The Role Of SPARC-induced Oxidative Stress In Human Brain Vascular Smooth Muscle Cells In The Occurrence And Development Of Intracranial Aneurysms

Background:Intracranial aneurysms(IAs)is a pathological condition in which the blood vessels in the brain are abnormally expanded,and the blood vessel walls become weak and easy to rupture.IAs rupture and bleed into the subarachnoid space around the brain,causing subarachnoid hemorrhage(SAH).Despite modern neurosurgical intensive care methods,the prognosis of SAH patients is poor.SAH is a common consequence of the rupture of IAs.The mortality rate of aneurysmal SAH is still around 30-40%,which is an important cause of stroke death in relatively young people;more than one-third of survivors also have permanent neurological deficits;therefore,IAs is a potential and destructive clinical problem.Despite in-depth research on IAs,our understanding of the mechanisms leading to the occurrence,progression and rupture of IAs is still not fully established.It is generally believed that the common pathway of IAs formation begins with hemodynamically induced endothelial dysfunction,and then stimulates the phenotype of cerebral artery smooth muscle cells(vascular smooth muscle cells,VSMCs)to secrete pro-inflammatory factors to induce inflammation in the arterial wall Eventually,the extracellular matrix is decomposed by various proteases and VSMCs apoptosis,which leads to vascular degeneration,dilation and rupture.VSMCs are the main cellular components of blood vessel walls.Under normal circumstances,when arterial blood vessels are damaged by various factors,smooth muscle cells in the middle layer of the blood vessel wall will be induced to migrate to the intima,proliferate and synthesize new matrix to repair and maintain the health of blood vessels.If the vascular wall loses mural cells,the "repair and maintenance"process maintained by matrix synthesis and VSMCs proliferation is disrupted,and the blood vessel will undergo pathological changes.Therefore,changes in the function and quantity of VSMCs,including phenotypic transformation and apoptosis,must play an important role in the pathogenesis of IAs.Secreted protein acidic and rich in cysteine(SPARC)is a glycoprotein located in the cell matrix and regulates the interaction between cells and the surrounding extracellular matrix.SPARC controls basic cell functions,such as cell adhesion,proliferation and differentiation,regulating the activity of a variety of growth factors and matrix metalloproteinases necessary for the degradation and turnover of extracellular matrix.However,it is reported in the literature that SPARC also plays a key role in certain cell pathophysiological mechanisms,such as oxidative stress,inflammation,apoptosis,and autophagy in cancer and liver diseases in a non-diabetic environment.Recently,it has been found that SPARC is widely expressed in human IAs,especially in VSMCs.This suggests that SPARC may be involved in the formation and development of IAs.Many studies have found that the formation of IAs is a chronic inflammatory process,that has an important factor in the pathophysiology is oxidative stress.Studies have found that oxidative stress can induce important processes in the formation of cerebral aneurysms,including direct damage to the endothelium,stimulating the conversion of VSMCs phenotype to inflammatory phenotype,and inducing the expression of chemotactic cytokines and adhesion molecules to regulate the recruitment of inflammatory cells,even inducing apoptosis of VSMCs.In addition,matrix metalloproteinases can be activated by free radicals,leading to remodeling and decomposition of blood vessel walls.Free radical-mediated lipid peroxidation leads to atherosclerosis,which is the main pathological manifestation of cystic IAs.Studies have shown that reactive oxygen species(ROS)are mainly derived from NADPH oxidase.In cerebral artery endothelial cells,VSMCs and adventitia cells,NADPH oxidase is widespread and active.Under the influence of blood flow impact and various cytokines and other factors,NADPH oxidase in cerebral arteries is induced and activated to produce oxygen free radicals,such as superoxide free radicals and hydrogen peroxide(H2O2).In addition,NADPH oxidase can also increase production through the feedforward effect of free radicals,which causes the rapid generation and accumulation of free radicals and triggers oxidative stress.Recent studies have clarified the pathogenesis of reactive oxygen free radicals derived from NADPH oxidase and SPARC in kidney and epithelial damage,as well as mature adipocytes and liver cells.Among these mechanisms,SPARC,NADPH oxidase and ROS have strong interactions,especially in terms of oxidative stress.NADPH Oxidase 4(NOX4)has been studied and found to be widely expressed in vascular smooth muscle and can be induced by transforming growth factor-?1(TGF-?1).It has been clear that both are involved in the pathological process of oxidative stress in Marfan syndrome,and patients with Marfan syndrome are prone to have intracranial aneurysms.Therefore,we speculate that SPARC may induce a series of pathophysiological processes of cerebrovascular smooth muscle cells through NOX4-induced oxidative stress,which is involved in the development of IAs.To sum up,in order to explore the relationship between SPARC and human brain vascular smooth muscle cells(human brain vascular smooth muscle cells,HBVSMCs)oxidative stress,pro-inflammatory behavior and apoptosis,we collected the The IAs tissue excised during the operation and the superficial temporal artery vascular tissue of random craniotomy patients were also collected as a control.After processing the tissue samples and imaging by immunohistological staining,analyze the expression and localization of SPARC,NOX4 and certain cytokines in the wall of IAs(intracellular or extracellular)to determine the correlation between the two.We chose HBVSMCs as a model of the arterial media.By directly supplementing SPARC to stimulate HBVSMCs in vitro,we used fluorescence microscope,flow cytometry and western blot(western blot,WB)techniques to analyze intracellular ROS products and corresponding proteins.Changes in expression and apoptosis,and finally through inhibitors and small interfering RNA(small interfering RNA,siRNA)gene knock-down methods to explore the mechanism of action between the above molecules.The purpose of this study is to explore the pathophysiological process of oxidative stress that SPARC is involved in the formation of IAs,which can enable us to further understand the relationship between SPARC and IAs.In the future,SPARC and its regulatory molecules may be used as therapeutic targets,providing a new idea and new method for the treatment of IAs.Objective:1.To explore the role of SPARC participating in oxidative stress in promoting the occurrence and development of IAs.2.To explore the molecular mechanism of SPARC induced oxidative stress,phenotypic changes of pro-inflammatory and matrix remodeling,and apoptosis of HBVSMCs.Methods:1.Clinical specimens and immune-histochemical stainingTwelve cases of clinical aneurysm tissue samples were collectd by IAs surgical resection through microsurgery,and 10 cases of superficial temporal artery at the scalp incision were collected as a control.The sample tissues were made into paraffin sections and immune-histochemical staining was used to detect the expression of SPARC and NOX4 in the aneurysm tissue and control samples.2.Cell culture and processingThe primary generation of HBVSMCs purchased from the Sciencell Research Laboratory were cultured under the standard culture conditions specified by the manufacturer and regularly observed and subcultured.HBVSMCs cultured to 3 to 6 generations were used as experimental objects.Cells were treated with SPARC at a concentration of 6?g/mL to simulate its overexpression,and cells were stimulated with TGF-?1 at a concentration of 1ng/mL to detect its effect.3.Detection of intracellular ROSAfter processing the cells for a specific experiment,follow the instructions of the instructions of the superoxide fluorescence kit(ENZ-51012,Enzo-LifeScience,Farmingdale,NY)and the intracellular hydrogen peroxide detection kit(MAK164,Sigma-Aldrich).Nuclear staining was performed with Hoechst 33342,the fluorescence intensity of ROS in the cells was checked with a fluorescence microscope and photographed,and ROS was analyzed by flow cytometry.4.Detection of oxidative stress related proteinsThe cells were seeded in a 6-well plate,and the total protein of VSMCs was extracted after the cells were treated in a specific experiment.The expression changes of the corresponding NOX4,NOX1,TGF-?1,SPARC protein and antioxidant proteins SOD1,CAT,PRDX1,Nrf2 were detected by WB,and then the expression changes of related proteins were qualitatively detected by immune-fluorescence.5.Detection of phenotypic transformationThe The cells were seeded in a 6-well plate,and the total protein of VSMCs was extracted after the cells were treated in a specific experiment.The phenotypic transformation marker proteins MCP-1 and MMP-9 were detected by WB,and the expression changes of related proteins were qualitatively detected by immunofluorescence.6.Detection of ApoptosisUing the TUNEL experiment to detect cell apoptosis,and the WB method was used to detect apoptosis-related proteins BAX,BCL2,casepase3,etc.By measuring the mitochondrial membrane potential(??m)and observing the cell mitochondrial morphology and ultrastructure under a transmission electron microscope,the SPARC-induced apoptosis of the mitochondria was proved.7.Inhibitor treatment and cell transfection with small interfering RNAPre-treatment with specific protein inhibitors or specially designed interfering sequence siRNAs targeting genes SPARC,NOX4 and TGF-?1(three for each gene)to prepare siRNA transfection mixture for cell transfection,aiming to block the transmission pathway and knock down the corresponding protein expression.Then according to the needs of the experiment,the corresponding treatment is given to study the molecular pathway mechanism.8.Statistical analysisWith SPSS26.0 software and GraphPad Prism 6.0,Statistical analysis was performed.Spearman rank correlation analysis was used for the correlation between SPARC and NOX4.Use unpaired t-test or one-way analysis of variance(ANOVA)to analyze two and multiple sets of data respectively.p<0.05 was considered statistically significant.Results:1.The expression and relationship of SPARC and NOX4 in aneurysm tissue1.1 The results of immunohistochemical analysis showed that the expression of SPARC and NOX4 in the vascular wall of IAs increased,compared with normal blood vessels.1.2 The results of immunofluorescence double staining showed that SPARC and NOX4 were both significantly expressed and co-localized in the middle layer of the vessel wall in the blood vessels of IAs.1.3 Spearman analysis showed that the overexpression of SPARC and NOX4 in the vessel wall of IAs was positively correlated.2.SPARC induces oxidative stress in HBVSMCs and promotes phenotypic transformation and apoptosis of HBVSMCs2.1 The mechanism that SPARC induces oxidative stress in HBVSMCs through the TGF-?1-NOX4 axis2.1.1 Direct supplementation of recombinant SPARC(rSPARC)can simulate the effect of increased SPARC expression.2.1.2 SPARC induces the accumulation of H2O2 in HBVSMCs,leading to oxidative stress.2.1.3 SPARC induces H2O2 production by up-regulating NOX4 in HBVSMCs.2.1.4 SPARC regulates the expression of NOX4 through TGF-?1 to control ROS production.2.1.5 SPARC and TGF-?1 are positive feedback regulation in HBVSMCs.SPARC is a necessary condition for NOX4 induced by TGF-?1 to have biological activity.2.2 SPARC induces oxidative stress to promote the phenotypic transformation mechanism of HBVSMCs2.2.1 SPARC induces the pro-inflammatory phenotype and matrix phenotype transformation of HBVSMCs.2.2.2 ROS participates in SPARC-induced MCP-1 and MMP-9 expression of HBVSMCs.2.2.3 NF-?B participates in ROS-mediated expression of MMP-9 and MCP-1.2.3 SPARC induces oxidative stress to promote mitochondrial apoptosis of HBVSMCs2.3.1 SPARC induced mitochondrial apoptosis of HBVSMCs.2.3.2 ROS is involved in SPARC-induced mitochondrial apoptosis of HBVSMCs.Conclusion:1.SPARC and NOX4 are overexpressed in aneurysm tissues and are positively correlated.2.SPARC induces the oxidative stress response of HBVSMCs through the TGF-?1/NOX4 axis.And it promotes the transformation of HBVSMCs to inflammatory phenotype and matrix remodeling phenotype to induce inflammation and vascular remodeling.By further inducing the apoptosis of HBVSMCs mitochondrial pathway,thereby weakening the structure and repair function of blood vessel wall.3.The disorder of SPARC and TGF-?1 autocrine positive feedback loop regulation amplifies the cascade of local oxidative stress and inflammation in the blood vessel wall,which may be the key mechanism for the local formation of aneurysms.4.SPARC overexpression promotes the development of IAs.In the future,diagnosis and treatment methods targeting SPARC and its regulatory molecules may become a new direction and new method for us to treat intracranial aneurysms.