| BackgroundAcute coronary syndrome(ACS)is the major mortality of coronary heart disease.Coronary thrombosis,induced by plaque rupture and plaque erosion,can cause acute coronary syndromes(ACS).Histopathologically,the plaque vulnerability is enhanced if plaque contains a large lipid-rich necrotic core covered by a thin fibrous cap.As the main components of plaque's fibrous cap,collagen determines the plaque vulnerability.Furthermore,the physical state of collagen can have a great impact on the development of atherosclerotic plaque by interfering with SMCs and macrophages.Prolyl-4-hydroxylase(P4H),composed of two alpha subsets and two beta subunits,plays a central role in the synthesis of all known types of collagens.As an isoenzyme of P4H,P4H?1 is a rate-limiting enzyme and is essential for collagen maturation and secretion.It has been shown that manipulating expression of P4H?1 affects atherosclerotic plaque stabilization,while different manipulations of P4H?1 have different influences on lesional collagen synthesis.The frictional force of vascular endothelial cells caused by blood flow is called shear stress.Atherosclerotic plaque formation occurs preferentially in areas of vascular bending,bifurcation,blockage or eddy flow,where wall shear stress is disordered and low.Conversely,straight arterial segments with laminar flow appear to be protected from atherosclerosis.In some studies of shear stress and atherosclerotic plaque,the size,composition and vulnerability of atherosclerotic plaques were different in different shear stress modes.Cheng et al.developed a perivascular shear stress modifier(called a cast)which could induce changes in shear-stress patterns in a straight vessel for studying the development of carotid atherosclerotic lesions of stable and vulnerable phenotypes in apolipoprotein E deficient(ApoE-/-)mice.Using this technique,the carotid artery was artificially under low shear stress or oscillatory shear stress.The changes of lipid metabolism,collagen metabolism and inflammatory responses were observed in the formation of atherosclerotic plaques under different shear stress induced atherosclerotic plaque by overexpressing P4H?1.Objectives1.To study the effects of P4H?1 overexpression on stability of shear stress induced atherosclerotic plaques in ApoE-/-mice;2.To study the influence of P4H?1 overexpression on shear stress induced atherosclerotic plaques.Methods1.animal experimental model and GroupingAll 60 mice,8 weeks old,were given a high-fat diet containing 0.25%cholesterol and 15%cocoa butter,starting at 2 weeks before surgery.As described previously by Cheng etal,we used a shear stress cast that imposed a fixed geometry on the right common carotid artery.Two weeks after cast placement,sixty mice were randomly assigned to three groups(n=20 in each group):Mock group;Lenti-EGFP group and Lenti-P4Hal group.the mice in each group were given a tail vein injection of saline,empty Lentivirus(3.5x106TU null lentivirus)or Lentivirus-P4Hal(3.5x106 TU lentivirus containing P4H?1)transgenic therapy,respectively.All mice were euthanized at 2 weeks after transgenic therapy.2.Hematological indicators measurementBlood samples were taken from the tail vein before euthanized,then centrifuged for 15 minutes(2500 r/min)at 4?.The enzyme method detected the serum levels of Triglycerides(TG),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol.The serum levels of hydroxyproline detected by ELISA kit.3.Immunohistochemistry and HistologyThe right common carotid arteries were fixed in 4%paraformaldehyde.The tissues were frozen in OCT compound and then cut at 5 ?m thickness continuously.The serial cryosections were stained with hematoxylin and eosin;oil-red O staining was used for lipid-rich lesions;The sirius red staining was used for collagen;The macrophages were detected with anti-rabbit CD68 by immunohistochemistry.4.Quantitative Real-Time PCR measurementTotal RNA was collected from the carotid artery specimens.The RT-PCR was performed to determine the reletive mRNA expression level of P4Ha1.5.Western Blot AnalysisThe expression of P4H?1 and TGF-?1 of the right carotid arteries in three groups were detected by Western Blot.6.Statistical analysisAll data are expressed as meanąSD.Unpaired t test was used for the analysis of two groups.When comparing more than two groups,one-way ANOVA was used.SPSS 16.0 was used for statistical analysis.A group difference was considered significant when p<0.05.Results1.General condition of the miceThe body weight and level of serum lipids did not differ between the three groups(p>0.05).However,the level of hydroxyproline in Lenti-P4Hal group was much higher than the other two groups(p<0.05).Therefore,overexpression of P4Hal had no significant effect on lipid levels,suggesting that diet or lipid levels did not appear to play a role in the atherosclerotic lesion differences between the three groups.2.The transfection efficiency of Lentivirus-P4HalWe measured the expressions of P4Ha1 in different regions using RT-PCR and western blot.The mRNA and protein expressions of P4Hal were higher in Lenti-P4Ha1 mice than in Mock or Lenti-EGFP mice(P<0.01).3.Plaque Size in different shear stress regions of three groupsCryosections of plaque tissues among all three groups in different shear stress regions stained with hematoxylin eosin.We measured H&E staining revealed that the lesion size under OSS regions was decreased than under LSS regions in Mock and Lenti-EGFP mice(P<0.05);the lesion size under LSS and OSS regions was increased in Lenti-P4Hal mice(P<0.01).4.Plaque composition in different shear stress regions of three groupsThe sirius red staining was used for collagen;Sirius red staining showed that the Collagen content under OSS regions was increased than under LSS regions in Mock and Lenti-EGFP mice(P<0.01);P4H?1 overexpression increased the thickness of fibrous cap and collagen content in LSS regions as compared to controls(P<0.01,P<0.05);P4H?1 overexpression increased the thickness of fibrous cap and collagen content in OSS regions as compared to controls(P<0.01).Oil red O staining was used to measure the lipid content.The lipid accumulation did not differ among all three groups in both LSS and OSS regions(P>0.05).We investigated the infiltration of macrophages in plaque tissues by stained with macrophage marker CD68.Compared to controls,overexpression of P4H?1 significantly reduced the expression of CD68 in LSS regions(P<0.01),which significantly decreased the expression of CD68 in OSS regions(P<0.01).5.The effects of TGF-?1 in conjunction with lenti-P4Hal treatmentWe measured the expression levels of TGF-?1 in carotid plaques using western blot.The protein expression of TGF-?1 was higher in Lenti-P4Hal mice than in Mock or Lenti-EGFP mice(P<0.01).Conclusions1.Lenti-P4H?1 treatment in both low and oscillatory shear stress-induced plaques increased collagen and the thickness of fibrous cap,and decreased macrophage accumulation but no change in lipid accumulation;2.Overexpression of P4Hal increased plaque size and lenti-P4H?1 treatment increased the expression of TGF-?1.BackgroundThe mortality of cardiovascular disease is the highest among all kinds of diseases.The common pathological basis is the changes of proliferation,migration,hypertrophy,apoptosis,cell morphology and function of vascular wall cells.SMCs(smooth muscle cells)and ECs(endothelial cells)are important cell components of the vascular wall and play an important role in the development of diseases.Under physiological conditions,SMCs are not exposed directly to fluid flow,however,SMCs are exposed to fluid shear stress due to transmural interstitial flow.Under pathological conditions such as angioplasty,atherosclerosis,intima and internal elastic lamina(IEL)injury,as a result,SMCs are directly exposed to fluid shear stress.Experiments in animal models have shown that SMCs may be present from days to months after a vascular impairment.Although the effects of shear stress on SMCs are not as well characterized as those on ECs in vitro.there have been several studies suggesting that elevated shear stress inhibit SMC proliferation rates.Collagen is an important component of extracellular matrix,which is mainly derived from SMCs.Under pathological conditions such as atherosclerosis,the composition of extracellular matrix was significantly altered.As the main components of plaque's fibrous cap,collagen determines the plaque vulnerability.P4Hal is a rate-limiting enzyme in collagen synthesis and the changes in expression can cause changes in collagen synthesis.Atherosclerotic plaques occur primarily at the curves and bifurcations of arteries where blood flow was distured.Cells are subjected to pathological oscillatory shear stress in these regions.The changes of P4H?1 under oscillatory shear stress have not been reported.c-Jun N-terminal kinases(JNK)is one member of mitogen-activated protein kinases(MAPK)in mammals.JNKs are encoded by three genes,namely JNK1,JNK2 and JNK3.JNK1 and JNK2 are widely expressed in all tissues,while expression of JNK3 is mainly in heart,brain and testicles.It has been showed that JNK was activated by environmental stimuli,such as oxidative stress and mechanical stress.The activated JNKs phosphorylate transcription factors including c-jun?ATF2?AP-1,which subsequently causes the change of gene expression.It had been found that TNF a could inhibit P4H a 1 expression by activating the ASK1-JNK signaling pathway in human aortic smooth muscle cells.FOXO1 is one of the members of FOXO family in mammals,which is expressed in most tissues and cells.It has been reported that FOX plays a key role in the development and maturity of blood vessels.FOXO1 deficiency in embryonic mice has been shown to be lethal,causing abnormal vascular development.Several studies found that JNK could regulate the expression of FOXOs in many human cancer cells.This study intends to study the changes of SMCs P4H?1 expression and the infulence of collagen content under oscillatory shear stress,and further explore the downstream signaling pathways of oscillatory shear stress.Objectives1.To observe the changes of P4Hal expression under oscillatory shear stress in smooth muscle cells;2.To study the effects of P4Hal interference on collagen under oscillatory shear stress in smooth muscle cells;3.To explore the role of JNK-FOXO1 pathway in the changes of P4H?1 expression by oscillatory shear stress in smooth muscle cells.Methods1.Cell cultureWhen newly purchased human aortic smooth muscle cells(HASMCs)overgrew the bottom of culture bottle,sucked out of the culture medium and washed wih PBS twice,then added suitable amount of pre-heated 0.25%Trypsin in the culture bottle.when the cells were observed to be in a round shape under the microscope,the appropriate medium poured into the culture bottle,beated cells,removed the mixture to the centrifugal tube,centrifuged for 5 minutes(800r/min),then discarded the supernatant and added the new cell suspension to the culture bottle,tnoroughly incorporated,cultured in an incubator with 5%CO2 at 37 ?.2.Shear stress interventionHASMCs from 4 to 7 generations were seeded onto glass slides precoated with rat tail collagen I after high pressure disinfection.When cells reached 90%confluence,the cells exposed to oscillatory shear stress(0ą4dyne/cm2)for 3?6?12?24 hr or maintained in a static state in an incubator with 5%CO2 at 37 ?.To investigate the effects of oscillatory shear stress on the expression of P4H?1?collagen?FOXO1 and JNK to carry out further study.3.Transfection of P4H?1 overexpression lentivirus vectorP4H?1 gene overexpression lentivirus vector and empty vector were purchased from Shanghai Genechem Company.The transfection procedure was in accordance with lentivirus transfection manual.48 hours after transfection,the fluorescence expression was observed and the transfection efficiency was evaluated.4.Quantitative real-time PCRTotal RNA was extracted from human aortic smooth muscle cells by TRIzol method after stimulation,RNA concentration was determined by spectrophotometry and reverse-transcribed into cDNA by using a TaKaRa's cDNA synthesis kit.The SYRB Premix Ex Taq kit(TaKaRa Bio,Japan)was used for qPCR analysis of gene expression.The reletive mRNA expression level of P4Hal of different stimulation was assessed.5.Western Blotting analysisProtein from HASMCs was extracted by RIPA after stimulation.Protein was heated for denaturation and the concentration of protein was determined by BSA,then cell lysates were separated on SDS-PAGE and transferred to PVDF membranes,incubated with 5%nonfat milk and with the antibodies overnight at 4?.Being washed by TBST,The bands were recorded by using enhanced chemiluminescence after incubated with secondary antibodies.Band densities were analyzed by use of Adobe Photoshop CS5.6.Statistical analysisData are expressed as mean?SD.The data were analyzed by unpaired t-test or one-way ANOVA as appropriate.SPSS v16.0 was used for statistical analysis.P<0.05 was considered significant.Results1.The expression of P4H?1 decreased in HASMCs by oscillatory shear stressCompared with static controls,P4H?1 mRNA and protein levels in HASMCs were both decreased in the setting of oscillatory shear stress,P4H?1 expression was decreased at 6h?12h and 24h(P<0.05)and peaked at 12h.2.Oscillatory shear stress decreased collagen ? and collagen ? expression in HASMCsCompared with static controls,the expression of collagen ? and collagen ? in HASMCs were significantly decreased under oscillatory shear stress,the expression of collagen ? and collagen ? were decreased at 6h and 12h(P<0.05).3.P4H?1 participated in the modulation of oscillatory shear stress on the Collagen metabolism in HASMCsP4Hal overexpression increased the expression of collagen ? and collagen ? by oscillatory shear stress(P<0.05).It indicated that P4H?1 participated in the regulation of collagen metabolism in HASMCs under oscillatory shear stress.4.JNK pathway was responsible for oscillatory shear stress induced P4H?1 expressionCompared with static controls,the protein expression of FOXO1 in HASMCs was significantly decreased,which decreased at 3h?6h and 12h(P<0.05).Compared with static controls,Western Blotting results indicated the protein expression of P-JNK/JNK in HASMCs was significantly increased at 3h?6h and 12h(P<0.05).Pretreated HASMCs with JNK inhibitor SP600125 for 1h,then cells were given oscillatory shear stress stumuli for 12h,Western Blotting results indicated the expression of P4Halwas significantly increased(P<0.05)and the expression of FOXO1 had no change(P>0.05).These results demonstrated JNK pathway was involved in oscillatory shear stress induced P4Hal expression.Conclusions1.Oscillatory shear stress decreased the expression of P4H?1;2.P4H?1 participated in the modulation of oscillatory shear stress on the collagen metabolism;3.JNK pathway mediated the downregulation of P4H?1 in HASMCs by oscillatory shear stress. |
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