post-wounding day 35, compared to 62% and 85% on the same days in animals treated with vehicle control, consisting of ELP alone. This coacervate system topically delivering a competitive inhibitor of AGEs has potential for the treatment of diabetic wounds.Ph.D.Includes bibliographical reference