低氧预处理hUCMSC-Exos通过抑制肺血管EndMT缓解低氧性肺动脉高压

Hypoxia-preconditioned hUCMSC-Exos relieve hypoxic pulmonary hy⁃pertension by inhibiting pulmonary vascular EndMT

目的:探讨低氧预处理人脐带间充质干细胞来源的外泌体(hUCMSC-Exos)对低氧性肺动脉高压(HPH)肺血管内皮-间充质转化(EndMT)的影响。方法:(1)用组织块贴壁法分离培养原代hUCMSCs,用超滤法提取hUCMSC-Exos并鉴定。(2)将24只SPF级雄性SD大鼠随机分为常氧(N)组、低氧(H)组、低氧+常氧hUCMSC-Exos组和低氧+低氧预处理hUCMSC-Exos组,每组6只。H组及各干预组大鼠置于模拟海拔5000 m低氧环境的低压氧舱中,并于低氧第3、5、7、10和14天经尾静脉注射常氧hUCMSC-Exos、低氧预处理hUCMSC-Exos或等体积PBS。造模21 d后检测各组大鼠右心室收缩压(RVSP)和右心室肥厚指数(RVHI),HE染色观察肺组织病理改变。(3)将人肺小动脉内皮细胞(HPAECs)饥饿12 h后随机分为常氧对照(N-Con)组、低氧模型(H-Con)组、低氧+常氧hUCMSC-Exos组和低氧+低氧预处理hUCMSC-Exos组。Transwell实验和小管形成实验检测HPAECs迁移和小管形成能力;免疫荧光双染法检测HPAECs中CD31和α-平滑肌肌动蛋白(α-SMA)的表达;Western blot法检测肺血管和HPAECs中CD31、VE-cadherin、α-SMA和vimentin蛋白水平。结果:(1)低氧21 d可成功构建HPH大鼠模型,且肺血管发生EndMT。与N组相比,H组大鼠RVSP、RVHI、血管壁面积占血管总面积的百分率(WA%)和血管壁厚度占血管外径的百分率(WT%)显著升高(P<0.01),肺血管管壁增厚,且肺血管中CD31和VE-cadherin蛋白表达显著减少(P<0.01),α-SMA和vimentin蛋白表达显著增加(P<0.05或P<0.01)。与H组相比,经常氧或低氧预处理hUCMSC-Exos干预后大鼠RVSP、RVHI、WA%和WT%显著降低(P<0.05或P<0.01),肺血管重构减轻,且低氧预处理hUCMSC-Exos显著抑制HPH肺血管重构和EndMT的形成。(2)低氧处理HPAECs 48 h可诱导细胞发生迁移、小管形成和End-MT。与H-Con组相比,经低氧预处理hUCMSC-Exos干预后细胞迁移和小管形成显著减少(P<0.01),CD31和VE-cadherin蛋白表达显著增多(P<0.05或P<0.01),α-SMA和vimentin蛋白表达显著减少(P<0.01)。结论:低氧预处理hUCMSC-Exos通过抑制肺血管EndMT而缓解HPH肺血管重塑。

AIM:To investigate the effect of hypoxia-preconditioned human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSC-Exos)on pulmonary vascular endothelial-mesenchymal transition(EndMT)in hypoxic pulmonary hypertension(HPH).METHODS:(1)Primary hUCMSCs were isolated and cultured by tissue adhesion method,and hUCMSC-Exos were extracted by ultrafiltration and identified.(2)Twenty-four SPF male SD rats were ran-domly divided into normoxia(N)group,hypoxia(H)group,hypoxia+normoxic hUCMSC-Exos group and hypoxia+hypoxia-preconditioned hUCMSC-Exos group,with 6 rats in each group.The rats in H group and intervention groups were placed in a cabin that simulated the hypoxic environment at an altitude of 5000 m,and normoxic hUCMSC-Exos,hypoxia-precon-ditioned hUCMSC-Exos or equivalent volume of PBS were injected through the tail vein on the 3rd,5th,7th,10th and 14th days in hypoxia environment.After 21 d of modeling,the right ventricular systolic pressure(RVSP)and right ven-tricular hypertrophy index(RVHI)of the rats were detected,and the pathological changes of lung tissues were observed by HE staining.(3)After starvation for 12 h,human pulmonary arteriole endothelial cells(HPAECs)were randomly di-vided into normoxic control(N-Con)group,hypoxic model(H-Con)group,hypoxia+normoxic hUCMSC-Exos group and hypoxia+hypoxia-preconditioned hUCMSC-Exos group.The migration ability and tube formation ability of HPAECs were detected by Transwell assay and tube formation experiment.The expression of CD31 andα-smooth muscle actin(α-SMA)in HPAECs was detected by immunofluorescence double-staining.The protein levels of CD31,VE-cadherin,α-SMA and vimentin in pulmonary vessels and HPAECs were assessed by Western blot.RESULTS:(1)The HPH rat model was suc-cessfully established after 21 d of hypoxia,and EndMT occurred in pulmonary vessels.Compared with N group,the levels of RVSP,RVHI,percentage of vascular wall area(WA%)and percentage of vascular wall thickness(WT%)in H group were significantly increased(P<0.01),pulmonary vascular wall thickening and the protein levels of CD31 and VE-cad-herin were significantly decreased(P<0.01),while the protein levels ofα-SMA and vimentin were significantly increased in pulmonary vessels(P<0.05 or P<0.01).Compared with H group,the RVSP,RVHI,WA%and WT%(P<0.01)were significantly decreased(P<0.05 or P<0.01),and pulmonary vascular remodeling was attenuated after normoxic or hypoxia-preconditioned hUCMSC-Exos intervention.After hypoxia-preconditioned hUCMSC-Exos intervention,HPH pul-monary vascular remodeling and EndMT formation were significantly inhibited.(2)After 48 h of hypoxic treatment,the migration,tubule formation and EndMT of HPAECs were induced.Compared with H-Con group,cell migration and tube formation were significantly decreased after hypoxia-preconditioned hUCMSC-Exos intervention(P<0.01).The protein levels of CD31 and VE-cadherin were increased,while the protein levels ofα-SMA and vimentin were decreased(P<0.05 or P<0.01).CONCLUSION:Hypoxia-preconditioned hUCMSC-Exos attenuate the formation of HPH pulmonary vascu-lar remodeling by inhibiting pulmonary vascular EndMT.