The Role of FXR1 and Senescence in Vascular Biology and Intimal Hyperplasia

Abstract

IntroductionDespite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle to success. Vascular smooth muscle cells (VSMC) play a critical role in the development and pathogenesis of intimal hyperplasia indicative of restenosis; therefore regulation of gene expression in VSMC represents a logical intervention point to attenuate this syndrome. FXR1 is a muscle‐enhanced RNA binding protein and expression is increased in injured human and mouse arteries. We have shown that modulation of FXR1 levels affects abundance and stability of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and acts as a crucial regulator of inflammatory and proliferative mRNA in VSMC.Approach/ResultsBecause FXR1 expression is increased in injured arteries, we have developed a novel VSMC‐specific conditional knockout mouse (FXR1VSMC/VSMC). In a carotid artery ligation model of intimal hyperplasia, FXR1VSMC/VSMC mice have significantly reduced neointima formation (p<0.001) post‐ligation compared to several controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC by siRNA and observed decreased proliferation (p<0.05) as well as an increase in beta galactosidase (p<0.05) and gamma H2AX (p<0.01) staining compared with controls, indicative of senescence. Senescent cells exhibit phenotypic changes called the senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RIP‐sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and RNA stability of these transcripts is decreased with FXR1 KO. qPCR analysis from FXR1 KO mouse VSMC show an increase in transcripts associated with senescence (p21, p16, p53) as well as an increase in SASP‐associated mRNA compared with controls. Furthermore, wild‐type human VSMC cultured in conditioned media from cells transfected with FXR1 siRNA show an increase in SASP mRNA and an increase in proliferation compared with cells cultured in conditioned media from cells transfected with a scrambled control.Summary & ConclusionsOur results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP gene expression, and reduction of intimal hyperplasia.