The Role Of LMCD1 In The Occurrence And Development Of Thoracic Aortic Dissections

BackgroundAcute thoracic aortic dissection(TAD)is a life-threatening cardiovascular disease with a high mortality and should be paid more attention to.Most patients still have high mortality and postoperative complications after emergency surgery.Due to the rapid onset and development of TAD,if it is not detected and treated in time,it can be fatal caused by the rupture.At present,there is no effective drug treatment to delay the progression of thoracic aortic dissection,and surgical treatment is the most effective treatment,but there is also a higher risk of postoperative complications,especially for the older people.Therefore,further exploration of the pathological basis and molecular biological mechanism of thoracic aortic dissection will provide more possibilities for the treatment of thoracic aortic dissection.The pathological changes of TAD are mainly manifested by the loss of aortic media smooth muscle cells(VSMCs),and the excessive degradation of extracellular matrix(ECM)such as elastic fibers and collagen fibers.Relevant studies have pointed out that proliferation,migration,phenotypic switching,and ECM degradation and destruction play an essential role in the progression of TAD.Under the stimulation of external pathological factors,the morphology of vascular smooth muscle cells changes from long-spindle type to short-spindle ones.This process is called dedifferentiation process.Meanwhile,synthetic secretion function of VSMCs increases while its contractile function weakens.The expression of such smooth muscle cell specific marker molecules as ?-SMA,SM22 a is reduced whereas the synthesis and secretion of extracellular matrix components such as collagens,proteoglycan,and certain proteases increases.Among many proteases,matrix metalloproteinases(MMPs),including MMP-2 and MMP-9,are particularly critical.They not only involve the increase of expression levels,but also the enhancement of activity,which leads to the degradation of ECM,causing the destruction of vascular wall tension.However the specific molecular mechanism of VSMCs proliferation,migration and phenotypic switching have not been elucidated to date.Among many proteases,matrix metalloproteinases(MMPs),including MMP-2 and MMP-9,are particularly critical.They not only involve the increase of expression levels,but also the enhancement of activity,which leads to the degradation of ECM.LIM domain proteins(named after the first 3 LIM proteins identified: Lin11,Isl-1and Mec-3),are found involved in cell growth and development,differentiation,cytoskeletal remodeling and cardiovascular disease through interaction with transcription regulators,kinases and structural proteins.Among them,related studies have found that LIM and cysteine-rich domain 1(LMCD1)can promote the proliferation and migration of human vascular smooth muscle cells(HVSMCs).Recent researchers showed that LMCD1 can promote vessel restenosis,and the expression level of LMCD1 can be detected in human atherosclerosis tissues.However,there is no report on whether LMCD1 is involved in the pathogenesis of TAD and how LMCD1 is involed in the progression of TAD..Objectives1.To Clarify the expression of LMCD1 in human TAD and the pathological mechanism involved;2.To Clarify the expression of LMCD1 in the mouse TAD model,as well as the effect of knocking out LMCD1 in the mouse TAD model and related pathological changes;3.To clarify the effect of LMCD1 on VSMCs phenotype switching,proliferation,migration and MMPs secretion;4.To Explore the specific molecular mechanism of LMCD1 in the process of biological function transformation of human vascular smooth muscle cells Methods1.Collection and processing of human aortic specimensAortic specimens of patients with TAD were collected,genetic diseases such as Marfan syndrome,and other diseases such as syphilitic TAD were excluded.The aorta of the normal control group could be taken from the recipient of the heart transplantation,or from the aorta that underwent aortic valve replacement with a normal aortic diameter.The specimens were divided into two parts and placed in formalin and liquid nitrogen,respectively,for pathological experiments(HE,VB,immunohistochemistry),PCR and Western Blot experiments,to detect the expression levels of LMCD1,MMPs and other related proteins in human TAD tissue and normal aortic tissue.2.Construction of mouse thoracic aortic dissection modelAfter being fed with BAPN water for 28 days,the TAD model was constructed in C57BL/J mice and LMCD1 knockout mice by subcutaneously embedding Ang II(1000ng/kg/min)mini pumps for 24 hours,and tissue samples were obtained in 4%paraformaldehyde and liquid nitrogen for pathological staining(HE,VB and immunofluorescence),PCR and Western Blot experiment.To detect the expression of LMCD1,contractile phenotype related proteins and MMPs in mouse TAD model and normal aortic tissue.To explore the role of knocking out LMCD1 in the development of mouse TAD model and related pathological mechanisms.3.The exploration of the function and mechanisms of LMCD1 in VSMCs1.Western Blot: According to the experimental method,the cell protein was extracted and the concentration was matched according to the BCA quantitative method,and the protein expression level including contraction phenotype markers and extracellular matrix proteins were detected.2.Cell model: Ang II(1umol/L)was used to stimulate HVSMCs cells for 24 hours to construct a phenotypic transformation model.3.Interfering RNA transfection: Using Lip02000 or RNAi MAX to introduce si-LMCD1 into HVSMCs according to the instructions,and replace the culture medium after 6 hours.4.Virus transfection: Construct LMCD1 overexpressing adenovirus,after determining the virus titer and concentration,appropriate amount of virus was added to infect cells.5.CCK-8: :Using CCK-8 kit based on WST to detect the proliferation ability of human aortic smooth muscle cells.6.Wound healing assay:Under different treatment conditions,the cell scratch experiment was used to determine the migration ability of vascular smooth muscle cells.4.Statistical analysis methodsWhen the statistical data were continuous variables,they were shown in the form of mean ± standard error.when the statistical data were categorical variables,they were shown in the form of frequencies.Normality of the data was examined by using the Kolmogorov-Smirnov test.Comparisons of continuous variables were performed using unpaired two-tailed Student's t-test,one-way analysis of variance with Turkey's post-test or two-way analysis of variance with Bonferroni test,or the non-parametric Kruskal-Wallis test with Dunn's multiple comparison,wherever appropriate,while the chi-square test was used to evaluate categorical variables.Kaplan-Meier survival curves were plotted to examine mouse survival rates,and the differences were analyzed by using the log-rank(Mantel–Cox)test.Software SPSS 22.0 and Prism 6.0 were used for statistical analysis,P<0.05 was considered statistically different.Results1.Elevated expression of LMCD1 in thoracic aortic dissection accompanied by increased expression of MMP-2 / 9The results of pathological staining showed that compared with the normal aorta,the vessel wall of the TAD was destroyed dramatically.PCR and Western Blot results showed that the expression of LMCD1 in human thoracic aortic dissection tissue was increased.Immunohistochemistry confirmed the increase of LMCD1 expression and also the elevated expression of MMP-2 / 9,and also showed that LMCD1 was localized in the aortic media.2.Knockout of LMCD1 significantly alleviated the pathological destruction and reduced mortality in TAD mice modelBy analyzing the incidence,mortality,survival curve and general data characteristics of TAD in each group of mice(including wild-type control group,wild-type TAD group,LMCD1 knockout control group,LMCD1 knockout TAD group),results showed that knocking out LMCD1 can significantly reduce the mortality of TAD.According to the HE and VB staining,results showed that compared with wild-type TAD mice,LMCD1 knockout TAD mice significantly reduced the destruction to the aortic wall integrity.We performed further analyses of mice aortas to determine the role of LMCD1 in VSMCs phenotypic switching,MMP-2 and MMP-9 production.In WT mice,aortic challenged with BAPN and Ang II markedly increased LMCD1 activation in the aortic wall.Furthermore,aortic challenged with BAPN and Ang II in WT mice also decreased SM22 and ?-SMA expression in the aorta.However,these phenotypic switching proteins were upregulated in LMCD1 knockout TAD mice,confirming the involvement of LMCD1 in inducing phenotypic switching.Aortic challenged with BAPN and Ang II in WT mice significantly increased MMP-2 and MMP-9 production,including MMP-2expression in SMCs,MMP-9 expression in macrophages in the aortic wall.BAPN and Ang II-induced upregulation of MMP-2 and MMP-9 observed in the aortas of WT TAD mice was downregulated in LMCD1 knockout TAD mice,suggesting that LMCD1 is involved in promoting MMP production and the aortic inflammatory response.Together,these findings suggest that LMCD1 may contribute to aortic degeneration and TAD formation in part by promoting SMC phenotypic switching,MMP-2 production in VSMCs and MMP-9 production in macrophages.3.LMCD1 can promote smooth muscle cell phenotypic transformation,proliferation and migration,and increases the secretion of MMP-2/9After Ad-LMCD1 treatment,cells were stimulated with Angll.Western Blot results showed that compared with Ad-GFP group,vascular smooth muscle cell contraction phenotype markers such as ?-SMA and SM22 were significantly reduced after overexpression of LMCD1 while elevation of the expression of MMP-2 / 9.CCK-8results showed that overexpression of LMCD1 could increase the cell proliferation ability,and through the scratch experiments in vascular smooth muscle cells,results showed that treated with Ad-LMCD1 to overexpress LMCD1 significantly promoted the migration to the center of the scratch.Western Blot results showed that compared with the Si-Control transfection,inhibition of LMCD1 resulted in the increased expression of such contractile phenotypic markers as ?-SMA and SM22 while in inhibition of MMP-2/9 expression.CCK-8 results showed that the cell proliferation activity decreased after inhibiting LMCD1 with Si-LMCD1,and through the scratch experiments in vascular smooth muscle cells,results showed that treated with Si-LMCD1 to inhibit LMCD1 significantly inhibited the migration to the center of the scratch.4.LMCD1 can promote VSMCs smooth muscle cell phenotypic transformation,proliferation,migration and MMPs secretion through the downstream PI3K/AKT/GSK3? signaling pathwayThe RNA sequence analysis report speculated that the downstream signaling pathway of LMCD1 involved in the activation of PI3K/AKT/GSK3?signaling pathway.Overexpression of LMCD1 can activate PI3K/AKT/GSK3?signaling pathway,while inhibiting LMCD1 can significantly reduce the downstream PI3K/AKT/GSK3 ?signaling pathway.Furthermore,we used Western Blot to verify the regulatory relationship between LMCD1 and downstream PI3K/AKT/GSK3?on VSMCs biological function.Overexpression of LMCD1 accompanied by the application of PI3 K inhibitor LY294002 can significantly reversed the VSMCs biological function such as increased proliferation and migration ability caused by overexpression of LMCD1.ConclusionsThe expression of LMCD1 is significantly increased in TAD tissues.Knockout of LMCD1 can significantly alleviate the pathological destruction of the aorta and reduce mortality in TAD mice model.LMCD1 can promote the proliferation,migration,phenotypic transformation and MMPs secretion of VSMCs through PI3K/AKT/GSK3?signaling pathway.