IGFBP3 Improves The Development Of Type A Acute Aortic Dissection By Regulating The P53 Pathway

Type A acute aortic dissection（TAAAD）is a severe aortic disease with low morbidity and high mortality.Pathological changes of TAAAD mainly include degradation of extracellular matrix（ECM）,apoptosis of vascular smooth muscle cell（VSMC）and structural disorder of ECM,Apoptosis of VSMC is the focus in the study of aortic dissection.However,the involvement of IGFBP3 in the pathogenesis of TAAAD and related mechanisms need to be further explored.P53 induces apoptosis of arterial smooth muscle.We hypothesized that IGFBP3 may promote the apoptosis of VSMC through P53.Part1.IGFBP3 and its downstream protein expression in the arterial wall of TAAADObjective:To test the expression of IGFPB3 and its downstream protein in the aortic dissection tissue of TAAADMethods:From September 2019 to January 2021,we collected aortic tissue of patients underwent surgery in the First Affiliated Hospital of China Medical University.The study was approved by the Ethics Committee of the First Affiliated Hospital of China Medical University,and all patients signed informed consent.12 aortic specimens were taken from the punctured aortic tissue of coronary artery bypass surgery,without obvious atherosclerosis changes.The aortic tissue of 24 patients with acute type A aortic dissection were enrolled as TAAAD group.Samples were collected and stored in 4%paraformaldehyde or liquid nitrogen respectively.The mRNA expression of IGFBP3 was measured by quantitative real-time polymerase chain reaction（qRT-PCR）,and the expressions of IGFBP3,p53,Bax,Bcl-2 and Caspase3-3 proteins were measured by western blot.HE staining and Masson staining were used for pathological evaluation.The expression of IGFBP3 in aorta was investigated by immunohistochemistry.Results:The mRNA expression of IGFBP3 in aortic dissection was higher than that of control group（P<0.05）,the expression of IGFBP3,p53,Bax and Caspase3 was higher than that of control group（P<0.05）,and the expression of Bcl-2 was lower than that of control group（P<0.05）.By HE staining and Masson staining,it can be observed that the vascular wall structure of human aortic dissection changed obviously,its integrity was seriously damaged,red blood cells infiltrated into the media space,smooth muscle cells were lost,and elastic fibers were disordered.Immunohistochemistry showed that IGFBP3 expression in aortic dissection was higher than that in control group（P<0.05）.Conclusion:The expression of IGFBP 3 and p53 in aortic tissue of patients with aortic dissection is higher than that of non-aortic dissection patients in control group,suggesting that IGFBP 3 and p53 may be involved in the occurrence and development of aortic dissection.Part 2.IGFBP3 promotes apoptosis in VSMC by regulating p53 and its downstream pathway.Objective:To clarify whether IGFBP3 can promote apoptosis in VSMC by regulating p53 and its downstream pathway.Methods:Human aortic smooth muscle cells（HVSMC）were cultured:VSMC was stimulated by hydrogen peroxide with five concentrations of 1x10^-5mol/L,5x10^-5mol/L,1x10^-6mol/L,1x10^-7mol/L and 1x10^-8mol/L.Transfect plasmid vector IGFBP3 or si IGFBP3 to up-regulate or down-regulate the expression of IGFBP3,and observe the expression of p53,Bax and Bcl-2 protein downstream.Transfected si p53to observe the expression of p53,Bax and Bcl-2 protein downstream.Tunel staining was used to detect apoptosis.Results:After H₂O₂treatment,HVSMC showed obvious apoptosis phenotype,and with the increase of H₂O₂concentration,the expression of IGFBP3 also increased（P<0.05）.When H₂O₂concentration was 1x10 5 mol/L,the apoptosis phenotype was the most obvious,and p53（P<0.05）,BAX increased significantly（P<0.05）.After 12h treated by H₂O₂,the RNA of IGF-BP3 changed most significantly（P<0.05）,and the protein level changed most significantly at 36h（P<0.05）.Up-regulation of vector IGFBP3 can increase the expression of p53 and Bax proteins and decrease Bcl2 protein（P<0.05）.Down-regulation of si IGFBP3 can decrease the expression of p53 and Bax proteins and increase Bcl2 protein（P<0.05）.IGFBP3 can be inhibited by si p53.Tunel experiment shows that the apoptosis of VSMC is significant in vector IGFBP3,and si p53 can reduce the apoptosis of VSMC（P<0.05）.Conclusion:IGFBP3 can promote apoptosis in VSMC by regulating p53 and its downstream pathway.Part3.Expression of IGFBP3 in aortic tissue of mouse dissected modelObjective:A mouse aortic dissection model was established and the expression of IGFBP3 in this model was detected.Methods:Three-week-old C57BL/6 male mice were divided into two groups,10 in the control group and 24 in the aortic dissected group.The mice in the aortic dissected group were fed with drinking water containing 0.4%β-aminopropionitrile（BAPN）for 28 days.Then,a micropump was implanted subcutaneously to pump angiotensin Ⅱ（AngⅡ）at a rate of 1000ng/（kg.min）for 72 hours.After the mice were killed in the two groups,the aorta tissue was taken,and the tissue samples were put in 4%paraformaldehyde and liquid nitrogen respectively for HE staining,Masson staining,Immunofluorescent staining（IF）and Western Blot experiments.The expressions of IGFBP3 and p53 protein in aorta of mice in control group and aortic dissected group were detected respectively,and the pathological changes were evaluated.Results:Through autopsy,the dissection of aorta was clearly observed in pathological dissection,indicating that the dissection was successfully modeled,and the success rate of modeling was 89.5%.HE and Masson staining results showed that the aortic wall’s structure in aortic dissected group was severely damaged.The results of Western Blot showed that compared with the control group,the levels of IGFBP3,p53 and Bax were significantly increased（P<0.05）,and the expression of Bcl-2 was decreased（P<0.05）,and the apoptosis was aggravated.In addition,the immunofluorescence results showed that IGFBP3 and p53 were co-located in aortic smooth muscle cells,and there was a spatial relationship between them.Conclusion:It is simple and feasible to establish the mouse aortic dissection model with BAPN and AngⅡ pump,and the success rate is high,which can provide the model basis for the study of the pathogenesis of aortic dissection.The expression of IGFBP3 in the aorta of aortic dissected group mouse model increased significantly,and the expression of p53 and its downstream protein also increased,suggesting that it was involved in the occurrence of aortic dissection.