Effects And Mechanisms Of PRMT5 Expression Induced By The Gut-derived TMAO On Neointima Formation

Cardiovascular diseases（CVD）are the leading cause of human death worldwide.Neointima formation is a common pathological form of vascular diseases,including atherosclerosis,arterial stenosis and vein bypass graft failure.Vascualr smooth muscle cells（VSMC）,a major component of the arterial wall,present differentiated/contractile phenotype normally.During vascular injury or other physiological stimulus,VSMC dedifferentiate from a contractile state to a synthetic state,along with enhanced proliferation and migration abilities,thereby contributing to the intimal hyperplasia in many cardiovascular diseases.Therefore,deciphering the regulatory mechanisms underlying VSMC proliferation and migration is key to the development of novel therapeutic strategies for vascular diseases.Plasma trimethylamine-N-oxide（TMAO）,a metabolite of the dietary lipid phosphatidylcholine,has been proven to be associated with various cardiovascular pathologies.Accumulating studies have highlighted the importance of epigenetic modification in the pathological processes of VSMC phenotypic conversion and arterial neointima formation.As a major typeⅡmethyltransferase,the protein arginine methyltransferase 5（PRMT5）has been implicated in the control of proliferation and transformation.However,whether PRMT5 is involved in modulating VSMC proliferation and neointima formation remains to be explored.However,the molecular mechanisms underlying this small molecule cause vascular diseases remain to be further elucidated.In the present study,the effects of high-fat diet on wire injury-induced neointima formation was firstly explored.The results show that high-fat diet lead to accelerated arterial intima hyperplasia is partially mediated via modulation of gut microbiota composition and TMAO production.Next,the expression of protein arginine methyltransferase（PRMT）was studied in the human carotid aorta with stenosis.The results showed that expression of PRMT5 was significantly elevated in human carotid aorta with stenosis.The expression of PRMT5 was also elevated in mouse carotid artery injury model.Further studies determined the role and mechnasim of TMAO in injury-induced vascular inflammation via upregulating PRMT5 expression.At last,the effects of PRMT5 in injury-induced neointima formation was explored by VSMC specific knockout mice and lentivirus-mediated PRMT5 knockdown or overexpression,and its mechanism on regulating VSMC phenotypic switching were studied in PDGF-stimulated VSMC proliferation model.The main results are as follows:（1）Mice fed with high fat diet（TD88137+1%choline）underwent carotid artery injury and the gut microbiota and intestinal permeability were test to determined the effects of high-fat diet on neointima formation.The result showed that a high fat diet promoted the development of neointimation formation,which was accompanied by reduced expression ofα-SMA and upregulated expression of the proliferative marker PCNA,partially via increasing intestinal permeability,decreasing the expression of zona occludens（ZO-1）,occludin and claudin-1 and altering microbiota composition with a profile characterized by the expansion of TMAO producing bacteria.（2）The expression of PRMT1～PRMT7 in human carotid aorta with stenosis was tested respectively.The results showed the expression of PRMT5 was obviously upregulated in human carotid aorta with stenosis.Furthermore,the study showed that the expression of PRMT5 was elevated in VSMC.In order to verify the correlation between PRMT5 and neointima formation,the expression of PRMT5 was detected in mouse carotid artery injury model,the results showed a time-dependent induction of PRMT5 expression in response to injury.VSMC-specific PRMT5 knockout mice were used to reveal the role of TMAO in injured-induced vascular inflammation.The results showed that exogenous TMAO administration TMAO treatment significantly increased the expression of VCAM-1 and the macrophage marker CD68 in injured carotids of mice.The mechanical studies show that Nox4-generated H₂O₂ was involved in TMAO-induced upregulation of PRMT5.（3）VSMC-specific PRMT5 knockout mice and lentivirus-mediated PRMT5knockdown or overexpression in mice were adopted before vascular injury,so as to reveal the role of PRMT5 in mice with neointima formation.The results showed that VSMC-specific PRMT5 knockout inhibited intimal hyperplasia with enhanced expression of contractile markers（α-SMA and SM22α）in mice.Conversely,PRMT5 overexpression inhibited SMC contractile markers and promoted intimal hyperplasia.Transfection with PRMT5 si RNA,but not scramble si RNA,dramatically reduced PDGF-induced PRMT5 expression in VSMC,along with upregulatedα-SMA and SM22αexpression and reduced PCNA and cyclin D1expression.Moreover,PRMT5 knockdown by si RNA suppressed VSMC migratory ability.However,PRMT5 overexpression by transfection with a PRMT5 over-expressing plasmid exerted the opposite effect on the expression of marker proteins and enhanced capacities to proliferate and migrate.（4）To elucidate the molecular mechanism underlying the neointima-promoting effect of PRMT5,VSMC were treated with PDGF,accompained si RNA transfection.The results showed a strong correlation between PRMT5 and KLF4 expression.Further studies showed that PRMT5-mediated KLF4 methylation（R374,376 and 377）inhibited ubiquitin-dependent proteolysis of KLF4,leading to a disruption of MYOCD-SRF interaction and MYOCD-SRF-mediated the transcription of SMC contractile markers,such as SM22α.Taken together,we demonstrate the mechanism of a high fat diet promoted neointima formation via increasing production of TMAO.Intriguingly,Nox4-derived H₂O₂ production play an important role in TMAO-induced PRMT5 upregulation,thus promoting the injury induced inflammation and neointima formation.Mechanistically,PRMT5 mediated methylation of KLF4 inhibits its degradation,which is a critical factor in modulating SMC phenotypic modulation,through competition with MYOCD for binding to SRF,resulting in decreased expression of differentiation-related genes,such as SM22α.