Expression And Role Of Chemokine Like Factor 1(CKLF1) In Aortic Dissection/Thoracic Aortic Aneurysm

Background and objectiveAortic dissection(AD)and thoracic aortic aneurysm(TAA)are common thoracic aortic diseases.The annual incidence of thoracic aortic diseases(AD and TAA)is 9.1 cases of /10 million in females,16.3 cases/10 million in males,and the incidence is increasing year by year.Once they lead to the rupture of thoracic aorta,the mortality is more than 90%.The risk and mortality of open surgery for AD and TAA remains high.Despite the rapid development of endovascular therapy,only certain patients with AD and TAA can be treated with this method,which is associated with some challenges such as high rate of surgical complications and reintervention.Therefore,the research on its etiology is conducive to improving our understanding of these diseases,which is of great importance for the prevention of AD occurrence and TAA enlargement and rupture in high-risk groups and the development of new early diagnostic biomarkers and therapeutic targets.Aortic medial degeneration is a common vital pathological feature of AD and TAA.It is mainly manifested in apoptosis and loss of smooth muscle cells,infiltration of inflammatory cells,degradation of extracellular matrix.The apoptosis of human AD and TAA smooth muscle cells is associated with inflammatory cell infiltration and endoplasmic reticulum stress caused by mechanical stress and so on.After invading the aortic wall,inflammatory cells secrete proapoptotic perforin and cytokines,such as monocyte chemoattractant protein-1(MCP-1),which can promote the apoptosis of smooth muscle cells.MCP-1 can in turn chemotactic monocytes and lymphocytes to the aortic wall,further aggravate inflammation,form a vicious circle and aggravate the occurrence and progress of AD and TAA.Chemokine like factor 1(CKLF1)is a new molecule discovered by Chinese scientists in 2001,with slightly different structure from the classical chemokine,has broad-spectrum chemotactic activity for neutrophils,monocytes and lymphocytes.CKLF1 also enhances the proliferation and anti-apoptosis capacity of smooth muscle cells and promote the occurrence and progress of arterial intimal hyperplasia.A study showed that CKLF1 is highly expressed in rat abdominal aortic aneurysm tissue,but its role in AD and TAA has not been fully revealed.Therefore,our project aims to verify the expression of CKLF1 in AD and TAA and explore its role in these diseases.MethodsThe tissue sections of human normal thoracic aorta and AD as well as TAA were stained with elastic fiber EVG staining and immunohistochemical staining of ACTA2 and CKLF1 to verify whether there were aortic degenerative changes such as degradation of elastic fiber,loss of smooth muscle cells and apoptosis in AD and TAA,and whether the protein level of CKLF1 was increased.Subsequently,the murine AD model was induced by adding 0.5% β-aminopropiononitrile fumarate salt in drinking water,the proximal TAA model was induced by exposing the proximal thoracic aorta through the anterior median incision of the mouse neck and periaortic application of elastase.Then,the elastic fiber EVG staining and ACTA2 immunohistochemical staining were performed to determine whether these models had the characteristics of aortic degeneration.Activated Caspase 3 and Ki67 immunofluorescence staining were used to verify the apoptosis and proliferation of smooth muscle cells in the AD and TAA model tissues,and RT-q PCR was performed to verify the expression of inflammatory factors,matrix metalloproteinases,collagen in the AD and TAA model tissues.After confirming the successful construction of the murine AD and TAA models,RT-q PCR,immunohistochemistry or Western blot were used to verify the changes of Cklf1 in m RNA and protein levels,and the cell localization of CKLF1 was determined by immunofluorescence staining.The microarray data published by NCBI GEO database were retrieved to further verify the expression of CKLF1 in human and murine AD and TAA tissues.The murine CKLF1 overexpression and knockdown plasmids were constructed,and the overexpression and knockdown lentivirus vectors were packaged(named Cklf1-LV and sh Cklf1-LV respectively,and the control groups were GFP-LV and sh NT-LV respectively).These lentiviral vectors were used to infect murine primary smooth muscle cells to regulate the expression of CKLF1.CCK-8,cell scratching test,transwell migration test and TUNEL staining were used to verify the changes of cell proliferation and migration ability and anti-apoptosis capacity after CKLF1 overexpression or knockdown.RT-q PCR and Western blot were used to detect the expression changes of phenotype transformation marker genes of smooth muscle cells after CKLF1 overexpression,and transcriptional sequencing was carried out to understand the changes of overall gene expression of smooth muscle cells caused by CKLF1 overexpression.Subsequently,the Cklf1-LV or GFP-LV virus gel was incubated on the proximal thoracic aorta on the fifth day of the mouse AD model construction,and the sh Cklf1-LV or sh NT-LV solution was injected by tail vein to explore the effect of CKLF1 overexpression and knockdown on the murine AD formation,respectively.The maximum diameter of the thoracic aortas were determined by ultrasonic examination on the twentieth day and the mortality of aortic rupture were recorded during the 28 days.The aorta of GFP-LV infection group was stained with GFPantibody immunofluorescence to verify the effect of virus infection.Activated Caspase 3 and Ki67 immunofluorescence staining were used to verify the effect of CKLF1 overexpression on the apoptosis and proliferation of smooth muscle cells in AD tissue.RT-q PCR was used to verify the change of expression of apoptosis related genes Bax and Bcl2 after CKLF1 overexpression.Systemic knockdown of CKLF1 by injecting the sh Cklf1-LV or sh NT-LV solution into the mice via tail vein to observe its effect on the formation of proximal TAA in mice.The ultrasonic examination was performed to determine the expansion rate of thoracic aorta from day 2 to day 7 after operation.ResultsEVG staining and ACTA2 immunohistochemical staining showed that there were features of aortic degeneration in human AD and TAA tissues such as degradation and rupture of elastic fibers and loss of smooth muscle cells.CKLF1 immunohistochemical staining was performed on 8 human normal thoracic aorta and 16 AD tissues.It was found that CKLF1 was mainly located in the aortic media and significantly increased in AD,which was verified by the data of GSE52093 AD microarray.The increased expression of CKLF1 in TAA was also observed by CKLF1 histochemical staining of TAA tissue,which was verified by GSE7084 abdominal aortic aneurysm microarray data.Immunofluorescence staining of CKLF1 showed that CKLF1 was located in and around smooth muscle cells in AD and TAA tissues.EVG staining,ACTA2 immunohistochemistry and activated Caspase 3immunofluorescence staining in mouse AD model suggest that there are phenomena of aortic degeneration such as the degradation and rupture of elastic fibers and the loss and apoptosis of smooth muscle cells.Ki67 immunofluorescence staining suggests the existence of proliferating smooth muscle cells in murine AD tissues.RT-q PCR showed that the expression of Opn,Mmp2,Mmp9 and Ccl2,the marker genes of synthetic transformation of smooth muscle in mouse AD model,were increased,and the expression of collagen molecules Col1a1 and Col3a1 were also up-regulated.RT-q PCR,immunohistochemistry and Western blot of murine AD tissue showed that Cklf1 increased significantly in mouse AD model tissue,which was further verified in the public microarray data GSE147078 and GSE116434.Immunofluorescence staining of CKLF1 and ACTA2 showed that CKLF1 was located in smooth muscle cells in AD tissues of mice.After consecutive ultrasonic examination for 28 days,it was found that the elastaseinduced proximal TAA model could be successfully induced by exposing the proximal thoracic aorta through the anterior median cervical incision of mice.ACTA2 immunohistochemistry and activated Caspase 3 immunofluorescence staining,EVG staining suggested that there were also phenomenon of loss and apoptosis of smooth muscle cells,degradation and rupture of elastic fibers in the proximal TAA model tissue.Ki67 immunofluorescence staining suggested that there were proliferating smooth muscle cells in TAA model tissues.RT-q PCR suggested that the inflammatory factor Tnfα,Il-6,Il-1β,and Mmp2,Mmp9 as well as collagen molecules Col1a1 and Col3a1 were increased in murine TAA tissues.RT-q PCR,Western blot and immunohistochemistry showed that Cklf1 was significantly increased in TAA tissue.The immunofluorescence staining of CKLF1 in mouse TAA tissue also suggested that it was located in smooth muscle cells.CCK-8,cell scratching test,Transwell migration test and TUNEL staining suggested that CKLF1 knock-down significantly decreased proliferation,migration,and anti-apoptosis capacity of smooth muscle cells.CKLF1 overexpression significantly enhanced cell proliferation,migration and anti-apoptosis capacity in mouse primary smooth muscle cells.RTq PCR showed that overexpression of CKLF1 could promote the phenotypic transformation of murine smooth muscle cells to synthetic direction.Transcriptome sequencing showed that CKLF1 overexpression was related to extracellular matrix organization,inflammation,chemotaxis and activation of ERK pathway in smooth muscle cells.Cklf1-LV or GFP-LV virus gel was incubated locally in the proximal thoracic aorta during the construction of mouse AD model.Ultrasound examination of thoracic aorta on the 20 th day showed that the maximal diameter of thoracic aorta in Cklf1-OE group was significantly reduced compared with GFP group.The survival rate of mice over time in Cklf1-OE group was also significantly improved.The representative gross anatomy showed that there was no obvious dissection in Cklf1-OE group,while in GFP group,aortic dissection was formed and aortic dilatation was more serious.Histological analysis showed that GFP positive region could be detected in GFP group,suggesting that lentivirus gel can control the gene expression effectively.In addition,Ki67 or cleaved Caspase 3 and ACTA2 immunofluorescence staining indicated the number of proliferative smooth muscle cells in Cklf1-OE AD tissue was significantly higher than that in the control group,and there were fewer apoptotic smooth muscle cells in Cklf1-OE AD tissue,which did not reach statistical significance.However,the ratio of Bax/Bcl2 m RNA in aortic media of Cklf1-OE group decreased significantly,suggesting the strengthened anti-apoptotic ability of cells in Cklf1-OE group.During the construction of mouse AD model,sh Cklf1-LV or sh NT-LV was injected into the mice via tail vein.The ultrasonic results showed that the maximal diameter of thoracic aorta of mice in the sh Cklf1 group was significantly higher than that in the sh NT group on the 20 th day.The survival rate of sh Cklf1 group was worse than that of sh NT group,the representative gross anatomy suggested that the mice in sh Cklf1 group formed severe aortic dissection.When the mice underwent the surgery to induce proximal TAA,sh Cklf1-LV or sh NT-LV was injected into the mice via caudal vein.The ultrasonic results showed that the expansion rate of thoracic aorta in sh Cklf1 group was higher than that in sh NT group from day 2 to day 7after operation.Conclusion1.Aortic degeneration exists in human and mouse AD and TAA tissues,and the expression of CKLF1 in human and mouse AD and proximal TAA tissues is significantly elevated.2.Overexpression of CKLF1 enhances the proliferation,migration,and anti-apoptosis ability in mouse primary smooth muscle cells,and promote their transformation into a synthetic or secretory phenotype.CKLF1 knockdown decreased the proliferation,migration and antiapoptosis capacity of mouse smooth muscle cells.3.The overexpression of CKLF1 in mice reduces dilation of thoracic aorta and the mortality rate of AD rupture induced by β-aminopropiononitrile significantly,at least partly by promoting the proliferation and anti-apoptosis capacity of smooth muscle cells in murine AD model.Systematic knockdown of CKLF1 can increase the degree of thoracic aortic dilatation and mortality in murine AD model.4.We established a simple,reproducible in vivo murine model of proximal TAA through a midline incision in the anterior neck by peri-adventitial application of elastase.Systematic knockdown of CKLF1 may promote the occurrence and progress of TAA.