H. Albadawi1,2, M. W. Koulopoulos1, L. M. Crowley1, H. Yoo1, M. T. Watkins1,2 1Massachusetts General Hospital,Division Of Vascular And Endovascular Surgery, Department Of Surgery,Boston, MA, USA 2Harvard School Of Medicine,Brookline, MA, USA
Introduction: Delayed wound healing is a significant comorbidity associated with peripheral vascular disease. Autophagy is an important cellular process for homeostasis during tissue repair. The role of autophagy in the wound healing process has not been investigated. This study evaluated the effect of the autophagy inhibitor, chloroquine, on cutaneous wound healing in mice.
Methods: Two groups of mice were subjected to excisional full-thickness splinted wounds created on the upper dorsum using a 5mm biopsy punch. The first group of mice were treated daily with an intraperitoneal injection of 50mg/kg chloroquine (CQ, n=8) starting 24hrs before wounding. The second group received saline as an untreated control (CON, n=10). Wound closure was assessed using serial photography and image analysis at days 0, 3, 7, 11 and 14, and expressed as a percentage of the original wound size. On day 14, the mice were euthanized and the wounds were harvested and analyzed by western blotting for markers of autophagy, the ratio of the lipidated autophagosome localized isoform of the microtubule-associated protein Light Chain-3 (LC3-II) to its non-lipidated cytosolic localized isoform (LC3-I) and the expression of Sequestosome-1 (p62). Western blot data was expressed as arbitrary units (AU). Statistical analysis was performed using student-t test and Pearson correlation test.
Results: Wound healing was significantly delayed at days 11 (CQ: 78.0±4.9, CON: 55.7±6.4 percent, p<0.0001) and 14 (CQ: 37±6.8; CON: 18.1±4.8 percent, p=0.028) in the CQ group. Western blot analysis showed significant increase in p62 protein levels (CQ: 0.61±0.06; CON: 0.44±0.04 AU, p=0.02) and LC3-II/I ratio (CQ: 0.61±0.04; CON: 0.37±0.08, p=0.024) in the CQ group. There was a positive correlation between the wound size at day 14 and the p62 protein levels in the same wound (Pearson r=0.83, p=0.02).
Conclusion: These data suggest that autophagy plays a significant role in the wound healing process. The significant increase in the LC3-II/I and p62 protein accumulation demonstrated chloroquine’s inhibitory effect on autophagy. This finding was supported by the positive correlation between wound size at day 14 and the corresponding p62 levels.