Role Of Methionine-induced Hyperhomocysteinemia In The Pathogenesis Of Abdominal Aortic Aneurysm And Its Molecular Mechanisms

Introduction: Abdominal aortic aneurysm(AAA)is a degenerative disease characterized by persistent dilatation of the abdominal aorta and degeneration of the blood vessels caused by degradation of the middle layer,disintegration of elastic fibers and collagen.When the abdominal aorta diameter of more than 3cm or more than 50%expansion rate can be diagnosed as abdominal aortic aneurysm,and become elderly men(65 years of age or more)one of the leading causes of sudden death 1-4.Smoking,high blood pressure,males,seniors and family genetic predispositions are considered to be the primary risk factors for the development of abdominal aortic aneurysms2.Small aneurysms(less than 5.5 cm in diameter)are usually found by imaging studies.Open surgery or endovascular repair of aortic aneurysm(diameter greater than 5.5cm)better.Although current advances in medical technology can provide effective surgical intervention,survival of abdominal aortic aneurysms over 6 years is less than 70% 7.We still lack of proven strategies to predict the development of abdominal aortic aneurysms and the risk of rupture.The pathological features of abdominal aortic aneurysm include degradation of extracellular matrix(ECM),apoptosis of vascular smooth muscle cells(VSMCs)and infiltration of inflammatory cells in the vascular middle and outer layers.The above processes lead to vascular remodeling and decreased vascular wall tension.Homocysteine(Hcy),as a sulfur-containing amino acid,is an intermediate in the normal biosynthesis of methionine and cysteine.Hcy metabolic disorders lead to abnormal accumulation in the body,which in turn leads to high homocysteine cycle levels,commonly known as hyperhomocysteinemia(HHcy)9,10.In general,HHcy is caused by abnormal metabolism of methionine(Met)or homocysteine due to metabolic enzyme deficiency and / or nutritional disorders.Plasma Hcy levels above10μmol / l can be diagnosed as HHcy.Hcy is an important,independent risk factor involved in a variety of vascular diseases,including: coronary disease,stroke and peripheral vascular disease11,12.A large number of clinical observations revealed the association between Hcy and AAA.Our previous study also confirmed that HHcy can be used as an independent risk factor for the pathogenesis of AAA.Patients with abdominal aortic aneurysm plasma homocysteine levels were significantly higher than the control group,the current Hcy has been admitted as a routine abdominal aortic aneurysm in patients with routine examination items13.In the meantime,a meta-analysis study by our team also confirmed that Hcy predicts an increased risk of AAA.Subjects with hyperhomocysteinemia are at a higher AAA risk14.Although the histopathological features of AAA have been widely reported,the exact mechanism by which Hcy participates in the pathogenesis of abdominal aortic aneurysms is not fully understood15.There is currently a large body of research that deals with the association between Hcy and AAA,but these studies are mostly limited to clinical studies and merely confirm that Hcy increases the risk of developing AAA.We still need further research and evidence to confirm how Hcy aggravates the occurrence and progression of AAA.This study was intended to establish the model of HHcy rat induced by methionine feeding,to verify the role of Hcy in promoting AAA and to explore the inflammatory response,whether MMP-2 activation participates in this process and to observe the level of autophagy.Then,The cultured VSMCs were treated with Hcy to observe the autophagy and cell phenotype changes and to explore the role of autophagy in this process.Finally,AMPK was used to observe the induction of Hcy Of VSMC autophagy is suppressed,Hcy VSMC induced cell-specific autophagy mechanism.Experimental Materials and Methods: 1.Experimental Reagents and Materials:porcine pancreatic elastase(Sigma-Aldrich,USA),Methionine(Beijing Solaray Technology Co.,Ltd.),Cell Biolabs Sirius Red Dye(Beijing Solei Bao Technology Co.,Ltd.),elastic fiber staining solution(Beijing Regen Biological Technology Co.,Ltd.),Sirius red dye(Beijing Regen Biological Technology Co.,Ltd.),SP immunohistochemical kit Zhonghua Jinqiao Biotechnology Co.,Ltd.),DAB color reagent(Fuzhou Mai new biotechnology Development Co.,Ltd.),FITC fluorescent secondary antibody(Wuhan proteintech),human aortic vascular smooth muscle cells(HA-VSMC)were purchased from Shanghai Chinese Academy of Sciences,High glucose DMEM,fetal bovine serum,trypsin,blue-streptomycin(Hyclone),homocysteine,Dorsomorphin(sigma USA),rapamycin(Beijing Solvay Technology Co.,Ltd.)3-MA(Shanghai selleck company),CCK-8 kit(Beijing Solei Bao Technology Co.,Ltd.).Anti-a-SMA(Wuhan proteintech),MMP-2,IL-6,LC-3,Beclin-1,OPN rabbit polyclonal antibody(Wuhan proteintech),Anti-AMPK AMPK(US CST).2.Animal model: Forty-two male Sprague-Dawley rats of 280-300 g were injected with elastase to establish AAA model.The tension was maintained for 10 minutes.The suture on the blood vessel was sutured with 10-0 vascular suture.Forty Sprague-Dawley rats were randomly divided into four groups: untreated group(n = 8),control group(200μl of 30% F-127 gel coated on AAA outer wall,n = 8)The control group(NC group,AAA outer membrane was coated with 50 μl of negative control si RNA # 1(Ambion 200 μl gel,n = 8)),the treatment group(ANRIL-si RNA group,AAA outer membrane smear dissolved 50 μl ANRIL-si RNA Mu] l gel,n = 16).Postoperative cage marker,unified feeding.The diameter of the abdominal aorta was examined by color Doppler to calculate the dilation rate.After 4w,the specimens were collected and fixed in 4%paraformaldehyde or frozen in liquid nitrogen.Morphological examination: The paraffin sections were made and the expression of p14 ARF,bcl-2,bax and caspase-3 in human abdominal aorta or AAA tissues were detected by immunohistochemistry.Frozen sections of the abdominal aorta were made and the in situ hybridization staining method was used to detect the gene expression and quantification of ANRIL in human abdominal aorta.Transfection efficiency of VSMC with immunofluorescence technique.Electron microscope observation of the abdominal aorta and AAA,understand the VSMC phenotype conversion.4,plasma Hcy detection Peripheral blood collected immediately 3000 rpm centrifuged10min,collected plasma and stored at-80 ℃ to use.Hcy levels were measured using an ELISA kit.Dilute the standards in multiples of the sample dilution to give 8 standard points of 1000,500,125,62.5,31.2,15.6 and 0 pg / ml.Sample Dilution Plasma samples are diluted.Add 100 ul of standard or test sample to each well,add 100 ul of sample diluent to blank wells and incubate at 37 ℃ for 90 min.Discard the hole liquid to join the cleaning fluid 3 times and pat dry.Add dilute diluted secondary antibody,each hole100 ul,incubated for 1h at room temperature.Enzyme conjugate,each hole 100 ul,incubated for 30 min at room temperature.Add enzyme substrate,each hole 100 ul,incubated for 5-15 min at room temperature to appear standard color gradient,immediately add stop solution 100 ul.Microplate reader at 450 nm wavelength measured absorbance.Calculate the standard curve to obtain Hcy concentration.Western-blot detection of ANRIL-related protein product expression: Human and rat abdominal aortic aneurysm tissue and non-tumor tissue after cutting placed in the EP tube,adding an appropriate amount of protein lysate and protease inhibitors,homogenate Homogenate or sonicate the cells.After centrifugation,the protein was extracted and the protein concentration was determined by Coomassie brilliant blue method.The polyacrylamide gel,electrophoresis,transfection,blocking,plus primary antibody,secondary antibody and MF-Chemi Bis were prepared and the optical density values were calculated.6,immunofluorescence detection: 4% paraformaldehyde fixed arterial specimens.1%bovine serum albumin,1: 200 dilution of MMP-2 and LC3 primary antibody were added dropwise,4 ℃ overnight,FITC fluorescent secondary antibody was added dropwise the next day,DAPI nuclei were counterstained with anti-quenching capsule The capsulation was observed under a fluorescence microscope and the images were collected.7,transmission electron microscopy: fresh tissue to determine the site of drawing,quickly put into the electron microscope fixation solution 4 ℃ fixed 2-4h.0.1M phosphate buffer PB(PH7.4)rinse three times,each 15 min.1% osmium tetroxide · 0.1M phosphate buffer solution PB(p H 7.4)for 2 h at room temperature(20 ℃).0.1M phosphate buffer PB(PH7.4)rinse three times,each 15 min.Followed by the organization into the gradient of alcohol and 100% acetone dehydration,each 15 min.Acetone: 812 embedding agent = 1: 1 2-4h,acetone: 812 embedding agent = 2: 1 infiltration overnight,pure 812 embedding agent 5-8h,the pure 812 embedding agent into the embedded plate,the sample Insert oven at 37 ℃ overnight after embedding.60 ℃ oven polymerization48 h.Ultra-thin section slicing 60-80 nm ultra-thin slices.Uranium and lead double staining(2% uranyl acetate saturated aqueous solution,lead citrate,each staining 15min),the slices were dried overnight at room temperature.Transmission electron microscope observation,acquisition of image analysis.8,cell culture: Adherent method to cultivate HA-VSMC cells,cell passage 3-4 times.Placed in a 37 ℃,5% CO 2 incubator and cultured in high glucose DMEM containing10% serum.9.Cell Migration Experiment: The cells in logarithmic growth phase were inoculated into24-well culture plates.After transfection for 96 hours,when the cells were fused to about 90%,the cells were counted under inverted microscope(× 100)24 h after scratching Number of cells migrated in the scratch area.10,statistical processing: using IBM SPSS Statistics 21.0 for statistical analysis.Aneurysm diameter measurement unit mm,take a decimal point after 1.Metrological results were expressed as mean ± standard deviation(`x ± s),and the comparison between the two groups was statistically significant assessed using the t-test.Chi-square test was used to assess the incidence of aneurysms.P values <0.05 were considered to have significant statistical differences.Results:1,methionine feeding induced mild hyperhomocysteine aggravate elastase-mediated abdominal aortic aneurysm and its progression: the performance of the largest diameter of aortic aneurysm was significantly increased and the arterial wall Thickened.Hyperhomocysteinemia induced related protein expression: inflammatory infiltration(increased IL-6),increased MMP-2 activity,elastin fiber disintegration and collagen deposition,smooth muscle cell phenotype changes(OPN Increased expression)and autophagy(elevated levels of LC3 and Beclin-1).2,In vitro culture of VSMC: Hcy promoted the proliferation and migration of VSMC,and the phenotype(systolic phenotype);the expression of autophagy-related proteins LC3 and Beclin-1 increased(p <0.05),indicating that Hcy induced VSMC autophagy occurs.3,AMPK is involved in the process of Hcy-induced autophagy: Hcy activates AMPK expression in VSMCs,and AMPK inhibitor is given to cells.The expression of autophagy-related proteins LC3 and Beclin-1 is significantly decreased by western blot,indicating that Hcy-induced Phagocytosis is inhibited,AMPK involved in Hcy-induced autophagy.Conclusion:1,Methionine feeding can induce mild hyperhomocysteinemia in rats and can aggravate the occurrence and progression of AAA in rats,resulting in the expansion of maximal diameter of abdominal aorta and thickening of vessel wall.Hcy can cause AAA inflammatory infiltration,MMP-2 activity enhancement,VSMC phenotype conversion,elastin disintegration and collagen deposition,and autophagy reaction;2,Hcy can induce vascular smooth muscle cell phenotype conversion,proliferation and migration Induced autophagy phenomenon;3,AMPK involved in the process mediated by Hcy-induced autophagy phenomenon.