Microbe-derived Butyrate Activation Of Free Fatty Acid Receptor3 Reduces Neointimal Hyperplasia After Arterial Injury By Regulating Immune Response Transcription Networks In Endothelial Cells

Introduction: Microbe-derived butyrate has been shown to reduce neointimal hyperplasia after transluminal arterial injury, an effect that is dependent on free fatty acid receptor-3 (FFAR3). However, the mechanism by which FFAR3 affects the endothelial response to injury is not known. We hypothesize that FFAR3 regulates endothelial cell (EC) migration and proliferation by modulating transcriptional responses. Methods: FFAR3 wildtype (WT) and knockout (KO) mice were fed a high fiber diet to promote microbial butyrate production followed by femoral artery injury. Arterial morphometric analysis was performed after 4 weeks. Stable shRNA-mediated knockdown of FFAR3 in primary HUVEC lines were generated using 3 rd generation lentivirus. In scratch assays, confluent HUVEC monolayers were subjected to a longitudinal “scratch” which was quantified at 0 and 8 hours. Proliferation (Ki67) and cell cycle (propidium iodide) analysis was assayed by flow cytometry. Bulk RNA-seq was performed on HUVEC and endothelial fractions of FFAR3 WT and KO mouse aortas. Gene set enrichment analysis (GSEA) was used to identify transcriptional pathways affected by FFAR3. Results: FFAR3 WT mice on a high fiber diet had 18.3% reduced neointimal hyperplasia 2 weeks after injury compared to KO mice (p<0.01) despite similar stool concentrations of butyrate. Compared to shScramble, shFFAR3 knockdown in HUVEC inhibited cell migration by 40.4% (p=0.02), which was not rescued by 1-methylcyclopropane carboxylate, a FFAR3 agonist. shFFAR3 knockdown reduced Ki67 by 50.7% (p<0.01) and increased the proportion of cells in G0/G1 phase by 15.3% (p<0.01), while reducing the proportion in S and G2 phases by 39.5% (p<0.01) and 18.7% (p=0.02), respectively. Pathway analysis of the transcriptomes from shFFAR3-HUVEC and ECs from FFAR3 KO aortas revealed significant overlap in immune regulatory pathways. Conclusions: FFAR3 activation by microbe-derived butyrate may promote EC migration and proliferation by stimulating progression through the cell cycle and modulating immune-related transcriptional pathways to limit neointimal hyperplasia after arterial injury.