AEBP1 Promotes The Occurrence And Development Of Abdominal Aortic Aneurysm By Modulating Inflammation Via NF-?B Pathway

Objective: Abdominal aortic aneurysm(AAA)is a vascular disease characterized by permanent localized full-thickness dilation of the abdominal aorta,with high incidenecs of morbidity and mortality.It is common in elderly people over 60 years old.With the aging of the global population,the incidence of AAA has increased 7-fold in the past 30 years.There are no special symptoms in the early stage of AAA.As the aneurysm progresses,once the rupture occurs,the emergency mortality rate is as high as 90%,which seriously threatens the health of the middle-aged and elderly people.The typical pathological signatures of AAA include inflammatory infiltration in the adventitia and tunica media,apoptosis of middle smooth muscle cells,aortic elastin proteolytic degradation,deposition of collagen fibers and pathological remodeling.Infiltration of chronic inflammatory cells has been considered as the primary factor of AAA,during which,a large number of inflammatory cells gardually infiltrate into the tissues from adventitia to intima,secrete various of inflammatory cytokines and proteolytic enzymes and simulate vascular smooth muscle cells to secrete matrix metalloproteinases(MMPs)to degrade elastic fibers.Our proteomic microarray results showed that Adipocyte enhance binding protein 1(AEBP1)was significantly increased in AAA patients compared with Aortic Atherosclerotic Acclusive Disease(AOD)patients and Healthy Control(HC)individuals.AEBP1 was a ubiquitous protein that is highly expressed in the extracellular matrix rich tissues,such as preadipose tissue,vascular wall,skin and other organs.AEBP1 was a transcriptional repressor that interact with the adipogensis aP2 gene activity which was first identified during adipocyte differentiation.Recently,AEBP1 was identified as a novel proinflammatory mediator in macrophages,involved in macrophage cholesterol homeostasis and inflammatory responsiveness.More recently,Majdalawieh et al.reported that AEBP1 manifests its pro-inflammatory effects by promoting NF-?B activity via impeding the inhibitory function of I?B? in macrophages,which is likely dependent on AEBP1-I?B? physical interaction.In canonical NF-?B pathway,the various stimuli that activate NF-kB cause phosphorylation of I?B?,which is followed by its ubiquitination and degradation.Subsequently,P-p65 translocates from the molecule tothe nucleus and binds with ?B sequence of various genes thereby activating their transcription.NF-?B pathway can promote chronic inflammation of vascular walls and regulate the transcription of MMPs.Studies using animal experiments have shown that inhibiting the activity of NF-?B can delay the occurrence and development of AAA.At present,there is no effective treatment for asymptomatic and aortic diameter less than5.5cm patients,and who can not tolerate surgery due to various factors.Gene therapy should be one of the main treatment methods,and the relationship between AEBP1 and AAA remains unknown.In the present study,we clarified the exact relationship between AEBP1 and AAA and its molecular mechanism through AAA clinical specimens,in vivo AAA animal models and in vitro cell experiments,aiming to provide a new pathogenesis of AAA,and to provide new targets for AAA gene therapy.Methods: 1.Serum and clinical tissue specimens are strictly in accordance with the inclusion and exclusion criteria,from our hospital AAA patients,AOD patients and HC individuals.Three groups of selected clinical tissue samples were subjected to ITRAQ for differentially expressed proteins,and results were selected from AEBP1.To expand the reliability of the selected AEBP1 gene,clinical samples were selected for ELISA,Real time PCR,western blot,immunohistochemistry,and immunofluorescence to detect the expression of AEBP1 and the activation of NF-?B pathway.2.The AAA model was established by porcine pancreatic elastase perfusion method,and the expression of AEBP1 gene was silenced by intra-adventitial injection of adenovirus.55 SD rats were randomly divided into sham group(only operation n=10),control group(only elastase perfusion n=25),con+AV group(elastase perfusion+empty adenovirus n=10)and con+shAEBP1 group(elastase perfusion+adenovirus with AEBP1 shRNA n=10);The diameter of the arteries after modeling was detected by vernier calipers.The pathological changes of arteries were detected by HE,EVG and Masson staining to confirm the successful modeling.The expression of AEBP1 and the activation of NF-?B pathway and aneurysm associated inflammatory factors TNF?,IL-10,IL-6,IL-1? and expression of MMP-2,MMP-9 were detected by western blot and immunofluorescence technique.3.Immunofluorescence co-localization was used to locate the expression of AEBP1 inAAA tissue samples.Vascular smooth muscle cells(VSMC)were selected according to the results.Lentiviral transfection was used to interfere with AEBP1 gene silence and overexpression,and NF-?B pathway inhibitor BAY11-7082 was used to inhibit NF-?B pathway activity.Based on this,we grouped the cell experiments as follows: blank group(Blank),negative control group(NC),AEBP1 silenced group(Sh),AEBP1 overexpression group(Over),and each group added NF-?B inhibitor group: Blank+BAY group,NC+BAY group,Sh+BAY group,and Over+BAY group.qRT-PCR and western blot were used to detect the transfection efficiency of AEBP1.ELISA detected the expression of inflammatory factors TNF?,IL-10,IL-6 and IL-1? in cell culture supermatants of each group.The expression of AEBP1 and the activation of NF-?B pathway and aneurysm associated inflammatory factors TNF?,IL-10,IL-6,IL-1? and expression of MMP-2,MMP-9 were detected by western blot technique.Results: 1.Proteomic microarray detection of differentially proteins showed that the expression of AEBP1 was significantly upregulated in AAA patients compared with AOD patients and HC individuals.qRT-PCR showed that AEBP1 is significantly upregulated in AAA patients.Western blot and immunohistochemistry(IHC)results showed the same trend as those from qRT-PCR.Serum samples from AAA,AOD and HC groups were collected for ELISA,and the circulationg level of AEBP1 was found to be significantly upregulated in AAA patients.Western blot and immunofluorescence results showed that the NF-?B pathway is aberrantly activated in AAA patients compared with AOD patients and HC individuals.2.Animal experiment: compared with the sham group,the aortic diameter of the aneurysm was clearly expanded,the inflammatory cells infiltration increased,elastic fibers in the artery medial layer were badly damaged,and deposition of collagen fibrils in the control group and the con+AV group.The con+shAEBP1 groups can partly alleviated the symptoms.Subsequently,we measured the protein expression of MMP-2 and MMP-9,as well as inflammatory cytokines TNF?,MCP-1,IL-6,and IL-1?,in the vascular wall tissue of rat models.We found that the expression levels of MMPs and inflammatory markers were clearly elevated in the control and con+AV groups,whereas AEBP1 silencing group con+AEBP1 blocked this effect.Immunofluorescence and nuclear protein western blot results showed that the expression of P-p65 into the nucleus was alsoreduced in the con+shAEBP1 group.3.The co-localization result showed that AEBP1 was co-expressed with media layer smooth muscle cell marker SMA in the human AAA vascular wall tissues.AEBP1-sh and AEBP1-Over VSMC were established by lentivirus-mediated AEBP1 overexpression and knockdown.AEBP1 expression was measured by qRT-PCR and western blot analysis in AEBP1-sh,AEBP1-Over,and control VSMC cells,and the AEBP1 expression was found to be significantly upregulated and downregulated in AEBP1-Over and AEBP1-sh VSMCs.The secretion of inflammatory cytokines TNF?,IL-10,IL-6 and IL-1? in cell culture supernatant was measured by ELISA,and the results showed that AEBP1 overexpression and knockdown respectively upregulated and downregulated the secretion of TNF?,IL-10,IL-6 and IL-1?.In contast,the addition of NF-?B inhibitor BAY11-7082 weakened the effect of AEBP1 on inflammatory cytokine secretion.Subsequent measurement of the expression of inflammatory cytokines TNF?,MCP-1,IL-6,and IL-1?,as well as that of MMP-2 and MMP-9 proteins using western blots also revealed results consistent with the previous ELISA results.Nuclear protein western blot also showed that AEBP1 knockdown in VSMC cells decreased the translocation of P-p65 into nucleus,whereas AEBP1 overexpression showed contrasting results.All the expression changes disappeared after the addtion of NF-?B pathway inhibitor BAY11-7082.In VSMCs,we found that AEBP1 binds to I?B? during Co-IP process,indicating that AEBP1 could successfully bind to I?B?,thereby activating the NF-?B pathway.Conclusion: Taken together,the results of our study demonstrate that AEBP1 has a very close correlation with AAA in clinical samples,whereas in mouse models,AEBP1 was shown to promote the occurrence and development of AAA.All these processes were accompanied with the activation of NF-?B pathway.Functional verification revealed that AEBP1 overexpression or knockdown changed the expression of inflammatory cytokines and MMPs,whereas NF-?B pathway inhibitor BAY11-7082 eliminated the effect of AEBP1,suggesting that AEBP1 promotes the development of AAA by regulating the NF-?B pathway.