Bone Marrow Mesenchymal Stem Cells Overexpressing Klotho Suppress The Development Of Abdominal Aortic Aneurysm

Objective:In this study,Secreted Klotho(sKL)was transfected into bone marrow mesenchymal stem cells(BMSCs),and BMSCs overexpressing sKL were transplanted into established abdominal aortic aneurysm(AAA)model by intravenous injection.Cell culture,virus preparation,histomorphology and Western blot were used to observe the changes of abdominal aortic wall and the expression of proteins related to aging,inflammation and oxidative stress.This study aims to investigate function of BMSCs overexpressing sKL in the repair of AAA and related mechanisms,which may provide a new theoretical basis for the future treatment of AAA by BMSCs.Methods:1.BMSCs were cultured in vitro and were divided into three groups:N group,GFP group and sKL group.N group was regarded as control group without any treatment.GFP group and sKL group were respectively infected with GFP empty recombinant lentivirus and Klotho gene lentiviral expression vector.The infection efficiency was observed by fluorescence microscopy.The expression of sKL,P21 and P53 in cultured cells and sKL in cultured medium were quantified by western blot.2.Wistar rats were randomly divided into five groups:N group,AAA group,BMSCs group,GFP group,sKL group.N group was regarded as control group without any treatment;The AAA group was not treated after the AAA model was established;BMSCs group,GFP group,sKL group was received intravenous injection of BMSCs,BMSCs with GFP,BMSCs overexpressing sKL after the AAA model was established,respectively.BMSCs(1x10~6)were injected once a week.Four weeks later,serum and abdominal aortic were taken for subsequent experiments.3.The size of the aortic diameter was measured.The fluorescence microscope was used to observe whether BMSCs overexpressing sKL infiltrated the arterial lesion area.The changes of arterial wall,morphology of elastic plates and collagen fibers were observed by HE staining,Orcein staining and Masson staining,respectively.?-Gal staining was performed to localize senescent cells.Localization and semi-quantitative analysis of SIRT1,CD45,CD68,and?-SMA in each group of arterial tissues were performed by immunohistochemistry.Western blot was used to analyze the expression of sKL in serum and SIRT1,P21,P53,NF-?B,P-AMPK?,AMPK?,MMP-2 and MMP-9 in arterial tissues.Results:1.The infection efficiency was observed under fluorescent microscope.Western blot was used to detect the expression of sKL in BMSCs and the medium.The expression of sKL in sKL group was significantly higher than the N group and GFP group.MTT results showed that sKL had no significant effect on the proliferation of BMSCs.P53 and P21 in BMSCs,aging markers,were detected by Western blot.The results showed that sKL could decreased the expression of P53 and P21 in BMSCs.2.Four weeks after operation,the the aorta tissues were collected.The results of measurement about the size of the arterial diameter showed a significant expansion and serious calcification in the AAA group compared with the N group.In addition,the MSC group,the GFP group,and the sKL group were significantly relieved compared with the AAA group,and the sKL group obtained a better therapeutic effect.HE staining was used to observe the morphology change of the abdominal aorta wall.The diameter of the aorta in AAA group was larger than that of the N group accompany the thiner aortic wall and the rupture of the media.The treatment group had a great improvement compared with the AAA group.The sKL group had the best effect.Orcein staining showed that the fragmentation of the elastic fibers in the AAA group was obvious,and the sKL group significantly inhibited the elastin fragmentation.The results of Masson staining showed that the number of smooth muscle cells in the aortic wall of the AAA group was significantly reduced,and the collagen fibers in media were significantly increased.The number of smooth muscle cells in the sKL group was significantly increased than that in AAA group.3.The sKL level in serum was detected by Western blot.The expression of sKL in AAA group was significantly lower than that in N group,and the treatment group was significantly higher than that in AAA group.4.The results of immunohistochemistry and Western blot showed that SIRT1 was mainly located in vascular media.SIRT1 expression decreased significantly in the AAA group and increased in treatment group.The level of SIRT1 in the sKL group was increased significantly compared with that in the AAA group.5.The results of?-Gal senescence cell staining showed that there were a large number of senescent cells in the AAA group,and the senescent cells in the treatment group were reduced,the sKL group significantly reduced cell senescence.The results of immunofluorescence showed that the number of smooth muscle cells in the AAA group was decreased compared with the N group,and MSC group and the GFP group was higher than that in the AAA group,the sKL group is close to the number of N group.Western Blot was used to detect the expression of P53 and P21 in AAA tissues.The results showed that P53 and P21 were highly expressed in AAA group and gradually decreased in the treatment group,sKL group obviously decreased compared with the AAA group.6.The expression of CD45 and CD68 were detected by immunohistochemistry.The results showed that the positive cells were mainly located in adventitia.Adventitia in AAA group was infiltrated by numerous inflammatory cells,and the number of inflammatory cells was decreased after treatment.Its number was the least in the sKL group.The results of Western blot showed that the expression of NF-?B was a small amount in the N group compared with in the AAA group.The expression of NF-?B was decreased in the treatment group and the trend of decline in the sKL group was the most obvious.7.The results of DHE staining showed that there was a large amount of ROS level in the AAA group,and was gradually decreased in the treatment group.The ROS level in the sKL group was close to the N group.Western blot was used to detect the expression of P-AMPK?and AMPK?.The results showed that the P-AMPK?level in the AAA group was lower than that in the N group.However,P-AMPK?expression in the treatment group was relatively increased,and it was significantly increased in the sKL group.8.Western blot was used to detect the expression of MMP-2 and MMP-9.The results showed that the MMP-2 and MMP-9 in the AAA group was higher compared with the AAA group,meanwhile,the treatment group was decreased,and the decreased level in the sKL group was most obvious.Conclusions:BMSCs overexpressing sKL have the function of inhibiting the pathological expansion of AAA,which may be achieved by inhibiting cell senescence,inflammatory response and oxidative stress in aortic wall..