The Roles Of Mitochondrial Function Related Factors In Phenotype Reverse Of Vascular Smooth Muscle Cells

Atherosclerosis plays a important role in coronary artery disease and cerebrovascular disease,though its mechanism is not known exactly up to now. Historically,vascular smooth muscle cells(VSMC) were thought that local factors caused the normally contractile cells of the vessel media to modulate to a synthetic state,and these cells migrated and proliferated in the vessel intima,ultimately to generate an atheromatous plaque.But it is not known that what factors are involved in VSMC phenotypes convert.synthetic state of VSMC contains a large number of cellular organelles such as ribosomes,rough endoplasmic reticulum,Golgi complexes,and mitochondria in its cytoplasm.In particular,an enhancement of cell growth and division is accompanied by an increase in mitochondria,because under such circumstances cells,need to generate high energy through oxidative phosphorylation.Thus,mammalian mitochondria must enhance the transcription and replication of mitochondrial DNA (mtDNA) to respond to the increased cellular ATP demands and mitogenesis.mitochondrial transcription factor A(mtTFA) is critical to transcription and replication of mitochondrial DNA(mtDNA),and is regulated by nuclear gene,such as nuclear respiratory factor-1 and peroxisome proliferator-activated receptor gamma coactivator 1(PGC-1),which play an important role in binding to or coactivating with a number of transcription factors involved in mitochondrial biogenesis.To investigate the relationship between mitochondria function and VSMC phenotype, PGC-1,NRF and mtTFA of VSMC,in vivo and in vitro,were evaluated in this article.Methods1.Anminal motile study of vascular smooth muscle cells phenotype alternation and adjust of mitochondria function Model groupsAS model:38Japan white rabbit were subject to two group randomly:control group(n=8) fed with common diet,model group(n=30)was randomly divided into three group(n=10) fed with common diet and 2%Cholesterol 8%,pork fat,4% eggyolk,we collect blood,aorta respective,when before experiment,4 week,8 week,12 week.(1) Blood lipid assay:Enzymatic colorimetic test was principle.Serum lipid concentrations were detected(2) Reverse transcription-PCR technique:Total RNA was isolated using Trizol reagent,following the manufacture instructions,synthesis of single stranded cDNA according to "First strand cDNA synthesis Kit".Then Polymerase chain was react GAPDH was used as an internal control.The primers were synthesized by Takara Technologies,PCR products were phoresed in 2%agarose gels in TBE buffer stained in 0.5%μg/ml ethidium bromide and photographed.(3) Western blotting:tissue lysates were prepared in lysis buffer.Whole lysates were collected and then mixed with sample buffer,each were subjected to SDS -polyacrylamide gel electrophoresis,and the proteins were then transferred to a polyvinylidene difluoride membrane using a Semi-Dry Transblot,and then it was blocked by incubation with skim milk for 2h at room temperature.The blockde membrane was subsequently incubated with first antibody for 12h at 4℃after washing with TTBs,the membrane was incubated for 2h at room temperature with alkaline phosphatase-conjugated secondary antibody IgC,bands of protein on the membrane were visualized with NBT/BCIP kit. 2.The relationship between vascular smooth muscle cells proliferation and mitochondria function(1) cell culture human vascular smooth muscle cells were incubated with ox-LDL,palmitate,NAN₃ for 24h.(2) Activity of cells:The growing state of cultured endothelial cells were examined by MTT.(3) Immunocytochemistry examination:All cells were fixed with pure acetone. SP method was used to detect the expression of PNCA.(4) Western blotting:Cell lysates were prepared in lysis buffer.The experimental process is similar to 1.3(5) Mitochondria extraction:performanced according to the instruction of kit(6) cytochrome c oxidase activity,performanced according to the instruction of kit3.statical AnalysisAll sata were expressed as mean±standard deviation,and SPSS11.5 software was employed to analyzed the data.Statistical evaluation was performed using one- way ANOVA and correlation analysis.P＜0.05 was considered significant.Result1.The detection of the marker of vascular smooth muscle cells phenotype and regulative factors of mitochondria function.(1) Animal conditionsIn the experiment,there was no difference in weight between experimental groups (0.85±0.10)and the control(1.0±0.22)(P＞0.05).(2) The results of blood lipid assayThe blood lipid had an increasing tendency with the time of feeding high-fat food going.The levels of TC and LDL increased significantly(p＜0.05).(3) The results of RT-PCR for detecting mRNA of PGC-1,NRF-1,mtTFA,SMembThere was a significant difference among the level of PGC-1,NRF-1 and mtTFA after 4W,8W and 12W(p＜0.05).they were upregulated with long time of high fat feeding.The expression of mtTFA had a consistant increasing with that of NRF-1 (p＜0.05).As the maker of synthetic cells,A high level of SMemb was found after 8Ws which presented increasing synthetic cells(p＜0.05)(4) the result of western blot test for detection the protein of PGC-1,NRF-1,mtTFA,SMembSignificant difference were seen among the protein level of PGC-1 after 4W,8W and 12W(p＜0.05),the same result can be found in NRF-1 and mtTFA(p＜0.05).they increased as time of high fat feeding gone.There had a consistant increasing of protein of PGC-1,NRF-1 and mtTFA.With synthetic cells increased,A high level of SMemb was found(p＜0.05)2.The result of interfering proliferation of VSMC and the change of mitochondria function.(1) The protein level of PGC-1,NRF and mtTFA under various interfere factors.when ox-LDL existed,protein level of PGC-1,NRF-1 and mtTFA increased (p＜0.05),but palmitate was given,protein level of them decreased(p＜0.05).In another group,NAN₃ was given,the increasing proteins of PGC-1,NRF-1 and mtTFA could be found(p＜0.05).(2) The result of cytochrome c oxidase activity test Under the condition of ox-LDL given,the cytochrome c oxidase activity has enhanced (p＜0.05),but in palmitate group and NAN₃ group,the activity of cytochrome c oxidase were supressed(p＜0.05).(3) The result of proliferation activity of VSMCOx-LDL can promote the activity of proliferation(p＜0.05),while palmitate and NAN₃ has a reverse result(p＜0.05).(4) The result of detection for PCNACompared with the control,PCNA protein revealed high level in ox-LDL group (p＜0.05),but low level in palmitate and NAN₃ grou(p＜0.01)ConclusionGenerally,the change of VSMC Phenotype from contractile cells to synthetic cells contributes to atherosclerotic process.1.In our study,the expression of NRF-1 was up-regulated with synthetic cells increasing,the same finding was seen in expression of mtTFA.This suggested that there were definite relationship between NRF-1 and mtTFA,and that coactivation of them controlled the function of mitochondria and transform of VSMC phenotype.2.Positive correlation between PGC-1 and NRF-1 was found in VSMC proliferation.Furthermore,interfering expression of PGC-1 might down-regulate the expression of NRF-1.so it can be supposed that in atherosclerotic process,some factors switch on the increasing expression of PGC-1,which up-regulate some receptors, especially NRF-1 in nucleus,then activate mtTFA.3.So the pathway of PGC-NRF-mtTFA may play critical roles in VSMC proliferation and transform of phenotype that contribute to atherosclerotic.