Abstract 127: Arterial Inflammation and Neointimal Hyperplasia Development in a Germ-free Mouse Model of Carotid Ligation

Abstract

Backgound: The microbiome plays a functional role in a number of inflammatory processes and disease states. While neointimal hyperplasia has been linked to inflammation, there has not been direct demonstration of the role of microbiota in this process. Germ-free (GF) mice raised in complete absence of microbes are an invaluable tool for studying causative links between commensal organisms and the host. We hypothesized GF mice would exhibit altered neointimal hyperplasia following carotid ligation compared to conventionally-raised (CONV-R) mice. Methods: Twenty-week-old male C57BL/6 GF mice underwent left carotid ligation under sterile conditions. Maintenance of sterility was assessed by cultivation and 16S rRNA qPCR of stool. Neointimal hyperplasia was assessed by morphometric and histologic analysis of arterial sections after 28 days. Local arterial cell proliferation and inflammation was assessed by immunofluorescence for Ki67 and inflammatory cell markers after 5 days. Systemic inflammation was assessed by multiplex immunoassays of serum. Identically treated CONV-R mice served as controls. GF and CONV-R outcomes were compared using standard statistical methods. Results: All GF mice remained sterile during the study period. Twenty-eight days after carotid ligation, CONV-R mice had significantly more neointimal hyperplasia compared to GF mice, as assessed by intima area, media area, intima+media area, and intima area/(intima+media area). Collagen content of neointimal lesions appeared qualitatively similar on Masson’s trichrome staining. Ki67 immunoreactivity was significantly reduced in GF carotid arteries at 5 days with increased arterial infiltration of neutrophils and M2 macrophages compared to CONV-R. GF mice also had significantly reduced serum IL-17 concentration and nearly undetected IL-10 at 5 days but not 28 days. Conclusions: GF mice have attenuated neointimal hyperplasia development compared to CONV-R mice, which may be related to altered kinetics of wound healing and acute inflammation. Understanding how commensal microbes regulate arterial remodeling could provide new directions in pathophysiology of restenosis and offer novel strategies to reduce restenosis risk via microbiota manipulation.