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 a regulatory roles in vasculogenic pathways. We have previously described a novel method to topically silence target genes in a wound bed with small interfering (si)RNA. 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 postwound day 1 and 7 (matrix alone and nonsense siRNA served as controls). Animals were sacrificed at postwound 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, reverse transcription-polymerase chain reaction, and enzyme-linked immunosorbent assay. The ANOVA/t-test was used to determine significance (p≤ 0.05). Local p53 silencing resulted in faster wound healing with wound closure at 18±1.3 d in the treated group vs. 28±1.0 d 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 (enzyme-linked immunosorbent assay) was significantly increased in treated wounds (109.3±13.9 pg/mL) vs. controls (33.0±3.8 pg/mL) while reverse transcription-polymerase chain reaction demonstrated a 1.86-fold increase in SDF-1 expression in treated wounds vs. controls. This profile was reversed after the treated wounds healed and before 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.

Figure 1

a) Western blot of wound tissue homogenate displaying marked knockdown of p53 after topical application of agarose matrix containing siRNA against p53. b) p53 mRNA expression in p53 siRNA treated wounds compared to nonsense siRNA treated wound and nontreated control wounds.

Figure 2

Immunohistochemical staining for p53 demonstrates reduced expression of p53 within p53-silenced wound beds compared to nonsense-treated wounds at post-wounding day 10.

Figure 3

Vasculogenic cytokine (HIF-1, SDF, and VEGF) mRNA expression in p53 siRNA treated wounds compared to nonsense siRNA treated wounds at post-wounding day 10. All cytokines demonstrate an upregulation of expression in p53-silenced wounds compared to nonsense siRNA treated wounds.

Figure 4

Vasculogenic cytokine (HIF-1, SDF, and VEGF) mRNA expression in p53 siRNA treated wounds compared to nonsense siRNA treated wounds at post-wounding day 24. Wounds no longer demonstrate the increase in vasculogenic cytokines observed in the open wound at the earlier time point.

Figure 5

H&E (top), and CD31 (bottom) immunofluorescent stained histologic sections of dorsal wound beds with nonsense control or p53 siRNA treatment, demonstrating 7.63 fold increase in CD31 staining in p53 siRNA treated wounds compared to controls.

Figure 6

Photometric image of diabetic stented wounds at day 14 (top) and graph of time to closure (bottom) in p53 siRNA-treated versus nonsense-treated siRNA wounds.