Abstract Body:
Atherosclerosis, an age-driven cardiovascular disease, results in the formation and accumulation of plaque in the arterial walls, increasing the risk of thrombus formation and rupture. The progression of atherosclerosis involves lipid deposition and an inflammatory response. Recent studies have highlighted the importance of the accumulation of senescent cells in atherosclerotic lesions as a key contributor to the disease progression. Cellular senescence is a persistent cell cycle arrest marked by the release of senescence-associated secretory phenotype (SASP), including many inflammatory cytokines and extracellular matrix remodeling components. In a recent study from our lab, we identified Osteopontin (OPN) as an mRNA marker of vascular cell senescence using single-cell RNA sequencing in a mouse model of atherosclerosis. OPN plays a crucial role in processes such as bone remodeling and cell-mediated immune responses; however, its role in cellular senescence is poorly characterized. To understand the role of OPN in senescence, we first performed a time course using senescent human vascular smooth muscle cells (VSMCs) which revealed that intracellular OPN is elevated early and remains highly expressed throughout senescence; surprisingly, we did not detect significant secretion of OPN, suggesting that it may not be a SASP factor produced by senescent VSMCs. We detected strong OPN secretion by LPS-stimulated macrophages, suggesting VSMCs may receive the OPN signal rather than produce it. This finding was further supported by increased expression of OPN receptor CD44 in senescent VSMCs. Next, we silenced OPN in senescent VSMCs and performed RNA-seq to obtain comprehensive information about processes and pathways that might be affected by the absence of OPN. The analysis revealed that mitochondrial-related processes were downregulated with the loss of OPN; we also observed a profound reduction in subunits related to Complex I and IV and general oxidative phosphorylation (OXPHOS) markers using RT-qPCR and western blot analyses, respectively. Notably, using a Seahorse assay, we found that the oxygen consumption rate (OCR) was decreased in OPN siRNA compared to CTRL siRNA, further confirming mitochondrial dysfunction appears mediated by OPN. We hypothesize that OPN plays a role in maintaining mitochondrial function in senescent VSMCs, preventing oxidative stress and premature cell death, allowing these cells to contribute to disease development.