Molecular Mechanism Of Mep-1A Expression In Mast Cells Regulating The Development Of AAA By Improving Inflammatory Response

Background:Abdominal aortic aneurysm(AAA)is common in seniors,and aneurysm rupture often causes sudden death.The current treatments for AAA are mainly open surgery and interventional treatment.However,these two treatment methods have their own limitations,and they are not fully applicable to elderly patients and early patients.Therefore,in-depth exploration of the mechanism of AAA is of great significance for finding new targets for diagnosis and treatment,and for early diagnosis,early prevention,and early drug treatment.At present,the mechanism research of AAA has done a lot of work and has made great achievements.However,there are still no effective drugs used in clinical treatment.Similar to atherosclerosis,chronic inflammation is an important mechanism of AAA.The arterial wall inflammation and extracellular matrix(ECM)degradation caused by mast cell(MC)and immunoglobulin E(IgE)play an important role in this process.The literature suggests that Meprin-a(Mep1A gene expression)which is a member of metalloendopeptidase and plays an important role in the pathogenesis of atherosclerosis by changing the activity of proteins and inflammatory factors in various diseases.Meprin A in the aortic wall can decompose vasoactive peptides,and also activate inflammatory cytokines to promote inflammation,and ultimately accelerate ECM degradation and promote vascular remodeling.Inhibiting or knocking out of Meprin A may attenuate arterial wall inflammation and inhibit remodeling of the aortic wall.Based on the above research report,we speculated that if the mouse Mep1A gene is knocked out,the inflammatory response should be significantly reduced,and the formation and progression of AAA in mice will be inhibited.Objective:We sought to investigate the functional role of Mep1A in AAA formation and rupture and the physiological mechanism.Materials and methods:(1)Human specimen detection.Aortic wall tissues of AAA patients and normal adult aortic wall tissues were collected.Immunohistochemical staining was used to detect whether the expression of Meprin-a was different between the tissue samples of AAA patient and healthy people.Serum samples of normal and AAA patients were also collected.The differences in serum inflammatory factors in the patients treated with and without statins,and the changes in inflammatory factors before and after treatment with statins in AAA patients were determined.The aim of this step is to reveal the importance of Meprin-a and inflammatory factors in the pathogenesis of human AAA.(2)Apoe-/-Mep1A-/-mouse generation and experimental AAA.This study used both Apoe-/-Mep1A-/-mice and their littermates,Apoe-/-Mep1A-/-mice,as experimental controls.Mice were weighed,and blood pressures were measured using the CODA non-invasive blood pressure system before and after AAA molding.Aneurysm formation was then induced by subcutaneous insertion of a mini-osmotic pump containing Ang-II for 28 days.(3)Mouse aortic tissue immunohistochemical analysis.Serial cryostat cross-sections(6μm)were used for immunostaining for macrophages(Mac-3),T cells(CD4),mast cells(CD117),major histocompatibility complex-Ⅱ(MHC-Ⅱ),MCP-1,elastin,and CD31,terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling(TUNEL)and Mep1A,TNF-a.The relative macrophage,and MCP-1 contents within the aortas were quantified by measuring the immunostaining signal-positive area.Lesion CD31+microvessels and CD4+ T cells were quantified as cell numbers per square millimetres.Elastin degradation was graded according to the grading keys reported by Deckert.(4)Real-Time Polymerase Chain Reaction.Total cellular RNA was extracted from AAA tissue using a Trizol reagent.The RNA samples were then treated with a RNase-free DNase to remove genomic DNA contaminants.Equal amounts of RNA were reverse transcribed,and quantitative PCR was performed in a single-color real-time polymerase chain reaction(RT-PCR)detection system.The mRNA levels of TNF-a and Mep1A were normalized to those of housekeeping gene-GAPDH.(5)Western Blot analysis.Aortic tissues or cells were extracted in a lysis buffer.Equal amounts of protein extracts(30 mg/lane)were loaded on an8%or 12%SDS-PAGE gel,transferred to nitrocellulose membrane,and probed with goat anti-rabbit Mep1A polyclonal antibodies and goat anti-rabbit TNF-a polyclonal antibodies.Immunoblot for housekeeping protein glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was performed to assure equal protein loading.Western blot analyses were performed to measure the signaling molecules in IgE-stimulated production.(6)Enzyme-linked immuno sorbent assay(ELISA).We determined TNF-a levels in the plasma from Apoe-/-Mepl A+/+ and Apoe-/-Mep1A-/-mice and supernatant from cell culture using ELISA kits,according to the manufacturer’s instructions.The reaction was stopped with 0.5mmol/L H2SO4 and the absorbance read at 450 nm.A standard curve was calculated by use of the graphical package Graphpad Prism 5.0.The target gene concentration was then determined by referring to the standard curve.(7)Cell culture and small-interfering RNA(siRNA)assay.Mast cells were cultured,and siRNA transfection was carried out according to the manufacturer’s protocol.After growth for 24 h,cells at 80%adherence were were washed exhaustively and harvested.Results:1.The expression of MeplA was increased in human AAA tissues and AngII-induced mouse AAA tissues using immunohistochemical staining.2.AAA animal model was successfully established using Ang II induced ApoE-/-mice.3.The defect of MeplA increased the survival rate of AAA mice.4.Pathological analysis showed that the loss of MeplA reduced the degradation of the elastic layer and vascular smooth muscle cell(VSMC)apoptosis in AAA tissue.5.Through in vitro rt-PCR,Western Blot and ELISA methods,it was found that Mep1A is mainly expressed in mast cells(MCs)and mediates TNF-a expression.6.TNF-a secreted by MCs promotes the expression of MMPs in VSMCs and promotes VSMC apoptosis.7.In human serum samples,it was confirmed that the expression of Meprin-α and TNF-a in serum of AAA patients was also increased,and after short-term treatment with statins,the inflammatory factor TNF-a was significantly reduced.8.Overall,this study found that Mep1A mediates AAA disruption in mast cells by secreting tumor necrosis factor alpha(TNF-a),which promotes the expression of matrix metalloproteinases(MMPs)in vascular smooth muscle cells(VSMC)and promotes elastin degradation.Apoptosis.