MicroRNA-21介导非对称性二甲基精氨酸诱导的内皮细胞衰老

目的：观察非对称性二甲基精氨酸（ADMA）对内皮细胞中microRNA-21（miR-21）表达的影响,探讨microRNA-21在ADMA诱导的内皮细胞衰老中的作用。方法：人脐静脉内皮细胞（HUVEC）与10uM的ADMA孵育48小时后收集细胞提取总RNA及蛋白,荧光定量实时RT—PCR检测miR-21表达,Westernblot检测超氧化物歧化酶2（SOD2）表达,衰老相关半乳糖苷酶（SA-β-gal）染色鉴定衰老的内皮细胞;然后HUVEC与miR-21抑制剂转染6小时后继续与10uM的ADMA孵育48小时留取细胞按上述方法检测相关指标。结果：HUVEC与ADMA孵育后miR-21表达量明显增加（P〈0．01）,同时衰老的内皮细胞数量增多（P〈0．05）,而SOD2表达减少（P〈0．01）;MiR-21抑制剂转染HUVEC后ADMA诱导的miR-21表达明显减少,同时衰老的内皮细胞减少,而SOD2表达明显增加（所有P〈0．01）。结论：ADMA诱导了HUVEC中miR-21表达及细胞衰老,miR-21介导了ADMA诱导的内皮细胞衰老作用,其机制可能与其抑制SOD2表达有关。     

MicroRNA-18a通过HIF-1α调控缺氧引起的人肺动脉平滑肌细胞增殖

目的:研究microRNA-18a(miR-18a)对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,hPASMCs)增殖的调控作用及其可能机制。方法:体外培养hPASMCs,分为未转染组、miR-18a模拟物对照组、miR-18a模拟物组、miR-18a抑制剂对照组、miR-18a抑制剂组、si RNA control组、si HIF-1α组、miR-18a抑制剂和si HIF-1α共转染组。分别于常氧(21%O2)和低氧(3%O2)作用24小时。采用CCK-8法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1α(HIF-1α)是否为miR-18a的靶基因,并通过western-blot以及实时荧光定量PCR技术检测相关蛋白和基因的表达。结果:缺氧可促进hPASMCs增殖,使miR-18a表达减少;miR-18a模拟物可抑制hPASMCs增殖,而miR-18a抑制剂可促进hPASMCs增殖;抑制miR-18a可使HIF-1α的表达上调。同时抑制miR-18a和HIF-1α,可使miR-18a对hPASMCs增殖调控的能力消失。结论:缺氧通过抑制miR-18a,上调HIF-1α的表达,促进h PASMCs增殖。     

过表达miR-455促进肝癌细胞诱导的管型形成

目的:构建miR-455前体的真核表达载体,探讨其对肝癌细胞诱导管型形成的影响。方法:采用PCR扩增miR-455前体序列,通过双酶切将其连接到真核表达载体pcDNA3中。连接产物转化入大肠杆菌内进行扩增,采用菌落PCR、双酶切和测序鉴定重组子。将构建的质粒转染SMMC-7721细胞,通过real-time PCR检测成熟miR-455的表达,采用ELISA方法检测培养液上清中VEGF的表达。用该上清处理人脐静脉内皮细胞HUVEC后,检测管型形成的情况。结果:本实验成功构建了miR-455前体的真核表达载体,将其瞬时转染SMMC-7721细胞后,real-time PCR检测结果显示miR-455表达水平显著升高(P0.01)。过表达miR-455后,SMMC-7721细胞上清VEGF表达水平呈时间依赖性升高(24 h P0.05,48 h和72 h P0.01)。人脐静脉内皮细胞分别用转染pcDNA3和pcDNA3-pre-455的肝癌细胞培养液上清(72 h)重悬,接种于基质胶Matrigel上,转染pcDNA3-pre-455组形成典型的微管,管样结构明显完整。结论:过表达miR-455可促进肝癌细胞诱导的微管形成。     

DDAH/ADMA通路在HDL促进内皮细胞NO生成中的作用

目的:观察高密度脂蛋白(HDL)对氧化低密度脂蛋白(ox-LDL)抑制内皮细胞一氧化氮(NO)生成的保护作用,并探讨其与二甲基精氨酸二甲胺水解酶(DDAH)/非对称性二甲基精氨酸(ADMA)通路的关系.方法:Ox-LDL(1000 g/ml)孵育人脐静脉内皮细胞(HUVECs)24小时或不同浓度的HDL(10、50、100μ g/ml)预处理HUVECs细胞1小时,再与ox-LDL(100μ g/ml)共孵育细胞24小时,收集细胞培养上清液检测NO、ADMA的浓度,收集内皮细胞检测DDAH-Ⅱ的mRNA和蛋白表达以及DDAH的活性.结果:Ox-LDL(100μ g/ml)孵育HUVECs24小时后,细胞培养上清液中NO的浓度显著降低,ADMA水平显著增高,细胞内DDAH-Ⅱ的mRNA和蛋白表达以及DDAH的活性均显著降低.HDL(10、50、100μ g/ml)可以拮抗ox-LDL(100μ g/ml)的上述作用.结论:HDL能显著抑制ox-LDL诱导的内皮细胞NO产生减少,其保护作用与其调节DDAH/ADMA通路有关.     

microRNA-195 在棕榈酸诱导肝细胞L02 葡萄糖摄取障碍中的作用

目的:探究microRNA-195对棕榈酸诱导肝细胞L02葡萄糖摄取障碍中的影响及其作用机制。方法:体外培养人肝细胞L02,用不同浓度棕榈酸(0.6 mmol/L,1 mmol/L)诱导人肝细胞L02(10 h),显微镜观察其形态学变化。应用葡萄糖氧化酶法检测细胞培养液中剩余的葡萄糖含量,Real Time-PCR检测棕榈酸诱导肝细胞L02葡萄糖摄取障碍后microRNA-195(miR-195)表达变化,Real Time-PCR检测转染miR-195 mimics及miR-195 inhibitor后miR-195的表达水平,激光共聚焦检测转染效率,miR-195过表达或低表达后葡萄糖摄取的变化。结果:高浓度棕榈酸可诱导肝细胞L02葡萄糖摄取障碍,培养液上清糖浓度升高约2.5 mmol/L,miR-195表达量明显升高;转染miR-195 mimic后miR-195表达升高约200倍,转染miR-195 inhibitor后miR-195表达降低约40倍;过表达miR-195后细胞培养液上清中葡萄糖量升高2.5 mmol/L,即葡萄糖摄取降低;低表达miR-195后上清液中葡萄糖量降低约1 mmol/L,即葡萄糖摄取作用加强。结论:miR-195有可能参与调控肝细胞L02葡萄糖摄取障碍。     

桂皮醛上调Nrf2改善衰老大鼠血管内皮功能障碍

目的:探讨桂皮醛对衰老大鼠模型血管内皮功能的影响及相关机制。方法:通过建立24月龄自然衰老的Sprague Dawley(SD)大鼠模型及第14代的衰老人脐静脉内皮细胞模型(HUVECs),以桂皮醛(10μM)进行体外干预,分别以DHE染色和DAF-2DA染色观察桂皮醛对颈动脉内膜和HUVEC中超氧阴离子、一氧化氮水平的影响。以微血管张力测定仪观察桂皮醛对乙酰胆碱诱导的颈动脉内皮依赖性舒张功能和硝酸甘油诱导的非内皮依赖性舒张功能的影响。Western blotting观察磷酸化eNOS水平及Nrf2表达。结果:桂皮醛孵育显著减少改善衰老大鼠颈动脉内膜和衰老HUVEC的ROS水平,促进HUVEC中eNOS的磷酸化,增加NO水平,改善乙酰胆碱诱导的血管内皮依赖性舒张功能,对硝酸甘油诱导的非内皮依赖性舒张功能无显著的影响;Nrf2的抑制剂鸦胆子苦醇可显著阻断桂皮醛的作用。结论:桂皮醛通过Nrf2通路减少衰老相关的ROS生成,增加NO水平从而改善衰老大鼠血管内皮依赖性舒张功能。     

miR-133b靶向抑制SGTB对oxLDL诱导的血管内皮细胞损伤的影响*

目的:探讨微小RNA-133b(miR-133b)靶向抑制富含谷氨酰胺三十四肽重复序列的小蛋白质分子(SGTB)对氧化低密度脂蛋白(oxLDL)诱导的血管内皮细胞损伤的影响。方法:采用100 μg/ml的oxLDL诱导人脐静脉血管内皮细胞(EVC-304)24 h构建血管内皮细胞损伤模型。将EVC-304细胞分为对照组、oxLDL组(oxLDL处理)、oxLDL+miR-NC组(转染20 nmol/L miR-NC+oxLDL处理)、oxLDL+miR-133b组(转染20 nmol/L miR-133b mimics+oxLDL处理)、oxLDL+si-NC组(转染20 nmol/L si-NC+oxLDL处理)、oxLDL+si-SGTB组(转染20 nmol/L si-SGTB+oxLDL处理)、oxLDL+miR-133b+pcDNA组(转染20 nmol/L si-SGTB和pcDNA+oxLDL处理)、oxLDL+miR-133b+pcDNA-SGTB组(转染20 nmol/L si-SGTB和pcDNA-SGTB处理)。实时荧光定量PCR(qRT-PCR)和蛋白质印记(Western blot)检测miR-133b和SGTB的表达水平;流式细胞术检测细胞凋亡;试剂盒检测丙二醛(MDA)含量、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)的活性;Western blot检测B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。双荧光素酶报告基因实验和Western blot验证miR-133b对SGTB的靶向调控关系。结果:与对照组比较,oxLDL诱导后EVC-304细胞miR-133b、Bcl-2的表达水平显著降低(P＜0.05),SGTB、Bax的表达水平显著升高(P＜0.05),MDA含量和细胞凋亡率显著增加(P＜0.05),SOD和GSH-Px活性显著降低(P＜0.05)。过表达miR-133b或干扰SGTB均可抑制oxLDL诱导的EVC-304细胞凋亡和氧化应激损伤(P＜ 0.05)。miR-133b与SGTB直接结合,过表达miR-133b显著下调SGTB表达(P＜0.05),抑制miR-133b显著上调SGTB表达(P＜0.05)。过表达SGTB可逆转过表达miR-133b对oxLDL诱导的血管内皮细胞损伤的影响(P＜0.05)。结论:miR-133b通过靶向抑制SGTB的表达,可减轻oxLDL诱导的血管内皮细胞氧化应激损伤和细胞凋亡。     

MicroRNA-18a通过HIF-1-alpha调控缺氧引起的人肺动脉平滑肌细胞增殖

目的：研究microRNA-18a (miR-18a) 对缺氧引起的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, hPASMCs)增殖的调控作用及其可能机制。方法：体外培养hPASMCs,分为未转染组、miR-18a 模拟物对照组、miR-18a 模拟物组、 miR-18a 抑制剂对照组、miR-18a 抑制剂组、siRNAcontrol组、siHIF-1-alpha组、miR-18a 抑制剂和siHIF-1-alpha共转染组。分别于常氧(21% O2)和低氧(3%O2)作用24小时。采用CCK-8 法检测细胞的增殖情况,萤光素酶报告基因系统验证缺氧诱导因子-1-alpha(HIF-1alpha)是 否为miR-18a 的靶基因,并通过western-blot 以及实时荧光定量PCR 技术检测相关蛋白和基因的表达。结果：缺氧可促进 hPASMCs 增殖,使miR-18a 表达减少;miR-18a 模拟物可抑制hPASMCs 增殖,而miR-18a 抑制剂可促进hPASMCs 增殖;抑制 miR-18a 可使HIF-1-alpha的表达上调。同时抑制miR-18a 和HIF-1-alpha,可使miR-18a 对hPASMCs 增殖调控的能力消失。结论：缺氧通 过抑制miR-18a,上调HIF-1-alpha的表达,促进hPASMCs增殖。     

MicroRNA-21靶向调控β-catenin促进骨肉瘤细胞U2OS的增殖与侵袭

目的:研究微小RNA-21(microRNA-21,miR-21)对骨肉瘤细胞U2OS增殖与侵袭的影响及其可能机制。方法:采用Real-time PCR (RT-PCR)检测miR-21在骨肉瘤和临近正常骨组织中的表达差异。通过脂质体转染法将miR-21模拟物(microRNA-21mimics,即mimics组)及microRNA无关序列(microRNA-NC,即NC组)转染入骨肉瘤细胞U2OS,real-time PCR(RT-PCR)检测miR-21和β-catenin m RNA在U2OS细胞中的表达,Western blot检测β-catenin蛋白在U2OS细胞中的表达,并通过双荧光素酶报告基因验证miR-21与β-catenin基因3'-非编码区(3'-untranslated region,3'-UTR)的特异性结合作用。MTT法检测U2OS细胞体外增殖能力;Transwell侵袭模型探查U2OS细胞侵袭潜能。结果:骨肉瘤组织中miR-21水平显著高于正常骨组织(P0.05)。过表达miR-21能够增强细胞增殖与侵袭,上调U2OS细胞β-catenin m RNA和蛋白的表达。双荧光素酶报告基因结果表明miR-21可与β-catenin基因3'-UTR结合,从而对β-catenin的表达起调控作用。结论:miR-21可能通过调节β-catenin的表达促进骨肉瘤细胞U2OS的增殖与侵袭。     

MicroRNA-1在心肌肥大中对L-型钙通道β2亚基的负性调控作用

目的:研究微小RNA-1(microRNA-1,miR-1)在心肌细胞肥大中对L-型钙通道β2亚基(Cavβ2)的负性调控作用及机制。方法:应用异丙肾上腺素(ISO)诱导心肌细胞肥大;采用HJ2000通用图像分析系统测定心肌细胞表面积;应用数据库microCosm预测miR-1的靶基因;构建含Cavβ23’UTR报告基因质粒和miR-1瞬时共转染HEK293细胞,验证Cavβ2为miR-1靶基因;应用qRT-PCR或Western blot方法检测心房钠尿肽(ANP)、β-肌球蛋白重链(β-MHC)、miR-1和Cavβ2mRNA和蛋白表达水平;转染miR-1模拟物上调miR-1或应用Cavβ2RNAi干扰Cavβ2蛋白的表达,观察对心肌细胞肥大的影响。结果:①在ISO诱导的心肌细胞肥大中,miR-1表达显著下降;应用miR-1 mimic转染心肌细胞使miR-1表达上调,心肌细胞表面积、ANP和β-MHC mRNA表达均显著低于ISO组(P<0.05)。②网络数据库预测显示Cavβ2为miR-1的潜在靶点;将miR-1和含Cavβ23’UTR报告基因质粒共转染HEK293细胞,其萤光值显著降低(P<0.01)。转染miR-1 mimic使心肌细胞miR-1表达上调,可以明显抑制Cavβ2蛋白的表达。③在ISO诱导心肌细胞肥大中Cavβ2表达较对照组显著增加;应用RNAi技术下调Cavβ2表达可明显抑制心肌细胞表面积、ANP和β-MHC mRNA表达的增加。结论:预测并验证L-型钙通道β2亚基为miR-1的靶基因。miR-1可能通过抑制其靶基因Cavβ2蛋白的表达,降低细胞内钙离子浓度,抑制心肌细胞肥大。     

MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells

The mechanisms by which oscillatory shear stress (OS) induces, while high laminar shear stress (LS) prevents, atherosclerosis are still unclear. Here, we examined the hypothesis that OS induces inflammatory response, a critical atherogenic event, in endothelial cells by a microRNA (miRNA)-dependent mechanism. By miRNA microarray analysis using total RNA from human umbilical vein endothelial cells (HUVECs) that were exposed to OS or LS for 24 h, we identified 21 miRNAs that were differentially expressed. Of the 21 miRNAs, 13 were further examined by quantitative PCR, which validated the result for 10 miRNAs. Treatment of HUVECs with the miR-663 antagonist (miR-663-locked nucleic acids) blocked OS-induced monocyte adhesion, but not apoptosis. In contrast, overexpression of miR-663 increased monocyte adhesion in LS-exposed cells. Subsequent mRNA expression microarray study using HUVECs treated with miR-663-locked nucleic acids and OS revealed 32 up- and 3 downregulated genes, 6 of which are known to be involved in inflammatory response. In summary, we identified 10 OS-sensitive miRNAs, including miR-663, which plays a key role in OS-induced inflammatory responses by mediating the expression of inflammatory gene network in HUVECs. These OS-sensitive miRNAs may mediate atherosclerosis induced by disturbed flow.     

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.     

Involvement of DDAH/ADMA/NOS pathway in nicotine-induced endothelial dysfunction

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase (NOS) inhibitor, is a key contributor for endothelial dysfunction. Decrease in activity of dimethylarginine dimethylaminohydrolase (DDAH), a major hydrolase of ADMA, causes accumulation of ADMA under cardiovascular abnormalities. The study was to determine whether nicotine-induced endothelial dysfunction is related to modulating DDAH/ADMA/NOS pathway. Four-week oral nicotine treatment (5 mg/kg/day) significantly increased the plasma level of ADMA and decreased aortic DDAH expression as well as impaired endothelial function in Sprague-Dawley rats. Similarly, the medium levels of both ADMA and lactate dehydrogenase were markedly elevated in umbilical vein endothelial cells (HUVECs) treated with nicotine (10 microM) for 48 h. Nicotine-induced endothelial damages were markedly attenuated by L-arginine or overexpression of DDAH-II. Nicotine greatly downregulated both mRNA and protein levels of DDAH-II, and decreased DDAH activity in HUVECs. HUVECs express alpha7 nicotinic acetylcholine receptor (alpha7 nAChR), whose antagonists could block these effects of nicotine mentioned above. Intracellular Ca2+ chelator did not affect nicotine-induced decrease in DDAH-II mRNA level. In conclusion, nicotine modulates DDAH/ADMA/NOS pathway of endothelial cell via activation of alpha7 nAChR, which may be involved in endothelial dysfunction associated to smoking.     

Apelin-13 passes through the ADMA-damaged endothelial barrier and acts on vascular smooth muscle cells

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is associated with vascular dysfunction. The polypeptide apelin mediates two major actions on blood vessels. However, their combined effects on vascular function are not fully understood. The present study aimed to determine the effect of apelin-13 on myosin light chain (MLC) phosphorylation in vascular smooth muscle cells (VSMCs) under ADMA-induced endothelial leakage conditions. To assess the increased permeability induced by ADMA, human umbilical vein endothelium cells (HUVECs) were plated in transwell dishes. The FITC-dextran flux and FITC-apelin-13 flux through the endothelial monolayer were measured. To examine the effect of leakage of apelin-13 on MLC phosphorylation in HUVSMCs, transwell dishes were used to establish a coculture system with HUVECs in upper chambers and HUVSMCs in lower chambers. Western blot was performed to assess the phospho-MLC levels. ADMA increased endothelial permeability in a concentration- and time-dependent manner, accompanied by actin stress fiber assembly and intercellular gap formation. When HUVECs were treated with ADMA, the permeability to both macromolecular dextran and micromolecular apelin-13 increased significantly. Both p38 MAPK inhibitor and NADPH oxidase inhibitor could prevent HUVECs from the increased permeability, and the changes of cytoskeleton and intercellular junction, which were induced by ADMA. Apelin-13 passed through the ADMA-stimulated endothelial monolayer and increased the expression of phospho-MLC in VSMCs. These results suggest that ADMA increases endothelial permeability, which may involve the p38 MAPK and NADPH oxidase pathway. Apelin-13 can pass through the damaged endothelial barrier, and acts directly on VSMCs to increase MLC phosphorylation.     

Protective effect of HDL on endothelial NO production: the role of DDAH/ADMA pathway

Accumulating studies have demonstrated that the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine (DDAH/ADMA) system is a novel pathway for modulating nitric oxide (NO) production. The aim of this study was to investigate whether the protective effect of high density lipoprotein (HDL) on endothelial NO production was related to its effect on DDAH/ADMA pathway. Human umbilical vein endothelial cells (HUVECs) were prior exposed to HDL (10, 50, or 100 μg/ml) for 1 h, and then incubated with oxidized low density lipoprotein (ox-LDL) (100 μg/ml) for 24 h. The cultured medium was collected for measuring the concentration of NO and ADMA. The cells were collected for measuring the mRNA and protein expression of DDAH-II as well as DDAH activity. HUVECs treated with ox-LDL (100 μg/ml) for 24 h significantly decreased the concentration of NO, the mRNA and protein expression of DDAH-II as well as DDAH activity and increased the level of ADMA. Pretreatment with HDL (10, 50, or 100 μg/ml) could counteract these changes induced by ox-LDL (100 μg/ml). HDL significantly increased the attenuated endothelial cell NO production induced by ox-LDL, which was attributed to its effect on DDAH/ADMA pathway.     

Dihydromyricetin increases endothelial nitric oxide production and inhibits atherosclerosis through microRNA-21 in apolipoprotein E-deficient mice

Natural products were extracted from traditional Chinese herbal emerging as potential therapeutic drugs for treating cardiovascular diseases. This study examines the role and underlying mechanism of dihydromyricetin (DMY), a natural compound extracted from Ampelopsis grossedentata, in atherosclerosis. DMY treatment significantly inhibits atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4-positive T cells in the vessel wall and hepatic inflammation, whereas increases nitric oxide (NO) production and improves lipid metabolism in apolipoprotein E-deficient (Apoe^−/−) mice. Yet, those protective effects are abrogated by using NOS inhibitor L-NAME in Apoe^−/− mice received DMY. Mechanistically, DMY decreases microRNA-21 (miR-21) and increases its target gene dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression, an effect that reduces asymmetric aimethlarginine (ADMA) levels, and increases endothelial NO synthase (eNOS) phosphorylation and NO production in cultured HUVECs, vascular endothelium of atherosclerotic lesions and liver. In contrast, systemic delivery of miR-21 in Apoe^−/− mice or miR-21 overexpression in cultured HUVECs abrogates those DMY-mediated protective effects. These data demonstrate that endothelial miR-21-inhibited DDAH1-ADMA-eNOS-NO pathway promotes the pathogenesis of atherosclerosis which can be rescued by DMY. Thus, DMY may represent a potential therapeutic adjuvant in atherosclerosis management.     

Endothelial Cells Can Regulate Smooth Muscle Cells in Contractile Phenotype through the miR-206/ARF6&NCX1/Exosome Axis

Active interactions between endothelial cells and smooth muscle cells (SMCs) are critical to maintaining the SMC phenotype. Exosomes play an important role in intercellular communication. However, little is known about the mechanisms that regulate endothelial cells and SMCs crosstalk. We aimed to determine the mechanisms underlying the regulation of the SMC phenotype by human umbilical vein endothelial cells (HUVECs) through exosomes. We found that HUVECs overexpressing miR-206 upregulated contractile marker (α-SMA, Smoothelin and Calponin) mRNA expression in SMCs. We also found that the expression of miR-206 by HUVECs reduced exosome production by regulating ADP-Ribosylation Factor 6 (ARF6) and sodium/calcium exchanger 1 (NCX1). Using real-time PCR and western blot analysis, we showed that HUVEC-derived exosomes decreased the expression of contractile phenotype marker genes (α-SMA, Smoothelin and Calponin) in SMCs. Furthermore, a reduction of the miR-26a-containing exosomes secreted from HUVECs affects the SMC phenotype. We propose a novel mechanism in which miR-206 expression in HUVECs maintains the contractile phenotype of SMCs by suppressing exosome secretion from HUVECs, particularly miR-26a in exosomes, through targeting ARF6 and NCX1.