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Objective:To investigate the inhibitory effect and the possible mechanism of essential oil from fructus Alpinia zerumbet （EOFAZ） on endothelial-to-mesenchymal transition （EndMT） induced by high glucose （HG）. Method:Human umbilical vein endothelial cells （HUVECs） was cultured in vitro to analyze the pharmacodynamic effects of EOFAZ on EndMT and oxidative stress damage induced by HG. The experiment was set the blank group， HG group （35 mmol·L-1）， EOFAZ low dose group （1 μg·L-1） and EOFAZ high dose group （4 μg·L-1）. After EOFAZ intervention for 2 h， HG was added to incubate for 72 h in order to establish EndMT cell model. Western blot was used to detect the protein expression of vimentin and platelet endothelial cell adhesion molecule （CD31）. Angiogenesis experiment was used to detect the ability of cell migration ability in order to analyze the effect of EOFAZ on EndMT. The changes of reactive oxygen species （ROS） levels were detected by 2'，7'-dichlorodihydrofluorescein diacetate （DCFH-DA） fluorescence probe and the contents of malondialdehyde （MDA）， superoxide dismutase （SOD） and catalase （CAT） in cells were detected by the kit method to analyze the effect of EOFAZ on oxidative stress. Western blot was used to detect the protein expression levels of nuclear transcription factor E2 related factor 2 （Nrf2） and Notch1. The overexpression of Nrf2 was achieved by adenovirus （AD） transfection and the mechanism of EOFAZ inhibiting EndMT was further analyzed. The experiment was set the blank group， HG group （35 mmol·L-1）， AD-Nrf2 group， EOFAZ group （4 μg·L-1）， AD-Nrf2+EOFAZ group （4 μg·L-1）. The cells were infected with recombinant adenovirus overexpression plasmid of Nrf2 gene for 6 h， then replaced with normal medium for 24 h. After EOFAZ intervention for 2 h， HG was added to co-incubate for 72 h to induce EndMT. Western blot was used to detect the protein expressions of Nrf2， CD31， vimentin， Notch1 and Snail. Result:Compared with the HG group， after treatment with EOFAZ， the protein expression of CD31 was significantly up-regulated （P<0.05）， the protein expression of vimentin was significantly down-regulated （P<0.01）， the ability of cell migration was decreased （P<0.01）， and the contents of ROS and MDA were decreased （P<0.05， P<0.01）， the levels of CAT and SOD were increased （P<0.01）. In addition， EOFAZ could significantly up-regulate the protein expression of antioxidant signal Nrf2 （P<0.01） and down-regulate the protein expression of Notch1 （P<0.01）. High expression of Nrf2 was achieved by stable AD transfection into HUVECs. The results of Western blot showed that， compared with the HG group， the protein expression levels of Nrf2 and CD31 in each treatment group were significantly increased （P<0.01）， while the protein expression levels of vimentin， Notch1 and Snail were down-regulated （P<0.01）. At the same time， compared with the AD-Nrf2 group， the AD-Nrf2+EOFAZ group could further up-regulate the protein expressions of Nrf2 and CD31 （P<0.05， P<0.01）， while decrease the protein expression levels of vimentin， Notch1 and Snail （P<0.01）. Conclusion:EOFAZ ameliorates oxidative stress injury of vascular endothelial cells induced by HG and inhibits EndMT， which is related to Nrf2/Notch1 signaling pathway.
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Objective:To investigate the regulatory effect of serum containing Buyang Huanwu Tang on endothelial-to-mesenchymal transition(EndMT) in human pulmonary artery endothelial cells (HPAEC), and make further analysis on its mechanism from the perspective of the signal transduction of Jagged1/Notch1. Method:Rabbit serum containing Buyang Huanwu Tang was prepared by gavage with dosage of 53.36 g·kg-1·d-1, and blank serum was prepared by gavage with same volume of normal saline. The HPAECs cultured in vitro, EndMT model was established by the transforming growth factor-β1(TGF-β1) induced, which were divided into five groups:the control group (10%blank serum), the model group (10%blank serum+TGF-β1), the serum containing high-dose Buyang Huanwu Tang group (10%medicated serum + TGF-β1), the medium-dose group (5%medicated serum + 5%blank serum medicated + TGF-β1) and the low-dose group(2.5%medicated serum+7.5%blank serum+ TGF-β1). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) method. Cell migration was detected by transwell and scratch assay. The endothelial markers platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), vascular endothelial cadherin (VE-cadherin) and the mesenchymal markers fibroblast-specific protein 1 (FSP1), α-smooth muscle actin (α-SMA) were observed by immunofluorescence assay. The expression levels of Notch1, Jagged1 and CBF1 were detected by Western blot assay. Result:Compared with the control group, the proliferation and migration abilities of the HPAEC cells in model group were enhanced (Pα-SMA were increased. Further study found that the expressions of Notch1, Jagged1 and CBF1 were up-regulated (PPPα-SMA were on the decline. The expressions of Notch1, Jagged1 and CBF1 were also significantly lower than those in model group (PPConclusion:The serum containing Buyang Huanwu Tang can partly inhibit the EndMT in human pulmonary artery endothelial cells, which may be related to the regulation effect of Jagged1/Notch1 signaling.
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Objective@#To investigate the role of fibroblast growth factor receptor(FGFR) 1 in endothelial to-mesenchymal transition(EndMT) and epithelial-to-mesenchymal transition(EMT), and to find out a new strategy to study the vascular endothelial function of diabetic renal fibrosis.@*Methods@#Culture media from FRS2 knockdown HMVECs was transferred to HK-2 cells. Western blot and immunofluorescence staining were used to measure EMT markers and key moleculars of transforming growth factor(TGFβ).@*Results@#It was found that the medium from FRS2 siRNA-transfected HMVECs reduced E-cadherin protein levels, increased EMT markers levels, and activated TGFβ signal pathway in HK-2 cells.@*Conclusion@#Endothelial FGFR1 deficiency-induced EndMT leads to EMT in neighboring cells in a manner dependent on TGFβ1 signaling. Endothelial cell FGFR1 is an important molecule for maintaining endothelial homeostasis and epithelial homeostasis, and seems to be a key target for anti-diabetic renal fibrosis.
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Corneal endothelial cells (CECs) are essential for maintenance of corneal transparency.Proliferative potency of human CECs is poor in vivo,so endothelial dysfunction is irreversible.Many studies have showed that CECs are able to proliferate in vitro.However,endothelial to mesenchymal transition (EndMT) will emerge with CECs acquire a myofibroblastic phenotype and lose their specific markers,which hinders the development of corneal regenerative medicine.EndMT participates in corneal disorders,but its process has not been clarified yet.Now,researchers have found that the mechanism of EndMT may include transforming growth factor-β (TGF-β)signaling pathway,fibroblast growth factors (FGFs),and Notch signaling pathway.Therefore,there are several ways to prevent EndMT,such as inhibiting TGF-β signaling pathway,using anti-phosphatidylinositol 3-kinase antibody,small interfering RNA (siRNA) against pl20,Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and LGR5 ligand,inhibiting Notch signaling pathway,making connexin43 knockdown,inhibiting matrix metalloproteinase activity and using Asc-2P.This review elaborated the mechanism of EndMT and ways to prevent or reverse it.
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OBJECTIVE To investigate the pharmacological effect and mechanism of Salvianolic acid A (SAA) on pulmonary vascular remodeling. METHODS In current study, we conducted a series of experiments to clarify the effect of SAA,a kind of polyphenol compound,in the process of EndMT in human pulmonary arterial endothelial cells and in vivo therapeutic efficacy on vascular remodeling in monocrotaline (MCT)-induced EndMT. EndMT was also induced by TGF-β1in human pulmonary arterial endothelial cells(HPAECs) in vitro.RESULTS SAA significantly attenuated EndMT,simul-taneously inhibited cell migration and reactive oxygen species(ROS)formation.In MCT-induced pulmonary arterial hypertension(PAH)model,SAA improved vascular function,decreased TGF-β1level and inhib-ited inflammation. Mechanistically, SAA stimulated Nrf2 translocation and subsequent heme oxygen-ase-1(HO-1)up-regulation.The effect of SAA on EndMT in vitro was abolished by ZnPP,a HO-1 inhibitor. CONCLUSION This study indicates a deleterious impact of oxidative stress on EndMT. Polyphenol antioxidant treatment may provide an adjunctive action to alleviate pulmonary vascular remodeling via inhibiting EndMT.
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Pulmonary artery hypertension is a disease with complicated pathogenesis, which is characterized by enhanced pulmonary artery constriction and arterial wall remodeling, leading to progressive increase of pulmonary vascular resistance and pulmonary artery pressure, then resulting in right heart failure.Many studies have shown that transforming growth factor-β1(TGF-β1) plays an important role in the development of various diseases, especially in cardiovascular and cerebrovascular diseases.TGF-β1 is involved in multiple cellular responses including cell proliferation, differentiation, migration and apoptosis.TGF-β1 participates in pulmonary artery hypertension mainly via promoting the proliferation of pulmonary artery smooth muscle cells as well as inducing the deposition of extracellular matrix and endothelial-to-mesenchymal transition(EndMT) through many signaling, which is mainly dominated by pulmonary artery smooth muscle cells and pulmonary artery endothelial cells.This review mainly introduces the role of TGF-β1 in pulmonary artery hypertension in order to provide potential drug targets and therapeutic strategies for pulmonary artery hypertension.
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Endothelial to mesenchymal transition(EndMT) plays a major role during organism development, and also contributes to several adult cardiovascular diseases.EndMT-derived fibroblast-like cells are common in atherosclerotic lesions.Pro-atherosclerosis factors, such as oxidative stress, hypoxia, inflammatory cytokines and oscillatory fluid shear stress can promote EndMT.EndMT is closely associated with plaque calcification, and unstable and ruptured plaque phenotype that may prone to cause clinical events.EndMT may be another key step in the prevention and treatment of atherosclerosis.Here, we reviewed the role played by endothelial-to-mesenchymal transition(EndMT) and its key regulators in atherosclerosis.
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OBJECTIVE To investigate the effect of asiaticoside (AS) on endothelial-to-mesenchymal transition (EndoMT) in hypoxia pulmonary hypertension (HPH). METHODS Male Sprague-Dawley (SD) rats were divided into normoxia control group, hypoxia model group, and AS 25 and 50 mg · kg-1 group. Hypoxia model group and AS group were subjected to intermittent hypoxia exposure. Control group and model group received 1-1.5 mL saline daily, and AS groups were ig administrated with AS 25 and 50 mg·kg-1 for 4 weeks. Human pulmonary artery endothelial cells (HPAECs) were divided into normoxia control group and hypoxia AS groups. Hypoxia groups were cultured with AS 0, 25, 50, 100 and 200 mg·L-1 for 72 h under hypoxic (5%O2, 5%CO2) conditions. Anti-proliferation effect of AS was investigated by CCK-8 assay. Then, HPAECs were divided into normoxia control group, normoxia AS 100 mg · L-1 group, hypoxia model group, and hypoxia AS 100 mg · L-1 group. After five days of culture, migration ability of cells was detected by Transwell test. Expression of CD31 andα-SMA was detected by immunofluorescence and Western blotting in both in vivo and in vitro experiments. RESULTS In both in vivo and in vitro experiments, compared with normoxia control group, expression of CD31 was reduced (P<0.01) andα-smooth muscle actin (α-SMA) was increased (P<0.01) in hypoxia model group in both immunofluorescent analysis and Western blotting. Compared with hypoxia model group, expression of CD31 was increased andα-SMA was decreased (P<0.05, P<0.01) in AS treatment groups. Compared with normoxia control group, proliferation and migration ability of HPAEC were elevated in hypoxia model group (P<0.05). Compared with hypoxia group, AS 100 mg · L-1 depressed proliferation and migration of HPAEC under hypoxia exposure up to 72 h (P<0.05). CONCLUSION EndoMT might be involved in HPH and could be partly inhibited by AS.
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Objective To investigate the effectsof quercetin on human umbilical vein endothelial cell (HUVEC)-12 undergoing endothelial-to-mesenchymal transition (EndMT) induced by TGF-β1; To discuss its mechanism of action. Methods Cell activity of intervening by quercetin with different concentrations and TGF-β1 for 72 h was detected by CCK-8 method;RT-PCR and immunofluorescence staining were used to detect the transition of endothelial and stromal markers; Western blot was used to detect the signal transduction pathway; RT-PCR was performed to detect the transcription factors that play crucial roles in the process of transformation. Results The results of RT-PCR and immunofluorescence double staining showed that TGF-β1 (10 ng/mL) stimulated HUVEC-12 cells for 72 h to induce fibroblast phenotype, showing more interstitial markers and less endothelial markers;Western blot and RT-PCR results showed that quercetin inhibited the phosphorylation of smad2/3 in a concentration-dependent manner;After TGF-β1 stimulation, the downstream transcription factors EndMT of snail1, twist1, twist2, ZEB1, and ZEB2 significantly increased, while 100 μmol/L quercetin could down-regulate the five downstream transcription factors. Conclusion Quercetin has anti-fibrosis effects through inhibiting HUVEC-12 cells undergoing EndMT.
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BACKGROUND: Soluble epoxide hydrolase (sEH) expressed by endothelial cells catalyzes the metabolism of epoxyeicosatrienoic acids (EETs), which are vasoactive agents. METHODS: We used a unilateral ureteral obstruction mouse model of kidney fibrosis to determine whether inhibition of sEH activity reduces fibrosis, the final common pathway for chronic kidney disease. RESULTS: sEH activity was inhibited by continuous release of the inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) for 1 or 2 weeks. Treatment with AUDA significantly ameliorated tubulointerstitial fibrosis by reducing fibroblast mobilization and enhancing endothelial cell activity. In an in vitro model of endothelial-to-mesenchymal transition (EndMT) using human vascular endothelial cells (HUVECs), AUDA prevented the morphologic changes associated with EndMT and reduced expression of fibroblast-specific protein 1. Furthermore, HUVECs activated by AUDA prevented the epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in a co-culture system. CONCLUSION: Our findings suggest that regulation of sEH is a potential target for therapies aimed at delaying the progression of kidney fibrosis by inhibiting EndMT and EMT.
Subject(s)
Animals , Humans , Mice , Coculture Techniques , Endothelial Cells , Epithelial Cells , Epithelial-Mesenchymal Transition , Fibroblasts , Fibrosis , In Vitro Techniques , Kidney , Metabolism , Renal Insufficiency, Chronic , Ureteral ObstructionABSTRACT
BACKGROUND: Soluble epoxide hydrolase (sEH) expressed by endothelial cells catalyzes the metabolism of epoxyeicosatrienoic acids (EETs), which are vasoactive agents. METHODS: We used a unilateral ureteral obstruction mouse model of kidney fibrosis to determine whether inhibition of sEH activity reduces fibrosis, the final common pathway for chronic kidney disease. RESULTS: sEH activity was inhibited by continuous release of the inhibitor 12-(3-adamantan-1-ylureido)-dodecanoic acid (AUDA) for 1 or 2 weeks. Treatment with AUDA significantly ameliorated tubulointerstitial fibrosis by reducing fibroblast mobilization and enhancing endothelial cell activity. In an in vitro model of endothelial-to-mesenchymal transition (EndMT) using human vascular endothelial cells (HUVECs), AUDA prevented the morphologic changes associated with EndMT and reduced expression of fibroblast-specific protein 1. Furthermore, HUVECs activated by AUDA prevented the epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells in a co-culture system. CONCLUSION: Our findings suggest that regulation of sEH is a potential target for therapies aimed at delaying the progression of kidney fibrosis by inhibiting EndMT and EMT.
Subject(s)
Animals , Humans , Mice , Coculture Techniques , Endothelial Cells , Epithelial Cells , Epithelial-Mesenchymal Transition , Fibroblasts , Fibrosis , In Vitro Techniques , Kidney , Metabolism , Renal Insufficiency, Chronic , Ureteral ObstructionABSTRACT
Recent studies have demonstrated that endothelial-to-mesenchymal transition ( EndMT) plays a pivotal role in the cardiovascular system, spanning cardiovascular development through to cardiovascular diseases (CVD).In this review, we describe the common signaling mechanisms that promote EndMT participating in the pathological process of CVD, and we discuss how these in-tracellular cascades participate in crosstalk to integrate cues to reverse EndMT, which potentially may provide novel therapeutic oppor-tunities for CVD.
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Cardiovascular complications dominate the landscape of chronic kidney diseases (CKD). Endothelial cell dysfunction (ECD) is a well-known culprit of cardiovascular morbidity and it develops in CKD with remarkable frequency. This brief overview of ECD in CKD scans two decades of studies performed in my laboratory, from genetic analyses to proteomic and metabolomics screens. I provide a detailed description of findings related to the premature senescence of endothelial cells, cell transition from the endothelial to mesenchymal phenotype, and stages of development of ECD. Clinical utility of some of these findings is illustrated with data on laser-Doppler flowmetry and imaging in patients with CKD. Some currently available and emerging therapeutic options for the management of ECD are briefly presented.
Subject(s)
Humans , Aging , Endothelial Cells , Laser-Doppler Flowmetry , Metabolomics , Phenotype , Renal Insufficiency, Chronic , Retrospective StudiesABSTRACT
Objective To observe hypoxia-induced pulmonary arterial endothelial-to-mesenchymal transition and investigate the role of transforming growth factor β1 (TGF-β1) in the process. Methods Pulmonary arterial cells improved by adherence method were cultured in normoxia (containing 21%O2,5%CO2 and 74%N2) or hypoxia (containing 1%O2,5%CO2 and 94%N2) for 1,4,or 7 days,respectively. Endothelial-to-mesenchymal transition was confirmed with morphological observation and expression of α-smooth muscle actin (α-SMA) by immunocytochemistry. Expression of TGF-β1 was evaluated by RT-PCR and Western blot,and α-SMA by Western blot. Results Hypoxia-induced paving-stone-like pulmonary arterial endothelial cells transdifferentiating to polygonal cells with high-expression of α-SMA. TGF-β1 expression was increased significantly after 7 days of hypoxia. TGF-β1 stimulating alone increasedα-SMA expression of pulmonary arterial en-dothelial cells;while,SD-208,inhibitor of TGF-β1,abolished the above effect. Conclusion Hypoxia can induce endothelial-to-mesenchymal transition. And TGF-β1 plays an important role in the process.