The Mechanism Of Interferon Regulatory Factor8in Phenotypic Switching Of Vascular Smooth Muscle Cells And Neointima Formation

BackgroundNeointima formation is a common pathological process in a variety of proliferative vascular diseases including atherosclerosis (AS), pulmonary arterial hypertension and resternosis after percutaneous transluminal coronary intervention (PCI). The phenotypic switching of vascular smooth muscle cells (VSMCs) plays an important role in neointima formation. VSMCs have differentiated/contractile phenotype and dedifferentiated/synthetic phenotype, which can transform into each other under certain conditions. The proliferation and migration of VSMCs are increased in the process from contractile phenotype to synthetic phenotype. Synthetic phenotype VSMCs could secrete a large amount of extracellular matrix to form neointima, which leads to a series of proliferative vascualr diseases. Therefore, if VSMC phenotypic switching were prevented or even reversed, we could curb the early progression of the proliferative vascualr diseases mentioned above. VSMCs phenotypic switching is mainly induced by growth factors and mechanical stimulation, which actives a number of signaling pathways and causes the smooth muslce specific gene changes in the nucleus. However, the mechanisms of VSMC phenotypic switching have not yet been fully elucidated, and there is still a lack of effective interventions.Interferon regμlatory factor8(IRF8, also well-known as interferon consensus sequence binding protein, ICSBP), is one of the members of interferon regulatory factor family (IRFs) in mammals. IRF8locates at16q24.1of chromosomes. An increasing number of investigations at home and abroad have indicated that IRF8plays an important role in the differentiation of immune cells. Recently studies have demostrated that IRF8was involved in the formation of carotid plaque and could increase the thickness of intima area and media area. Therefore, IRF8may have a crucial effect on the regulation of SMCs phenotypic switching and neointima formation. This study establised the left carotid guid wire injury model on IRF8deficiency mice and SMC-specific IRF8transgenic mice, respectively, to examine the effect of IRF8on SMCs phenotypic switching and neointima formation, and explored the underlying mechanisms.AimsThe aim of this study was to explore the effect of Interferon regulatory factor8on injury-induced VSMC phenotypic switching and intima hyperplasia and the underlying molecular mechanisms.MethodsPart one:The male wild-type C57BL/6(WT) mice with body weight ranged from24-28g and aged10to12weeks were used to establish left carotid guid wire injury model. The WT mice were randomly divided into four groups, respectively, e.g. sham operation group (sham),7days post injury,14days post injury and28days post injury. The Western Blot Analysis and immunofluorescence staining were used to determine the expression of IRF8in mice carotid artery tissue at different time points. Rat aortic smooth muscle cells (RASMCs) and Human aortic smooth muscle cells (HASMCs) were treated with (Platelet-derived growth factor, PDGF-BB) to mimic the process of intima hyperplasia in vitro. Western Blot Analysis was used to detected the expression of IRF8before and after PDGF-BB stimulation.Part two:The male WT and IRF8Knockout mice (IRF8-KO) with body weight ranged from24-28g and aged10to12weeks were included to establish left carotid guid wire injury model. WT and IRF8-KO mice were radomely divided into Sham and injured group. At14and28days post injury, we stained carotid sections with hematoxylin and eosin (H&E) for morphometric analysis. The immunofluorescence staining and Western Blot Analysis were used to determine the expression of VSMCs specific gene. Simultaneously, VSMCs were treated with PDGF-BB (20ng/ml), Western Blot analysis was used to detected the effect of IRF8knockdown or knockout on the phenotypic marker expression of VSMCs. BrdU incorporation was used to assess the regulatory function of IRF8deficiency to the proliferation of mouse VSMCs.Part three:SMC specifc IRF8transgenic mice were constructed using microinjection techniques. The male NTG and IRF8transgenic mice (IRF8-TG) with body weight ranged from24-28g and aged10to12weeks were used and established left carotid guide wire injury model. NTG and IRF8-TG mice were radomely divided into Sham and injured group. At14and28days post injury, Hematoxylin and eosin (H&E) was performed to morphometric analysis. The immunofluorescence staining and Western Blot analysis were used to evaluate the expression of VSMCs specific gene. Following that, VSMCs were treated with20ng/ml of PDGF-BB, Western Blot analysis was used to detected the influence of IRF8overexpression on the phenotypic marker expression of VSMCs. BrdU incorporation was used to assess the effect of IRF8 overexpression on the VSMCs proliferation.Part four:Using dual-luciferase reporter assay system to detect the effect of IRF8overexpression on luciferase activity of three tandem repeats conserved CArG elements present in the SM22a promoters of SMC-specific genes and SM22a itself.Using point mutation method, the CArG elements were mutated where have been identified at approximately-107and+16of the SM22a transcription start site, and constructed the corresponding mutant SM22a-luc plasmids, respectively. We employed the Gal4-UAS system and constructed a chimeric transcription factor with the Gal4DNA-binding domain fused to Myocardin and Gal4-Myocardin adenovirus infection in A7r5cells and RASMCs to examine IRF8overexpression on luciferase activity of UAS. Co-Immunoprecipitation and GST-Pulldown were employed to examined whether IRF8could directly interact with Myocardin and confocal microscopy imaging was performed to determine whether IRF8could co-localizated with Myocardin. Using truncated IRF8and Myocardin, the domains where they interact with each other were detected. Moreover, adenovirus with a mutant IRF8domain which lacks the N-terminal DNA-binding domain and the intermediate region required for the interaction with myocardin was constructed and infected primary RASMCs to determine the effect of IRF8on VSMC specific gene expression.ResultsPart1:H&E staining analyses showed that intima area and ratio of intima to media gradually increased at7,14and28d post-injury in the WT mice. The Western blot analyses showed that the expression of carotid IRF8protein at a low level in Sham groups and was mildly elevated at7day after injury. However, IRF8expression increased strikingly at14day and returned to lower level at28day post injury. Immunofluorescence staining analysis showed that IRF8was highly expressed in the VSMCs, especially at14days post injury. Western blot results in vitro showed that, before PDGF-BB treatment, IRF8protein at a low level in two cell type. After PDGF-BB (20ng/ml) administration, the expression of IRF8was markly induced and peaked at24hours after PDGF-BB treatment.Part2:H&E staining analyses showed that intima area and ratio of intima to media in the IRF8-KO mice were much lower at14days and28days post injury than those of WT mice. Immunofluorescent staining and Western Blot results showed the expression of SMC-specific genes, including a-SMA, SM22a, smoothelin and desmin in IRP8-KO mice were significantly higher than that in WT cells at day14and28post injury. The expression of Osteopontin, a marker of synthetic VSMCs, however, was significantly higher in WT SMCs than that in IRF8-KO cells. Western Blot analysis showed that the expression of SMC-specific genes including a-SMA, SM22a, smoothelin and desmin were significantly higher in the knocking down IRF8expression using shRNA or IRF8deficiency SMCs compared with WT SMCs after PDGF-BB stimulation. The expression of PCNA, CyclinDl and MMP9was significantly higher in WT SMCs than that in AdshIRF8and IRF8-KO SMCs. The incorporation of5’-bromo-2’-deoxyuridine (BrdU) results showed that IRF8-KO SMCs incorporated less BrdU than WT SMCs after PDGF-BB treatment.Part3:we successfully generated four lines of SMC specific IRF8transgenic mice in a C57BL/6background under the control of a SMC-specific mouse minimal SM22promoter. Western Blot analysis showed line4of which VSMC express the highest IRF8level (approximately14.3-fold compared to WT mice). H&E staining analyses showed that the intima area and the ratio of intima to media were higher in IRF8-TG mice than in NTG controls at14and28days post injury. Immunofluorescent staining and Western blotting showed that the expression of a-SMA, SM22a, smoothelin and desmin were decreased while the osteopontin increased more significant after injury in the IRF8-TG mice compared to NTG mice. Compared with control SMCs, the expression of SMC-specific genes including a-SMA, SM22a, smoothelin and desmin were lower in the IRF8overexpressing RASMCs and IRF8-TG SMCs after PDGF-BB stimulation. The expression of PCNA, CyclinDl and MMP9was significantly higher in AdIRF8and IRF8-TG SMCs than that in WT SMCs. BrdU incorporation results showed that IRF8-TG SMCs incorporated much BrdU than NTG SMCs upon PDGF-BB challenge..Part4:Dual-luciferase reporter assay results showed that3×CArG-luc and SM22a-luc activity were greatly induced after RASMCs were infected with a Myocardin containing adenovirus, while after co-infected with IRF8containing adenovirus and Myocardin adenovirus, the activity of3×CArG-luc and SM22a-luc could not be induced. The CArG elements mutation results indicated that the mutant SM22a-luc activity was lower than that of the wild-type SM22a-luc after RASMCs infected with Myocardin containing adenovirus. In cells with shRNA induced downregulation IRF8expression, the activities of the wild-type and single-mutation SM22α-luc increased, but the double-mutation SM22α-luc did not affect. In Ga14-UAS system, the results showed that UAS-luc activity was significantly induced when A7r5cells and RASMCs were infected with Gal4-Myocardin adenovirus. However, the induction of UAS-luc activity by Ga14-Myocardin was largely inhibited when we co-infected with the IRF8-containing adenovirus. Co-IP assays showed that IRF8direct interacted with Myocardin, which was further conformed by GST-pulldown assay. Fluorescent confocal microscopy demonstrated that IRF8and Myocardin were predominantly colocalized in the nucleus. A seried of truncated IRF8and Myocardin and the IP results demonstrated that the C-terminal transcriptional activation domain (TAD) of Myocardin can interact with IRF8, both the N-terminal DNA-binding domain (DBD) and the intermediate region of IRF8can interacted with Myocardin. Dual-luciferase reporter assay results indicated that wild-type IRF8inhibited the induction of3×CArG-luc and SM22α-luc activities by Myocardin. However, overexpression of IRF8with a mutant IRF8domain failed to repress the luciferase activity induced by Myocardin.Conclusion1. IRF8expression is significantly up-regulated in carotid artery wire injury model, and was highly expressed in VSMCs.2. IRF8deficiency represses injury-induced VSMC phenotypic switching and neointima formation, while SMC specific overexpression of IRF8promotes injury-induced VSMCs phenotypic switching and neointima formation.3. IRF8is crucial in modulating injury-induced VSMC phenotype switching and neointima formation via direct interaction with Myocardin.