Novel Mechanisms for Augmenting Diabetic Wound Healing

Abstract

Introduction: Impaired diabetic wound healing is multi-factorial and incompletely understood. Reactive oxygen species (ROS) and p53, a master gene regulator, are upregulated in diabetic wounds. We have demonstrated improved diabetic wound healing through a novel topical p53 silencing system. We hypothesized that topical silencing of p53 improves diabetic wound healing through a decrease in ROS. Methods: In vitro, p53 was silenced in Leprdb/db diabetic murine dermal fibroblasts in normoxia (21% O2) and hypoxia (< 1% O2) to simulate injury. In vivo, a murine stented-wound model was used to create two 4 mm wounds on the dorsum of Leprdb/db diabetic mice. Topically applied p53 siRNA or nonsense siRNA, distributed evenly in an agarose matrix, was applied to wounds at post-wound day 1 and 7. In addition, N-acetylcysteine, a ROS scavenger, suspended in agarose gel was applied to wounds from post-wound day 1 and every other day for 10 days (agarose gel alone served as control). Animals were sacrificed at post-wound day 10. Wound time-to-closure was photometrically assessed, and wounds were harvested for histology, immunohistochemistry, and immunofluorescence. Vasculogenic cytokine expression was evaluated via Western blot, RT-PCR, and ELISA. Results: FACS demonstrated a 212.1% increase in caspase-3 expression in db fibroblasts exposed to hypoxia compared to a 74.2% increase in wt animals. RT-PCR demonstrated a 766% increase in pro-apoptotic Bax expression and a 319% increase in anti-apoptotic Bcl-2 expression in diabetic fibroblasts exposed to hypoxia. After p53 silencing, there was a substantial improvement in cellular tolerance to hypoxia with an 11.97 fold decrease in Bax expression and 6.02 fold decrease in Bcl-2 expression. Silencing of p53 also negated Bax or Bcl-2 upregulation in wt cells exposed to hypoxia. Western Blot analysis confirmed p53 knockdown in treated wounds harvested at post wound day 10. Immunohistochemistry confirmed decreased ROS in NAC treated wounds harvested at post wound day 10. Wounds closed significantly faster in local p53 silenced wounds and NAC treated wounds (18±1.3d and 17±1d respectively) versus controls (28±1d) (p<0.05). Histology of untreated db animals revealed scant tissue within the wound bed, while the treated siRNA p53 group showed near complete local p53 knockdown and abundant granulation tissue. Similar results were obtain with NAC application. siRNA p53 treated wounds showed a 7.63 fold increase in CD31 endothelial cell staining over controls. PCNA immunohistochemical staining showed increased proliferation and cytochrome-c staining demonstrating decreased apoptosis in treated wounds versus controls at both time points. Western blot analysis confirmed near complete p53 knockdown in treated wounds. At day 10, VEGF secretion (ELISA) was significantly increased in treated wounds (109.3±13.9 pg/ml) versus controls (33.0±3.8 pg/ml) while RT-PCR demonstrated a 1.86 fold increase in SDF-1 expression in treated wounds versus controls. Conclusions: Our data suggest that p53 silencing improves diabetic wound healing by abrogating apoptosis, augmenting vasculogenesis and modulating reactive oxygen species. Elucidating these mechanisms is a fundamental step towards evolution of targeted therapies for diabetic wound healing and improved patient care.