Regulation of Angiogenesis in Healing Wounds

Hypothesis: Pigment epithelium derived factor (PEDF) interacts with low-density lipoprotein receptor-related protein 6 (Lrp6) to modulate wound angiogenesis. Objective: Previous studies demonstrate that anti-angiogenic proteins such as pigment epithelium-derived factor (PEDF) cause the pruning of capillaries in wounds and are critical to wound resolution. This study assesses the function of PEDF and its likely receptor, Lrp6, in wound angiogenesis. Methods: Adult PEDF -/- mice and wild-type littermate controls (C57 BL/6) (WT) were anesthetized, 3mm diameter skin wounds were prepared, and harvested at days 1, 3, 5, 7, 10, and 21 post-wounding. Vessel permeability was analyzed using fluorescein isothiocyanate (FITC) dextran. The effectiveness of siRNA transfection in reducing Lrp6 was demonstrated in vitro. Wounds were treated with Lrp6 siRNA or scrambled siRNA to examine the effect of Lrp6 knockdown on angiogenesis in vivo. Immunohistochemistry was performed to localize Lrp6 and to determine CD31+ blood vessel density in the wound bed. Results: Wound vascularity was significantly elevated in PEDF -/- compared to WT mice at days 0, 7, and 21. Vascular permeability, quantified using extra-vascular dextran, was also higher in PEDF -/- vs WT mice. Treatment of wounds with rPEDF caused an increase in Lrp6 expression on endothelial cells (EC) at day 10 (p=0.05). Lrp6 knockdown by siRNA caused a significant increase in wound angiogenesis in vivo (p<0.05). Conclusion: PEDF is required for proper vascular pruning and maturity in the resolving wound. PEDF stimulates the expression of Lrp6 on wound vasculature, a response that allows for appropriate endothelial responsiveness to PEDF. Inhibition of Lrp6 leads to increased wound vascularity, suggesting that PEDF acts through this receptor. The findings indicate that the interaction of PEDF with LRP6 is critical for the regulation of wound angiogenesis.