Histone modification and chromosome remodeling regulate VSMC phenotypes in aging-associated aortic aneurysm

Aortic aneurysm is a life-threatening vascular disease that has high morbidity and mortality after acute rupture. The development of aortic aneurysm involves the loss of vascular smooth muscle cells (VSMCs), extracellular matrix degradation, and inflammation. The pathogenesis of aortic aneurysm is poorly defined, and current studies have mainly focused on genetic factors, especially mutations in extracellular matrix proteins and components of vascular smooth muscle cells. The major risk factor for aortic aneurysm is aging. However, how aging contributes to the mechanism of aortic aneurysm is not fully understood. In this study, we found that aging mice developed aortic aneurysm naturally which was exacerbated by angiotensin II. Aging downregulated VSMC contractile proteins (Myh11, α-SMA, SM22α, and MLC2), decreased VSMC proliferation, and induced VSMC apoptosis. Consistently, aging impaired vascular contractile responses to norepinephrine, indicating decline of vascular contractile function. Protein expression of KDM4A, a specific demethylase of histone 3 lysine 9 trimethylation (H3K9me3) was decreased while levels of H3K9me3 (often associated with heterochromatin) were upregulated significantly in aortic VSMCs isolated from aging mice, suggesting that aging methylates histone 3 (H3K9) which causes chromosome remodeling. We then performed ChIP-seq analysis to identify genes that are physically associated with and regulated by H3K9me3. The ChIP-seq data clearly distinguished the upregulated and downregulated genes in VSMCs between young and old mice. Of note, the promoter region (TSS) of the cell division cycle and apoptosis regulator 1 (CCAR1) gene was highly methylated in aged VSMCs. Western blot analysis confirmed downregulation of CCAR1 protein expression in aged VSMCs, indicating that increased H3K9me3 in the CCAR1 promoter suppressed CCAR1 gene expression. This finding provides the first evidence that CCAR1 gene expression is regulated by H3K9me3. To explore whether CCAR1 plays a role in the regulation of VSMC phenotypes, we silenced CCAR1 using siRNA in VSMCs. Interestingly, silencing of CCAR1 resulted in a decrease in contractile proteins (Myh11, α-SMA, SM22α, and MLC2), suggesting a loss of VSMC contractile phenotype. Knockdown of CCAR1 decreased cell proliferation marker (PCNA), cell cycle markers (Cyclin D1, Cyclin E1, and CDK2) and cell migration. Importantly, knockdown of CCAR1 induced apoptosis as evidenced by increases in cleaved caspase-3 and cytochrome C. Overall, these findings demonstrate that downregulation of CCAR1 alters VSMCs phenotypes including impaired contractile function and decreased proliferation and migration. CCAR1 deficiency may cause VSMC apoptosis. We are in the process of determining whether downregulation of CCAR1 mediates aging-associated aortic aneurysm by in vivo VSMC-specific expression of CCAR1 gene in old mice. Our hypothesis is that lentiviral-based VSMC-specific delivery of CCAR1 gene would attenuate or prevent aging-associated aortic aneurysm. Our ultimate goal is to develop CCAR1 as a novel therapeutic target for aortic aneurysm. This work was supported by 1R01 DK138872-01, R01HL154147-01, R01AG062375, and R073037044. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.