Vascular smooth muscle aging and the epigenetic clock

/ ABSTRACT Cardiovascular disease (CVD) is the leading cause of mortality worldwide, disproportionately affecting older adults. Indeed, aging results in vascular stiffening and is the dominant risk factor for atherosclerosis. Although traditionally viewed as a disease of chronological aging, emerging evidence supports that CVD is driven by accelerated biological aging within the arterial wall. Among the cellular players involved, smooth muscle cells (SMCs) are pivotal in maintaining vessel integrity and contributing to atherosclerotic plaque structure. However, with aging, SMCs undergo epigenetic alterations particularly in DNA methylation that disrupt phenotypic plasticity, promote maladaptive remodeling, and compromise reparative functions. We recently reported that age is a key factor regulating SMC expansion in atherogenesis. Despite this central role, the biological age of SMCs and their potential for rejuvenation remain unexplored. To address this gap, our group has recently generated novel in vivo and in vitro SMC-specific epigenetic clocks using the Infinium Mouse Methylation platform. These clocks capture chronological aging of SMCs with high fidelity and provide a powerful platform to assess molecular and functional rejuvenation. We now leverage this tool to test the rejuvenation potential of transient epigenetic reprogramming with pluripotency factors OSKM (octamer-binding transcription factor 4 [OCT4], sex determining region Y-box 2 [SOX2], Kruppel-like factor 4 [KLF4], and c-MYC) to reverse SMC aging and mitigate CVD risk. Previous studies in the literature have briefly expressed pluripotency factors in other aged somatic cell types to reset epigenetic age and restore gene expression and functional capacities. Our central hypothesis is that transient epigenetic reprogramming of aged SMCs reverses molecular and functional hallmarks of aging, preserves cell identity, and mitigates vascular stiffness and atherosclerosis. In this R21 proposal, we aim to: 1) evaluate the effects of OSKM reprogramming on aged SMC function and epigenetic state in vitro and in vivo; and 2) determine how transient reprogramming alters vascular stiffness and atherosclerosis. By integrating DNA methylation profiling, gene expression analysis (including single cell RNA-sequencing), SMC and vascular functional assays and vascular disease models, this project will provide critical insights into the plasticity and therapeutic potential of aged SMCs. Our studies will serve as a foundation for novel vascular rejuvenation strategies and generate high impact preliminary data for subsequent R01 proposals focused on combating age-associated vascular disease. Project Number: 1R21AG101308-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Daniel Greif (+1 co-PI) | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $461,311 | Activity Code: R21 | Study Section: Atherosclerosis and Vascular Inflammation Study Section[AVI] View on NIH RePORTER: https://reporter.nih.gov/project-details/11363308