The Study Of Meprin-α Aggravate Atheroscleosis Through Transactived EGFR And Promote Generation Of ROS

With the increasing improvement of the socio-economic level and the population agingsituation，Cardiovascular and cerebrovascular disease caused by atherosclerosis（AS）havebecome a serious threat to human health and one of the most important diseases lead todeath. AS is one vascular diseases characterized by reactive damage repair which caused bythe depositon of lipid，proliferation and calcification of smooth muscle cells and theinfiltration of inflammatory cells. The hardening of the arteries and atherosclerotic plaqueformation are the main pathologic changes of AS, can gradually lead to stenosis of theartery lumen, is the most major cause of heart, brain and distal limb ischemia. In recentyears, researchers understood more about the mechanism of formation of atherosclerosis,oxidative stress response in the form of AS function is attracting more and more attention,but the antioxidant drugs has not achieved the expected effect to prevent the development ofAS, the relationship between oxidative stress and the development of AS is need furtherresearchMetalloprotease meprin is the surface protein of mammalian cells containing a zincfinger protein hydrolase structure, contains α and β two subtype，widely distributed in avariety of cells including endothelial cells, macrophages and tumor cells. meprin usuallyplay a physiological role by activation or inactivation of cytokines in vivo. Studies havefound that meprin-α in closely related to the occurrence of tumor and chronic inflammatoryreaction. In our previous research we found that meprin-α play a role in AS formation anddevelopment, he chronic intervention of meprin inhibitor actinonin (AC)，the high fat-fedApoE-/-mice present less worse formation and progression of AS to control mice in arterywall, accompanied with the reduction in the number of macrophages in atherosclerotic andless generation of active oxygen species(ROS) in vessel wall in situ. These results suggestthat meprin-α may be involved in regulation of AS by involved in oxidative stress reaction.But because of the expression of meprin-α in ApoE-/-mice vascular wall was too low to make experiment to clarify the specific mechanism further.Epidermal growth factor receptor (EGFR) is a widely distributed cell surfacetransmembrane glycoprotein receptors in a variety of animal tissue, mainly is activatedthrough the ligand effect and then play a physiological function, promote the generation ofROS in cells. The study found the metalloproteinase family of zinc metalloprotease couldactivate the three main ligand in vivo which could activate EGFR, including epidermalgrowth factor (EGF), heparin binding epidermal growth factor (HB-EGF) and transforminggrowth factor alpha (TGF-α). EGFR ligands are activated vary in different cell types anddifferent substrate. Metalloproteinase meprin-α belongs to zinc metalloprotease, we supposethat the meprin-α maybe activated the EGFR through activated the ligand of EGFR inmacrophages，induce ROS production in macrophage and participate in AS development.Based on the above assumptions, in this study we plan to construct a lentiviral vectorcontaining meprin-α and chronic intervene the high fat-fed ApoE-/-mice，using molecularbiology techniques to observe the impact of meprin-α in AS formation and macrophageROS production from animal and cell level，further explore its mechanism. Through thisstudy we can initial clarify the effect and mechanism of meprin-α induced vascular ROSproduction to promote the AS development，and the role of EGFR in macrophages in theformation process of ROS and its downstream signaling pathways. There results shouldhelp us understanding the relationship between oxidative stress and AS from a new angleand provide the experimental basis for further study on the regulation of oxidative stressresponse in AS formation, provides the theory support for the new drug target research.Method1. Construct MEP1A lentiviral vector⑴Shanghai Jierui Biotechnology Co was commissioned to synthesis the MEP1Agene.⑵The MEP1A gene synthesized was PCR amplified of target gene as PCR template.Expression vector digested plastic recycling after the vector fragment. Objective genefragment homologous recombination and transformed into E.coli competent cells.Transformants with colony PCR identification, the positive clones were sequenced. Cloningand sequencing of the correct plasmid.⑶Virus particles was packed using HEK-293T cells and titer determination. 2.animal experiment⑴the experimental animal and grouping:150male ApoE-/-mice (C57/BL6genebackground),6weeks old, weighing18-20g, were randomly divided into6groups,25ratsin each group. The control group (group CON) was under the ordinary feed feeding. Theother5groups were under high-fat diet, according to the different intervention: high fatgroup (HF group): treated with sterile saline (0.5ml/days) intraperitoneal injection fromeleventh weeks， continuous injection for4weeks. High fat+L-meprin-α group (HF+LMgroup): intravenous injection of L-meprin-α (1.84x108TU) through caudal vein at eleventhweeks, then treated with sterile saline (0.5ml/days) intraperitoneal injection, continuousinjection for4weeks. High fat and+L-meprin-α+AC group (group HF+LM+AC):intravenous injection of L-meprin-α (1.84x108TU) at eleventh weeks through caudal vein,then treated with AC (5mg/kg/day) intraperitoneal injection from eleventh weeks,continuous injection for4weeks. High fat and+L-meprin-α+AG1478group (groupHF+LM+AG): intravenous injection of L-meprin-α (1.84x108TU) at eleventh weeksthrough caudal vein, then treated with AG1478(20mg/kg/day) intraperitoneal injectionfrom eleventh weeks， continuous injection for4weeks. High fat group+L-EGFP(HF+LGFP group): intravenous injection of L-EGFP (1.92x108TU) at eleventh weeksthrough caudal vein, then treated with sterile saline (0.5ml/days) intraperitoneal injection,continuous injection for4weeks. The HF+LGFP group has3mice were executed beforethe tail vein injection of L-EGFP and after the injection one or four weeks，in order toconfirm the efficiency of transfection of lentiviral vector in atherosclerotic plaques. Allmice were killed after fourteenth weeks for each group.⑵Observe the size and number of atherosclerotic plaque in pathology: ApoE-/-mousewere executed by intraperitoneal anesthesia with overdose of pentobarbital sodium. thoracicaortic were peeled and removed from body and then fixed，embeddinged and cutted into5um thick slices. After conventional hematoxylin and eosin (hematoxylin and eosin, HE)staining， tissue section were observed under microscope to observe formation ofatherosclerotic plaque；oil red O staining, observe the number and size of atheroscleroticplaque in the general level.⑶Detection of the generation of ROS in vascular intima in situ by DHE: mousethoracic aorta section treatment with DHE after dewaxing, confocal microscopy can detect the fluorescence of ROS and obtain image. using IPP6image analysis software was used toanalysis results.⑷Detection of meprin-α expression in endothelium by Western blot assay (WesternBlot, WB): extraction of-80℃frozen mouse thoracic aortic atherosclerotic plaquepreservation organization50micrograms of protein by SDS-PAGE method, extracted andindexed to the PVDF membrane, antibody closed and incubation overnight. Secondantibodies with horseradish peroxidase detection protein strips and chemiluminescence.⑸Detection of EGFR expression in endothelium by immunohistochemistry: mouseaorta sections with antibodies embedding and incubation at4℃overnight, and thenincubated with biotinylated second antibody30min, adding DAB directly under themicroscope.3. Cell experiment⑴Detection of generation of ROS in cell by DHE fluorescent probe labeled: J774A.1cells were pretreat with different factors，intracellular ROS generation were detected，theresults should make it clear that the factors affected the intracellular ROS generation andtheir relationship.⑵Detection of protein concentration in cell culture fluid by ELISA: J774A.1cellswere pretreat with different factors，HB-EGF protein concentration in cell culture fluidwere detected by ELISA，the results should make it clear that the influence factors ofJ774A.1cells to release HB-EGF.⑶Detection of intracellular protein expression by: J774A.1cells were pretreat withdifferent factors，EGFR and its downstream signal factors were detection by WB, the resultsshould make it clear that the factors affected the protein expression and their relationship.4.Statistical analysisMeasurement data as mean±standard deviation (mean±SD) said. Statistical datausing single factor analysis of variance (One-way ANOVA), there were significantdifferences in P<0.01. Statistical analysis using SPSS13.0software.Result1. Construction of Lentivirus vectorLentivirus vector containing Merin1A gene and pSB1control vector were successfullyconstructed. 2.animal experiment⑴Results of histopathological observation①HE staining: The three layer structure of vascular wall in CON group is clear, innermembrane is smooth, smooth muscle cells arranged in neat rows, the artery wall slightlyuplift and form very small amount atherosclerotic plaque. A small amount of lipid andinflammatory cells infiltration under the endothelial. Middle and outer membrane structureare clear, no pathological change. Vessel wall were diffuse swelling in high fat-diet HFgroup, protruding into the lumen surface formed obvious plaque, the inner membrane wasthickening and structure was serious damaged. A large number of irregular arrangement ofsmooth muscle cell proliferation under inner membrane, with lipid deposition, the formationof unequal size, irregular shape of the foam cells, a massive infiltration of macrophages,disorder of middle structure, smooth muscle hyperplasia, necrosis, and inflammatory cellinfiltration. Clearance elastic plate and cells showed large amounts of lipid deposition.Compared with HF+LM group and HF group, plaque area, neonatal formation of larger,intimal thickening was more obvious, more lipid deposition, plaque macrophage infiltrationis more obvious; HF+LM+AG group and HF+LM+AC group compared with HF+LMgroup, the plaque area decreased, inner membrane thickening, plaque decreases lipiddeposition, a small amount of macrophages and inflammatory cells; compared with HFgroup, the change of vessel wall in HF+LGFP group is similar.②The oil red O staining: The thoracic aorta in CON group were soft elastic, innermembrane were smooth, with only a local dyeing damage area. The visible flake darkerdamage area of vascular inner membrane in high fat-diet HF group, inner membrane werenot complete and large amounts of membrane structure was destroyed (P<0.01). Comparedwith HF group, the number and size of lesions were significantly increased in HF+LMgroup (P<0.01); HF+LM+AG group and HF+LM+AC group compared with HF group, thenumber and size of lesions were significantly reduced (P<0.01); compared with HF+LGFPgroup and HF group, the number and size of the injury site has no obvious difference.⑵Each group ofApoE-/-mouse vascular intima in situ ROS generationThere were very small amounts of ROS generation thoracic aortic intimal in situ inCON group.Compared with CON group, HF group have increased ROS generation (P<0.01)in situ. Compared with HF group, HF+LM group ROS generation in situ increased significantly (P<0.01).Compared with HF+LM group, HF+LM+AC group ROS generationin situ was significantly reduced (P<0.01).⑶Each group ofApoE-/-mouse expression of meprin-α in vascular intimalOnly a small amount of the expression of meprin-α in CON group.Compared withCON group, expression of meprin-α increased significantly in HF group (P<0.01).⑷Each group ofApoE-/-mouse expression of EGFR in vascular intimalThere were almost no expression of EGFR in CON group thoracic aortic vascularintimal and a small amount expression of EGFR was visible in HF group. Compared withHF group, the expression of EGFR in HF+LM group was significantly increased (P<0.01).Compared with HF+LM group, the expression of EGFR in HF+LM+AC group wassignificantly reduced (P<0.01).3.cell experiment⑴Different intervention treatment impact on J774A.1intracellular ROS generation.①In the comparison of the groups, compared with the control group, OxLDLtreatment significantly increased the intracellular ROS generation at all time.②Compared with OxLDL treated cells: OxLDL+AC, Ox-LDL+meprin-α siRNA,OxLDL+AG1478, Ox-LDL+HB-EGF and p38inhibitor SB203580treatment ofintracellular ROS generation was significantly reduced（P<0.01），while ROS generatesOxLDL+meprin-α treated cells increased (P<0.01).③Compared with OxLDL+meprin-α treated cells, ROS significantly reduced inOxLDL+meprin-α+AG1478treated cell (P<0.01).⑵Different intervention treatment impact on J774A.1release HB-EGF①Compared with group control，the cell culture fluid of OxLDL and OxLDL+meprin-α treatment cells had increased concentration of HB-EGF (P<0.01).②Compared with cells treated with OxLDL, the cell culture fluid of OxLDL+AC andOxLDL+meprin-α siRNA treated cells had reduced concentration of HB-EGF(P<0.01).⑶Different intervention treatment impact on J774A.1intracellular proteinexpression.Compared with control group, OxLDL treated cells had increased expression of EGFR,p38and PI3K-Akt phosphorylation(P<0.01), increased expression of GTP-rac1(P<0.01).Compared with OxLDL treated cells, expression of EGFR phosphorylation were reduced in cell treated with antibodies against HB-EGF, AC or meprin-α siRNA (P<0.01), expressionof p38phosphorylation were reduced in cell treated with AC, meprin-α siRNA, Rac1inhibitor EHT1864and PI3K inhibitor wortmsnin (P<0.01), intracellular expression ofGTP-rac1were significantly reduced in cells treated with wortmsnin (P<0.01), intracellularexpression of PI3K-Akt phosphorylation in cell treated with AC，meprin-α siRNA andAG1486were decreased significantly (P<0.01).Conclusion1.Meprin-α promote formation and development of thoracic aortic atheroscleroticplaque in high fat-fed ApoE-/-mouse through induce oxidative stress. meprin-α inhibitor ACand EGFR inhibitor AG1478can counteract the effect of meprin-α. meprin-α is theupstream signal molecule of EGFR, affecting the expression and activity of EGFR.2.Meprin-α could promote macrophages release the extracellular part of thetransmembrane structure of HB-EGF out of cell, as a combination of ligand oftransmembrane structure of the extracellular domain of EGFR structure, activation of EGFRby promoting and tyrosine kinase phosphorylation patterns, initiate downstream signalingpathways and promote the generation of ROS in cells.3. Meprin-α transactivation EGFR start receptor signaling in macrophages, via thePI3K-Rac1-p38pathway induce the generation of ROS in cells.