Expression And Mechanism Of Cold-inducible RNA Binding Protein In Abdominal Aortic Aneurysm

Background and Objective:Abdominal aortic aneurysm(AAA)is a fairly common condition in the elderly population,with prevalence estimates ranging from 5%-10%.It is a significant medical problem with an associated mortality rate due to rupture that is as high as 90%.With the arrival of the elderly population in our country.the incidence of AAA is increasing every year,and AAA has a high mortality rate,which has become a serious threat to the health of the elderly population.The rupture of AAA is a devastating event with high mortality potentially preventable with therapies that inhibit the growth of small aneurysms.Although open or endovascular surgical therapies are recommended for large AAA(diameter?5.5cm).the repair of small aneurysms(diameter<5.5 cm)does not provide a significant benefit.The intently clinical follow up is recommended for small abdominal aortic aneurysms(sAAA).But,previous studies have shown that more than 60%of sAAA need open or endovascular surgical therapies due to presenting symptom of rupture.Therefore,effective pharmacological therapy is considered to be a good option for small abdominal aortic aneurysms(sAAA).Therapeutic strategies for altering the course of AAA must be aimed at the underlying events that promote AAA development.Despite an improved understanding of the pathophysiology of AAA.physicians remain incapable of modifying the natural history of AAA development.Therefore,the identification of novel targets for the development of therapeutic interventions to treat this life-threatening vascular disease remains a subject of intensive research.Now,the pathophysiology of AAA has not been fully elucidated.Recent research have found that the pathological changes of AAA wall include apoptosis of vascular smooth muscle cells,accumulation of inflammatory cells,degradation and remodeling of extracellular matrix(ECM)and so on.It is well known that chronic inflammation of the aortic wall is a major characteristic of AAA that has been implicated in its development,expansion,and rupture.The inflammatory cells in the aortic wall can secrete TNF-a,IL-?,IL-6,MCP-1 and other inflammatory cytokines which cause aortic wall damage.Simultaneously,these inflammatory cytokines can recruit more inflammatory cells into the aortic wall and then aggravate the aortic wall damage.Inflammatory cells in the media and adventitia are also significant sources of cytokines that stimulate proteolytic enzymes,such as matrix metalloproteinases(MMPs).MMPs mediate the fragmentation of ECM elastin fibers,reduce the concentration of these fibers in the ECM,and are considered important mediators of the development of aneurysms.So,chronic inflammation of the aortic wall is a important aspect of AAA development.Cold-inducible RNA binding protein(CIRP)is an 18-kDa protein that belongs to the f'amily of cold shock proteins.CIRP is widely expressed at low levels in various tissues and is upregulated in response to a wide variety of cellular stressors which suggests that it is a general stress-responsive protein.CIRP was originally identified as a facilitator of translation that stabilizes mRNAs in cells under certain stressful conditions,including hypothermia,exposure to ultraviolet irradiation.H2O2 and hypoxia.However,increasing numbers of studies have indicated that CIRP displays proinflammatory cytokine-like properties in CIRP-associated diseases.such as hemorrhagic and septic shock,liver ischemia/reperfusion(IR)injury,brain IR injury.CIRP,act as a novel proinflammatory cytokine,can directly upregulate expression of TNF-a and IL-6 and then induce organ injury.Previous studies have demonstrated that CIRP participated the inflammatory response of heart,brain,liver.kidney,lung,intestine and skin.Additionally,anti-CIRP-based therapy has exhibited beneficial effects in diseases involving CIRP that are mediated by the attenuation of the inflammatory response.But the expression of CIRP and its role during aneurysmal aortic wall inflammation have not been clarified.In this study,we will examined the expression levels of CIRP in the sera and aortic tissues of AAA and then evaluated whether the blockade of CIRP attenuates aneurysm formation.The AAA model was induced by intra-aortic elastase infusion as described previously.In brief,under intraperitoneal anesthesia,the abdominal aorta was isolated from the level of the left renal vein to the iliac bifurcation via a median laparotomy in sterile conditions.Subsequently,the left iliac artery was exposed,and a PE-10 polyethylene tube was introduced through the iliac artery to the distal aorta.The aorta was clamped below the renal artery and above the tip of the PE tube and then ligated with a 4-0 silk suture near the aortic bifurcation,which was followed with the infusion with type I porcine pancreatic elastase.After infusion,the aortic walls immediately exhibited substantial expansion and then exhibited degradation of elastic lamellae and infiltration of inflammatory cell(macrophage,T cells,B cells ect.).This AAA model is widely accepted and very stable.So,we will choose this AAA model in our study of CIRP research.In this study,we will investigate the expression levels of CIRP in human AAA,and the correlation of CIRP with aortic inflammation.And we will also investigate the role of CIRP in aneurysm formation in the rat AAA model.This study will help clarify the role of aortic inflammation in the pathophysiology of AAA,and provide experimental support and clues for treatment of AAA.Content:Part I:We investigated CIRP expression in the sera and aneurysmal tissues of human AAA patients.Part II:We investigated CIRP expression in the sera and aneurysmal tissues of elastase-induced AAA rats.To further examine the role of CIRP in the development of AAA,anti-CIRP antibody or non-immunized control IgG was injected via the caudal vein in the experimental AAA model.Part?:We did further investigation about the underlying mechanisms of CIRP in the development of AAA.Methods:Part ?:1.Human blood samples were collected from AAA patients who were admitted to the Department of Vascular Surgery and/or healthy volunteers(age>50 years).Human AAA specimens were obtained from patients undergoing surgical AAA repair by members of the Department of Vascular Surgery of the Shandong Provincial Hospital affiliated to Shandong University in Jan,2014—Jun,2015.As a control,normal human aorta samples were obtained from multiorgan donors(age>50 years).None of the donors were known to have connective tissue disorders.2.The level of serum CIRP were measured via ELISA and Westcrn blotting.And the level of aneurysmal CIRP were measured via RT-PCR?Western blot?immunohistochemical and immunofluorescence staining.3.Spearson correlation analysis was used to evaluate the correlation of serum CIRP and aneurysmal proinflammatory cytokines.Part ?:1.Male Wistar rats were used in this study and were randomly divided into Sham and AAA groups.The AAA model rats were induced by intra-aortic elastase infusion as described previously.In brief,under intraperitoneal anesthesia with 10%chloral hydrate,the abdominal aorta was isolated from the level of the left renal vein to the iliac bifurcation via a median laparotomy in sterile conditions.Subsequently,the aorta was clamped below the renal artery and above the tip of the PE tube and then ligated with a 4-0 silk suture near the aortic bifurcation,which was followed with the infusion with type I porcine pancreatic elastase.The maximum inner luminal diameters of the abdominal aortas were measured by ultrasound after the operations.HE and EVG staining were did in the 4th week after the operations.2.The level of serum CIRP were measured via Western blotting.And the level of aneurysmal CIRP were measured via RT-PCR.Western blot?immunohistochemical and immunofluorescence staining.3.In the CIRP blockade study,the AAA rats were randomly divided into vehicle-treated(Ig G)and anti-CIRP antibody-treated(anti-CIRP)groups:Non-immunized control IgG or anti-CIRP antibody was injected via the caudal vein 24 h or 2 weeks after the operation and subsequently every 3 days for 4 weeks.4.The changes of aortic wall were measured by HE staining?EVG staining?RT-PCR?Western blot and gelatin zymography after anti-CIRP antibody treatment.Part?:1.The changes of TLR4/NF-?B signal pathway and inflammation response of aortic wall were measured by RT-PCR and Western blot after anti-CIRP antibody treatment.2.For the cell culture experiments,the RAW 267.4 cells were randomly divided into Control,rmCIRP and rmCIRP + anti-CIRP groups.3.The level of MMP-9 were measured via RT-PCR and Western blot after rmCIRP stimulation.4.The activity of MMP-9 were measured via gelatin zymography after rmCIRP stimulation.5.The migration of RAW 264.7 cells were measured via transwell migration assay after rmCIRP stimulation.6.The TLR4/NF-?B signal pathway were measured via RT-PCR and Western blot after rmCIRP stimulation.Result:Part ?:1.The characteristic of aortic aneurysmal wall:HE staining revealed that the aortic walls exhibited inflammatory cell infiltration.And EVG staining revealed that the elastic lamellae were disrupted in the AAA tissue in contrast to the integrated wavy appearance observed in the normal aortic tissue.The RT-PCR showed that TNF-??HMGB1 and TLR4 expression was elevated in the human AAA tissue compared with the normal aortic tissue.The RT-PCR and Western blot showed that MMP-2/9 expressions were both elevated in the human AAA tissue.And the gelatin zymography showed that the activities of MMP-2/9 were elevated in the human AAA tissue.2.CIRP expression in human AAA:The result of ELISA and Western blot revealed that serum CIRP was elevated in the human AAA compared with the health people.The RT-PCR and Western blot revealed that CIRP expression was elevated in the human AAA tissue compared with the normal aortic tissue.The immunohistochemical staining showed that CIRP was expressed in nucleus?cytoplasm and extracellular.Double immunostaining showed that CIRP was primarily found in the a-SMA-labeled and CD68-labeled cells.3.The correlation of CIRP and aortic wall inflammation:Spearson correlation analysis showed that the concentration of serum CIRP was positively correlated with the expression of aneurysmal TNF-a(P=0.038 R=0.736).Part II:1.Rat AAA model:In our study.elastase-induced AAA were observed at the macroscopic and morphological levels in the 4th postoperative week.The ultrasound system showed that the maximum aortic diameter of AAA group expanded more than 50%of the aortic diameter in the 4th week after operation.Histopathological analysis revealed that the aortic walls stained with HE exhibited substantial expansion and inflammatory cell infiltration in the AAA group.IEVG staining revealed that the elastic lamellae were disrupted in the AAA group as demonstrated by the appearance of elastin fragmentation in contrast to the integrated wavy appearance observed in the sham group.2.CIRP expression in the rat AAA model:The Western blot revealed that serum CIRP were elevated in all of the AAA mice.The RT-PCR and Western blot revealed that CIRP expression was enhanced in the aortas of the elastase-infused mice compared with the aortic tissues of the mice infused with saline.The immunohistochemical staining showed that CIRP was expressed in nucleus?cytoplasm and extracellular.Simultaneously,double immunostaining showed that CIRP was primarily found in the a-SMA-labeled and CD68-labeled cells.3.The treatment of anti-CIRP antibody on the rat AAA model:In the 4th week after antibody treatment,the maximum aortic diameter of the anti-CIRP group,as measured with an ultrasound system,was significantly diminished compared with that of the IgG group.The EVG-stained elastic lamellae of the anti-CIRP group exhibited partial recovery of the wavy structures compared with the elastin fragmentation observed in the IgG group.Moreover,treatment with anti-CIRP antibody significantly reduced the MMP-2 and MMP-9 levels in the aneurysmal tissue.Simultaneously,the activities of MMP-2 and MMP-9 in the aneurysmal tissue were significantly attenuated in the anti-CIRP group compared with those observed in the IgG group.Part?:1.The changes of TLR4/NF-?B signal pathway and inflammation of aortic wall:In the 4th week after antibody treatment,treatment with anti-CIRP antibody significantly reduced TLR4 and nuclear NF-?B levels in the aneurysmal tissue.Simultaneously,the expression of TNF-a in the aneurysmal tissue were significantly attenuated in the anti-CIRP group compared with that observed in the IgG group.CD68 staining revealed that the macrophages infiltration into the aortic wall was attenuated in the anti-CIRP group compared with the IgG group.Simultaneously,RT-PCR and Western blot revealed that the neutralizing anti-CIRP antibody treatment also significantly reduced the mRNA and protein expression of MCP-1 in the aneurysmal tissue.2.The rmCIRP stimulation on the migration of RAW 264.7 cells:The transwell migration assay results revealed that rmCIRP significantly promoted RAW 264.7 cell migration,and this effect was prevented by the anti-CIRP antibody.3.The rmCIRP stimulation on TLR4/NF-?B signal pathway of RAW 264.7 cells:RT-PCR and Western blot revealed that rmCIRP significantly increased TLR4 mRNA and protein expression.Simultaneously,Western blot revealed that rmCIRP significantly increased nucleus NF-?B expression which indicated that rmCIRP stimulated cytoplasm NF-?B transferred into the nucleus.4.The rmCIRP stimulation on the expression and activity of MMP-9 of RAW 264.7 cells:RT-PCR revealed that rmCIRP significantly increased MMP-9 mRNA expression in a dose-dependent manner.However,Western blot and gelatin zymography revealed that its expression and activity were inhibited by the anti-CIRP antibody.Conclusion:1.CIRP is highly expressed in serum and aortic tissue of human AAA.Simultaneously,the concentration of serum CIRP was positively correlated with the expression of aneurysmal TNF-?.2.The anti-CIRP antibody treatment suppressed the rat AAA development:CIRP mediated experimental AAA development.3.CIRP mediated AAA development through promoting the inflammatory cell infiltrate into the aortic wall and activate the aortic TLR4/NF-?B signal pathway.