Inhibition of prostaglandin E2 receptor 4 by lnc000908 to promote the endothelial‐mesenchymal transition participation in cardiac remodelling

Long non‐coding RNAs (lncRNAs) have emerged as potent regulators of cardiac disease; however, the role of lncRNA in cardiac fibrosis remains partially understood. In this study, we identified a cardiac endothelial‐enriched lncRNA‐lnc000908, which was markedly up‐regulated in rats with cardiac fibrosis. In addition, the expression of prostaglandin E2 receptor 4 (EP4) was decreased in cardiac fibrosis. In vivo lnc000908 silencing by lentivirus increased the EP4 level, decreased endothelial‐mesenchymal transition (EndMT) and improved cardiac fibrosis and cardiac function. Consistently, the lnc000908 knockdown also up‐regulated EP4 and suppressed transforming growth factor‐beta (TGF‐β)‐induced EndMT in cardiac microvascular endothelial cells. In contrast, the lnc000908 overexpression by lentivirus decreased the EP4 level and induced EndMT. Of note, these pro‐ or anti‐EndMT effects were reversed by the EP4 overexpression or the EP4 antagonist AH‐23848, respectively. This study demonstrates that lnc000908 is a novel regulator of cardiac fibrosis by modulating the EP4 expression and EndMT.     

Inhibition of miR‐21 alleviated cardiac perivascular fibrosis via repressing EndMT in T1DM

In type 1 and type 2 diabetes mellitus, increased cardiac fibrosis, stiffness and associated diastolic dysfunction may be the earliest pathological phenomena in diabetic cardiomyopathy. Endothelial‐mesenchymal transition (EndMT) in endothelia cells (ECs) is a critical cellular phenomenon that increases cardiac fibroblasts (CFs) and cardiac fibrosis in diabetic hearts. The purpose of this paper is to explore the molecular mechanism of miR‐21 regulating EndMT and cardiac perivascular fibrosis in diabetic cardiomyopathy. In vivo, hyperglycaemia up‐regulated the mRNA level of miR‐21, aggravated cardiac dysfunction and collagen deposition. The condition was recovered by inhibition of miR‐21 following with improving cardiac function and decreasing collagen deposition. miR‐21 inhibition decreased cardiac perivascular fibrosis by suppressing EndMT and up‐regulating SMAD7 whereas activating p‐SMAD2 and p‐SMAD3. In vitro, high glucose (HG) up‐regulated miR‐21 and induced EndMT in ECs, which was decreased by inhibition of miR‐21. A highly conserved binding site of NF‐κB located in miR‐21 5′‐UTR was identified. In ECs, SMAD7 is directly regulated by miR‐21. In conclusion, the pathway of NF‐κB/miR‐21/SMAD7 regulated the process of EndMT in T1DM, in diabetic cardiomyopathy, which may be regarded as a potential clinical therapeutic target for cardiac perivascular fibrosis.     

MicroRNA-92a -mediated endothelial to mesenchymal transition controls vein graft neointimal lesion formation

PurposeLong-term failure of vein grafts due to neointimal hyperplasia remains an important problem in coronary artery bypass graft surgery. Endothelial to mesenchymal transition (EndMT) contributes to vein graft vascular remodeling. However, there is little study on microRNA-mediated EndMT contributions to neointimal formation in vein graft. We hypothesized that microRNA-92a (miR-92a) might play an important role in determining EndMT contributions to neointimal formation.MethodsmiR-92a and EndMT-related proteins detected by qRT-PCR and Western blot in vitro and in vivo. Adeno-associated virus 6 (AAV6) delivery gene therapy was used to inhibit neointimal formation in vivo. The intimal hyperplasia of vein grafts was measured by HE staining, the expression of EndMT-related protein in vein grafts was measured by immunofluorescence. Immunohistochemistry and luciferase assay were used to detect potential targets of miR-92a.ResultsThe expression of miR-92a was found to be upregulated in neointimal hyperplasic lesions after vein grafting. Using cultured human umbilical vein endothelial cells (HUVECs), we show that TGF-β1 treatment of HUVECs significantly increased miR-92a expression and induced EndMT, characterized by suppression of endothelial-specific markers (CD31 and VE-cadherin) and an increase in mesenchymal-specific markers (a-SMA and vimentin), while inhibition of miR-92a expression blunted EndMT in cultured HUVECs. Furthermore, AAV6 mediated miR-92a suppression gene therapy effectively resulted in decreased EndMT and less neointimal formation in vein grafts in vivo. We further identified that integrin alpha 5 (ITGA5) is a potential target gene involved in the development of neointima formation in these vein grafts.ConclusionThis data suggests that neointimal formation does not solely rely on vascular smooth muscle cell phenotypic switching but is also related to EndMT, and miR-92a-mediated EndMT is an important mechanism underlying neointimal formation in vein grafts.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

SIRT1 inhibits TGF‐β‐induced endothelial‐mesenchymal transition in human endothelial cells with Smad4 deacetylation

Endothelial‐mesenchymal transition (EndMT) plays a pivotal role in organ fibrosis. This study examined the effect of SIRT1 on transforming growth factor beta (TGF‐β)‐induced EndMT in human endothelial cells (ECs) and its probable molecular mechanism. We assessed EndMT by immunofluorescence staining, quantitative real‐time polymerase chain reaction, Western blotting, and migration and invasion assays. Adenovirus was used to overexpress or knockdown SIRT1 in ECs. The regulatory relationship between SIRT1 and Smad4 was analyzed by coimmunoprecipitation assay. We found that SIRT1 was decreased in TGF‐β‐induced EndMT, and SIRT1 inhibited TGF‐β‐induced EndMT through deacetylating Smad4. Our findings suggest that SIRT1 has an important role in inhibiting EndMT by regulating the TGF‐β/Smad4 pathway in human ECs and, thus, protecting against fibrosis.     

miR-448-3p alleviates diabetic vascular dysfunction by inhibiting endothelial–mesenchymal transition through DPP-4 dysregulation

Diabetes mellitus (DM) often causes vascular endothelial damage and alters vascular microRNA (miR) expression. miR-448-3p has been reported to be involved in the development of DM, but whether miR-448-3p regulates diabetic vascular endothelial dysfunction remains unclear. To investigate the molecular mechanism of diabetic vascular endothelial dysfunction and the role of miR-448-3p therein, Sprague-Dawley rats were injected with streptozotocin (STZ) to establish diabetic animal model and the rat aortic endothelial cells were treated with high glucose to establish diabetic cell model. For the treatment group, after the induction of diabetes, the miR-448-3p levels in vivo and in vitro were upregulated by adeno-associated virus serotype 2 (AAV2)-miR-448-3p injection and miR-448-3p mimic transfection, respectively. Our results showed that AAV2-miR-448-3p injection alleviated the body weight loss and blood glucose level elevation induced by STZ injection. The miR-448-3p level was significantly decreased and the dipeptidyl peptidase-4 (DPP-4) messenger RNA level was increased in diabetic animal and cell models, which was reversed by miR-448-3p treatment. Moreover, the diabetic rats exhibited endothelial damage and endothelial–mesenchymal transition (EndMT), while AAV2-miR-448-3p injection relieved those situations. In vitro experiments demonstrated that miR-448-3p overexpression in endothelial cells alleviated endothelial damage by inhibiting EndMT through blocking the transforming growth factor-β/Smad pathway. We further proved that miR-448-3p negatively regulated DPP-4 by binding to its 3′-untranslated region, and DPP-4 overexpression reversed the effect of miR-448-3p overexpression on EndMT. Overall, we conclude that miR-448-3p overexpression inhibits EndMT via targeting DPP-4 and further ameliorates diabetic vascular endothelial dysfunction, indicating that miR-448-3p may serve as a promising therapeutic target for diabetic endothelial dysfunction.     

Knockout RAGE alleviates cardiac fibrosis through repressing endothelial-to-mesenchymal transition (EndMT) mediated by autophagy

Endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to cardiac fibrosis and heart failure (HF). Recent studies have demonstrated that EndMT is regulated by autophagy, and we previously showed suppression of excessive autophagy and alleviation of cardiac fibrosis in HF mice with inactivated receptor for advanced glycation end products (RAGE). Thus, we investigated whether reduced cardiac fibrosis due to RAGE knockout occurred by inhibiting EndMT mediated by excessive autophagy. We found a decrease in endothelial cells (CD31⁺/VE-Cadherin⁺) and an increase in cells co-expressing CD31 and α-smooth muscle actin (α-SMA, myofibroblast marker) at 8 weeks in heart tissue of mice subjected to transverse aortic constriction (TAC), which implied EndMT. Knockout RAGE decreased EndMT accompanied by decreased expression of autophagy-related proteins (LC3BII/I and Beclin 1), and alleviated cardiac fibrosis and improved cardiac function in TAC mice. Moreover, 3-methyladenine (3-MA) and chloroquine (CQ), inhibitors of autophagy, attenuated EndMT, and cardiac fibrosis in TAC mice. Importantly, EndMT induced by AGEs could be blocked by autophagy inhibitor in vivo and in vitro. These results suggested that AGEs/RAGE-autophagy-EndMT axis involved in the development of cardiac fibrosis and knockout RAGE ameliorated cardiac fibrosis through decreasing EndMT regulated by autophagy, which could be a promising therapeutic strategy for HF.Subject terms: Heart failure, Experimental models of disease     

The role of endothelial-mesenchymal transition in development and pathological process

Epithelial-mesenchymal transition is an important developmental process, participates in tumor's formation, invasion, and metastasis and has been extensively studied. Recently, endothelial-mesenchymal transition (EndMT), a newly recognized type of cellular transdifferentiation, has been demonstrated to participate in a number of diseases by causing morphology changes and pathological processes. Previous studies showed that EndMT was a critical process of embryonic cardiac development. Not only that recent advances also suggested that EndMT occurred postnatally in cancer and cardiac fibrosis and emerged as a possible source of cancer-associated fibroblasts (CAFs). CAFs were found to acquire properties that promoted tumor development and metastasis formation. Resident endothelial cells undergoing EndMT lose their endothelial markers, acquire a mesenchymal or myofibroblastic phenotype, express mesenchymal cell products such as α-smooth muscle actin and type I collagen and develop invasive and migratory abilities. EndMT-derived cells are believed to function as fibroblasts in damaged tissue and may therefore have an important role in pathological process. However, little is known about the signaling mechanisms that cause endothelial cells to transform into mesenchymal cells. Transforming growth factor-β, Notch, or other signaling pathways could direct or interact to mediate EndMT. Therefore, to explore the signaling mechanisms of EndMT may provide novel therapeutic strategies for treating cancer. ? 2012 IUBMB IUBMB Life, 64(9): 717-723, 2012.     

Cigarette smoke extract induces HO‐1 expression in mouse cerebral vascular endothelial cells: Involvement of c‐Src/NADPH oxidase/PDGFR/JAK2/STAT3 pathway

Several chemicals present in cigarette smoke (CS) have been reported to induce heme oxygenase‐1 (HO‐1) expression, which represents a prime defense mechanism in protecting the cells from stress‐dependent adverse effects on peripheral vascular system. However, the effects of cigarette smoke extract (CSE) on HO‐1 induction and the mechanisms underlying CSE‐induced HO‐1 expression in brain vessels are not completely understood. Here, we used a mouse brain endothelial cell culture (bEnd.3) to investigate the effect of CSE on HO‐1 induction and the mechanisms underlying CSE‐induced HO‐1 expression in cerebral vessels. We demonstrated that sublethal concentrations of CSE (30 µg/ml) induced submaximal HO‐1 expression in bEnd.3 cells. NADPH oxidase‐dependent ROS generation played a key role in CSE‐induced HO‐1 expression. CSE‐induced HO‐1 expression was mediated through PDGFR/JAK2/STAT3 cascade, which was observed by pretreatment with the respective pharmacological inhibitors or transfection with PDGFR shRNA. CSE activated NADPH oxidase through c‐Src in bEnd.3 cells. Taken together, these results suggested that, in bEnd.3 cells, CSE‐induced HO‐1 expression was mediated through PDGFR/JAK2/STAT3 cascade, which was regulated by c‐Src or c‐Src activated‐NADPH oxidase/ROS. J. Cell. Physiol. 225: 741–750, 2010. © 2010 Wiley‐Liss, Inc.     

Identification of human placenta‐derived mesenchymal stem cells involved in re‐endothelialization

Human placenta is an attractive source of mesenchymal stem cells (MSC) for regenerative medicine. The cell surface markers expressed on MSC have been proposed as useful tools for the isolation of MSC from other cell populations. However, the correlation between the expression of MSC markers and the ability to support tissue regeneration in vivo has not been well examined. Here, we established several MSC lines from human placenta and examined the expression of their cell surface markers and their ability to differentiate toward mesenchymal cell lineages. We found that the expression of CD349/frizzled‐9, a receptor for Wnt ligands, was positive in placenta‐derived MSC. So, we isolated CD349‐negative and ‐positive fractions from an MSC line and examined how successfully cell engraftment repaired fractured bone and recovered blood flow in ischemic regions using mouse models. CD349‐negative and ‐positive cells displayed a similar expression pattern of cell surface markers and facilitated the repair of fractured bone in transplantation experiments in mice. Interestingly, CD349‐negative, but not CD349‐positive cells, showed significant effects on recovering blood flow following vascular occlusion. We found that induction of PDGFβ and bFGF mRNAs by hypoxia was greater in CD349‐negative cells than in CD349‐positive cells while the expression of VEGF was not significantly different in CD349‐negative and CD349‐positive cells. These findings suggest the possibility that CD349 could be utilized as a specialized marker for MSC isolation for re‐endothelialization. J. Cell. Physiol. 226: 224–235, 2010. © 2010 Wiley‐Liss, Inc.     

Role of endothelial‐to‐mesenchymal transition induced by TGF‐β1 in transplant kidney interstitial fibrosis

Chronic allograft dysfunction (CAD) induced by kidney interstitial fibrosis is the main cause of allograft failure in kidney transplantation. Endothelial‐to‐mesenchymal transition (EndMT) may play an important role in kidney fibrosis. We, therefore, undertook this study to characterize the functions and potential mechanism of EndMT in transplant kidney interstitial fibrosis. Proteins and mRNAs associated with EndMT were examined in human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor‐beta1 (TGF‐β1) at different doses or at different intervals with western blotting, qRT‐PCR and ELISA assays. Cell motility and migration were evaluated with motility and migration assays. The mechanism of EndMT induced by TGF‐β1 was determined by western blotting analysis of factors involved in various canonical and non‐canonical pathways. In addition, human kidney tissues from control and CAD group were also examined for these proteins by HE, Masson's trichrome, immunohistochemical, indirect immunofluorescence double staining and western blotting assays. TGF‐β1 significantly promoted the development of EndMT in a time‐dependent and dose‐dependent manner and promoted the motility and migration ability of HUVECs. The TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways were found to be associated with the pathogenesis of EndMT induced by TGF‐β1, which was also proven in vivo by the analysis of specimens from the control and CAD groups. EndMT may promote transplant kidney interstitial fibrosis by targetting the TGF‐β/Smad and Akt/mTOR/p70S6K signalling pathways, and hence, result in the development of CAD in kidney transplant recipients.     

内皮细胞向间充质细胞的转分化

内皮细胞向间充质细胞转分化(endothelial-to-mesenchymal transition,EndMT)过程中,内皮从内皮层分离,获得间充质细胞表型,其特征是细胞连接能力下降,内皮标志物减少,获得间充质细胞标志物,并具有扩散和迁移特性。在受损组织中,内皮细胞能够通过EndMT转分化为成纤维细胞,因此具有EndMT特性的内皮细胞在组织重构和纤维化中发挥重要作用。EndMT不仅参与心脏发育这一重要过程也参与许多病理性疾病如癌症,心脏的纤维化和肺动脉高压等。所以,靶向EndMT对多种疾病具有潜在的治疗意义。     

Differentiation of human umbilical cord Wharton's jelly‐derived mesenchymal stem cells into germ‐like cells in vitro

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly‐derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all‐trans retinoic acid, testosterone and testicular‐cell‐conditioned medium prepared from newborn male mouse testes. HUMSCs formed “tadpole‐like” cells after induction with different reagents and showed both mRNA and protein expression of germ‐cell‐specific markers Oct4 (POUF5), Ckit, CD49_f (α6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage. J. Cell. Biochem. 109: 747–754, 2010. © 2010 Wiley‐Liss, Inc.     

Irisin inhibits high glucose‐induced endothelial‐to‐mesenchymal transition and exerts a dose‐dependent bidirectional effect on diabetic cardiomyopathy

Emerging evidence indicates that irisin provides beneficial effects in diabetes. However, whether irisin influences the development of diabetic cardiomyopathy (DCM) remains unclear. Therefore, we investigated the potential role and mechanism of action of irisin in diabetes‐induced myocardial dysfunction in mice. Type 1 diabetes was induced in mice by injecting streptozotocin, and the diabetic mice were administered recombinant r‐irisin (low or high dose: 0.5 or 1.5 μg/g body weight/day, I.P.) or PBS for 16 weeks. Irisin treatment did not alter blood glucose levels in the diabetic mice. However, the results of echocardiographical and histopathological assays indicated that low‐dose irisin treatment alleviated cardiac fibrosis and left ventricular function in the diabetic mice, whereas high‐dose irisin failed to mitigate the ventricular function impairment and increased collagen deposition. The potential mechanism underlying the effect of low‐dose irisin involved irisin‐mediated inhibition of high glucose‐induced endothelial‐to‐mesenchymal transition (EndMT); conversely, high‐dose irisin treatment enhanced high glucose‐induced MMP expression by stimulating MAPK (p38 and ERK) signalling and cardiac fibroblast proliferation and migration. Low ‐ dose irisin alleviated DCM development by inhibiting high glucose‐induced EndMT. By contrast, high‐dose irisin disrupted normal MMP expression and induced cardiac fibroblast proliferation and migration, which results in excess collagen deposition. Thus, irisin can inhibit high glucose‐induced EndMT and exert a dose‐dependent bidirectional effect on DCM.     

ALK2使内皮细胞转化为类干细胞的机制

内皮细胞向间充质细胞转分化（endothelial-to-mesenchymal transition,EndMT）过程是血管内皮细胞转化为类干细胞的基础,持续激活状态的ALK2在EndMT过程起到关键的促进作用,EndMT发生后内皮细胞能获得类干细胞的表现型.TGF-β2和BMP4作为特异性配体激活ALK2,下调某些内皮细胞标志物并上调某些间质细胞标志物,促进EndMT过程.此外,编码ALK2的基因序列在206号氨基酸上突变形成R206H ALK2后作用类似持续激活状态ALK2,同样也能达到促进内皮细胞向间充质细胞转分化的目的.