Pax9 Promotes PDGF-BB-induced Vascular Smooth Muscle Cell Dedifferentiation,Proliferation And Migration And Its Mechanism

Objective: Atherosclerosis(AS)is a chronic inflammatory vascular disease characterized by the obstruction of arterial walls with atherosclerotic plaques.It has been well-established that AS is the main cause and pathological process associated with the development of cardiovascular diseases(e.g.,angina pectoris,myocardial infarction and peripheral vascular disease).Moreover,AS has been a primary cause of morbidity and mortality in Western countries for more than half a century and is currently the leading cause of death worldwide.Therefore,it is important to study the underlying mechanism of AS initiation and development.Normally mature vascular smooth muscle cells(VSMCs)exhibit the contractile phenotype with weak proliferation,poor synthesis,and low secretion,and highly express the contractile phenotypic markers,calponin,SMMHC,MLCK,?-SMA,and SM22?,but hardly express the synthetic phenotypic marker OPN.In the pathological process of AS formation and development,VSMCs change from contractile phenotype to synthetic phenotype by down-regulating the expression of contractile phenotypic markers and up-regulating the expression of synthetic phenotypic markers.This process is called dedifferentiation or phenotypic transformation of VSMCs.VSMCs dedifferentiation is widespread in cardiovascular diseases such as atherosclerosis,intimal hyperplasia,arteriosclerosis,hypertension,restenosis after angioplasty,and arterial dissection.Elucidating the underlying mechanism of VSMCs dedifferentiation could provide new targets for the prevention and treatment of these diseases.Abnormal proliferation and migration of VSMCs are key cellular events in the pathophysiology of many cardiovascular diseases including AS and restenosis.VSMCs hyperproliferation and migration are closely related to VSMCs dedifferentiation,and are common pathological conditions of AS,restenosis,and failure of venous bypass grafts.Therapeutic measures for the dedifferentiation of VSMCs are conducive to improving these conditions related to the excessive proliferation and migration of VSMCs.In response to vascular injury stimulation in AS,inflammatory cells,platelets,and VSMCs will release inflammatory and growth factors,especially platelet-derived growth factor(PDGF),causing VSMCs to change from the differentiated phenotype to the dedifferentiated phenotype.PDGF family is a group of endogenous growth factors that regulate cell growth and division.Among them,PDGF-BB is considered to be the most effective stimulating protein for studying VSMCs dedifferentiation,proliferation and migration in AS.Therefore,this study used PDGF-BB to induce human aortic smooth muscle cells(HASMC)in vitro in order to explore the molecular mechanism of dedifferentiation,proliferation and migration of VSMCs in AS.The paired box(Pax)gene family encodes a group of transcription factors with a highly conserved paired-box DNA-binding domain.In addition,Pax genes play a significant role in morphogenesis,organogenesis,cell proliferation,cell differentiation,and tumorigenesis.As a member of the Pax gene family,Pax9 is one of the most important transcription factors regulating tooth development.It also plays an indispensable role in the normal development of thymus,parathyroid gland,posterior gill body,skeletal elements of skull and larynx as well as distal limbs.In addition,Pax9-deficient mice display developmental defects that affect the aortic arch arteries and the vascular outflow tract,revealing that Pax9 can also affect cardiovascular development.however,it is unclear whether Pax9 is involved in the biological behavior changes of VSMCs in AS.Shh is a type of morphogen that belongs to the hedgehog protein family.In addition,Shh signaling has been linked to cell survival,proliferation,and differentiation in various target tissues and organs.As an angiogenic agent,Shh induces angiogenesis under physiological and pathological conditions.Moreover,Shh can also promote PDGF-BB-induced VSMCs phenotype transformation by regulating KLF4.Hence,we hypothesized that Pax9 might modulate the phenotypic transformation,proliferation,and migration of VSMCs via Shh,thereby regulating AS.This study aimed to investigate the role of Pax9 in the phenotypic transformation,proliferation,and migration of atherosclerotic VSMCs and its underlying molecular mechanism.Methods: Part ?: Twenty-four male Apo E-/-mice aged 6 weeks and weighing 16-18 g were randomly divided into 2 groups,the control group and the AS group,which were fed with standard chow and high fat chow,respectively.The mice were weighed once a week for 12 weeks.After the experiment,peripheral blood samples were taken byremoving unilateral eyeball to determine serum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C).Next,all mice were sacrificed and thoracic and abdominal aortic specimens were taken.H & E staining was used to observe the degree of vascular stenosis,vascular plaque and vascular wall thickness.Finally,immunofluorescence staining and Western blot were used to detect the differential expression of Pax9 in normal and atherosclerotic aorta.Part ?: Firstly,PDGF-BB was selected as an inducer of HASMC dedifferentiation,proliferation and migration.HASMC was treated with four concentrations of PDGF-BB(5ng / ml,10 ng / ml,15 ng / ml,and 20 ng / ml)for 24 hours.Cell proliferation activity was detected by CCK8,cytoskeleton changes were detected by phalloidin staining,cell migration ability was detected by Transwell assay,Pax9 and expression of contractile phenotypic markers ?-SMA and SM22?,proliferation marker PCNA,and migration-related proteins MMP2 and MMP9 were detected by Western Blot.Control siRNA and Pax9 siRNA fragments were used to perform RNA interference on HASMC,and HASMC was divided into four groups: Control siRNA group;Control siRNA + 20ng/ ml PDGF-BB group;Pax9 si RNA group;Pax9 siRNA + 20 ng / ml PDGF-BB group.Changes in the proliferation and migration ability of HASMC were detected using CCK8,phalloidin staining and Transwell assay.Knockdown efficiency and protein expression changes of the dedifferentiation,proliferation,and migration-related indicators were detected by Western Blot.Further,the over-expressing Flag-Pax9 plasmid was used to overexpress Pax9 in HASMC,and HASMC was divided into four groups: vector plasmid group;vector plasmid + 20 ng / ml PDGF-BB group;Flag-Pax9 group;Flag-Pax9 + 20ng/ ml PDGF-BB group.CCK8,phalloidin staining,and Transwell assay were also used to detect changes in the proliferation and migration ability of HASMC,and Western Blot was used to detect Flag overexpression level and protein expression changes of the dedifferentiation,proliferation,and migration-related indicators.Part ?: HASMC was treated with four concentrations of PDGF-BB(5ng / ml,10ng/ ml,15 ng / ml,and 20 ng / ml)for 24 hours.Shh expression change was detected by Western Blot.HASMC was pretreated with the Shh signaling agonist SAG(100nM for24h)or the Shh signaling pathway inhibitor cyclopamine(5uM for 2h),then HASMCwas stimulated with 20 ng / ml PDGF-BB for 24 h.Changes in the proliferation and migration ability of HASMC were detected by CCK8,phalloidin staining,and Transwell assay.The protein expression changes of the dedifferentiation,proliferation and migration-related indicators were detected by Western Blot.Next,Western Blot was used to detect the effect of knockdown and overexpression of Pax9 on the expression of Shh in PDGF-BB-induced HASMC.Then,the Shh signaling pathway agonist SAG was used to treat Pax9 knockdown HASMC,and HASMC was divided into six groups: Control siRNA group;Control siRNA + 20 ng / ml PDGF-BB group;Pax9 siRNA group;Pax9siRNA + 20 ng / ml PDGF-BB group;Pax9 siRNA + SAG group;Pax9 siRNA + SAG +20ng / ml PDGF-BB group.Changes in the proliferation and migration ability of HASMC were detected by CCK8,phalloidin staining,and Transwell assay.The protein expression changes of Pax9,Shh and the dedifferentiation,proliferation and migration-related indicators were detected by Western Blot.Finally,the shh signaling pathway inhibitor cyclopamine was used to treat Pax9 over-expressed HASMC,and HASMC was divided into six groups: vector plasmid group;vector plasmid + 20 ng / ml PDGF-BB group;Flag-Pax9 group;Flag-Pax9 + 20 ng / ml PDGF-BB group;Flag-Pax9+ cyclopamine group;Flag-Pax9 + cyclopamine + 20 ng / ml PDGF-BB group.CCK8,phalloidin staining,and Transwell assay were used to detect changes in the proliferation and migration ability of HASMC.Western Blot was used to detect the protein expression changes of Flag,Shh and the dedifferentiation,proliferation and migration-related indicators.Results: Part ?: 1.After 12 weeks of feeding,the weight of Apo E-/-mice in the control(standard chow fed)group and the AS(high fat chow fed)group increased significantly compared with that before the experiment(P<0.001),and the weight of mice in the AS group increased significantly compared with that in the control group(P<0.001).2.Serum TG,TC and LDL-C levels of Apo E-/-mice in the AS group were significantly higher than those in the control group(P<0.001).The HDL-C level of Apo E-/-mice in AS group was significantly lower than that in control group(P<0.001).3.H&E staining results showed that the intima of the thoracic aorta of Apo E-/-mice in the control group was intact,without the formation and accumulation of foam cells,and no AS lesions were observed.In the AS group,the vascular wall was thickened,the intimal integritywas destroyed,and the formation and aggregation of foam cells and atherosclerotic plaque were observed under the intima.Compared with the control group,the vessel wall thickness of the AS group increased significantly(P<0.001).4.Immunofluorescence staining showed that the expression of Pax9 in Apo E-/-mice in the AS group was significantly higher than that in the control group,and it was mainly distributed in the middle membrane of the artery.5.Western blot results showed that the expression level of Pax9 protein in the AS group was significantly higher than that in the control group(P<0.01).Part ?: 1.With the increase of the intervention concentration of PDGF-BB,the proliferation activity of HASMC gradually increased,and the microfilaments in the cytoskeleton were reconstructed from irregular distribution to uniform and tight distribution,and the migration ability gradually increased.Moreover,with the increase of PDGF-BB intervention concentration,the expression of HASMC contractile phenotype marker proteins ?-SMA and SM22? gradually decreased,the expression of proliferation marker proteins PCNA gradually increased,and the expression of migration-related proteins MMP2 and MMP9 gradually increased.Meanwhile,Pax9 expression gradually increased and showed a significant dose-effect relationship.2.Knockdown of Pax9 in HASMC significantly inhibited the increase in PDGF-BB-induced HASMC proliferation activity,cytoskeletal remodeling and the enhancement of migration ability.Furthermore,knocking down Pax9 increased the expression of ?-SMA and SM22? in PDGF-BB-induced HASMC,and significantly inhibited the expression of PCNA,MMP2 and MMP9 in PDGF-BB-induced HASMC.3.Overexpression of Pax9 in HASMC significantly promotes the increase of PDGF-BB-induced HASMC proliferation activity,cytoskeletal remodeling and the enhancement of migration ability.Moreover,overexpression of Pax9 further reduced the expression of ?-SMA and SM22? in PDGF-BB-induced HASMC,and significantly promoted the expression of PCNA,MMP2 and MMP9 in PDGF-BB-induced HASMC.Part ?: 1.With the increase of PDGF-BB intervention concentration,Shh expression gradually increased and showed a significant dose-effect relationship.2.Treatment of HASMC with Shh signaling pathway agonist SAG before PDGF-BB stimulation significantly promotes PDGF-BB-induced increase in cell proliferationactivity,cytoskeletal remodeling and migration ability enhancement.At the same time,the expression of ?-SMA and SM22? in PDGF-BB-induced HASMC was more significantly reduced,and the expression of PCNA,MMP2 and MMP9 in PDGF-BB-induced HASMC was also more significantly increased.3.Treatment of HASMC with Shh signaling pathway inhibitor cyclopamine before PDGF-BB stimulation significantly inhibited PDGF-BB-induced increase in cell proliferation activity,cytoskeletal remodeling and migration ability enhancement.At the same time,the expression of ?-SMA and SM22? in PDGF-BB-induced HASMC increased,and the expression of PCNA,MMP2 and MMP9 in PDGF-BB-induced HASMC decreased.4.Knocking down Pax9 reduced the expression of Shh in HASMC induced by PDGF-BB.overexpression of Pax9 increased the expression of Shh in HASMC induced by PDGF-BB.5.Administration of SAG significantly reversed the effects of knockdown Pax9 on PDGF-BB-induced HASMC proliferation,cytoskeleton and migration.SAG significantly reversed the changes in the expression of PDGF-BB-induced HASMC dedifferentiation,proliferation and migration-related indicators induced by Pax9 knockdown.6.The administration of cyclopamine significantly attenuated the effects of overexpression of Pax9 on PDGF-BB-induced HASMC proliferation ability,cytoskeleton and migration ability.Cyclopamine significantly attenuated the changes in the expression of PDGF-BB-induced HASMC dedifferentiation,proliferation and migration-related indicators induced by the overexpression of Pax9.Conclusions: Part ?: 1.Apo E-/-mice fed with high-fat diets for 12 weeks successfully established AS animal model.2.The expression of Pax9 in the aortic tissue of atherosclerotic Apo E-/-mice increased,and it was mainly distributed in the arterial media.Part ?: 1.Pax9 promotes PDGF-BB-induced VSMCs dedifferentiation and cytoskeletal remodeling,and reduces the expression of contractile VSMCs markers?-SMA and SM22?.2.Pax9 promotes the proliferation of VSMCs induced by PDGF-BB,and increases the expression of the proliferation marker PCNA.3.Pax9 promotes the migration of PDGF-BB-induced VSMCs and increases the expression of migration-related proteins MMP2 and MMP9.Part ?: 1.Pax9 can promote PDGF-BB-induced VSMCs dedifferentiation,proliferation and migration by increasing Shh expression.2.Shh signaling pathway agonist SAG reverses the effect of knockdown Pax9 on dedifferentiation,proliferation and migration of PDGF-BB-induced VSMCs.3.Shh signaling pathway antagonist cyclopamine attenuates the effect of overexpression of Pax9 on dedifferentiation,proliferation and migration of PDGF-BB-induced VSMCs.