| Objective:Abdominal aortic aneurysm(AAA)is an aortic dilatation disease caused by irreversible dilatation of the local wall of the abdominal aorta,in which the maximum diameter is more than 50%of the normal segment or the local maximum diameter is more than 3cm.Abdominal aortic aneurysm is a relatively hidden disease.Without intervention,the aneurysm will gradually expand,eventually leading to rupture of the aneurysm,accompanied by a mortality rate of more than 80%.Therefore,the best way to prevent and treat abdominal aortic aneurysm is to detect the disease in the early stage and choose appropriate intervention measures to deal with the aneurysm in time to prevent the risk of aneurysm rupture.Large population-based randomized screening trials abroad have shown that ultrasonic screening of abdominal aortic aneurysms in elderly men is cost-effective and can reduce the overall mortality of abdominal aortic aneurysms.At present,surgical intervention is mainly used for abdominal aortic aneurysms with a diameter larger than5cm,including the traditional open operation(open surgery repair,OSR),namely artificial vascular replacement,and the new endovascular aneurysm repair(EVAR).Studies have shown that the above surgical interventions are effective to prevent the rupture of abdominal aortic aneurysm.Although EVAR has lower perioperative complications,the results of several clinical randomized controlled trials show that EVAR does not have much advantage over OSR in terms of medium-and long-term complications and long-term prognosis.For patients with abdominal aortic aneurysm,there is no effective drug to slow down or even reverse the further expansion of the tumor.Therefore,it is very important to continue to study the pathological mechanisms of abdominal aortic aneurysm,clarify the internal factors of the occurrence and development of abdominal aortic aneurysm,and develop a new type of non-operative treatment.Current evidence shows that the occurrence and development of abdominal aortic aneurysm is the result of a variety of risk factors,including sex,age,genetic susceptibility,hyperlipidemia,atherosclerosis and hypertension.The typical pathological changes of abdominal aortic aneurysm include the infiltration of a large number of inflammatory cells and inflammatory factors such as neutrophils,macrophages and lymphocytes,and the degradation of extracellular matrix such as collagen fibers and elastic fibers.phenotypic transformation and apoptosis of middle vascular smooth muscle cells.Vascular smooth muscle cells(VSMC)are the main cell types that make up the normal tissue structure of vascular wall and maintain the elasticity of vascular wall.VSMC dysfunction is the pathological basis for the occurrence and development of abdominal aortic aneurysm.The contractile phenotype of VSMC is the main form in normal arteries.At this time,VSMC highly expresses proteins related to cell contraction,such as SMA-αand SM-22,which is highly differentiated and maintains the normal contractile elasticity of blood vessels.When VSMC have phenotypic transformation and dysfunction with the progression of the disease,they change from contractile phenotype to synthetic phenotype with the function of proliferation and migration.At this time,cells synthesize and secrete a large number of extracellular matrix and matrix metalloproteinases,and highly express synthetic phenotypic proteins such as OPN.Restoring the expression level of VSMC contractile proteins will contribute to the vascular remodeling process of abdominal aortic aneurysm,maintain the normal elastic retraction ability of vascular wall,improve the arterial tissue structure,and then delay the progression of abdominal aortic aneurysm.Transforming growth factor-β(TGF--β)signaling pathway plays an important role in maintaining vascular integrity and homeostasis,and is a powerful activator of vascular smooth muscle cell differentiation.It can promote the expression of contractile phenotypic markers of vascular smooth muscle cells by activating downstream transcription factors and protect abdominal aortic aneurysms.Weighted gene coexpression network analysis(WGCNA)is a new systematic bioinformatics research method,which is used to describe and characterize the correlation patterns of gene expression profiles among different samples.The WGCNA algorithm can classify the co-expressed genes into gene modules with different color attributes,and then associate the module with the sample traits,and use the module eigengene(ME)or the hub genes in the module to characterize the module features,so as to systematically and deeply mine the key signal pathways and genes potentially related to clinical traits.In recent years,WGCNA algorithm is more and more widely used in nervous system diseases,tumor molecular regulation and so on.This study will deeply analyze the tissue microarray data of abdominal aortic aneurysm by WGCNA method to further clarify the specific mechanism and signal pathway in the occurrence and development of abdominal aortic aneurysm.Mesenchymal stem cells(MSC)are a kind of adult stem cells derived from mesoderm,which have the ability of low immunogenicity,multipotential differentiation and self-renewal and replication.Studies have shown that mesenchymal stem cells can not only secrete a variety of cytokines and exosomes,regulate the inflammatory response of abdominal aortic aneurysm,but also differentiate into functional VSMC,locally to promote local tissue regeneration and repair.Umbilical cord mesenchymal stem cells(UC-MSC)are a kind of mesenchymal stem cells derived from umbilical cord Wharton jelly,which have a wide range of sources,relatively low immunogenicity,can be expanded in vitro,and their stem cell characteristics can be well maintained.Most of the studies on mesenchymal stem cells in the treatment of abdominal aortic aneurysms focus on the mechanism of regulating inflammatory response,but there are no reports on whether mesenchymal stem cells can promote the phenotypic transformation of VSMC.Therefore,the purpose of this study is to clarify the role and mechanism of umbilical cord mesenchymal stem cells in promoting the phenotypic transformation of VSMC in abdominal aortic aneurysms,and to further clarify the possibility of MSC as seed cells in the treatment of abdominal aortic aneurysms and the internal mechanism of promoting VSMC phenotypic transformation,so as to provide a new direction and possibility for the non-operative treatment of abdominal aortic aneurysms.Methods:The main results are as follows:(1)the original microarray data set GSE57691,was used to analyze the gene module significantly related to abdominal aortic aneurysm by WGCNA method,and the genes in the module were further analyzed by biological function enrichment analysis and signal pathway enrichment analysis to identify the signal pathways and main molecules related to abdominal aortic aneurysm.(2)Primary human umbilical cord mesenchymal stem cells were extracted and expanded in vitro.P3and P5 cells were detected,cell morphology was detected under light microscope,growth curve was detected by CCK-8 method,cell cycle was detected by flow cytometry,adipogenic differentiation ability was detected by oil red O staining,osteogenic differentiation ability was detected by alizarin red staining,and chondrogenic differentiation ability was detected by HE staining.Flow cytometry was used to detect the expression of cell surface markers such as CD73,CD90,CD105,CD34,CD45 and HLA-DR.It was confirmed that umbilical cord mesenchymal stem cells had qualified stem cell characteristics between P3 and P5 generations,which could be used in follow-up experiments.(3)the abdominal aortic aneurysm model of SD rats was established by elastase perfusion.The rats were divided into three groups:Control group(saline perfusion,tail vein injection of saline 1mL),Elastase group(elastase perfusion,tail vein injection of saline 1mL),and Elastase+MSCs group(elastase perfusion,1cm 10~6 cells/m L tail vein injection of 1mL).Abdominal ultrasonography was performed on the 0th day,7th day and14th day after the establishment of the model,and the maximum diameter of abdominal aorta and related hemodynamic parameters were measured.After that,elastic fiber staining was used to detect the total amount of elastic fiber and the degree of elastic fiber fracture in each group,and to explore the effect and therapeutic effect of umbilical cord mesenchymal stem cells on the diameter dilatation of abdominal aortic aneurysm.Immunohistochemical method was used to detect the expression of SMA-αand SM-22 in arterial wall,immunofluorescence method was used to detect the expression of SMA-αand SM-22 in arterial wall,and western blot method was used to detect the expression of SMA-α,SM-22,OPN,MMP-2,MMP-9,TGF-β,t-smad2,p-smad2 and TNF-αin arterial wall of each group.To investigate the role of umbilical cord mesenchymal stem cells in promoting the phenotypic transformation of VSMC in abdominal aortic aneurysms and the function of TGF-βsignal pathway in it.(4)the experiment of human primary aortic VSMC,was divided into 6 groups:Control group,Control+LY2157299 group,CM group(conditioned medium derived from umbilical cord mesenchymal stem cells),CM+LY2157299 group,Co-culture group and Co-culture+LY2157299 group.Immunofluorescence and Western Blot methods were used to detect the expression of SMA-α,t-smad2 and p-smad2 in the arterial wall of each group,and to explore the role of umbilical cord mesenchymal stem cells in promoting the phenotypic transformation of VSMC in vitro.To clarify the mechanism of umbilical cord mesenchymal stem cells promoting VSMC epigenetic transformation by secreting TGF-β.Results:The main results are as follows:(1)A total of 13 gene modules were identified in the GSE57691 microarray data set of WGCNA analysis,in which the green module was positively correlated with abdominal aortic aneurysm and the brown module was negatively correlated with abdominal aortic aneurysm.The genes in the two modules were analyzed by GO enrichment analysis and KEGG signal pathway enrichment analysis,respectively.It was found that the genes of green module were mainly related to the activation of inflammatory cells including T lymphocytes and B lymphocytes and inflammation-related signal pathways.The results showed that inflammatory cells and inflammatory signal pathway were closely related to the occurrence and development of abdominal aortic aneurysm.The genes in brown module were mainly related to myofilament and various cytoskeleton proteins of smooth muscle cells.KEGG pathway enrichment analysis showed that TGF-βsignal pathway was significantly enriched,indicating that smooth muscle cell contractile phenotypic markers and TGF-βsignal pathway were significantly inhibited in the occurrence and development of abdominal aortic aneurysm.(2)the P3 and P5 generation umbilical cord mesenchymal stem cells adhered to the wall and grew in a swirl shape under the light microscope,and the cell growth curve showed that the P3 and P5 generation umbilical cord mesenchymal stem cells entered the logarithmic growth phase on the 1st day and the plateau stage on the 4th day,which indicated that the P3 and P5 generation umbilical cord mesenchymal stem cells had great growth potential.The results of cell cycle showed that about 90%of P3 and P5generation umbilical cord mesenchymal stem cells were concentrated in G0/G1 phase,8%in S phase,and 2%in G2max M phase.Multi-directional differentiation test showed that umbilical cord mesenchymal stem cells had the ability of adipogenesis,osteogenesis and chondrogenesis.The detection of cell surface markers showed that the positive rates of CD73,CD90 and CD105 of P3 and P5 generation cells were more than 95%,while the positive rates of CD34,CD45 and HLA-DR were less than 5%,which accorded with the characteristics of mesenchymal stem cells.(3)the in vivo experimental results showed that umbilical cord mesenchymal stem cells could significantly reduce the diameter of abdominal aortic aneurysm and reduce the degree of elastic fiber rupture and degradation of abdominal aortic aneurysm.Immunohistochemistry and immunofluorescence experiments showed that compared with Elastase group,umbilical cord mesenchymal stem cells significantly increased the expression of contractile phenotypic markers SMA-αand SM-22 in aortic wall smooth muscle cells.The results of western blot assay showed that compared with Elastase group,umbilical cord mesenchymal stem cells significantly increased the expression of contractile phenotypic markers SMA-αand SM-22,decreased the expression of secretory phenotypic markers OPN,significantly increased the expression levels of TGF-βand p-smad2 proteins,and decreased the expression levels of matrix metalloproteinases MMP-2 and MMP-9 and inflammatory factor TNF-αin aortic wall.(4)the results of cell experiment in vitro showed that the expression levels of SMA-αand p-smad2 in CM group and Co-culture group were significantly higher than those in Control group.After the intervention of LY2157299,the expression levels of SMA-αand p-smad2 were significantly decreased.Conclusion:This study showed that during the occurrence and development of abdominal aortic aneurysm,inflammatory cell-related signal pathways were significantly activated,smooth muscle cells changed from contractile phenotype to synthetic epiphenotype,and TGF--βsignaling pathway was inhibited;UC-MSC could significantly reduce the diameter of abdominal aortic aneurysm,reduce the level of elastic fiber rupture and degradation,and promote the transformation of medial smooth muscle cells to contractile phenotype.UC-MSC can promote the transformation of smooth muscle cells to contractile phenotype by secreting TGF-β. |
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