Research On The Effects And Mechanism Of Smooth Muscle LGL1 In Neointimal Hyperplasia

BackgroundNeointimal hyperplasia is a significant type of vascular remolding defined as the pathological accumulation of vascular smooth muscle cells(VSMCs)and extracellular matrix(ECM)in the intima.It is a process of excessive repair in the vascular wall caused by various activated cells and recycling substances responding to vessel injury.Vascular injury inevitably occurs with various clinical procedures.Percutaneous coronary interventions such as balloon angioplasty and stents to treat ischemic coronary artery disease,vein grafts for coronary artery bypass graft surgery,and vascular access in hemodialysis can result in lumen re-narrowing and restenosis in a year after the operation.The behavior of VSMCs plays a vital role in neointimal hyperplasia.Gathering around the vessel lesion,activated inflammatory cells,and disturbed endothelial cells release a number of factors such as platelet-derived growth factor(PDGF)to stimulate the proliferation of VSMCs and subsequent migration from the media layer of the vessel to the intima.Previous studies have revealed that many proteins regulate neointimal hyperplasia,including signal transducer and activator of transcription 3(STAT3).When VSMCs are activated by cytokines or growth factors,STAT3 is phosphorylated and translocates into the nucleus to regulate the expression of target genes involved in proliferation and migration.Earlier researches have shown that interfering the STAT3 gene or administrating STAT3 inhibitor could suppress VSMCs proliferation and migration in vitro,thus decreasing neointimal formation in animal models of neointimal hyperplasia.Although the role of STAT3 in neointimal hyperplasia is clear,the regulation of STAT3 expression and activity needs further exploration.Lethal giant larvae(LGL)proteins are a group of highly conserved proteins first discovered in Drosophila.LGL1 maintains cell polarity and acts as a tumor suppressor.In our recent study,LGL1 could inhibit vascular calcification via high mobility group box 1.Nonetheless,the role of LGL1 in neointimal hyperplasia after the vascular injury has not been elucidated.In this study,we used smooth muscle-specific LGL1 knockout(LGL1SMKO)mice to explore the role of smooth-muscle LGL1 in neointimal hyperplasia to discover possible clinical intervention targets for vascular remodeling after injury.Dissertation IThe Role and Mechanism of Smooth Muscle LGL1 in Vascular Smooth Muscle Cell Proliferation and MigrationObjectives1.To determine the change of LGL1 expression in VSMCs after pathological proliferation stimulation;2.To investigate the effect of LGL1 on the proliferation and migration of VSMCs;3.To explore the specific mechanism of LGL1 modulating the proliferation and migration of VSMCs.Methods1.Extraction of mouse primary aortic vascular smooth muscle cellsLGL1SMKO mice were generated by cross-breeding LGL1flox/flox mice with transgenic Cre mice controlled by α-smooth muscle actin(α-SMA)promoter.Littermate LGL1flox/flox/Cre-mice were used as control(CTR).Mice of 4-6 weeks old were chosen to extract primary aortic VSMCs by tissue block adherent method.Cell immunofluorescence was used to detect the smooth muscle cell markers(SM22α,αSMA)in the extracted cells.2.Explore the change of LGL1 expression in VSMCs after pathological proliferation stimulationVSMCs were treated with PDGF-BB(20ng/mL)for different times(0h,12h,24h,48h and 72h).Western Blot and qRT-PCR were used to detect protein and mRNA levels of LGL1,respectively.3.Explore the effect of LGL1 on the proliferation and migration of VSMCsVSMCs were infected with adenovirus-expressing GFP or LGL1,then treated with PDGF-BB(20ng/mL)or Vehicle(PBS)for 48 h.VSMCs were divided into 4 groups:①GFP+Vehicle;②LGL1+Vehicle;③ GFP+PDGF-BB;④ LGL1+PDGF-BB.Western Blot and qRT-PCR were used to detect protein and mRNA levels of cell cycle related-genes Cyclin D1 and proliferating cell nuclear antigen(PCNA).Cell proliferation was measured by a CCK-8 assay.A scratch wound healing assay was conducted to evaluate cell migration.4.Explore the effect of LGL1 deletion on the proliferation and migration of VSMCsVSMCs extracted from CTR and LGL1SMKO mice were treated with PDGF-BB(20ng/mL)or Vehicle(PBS)for 48 h.VSMCs were divided into 4 groups:① CTR+Vehicle;② LGL1SMKO+Vehicle;③ CTR+PDGF-BB;④ LGL1SMKO+PDGF-BB.Western Blot and qRT-PCR were used to detect protein and mRNA levels of Cyclin D1 and PCNA.Cell proliferation was measured by a CCK-8 assay.A scratch wound healing assay was conducted to evaluate cell migration.5.Explore the regulation of LG1 on STAT3VSMCs were infected with adenovirus-expressing LGL1.Western Blot and qRTPCR were used to detect protein and mRNA levels of STAT3 and P-STAT3(Y705).Immunoprecipitation was conducted to determine whether LGL1 could bind with STAT3.Furthermore,autophagic inhibitor 3-methyladenine(3-MA),lysosomal inhibitor chloroquine(CQ)and proteasome inhibitor MG132 were used to confirm how LGL1 influences STAT3 in protein degradation pathway experiments.6.Explore the effect of STAT3 inhibitor on the proliferation and migration of LGL1 deficient VSMCsVSMCs extracted from CTR or LGL1SMKO mice were pretreated with the STAT3 inhibitor SH-4-54(10μM)or Vehicle(DMSO)for 24h and then treated by PDGF-BB(20ng/mL)or Vehicle(PBS)for 48h.VSMCs were divided into the following 8 groups:①CTR+Vehicle+Vehicle;②LGL1SMKO+Vehicle+Vehicle;③CTR+Vehicle+SH-4-54;④ LGL1SMKO+Vehicle+SH-4-54;⑤ CTR+PDGF-BB+Vehicle;⑥ LGL1SMKO+PDGF-BB+Vehicle;⑦CTR+PDGF-BB+SH-4-54;⑧LGL1SMKO+PDGF-BB+SH-4-54.Western Blot and qRT-PCR were used to detect protein and mRNA levels of Cyclin D1 and PCNA.Cell proliferation was measured by a CCK-8 assay.A Scratch wound healing assay was conducted to evaluate cell migration.7.Statistical analysisAll data are presented as mean± SEM.The normality assumption of the data distribution was assessed by the Kolmogorov-Smirnov test.For the normal distribution,a two-tailed Student unpaired t-test was used to compare the two groups.Differences between multiple groups with one variable were analyzed by one-way ANOVA followed by Bonferroni’s post-hoc test.P<0.05 was considered statistically significant.Results1.LGL1 expression was inhibited by PDGF-BB in VSMCsWestern Blot and qRT-PCR measured the protein and mRNA expression of LGL1 in VSMCs treated by PDGF-BB at different times.The results indicated that PDGFBB inhibited both protein and mRNA expression of LGL1 in VSMCs,and the level decreased with the increment of treatment time.2.LGL1 overexpression inhibited the proliferation and migration of VSMCsWestern Blot and qRT-PCR showed that PDGF-BB increased the Cyclin D1 and PCNA protein and mRNA levels,while LGL1 overexpression inhibited the protein and mRNA expression of Cyclin D1 and PCNA.CCK-8 assay also showed that LGL1 overexpression could inhibit the proliferation of VSMCs.In addition,the wound healing experiment showed that LGL1 overexpression could significantly inhibit the migration of VSMCs.3.LGL1 deletion aggravated the proliferation and migration of VSMCsCompared with the CTR group,the LGL1SMKO group upregulated the expression of Cyclin D1 and PCNA protein as well as mRNA in the VSMCs.LGL1 deletion exacerbated the increased expression of cell cycle-related proteins induced by PDGFBB.Cell proliferation assay and wound healing assay indicated that LGL1 deletion could promote the proliferation and migration of VSMCs,respectively.4.LGL1 could bind with STAT3 and promote its degradationWestern Blot and qRT-PCR showed that LGL1 overexpression reduced both STAT3 and P-STAT3(Y705)protein levels but not STAT3 mRNA levels.Thus,LGL1 may affect the STAT3 protein expression by regulating its degradation.Immunoprecipitation assay revealed that LGL1 could bind with STAT3.MG132 but not 3-MA or CQ could reverse the degradation of STAT3 induced by LGL1 overexpression.Thus,LGL1 could bind with STAT3 and promote its degradation via the proteasomal pathway.5.LGL1 inhibited the proliferation and migration of VSMCs via STAT3LGL1 deficiency increased the protein and mRNA levels of Cyclin D1 and PCNA,which were attenuated by SH-4-54.Consistently,LGL1 deletion promoted cell proliferation under PDGF-BB stimulation,which was significantly suppressed by SH4-54 treatment.Furthermore,LGL1 deficiency-enhanced cell migration was also attenuated by the STAT3 inhibitor.Therefore,via STAT3,LGL1 inhibited VSMC proliferation and migration.Conclusion1.PDGF-BB inhibits the expression of LGL1 in VSMCs;2.LGL1 overexpression in VSMCs reduces the expression of cell cycle-related proteins,thus inhibiting the proliferation and migration of VSMCs;3.LGL1 deletion in VSMCs enhances the expression of cell cycle-related proteins and promotes the proliferation and migration of VSMCs;4.LGL1 downregulates STAT3 protein without affecting STAT3 mRNA level by binding to STAT3 and promoting STAT3 degradation through the proteasomal pathway.5.Inhibition of STAT3 in VSMCs significantly counteracts the enhanced proliferation and migration of VSMCs caused by LGL1 deficiency.Dissertation ⅡThe Role and Mechanism of Smooth Muscle LGL1 in Mouse Neointimal Hyperplasia ModelObjectives1.To determine the change of LGL1 expression during vascular remodeling after injury in vivo;2.To investigate the effect of smooth muscle LGL1 in neointimal hyperplasia;3.To verify the specific mechanism of smooth muscle LGL1 regulating neointimal hyperplasia in vivo.Methods1.Mice model for neointimal hyperplasiaLGL1SMKO mice were generated by cross-breeding LGL1flox/flox mice with transgenic Cre mice controlled by α-smooth muscle actin(α-SMA)promoter.Littermate LGL1flox/flox/Cre-mice were used as CTR.The expression of the smooth muscle LGL1 gene in mice vascular system was identified by immunofluorescence colocation staining(α-SMA,LGL1).8-week-old mice were subjected to left common carotid artery(LCCA)ligation to induce neointimal hyperplasia and labeled as Ligated.The right common carotid artery(RCCA)as a Sham was processed as for the LCC except for ligation.After 3 weeks,mice were euthanized to collect tissues.2.Explore the change of LGL1 expression during vascular remodeling after injury in vivo8-week-old wild mice were undergone LCCA(Ligated)to induce neointimal hyperplasia.The RCCA acts as control(Sham).Mice were euthanized to collect tissues after 3 weeks.Western Blot and immunohistochemistry were used to test the change of LGL1 protein content in Ligated and Sham.mRNA level was measured by qRT-PCR.Experiment groups are Ligated and Sham.3.Explore the role of vascular smooth muscle LGL1 in mice with neointimal hyperplasia8-week-old wild mice were undergone LCCA(Ligated)to induce neointimal hyperplasia.The RCCA acts as control(Sham).Mice were euthanized to collect tissues after 3 weeks.Western Blot and immunohistochemistry were used to detect the level of cell cycle related-protein Cyclin D1 and PCNA.HE staining of common carotid arteries was conducted to evaluate the degree of neointimal hyperplasia.The mice divided into 4 groups were as follows:① CTR+Sham;② LGL1SMKO+Sham;③ CTR+Ligated;④ LGL1SMKO+ Ligated.4.Verify the specific mechanism of smooth muscle LGL1 regulating neointimal hyperplasia in vivo8-week-old wild mice were undergone LCCA(Ligated)to induce neointimal hyperplasia.The RCCA acts as control(Sham).The mice were administrated with STAT3 inhibitor SH-4-54(1Omg/Kg/day)or Vehicle during the 3 weeks.Mice were euthanized to collect tissues after 21 days.HE staining of common carotid arteries was conducted to evaluate the degree of neointimal hyperplasia.The mice divided into 8 groups were as follows:①CTR+Vehicle+Sham;② LGL1SMKO+Vehicle+ Sham;③ CTR+SH-4-54+Sham;④LGL1SMKO+SH-4-54+Sham;⑤ CTR+Vehicle+Ligated;⑥ LGL1SMKO+Vehicle+Ligated;⑦ CTR+SH-4-54+Ligated;⑧LGL1SMKO+SH-4-54+Ligated.5.Statistical analysisThe same as Dissertation I.Results1.LGL1 expression was decreased during vascular injuryWestern Blot and qRT-PCR manifested that LGL1 protein and mRNA expression in Ligated were decreased compared with Sham.Immunohistochemical staining also demonstrated that LGL1 expression was decreased during neointimal hyperplasia.2.Smooth muscle cell-specific deletion of LGL1 promoted neointimal hyperplasiaWestern Blot and immunohistochemical staining showed that the protein levels of Cyclin D1 and PCNA in the Ligated group were significantly increased than those in the Sham group.The protein content of Cyclin D1 and PCNA in the LGL1SMKO group was further upregulated than in the CTR group,which agreed with the severe neointimal hyperplasia.HE staining reflected that LGL1 deficiency significantly aggravated neointimal formation,as reflected by enlarged intima area and increased intima/media ratio.3.LGL1 regulated the development of neointimal hyperplasia via STAT3 in vivoHE staining was used to reveal the morphological changes in neointimal hyperplasia of common carotid arteries.Compared with control,LGL1SMKO mice displayed aggravated neointimal hyperplasia,which was implied by the intimal area and intima/media ratio.Moreover,this phenomenon was partly reversed by SH-4-54,which confirmed that SH-4-54 could significantly attenuate neointimal hyperplasia in LGL1SMKO mice.Conclusion1.The protein and mRNA levels of LGL1 were decreased in neointimal hyperplasia;2.Smooth muscle cell-specific deletion of LGL1 promotes neointimal hyperplasia and lumen restenosis after vascular injury;3.LGL1 regulates neointimal hyperplasia through STAT3,and STAT3 inhibitor can significantly attenuate neointimal hyperplasia and vascular remodeling in LGL1SMKO mice.