Mechanism Of SPARC Regulating Apoptosis And Phenotypic Transformation Of Human Brain Vascular Smooth Muscle Cells In Vitro

BackgroundIntracranial aneurysms(IAs)are cerebrovascular degenerative diseases.They are characterized by tumor-like protrusions formed by the abnormal expansion of the intracranial arteries and the thinning of the tube wall.IAs rupture and bleeding cause subarachnoid hemorrhage(SAH),which has a high lethality and disability.Therefore,early diagnosis and treatment of IAs is crucial.Epidemiological studies show that the prevalence of IAs ranges from 0.2%to 9%,and their 1-year and 5-year rupture risks are 1.4%and 3.4%,respectively.At present,it is believed that the degradation of extracellular matrix(ECM),the destruction of arterial vessel wall homeostasis caused by various factors,inflammatory response,apoptosis and phenotypic transformation of vascular smooth muscle cells(VSMCs)are involved in the pathophysiology and the pathogenesis of IAs.However,the specific mechanism of the occurrence and development of IAs is not completely clear.Elucidating the relevant molecular mechanisms of the occurrence,development and rupture of IAs will help us to find new interventions and treatment strategies.Along with the infiltration of various inflammatory cells,the secretion of cytokines and inflammatory factors,VSMCs will undergo apoptosis,proliferation and phenotypic transformation,and play an important role in the occurrence,development and rupture of IAs.Studies found that VSMCs are lost in IAs tissue,and numerous apoptotic VSMCs exist in ruptured IAs.VSMCs have two types of phenotypes:contractile phenotype and synthetic phenotype.The phenotypic transformation of VSMCs refers to the process of converting from a contractive phenotype to a synthetic phenotype.Autophagy can regulate the phenotypic transformation of VSMCs,while autophagy plays a key role in the formation and ruptures of IAs.In non-ruptured and ruptured IAs,the expression levels of autophagy-related genes such as microtubule associated protein light chain 3(LC3),autophagy related protein-5(ATG5),and Beclin-1 were significantly up-regulated.Therefore,autophagy-induced phenotypic transformation of VSMCs is closely related to the formation of IAs.Secreted protein acidic and rich in cysteine(SPARC)is a secreted glycoprotein widely existing in the extracellular matrix of various tissues and organs.SPARC participates in multiple biological processes such as cell migration,proliferation and apoptosis and tissue repair.It is related to the processes of ECM remodeling,growth,cell differentiation,wound repair and is involved in the pathophysiological processes of tumors,vascular diseases and metabolic diseases.The expression of SPARC in the IAs tissue has been significantly increased.However,the specific mechanism by which SPARC participates in the formation and development of IAs is not clear.SPARC may be involved in the formation and development of IAs through the following pathways.(1)Regulates the mitochondrial pathway apoptosis:over-expression of SPARC can up-regulate expression levels of cleaved Caspase 3(CC3)and cleaved poly ADP-ribose polymerase(CPARP),and initiate apoptosis in the mitochondrial pathway;(2)Regulates autophagy:SPARC can induce increased autophagy in various cells;(3)Regulates glucose and lipid metabolism:SPARC expression levels in vascular endothelial cells and VSMCs of atherosclerosis(As)are significantly up-regulated.As the first rate-limiting enzyme in the glycolytic pathway,the expression of hexokinase 2(HK2)is obviously down-regulation in As plaques.The expression of SPARC in the plasma of type 2 diabetes patients is significantly higher than that of normal people.SPARC can negatively regulate the expression of HK2 and affect the glycolysis process of liver cancer cells.Both ox-LDL and high glucose can induce phenotypic transformation of VSMCs,thereby participating in the formation of IAs.Therefore,it is speculated that SPARC may affect the biological function of VSMCs by regulating HK2 expression and participate in the occurrence of IAs;(4)Anti-adhesion effect:SPARC has a significant ability to resist local adhesion between cells;(5)Regulates ECM:SPARC can interact with ECM proteins,promote the production of matrix metalloproteinases(such as MMP-2,MMP-9 etc.)with their activity increasing,regulate remodeling of ECM;(6)Regulates inflammatory response:SPARC can regulate a variety of inflammatory responses.An increased expression of SPARC indicates that the vessel wall is in an inflammatory state,thereby promoting the occurrence of IAs.In summary,as a secreted glycoprotein in the extracellular matrix,SPARC may be a direct participant in affecting the onset,progression and even prognosis of IAs.However,the effects of SPARC on the apoptosis and phenotypic transformation of VSMCs and the specific mechanism are still unclear.The related molecular mechanisms and the inter-regulatory relationship between genes need to be further studied.Based on the above research background,this topic investigated the role of SPARC protein in inducing apoptosis and phenotypic transformation of the mitochondrial pathway in human brain vascular smooth muscle cells(HBVSMCs).We first analyzed the mRNA expression profile of HBVSMCs treated with SPARC in vitro,and found that HK2,a gene whose expression was significantly down-regulated,may be involved in SPARC-induced HBVSMCs apoptosis.Then,by using immunofluorescence staining,Western blot(WB),small interfering RNA(siRNA)to knock down HK2 gene,liposome transfection,RT-PCR,flow cytometry,Hoechst 33342/PI staining,JC-1 staining,transmission electron microscope(TEM)and other techniques and methods,we proved that SPARC regulates mitochondrial apoptosis of HBVSMCs by down-regulating HK2 expression.Finally,by using immunofluorescence staining,WB,siRNA to knock down ATG5 gene and liposome transfection,RT-PCR,Hoechst 33342/PI staining,autophagosome staining,TEM and other techniques and methods,we demonstrated that SPARC mediates increased autophagy of HBVSMCs through the AMPK/mTOR pathway,thereby regulating phenotypic transformation and apoptosis of HBVSMCs.Part I:Detection and analysis of HBVSMCs whole gene expression profile chip after SPARC treatmentObjectiveTo explore the molecular mechanism of SPARC-induced apoptosis of HBVSMCs and find the differentially expressed genes of HBVSMCs regulated by SPARC through detecting the changes of the whole gene expression profile of HBVMSCs after SPARC treatment.Methods1.The morphology of HBVSMCs was observed under an optical microscope and the contractile phenotype of HBVSMCs was identified by immunofluorescence staining.2.A control group and SPARC(2 ?g/mL)treatment group were set.After 24 hours of SPARC treatment,the total RNA of two groups of HBVSMCs were extracted,and detailed raw data and analysis reports were obtained by detecting the whole gene expression profile.3.The quality of the chip data was evaluated.Gene Ontology analysis,KEGG and BioCarta pathway enrichment analysis on the expression profile chip results were performed.4.To verify the stability of the chip results,mRNA expression of randomly selected genes with significantly up-regulated and down-regulated genes were performed by RT-PCR.Results1.The HBVSMCs of the 5th generation were spindle-shaped with multiple cell protrusions under the light microscope.Immunofluorescence staining showed that both the contractile phenotypes SMA and SM22-a were significantly stained,the cell nuclei were round,and fiber filaments parallel to the long axis of the cell were visible.2.The signal intensity distribution curve,box plot and principal component analysis plot showed that the chip data were reproducible and met the criteria for continued analysis.The scatter plot,volcano plot and cluster analysis results showed that the data distribution of this chip was reasonable.In this project,a total of 21,449 known gene loci were detected by the chip.A total of 368 differential genes were screened that met the expression change | Fold Change |>1.5 and P-value<0.05,with 165 up-regulated and 203 down-regulated.3.GO analysis showed that differentially expressed genes were mainly involved in the biological processes such as anatomical structure and morphology,cellular organism development and cytoskeleton expression of HBVSMCs.KEGG and BioCarta pathway enrichment analysis showed that the molecular pathways involved in differentially expressed genes were mainly mitogen-activated protein kinase(MAPK)pathway,JAK-STAT signaling pathway and local adhesion pathway.4.RT-PCR results showed that among the 6 genes selected randomly,the mRNA changes of 5 genes were consistent with the chip results,indicating that the chip results have high reliability.5.Based on the results of the chip and through large numbers of literature readings,we found that HK2 may play an important role in SPARC-induced apoptosis of HBVSMCs.Conclusions1.SPARC treatment can cause changes in the gene expression profile of HBVSMCs.There are 368 differentially expressed genes,with 165 up-regulated and 203 down-regulated.2.Differentially expressed genes are mainly involved in biological processes such as the anatomy and morphology,the development of cellular organisms,and the expression of cytoskeleton in HBVSMCs.The enriched molecular pathways are mainly involved in MAPK pathway,JAK-STAT signaling pathway,focal adhesion pathway and so on.3.SPARC may induce mitochondrial apoptosis of HBVSMCs by down-regulating expression of HK2 and may promote apoptosis of HBVSMCs by inducing excessive autophagy.Part II:SPARC regulates mitochondrial pathway apoptosis of HBVSMCs by down-regulating HK2 expressionObjectiveTo investigate that SPARC can regulate mitochondrial pathway apoptosis of HBVSMCs by down-regulating HK2 expressionMethods1.Immunofluorescence staining and WB were used to verify that SPARC can induce HK2 expression down-regulation in HBVSMCs2.The siRNAs were constructed to knock down HK2 expression of HBVSMCs,and their knockdown effects were detected by RT-PCR and WB.Appropriate knockdown sequence was selected for subsequent experiments.3.The effects of SPARC treatment and HK2 knockdown on the apoptosis and cell cycle of HBVSMCs were detected by Hoechst33342/PI double staining,flow cytometry and WB method,respectively.4.The effects of SPARC treatment and HK2 knockdown on mitochondrial membrane potential of HBVSMCs were detected by JC-1 staining,respectively5.The mitochondrial damage of HBVSMCs by SPARC treatment was observed under transmission electron microscopy.Results1.Immunofluorescence staining and WB results showed that SPARC can regulate HK2 expression and induce HK2 expression down-regulation in HBVSMCs.2.The siRNAs to knock down HK2 gene were successfully constructed.The siRNA HK2 3#was selected for subsequent experiments because it had the strongest inhibitory effect detected by RT-PCR and WB.3.Hoechst 33342/PI double staining,flow cytometry and WB results showed that both SPARC treatment and HK2 knockdown can induce apoptosis of HBVSMCs.Cell cycle tests showed that both SPARC and HK2 knockdown can change the cell cycle distribution of HBVSMCs and arrest cells in G0/G1 phase.4.JC-1 staining showed that both SPARC treatment and HK2 knockdown can reduce mitochondrial membrane potential of HBVSMCs cells.5.The mitochondria of HBVSMCs were significant damaged due to SPARC treatment,because of mitochondrial swelling,mitochondrial crista rupture and even disappearance observed under TEM.Conclusions1.SPARC induces down-regulation of HK2 expression in HBVSMCs.2.SPARC can induce mitochondrial pathway apoptosis in HBVSMCs,inhibit cell cycle,reduce mitochondrial membrane potential and cause mitochondrial ultrastructure changes.3.The knockdown of HK2 in HBVSMCs can also induce mitochondrial pathway apoptosis,inhibit cell cycle and reduce mitochondrial membrane potential in HBVSMCs.4.HK2 is involved in SPARC-induced mitochondrial pathway apoptosis in HBVSMCs.Part ?:SPARC regulates phenotypic transformation and apoptosis of HBVSMCs through AMPK/mTOR-mediated autophagyObjectiveTo explore the change of autophagy level in HBVSMCs after SPARC treatment and its relationship with phenotypic transformation and apoptosis.Methods1.The expression of contractile phenotype SMA and SM22-?,as well as synthetic phenotype OPN of HBVSMCs after SPARC treatment were detected by immunofluorescence staining,RT-PCR and WB.2,The protein expression of autophagy-related genes LC3 ?,p62,ATG5 and Beclin-1 in HBVSMCs induced by different concentrations of SPARC treatment and at different time points after treatment were detected by WB.The most appropriate treatment concentration and observation time for subsequent experiment were determined.3.The effects of SPARC and 3-MA on the number of autophagosome in HBVSMCs were observed under confocal microscopy and TEM.The autophagy inhibitor 3-MA and lysosomal inhibitor CQ were administered to detect changes in autophagy flow of HBVSMCs induced by SPARC.4.The expression of p-AMPK and p-mTOR in HBVSMCs before and after SPARC treatment was detected by WB.The expression of p-AMPK,p-mTOR and autophagy related proteins LC3?,p62,ATG5,Beclin-1 was detected after administration of AMPK inhibitor Compound C by WB.5.The siRNAs to knock down ATG5 expression of HBVSMCs were constructed,and their knockdown effects were detected by RT-PCR and WB.Appropriate knockdown sequence was selected for subsequent experiments.The effects of ATG5 knockdown on the expression of autophagy-related(LC3 II,p62,ATG5,and Beclin-1)and apoptosis-related(Bax,Bcl-2,and CPARP)proteins in SPARC-treated HBVSMCs were detected by WB.The effects of ATG5 knockdown on HBVSMCs apoptosis before or after SPARC treatment were detected by Hoechst 33342/PI double staining.6.The effects of autophagy inhibition by 3-MA on the phenotypic transformation of HBVSMCs before and after SPARC treatment were detected by WB.Results1.Immunofluorescence staining,RT-PCR,and WB results showed that SPARC can induce phenotype transformation of HBVSMCs,with expression of contractile phenotypes SMA and SM22-a down-regulated and synthetic phenotype OPN up-regulated.2.WB results showed that SPARC induces autophagy increase of HBVMSCs in a time-and dose-dependent manner.The increase of autophagosome in HBVSMCs induced by SPARC was observed under confocal microscopy and TEM,while 3-MA could partially prevent this process.SPARC induced an increase of autophagy flow in HBVSMCs detected by pretreatment with 3-MA and CQ.3.WB results showed that SPARC treatment can induce the up-regulation of p-AMPK expression and the down-regulation of p-mTOR expression in HBVSMCs.Compound C pretreatment could partially prevent SPARC-induced autophagy increase of HBVSMCs by inhibiting of p-AMPK expression.4.The siRNAs to knock down ATG5 gene were successfully constructed.The siRNA ATG5 3#was selected for subsequent experiments because it had the strongest inhibitory effect detected by RT-PCR and WB.WB results showed that ATG5 gene knockdown can significantly inhibit SPARC-induced autophagy increase of HBVSMCs,and simultaneously inhibit SPARC-induced apoptosis increase of HBVSMCs.Hoechst 33342/PI double staining results also showed that ATG5 gene knockdown can partially prevent SPARC-induced apoptosis increase of HBVSMCs.5.WB results showed that inhibition of autophagy with 3-MA can partially prevent SPARC-induced HBVSMCs phenotypic transformation.Conclusions1.SPARC-induced autophagy can mediate the conversion of HBVSMCs from a contracted phenotype to a synthetic phenotype,and inhibition of autophagy can partially prevent the phenotype transformation of HBVSMCs.2.SPARC induces increased autophagy and autophagic flux in HBVSMCs,and this process is mediated by the AMPK/mTOR signaling pathway.3.SPARC-induced excessive autophagy of HBVSMCs leads to apoptosis of HBVSMCs,and inhibition of autophagy can partially prevent SPARC-induced apoptosis of HBVSMCs.