心肌炎小鼠心肌纤维化与心脏上皮/内皮间充质转化的关系

目的: 探讨心脏上皮/内皮间充质转化(EMT/EndMT)与病毒性心肌炎心肌纤维化的关系。方法: 40只BALB/c 小鼠随机分为2组,分别为对照组(n=16)和心肌炎组(n=24)。2组小鼠每周1次分别腹腔注射无病毒培养液或柯萨奇病毒B3(CVB3);于第7 d后随机处死2组小鼠各8只,30 d后处死其余存活小鼠。以苦味酸天狼星红染色检测小鼠心脏胶原容积分数(CVF),ELISA法检测血清胶原前肽的含量变化,以实时RT-PCR法和Western blotting方法检测小鼠心脏中EMT/EndMT蛋白组包括启动因子转化生长因子β₁(TGF-β₁)和Wnt1、转录因子Twist1、上皮细胞标志物上皮细胞钙黏素(E-cadherin)、内皮细胞表面标志血管内皮钙黏素(VE-cadherin)、间充质蛋白如成纤维细胞特异蛋白(FSP-1)和α-平滑肌肌动蛋白(α-SMA)等的基因和蛋白表达情况。结果: 急性病毒性心肌炎时,出现EMT/EndMT现象,其特征为VE-cadherin和E-cadherin丢失,FSP-1和α-SMA表达上调,启动因子TGF-β₁和Wnt1表达增高,转录因子Twist1表达增加,胶原合成增多;慢性期未发现上皮/内皮细胞表型丢失,间充质标志蛋白仍表达上调,胶原合成增多,TGF-β₁、Twist1和Wnt1表达依然增加。结论: EMT/EndMT参与了急性病毒性心肌炎心肌纤维化的形成,慢性期未发现EMT/EndMT参与心肌纤维化。     

c-SKI对冠脉内皮细胞增殖及内皮-间充质转化的影响

目的:探讨核蛋白c-SKI对冠状动脉内皮细胞增殖能力及内皮-间充质转化的影响。方法:使用不同浓度的转化生长因子β1(TGF-β1)作用人冠脉内皮细胞不同时点,Western blot法检测各组细胞中c-SKI、间充质细胞标志物波形蛋白和α-平滑肌肌动蛋白(α-SMA)及内皮细胞标志物E-钙黏蛋白的表达。使用携带c-ski基因的慢病毒转染冠脉内皮细胞后采用RT-qPCR验证转染效率。将细胞分为4组:对照组、TGF-β1(5μg/L)组、c-ski基因感染+TGF-β1组及对照病毒感染+TGF-β1组。过表达c-SKI后,MTT实验和集落形成实验检测冠脉内皮细胞的增殖能力,Western blot检测波形蛋白、α-SMA、E-钙黏蛋白、Smad2、Smad3、p-Smad2和p-Smad3蛋白的水平。结果:TGF-β1处理冠脉内皮细胞后,c-SKI在冠脉内皮细胞中的表达呈剂量和时间依赖性下降(P0.01)。MTT及集落形成实验结果显示,过表达c-SKI可显著抑制冠脉内皮细胞增殖(P0.01)。Western blot检测结果显示,与病毒对照组相比,过表达c-SKI可显著下调冠脉内皮细胞中α-SMA和波形蛋白的表达量(P0.01),上调E-钙黏蛋白的表达量(P0.01),同时抑制Smad2和Smad3蛋白的磷酸化(P0.01),逆转TGF-β1诱导的内皮-间充质转化。结论:内皮-间充质转化过程中c-SKI表达下调,而过表达c-SKI能够抑制冠脉内皮细胞增殖及内皮-间充质转化,其机制可能与调控TGF-β1/Smad信号通路活性有关。     

c-Ski调控p38信号通路在TGF-β1介导人冠状动脉内皮细胞内皮间充质转化中的作用研究

目的 探索c-Ski蛋白在人冠状动脉内皮细胞内皮间充质转化过程中的表达特征及其调控p38信号通路的机制,为心肌纤维化提供新的预防和治疗的靶点.方法 以人冠状动脉内皮细胞(human coronary artery endothelial cells,HCAECs)为研究对象,采用转化生长因子β1(transformin...     

心血管系统中内皮细胞向间充质细胞转分化的研究进展

细胞的转分化是一种细胞在某些理化因素作用下转化为另一种细胞的现象。在胚胎发育期或某些病理及压力条件下,心血管系统中内皮细胞会转分化成间充质细胞,并带来不同的生物学意义。本文就心血管系统中内皮细胞向间充质细胞转分化的机制及检测方法进行了综述。     

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

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ML...     

热休克内皮细胞诱导骨髓间充质干细胞向血管内皮细胞分化

文题释义：血管内皮细胞：研究中一般称内皮细胞,通常指衬于心、血管和淋巴管内表面的单层扁平上皮,它形成血管的内壁。它们具有吞噬异物、细菌、坏死和衰老组织的功能,还参与机体免疫活动功能。 热休克：组织工程中一种细胞处理方法,将细胞置于42 ℃(也有文献报道为47 ℃)中1 h,造成热休克状态。 背景：目前关于间充质干细胞向内皮细胞分化的研究,多采用细胞因子或2种细胞共培养的方法进行诱导,热休克处理的内皮细胞诱导间充质干细胞向内皮细胞分化尚未见报道。 目的：观察热休克处理的人脐静脉内皮细胞诱导骨髓间充质干细胞向血管内皮细胞分化的能力,并探究诱导骨髓间充质干细胞形成血管的能力。 方法：将热休克处理后的人脐静脉内皮细胞与人骨髓间充质干细胞体外非接触共培养,诱导14 d后采用流式细胞仪和免疫荧光检测骨髓间充质干细胞中CD144、CD31、VEGFR2、vWF表型的表达;将诱导14 d后的骨髓间充质干细胞、未诱导的骨髓间充质干细胞移植到裸鼠皮下,14 d后取移植物做苏木精-伊红染色,观察体内血管形成能力;Matrigel成血管实验观察诱导14 d后的骨髓间充质干细胞和未诱导的骨髓间充质干细胞的体外血管生成能力。 结果与结论：①共培养后骨髓间充质干细胞形态改变,呈类似铺路石状排列,流式细胞仪及细胞免疫荧光结果显示共培养后骨髓间充质干细胞VEGFR2、CD31、CD144、vWF表达增加;②体内移植物苏木精-伊红染色显示诱导后的骨髓间充质干细胞排列较对照组规律,有成血管倾向;③体外Matrigel成血管实验显示诱导后骨髓间充质干细胞成血管能力较对照组有所增加;④结果表明,与热休克处理的人脐静脉内皮细胞共培养能促进人骨髓间充质干细胞向内皮细胞分化,内皮细胞特异性表型转化较明显,具有一定血管形成倾向。 ORCID: 0000-0003-4089-8882(曹百川) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

白血病间充质细胞体外转分化内皮细胞的研究

目的实体肿瘤抑或血液系肿瘤的生长和转移都是血管依赖性的 ,在血管新生过程中 ,内皮细胞的分化来源是其中的重要研究课题。已证实正常组织中的间充质细胞具有多分化潜能。为进一步探讨肿瘤血管新生的机制、肿瘤组织中内皮细胞的分化来源及其增殖机制 ,本文以初治白血病骨髓标本为研究对象 ,进行肿瘤状态下的骨髓间充质细胞转分化内皮细胞的研究。方法采集初治白血病骨髓标本 ,以淋巴细胞分离液1800r/min离心 ,分离出单个核细胞 ,以HumanMesenchymalStemCells基础及补充培养基进行骨髓中间充质细胞的扩增培养 ,以上述培养基培养14d以上 ,后分选出间充质细胞转入含内皮细胞特异性生长因子的内皮细胞培养体系中进行培养。待培养瓶内长满细胞后 ,进行内皮细胞特性鉴定。结果经扩增培养后的骨髓间充质细胞转入至内皮细胞培养系统中 ,再经7～14d培养后 ,收集细胞进行鉴定 ,结果显示在普通光镜下上述细胞形态学符合内皮细胞形态特征 ,收集上述细胞进行内皮细胞特异性Ⅷ因子相关抗原的检测为阳性。结论虽然在骨髓中的间充质细胞所占比例较低 ,但经间充质细胞扩增培养基培养后 ,能够得到较多的间充质细胞用于作进一步的实验研究。本研究的上述初步结果表明在肿瘤时 ,肿瘤组织内部的血管新生除通过周边血管的内皮     

体外诱导羊膜来源间充质干细胞分化为血管内皮细胞

背景：羊膜来源间充质干细胞植入机体不同类型组织后是否可以分化为相应组织靶细胞呢？ 目的：检测血管内皮细胞生长因子在体外诱导人羊膜间充质干细胞分化为血管内皮细胞的可行性。 方法：分离培养羊膜间充质干细胞,鉴定其表面抗原表达,用含体积分数2%胎牛血清以及50 μg/L血管内皮生长因子的条件培养基诱导,诱导后细胞通过内皮细胞标志物血管内皮生长因子受体2以及v-WF染色鉴定。 结果与结论：羊膜间充质干细胞表面抗原CD29、CD44、CD105阳性,CD34、CD45、CD106以及HLA-DR阴性。诱导后细胞形态明显改变,内皮细胞标志物血管内皮细胞生长因子受体2以及v-WF染色结果阳性。提示羊膜间充质干细胞在体外具有分化为血管内皮细胞的能力。     

骨髓间充质干细胞体外诱导向内皮和上皮细胞的分化

背景：骨髓来源的间充质干细胞在体外具有多系分化潜能,但其在体外向肺组织细胞的分化能力尚存在争议。 目的：体外诱导验证小鼠骨髓间充质干细胞向内皮细胞和上皮细胞分化的能力。 方法：分离小鼠骨髓来源的间充质干细胞,以内皮诱导液向内皮细胞分化。另外将小鼠骨髓间充质干细胞分别在以下诱导培养基中进行上皮诱导3周：单纯上皮诱导培养液,上皮诱导培养液加10 μg/L 转化生长因子β1,并以未经诱导的小鼠骨髓间充质干细胞作为阴性对照,肺泡上皮作为阳性对照。 结果与结论：小鼠骨髓间充质干细胞在上皮诱导培养液中诱导培养3周后,部分细胞由梭形变为典型的卵石样上皮细胞形态,诱导后约60%细胞表达广谱上皮细胞标志pan-CK,RT-PCR结果显示分化后的细胞表达上皮细胞特异标志CK18,未经诱导的间充质干细胞未表达。小鼠骨髓间充质干细胞在内皮诱导24 h后即出现了典型的血管网状结构,vWF免疫荧光染色显示约70%的细胞呈阳性,RT-PCR显示分化后的细胞表达内皮细胞特异性标志CD31、vWF和CD34。提示骨髓来源的间充质干细胞体外诱导培养具有跨胚层多系分化能力。     

人胎盘间充质干细胞促进血管生成

背景：构建组织工程化骨组织的同时,促进种子细胞与材料复合物内血液供应的重建成为研究的关键。胎盘间充质干细胞可以作为骨组织工程研究的种子细胞,研究其分化为血管内皮细胞以及促进血管生成有着重要意义。 目的：观察胎盘间充质干细胞在体外分化为血管内皮细胞以及体内促血管生成作用。 方法：分离培养人胎盘间充质干细胞,鉴定其表面抗原,经血管内皮生长因子和人碱性成纤维细胞生长因子联合体外诱导胎盘来源间充质干细胞向血管内皮细胞定向分化,诱导后细胞通过内皮细胞标志物KDR、v-WF染色鉴定。8只新西兰大白兔制成桡骨中段1.5 cm长的骨缺损模型,分别植入人胎盘间充质干细胞/丝素蛋白/羟基磷灰石和丝素蛋白/羟基磷灰石进行对照。植入后4,12周分别行大体观察、组织学观察和X射线观察,比较骨缺损修复以及血管生成情况。 结果与结论：胎盘间充质干细胞的诱导分化形态明显改变,胞体逐步回缩,立体感增强,内皮细胞标志物KDR、v-WF染色结果阳性。胎盘来源间充质干细胞与丝素蛋白/羟基磷灰石材料复合培养植入后,4周时新骨已开始形成,12周时有部分新生骨组织形成板层骨,骨小梁形成,内可见新生血管形成,而对照组支架材料降解较慢,未见新生血管。说明胎盘来源间充质干细胞体外可以分化为血管内皮细胞,与丝素蛋白/羟基磷灰石材料联合移植能够促进移植物内血管生成,较好的修复骨缺损。     

内皮-间充质转化在肺动脉高压发病机制中的研究进展

正肺动脉高压(pulmonary hypertension,PH)是由多种已知和未知原因引起的肺循环血压异常升高的一种病理生理综合症,主要累及心血管及呼吸系统~([1-2])。根据2015年最新PH的临床分型,PH分为动脉型肺动脉高压(pulmonary arterial hypertension,PAH)、左心疾病所致PH、肺部疾病或缺氧所致PH、     

The role of vascular endothelial cells in tumor metastasis

Vascular endothelial cells (VECs) are an integral component of the inner lining of blood vessels, and their functions are essential for the proper functioning of the vascular system. The tight junctions formed by VECs act as a significant barrier to the intravasation and extravasation of tumor cells (TCs). In addition to that, the proliferation, activation, and migration of VECs play a vital role in the growth of new blood vessels, a process known as tumor angiogenesis, which is closely related to the malignant progression of tumors. However, during tumor progression, VECs undergo endothelial-to-mesenchymal transition (EndMT), which further promotes tumor progression. Furthermore, VECs act as the first line of defense against effector immune cells and help prevent immune cells from infiltrating into tumor tissues. VECs also secrete various cytokines that can contribute to regulating the stemness of tumor stem cells. Thus, it has been increasingly recognized that dysfunction of VECs is one of the key driving forces behind tumor metastasis, and therapeutic strategies targeting VECs have the potential to be an effective means of antitumor therapy. This review aims to present a comprehensive overview of the role and mechanisms of VECs in regulating tumor progression and metastasis, providing insights into the possibilities for the development of novel antitumor therapies that target VECs.     

Vaspin contributes to autophagy and endothelial-to-mesenchymal transition via PI3K-/AKT-mTOR pathway

BackgroundVisceral adipose tissue–derived serine protease inhibitor (Vaspin) was found to have anti-inflammatory, anti-apoptosis, and pro-autophagy activities. Our investigation is aimed to ascertain the effect of Vaspin on hypoxia-evoked endothelial-mesenchymal transition (EndMT) in human cardiac microvascular endothelial cells (HCMECs).MethodsIn vitro assays including CCK8, TUNEL, western blots, RT-qPCR to assess the effect of Vaspin on hypoxia-induced cell injuries, endothelial-mesenchymal transition (EndMT) and the inflammatory state in HCMECs. Transmission electron microscopy (TEM) was used to monitor autophagosome formation in HCMECs. The autophagy related proteins coupled with the critical effectors of PI3K/AKT-mTOR signaling pathway were also detected by western blots. In vivo assays including HE and ELISA to assess the effects of Vaspin on myocardial fibrosis pathology and type I and type III collagen in rats.ResultsVaspin pretreatment dramatically dose-dependently restored the proliferative impairment and the induced EndMT in HCMECs by hypoxia. The Vaspin-pretreated HCMECs also presented with attenuated expression of increased IL-1β, TNF-α and IL-6 by hypoxia a dose-dependent manner. Vaspin alleviated rat MF. The impact of Vaspin is also related to the increased autophagy and activated PI3K/AKT-mTOR signaling pathway. The protective and pro-autophagy activity of Vaspin was antagonized by the PI3K/AKT-mTOR inhibitor LY294002.ConclusionVaspin ameliorated the hypoxia-stimulated cell injuries and EndMT by activating autophagy via PI3K/AKT-mTOR signaling pathway.     

Inflammation induces endothelial-to-mesenchymal transition and promotes vascular calcification through downregulation of BMPR2

Endothelial-to-mesenchymal transition (EndMT) has been unveiled as a common cause for a multitude of human pathologies, including cancer and cardiovascular disease. Vascular calcification is a risk factor for ischemic vascular disorders and slowing calcification may reduce mortality in affected patients. The absence of early biomarkers hampers the identification of patients at risk. EndMT and vascular calcification are induced upon cooperation between distinct stimuli, including inflammatory cytokines and transforming growth factor beta (TGF-β) family members. However, how these signaling pathways interplay to promote cell differentiation and eventually vascular calcification is not well understood. Using in vitro and ex vivo analysis in animal models and patient-derived tissues, we have identified that the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) induce EndMT in human primary aortic endothelial cells, thereby sensitizing them for BMP-9-induced osteogenic differentiation. Downregulation of the BMP type II receptor BMPR2 is a key event in this process. Rather than compromising BMP canonical signal transduction, loss of BMPR2 results in decreased JNK signaling in ECs, thus enhancing BMP-9-induced mineralization. Altogether, our results point at the BMPR2–JNK signaling axis as a key pathway regulating inflammation-induced EndMT and contributing to calcification. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Impairment of endothelial‐mesenchymal transformation during atrioventricular cushion formation in Tmem100 null embryos

Background: Endothelial‐mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin‐dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up‐regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively‐active calcineurin A, suggesting dysregulation of myocardial calcineurin‐VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium‐derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial‐specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium‐related signaling events. Developmental Dynamics 244:31–42, 2015. © 2014 Wiley Periodicals, Inc.     

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Background: Endothelial‐mesenchymal transformation (EndMT) is essential for endocardial cushion formation during cardiac morphogenesis. We recently identified Tmem100 as an endothelial gene indispensable for vascular development. In this study, we further investigated its roles for EndMT during atrioventricular canal (AVC) cushion formation. Results: Tmem100 was expressed in AVC endocardial cells, and Tmem100 null embryos showed severe EndMT defect in the AVC cushions. While calcineurin‐dependent suppression of vascular endothelial growth factor (VEGF) expression in the AVC myocardium is important for EndMT, significant up‐regulation of Vegfa expression was observed in Tmem100 null heart. EndMT impaired in Tmem100 null AVC explants was partially but significantly restored by the expression of constitutively‐active calcineurin A, suggesting dysregulation of myocardial calcineurin‐VEGF signaling in Tmem100 null heart. Moreover, Tmem100 null endocardial cells in explant culture did not show EndMT in response to the treatment with myocardium‐derived growth factors, transforming growth factor β2 and bone morphogenetic protein 2, indicating involvement of an additional endocardial‐specific abnormality in the mechanism of EndMT defect. The lack of NFATc1 nuclear translocation in endocardial cells of Tmem100 null embryos suggests impairment of endocardial calcium signaling. Conclusions: The Tmem100 deficiency causes EndMT defect during AVC cushion formation possibly via disturbance of multiple calcium‐related signaling events. Developmental Dynamics 244:31–42, 2015. © 2014 Wiley Periodicals, Inc.     

Inflammatory Stress Exacerbates the Progression of Cardiac Fibrosis in High-Fat-Fed Apolipoprotein E Knockout Mice via Endothelial-Mesenchymal Transition

Background Chronic inflammation plays a crucial role in the progression of cardiac fibrosis. This study investigated whether inflammation exacerbated the progression of cardiac fibrosis in high-fat-fed apolipoprotein E knockout (ApoE KO) mice via endothelial-mesenchymal transition (EndMT).Methods Twenty-four male ApoE KO mice were divided into normal chow diet (Control), high-fat diet (HFD), or high-fat diet plus 10% casein injection (inflamed) groups for 8 weeks. The body weight of ApoE KO mice was measured at each week. The lipid profile and serum amyloid A (SAA) levels were examined using clinical biochemistry and enzyme-linked immunosorbent assays, respectively. Cardiac lipid and collagen accumulation was visualised with haematoxylin-eosin (HE) and Masson''s trichrome staining. EndMT-related molecule expression was examined by immunohistochemistry and Western blotting.Results SAA levels were increased in the inflamed group compared with the HFD and control groups, suggesting that inflammation was successfully induced. There were no differences in body weight among three groups at each week. Interestingly, inflammation significantly reduced serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels compared with the HFD mice. However, both foam cell formation in cardiac blood vessels and cardiac collagen deposition were increased in the inflamed group, as demonstrated by HE and Masson trichrome staining. Furthermore, inflammation reduced protein expression of CD31 and increased protein expression of alpha-smooth muscle actin (α-SMA) and collagen I, which contribute to cardiac EndMT.Conclusions Inflammatory stress exacerbates the progression of cardiac fibrosis in high-fat-fed ApoE KO mice via EndMT, suggesting that hyperlipidaemia and inflammation act synergistically to redistribute plasma lipids to cardiac tissues and accelerate the progression of cardiac fibrosis.