| BackgroundAbdominal aortic aneurysm(AAA)is a potentially fatal vascular disease defined as a localized dilation of the abdominal aorta with a diameter greater than 3 cm or exceeding the normal aortic diameter by 50%.AAA mainly involves the abdominal aorta below the branches of the renal artery.Patients are usually asymptomatic and it is often difficult to detect the dilated abdominal aorta even during the course of physical examination by doctors.Patients are often found accidentally to have AAA during abdominal ultrasound or CT scanning because of other clinical signs.Therefore,early detection of AAA is extremely difficult.Once AAA ruptures,the death rate is as high as 85% to 90%,and the patients is almost incurable.Thus,a major challenge is the early detection and inhibition of the rapid expansion of AAA in clinical medicine.Unfortunately,although studies exploring the pathogenesis of AAA have made a tremendous progress,no clinical predictor or pharmacologic treatments have yet been effective in reducing the risk of AAA development or limiting its progression.When the AAA diameters of male and female patients is greater than 55mm and 50mm respectively,surgical correction or intravascular interventional surgery can be performed,and the postoperative survival rate is over 95%.Therefore,surgical correction and stent intervention are the only effective methods for preventing AAA rupture,but these procedures have trauma and complications.Research and development of new drugs that can inhibit the occurrence and development of AAA has become an urgent task in AAA research field.Erythropoietin(EPO)is composed of 165 amino acids and four carbohydrate side chains with a compact spherical structure and a molecular weight of 3 4kd.EPO is essential for the production of normoblast which is synthesized primarily in the fetal liver and adult kidney.EPO mainly stimulates the EPO receptor(EPOR)in the hematopoietic system to promote erythropoiesis.EPO is produced by the renal interstitial cells during hypoxia and increases the number of red blood cells by inhibiting the apoptosis of erythrocyte progenitor cells.Meanwhile,the potential nonhematopoietic function of EPO has also attracted much attention.Studies have shown that EPO can also be produced in tissues other than kidneys,and EPORs not only exist in red line progenitor cells,but also widely distributed in other tissues of the body.EPO produced outside the kidney functions through paracrine/autocrine rather than hormone-like efects in hematopoietic function.EPO and receptor expression are significantly increased in conditions of trauma and inflammation,triggering a critical protective response in injured tissues/organs.The tissue protective effects of EPO have been demonstrated in multiple animal models,including focal cerebral ischemia,embolic stroke,traumatic brain injury,myocardial ischemia,acute renal injury,limb ischemia,wound healing and so on.Clinical studies have shown that the administration of EPO in patients with severe trauma reduces mortality.Recent studies have found that EPO can promote angiogenesis through promoting endothelial cell proliferation and migration and matrix metalloproteinase 2(MMP2)expression.Previous clinical studies have shown that about one-third of AAA patients undergoing endovascular repair have anemia,and circulating hemoglobin levels are independent and negatively correlated with AAA size,but the mechanism is unclear.A recent experimental study in hyperlipidemic mice infused with angiotensin ?(Ang ?)found that inhibition of hypoxia-inducible factor-1 alpha(HIF-la)attenuated the progression of AAA.It is well known that chronic anemia and HIF may trigger an increase in EPO production,and recent evidence suggests that Ang ? may affect hematopoiesis by directly stimulating receptors in hematopoietic progenitors or indirectly regulating EPO gene expression.An unusual case of AAA has been reported who underwent chronic hemodialysis and developed resistance to recombinant human EPO without any obvious reasons.These clinical and experimental studies strongly suggest that there is a relationship between EPO and AAA.Angiogenesis in the aortic wall,is an experimental and clinical marker of AAAs,which plays a key role in the progression and rupture of the aneurysm.Angiogenesis is more common in human aneurysm tissue,especially in areas adjacent to rupture and infiltrated by white blood cells.Drugs that inhibit the progress of AAA also reduce angiogenesis in arterial walls in animal models.The matrix metalloproteinases(MMPs)family is closely involved in the process of new angiogenesis and plays a key role in promoting angiogenesis,which also related with degradation and rupture of the aortic wall.Hypoxia and inflammation are two key stimulators of angiogenesis.The expressions of hypoxia-inducible factor 1(HIF-1?)and its target genes were increased in human and experimental AAAs.Inhibition of HIF-1? prevents the progression of experimental AAA and reduces leukocyte infiltration,angiogenesis,and overexpression of MMPs.Inflammation and immune related diseases associate with angiogenesis,because most of the white blood cells can produce a series of angiogenic factors,such as vascular endothelial growth factor(VEGF),platelet-derived growth factor(b-FGF),basic fibroblast growth factor(b-FGF),monocyte chemotactic protein 1(MCP-1)and protease,such as chymotrypsin,trypsin,MMPs.It has been reported that the mast cell-specific chymotrypsin and trypsin induce the expression of adhesion molecules and chemokines in endothelial cells(ECs),which use the chemokine receptor 2(CCR2)as the receptor to recruit chemokines,degrade matrix proteins,promote angiogenesis,and induce smooth muscle cell apoptosis.A variety of pathological processes are involved in the development of AAA,such as angiogenesis,inflammatory infiltration,oxidative stress and smooth muscle cell apoptosis,etc.Therefore,the pathophysiological process of AAA induced by EPO needs to be further explored.In the present study,we hypothesized that EPO may promote the formation of AAA via angiogenesis and inflammatory response.A series of in vitro and in vivo experiments were meticulously designed and conducted to test this hypothesis.Objective1.To investigate whether EPO promotes AAA formation in ApoE-/-mice and wild-type mice without Ang ? stimulation and high fat feeding;2.To investigate whether the effect of EPO on AAA in mice depended on high fat feeding and hypercholesterolemia;3.To explore the type of the activating EPO receptors by EPO;4.To investigate the pathophysiological mechanism of EPO-induced AAA in mice;5.To investigate the effect of EPO on three types of vascular wall cells(endothelial cells,smooth muscle cells and macrophages);6.To explore whether the level of serum EPO is associated with the occurrence of AAA in clinical patients.Methods1.Animal models grouping(1)60 male ApoE-/-mice were randomly divided into 4 groups for treatment(n=15 per group)and the whole process was fed with high fat diet:the vehicle group received intraperitoneal injection of saline and the low-,medium-and high-dose EPO groups received intraperitoneal EPO inj ection of 2,500 IU/kg/day,5,000IU/kg/day and 10,000IU/kg/day,respectively,for 4 weeks.All mice underwent euthanasia after 4 weeks' infusion.(2)60 male ApoE-/-mice were randomly divided into 4 groups for treatment(n=15 per group)and the whole process was fed with normal diet:the vehicle group received intraperitoneal injection of saline and the low-,medium-and high-dose EPO groups received intraperitoneal EPO injection of 2,500 IU/kg/day,5,000IU/kg/day and 10,000IU/kg/day,respectively,for 4 weeks.All mice underwent euthanasia after 4 weeks' infusion.(3)60 male wild-type mice were randomly divided into 4 groups for treatment(n=15 per group)and the whole process was fed with normal diet:the vehicle group received intraperitoneal injection of saline and the low-,medium-and high-dose EPO groups received intraperitoneal EPO injection of 2,500 IU/kg/day,5,000IU/kg/day and 10,000IU/kg/day,respectively,for 4 weeks.All mice underwent euthanasia after 4 weeks' infusion.(4)30 female ApoE-/-mice were randomly divided into 2 groups for treatment(n=15 per group)and the whole process was fed with high fat diet;30 female wild-type mice were randomly divided into 2 groups for treatment(n=15 per group)and the whole process was fed with normal feeding:the mice received intraperitoneal injection of saline and EPO 5,000IU/kg/day,respectively,for 4 weeks.All mice underwent euthanasia after 4 weeks' infusion.(5)45 male ApoE-/-mice were randomly divided into 3 groups for treatment(n=15 per group)and the whole process was fed with high fat diet;45 male wild-type mice were randomly divided into 3 groups for treatment(n=15 per group)and the whole process was fed with normal feeding:the mice received saline,pyroglutamate helix B surface peptide(pHBSP)30 mg/kg/day and pHBSP 300 mg/kg/day via an osmotic pump,respectively,for 4 weeks.All mice underwent euthanasia after 4 weeks' infusion.(6)30 male ApoE-/-mice and 30 male WT mice were randomly divided into 2 groups for treatment(n=15 per group),respectively.The whole process was fed with high fat diet or normal feeding.The mice received saline or Ang ?(1.44 mg/kg/day)via an osmotic pump,respectively,for 4 weeks.All mice underwent euthanasia after 4 weeks' infusion.2.Blood pressure measurementThe blood pressure was measured by the tail blood pressure meter of small animals.The measurement site was the tail artery of the mice.Each mouse was detected 3 times,and the average value was taken as the final value.3.Obtain tissue samplesThe mice were starved for 6-8 hours,and the whole blood,serum,heart,liver,spleen,kidney,adipose tissue and aorta were collected after anesthesia.Parts of the tissues were frozen in liquid nitrogen,stored in a refrigerator at-80?,and parts were fixed in 4%paraformaldehyde for subsequent experiments.4.Blood lipid,whole blood,liver and kidney function testSerum levels of total cholesterol,triglyceride,LDL-C,HDL-C,ALT,AST,Cr,BUN in each group were measured.The total red blood cell count,hemoglobin content and hematocrit of each group were measured.5.RNA-seqAortic tissues were isolated from ApoE-/-mice receiving medium-dose EPO(n=3)and vehicle(n=3)treatment for RNA-seq by Novogene Co..6.Pathological examinationParaffin sections of abdominal aortic tissues were prepared,and H&E staining,Masson and Verhoff staining were performed,respectively.Immunohistochemical staining was performed by using anti-Endomucin,MMP2,MMP9,MT1-MMP,CD68,ICAM-1,VCAM-1,IL-6,IL-1?,MCP-1 and TNF-?,and tissue immunofluorescence staining was performed by using anti-CD68,IL-6,IL-1?,MCP-1,TNF-?,CD144 and Ki67.7.Western blotAbdominal aorta tissue proteins and total cell proteins were extracted.Determine the concentration by BCA protein.SDS-PAGE gel was prepared and the protein contents of collagen ?,collagen ?,MMP2,MMP9,MT1-MMP,VEGF,TGF-?1,KDR,Tie2,CD68,ICAM-1,VCAM-1,IL-6?IL-1?,MCP-1 and TNF-? were detected by western blotting.8.ZymographySDS-PAGE gel containing 1%gelatin was prepared.The enzyme activity of MMP2 and MMP9 was detected by an MMP gelatin zymography kit.9.RNA extraction,reverse transcription of mRNA,and RT-PCRRNA from abdominal aorta tissues and total RNA from various cells were extracted,and mRNA reverse transcription was performed using the TaKaRa#RR047A reverse transcription kit.The Ct value of circulation threshold was obtained by RT-PCR,and the relative expression level of cDNA was calculated by formula 2-??CT.10.Cell culture and signaling pathways(1)The dose of 5 IU/mL EPO was selected because in our early studies,5 IU/mL EPO induced the highest activity of MMP2 and MMP9 in HUVECs;(2)HUVEC,HAEC,HASMC and macrophages:5 IU/mL of EPO was added to the experimental group,and lx PBS was added to the control group;Cells and medium were collected after 24h stimulation.(3)HASMCs were stimulated with EPO alone or the medium of EPO-pretreated HUVECs for 24 hrs.Collected cells and supernatant.(4)Macrophages were stimulated with EPO alone or the medium of EPO-pretreated HUVECs from 0 h to 10 hrs.Collected cells and supernatant.(5)HUVEC were stimulated with 10mg/L pHBSP or lx PBS,and collect cells and supernatant after 24 hours.(6)The experiment was divided into 4 groups:the vehicle group received DMSO stimulation,the EPO group received EPO and DMSO,the JAK2 inhibitor group received EPO and TG101348,and a STAT5 inhibitor group received EPO and CAS 285986-31-4,which was stimulated for 24 hours.11.EDU cell proliferation assayThe proliferation capacity of HUVEC or HASMC was detected by using the EDU proliferation kit.12.Endothelial cell migration experimentThe migration ability of HUVEC and HAEC induced by EPO was detected by scratch experiment and Transwell cell migration experiment.13.In vitro tube formation assayThe tube formation ability of HUVEC and HAEC in vitro induced by EPO was observed using growth factor-reduced Matrigel matrix.14.Aortic ring assayThoracic aortas from 3 WT mice were used and the dissected aortas were cut into 1mm pieces and embedded in growth factor-reduced Matrigel matrix.The number of sprouts was counted under a light microscope.15.Monocyte-endothelial cell adhesionMonocyte-endothelial cell interaction was examined by adherence of BCECF-AM-labelled THP-1 cells to EPO-stimulated HUVECs.16.Detection and quantitation of apoptosisHASMCs were stimulated with EPO alone or the medium of EPO-pretreated HUVECs for 24 hrs.The apoptosis of HASMC was detected by TUNEL detection kit.17.Extraction of peritoneal macrophages8-12 weeks male wild-type mice were selected and 1mL of 6%starch solution was intraperitoneally injected 3 days in advance.The mice were sacrificed,and tear the skin,and the peritoneum was fully exposed.DMEM was injected into the abdominal cavity with a sterile syringe and the fluid was withdrawn into a new 50mL centrifugal tube.Rinsed peritoneal cavity for 3 times until the DMEM became clear,then the peritoneal macrophage suspension was obtained.18.Matrigel plug assayMatrigel plug assay involved a high concentration of Matrigel matrix.After mixing Matrigel matrix with or without EPO,the Matrigel mixture was injected into WT mouse subcutaneously.After 14 days,mice were anaesthetized and a square segment of injected tissues was excised with scissors.Tissues were then fixed with formalin.The fixed Matrigel plug was embedded in paraffin and sectioned for histochemical staining.19.Enzyme-linked immunosorbent assaySerum EPO levels were detected by ELISA.20.Collection of serum from AAA patientsWe enrolled 40 AAA patients and collected blood samples within 24 hours of admission.Patients with anemia,heart failure,chronic respiratory disease,and renal failure were excluded.Serum from 45 healthy volunteers was included as the normal control group.21.Statistical analysis of dataAll data were expressed as mean ± standard error.Independent sample t test was used for two groups of measurement data,and one-way anova LSD post-test was used for many groups of measurement data.The counting data were tested by chi-square test.Log-rank test was used for survival analysis.P<0.05 was considered statistically significant.Results1.EPO dose-dependently induced AAA formation in both Apoe-/-and WT miceAfter 4 weeks of intraperitoneal injection of EPO,the results showed that EPO dose-dependent increased the incidence of AAA in mice and significantly increased the diameters of abdominal aorta.Moreover,EPO injection led to a dose-dependent increase in mortality.The AAA incidence of ApoE-/-mice induced by high-dose EPO was similar to that of Ang ?,while the AAA incidence of wild-type mice induced by high-dose EPO was significantly higher than that of Ang ?.2.EPO injection increased aortic wall thickness and enhanced elastic lamina degradation in these miceThe results showed that after EPO injection,the arterial wall thickened significantly,the elastic fiber broke,and the lumen was accompanied by thrombosis.3.EPO did not affect blood pressure and blood lipid in ApoE-/-mice and wild-type miceAfter 4 weeks of EPO intervention,there was no significant change in blood pressure of the low-,medium-and high-dose group compared with the vehicle group.Similarly,EPO intervention had no effect on lipid in mice.4.EPO did not affect liver and kidney functions of ApoE-/-mice and wild-type miceAfter 4 weeks of EPO intervention,there was no significant change in ALT,AST,Cr,BUN of the low-,medium-and high-dose group compared with the vehicle group.There was no significant difference in tissue morphology.Thus,from the perspective of drug toxicology,EPO injection for 4 weeks did not cause liver and kidney dysfunction.5.The high-fat diet did not affect the dose effect of EPO on the AAA formation of ApoE-/-miceThe results showed that EPO dose-dependent increased the incidence of AAA in ApoE-/-mice with normal diet,which was not significantly different from that in high-fat diet.The mortality of mice in high-dose EPO group with or without high-fat feeding was not significant statistical differences.Thus,the high-fat diet did not affect the dose effect of EPO on the AAA formation of ApoE-/-mice.6.EPO promotes AAA formation in female ApoE-/-mice and wild-type miceThe results showed that AAA occurred in both female ApoE-/-mice and female wild-type mice under the intervention of medium dose EPO,but the incidence rate was slightly lower than that of male mice,which was consistent with the characteristic that male was higher than female in the incidence rate of human AAA.7.pHBSP could not be dose-dependent to induce AAA in miceThe results showed that pHBSP could not induce AAA in both genotypes of mice,so it was inferred that EPO may induce AAA through the homodimer receptor.8.Serum EPO levels in AAA patientsThere were no significant differences in age,sex,kidney function,or medication between the two groups,but smokers were more common in the AAA group than in the normal control group.The analysis showed no significant differences in abdominal aortic diameters between ages>65 and<65 years,men and women,smokers and nonsmokers,suggesting that AAA diameters were unaffected by traditional atherosclerotic risk factors in this group of AAA patients.Serum EPO levels were significantly higher in AAA patients than in healthy group,suggesting that AAA patients produced more EPO into the circulating blood.9.Gene sequencing analysis of the effect of EPO on mRNA expression profile of ApoE-/-mice aortic tissueAccording to the analysis of gene ontology,the gene differences between the two groups were mainly concentrated in angiogenesis,cell cycle and migration,inflammatory response,leukocyte infiltration,and extracellular matrix remodeling.10.Angiogenesis and collagen metabolism are involved in the process of EPO-induced AAAThe results showed that compared with the control group,the EPO group had significantly less collagen in the abdominal aortic wall,especially the expression of collagen ? and collagen ?.The expression and activity of MMPs were significantly higher than that of the control group.In the EPO group,the microvascular density in the abdominal aortic wall increased significantly,and the expression of VEGF,TGF-?1,KDR and Tie2 proteins increased significantly.11.Inflammatory response is involved in the process of EPO-induced AAAThe results showed that the degree of CD68 positive cells infiltrating the arterial wall of the EPO group was significantly higher than that of the control group,and the mRNA and protein levels of inflammatory factors was significantly higher than that of the control group.CD68 positive cell-derived inflammatory cytokines were significantly increased.Serum levels of inflammatory factors were also significantly higher than in control mice.12.Effect of EPO on red blood cells,white blood cells and platelets in miceAfter 4 weeks of EPO injection,RBC,HGB and HCT were significantly increased in the EPO group compared with the control group.However,there was no significant difference in WBC,MC,LPC,GC and PLT between the two groups.13.EPOR mRNA expression level of ECs was the highest in the three types of cellsCompared with SMCs and macrophages,EPOR expression in ECs was the highest,suggesting that EPO may mainly target ECs.14.EPO promotes ECs proliferation and migrationThrough EDU experiment,scratch experiment and Transwell cell migration experiment,we found that EPO promoted HUVEC proliferation,HUVEC and HAEC migration.15.EPO promoted ECs tube formation in vitro and budding of aortic rings in miceThe results showed that ECs in the EPO group were more likely to form a closed tubular structure.Mouse thoracic aortic ring was planted in Matrigel matrix,and the aorta induced by EPO was easier to bud than the control group,and the number and length of sprouts increased significantly.16.EPO promotes the expression of angiogenesis related proteins in ECsThe results showed that the expression level and activity of HUVEC or HAEC MMPs after EPO stimulation were significantly higher than those in the control group.The expression of KDR and Tie2 in EPO group was significantly increased.17.pHBSP does not promote HUVEC migration,tube formation in vitro,nor MMPs expressionThe results showed that there was no significant difference in the number of cell migration between the control group and the pHBSP group,and the number and the total length of tube were not statistically significant.The MMPs protein expression and enzyme activity of HUVEC after pHBSP stimulation were not significantly different from those of the control group.18.EPO promotes adhesion between endothelium-mononuclear cellsThe results showed that the number of adherent mononuclear cells in the EPO group increased significantly compared with the control group.19.Effect of EPO on expression of inflammatory cytokines in macrophagesDirect EPO stimulation did not up-regulate the expression of inflammatory cytokines in macrophages.After 24 hours of solvent control or EPO stimulation of HUVEC,a cell culture medium was used to stimulate macrophages.The results showed that IL-6,IL-1? and TNF-? levels increased significantly at 6 hours and MCP-1 levels increased significantly at 8 hours,suggesting that ECs involvement is a key link in the up-regulation of EPO-induced inflammatory cytokines in macrophages.20.Effect of EPO on MMPs expression in macrophagesAfter 24h stimulation of macrophages by vehicle or EPO,there was no significant difference in MMP2 and MMP9 protein expression and enzyme activity.After 24 hours of vehicle or EPO stimulation of HUVEC,the cell culture medium was used to stimulate macrophages.The results showed that the protein expression and enzyme activity of MMP2 and MMP9 in the two groups were still not significantly different.These results suggested that EPO did not affect the expression of MMPs in macrophages.21.Effect of EPO on expression of collagen,MMPs and apoptosis-related proteins in SMCsThe expressions of collagen ?,collagen ?,MMP2 and MMP9 did not change significantly after HASMC being stimulated by EPO with concentration gradient.There was no significant difference in HASMC expression of BCL2 and BAX between EPO group and control group.After 24 hours of vehicle control or EPO stimulation of HUVEC,the cell culture medium was used to stimulate the SMCs.The results showed that the expression of collagen ? and collagen ? of HASMC in the EPO group was significantly decreased,while the expression and enzyme activity of MMP2 and MMP9 were significantly increased.The expression of anti-apoptotic protein BCL2 in the EPO group was significantly less than that in the control group,while the expression of pro-apoptotic protein BAX was significantly more than that in the control group.22.Effect of EPO on proliferation and apoptosis of SMCsThe results showed that the number of HASMC proliferation and apoptosis induced by direct EPO stimulation was not significantly different from that of the control group.After 24 hours of vehicle control or EPO stimulation of HUVEC,HASMC was stimulated in cell culture medium.The results showed that the proportion of TUNEL positive cells in EPO group was significantly higher than that in the control group,while the proportion of EDU positive cells was significantly lower than that in the control group.23.EPO promotes angiogenesis and inflammatory infiltration in vivoThe Matrigel plugs were taken from the mice after 14 days and showed clear yellow color of the vehicle group,while red color in Matrigel plugs containing EPO,indicating the formation of blood vessels in the Matrigel plugs.H&E staining results showed that the number of cells and blood vessels invaded in EPO group significantly.The number of CD 144 and Ki67 positive cells in the EPO group was significantly higher than that in the control group.Moreover,the expression of CD68 positive cells and inflammatory factors in the EPO group was significantly higher than that in the control group.24.EPO induces angiogenesis via JAK2/STAT5 pathwayThe results showed that EPO significantly enhanced the phosphorylation levels of JAK2 and STAT5,while JAK2 inhibitor significantly inhibited the phosphorylation levels of JAK2 and STAT5.The STAT5 inhibitor only inhibited the phosphorylation level of STAT5,but did not affect the phosphorylation level of JAK2.The addition of JAK2 inhibitor and STAT5 inhibitor significantly inhibited the formation of closed tubular structures in ECs.According to the above results,EPO promotes ECs neovascularization through JAK2/STAT5 pathway.25.Mechanisms by which EPO induces the formation and development of AAAThrough the above in vivo in vitro experiments,we concluded the EPO-induced AAA mechanism as follows:EPO activates the JAK2/STAT5 pathway in vascular endothelial cells,promotes angiogenesis,MMPs secretion,SMCs apoptosis,inhibits SMCs in the synthesis of collagen,enhances inflammatory cells and inflammatory cytokines release,leading to the formation and development of AAA.Conclusions1.EPO dose-dependently promoted the formation of AAA in both ApoE-/-mice and wild-type mice.The AAA incidence of ApoE-/-mice caused by EPO was similar to that of Ang ?,while the AAA incidence of wild-type mice caused by EPO was significantly higher than that of Ang ?.2.EPO promoted the formation of AAA through(EPOR)2 homodimer;3.EPO leads to the formation of AAA by causing angiogenesis,inflammatory response and collagen degradation;4.EPO induces macrophage expressing inflammatory factors through endothelial cell mediation;EPO inhibits collagen secretion of HASMC,promotes HASMC apoptosis and inhibits HASMC proliferation through endothelial cell mediation.Background Abdominal aortic aneurysm(AAA)has been traditionally considered as a result of atherosclerosis because it is always associated with severe atherosclerotic injury to the aortic wall.This traditional view has been increasingly challenged in recent years.The OR values of AAA in patients with coronary heart disease,peripheral vascular disease,carotid artery disease and cerebrovascular disease were 1.72,1.59,1.51 and 1.18 respectively,compared with those without atherosclerotic cardiovascular disease.In the Tromse study of 6446 patients undergoing carotid,femoral and abdominal aorta ultrasound scanning,there was no dose-effect relationship between plaque load and the incidence of AAA,suggesting that the relation between atherosclerosis and AAA may be an association rather than causality.Diabetes is a significant risk factor for atherosclerosis,but paradoxically it is a protective factor for AAA.In the AAA epidemiological study of 3.1 million people in the United States,compared with non?diabetic patients,the OR value of AAA in diabetic patients was 0.75,indicating that diabetes can reduce the risk of AAA by 25%.This paradox again suggests that atherosclerosis and AAA may be two independent diseases.There is a strong clinical association between smoking and the occurrence of AAA.The risk of AAA among smokers was more than four times higher than among lifelong nonsmokers.A report comparing the relative risks of different diseases among chronic smokers found that the risk of AAA was three times higher than the risk of coronary artery disease and nearly five times higher than the risk of cerebrovascular disease.In 2011,ultrasound examinations of 26256 older men over 65 years old in Sweden showed that the incidence of AAA had dropped to 2.2%.The main reason may be the reduction in smocking populations.Thus,smoking control may be an effective strategy to prevent AAA.Based on these clinical observations,chronic smoking may be the most important environmental risk factor for the development of AAA.In addition to smoking,other risk factors include gender,age,hypertension,chronic obstructive pulmonary disease,hyperlipidemia,and family history.Clarification of the mechanisms underlying the development and progression of AAA is dependent on a reliable animal model of AAA.Currently,angiotensin ? injection model is the most commonly used animal model of AAA.In high-fat fed ApoE-/- or LDLR-/-mice,subcutaneous inflision pumps are implanted,and a large dose of angiotensin ? is continuously injected,which can lead to the development of AAA.Therefore,the cellular and molecular mechanisms underlying the development and progression of AAA are derived mainly from studies on this traditional animal model.Up to now,several mechanisms of AAA have been proposed: Oxidative stress : Some risk factors of AAA such as smoking,and hypertension,and angiotensin ? can induce increased pro-oxidant,whereas antioxidants are decreased.As a result,ROS level and oxidative stress are increased.ACE expression is stimulated and endogenous angiotensin ? increases.Therefore,inflammatory reaction is enhanced.Activation of renin-angiotensin ? system : In mice with a normal blood lipid level and hyperlipidemia,continuous drip angiotensin ? causes inflammation of the abdominal aorta,and then elastin degradation,medial rupture and lumen dilation.These effects are mediated by ATI receptor,as administration of angiotensin-converting enzyme inhibitors,angiotensin receptor antagonists,and angiotensin-converting enzyme 2 overexpression may alleviate AAA lesions in mice by reducing the production and action of angiotensin ?.AMPKa2 activation : Nicotine and angiotensin ? activated G protein coupled receptor,which is located on the surface of cell membrane,increased intracellular level of reactive oxygen species,which can activate AMPK and promote AMPKa2/AP-2a/MMP2 cascade reaction.The gene transcription of MMP2 was activated and ultimately leads to accelerated degradation of the extracellular matrix in the blood vessel wall and initiation of AAA.Collagen metabolism: Inflanunatory factors not only increased the degradation of extracellular matrix by stimulating the expression of MMPs,but also inhibited the synthesis of collagen,which accelerated the formation of AAA.Since AAA is a multifactorial disease with unknown etiology,prolonged course,disease progression,limited treatment,and sinister prognosis,and there is no successful intervention target in clinical trials,we are still in the early stage of the research on the mechanism of the occurrence and development of AAA.Ang ? promotes the proliferation of specific hematopoietic cell lines and involved in the regulation of EPO in vivo.Clinical studies on healthy volunteers have shown that Ang ? increases serum EPO concentrations by approximately 35% or more via AT1 R.In addition,in another study of healthy volunteers,captopril and enalapril significantly reduced plasma EPO levels by 20-30%.These findings suggest that Ang ? regulates EPO production through receptor-dependent signaling in vivo,but whether EPO mediates the biological role of Ang ? in vivo and AAA production has not been investigated.Combined with the conclusions ?rom part I and part ? that EPO could induce AAA,we speculate that EPO mediates Ang ?-induced AAA.Objective1.To explore the role of EPO/EPOR signaling pathway in Ang ?-induced AAA;2.To explore the reasons for the low incidence of Ang ?-induced AAA in wild type mice;3.To explore the molecular mechanism of EPO regulation by Ang ?.Methods1.Animal models grouping(1)60 male ApoE-/- mice were randomly divided into 4 groups: the vehicle group received continuous subcutaneous infusion of saline via an osmotic pump and caudal vein injection of saline,the Ang ? group received continuous subcutaneous infusion of Ang ? via an osmotic pump and caudal vein injection of saline,the Ang ? + IgG2a group received continuous subcutaneous in... |
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