Improved diabetic wound healing through topical silencing of p53 is associated with augmented vasculogenic mediators

Abstract

Diabetes is characterized by several poorly understood phenomena including dysfunctional wound healing and impaired vasculogenesis. P53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with siRNA. We hypothesized that silencing p53 results in improved diabetic wound healing and augmentation of vasculogenic mediators. Paired 4-mm stented wounds were created on diabetic db/db mice. Topically applied p53 siRNA, evenly distributed in an agarose matrix, was applied to wounds at post-wound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at post-wound days 10 and 24. 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. ANOVA/t-test was used to determine significance (p<=0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18 ± 1.3d in the treated group versus 28 ± 1.0d in controls. The treated group demonstrated improved wound architecture at each time point while demonstrating near complete local p53 knockdown. Moreover, treated wounds showed a 1.92 fold increase in CD31 endothelial cell staining over controls. 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. This profile was reversed after treated wounds healed and prior to closure of controls (day 24). Augmented vasculogenic cytokine profile and endothelial cell markers are associated with improved diabetic wound healing in topical gene therapy with p53 siRNA.

Inhibition of PAI-1 Via PAI-039 Improves Dermal Wound Closure in Diabetes

“…One of the most prominent diabetic complications is the impaired ability to heal wounds (14,(18)(19)(20)(21). In the current investigation, decreases in epidermal closure and CD31 content, as well as an increase in epidermal edge thickness exemplify the impairments in healing associated with diabetes.…”

siRNA-based spherical nucleic acids reverse impaired wound healing in diabetic mice by ganglioside GM3 synthase knockdown

“…Much attention has focused on vascular abnormalities in the pathogenesis of the delayed wound healing in diabetes (36)(37)(38). The observation that GM3S SNA treatment almost doubles CD31…”

Overexpression of SIRT1 Promotes High Glucose–Attenuated Corneal Epithelial Wound Healing via p53 Regulation of the IGFBP3/IGF-1R/AKT Pathway

“…A report showed that improved diabetic wound healing through the silencing of p53 is associated with augmented vasculogenic mediators. 34 Furthermore, Vollmar et al 35 found that the transient inhibition of p53 supports the early cell proliferation required for rapid tissue repair and that this may represent an attractive approach for the treatment of delayed wound healing. In addition, the direct effects of SIRT1 on p53 acetylation status have been shown to be indispensable for the repression of cell growth and apoptosis.…”