A. J. Godwin1, W. Yang1,2, A. Sharma2, J. Nicastro1, G. F. Coppa1, P. Wang1,2 1Hofstra North Shore-LIJ School Of Medicine,Surgery,Manhasset, NY, USA 2The Feinstein Institute For Medical Research,Manhasset, NY, USA
Introduction: Acute cutaneous wounds from trauma can become chronic non-healing wounds, resulting in a significant morbidity and mortality to the patients. Wound healing consists of a complex, dynamic process which involves three overlapping processes, namely; inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular levels is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) has been identified to involve in controlling programmed necrosis in response to TNF-α during inflammation. We then hypothesized that RIPK3 regulated the progress of cutaneous wound healing.
Methods: Full-thickness 2.0-cm diameter circular wounds were created on the dorsum of male wild-type (WT) and RIPK3-knockout (KO) mice on C57BL/6 background. Wound area was measured daily until day 14 post-wound and the pixel of the traced area was analyzed by NIH ImageJ. Skin tissues were collected from the wound sites at various days for histological evaluation and gene expression analysis by qPCR. Mouse embryonic fibroblasts (MEFs) were isolated from WT and RIPK3-KO mice for a transwell migration assay.
Results: The wound healing rate in RIPK3-KO mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in RIPK3-KO mice was 53% larger than WT mice (n=7/group, P < 0.05). H&E and Masson-Trichrome staining indicated that RIPK3-KO wounds had a worse quality of wound closure and less collagen deposition in comparison with WT wounds. The number of Gr-1-positive cells for indicating neutrophils infiltrating in RIPK3-KO wounds was much less than WT wounds at day 1. The expression of growth factors (VEGF and TGF-β) and proinflammatory cytokines (TNF-α and IL-1β) were less and delayed in RIPK3-KO wounds, compared to WT wounds (Table). Furthermore, the numbers of migrated MEFs from RIPK3-KO mice toward PDGF and TGF-β, as chemoattractants, were 62% and 73%, respectively, lower than those of MEFs from WT mice.
Conclusion: RIPK3-KO mice exhibit impairment of wound healing in association with reduced and delayed production of growth factors and proinflammatory cytokines. The chemotactic activity of RIPK3-deficient fibroblasts to growth factors is suppressed. Thus, RIPK3 is an important molecule required for normal progression of wound closure.
