Abstract Or103: Smooth Muscle Cells-Expressed Bmal1 Regulates Vascular Calcification Independent of the Canonical Circadian Pathway

Vascular calcification is a common complication of diabetes mellitus, which has been associated with the osteogenic differentiation of vascular smooth muscle cells (VSMC). Disrupted circadian rhythms have been linked to insulin resistance and the progression of diabetes. However, the mechanisms underlying the regulation of circadian proteins in diabetes, particularly in VSMC and its contribution to vascular calcification remain largely unknown. We found that the Basic Helix-Loop-Helix ARNT-like protein 1 (Bmal1), a key regulator of the circadian clock, was upregulated in mouse VSMC exposed to high glucose as well as in diabetic mice arteries. To elucidate the function of Bmal1 in VSMC in vivo, we generated a smooth muscle cell (SMC)-specific Bmal1 deletion (Bmal1 Δ/Δ ) mouse model, and we found SMC-specific Bmal1 deletion reduced vascular calcification in diabetes in vivo . RNA sequencing and Gene Ontology analysis revealed that downregulated genes in Bmal1 Δ/Δ VSMC were involved in bone trabecula formation, osteoblast differentiation, and cellular response to TGF-β stimulus, suggesting a potential pro-osteogenic role for Bmal1 in VSMC. Furthermore, Bmal1 Δ/Δ VSMC exhibited reduced expression of the master osteogenic regulator, Runt-related transcription factor 2 (Runx2), and its target gene osteocalcin. Mechanistic studies identified Bmal1 DNA binding sites in the Runx2 promoter region, in close proximity to Assay for transposase-accessible chromatin (ATAC), H3K4me3, H3K9ac, and H3K27ac peaks, suggesting potential Bmal1 binding and increased epigenetic activity and chromatin accessibility that facilitate Runx2 gene expression. Consistently, Bmal1 overexpression increased luciferase activity driven by the wild-type Runx2 promoter but not the Runx2 promoter containing a mutated Bmal1 binding site. Using CRISPR-Cas9 to disrupt the Bmal1 binding site in the Runx2 promoter in VSMC, this mutant VSMC showed the abolished Bmal1 overexpression-induced Runx2 expression. Collectively, these studies have demonstrated that increased Bmal1 mediates diabetes-induced vascular calcification through transcriptional regulation of Runx2 in VSMC, by directly binding to the Runx2 promoter. Our studies have uncovered a novel Bmal1-dependent non-canonical circadian pathway in regulating diabetes-induced vascular calcification. Therefore, maintaining normal levels of the clock regulator Bmal1 could improve VSMC function and vascular health in diabetes.