Abstract 3053: An Emerging Role Of The RNA Binding Protein Human Antigen R In Atherosclerotic Vascular Smooth Muscle Cell Heterogeneity

Abstract

Objective: Lineage tracing studies have shown that advanced atherosclerosis causes vascular smooth muscle cells (VSMCs) to become macrophage-like (MLCs), which increases inflammation. We investigated whether the RNA binding protein human antigen R (HuR) and the inflammatory receptor protease activated receptor 2 (PAR2) accelerate VSMC-MLC in a mouse model of atherosclerosis. Methods and Results: Downregulation of VSMC migration and cytokine release in Par2 -/- mice reduces atherosclerotic lesion area compared to proficient controls. To examine the role of PAR2 in the VSMC-MLC dedifferentiation, Par2 +/+ and Par2 -/- VSMCs were treated with 10μg/mL methyl-β-cyclodextrin cholesterol for 72 hours. While Par2 +/+ VSMCs demonstrated a loss of VSMCs markers and upregulated macrophage markers, Par2 -/- VSMCs remained stable. When comparing 101 mice strains with induced atherosclerosis, PAR2 was significantly correlated with Krüppel-like factor 4 (KLF4), a gene that regulates VSMC dedifferentiation. Also, Par2-/- VSMCs had lower basal KLF4 expression. Previous studies have demonstrated PAR2 mRNA is bound and stabilized by the RNA binding protein HuR. Our studies confirm these findings where Par2 -/- VSMCs have less HuR mRNA expression, suggesting a role of HuR in PAR2 mRNA stability. Our studies have also demonstrated a dampening of HuR activation in Par2 -/- VSMCs, suggesting a potential feedforward mechanism between HuRs stabilizing capabilities of PAR2 mRNA and PAR2s presence necessary for HuR activation. To delineate the role of HuR in VSMC dedifferentiation and atherosclerosis, a HuR flox mouse line has been bred on to a SM22 Cre as well as a Myh11 Cre lineage tracing reporter line. To date, we have determined male and female Ldlr -/- HuR flox/flox SM22 Cre+ mice have attenuated atherosclerosis compared to their littermate Cre - controls at both 12 and 24 weeks. Conclusions: These results suggest that VSMC PAR2 activation mediates the dedifferentiation of VSMCs via KLF4 and subsequent binding by HuR, which stabilizes PAR2 mRNA and upregulates PAR2. We also have established a definitive role of VSMC HuR in the development of atherosclerosis. Future studies will continue to elucidate the specific role of HuR in VSMC dedifferentiation.