The Effect And Mechanism Of Interferon Regulatory Factor 4 In Intima Hyperplasia

Background:Cardiovascular diseases are the most common causes of death in developed societies and widespread whole the world,and atherogenesis is the leading pathophysiological cause of the diseases.Surgical procedures,including angioplasty,stenting,atherectomy,and bypass surgery,remain indispensable treatments for patients with life-threatening coronary atherosclerosis;however,restenosis after mechanical injuries is the most prominent pathological issue limiting the prognoses of these patients.It is well known that the hallmark of restenosis is arterial neointima formation,during which the contractile vascular smooth muscle cells(SMCs)transform to dedifferentiate state and increase the ability of proliferation,migration,as well as secrete more extracellular matrix in response to various stimuli.However,the treatments for restenosis remain far from satisfactory mainly because of a lack of a clear understanding of the molecular mechanisms underlying restenosis development.Innate immune signaling networks can affect the progression of cardiovascular diseases by functioning in nonimmune cells.Interferon(IFN)regulatory factors(IRFs),which were originally identified as transcriptional regulators of type ?IFN,are recently well recognized as the key regulators of innate immune signaling.Accumulated papers have demonstrated that IRFs have also been found to play important roles in insulin resistance,heart disease,cell survival,oncogenesis and neointima formation.IRF4,the fourth member of the IRF family,was initially identified in B and T lymphocytes,in which it regulates host cell differentiation.Similar to other members of its family,IRF4 harbors a highly conserved DNA-binding domain(DBD)in its N-terminal and an IRF-association domain(IAD1)in its C-terminal.Under certain circumstances,IRF4 can regulate the transcriptional activity of its target genes by binding to IFN-stimulating response elements(ISREs)via its DBD after interacting with co-factors via its IAD1.Most previous studies have shown that IRF4 plays a critical role in regulating the differentiation of lymphoid,myeloid and dendritic cells;however,its tumor suppressing functions have also been addressed.Moreover,IRF4 acts as a protective regulator against metabolic diseases by regulating macrophage polarization and as a transcriptional driver of thermogenic gene expression and energy expenditure by interacting with peroxisome proliferator-activated receptor gamma coactivator 1 alpha(PGC-la).We recently found that IRF4 overexpression aggravates cardiac hypertrophy by promoting the transcriptional regulation of CREB but protects against ischemic stroke by enhancing the expression of serum response factor(SRF).These results indicate that IRF4 can play very different roles by regulating different downstream molecular signaling in nonimmune cells under different pathophysiological conditions.Therefore,it is very intriguing to explore whether and how the key regulator of innate immunity IRF4 functions in neointima formation,as understanding this issue is also critical for understanding the mechanisms underlying restenosis.Object:The aim of the present study was to clarify the effect of IRF4 on the vascular injury-induced VSMCs phenotypic switching and intimal hyperplasia,as well as to explore the underlying mechanism.Methods:Part one:To examine whether IRF4 affected the pathogenesis of intimal hyperplasia,the human coronary arteries with restenosis and wire injury-induced mouse carotid artery were utilized.The qPCR and Western blots analysis were performed to observe the expression of IRF4.Subsequently,we constructed the global IRF4 knockout rat and mouse,which were further used to perform morphological detection staining with EVG and to confirm the effect of changed IRF4 expression on intimal hyperplasia.Part two:By collecting the human coronary arteries with restenosis and wire injury-induced mouse carotid artery at different post-injury time,the double immunofluorescence staining and Western blots were performed to test the IRF4 expression in VSMCs.Part three:To investigate the important role of smooth muscle cell specific IRF4 knockout in intimal hyperplasia,the SMC-Cre and SMC-IRF4-KO mice were subjected to carotid artery wire injury.The size of neointima and the ratio of intima/media measured by EVG staining were used to morphological assessment,while the immunofluorescence staining and western blot were utilized to examine the protein expression of markers represented the ability of VSMCs proliferation and migration.Moreover,the mRNA expression of these markers tested by qPCR was further confirmed by the primary VSMCs upon PDGF-BB stimulation.The SMC proliferation was assessed via BrdU incorporation assay and the SMC migration were tested by Transwell-modified Boyden chamber in vitro.Part four:To understand whether SMC-specific IRF4 overexpression can reverse the pathogenesis of intimal hyperplasia,the SMC-IRF4-TG and the littermate mice were subjected to carotid artery wire injury.The size of neointima and the ratio of intima/media were used to morphological analysis,while the immunofluorescence staining,qPCR and western blot were used to observe the expression of VSMCs proliferation and migration markers.Moreover,the SMC proliferation and migration of primary VSMCs upon PDGF-BB stimulation were assessed via BrdU incorporation assay and Transwell-modified Boyden chamber,respectively.Part five:To investigate the mechanism underlying the effects of IRF4 on restenosis,we performed a genome-wide microarray screen and ingenuity pathway analyses of carotid arteries from WT and IRF4-KO mice at 14 days post-injury.qPCR,immunofluorescence staining and western blot were subjected to reveal the change of KLF4.We performed bioinformatics analysis and identified the potential ISREs in the KLF4 gene promoter for IRF4 directly binding.ChIP and luciferase assays were further used to confirm the transcription regulation of KLF4 by IRF4.Part six:To confirm whether KLF4 is the essential role for the effect of IRF4 on intimal hyperplasia,SMC-IRF4-KO-KLF4-TG and SMC-IRF4-KO-KLF4-TG were subjected to carotid artery wire injury.The size of neointima and the ratio of intima/media were used to morphological analysis by EVG staining,while the immunofluorescence staining were used to observe the expression of VSMCs proliferation,migration and differentiation markers.Part seven:The immunofluorescence staining,qPCR and western blot were used to observe the effect of IRF4 on the expression of VSMCs differentiated markers.Moreover,these genes were further confirmed in the primary VSMCs upon PDGF-BB stimulation by qPCR.Results:Part one:IRF4 was expressed at significantly lower levels at both the mRNA and protein levels in arteries with restenosis than in coronary arteries without restenosis.Using the mouse carotid artery injury model,we found that IRF4 expression levels changed in response to injury in a time-dependent manner,as IRF4 expression levels were markedly increased at both the mRNA and the protein level at 7 days and 14 days after injury but were significantly decreased at both the mRNA and protein level at 28 days after injury.More importantly,the neointima and intima-to-media ratio in the IRF4-KO mice and rat were significantly thicker than that in the control group at 7,14 and 28 days post-injury.Part two:By immunofluorescence staining with a-SMA(Green,VSMCs),IRF4(Red)and DAPI(blue,nucleus),we observed that IRF4 was positive in VSMCs of human coronary arteries and mouse carotid arteries,while IRF4 expression levels showed a result consistent with mRNA and protein expression mentioned in the part one.Western blot analysis of the proteins extracted from SMCs isolated from the control carotid arteries and injured carotid arteries at different time points exhibited a similar result.Part three:SMC-IRF4-KO mice exhibited more severe neointima formation and intima-to-media ratio than their SMC-Cre littermates at 14 and 28 days after carotid artery wire injury.Immunofluorescence staining and western blot analyses revealed that the expression levels of the proliferation-inducing genes Cyclindl and PCNA,the migration-inducing gene MMP2 were upregulated in SMC-IRF4-KO mice compared with SMC-Cre control mice at 14 and 28 days post-injury,while the proliferation suppressor P21 were downregulated.When the primary VSMCs were subjected to PDGF-BB stimulation,the qPCR revealed Cyclindl and PCNA transcript levels were upregulated in SMC-IRF4-KO mouse,but P21 transcript levels was downregulated.In primary SMCs,the absence of IRF4 significantly promoted PDGF-BB-induced VSMCs proliferation and migration tested by BrdU incorporation and Transwell migration assays.Part Four:SMC-IRF4-TG mice exhibited significantly decreased neointima formation and intima-to-media ratio than their WT littermates at 14 and 28 days after carotid artery wire injury.Immunofluorescence staining and western blot analyses revealed that the expression levels of the proliferation-inducing genes Cyclindl and PCNA,the migration-inducing gene MMP2 were downregulated in SMC-IRF4-TG mice compared with WT control mice at 14 and 28 days post-injury,while the proliferation suppressor P21 were upregulated.In primary SMCs,the overexpression of IRF4 significantly attenuated PDGF-BB-induced VSMCs proliferation and migration tested by BrdU incorporation and Transwell migration assays.Part Five:A genome-wide microarray screen and ingenuity pathway analyses demonstrated that the mRNA expression levels of genes related to proliferation(Cyclindl and PCNA),migration(MMP2),SMC differentiation(?-SMA,SM22a and Smoothelin)and inflammation development(TNF-?,iNOS and IL-6)were significantly increased,but the mRNA expression levels of genes reported to inhibit proliferation(P21,KLF4)and inflammation(Retnla,Arg-1,PPARy and Chi313)were decreased in the carotid arteries of IRF4-deficient mice compared with those in control mice at 14 days post-injury.Among the above genes,KLF4 is known to play key roles in SMC phenotype modulation and neointima formation.SMC-IRF4-KO inhibited the mRNA and protein expression of KLF4,especially in VSMCs.We performed bioinformatics analysis and identified 6 putative ISREs in the KLF4 gene promoter.Chromatin immunoprecipitation(ChIP)assays demonstrated that P3(-1544?-1435)and P4(-1003?-910)were the two significant ISREs that allowed IRF4 to bind to the KLF4 gene.In primary SMCs,PDGF-BB treatment enhanced KLF4 transcriptional activity(as demonstrated via KLF4-luc luciferase reporter assays),which was decreased by IRF4 ablation but increased by IRF4 overexpression.In MOVAS cells,KLF4 transcriptional activity was enhanced by adenovirus-medi-ated IRF4 overexpression.Mutating the IRF4-binding sites in the ISREs of the KLF4 promoter blunted these IRF4-specific effectsPart six:We investigated whether the effects of IRF4 on neointima formation are mediated by KLF4 in vivo,SMC-specific IRF4-KO-KLF4-TG mice and IRF4-TG-KLF4-KO were generated.The EVG and immunofluorescence staining demonstrated IRF4 functions in neointima formation was depended on KLF4.Part seven:The immunofluorescence staining and western blot analysis showed SMC-IRF4-KO promoted SMC differentiation during neointima formation after wire injury,but SMC-IRF4-TG attenuated the effect.Subsequent qPCR of primary VSMCs upon PDGF-BB stimulation further verified the results.