细胞分裂周期蛋白42通过内皮-间充质转化参与动脉型肺动脉高压小鼠右心室纤维化

Cell division cycle protein 42 is involved in right ventricular fibrosis in mice with pulmonary arterial hypertension by endothelial-mesenchymal transition

目的:探讨细胞分裂周期蛋白42(Cdc42)是否通过内皮-间充质转化(EndMT)参与动脉型肺动脉高压(PAH)小鼠右心纤维化。方法:健康雄性C57BL/6小鼠(5~8周龄)18只,随机分成常氧对照(NC)组、PAH模型[SU5416(血管内皮生长因子受体2抑制剂)+低氧,SuHx]组和SuHx+ML141(Cdc42抑制剂)组,每组6只。所有存活小鼠在4周后用异氟烷麻醉,行心脏超声检查及右心室收缩压(RVSP)监测,之后处死小鼠。用HE和Masson染色观察小鼠右室心肌细胞改变及纤维化程度。使用磁珠分选小鼠心脏内皮细胞并用不同条件处理。通过Western blot检测小鼠心脏组织Cdc42表达水平及内皮细胞Cdc42和EndMT相关蛋白[波形蛋白(vimentin)、α-平滑肌肌动蛋白(α-SMA)、血小板内皮细胞黏附分子1(PECAM-1/CD31)、血管内皮钙黏蛋白(VE-cadherin)和锌指蛋白Snail]表达水平,同时用倒置显微镜观察各组内皮细胞形态。结果:体内实验结果显示,与NC组相比,SuHx组小鼠心脏组织Cdc42表达水平显著升高(P<0.05);预防给予ML141(8 mg/kg)可降低小鼠RVSP,增大三尖瓣环平面收缩期位移(TAPSE),减轻心脏(尤其是血管旁)纤维化(P<0.01)。体外实验结果显示,SuHx组和低氧72 h组小鼠心脏内皮细胞Cdc42表达水平显著升高,EndMT增强,EndMT相关蛋白vimentin和α-SMA的表达显著增加,CD31和VE-cadherin的表达显著降低(P<0.05);预防给予ML141(10μmol/L)后可缓解低氧72 h诱导的EndMT。结论:Cdc42可能通过上调EndMT参与PAH小鼠右心纤维化。

AIM:To examine the potential involvement of cell division cycle protein 42(Cdc42)in the develop‐ment of right cardiac fibrosis induced by pulmonary arterial hypertension(PAH)in mice through the process of endothelial-mesenchymal transition(EndMT).METHODS:Eighteen healthy male C57BL/6 mice(5~8 weeks old)were randomly divided into 3 groups:normoxia control(NC)group,PAH model[SU5416(vascular endothelial growth factor receptor 2 inhibitor)+hypoxia,SuHx]group and SuHx+ML141(Cdc42 inhibitor)group,with 6 mice in each group.After 4 weeks,all surviving mice were anesthetized with isoflurane for echocardiography and monitoring of right ventricular systolic pres‐sure(RVSP).The morphological changes and fibrosis of mouse right ventricular myocardium were assessed using HE and Masson staining.Mouse heart endothelial cells were sorted using endothelial magnetic beads and treated under different conditions.The expression level of Cdc42 in cardiac tissues,and the expression levels of Cdc42 and EndMT-related pro‐teins[vimentin,α-smooth muscle actin(α-SMA),platelet endothelial cell adhesion molecule-1(PECAM-1/CD31),vas‐cular endothelial(VE)-cadherin and zinc finger protein Snail]in endothelial cells were detected by Western blot.The morphology of endothelial cells in different groups was observed under an inverted microscope.RESULTS:Compared with NC group,the Cdc42 level in mouse heart tissues of SuHx group,and that in cardiac endothelial cells of SuHx and hy‐poxia(72 h)groups were significantly increased.Additionally,EndMT was enhanced in cardiac endothelial cells of SuHx and hypoxia(72 h)groups,as evidenced by increased expression of vimentin andα-SMA,while the expression levels of CD31 and VE-cadherin were decreased(P<0.05).Subsequently,in vitro treatment with Cdc42 inhibitor ML141(10µmol/L)helped to restrain EndMT induced by endothelial hypoxia for 72 h.Furthermore,in vivo administration of ML141(8 mg/kg)decreased RVSP,increased tricuspid annular plane systolic excursion(TAPSE),and attenuated cardiac(espe‐cially para-vascular)fibrosis(P<0.01).CONCLUSION:Cdc42 may play a role in right ventricular fibrosis in PAH mice through the up-regulation of EndMT.