| BackgroundAbdominal aortic aneurysm(AAA)is degenerative and pathological dilation of the abdominal aorta.The prevalence rate of AAA reaches 4%in over 65 years old people.Once ruptured the mortality rate is as high as 65-85%,the rupture of AAA leads to fatal internal bleeding.At present,the main intervention methods for ruptured AAA are open surgical repair(OSR)and endovascular aneurysm repair(EVAR).Current guidelines recommend surgical criteria for men with AAA>5.5cm and women with AAA≥5.0cm in maximum diameter and abdominal or back pain that is likely to be attributed to the aneurysm.For surgical repair is invasive treatment,so the small AAAs do not meet the guidelines recommend standard,whether active surgical repair is controversial.Because it did not improve survival rate,early surgical repair don’t have any advantage.Recommend preventive surgical intervention is controversial and should undertake aneurysm surveillance closely,but frequent surveillance will take up a lot of medical resources.Followup finds the aneurysm volume have been increased state,as some AAA patients,increasing age and declining health,such as aneurysm cavity volume to reach the standard clinical guidelines recommend or ruptured aneurysm symptoms need emergency surgical intervention,however patients with poor physical condition can’t tolerate OSR,EVER has high requirements on the location and morphology of aneurysms,as well as the healthy vessels in the riveted area of the proximal and distal stents and whether the opening of the visceral artery is exhausted.As a result,minimally invasive endovascular treatment has great limitations and cannot be applied to all patients.Therefore,to explore the mechanism of occurrence and development of AAA,It is of greatly clinical significance to actively seek non-invasive treatment methods to inhibit the occurrence and development of AAA.With the development of molecular biology,it is generally believed that the pathology of the AAA is a variety of factors(such as smoking,hypertension,hyperlipidemia and wound infection etc.),Which damage the aortic wall,especially endothelial injury and induce aortic wall autoimmune response and activing the abnormal immune response.associated with adventitial and medial inflammatory cell infiltration.resulting in pathological features include extracellular matrix(ECM)degradation and loss of vascular smooth muscle cells(VSMCs),contributing to vascular remodelling and weakening of the aortic wall.With the impact of the arterial blood flow,the wall gradually expands.Therefore,the aortic wall inflammation is thought to be one of the important factors in the development of AAA.The study found the receptor for advanced glycation end products(RAGE)may play an important role in inflammatory vascular disease.RAGE is a transmembrane protein located in the cell surface and expressed in a variety of tissues.The RAGE is composed of three domains,including the extracellular domain,transmembrane domain and carboxy-terminal intracellular domain.After binding to ligand,the extracellular signal is transmitted to the intracellular domain through the transmembrane domain to complete signal conduction.Under physiological conditions,RAGE is expressed in the macrophages,smooth muscle cells,monocytes and lymphocytes at low level.But in pathological conditions(such as diabetes,inflammation,cancer,atherosclerosis,myocardial infarction),the expression will increase significantly,cause inflammation and promote a variety of pathological change.There is also soluble RAGE(sRAGE)in the serum,which is deficient in transmembrane structure.It is caused by proteolytic cleavage of metalloproteinase to produce cleaved RAGE(cRAGE),and alternative mRNA splicing results in multiple variants among which endogenous secretory RAGE(esRAGE).sRAGE lacks transmembrane domain and intracellular domain of complete RAGE structure.Acting as decoy receptors,it could competitively bind ligand to inhibit ligandRAGE interactions and attenuate the deleterious effects of the activation of the full-length RAGE.People are coming to realize that RAGE as the targeting therapy can reduce tissue damage.Previous studies have found that RAGE is highly expressed in the tissue of human AAA and the aortic wall of animal AAA model.The specific mechanism of action has not been clarified,and no literature has been reported.In this study,we will explore the role of RAGE in the inflammatory response process of aortic wall and investigate whether targeting therapy to RAGE can inhibit the development of AAA.ADAM10,as a membrane protease,is related to cleave the ectodomain of RAGE.It is a type I transmembrane endonuclease,which is responsible for the main enzymatic cleavage of the ectodomain.ADAM10 mediates the enzymatic cleavage of the ectodomain of transmembrane proteins.These transmembrane proteins include cell adhesion molecules,cadherin,chemokines,amyloid precursor proteins,Notch receptors and RAGEs.Therefore,ADAM10 mediates the shedding of these key proteins and plays an important role in regulating chemoattractors,inflammation,cell-cell adhesion and induction of apoptosis.AD AM 10 can potentially change the interaction between RAGE and ligand,and reduce the activity and signal transduction of RAGE by enzymatic cleavage of ectodomain on the cell surface.In this study,murine AAA model were intrapitoneally injected with ADAM10 to explore its role in the development of AAA.In the rodent model of AAA,using porcine elastin type I infused into the lumen of the infrarenal aorta through the perfusion tube under high pressure.Elastin infiltrates the aortic wall,degrades elastin,weakens the elasticity,and results in inflammatory response.A large number of inflammatory cells,such as T cells,B cells and macrophages,inflitrate the aortic wall.In addition,the materials required that this animal models are cheap,simple and easy to be obtained,and the model is stable,the operation is simple.The biggest advantage of this model is that the pathological changes are closest to those of human AAA,and the aneurysm wall has obvious inflammatory response,which is in line with the purpose of this study.RAGE is involved in the pathological progression of the wall inflammatory response of AAA and whether the related targeting therapy can inhibit the development of aneurysm.In this study,we explored the relationship between RAGE and aortic wall inflammation by detecting the expression of RAGE in human abdominal aortic aneurysm.At the same time,we will conduct validation in animal models to clarify its molecular mechanism in the development of abdominal aortic aneurysm.For the treatment of RAGE,we treated model mice with AD AM 10 to test its effect on the model of AAA.On this basis,the molecular mechanism of RAGE in the pathological process of the development of AAA was further clarified.Providing a new theoretical basis and therapeutic clues for the clinical treatment of AAA.Objective1.To explore the expression of RAGE in serum and abdominal aortic tissue of healthy adults and patients with AAA,and to explore the correlation between the expression of serum soluble RAGE(sRAGE)and inflammatory factors of aortic tissue and the correlation between the expression of RAGE in aorta and inflammatory factors of aortic tissue.2.The murine AAA model was used to further verify the expression of RAGE in murine serum and aortic specimens,the correlation between the concentration of sRAGE in serum and the expression of inflammatory factors in aortic wall,and the correlation between the aortic expression of RAGE and inflammatory factors of aortic tissue.As well as to further explore the mechanism of RAGE in the pathological development of AAA.3.To explore the influence of ADAM10 on the inflammatory response of murine AAA wall and the development of aneurysms,and to provide theoretical basis for the study of the treatment with AAA.MethodsPart one1.1 Human blood samples and human AAA specimens of 30 patients who underwent OSR in the department of vascular surgery of Shandong Provincial Hospital affiliated to Shandong University were collected.The aged-matched normal human aortic specimens and blood samples were obtained from organ donors(without AAA)as a control.The normal aortic samples were left after kidney and liver transplantation in Shandong Provincial Hospital.This study was approved by the Human Research Committee of Shandong Provincial Hospital affiliated to Shandong University,and the procedures conformed to the ethical guidelines of the Declaration of Helsinki.1.2 Human serum sRAGE concentration was detected by ELISA.The levels of mRNA and protein of RAGE were detected by RT-PCR and Western-blotting.The level of NF-κB in human AAA tissue was detected by immunohistochemistry and Western-blotting.The source and location of RAGE were detected by immunofluorescence double staining.1.3 Spearson correlation coefficient was used to determine the correlation between serum sRAGE concentration and the levels of inflammatory factors in human AAA specimens.Correlation between the expression of RAGE and the level of inflammatory factors in the AAA specimens.Part two2.1.1 Establishment and grouping of mouse AAA PPE-perfusion model:12 male C57BL/6 mouse were divided into Sham group and AAA model group with 6-0 musse wire ligated near the abdominal aorta at the distal end,the blood flow was blocked,and the anterior wall of the occluded aorta was punctured with 30G needle.The PE-10 tube was inserted,and 20 ul elastic protein solution were injected.The perfusion pressure was maintained 100mmHg for 5min.2.1.2 Mouse serum sRAGE,IL-1β and TNF-α were detected by ELISA.The levels of mRNA and protein of RAGE were detected by RT-PCR and Western-blotting.The levels of NF-κB in murine AAA tissues were detected by Western-blotting.The sources of RAGE in murine AAA model were detected by immunofluorescence.2.1.3 Spearson correlation coefficient was used to determine the correlation between mouse serum sRAGE concentration and the levels of inflammatory factors in the murine AAA specimens,and the correlation between the levels of RAGE and the inflammatory factors in the murine AAA tissues.2.2.1 The male mouse were divided into three groups:Sham group,model control group(AAA model group),RAGE-/-Sham group and RAGE-/-AAA group.2.2.2 The diameters of abdominal aorta were detected by ultrasound.The pathological changes of aneurysm tissues were detected by HE/EVG.The levels of CD68+macrophages,CD8+T cells,CD31+neovascularization,α-SMA were detected by immunohistochemistry.The mRNA levels of RAGE,TNF-α and IL-1β were detected by RT-PCR.The protein levels of RAGE,NF-κB,TNF-α and IL-1 β were detected by Western blotting.Part three3.1 Experimental group:AAA model control group(AAA model group),sham operation group(Sham group)and AD AM 10 treatment group(ADAM10 treatment group).3.2 Using ELISA assay was to detect the serum sRAGE concentrations of mouse in the three groups.Diameters of murine AAA were detected by ultrasound.Pathological changes were detected by HE/EVG,and the levles of CD68+macrophages were detected by immunohistochemistry.Real-time PCR was used to detect the change of RAGE TNF-α IL-1βmRNA expression.Western Blotting was used to detect RAGE,NF-κB,TNF-α,II-1β protein expression.Using real-time PCR,Western Blotting and gelatinase were to detect the levels of mRNA,protein and activity of MMP-2/9 in murine specimens respectively.Results1.1 Detection of human abdominal aortic aneurysm specimensTNF-α and IL-1β were highly expressed in the serum of AAA patients.HE and EVG staining showed obvious inflammatory cell infiltration and severe destruction of elastic fiber in the AAA specimens.Immunohistochemistry and Western blotting showed that NF-κB expression in the AAA specimens was significantly increased.RT-PCR and Western blotting showed that the expression of TNF-α and IL-1β in AAA specimens were significantly increased.The levels of mRNA and protein of MMP-2/9 in the AAA specimens were significantly increased.1.2 Expression of RAGE in serum and tissue of AAA patientsThe expression of sRAGE in the serum of patients were significantly decreased.the expression of RAGE was significantly increased in AAA tissues.RAGE was expressed in the cell membrane and intercellular space.1.3 Correlation between serum sRAGE concentration and the levels of inflammatory factors in human AAA specimensSpearson correlation analysis showed that serum sRAGE concentration was negatively and signifcantly correlated with the level of TNF-α mRNA(R=-0.5911),and negatively and signifcantly correlated with the level of IL-1β mRNA(R=-0.6422).1.4 The level of RAGE was correlated with the level of inflammatory factors in human AAA specimensSpearson correlation analysis showed that the level of RAGE was positively and signifcantly correlated with the level of TNF-α mRNA(R=0.6802),and positively and signifcantly correlated with the level of IL-1β mRNA(R=0.6495)in human AAA specimens.Part two 2.12.1.1 Modle evaluationAt 14 days after prefusion,ultrasound examination was performed,and the results showed that the maximum diameter was more than 50%of the preoperative diameter.The changes of abdominal aortic diameter showed that the mice AAA model was successfully made.HE staining of the murine AAA specimen showed that the structure of the three layers of the aortic wall was disordered,and the inner,middle and outer layers were destroyed seriously.The number of smooth muscle cells in the media was reduced,the arrangement was disordered,and the fibroblasts were increased.A large number of inflammatory cells were visible in each layer.EVG staining showed that the elastic plate and elastic fiber were damaged seriously.2.1.2 Expression of RAGE in murine AAA modelThe serum sRAGE concentration in the AAA model group was significantly decreased,and the level of RAGE in the murine AAA model was increased.Immunofluorescence double staining showed that RAGE was expressed in infiltrated macrophages and residual smooth muscle cells.2.1.3 Expression of inflammatory factors in murine AAA modlesThe levels of NF-κB,TNF-α and IL-1β were increased in AAA specimens.The levels of TNF-α and IL-1β in serum were increased in AAA model group.2.1.4 Expression of MMP-2/9 in murine AAA model specimens were increased2.1.5 Correlation between serum sRAGE and aortic inflammation in AAA model groupSpearson correlation analysis showed that serum sRAGE concentration in murine AAA model was negatively and signifcantly correlated with the level of TNF-α mRNA in murine AAA specimens(R=-0.5030),and negatively and signifcantly correlated with the expression of IL-1β mRNA in murine AAA specimens(R=-0.5566).2.1.6 Correlation between the level of RAGE and the inflammatory factors in murine AAA specimensSpearson correlation analysis showed that RAGE expression was positively and signifcantly correlated with TNF-α expression in murine AAA specimens(R=0.5222).As well positively and signifcantly correlated with the level of IL-1β(R=0.5018).Part two 2.22.2.1 Detection of aortic diameterThe aortic diameters in RAGE-/-murine AAA models were significantly decreased compared with the murine AAA models 2.2.2 Immunohistochemical resultsThe infiltration degree of CD68+macrophages,CD8+T cells in the murine AAA specimens of the RAGE-/-AAA model group was significantly reduced.The apoptosis degree of a-SMA was significantly reduced and the expression of CD31+ neovascularization was decreased.2.2.3 The levels of NF-κB,TNF-α and IL-1β in the specimens of RAGE-/-murine AAA model were lower than those of murine AAA model The experssions of NF-κB,TNF-α and IL-1β were signifcantly decreased.Part three3.1 Detection of the diameter of murine abdominal aortaThe diameter of the murine aorta in ADAM10 treatment group was significantly reduced.3.2 HE and EVG staining resultsThe pathological changes of the murine AAA specimens in AD AM 10 treatment group were significantly reduced.3.3 AD AM10 treatment can relieve the aortic inflammationThe infiltration of CD68+ macrophages in the murine specimens of the AD AM10 treatment group were significantly reduced.3.4 Serum sRAGE concentration was significantly increased but the TNF-α and IL1β expression were significantly decreased in the ADAM10 treatment group.3.5 The expressions of RAGE and NF-κB were lower in the aortic tissues of mouse treated with ADAM10.3.6 The levels of mRNA,protein and activity of MMP-2/9 were decreased in aortic tissues of the ADAM10 treatment group.Conclusion1.RAGE was involved in the pathological process of the development of AAA and activation of NF-κB signaling pathway played an important role in the pathophysiology of AAA.2.ADAM10 treatment significantly inhibited the inflammatory response of murine AAA wall and the progression of AAA through blocking signal transduction by extracellular proteolytic cleavage of RAGE.Meanwhile sRAGE,as a decoy receptor,plays competitively inhibiting ligands binding to RAGE activation to alleviate the deleterious injury and implicated in its pathogenesis of AAA. |
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