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The Role of FXR1 in Cell Cycle Control and Induction of Senescence in Vascular Smooth Muscle
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2023-08
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Organ Systems & Translational Medicine
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http://dx.doi.org/10.34944/dspace/9471
Abstract
Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle. Vascular smooth muscle cells (VSMC) play a critical role in the pathogenesis of intimal hyperplasia; therefore, regulation of VSMC gene expression is a logical intervention point. FXR1 is a muscle-enhanced RNA binding protein and its expression is increased in injured arteries. We have shown that modulation of FXR1 levels affects stability and abundance of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. RNA-sequencing analysis in FXR1-depleted human VSMC (hVSMC) identified a number of transcripts with decreased abundance, the overwhelming majority of which were associated with proliferation and cell division. This drives our hypothesis that FXR1 is involved in mitigating vascular disease by regulating inflammatory and proliferative mRNA in VSMC. The mRNA abundance and stability of a number of these transcripts was decreased in FXR1 depleted hVSMC, and RIP-sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and stability of these transcripts is decreased with FXR1 depletion. FXR1-depleted cells showed decreased proliferation (p<0.05), however, an increase in β-galactosidase (p<0.05) and γH2AX (p<0.01), indicative of senescence was noted. Senescent cells exhibit a senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. HVSMC depleted of FXR1 had increased transcripts abundance of many SASP genes, as well as an increase of both mRNA and protein expression of canonical senescence markers p53 and p21.
We developed a novel SMC-specific conditional knockout mouse (FXR1SMC/SMC) to further study these results in a more translational context. In a carotid artery ligation model of intimal hyperplasia, FXR1SMC/SMC mice have significantly reduced neointima formation (p<0.001) post-ligation compared to controls. qPCR analysis from FXR1 conditional knockout mouse VSMC (mVSMC) show increased transcripts associated with senescence (p21, p16, p53) as well as increased SASP-associated mRNA, a decrease in proliferation, and an increase in β-galactosidase staining.
Our 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 genes, and reduction of intimal hyperplasia.
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