Abstract 007: Athero-protective Flow Regulation of ITPR3: an Epigenetic Approach

Background: The topographic distribution of atherosclerosis in human vasculature underscores the role of shear stress in regulating gene expression and function in the endothelium. Recent advances in understanding of mechanotransduction reveal that epigenetic regulation is integral to shear stress-mediated gene expression. With the use of ChIP-seq and ATAC-seq, the current study aims to demonstrate the link between histone modifications and transcriptional regulation in endothelial cells (ECs) in response to shear stress. Methods and Results: We found that pulsatile shear (PS)- and oscillatory shear (OS)-induced differential H3K27ac enrichments were associated with adjacent gene expression in ECs, approximately 30% of which showed significant positive correlation (Pearson’s correlation coefficient >0.7). In silico prediction revealed that Krüppel-like factor 4 (KLF4) binding motifs were enriched in the PS-enhanced H3K27ac regions. By integrating genes that are induced by PS, have the KLF4 binding loci, and contain PS-associated H3K27ac in their promoter regions, we identified 18 novel PS-upregulated genes. Validating these results in mouse ECs isolated from intima of the thoracic aorta vs aortic arch, lung ECs from EC-KLF4-TG vs EC-KLF4-KO mice, and atorvastatin-treated vs control ECs, we found that Inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) had the most robust expression in multiple systems. Consistent with these findings, ATAC-seq and ChIP-qPCR demonstrated a specific locus in the promoter region of the ITPR3 gene that was essential for KLF4 binding, H3K27ac enrichment, chromatin accessibility, and ITPR3 transcription. Deletion of this KLF4 binding locus in ECs by using CRISPR-Cas9 resulted in blunted calcium influx, reduced eNOS expression, and diminished NO bioavailability. Furthermore, ITPR-KO mice fed an atherogenic diet resulted in exacerbated atherosclerosis compared to wildtype littermates. Conclusions: Using a multi-layer systems approach, we have demonstrated that KLF4 is crucial for the histone modifications that allow the transcriptional activation of ITPR3 in ECs. This novel mechanism contributes to Ca2 + -dependent eNOS activation and EC homeostasis by maintaining an athero-protective phenotype.