BACKGROUND:
The pathogenesis of impaired wound healing in diabetes is multi-factorial and not completely elucidated. P53, a master cell cycle regulator, is upregulated in diabetic wounds and has recently been shown to play regulatory roles in vasculogenic as well as apoptotic pathways. Previously, we have 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 by promoting vasculogenesis and abrogating apoptosis.
METHODS:
Dermal fibroblasts from diabetic (db/db) and wild-type C57/BL6 mice were explanted and grown in culture. Untransfected cells and cells transfected with nonsense siRNA or p53 siRNA were placed in normoxic (21% O2) or hypoxic (1%) conditions for 24 hours. Markers (caspase-3) and mediators (Bax, Bcl-2) of apoptosis were measured by real time quantitative PCR (RT-PCR) and FACS. Paired 4mm stented wounds were created on diabetic db/db mice. Nonsense siRNA or p53 siRNA, distributed evenly in an agarose matrix, was topically applied to wounds at post-wound day 1 and 7. 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).
RESULTS:
FACS demonstrated a 212.1% increase in caspase-3 expression in diabetic fibroblasts exposed to hypoxia compared to a 74.2% increase in wild-types. Real time quantitative PCR demonstrated a 7.5 fold increase in Bax expression compared to a 3.2 fold increase in Bcl-2 expression in diabetic fibroblasts exposed to hypoxia. After p53 silencing, there was a blunting of cellular response to hypoxia with a 12.0 fold decrease in Bax expression and 6.0 fold decrease in Bcl-2 expression. Silencing of p53 also negated Bax or Bcl-2 upregulation in wild-type cells exposed to hypoxia. Wounds closed faster in local p53 silenced wounds (18 ±1.3d) versus controls (28 ± 1d) (p<0.05). Histology of untreated animals revealed scant tissue within the wound bed, while the treated group showed near complete local p53 knockdown and abundant granulation tissue. Treated wounds showed a 7.6 fold increase in CD31 endothelial cell staining over controls. PCNA immunohistochemical staining showed increased proliferation while cytochrome-c staining demonstrated 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 1.9 fold increase in SDF-1 expression in treated wounds versus controls. This profile returned to baseline upon closure of both untreated and treated wounds.
CONCLUSIONS:
Improved diabetic wound healing is associated with abrogated apoptosis and augmented vasculogenic markers through topical gene therapy with p53 siRNA. These results aid in further elucidating targets for potential improvement of diabetic wound healing.