FERM、ARH/RhoGEF和pleckstrin结构域蛋白1在胸主动脉夹层中的表达及作用

Expression and role of FERM,ARH/RhoGEF and pleckstrin domain protein 1 in thoracic aortic dissection

目的探讨FERM、ARH/RhoGEF和pleckstrin结构域蛋白1(Farp1)在胸主动脉夹层中的表达及作用。方法胸主动脉夹层(TAD)组织来源于2022年12月至2023年3月在武汉大学中南医院因Stanford A型主动脉夹层行外科手术的患者,收集心脏移植供体残余的胸主动脉作为对照(CRTL)组。通过蛋白质印迹法(Western blot)和免疫组织化学检测TAD与CRTL组胸主动脉组织中Farp1的表达水平;3周龄雄性C57BL/6J小鼠喂养0.25%的β-氨基丙腈酯(BAPN)4周构建胸主动脉夹层模型,检测指标及方法同人胸主动脉组织;血管紧张素Ⅱ(AngⅡ,1×10^(-7) mol/L)刺激原代大鼠胸主动脉平滑肌细胞(RAVSMCs)24 h模拟胸主动脉夹层,构建Ad-sh-Farp1敲低腺病毒干扰RAVSMCs中Farp1的表达,通过Western blot检测RAVSMCs中收缩表型蛋白标志物。采用非配对t检验进行统计学分析。结果Western blot(1.723±0.084,t=5.667,P<0.05)及免疫组织化学染色(3.507±0.232,t=4.393,P<0.05)结果显示人TAD组织中Farp1的蛋白水平高于对照组;小鼠主动脉夹层组织Western blot(1.304±0.042,t=2.996,P<0.05)和免疫组织化学染色(0.458±0.030,t=4.060,P<0.05)显示了相同结果,且免疫组织化学染色进一步提示Farp1蛋白水平改变主要发生于主动脉中层(平滑肌);细胞实验显示AngⅡ促进RAVSMCs中Farp1蛋白表达上调(1.469±0.115,t=3.912,P<0.05),敲低Farp1抑制了RAVSMCs表型转换[α-平滑肌肌动蛋白(α-SMA):1.762±0.202,t=3.123,P<0.05;CNN1:1.615±0.101,t=5.288,P<0.05;SM22α:1.618±0.135,t=2.964,P<0.05]。结论Farp1与主动脉平滑肌细胞表型转换明显相关,Farp1蛋白水平增高可能促进胸主动脉夹层的发生发展。

Objective To investigate the expression and role of FERM,ARH/RhoGEF and pleckstrin domain protein 1(FARP1)in thoracic aortic dissection(TAD).MethodsPatients who underwent surgical intervention for Stanford type A aortic dissection at Zhongnan Hospital of Wuhan University from December 2022 to March 2023 were selected as the TAD group.The control(CRTL)group consisted of residual thoracic aortic tissues from heart transplant donors.The expression levels of FARP1 in TAD group and CRTL group were detected by Western blotting and immunohistochemical staining.The 3-week-old male C57BL/6J mice were fed on 0.25%β-aminopropionitrile(BAPN)for 4 weeks to establish a TAD model.The detection indexes and methods were the same as those in human thoracic aortic tissues studies.Primary rat aortic vascular smooth muscle cells(RAVSMCs)were stimulated with angiotensinⅡ(AngⅡ,1×10^(-7) mol/L,24 h)to mimic TAD.Ad-sh-FARP1 adenovirus was used to knock down FARP expression in RAVSMCs,and Western blotting was performed to detect the contractile phenotype protein markers.A non-paired t-test was utilized for statistical analysis of the data.ResultsThe results of Western blotting(1.723±0.084,t=5.667,P<0.05)and immunohistochemical staining(3.507±0.232,t=4.393,P<0.05)revealed significantly increased levels of FARP1 protein in human TAD tissues compared to the CRTL group.Similar results were observed in Western blotting(1.304±0.042,t=2.996,P<0.05)and immunohistochemical staining(0.458±0.030,t=4.060,P<0.05)of mouse aortic dissection tissues,with further indication that the change in FARP1 protein level primarily occurred in the medial layer(smooth muscle)of the aorta.Celluar experiments showed that AngⅡstimulation promoted upregulation of FARP1 protein expression(1.469±0.115,t=3.912,P<0.05),while knocking down FARPl suppressed phenotypic switch in RAVSMCs[α-smooth muscle actin(α-SMA):1.762±0.202,t=3.123,P<0.05;CNN1:1.615±0.101,t=5.288,P<0.05;SM22α:1.618±0.135,t=2.964,P<0.05].Conclusion FARP1 is significantly associated with phenotypic switch of aortic smooth muscle cells,and increased levels of FARP1 protein may promote the occurrence and development of TAD.