Abstract
Objective: People with combined ischemic and diabetic wounds of the lower extremities have the highest risk for limb loss, especially for those without surgical revascularization options. We have demonstrated that Poly-ADP-Ribose polymerase (PARP-1) is hyperactivated in hyperglycemic/hypoxic cells and in ischemic/diabetic murine wounds. This study elucidates the molecular mechanisms of PARP-1 mediated impairment of angiogenesis in diabetic/ischemic wounds. Methods: A model of dorsal bipedicle flap-ischemic wounds on diabetic mice was used. The wounds were treated topically with nanoparticle-encapsulated siPARP-1 or vehicle. Wound closure rate and perfusion was analyzed using digital photography and Laser Doppler scanning, respectively. Angiogenetic markers in the tissues were measured by immunohistochemistry. In-vitro endothelial tube formation assay was performed using HUVECs cultured under hyperglycemic and hypoxic conditions. Results: Wounds treated with topical siPARP-1 significantly accelerated wound healing compared to vehicle (from 25% ± 5% to 40%± 8% ( n =7, p < .05) by day 6 and from 50% ± 15% to 75%± 3% ( n =7, p < .05) by day 12, and also exhibited improved tissue perfusion (50%± 5% increase in perfusion units over control on day 6, n =47 p <0.05). Improved capillary density was also observed in the siPARP-1 treated wounds detected by immunohistochemistry for SMA (250%±35% increase in mean fluorescence intensity over control on day 12, n =4, p <0.05) and CD31 (125% ± 15% increase in mean fluorescence intensity over control on day 12, n =4, p <0.05). In-vitro angiogenesis assay showed that PARP-1-silencing significantly enhanced endothelial tube formation of hyperglycemic/hypoxic HUVECs (15± 4 complete polygons as compared to 0 in untreated, n =4, p <0.05). Human angiogenesis PCR-array analysis of pro-angiogenic factors revealed that PARP-1 silencing upregulated FOSL1 transcription by 5-fold ( n =4, p <0.05). Interestingly, co-silencing of FOSL1 in PARP-1 silenced HUVECs resulted in loss of endothelial tube formation. Conclusions: PARP-1 silencing is an effective strategy to promote ischemic-diabetic wound healing. Our data suggest that PARP-1-FOSL1 is a potential novel axis in angiogenesis and PARP-1 could be a promising therapeutic target for improving angiogenesis in these wounds.
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