| â… study of the relationship between IL-6 and vulnerability of vulnerable plaque and P4Hα1/MMP-14BackgroundAtherosclerosis,which causes ischemic cardiopathy and stroke,is the most common cause of mortality and morbidity in developed countries.Atherosclerotic disease of arteries leading to formation of the atherosclerotic plaque involves modulation of the normal functioning of the vessel wall endothelium,the entrapment of low-density lipoprotein(LDL) within the vessel wall and itsoxidative modification, the migration into the wall of monocytes,their conversion to activated macrophages by oxidized LDL and the release from macrophages of cytokines and proteases and the uptake of lipid by macrophages in the vessel wall to form the "foam" cells characteristic of the early lesion,the "fatty streak." The most dangerous plaque is rupture -prone plaques,also named vulnerable plaque which are characterized by possessing an easily disrupted/fissured and occlusive thrombus have a large lipid core and a thin fibromuscular cap accompanied by many macrophages and other inflammatory cells on or beneath the cap surface.Finding the mechanism of the formation of vulnerable plaque is a hot study recently years.In atherosclerotic arteries collagen is crucial for plaque stability and its removal from the plaque's fibrous cap area may result in plaque rupture.Since collagen plays key roles in plaque stability and cell migration properties,a comprehensive understanding of collagen expression and organization during the progression of atherosclerosis is essential.Inflammation directly contributes to the pathogenesis of most cardiovascular diseases,including heart failure,cardiomyopathy,atherosclerotic plaque rupture,and aortic aneurysm and degradation of extracellular matrix. Extracellular matrix(ECM) of the vascular is a major target of inflammatory cytokines.The ECM,which includes fibrillar(collagen and elastin) and adhesive proteins(laminin and fibronectin),serves as the structural framework for all 3 layers of the arterial wall.Of the more than subtypes of collagens,typesâ… andâ…¢are the most common in thearterial wall IL-6(a 26 kDa cytokine),produced by lymphocytes, monocytes,fibroblasts,vascular smooth muscle cells,and endothelial cells has been identified as a local and circulating marker of coronary plaque inflammation.IL-6 is a pleiotropic cytokine and its role in the modulation of inflammation-related processes, particularly cytokine responses and tissue inflammatory cell infiltration.IL-6 deficiency was reported to result in an enhanced formation of atherosclerotic lesions, reduced collagen metabolism,and elevated levels of serum cholesterolin an ApoE-/-model of atherosclerosisCollagen synthesis and secretion require posttranslational modifications of procollagens,including hydroxylation and glycosylation.Prolyl-4-hydroxylase(P4H), an essential enzyme involved in collagen modification,catalyzes the formation of hydroxyproline from proline residues locatedin repeating X-Pro-Gly triplets in the procollagens.This step is required for folding the polypeptide chains into stable triple-helical molecules.Therefore,adequate P4H expression is essential for the formation of functional collagen in the arterial wall and pathogenesis of vascular diseases.Some studies have examined inflammatory cytokines,such as TNF alpha,inducing suppression of prolyl-4 hydroxylase alphal(P4Halphal),the rate-limiting isoform of P4H responsible for procollagen hydroxylation,maturation,and organization.In the present study,we aim to find the effection of IL-6 to vulnerable plaque and the interaction between IL-6 and P4Halphal in the plaque.Objectives 1.Elucidate the interaction between MMP-14 and P4Halphal and the corresponding mechanisms during the process of the Atherosclerosis.2.Elucidate the effects of IL-6 on vulnerable plaque and the corresponding mechanisms.3.Elucidate the interaction between IL-6 and P4Halphal in the vulnerable plaque and the corresponding mechanisms.4.Elucidate the interaction between IL-6 and MMP-14 in the vulnerable plaque and the corresponding mechanisms.Methods1.Animal modelAnimal stress and received a model with AS vulnerable plaque was established LPS stimulation.Eighty male apoE-/- mice were divided into two groups,with Westem-type diet(0.25%cholesterol and 15%by combination mental 12 weeks of age,were cocoa butter) from the beginning of the study.Carotid atherosclerotic lesions were induced perivascular constrictive collars placement on the left common carotid arteries.Four weeks after surgery,mice were put into boxes independly to limited these actions,one hour a day,one day a week.2.Establishment of pLVX-AcGFP-N1-IL6 virus vectorSeprate RNA from smooth muscle cells,througeh reverse transcripting it into cDNA to synthetize DNA.Obtain the target segment by electrophoresis identification. Make combination of target segment and GFP.3.pLVX-AcGFP-N1-IL6 virus vector transfection into the vulnerable plaque4.1 Histological and Morphology AnalysesSections were stained with hematoxylin and eosin(H&E),Masson's trichrome,sirius red staining,Verhoeff staining,oil red O staining,Perl's staining. Immunostaining were performed for detecting MOMA-2,a-actin,P4Hα1,IL-6 expression in the vulnerable plaque.Plaque area,vessel area and cap thickness were measured.Smooth muscle cell,macrophage,lipid and collagen positive areas werequantified and the ratios correlated to the entimal areas were calculated.Plaque rupture rate and vulnerable index were calculated.4.2.Real-Time RT PCRThe mRNA levels of P4Hα1 mRNA in the fresh carotid lesions.were quantified by real-time reverse-transcriptase polymerase chain reaction(RT-PCR) using SYBR Green technology.4.3.Western-blotWestern-blot was performed for examining P4Hα1 protein expression in the fresh carotid lesions.Results1.Establishment of pLVX-AcGFP-N1-IL6 virus vectorPlasmid of pLVX-AcGFP-N1-IL6 virus vector,which binded the the sequence of GFP and IL-6 gene of mouse,was transfected into HK293 cells.Forty-eight hours later,pLVX-AcGFP-N1-IL6 virus vector was successfully constructed confirmed by PCR reaction.2.pLVX-AcGFP-N1-IL6 virus vector transfection in the vulnerable plaqueThree days after transfection,expression of green fluresence protein was 36.07%in plaque,and decreased to 18.81%fourteen days after transfection.3.Blood lipid profileNo statistic differences of serum lipid profiles were found brtween IL-6 group and control.4.1 Histological and Morphology AnalysesAtherosclerotic plaques of 10 animals of each group were analyzed for histology. No statistic difference of plaque area was found brtween IL-6 group and control,(71000±8000 vs 82000±11000um~2,P=0.073).Compared with control,mean fibrous cap thickness(6.73±0.96 vs 7.25±1.35um,P=0.029),cap/core ratio(0.06±0.02 vs 0.09±0.02,P=0.045) and collagen(11.23%±3.57%vs 16.34%±4.72%,P=0.005) were significantly decreased,and lipid content was significantly increased (21.23%±3.56%vs 12.25%±2.01%,P=0.0011) in IL-6group.Plaque rupture rates were 20%,significantly higher in IL-6 group(P=0.017).Immunostainning results showed that there more macrophages in the IL-6 group(15.32%±3.01%vs 7.67%±2.87%,P=0.004) than those in control.IL-6 expression increased by 72.33%in IL-6group compared with that in control(12.54%±2.76%vs63%±1.18%,P＜0.005).In addition,P4Hα1 expression were all significantly decreased in IL-6 group than those in control(all P＜0.05).Vulnerable index of IL-6 group was statistically higher than that of control(2.83±1.02 vs 0.81±0.43,P=0.009).4.2 Real time RT PCRThe mRNA expression of P4Hα1 in the vulnerable carotid plaques decreased 69.3%compared with that in controls(P=0.03).4.3 Westem-blot analysisThe proteins expression of P4Hα1 in the vulnerable carotid plaques decreased 61.9%compared with that in controls(P＜0.001).Conclusions1.pLVX-AcGFP-N1-IL6 virus vector was successfully established.2.In vivo,pLVX-AcGFP-N1-IL6 virus vector can reduced P4Hα1 expression in some reagion of vulnerable plaques,increased lipid content and the number of macrophage,decreased the expression of mooth muscle cells and collagens significantly.So IL-6 validated to increase the vulnerability of plaques markably.3.In vivo,pLVX-AcGFP-N1-IL6 virus vector can reduce P4Hα1 expression and increase MMP-14 expression in some reagion of vulnerable plaques. â…¡Interleukin-6 Decreases Proly-4-hydroxylaseα1 Expression via the Raf-Mek-Erk1/2-Ap-1 pathwayBackgroundInflammation plays a pivotal role in the pathogenesis of cardiovascular diseases, including heart failure and atherosclerosis.An advanced atherosclerotic lesion typically has a fibrous cap,which prevents the necrotic core from having direct contact with flowing blood.In the event of cap rupture,thrombosis occurs and causes acute coronary syndrome.Arterial collagen is the main extracellular matrix(ECM) constituent of the fibrous cap.Arterial interstitial collagen confers tensile strength on the fibrous cap and thus determines plaque stability and vulnerability to ruputure. Synthesis of collagen in the fibrous cap therefore may be directly responsible for the plaque rupture.ECM is the structural framework of all tissues,in which fibrillar proteins (collagen and elastin) and adhesive proteins(eg,laminin and fibronectin) form the structural backbone.In the arterial wall,various cells including smooth muscle cells, endothelial cells,fibroblasts and certain inflammatory cells,are the major cell source regulating ECM metabolism.Collagen,as one of the most metabolically active ECM components,has at least 39 subtypes.Typeâ… andâ…¢collagens are most commonly found in the arterial wall.Although ECM degradation is important for balanced ECM metabolism,ECM synthesis,mainly modified by Prolyl-4-hydroxylaseα1(P4Hα1),is also crucial.Considerable evidence suggests that P4Hα1 is involved in plaque remodeling.P4Hα1 is one of the key intracellular enzymes required for the synthesis of all known types of collagens.It is essential for folding the procollagen polypeptide chains into stable triple helical molecules.Inhibition of P4Hα1produces unstable collagen associated with collagen decrease.P4Hα1is regulated by various cytokines, such as TNF-A and cigarette smoking.Among many regulatory factors of ECM,cytokines such as interleukin 6(IL-6) may directly inhibit P4Hα1 and therefore participate in ECM metabolism.IL-6,a 26-kDa protein,is released by T cells,tumour-associated macrophages and fibroblasts, especially from activated CD4+T cells.It is one of the most crucial cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism.IL-6 acts through a membrane receptor composed of two subunits:anαchain,the IL-6 binding protein gp80;and aβchain,the signal-transducing protein gp130. Subsequently,gp130 can activate the Raf-dependent mitogen-activated protein kinase (Raf-MAPK) pathway,which leads to the activation of transcription factors such as AP-1 and NF-IL-6 The relation between IL-6 and P4Hα1 activity is unclear.In the current study,we investigated the effect of IL-6 on P4Hα1 expression and the underlying molecular mechanisms.IL-6 reduced P4Hα1 expression at the RNA and protein levels,with the RAF-MEK-ERK1/2 pathway involved in the process. Furthermore,we also showed that AP-1 was involved in the IL-6-mediated downregulation.Our study describes the molecular pathway responsible for IL-6-induced ECM disturbance in cardiovascular disease.Objectives1.Describes the effect of IL-6 on P4Hαexpression during the process of HASMCs.2.To investigate if IL-6 reduced P4Hαexpression through the molecular pathway RAF-MEK-ERK1/2-AP-1 during the process of HASMCs,and to elucidate the corresponding mechanisms.Methods1.Cell CultureHuman aortic smooth muscle cells(HASMCs) were obtained from ScienCell (Carlsbad,CA) and cultured in smooth-muscle-cell culture medium(Cat#311,Cell Application,San Diego,CA) containing 10%fetal bovine serum.Cells were cultured up to passage 4 before the experiments were conducted.2.Different Dose to P4Hα1 ExpressionHASMCs were treated with 0,10,20,50,and 100 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 24 hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6.3.different time point to P4Hα1 ExpressionMeasurement of target gene mRNA and protein levels were harvested at the time point of 0,6,12,24,48h independtly with 20ng/ml IL-6 treating the HASMCs).And find that the expression of P4Hα1 was mostly induced at time point of 24 hr.4.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.To investigate the effect of AP-1siRNA.5.the effect of NF-κBsiRNAFour groups were detected as control group,NF-κBsiRNA group,IL-6 group,IL-6+ NF-κBsiRNA group with Western blot.To investigate the effect of NF-κBsiRNA6.PD98059,the inhibiter of ERK1/2 to P4Hα1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.7.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.8.SB203580,the inhibiter of p38All the cells were devided into four groups as control group,SB203580 group,IL-6 group,IL-6+ SB203580 group.To investigate the effect of SB203580.9.SP600125,the inhibiter of JNKAll the cells were devided into four groups as control group,SP600125 group,IL-6 group,IL-6+ SP600125 group.To investigate the effect of SP600125.10.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsTo find compared with the control group,effection of IL-6 group to expression of protein RAF,MEK,ERK1/2 of HASMCs.Results1.At different dose of IL-6HASMCs were treated with 0,10,20,50,and 100 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 24 hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of P4Hα1 was mostly induced at the level of 20ng/ml IL-6.2.At different point of timeMeasurement of target gene mRNA and protein levels were harvested at the time point of 0,6,12,24,48h independtly with 20ng/ml IL-6 treating the HASMCs).And find that the expression of P4Hα1 was mostly induced at time point of 24 hr.3.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.Find that compared with the control group,protein levels of P4Hα1 in AP-1siRNA group decreased slightly but no significant,IL-6 group,IL-6+ AP-1siRNA group decreased significantly,and IL-6+ AP-1siRNA group is superior than IL-6 group.4.the effect of NF-κBsiRNAFour groups were detected as control group,NF-κBsiRNA group,IL-6 group,IL-6+ NF-κBsiRNA group with Western blot.Find that compared with the control group,protein levels of P4Hα1 in NF-κBsiRNA group have no changes, IL-6 group,IL-6+ NF-κBsiRNA group decreased significantly,and the two groups have no diffenence.5.PD98059,the inhibiter of ERK1/2 to P4Hα1All the cells were devided into four groups as control group,PD98059 group,IL-6 group,IL-6+ PD98059 group.Find that compared with the control group,protein levels of P4Hα1 in PD98059 group have no changes, IL-6 group,IL-6+ PD98059 group decreased significantly.IL-6 group is superior than IL-6+ PD98059 group.6.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group,IL-6 group,IL-6+ PD98059 group.Find that compared with the control group,protein levels of AP-1 in PD98059 group have no changes, IL-6 group,IL-6+ PD98059 group increased significantly.IL-6 group is superior than IL-6+ PD98059 group.7.SB203580,the inhibiter of p38All the cells were devided into four groups as control group,SB203580 group,IL-6 group,IL-6+ SB203580 group.Find that compared with the control group,protein levels of P4Hα1 in SB203580 group have no changes,IL-6 group, IL-6+ SB203580 group decreased significantly,and the two groups have no diffenence.8.SP600125,the inhibiter of JNKAll the cells were devided into four groups as control group,SP600125 group,IL-6 group,IL-6+ SP600125 group.Find that compared with the control group,protein levels of P4Hα1 in SP600125 group have no changes, IL-6 group,IL-6+ SP600125 group decreased significantly,and the two groups have no diffenence.9.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsFind that compared with the control group,I protein levels of P4Hα1 in L-6 group increased significantly. Conclusions1.IL-6 decrease P4Hαexpression at the level of mRNA and protein through the process of HASMCs.2.RAF-MEK-ERK1/2-AP-1 pathway responsible for IL-6 reduced P4Hα1 expression during the process of HASMCs.3.JNK,p38 and NF-κB have nothing to do with the process of IL-6 reducing P4Hα1 expression â…¢Interleukin-6 Enhances Matrix Metalloproteinase-14 Expression via the Raf-Mek-Erk1/2-Ap-1 pathwayBackgroundInflammation plays a pivotal role in the pathogenesis of cardiovascular diseases, including heart failure and atherosclerosis.An advanced atherosclerotic lesion typically has a fibrous cap,which prevents the necrotic core from having direct contact with flowing blood.In the event of cap rupture,thrombosis occurs and causes acute coronary syndrome.Arterial collagen is the main extracellular matrix(ECM) constituent of the fibrous cap.Arterial interstitial collagen confers tensile strength on the fibrous cap and thus determines plaque stability and vulnerability to ruputure. Synthesis of collagen in the fibrous cap therefore may be directly responsible for the plaque rupture.ECM is the structural framework of all tissues,in which fibrillar proteins(collagen and elastin) and adhesive proteins(eg,laminin and fibronectin) form the structural backbone.In the arterial wall,various cells including smooth muscle cells,endothelial cells,fibroblasts and certain inflammatory cells,are the major cell source regulating ECM metabolism.Collagen,as one of the most metabolically active ECM components, has at least 39 subtypes.Typeâ… andâ…¢collagens are most commonly found in the arterial wall.Although ECM synthesis is important for balanced ECM metabolism, ECM degradation,mainly catalyzed by matrix metalloproteinases(MMPs),is also crucial.Considerable evidence suggests that MMPs are involved in plaque remodeling. As one of the MMP family proteins,MT1-matrix metalloproteinase(MMP-14) is associated with the pathogenesis of cardiovascular disease.MMP-14 is an efficient ECM-degrading enzyme and is responsible for the proteolytic cleavage of multiple pericellular and membrane-associated substrates,including collagen and other ECM proteins,and is implicated in the development of vascular diseases.Among many regulatory factors of ECM,cytokines such as interleukin 6(IL-6) may directly inhibit P4Hα1 and therefore participate in ECM metabolism.IL-6,a 26-kDa protein,is released by T cells,tumour-associated macrophages and fibroblasts, especially from activated CD4+ T cells.It is one of the most crucial cytokines involved in cardiovascular pathogenesis and actively regulates ECM metabolism.IL-6 acts through a membrane receptor composed of two subunits:anαchain,the IL-6 binding protein gp80;and aβchain,the signal-transducing protein gp130. Subsequently,gp130 can activate the Raf-dependent mitogen-activated protein kinase (Raf-MAPK) pathway,which leads to the activation of transcription factors such as AP-1 and NF-IL-6 The relation between IL-6 and MMP-14 activity is unclear.In the current study,we investigated the effect of IL-6 on MMP-14 expression and the underlying molecular mechanisms.IL-6 enhanced MMP-14 expression at the RNA and protein levels,with the RAF-MEK-ERK1/2 pathway involved in the process.Furthermore,we also showed that AP-1 was involved in the IL-6-mediated upregulation.Our study describes the molecular pathway responsible for IL-6-induced ECM disturbance in cardiovascular disease.Objectives1.Describes the effect of IL-6 on MMP-14expression during the process of HASMCs.2.To investigate if IL-6 reduced MMP-14expression through the molecular pathway RAF-MEK-ERK1/2-AP-1 during the process of HASMCs,and to elucidate the corresponding mechanisms.Methods1.Cell CultureHuman aortic smooth muscle cells(HASMCs) were obtained from ScienCell (Carlsbad,CA) and cultured in smooth-muscle-cell culture medium(Cat#311,Cell Application,San Diego,CA) containing 10%fetal bovine serum.Cells were cultured up to passage 4 before the experiments were conducted.2.Different Dose to MMP-14 ExpressionHASMCs were treated with 0,5,10,20,and50 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 12hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of MMP-14 was mostly induced at the level of 10ng/ml IL-6.3.different time point to MMP-14 ExpressionMeasurement of target gene mRNA and protein levels were harvested at the time point of 0,6,12,24 independtly with 10ng/ml IL-6 treating the HASMCs).And find that the expression of MMP-14 was mostly induced at time point of 12 hr.4.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.To investigate the effect of AP-1siRNA.5.PD98059,the inhibiter of ERK1/2 to MMP-14All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.6.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.To investigate the effect of PD98059.7.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsTo find compared with the control group,effection of IL-6 group to expression of protein RAF,MEK,ERK1/2 of HASMCs.Results1.At different dose of IL-6HASMCs were treated with 0,5,10,20,and 50 ng/mL recombinant human IL-6(Cat#206-IL/CF,R&D Systems,Minneapolis,MN) for 12 hr before being harvested for measurement of target gene mRNA and protein levels.And find the expression of MMP-14 was mostly induced at the level of 10ng/ml IL-6.2.At different point of timeMeasurement of target gene mRNA and protein levels were harvested at the time point of 0,6,12,24 independtly with 20ng/ml IL-6 treating the HASMCs). And find that the expression of MMP-14 was mostly induced at time point of 12 hr.3.the effect of AP-1siRNAFour groups were detected as control group,AP-1siRNA group,IL-6 group, IL-6+ AP-1siRNA group with Western blot.Find that compared with the control group,expression of MMP-14 protein in AP-1siRNA group decreased slightly but no significant,IL-6 group,IL-6+ AP-1siRNA group decreased significantly, and IL-6+ AP-1siRNA group is superior than IL-6 group.4.PD98059,the inhibiter of ERK1/2 to MMP-14All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.Find that compared with the control group, expression of MMP-14 protein in PD98059 group have no changes,IL-6 group, IL-6+ PD98059 group decreased significantly.IL-6+ PD98059 group is superior than IL-6 group.5.PD98059,the inhibiter of ERK1/2 to AP-1All the cells were devided into four groups as control group,PD98059 group, IL-6 group,IL-6+ PD98059 group.Find that compared with the control group, expression of AP-1 protein in PD98059 group have no changes,IL-6 group, IL-6+ PD98059 group decreased significantly.IL-6+ PD98059 group is superior than IL-6 group.6.Effect of IL-6 to expression of protein RAF,MEK,ERK1/2 of HASMCsFind that compared with the control group,expression of protein RAF,MEK, ERK1/2 in IL-6 group increased. significantly.Conclusions 1.IL-6 increase MMP-14 expression at the level of mRNA and protein through the process of HASMCs.2.RAF-MEK-ERK1/2-AP-1 pathway responsible for IL-6 enhanced MMP-14 expression. |
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