An Agonistic Monoclonal Antibody Targeting cMet Attenuates Inflammation and Up-Regulates Collagen Synthesis and Angiogenesis in Type 2 Diabetic Mice Wounds

Abstract

Background: Diabetic wounds account for 25 to 50 percent of total diabetic health care costs annually, and present overall healing rates of less than 50 percent. Because delayed diabetic wound healing is associated with impaired fibroblast function, the authors hypothesize that tyrosine kinase Met (cMet) agonistic monoclonal antibody will promote diabetic wound healing by means of stable activation of hepatocyte growth factor/ cMet signaling. Methods: Two 6-mm dorsal wounds were created in each mouse (6-week-old, male BKS. Cg-Dock7(m)+/+Lepr(db)/J; n = 5). After subcutaneous injections of agonist (20 mg/kg) at 0 and 72 hours, the wound sizes were measured at days 0, 1, 3, 6, and 10. Histologic and immunohistochemical analyses were performed at day 10 (cMet, a-smooth muscle actin, CD68, and transforming growth factor-ss). In vitro cytotoxicity and migration tests with diabetic fibroblasts were performed with or without agonist treatment (1 or 10 nM). cMet pathway activation of fibroblasts was confirmed through p-p44/42 mitogen-activated protein kinase, p-mTOR, p-cMet, and ROCK-1 expression. Results: The cMet agonistic monoclonal antibody-treated group showed 1.60-fold lower wound area (p = 0.027), 1.54-fold higher collagen synthesis (p = 0.001), and 1.79fold lower inflammatory cell infiltration (p = 0.032) than the saline-treated control. The agonist increased cMet (1.86-fold; p = 0.029), a-smooth muscle actin (1.20-fold; p = 0.018), and vascular endothelial growth factor (1.68-fold, p = 0.029) expression but suppressed CD68 (1.25-fold; p = 0.043), transforming growth factor-ss (1.25-fold; p = 0.022), and matrix metalloproteinase-2 (2.59-fold; p = 0.029) expression. In vitro agonist treatment (10 nM) of diabetic fibroblasts increased their migration by 8.98-fold (p = 0.029) and activated the hepatocyte growth factor/cMet pathway. Conclusions: Tyrosine kinase Met agonistic monoclonal antibody treatment improved diabetic wound healing in mice and reduced wound-site inflammatory cell infiltration. These results need to be validated in large animals before piloting human trials.