EMMPRIN And UPA Expression In Atherosclerotic Plaque And Affect In Treated With Atorvastatin

Background and ObjectiveThe mechanism of Acute coronary syndrome (ACS) is the rupture of the vulnerableplaque,which has become the first threat to human health. The vulnerable plaque hasstructural characteristics as: eccentric thin fibrous cap, lipid-rich necrotic center ofatherosclerotic plaque, a large number of inflammatory cells infiltration. Previous studieshave confirmed that extracellular matrix metalloproteinase inducer (EMMPRIN) plays animportant role in the occurrence of ACS, recent reseaches found that, EMMRPIN caninduce the expression of matrix metalloproteinase (MMPs) in atherosclerotic plaques.MMPs can lead to plaque rupture according to degrade the fibrous cap matrix by lossing ofplaque stability. Large number of research data confirmed that EMMRPIN exerts itsbiological effects in the body mainly through the MMPs. Application of a number of largeclinical trials confirmed that statins can significantly reduce the cardiovascular mortalityand all-cause mortality of ACS and unstable angina patients, not dependent on cholesterollowering, but the relevant mechanism is not completely clear.MMPs play a key role indegradation extracellular matrix (ECM) of thin fibrous cap of vulnerable plaque. It isconfirmed that,because of being expressed in macrophages, smooth muscle cells inatherosclerotic plaque, and degradating extracellular matrix,MMPs are unavoidably linkedto the occurrence of atherosclerosis and ACS. Further studies have found that EMMPRINhighly express in endothelium, vascular smooth muscle cells in human atheroscleroticplaque which induce MMPs production and secretion. Studies also have shown thatEMMPRIN increased express in patients with acute myocardial infarction(AMI).EMMPRIN stimulated monocytes/macrophages expression MMP-9, smooth muscle cellsexpression MMP-2, but not increased expression in patients with stable angina. So, manystudies confirm that MMPs and its upstream factor-EMMPRIN are key influence inpromoting matrix degradation, leading to the occurrence of ACS. The urokinase-type plasminogen activator (urokinase-type plasminogen activator of uPA) is an important partof the plasminogen system, which role of causing atherosclerosis becoming a newunderstanding: play a key role to promote matrix degradation, cell adhesion, cell migration,proliferation, cytokine regulation in the arterial wall. Also directly involved in thestimulation of a variety of cytokines in the process of atherosclerosis, vascularremodeling.Quemener found that in the invasive process of tumor cell, EMMPRINstimulate the expression of MMP2, MMP9, uPA to improve invasion ability of tumorcell. In this study, overexpression of EMMPRIN stimulated the secretion of uPA, MMP2,MMP9and its participation in angiogenesis. Kienast, Joachim suggested that the content ofuPA in human coronary artery is clearly related to the presence and severity ofatherosclerotic lesions. Studies also mean that EMMPRIN and uPA in the plaque ofcoronary heart disease in patients with atherosclerosis and plaque rupture parts of the"shoulder" are highly expressed.There are studies have shown that uPA and MMPs aresubject to the regulation of the EGF/EGFR system, uPA can be induced by the expressionof MMPs play a role in the hydrolysis of the ECM. So, in the process of humanatherosclerotic lesions, EMMRPIN uPA there existed what kind of relationship, whetheratherosclerosis caused by MMPs in this way and play the role of the atherosclerotic plaqueto stabilize EMMRPIN whether uPA there is also induce the expression of the role.A number of large-scale clinical trials confirmed the application of statins can significantlyreduce cardiovascular mortality and all-cause mortality in stable angina and ACS patients,and not wholly dependent reduction in plasma cholesterol, but the mechanism isnot entirely clear. Atorvastatin (atorvastatin) is a hydroxymethyl glutaryl coenzyme A(HMG-CoA) reductase inhibitors, as a representative of statins, in addition to a lipidmechanisms, also inhibit the inflammatory response, anti-oxidative stress, and stability ofendothelial function, plaque stabilization, and promote apoptosis, inhibit smooth musclecell proliferation, inhibition of platelet aggregation does not depend on cholesterol toreduce the direct and indirect cardiovascular protective effects. WOSCOPS (West ofScotland Coronary Prevention Study) and AFCAPS/TexCAPS trial (Air Force/TexasCoronary sclerosis), the ASCOT LLA,4S (Scandinavian Simvastatin Survival Study),CARE (recurrent coronary events study) LIPID (pravastatin on coronary heart disease,long-term intervention) and large-scale international clinical trials, results have proved that statin use can make the fatal and non-fatal myocardial infarction incidence declined,reduced cardiovascular events and total mortality rate was lower vascularreconstruction/the CABG reduced demand.This experiment research EMMPRIN and uPA expression in atherosclerotic plaqueand affect in treated with Atorvastatin in vivo using apolipoprotein E knockout(ApoE-/-)mice to establish model of atherosclerosis by high-fat diet feeding which could be formedpathology of atherosclerosis similar to human:①If EMMPRIN and uPAover-coexpression in atherosclerotic plaques;②Whether expression of EMMPRIN and uPAconnect with the unstability of plaque,in different stage of the palque whether have thedifference;③Whether treated with Atrovastain in different time and dose make differenteffect on EMMPRIN and uPA expression.Give new theoretical basis and new drug targetsfor the prevention and treatment with ACS.Methods1.The establishment of atherosclerosis model and the experimental groups:62male8-week old ApoE-/-mice fed with a normal rodent diet for one week. Then pick out6micerandomly for backup feeded with high-fat diet which contents normal mice diet+15%lardand1.25%cholesterol.The other mice were distributed randomly and evenly into the earlystart (ES) group (n=28)(raise6weeks)and the late start (LS) group(n=28)(raise10weeks) toestablish atherosclerotic model. Mice of the two groups were both randomly divided into4groups, n=7. They are Normal diet control groups(A E); high-fat diet control groups(BF);Low-dose satins intervention groups(C G); high-dose satins intervention groups(DH).Drug intervention in mice: Low-dose satins intervention groups(C G)were interventedby Atrovastatin5mg.kg-1.d-1for8weeks. high-dose satins intervention groups(D H)wereintervented by Atrovastatin10mg.kg-1.d-1for8weeks. Normal diet control groups(A E);and high-fat diet control groups(B F) were treated with saline.2. The lipids detection: The serum of ApoE-/-mice was analyzed total cholesterol(TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C) levels by OlympusAu2700automatic biochemical.3. HE staining: The paraffin sections of aorta of ApoE-/-mice were stained by HEafter dewaxing with staining, then observed aortic atherosclerotic plaque morphology.4. RT-PCR: Total RNA from ApoE-/-mice aorta was isolated with RNAiso Plus according to the manufacturer’s protocol after grinding organization in Liquid nitrogen.The quality of RNA was examined by an Experion Automated Electrophoresis System.First-strand cDNA was synthesized using ReverTra Ace reverse transcriptase. All PCRprimers EMMPRIN uPA and housekeeping gene (β-actin) were designed using softwarePrimerExpress (Applied Biosystem) and using the sequence information of the NCBIdatabase. The forward and reverse primers were as follows:5/-GCA GAG GAC ACAGGC ACT TAC-3/and5/-ACA GGC TCA GGA AGG AAG ATG-3/for EMMPRIN;.5/-CCC CAA GGT TTA CTG ATG TGC TC-3/and5/-GAA GTG TGA GAC CCT CGTGTA GA-3/for uPA. The mRNA was measured by1.5%agarose gel electrophoresis afteramplify by RT-PCR system. The results were analyzed using Gel imaging analyzer.5. Real-time RT-PCR: Total RNA extraction, cDNA synthesis with the former. ThemRNA was measured by SYBR Green1fluorescence, and the PCR reaction monitored byan7500Real-Time PCR Detection System. The results were analyzed using7500software.6. Western blot: Protein from ApoE-/-mice aorta was isolated with RIPA lysateaccording to the manufacturer’s protocol grinding organization in Liquid nitrogen. Aftercentrifugation at20,000g for30min at4°C, the protein was obtained as the deposition.The protein concentration was determined by the Coomassie brilliant blue method usingBSA as a standard. Equal amounts of protein were separated by SDS-PAGE (10%polyacrylamide gels). The protein was subsequently transferred onto a polyvinylidenedifluoride membrane by electroblotting for0.5h at50V. The membrane was blocked in a6%non-fat milk solution in TBS with0.5%Tween20. The membrane was allowed to reactwith a specific antibody and the detection of specific proteins was carried out by enhancedchemiluminescence following the manufacturer’s instructions. X-pressed exposure, proteinexpression was evaluated by EMMPRIN uPA and β-actin band integral area ratio ofabsorption with the GSD8000density scanning system. β-Actin was used as a loadingcontrol.7. Immunohistochemistry:Serial4-μm paraffin sections were dewaxed and rehydrated.Endogenous peroxidase activity was inhibited by incubation with3%hydrogen peroxide.After sections were blocked with BSA for incubating, then incubated with antibody.Sections were then incubated at room temperature with the peroxidase-conjugated affin-ipure secondary antibodies. Sections were colored with diaminobenzidine (DAB) as a substrate and afterstained with hematoxylin to reveal nuclei. Brown particles seen under thelight microscope were considered positive. The positive staining integrated optical density(IOD) was measured by IPP6.0color microscopic image analysis software forsemi-quantitative analysis. In each group, five DAB stained paraffin sections wererandomly selected, taking the average of five measurements of the IOD values for eachgroup.Results1．Animal model of atherosclerosis establishment: ApoE-/-mice fed with the high-fatdiet that contained15%fat from lard and was supplemented with1.25%(wt/wt)cholesterolfor6weeks. Aortic gross specimens have observed numbers of atherosclerotic plaqueformations in aortic. After10weeks high-fat feed,HE stained paraffin sections haveobserved aortic fibrous plaque formation, many foam cells accumulation in the plaque,aortic intimal thickening, and suddenly into the lumen. The model of atherosclerosisestablishment successful.2. HE stained paraffin sections have shown that mice feed with high-fat diet for6weeks had more foam cells formation and accumulation in aortic atherosclerotic plaque,significantly thickened intima, and suddenly into the lumen, plaque stability is poor, morevulnerable,than which feed with ordinary diet obviously.With feeding time longer,the levelof atherosclerotic plaque more serious,the unstability more.Compared with the ordinary dietgroups they had obvious difference,the plaque formed earlier and stronger.3. Expression of EMMPRIN and uPA in aorta and atherosclerotic plaque in ApoE-/-mice: Used RT-PCR, Real-Time PCR, Western blot and immunohistochemistry to examineif EMMPRIN and uPA mRNA and protein were up-regulated in aorta and atheroscleroticplaque in ApoE-/-mice with high-fat diet feeding. The results showed that high-fat dietfeeding groups of EMMPRIN, uPA gene and protein expression significantly increasedcompared with those expressions in the control groups (P <0.05, both gene and proteinexpression to β-actin as internal control).4. Atrovastatin treatment reduce expression of EMMPRIN and uPA in in aorta andatherosclerotic plaque in ApoE-/-mice: To intervene the high-fat diet feeding mice withatrovastatin in high and low dose.Then used RT-PCR, Real-Time PCR, Western blot, andimmunohistochemistry to examine mRNA and protein expression of EMMPRIN and uPA in aorta and atherosclerotic plaque in ApoE-/-mice. The results showed that atrovastatinintervene groups of EMMPRIN, uPA gene and protein expression significantly decreasedcompared with those expressions in high-fat diet control group(P <0.05, both gene andprotein expression to β-actin as internal control). High-fat diet induced EMMPRIN anduPA upregulation in aorta and atherosclerotic plaques in ApoE-/-mice by formed plaqueeariler and more senious, and this effect could be blocked by atrovastatin.5. uPA and EMMPRIN in ApoE-/-mouse aorta and atherosclerotic plaque expressionincreased and showed the same trend, the use of atorvastatin decreased expression afterstatin intervention trend.Conclusion1.High-fat diet feeding could make more serious atherosclerotic plaque eariler inApoE-/-mice aorta.2. High-fat diet feeding could induce the EMMPRIN、uPA expression obviously inaorta and atherosclerotic plaque in ApoE-/-mice.3. The over-expression of EMMPRIN and uPA in aorta and atherosclerotic plaquewere connected with the level of plaque and unstabiliy.4. Atrovastatin treatment can reduce expression of EMMPRIN and uPA in aorta andatherosclerotic plaque in ApoE-/-mice and the unstability of the plaques.The earlier usedthe statins,the more effect could be observed.