The Role And Mechanism Of Mitochondrial DNA Polymerase In Angiotensin Ⅱ-Induced Vascular Remodeling

Background and objective: Vascular remodeling is an important pathological basis for human cardiovascular events,and also one of the main reasons for hypertension leading to body complications such as stroke,heart failure and renal failure.Vascular smooth muscle cells are the main components of arterial vessels,and the phenotypic transformation of vascular smooth muscle is a key process of vascular remodeling.Angiotensin Ⅱ(Ang Ⅱ)can act on smooth muscle cells through multiple signaling pathways,promote their phenotypic transition,and then promote vascular remodeling.Therefore,it is considered as a classic model drug for vascular remodeling in hypertension.Mitochondria are often regarded as the powerhouse of cells,and mitochondrial damage may make a major impact on cellular function and is implicated in the pathogenesis of normal aging and vascular remodeling.Mitochondrial DNA polymerase γ(mitochondrial DNA Polymerase gamma,POLG)is encoded by the POLG gene,which is widely expressed in tissues and plays an important role in maintaining the functional integrity of cells.It is located in mitochondria and is involved in mitochondrial DNA replication and repair.POLG is involved in the occurrence and development of various diseases.When POLG has a D257 A point mutation,its exonuclease activity will be impaired,resulting in abnormal and decreased mitochondrial DNA copy number and mitochondrial damage.The role of POLG in Ang Ⅱ-induced vascular remodeling and the underlying mechanism of its effect on vascular remodeling are still unclear.Mitochondrial STAT3 exists in mitochondria and plays a key role in regulating mitochondrial function.It is currently believed that STAT3 can enter mitochondria through phosphorylation at Ser727.Mitochondrial STAT3 can bind to mitochondrial DNA and regulate mitochondrial gene expression,and it can also regulate the activity of mitochondrial respiratory chain and affect ATP production.It is still unclear whether mitochondrial STAT3 interacts with POLG in maintaining mitochondrial function,and whether mitochondrial STAT3 participates in vascular remodeling by interacting with POLG.The role of POLG in vascular remodeling in hypertension and the specific mechanism of its effect on vascular remodeling in hypertension are still unclear.The purpose of this study is to analyze the role of POLG in the process of Ang Ⅱ-induced vascular remodeling in vivo and in vitro,and to further explore the potential mechanism of POLG’s regulation of vascular remodeling,so as to provide new ideas for clinical prevention and treatment of vascular remodeling.Methods: In the first part,we investigated the role of POLG in Ang Ⅱ-induced hypertensive vascular remodeling in mice.C57BL/6 wild type(wild type,WT)and POLG point mutation(point mutation at the 257 th amino acid,PolgD257A/D257 A,D257A)mice selected,angiotensin Ⅱ(Ang Ⅱ)was subcutaneously infused for 2 weeks,and the control group was subcutaneously infused with normal saline for 2 weeks.The blood pressure of the mice was detected every week,and the remodeling of the aorta of the mice was detected by a small animal ultrasound apparatus after 2 weeks of administration.Then the mice were sacrificed and thoracic aorta and abdominal aorta specimens were collected.H&E staining was performed to observe the thickness of the vessel wall and blood vessel diameter size,and Masson staining was performed to observe the degree of vascular fibrosis.Finally,Western blot detected the differential expression of POLG in normal aorta and aorta in vascular remodeling,as well as phenotype transformation marker proteins(α-SMA and SM22α)and proliferation and migration marker proteins(PCNA,MMP9 and MMP2).Part Ⅱ: 1.A model of Ang Ⅱ-induced phenotype transformation was constructed in human aortic vascular smooth muscle cells(HAVSMCs).First,we divided HAVSMCs into four groups,and stimulated them with Ang Ⅱ with increasing concentration gradients,and detected POLG expression and smooth muscle phenotype transformation marker proteins α-SMA and SM22α by Western blot.Cytoskeleton was detected by phalloidin staining,cell viability was evaluated by CCK8 assay.We performed Transwell assay and other examinations to measure proliferation and migration ability of HAVSMCs.2.To explore the effect of overexpression of POLG on Ang Ⅱ-induced phenotype transformation in HAVSMCs.We performed Western blot analysis to show the changes of Myc,smooth muscle cell dedifferentiation.Cytoskeleton changes were detected by phalloidin staining.We used CCK8 assay,Transwell assay and Western blot analysis to measure proliferation and migration ability of smooth muscle cells.3.To explore the effect of knockdown of POLG on Ang Ⅱ-induced phenotype transformation in HAVSMCs.The smooth muscle phenotype transformation marker proteins were detected by Western blot,and cytoskeleton changes were detected by phalloidin staining.4.To investigate the effect of overexpression of mutant POLG on AngⅡ-induced phenotype transformation in HAVSMCs.Myc and smooth muscle phenotype transformation marker proteins were detected by Western blot,and cytoskeleton changes were detected by phalloidin staining.Part Ⅲ: To explore the potential mechanism of POLG regulating vascular remodeling.1.First,we explored the relationship between POLG’s effect on mitochondrial function and vascular remodeling through the D257 A mouse model of mitochondrial damage.Co-immunoprecipitation experiments were used to detect the binding of mitochondrial STAT3 to POLG in normal condition and POLG mutation condition.2.By overexpressing POLG in different states,the possible mechanism of POLG’s effect on AngⅡ-induced smooth muscle phenotype transformation was explored.Mitochondrial function was evaluated by detecting ATP content and mitochondrial membrane potential.Co-immunoprecipitation experiments were used to detect the binding of endogenous POLG to mitochondrial STAT3 in normal condition and stimulated by AngⅡ condition.3.To explore the effect of POLG on mitochondrial STAT3 and the upstream and downstream relationship between them.Mitochondria were extracted to detect the expression of STAT3 and its various phosphorylated forms in mitochondria.In HAVSMCs given STAT3 inhibitor,POLG was overexpressed to determine whether overexpression of POLG could rescue the effect of inhibiting STAT3 on mitochondrial function,and then to determine the upstream and downstream relationship between them.Result: Part Ⅰ: POLG expression was decreased in AngⅡ-induced vascular remodeling in mice.The D257 A mutation in POLG did not affect the blood pressure levels of mice in the basal state and AngⅡ-induced state.After stimulation with AngⅡ,D257 A mice showed more obvious vascular wall thickening,more obvious lumen enlargement,and more severe fibrosis.The expression levels of marker proteins(α-SMA and SM22α)related to the phenotypic transformation of vascular smooth muscle in D257 A mice were significantly decreased after AngⅡ stimulation,while the expression levels of marker proteins related to proliferation and migration(PCNA,MMP2 and MMP9)were more significantly increased.Part Ⅱ: 1.With the increase of AngⅡ administration concentration,the expression level of POLG gradually decreased,the expression level of HAVSMCs phenotype transformation marker protein gradually decreased,and the expression of proliferation and migration marker protein gradually increased.With the increase of drug concentration,the skeleton of cells was rearranged,the distribution of myofilaments became more and more chaotic,and the number of cell viability and migration increased gradually.2.Overexpression of POLG in HAVSMCs significantly inhibited AngⅡ-induced vascular smooth muscle phenotypic transformation-related protein expression decline,cytoskeleton rearrangement and myofilament distribution disorder.At the same time,overexpression of POLG significantly inhibited the increase of AngⅡ-induced proliferation and migration-related marker protein expression levels and the increase in the number of cell migration.3.Knockdown of POLG in HAVSMCs aggravated the AngⅡ-induced decreased expression of vascular smooth muscle phenotype markers,cytoskeletal rearrangement and disordered myofilament distribution.4.Overexpression of mutant POLG in HAVSMCs didn’t inhibit AngⅡ-induced decreased expression of vascular smooth muscle phenotypic transformation markers,cytoskeleton rearrangement and disordered myofilament distribution.Part Ⅲ: 1.D257 A mice vascular smooth muscle tissue decreased ATP production,and spontaneously showed a decrease in the expression level of proteins related to phenotypic transformation of vascular smooth muscle,and an increase in the expression of marker proteins related to proliferation and migration.There was no obvious abnormality in vascular structure.In mouse aortic smooth muscle tissue,POLG normally bound to mitochondrial STAT3,and the binding was attenuated after the D257 A point mutation.2.Overexpression of wild-type POLG in HAVSMCs inhibited the reduction of mitochondrial membrane potential and ATP production induced by AngⅡ stimulation,while overexpression of mutant POLG didn’t achieve the abovementioned inhibitory effect.POLG bound to mitochondrial STAT3 under normal conditions,and the binding was enhanced in Ang Ⅱ-induced cell damage.3.STAT3 existed in mitochondria,and its main phosphorylation active form was p-STAT3 Ser727.Ang Ⅱ-induced mitochondrial dysfunction was exacerbated by STAT3 inhibitor administration,while POLG overexpression rescued STAT3 inhibitor-induced mitochondrial dysfunction.Conclusions: POLG inhibits Ang Ⅱ-induced aortic vascular remodeling.Mitochondrial damage and dysfunction caused by abnormal POLG is one of the causes of vascular remodeling.Overexpression of POLG but not mutant POLG could stabilize mitochondrial function and inhibit Ang Ⅱ-induced vascular smooth muscle injury.In the process of stabilizing mitochondrial function,POLG is the direct executive molecule,and mitochondrial STAT3 plays an auxiliary role.When mitochondria are damaged,POLG and STAT3 form a complex to maintain and repair mitochondrial damage.Reduced complex formation resulting from mutations in POLG aggravates vascular damage by failing to maintain mitochondrial function.This study expounds the regulation and molecular mechanism of POLG on vascular remodeling,and provides a new idea for clinical prevention and treatment of vascular remodeling-related diseases.