The Role And Mechanism Of Mitochondrial Dynamics And Redox In Mediating The Function Of Vascular Smooth Muscle Cells And The Pathogenesis Of Hypertension

BACKGROUNDS:Hypertension is the leading risk factor of premature morbidity and mortality,affecting about one-third adults worldwide.Essential hypertension account for more than 90%of total hypertensive population,however,its pathogenesis is complex and remains less clear.Vascular smooth muscle cells（VSMCs）,the main component of blood vessels,predominant vascular tension,blood flow and blood pressure.Mitochondria is the "power machine" of all eukaryotic cells,of which structural or functional disorder have been demonstrated involving in the evolution of various cardiovascular diseases.To date,the role of mitochondrial fission/fusion dynamics or redox homeostasis in mediating VSMCs function and blood pressure has not been fully investigated.Type Ⅱ angiotensin（AngⅡ）is a powerful stimulator of hypertension,has been reported promoting mitochondrial fission in adventitial fibroblasts and endothelium.On the other hand,hydrogen sulfide（H2S）is an endogenous gas transmitter,of which synthesis in VSMCs is mainly derived by cystathionine-y-lyase（CTH/CSE）.CTH-H2S is an endogenous scavenger of mitochondrial reactive oxygen species（ROS）.Conversely,excessive mitochondrial ROS contribute to the rise of VSMCs stiffness and thereby entire vascular stiffness and hypertension.OBJECTIVE:Based on previous evidence discussed above,this study aims to explore the possible regulatory roles and the underlying mechanism of mitochondrial dynamics and redox in VSMCs function and the pathogenesis of hypertension.The following scientific questions were raised:1.Whether mitochondrial fission/fusion disbalance is an integrated part,and thus a potential therapeutic target in the process of AngⅡ-induced VSMCs phenotypic transformation and blood pressure elevation?2.Whether VSMCs CTH-H2S plays a role in blood pressure regulating?And if so,what’s the underlying molecular mechanism,is mitochondrial ROS involved in this process?METHODS:In vivo,mice were planted with AngⅡ micro-osmotic pump（400 ng/kg/min）to induce hypertension.Hypertensive mice were randomly allocated into mitochondrial division inhibitor-1（Mdivi-1）group（20 mg/kg,intraperitoneal injection[i.p.],every other day）,or the dimethyl sulfoxide（DMSO）group（corresponding dose of vehicle solution by i.p.,every other day）.Throughout 10 days of treatment,the blood pressure of the mice was continuously recorded by a carotid artery intubated telemetry.Vessels were collected for transmission electron microscope（TEM）scanning of the media mitochondrial,dihydroethidium（DHE）staining,Haematoxylin-eosin（HE）staining,vasodilation detection.Plasma were collected for liver and kidney function assay.In vitro,mice primary aortic VSMCs were isolated and cultured,and then treated with AngⅡ and/or Mdivi-1 to assay their effects on VSMCs mitochondrial morphology,the expression and phosphorylation dynamin related protein 1（Drp1）,VSMCs proliferation,migration and phenotypic transformation,respectively.VSMCs-specific CSE knockout(cth^SMC-/-)mice were constructed,and Cre recombinase-negative(cth^flox/flox)littermates were used as controls.The blood pressure of the adult mice were recorded by carotid artery intubated telemetry and tail artery blood pressure measurements.The pulse wave velocity（PWV）was measured by ultrasound,and Young’s Modulus of the arterial media was measured by the nanoindentation.Vessels were collected for TEM scanning of the media mitochondria,DHE staining,HE staining,Sirius red staining,Verhoeff’s Van Gieson staining,etc.and vasodilation detection.In vitro,the mice primary aortic CSE knockout（KO）and wide type（WT）VSMCs were isolated and cultured.Comparisons of the elastic modulus detected by atomic force microscopy,Young’s modulus detected by nanoindentation,cell contractility detected by type I collagen gel and expression of phenotype markers detected by immunoblots between KO and WT VSMCs were made.Additionally,comparisons of mitochondrial respiratory function,mitochondrial ROS staining and mitoTracker staining,ect.were made between KO and WT VSMCs to detect their differences regarding mitochondrial morphology and function.In order to identify the potential downstream mitochondrial target of VSMCs CTH-H2S,mass spectrometry analysis of the mitochondrial proteins isolated from WT and KO VSMCs were performed.By performing gain-of-function and loss-of-function experiments of this presumed downstream target,its role in mediating VSMCs CTH-H2S regulated mitochondrial ROS,cell stiffness and contractility were further investigated.RESULTS:In vivo,Mdivi-1 i.p.successfully blocked the AngⅡ micro-osmotic pumpinduced elevation in blood pressure,and rescued AngⅡ-induced vascular media remodeling and vasodilation impairment.Importantly,plasma biochemical examinations indicated that Mdivi-1 treatment was not associated with noticeable liver or renal functional impairments,as compared with the control vehicle solution treatment.In vitro,AngⅡ induced Drpl mitochondrial translocation by increasing its phosphorylation of Ser616 site,and thus mitochondrial fission in VSMCs.Mdivi-1 treatment significantly inhibited AngⅡ-induced phosphorylation of Drpl Ser616 phosphorylation and mitochondrial fission,as well as VSMCs proliferation,migration and phenotypic transformation.Compared with the cth^flox/flox mice,cth^SMC-/-mice developed spontaneous hypertension and markedly increased vascular media stiffness,PWV acceleration,extracellular collagen deposition,vasoconstriction and vasodilation impairment.Consistently,as compared with WT VSMCs,KO VSMCs exhibited increased cellular stiffness and reduced cell contractility.Besides,KO VSMCs displayed obvious mitochondria structural and functional damage and increased mitochondrial ROS production.The application of mitochondrial ROS scavenger mito-TEMPO in KO VSMCs significantly reduced cell stiffness as well restored cell contractility.Mitochondrial protein profiling showed that Uridine diphosphate glucuronosyltransferase Family 1 Member A6（Ugt1a6）was significantly down-regulated in mitochondria of KO VSMCs.Down-regulation of Ugt1a6 in WT VSMCs increased mitochondrial ROS level,increased cell stiffness,and decreased cell contractility.Conversely,overexpression of Ugt1a6 in KO VSMCs reduced mitochondrial ROS,decreased cell stiffness,and improved cell contractility.CONCLUSIONS:Overall,this study suggested that mitochondrial fission/fusion and redox disturbance are important regulatory mechanisms that mediating VSMCs function and the pathogenesis of hypertension.Pharmacological interference of mitochondrial fission in VSMCs prevented AngⅡ-induced VSMCs phenotypic transformation and hypertension.In addition,VSMCs CTH-H2S inhibited VSMCs stiffness and blood pressure elevation via Ugt1a6-mediated mitochondrial ROS.These results complement the complex molecular network that controls blood pressure and reveal novel mitochondrial molecular targets and drug strategies in hypertension management.