Abstract MP02: Angiotensin II Mediates Microvascular Rarefaction
            In Vivo
            and Exacerbates Endothelial-To-Mesenchymal Transition
            In Vitro

Cardiac fibrosis accompanies numerous cardiovascular diseases (CVD) such as hypertension and myocardial infarction and increases myocardial stiffness leading to contractile dysfunction. Recently, endothelial-to-mesenchymal transition (EndMT) has been shown to contribute to myocardial fibrosis. EndMT describes a process by which endothelial cells transform into mesenchymal cells such as fibroblasts and has been implicated in many fibrotic diseases. Angiotensin II (AngII) plays a key role in myocardial fibrosis and has been associated with the activation of fibroblasts to myofibroblasts and an increase in myocardial collagen deposition. Here, we assessed the role of AngII in capillary loss and EndMT in vivo and in vitro . C57BL/6J mice were infused with H 2 O (control) or 24μg/kg/hr AngII for 4 weeks. Mice infused with AngII developed significant cardiac fibrosis characterised by the deposition of collagen I (2.5-fold vs. control; p<0.05) and III (1.9-fold vs. control; p<0.05). Capillary density was assessed by CD31 immunohistochemistry and revealed significant vascular rarefaction (control 2161±111 vs . AngII 838±132 capillaries/mm 2 ; p<0.05). To investigate whether AngII can induce EndMT in vitro , human coronary artery endothelial cells were stimulated with 10ng/mL TGFβ 1 alone or in combination with 1μM AngII for 10 days. AngII significantly enhanced TGFβ 1 -induced gene expression of α-smooth muscle actin (TGFβ 1 1.8-fold; TGFβ 1 ±AngII 4.3-fold vs . control; p<0.05) and collagen I (TGFβ 1 9.2-fold; TGFβ 1 +AngII 30.2-fold vs . control; p<0.05). Concomitantly, AngII significantly increased α-smooth muscle actin protein expression (TGFβ 1 3.9-fold; TGFβ 1 +AngII 23.6-fold vs . control; p<0.05) and significantly decreased CD31 expression (TGFβ 1 0.9-fold; TGFβ 1 +AngII 0.7-fold vs . control; p<0.05), suggesting AngII acts in concert with TGFβ 1 to enhance conversion of endothelial cells to myofibroblasts. Further studies investigating the underlying mechanism, including the role of the Smad pathway, are ongoing. These results demonstrate that AngII induces vascular rarefaction in vivo and potentiates TGFβ 1 -induced EndMT in vitro. Understanding the molecular basis for these observations may help to identify new therapeutic options in CVD.

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Evodiamine Prevents Isoproterenol-Induced Cardiac Fibrosis by Regulating Endothelial-to-Mesenchymal Transition

Planta Medica ◽

10.1055/s-0042-124044 ◽

2016 ◽

Vol 83 (09) ◽

pp. 761-769 ◽

Cited By ~ 13

Author(s):

Xiao-han Jiang ◽

Qing-qing Wu ◽

Yang Xiao ◽

Yuan Yuan ◽

Zheng Yang ◽

...

Keyword(s):

Smooth Muscle ◽

Cardiac Fibrosis ◽

Transforming Growth Factor ◽

Smooth Muscle Actin ◽

Transforming Growth Factor Β1 ◽

Heart Weight ◽

Muscle Actin ◽

Mesenchymal Transition ◽

Expression Levels ◽

Endothelial To Mesenchymal Transition

AbstractEvodiamine, a major component of Evodia rutaecarpa, can protect the myocardium against injury induced by atherosclerosis and ischemia-reperfusion. However, the effect of evodiamine against cardiac fibrosis remains unclear. This study aims to investigate the possible effect and mechanism involved in the function of evodiamine on isoproterenol-induced cardiac fibrosis and endothelial-to-mesenchymal transition. Isoproterenol was used to induce cardiac fibrosis in mice, and evodiamine was gavaged simultaneously. After 14 days, cardiac function was accessed by echocardiography. The extent of cardiac fibrosis and hypertrophy was evaluated by pathological and molecular analyses. The extent of endothelial-to-mesenchymal transition was evaluated by the expression levels of CD31, CD34, α-smooth muscle actin, and vimentin by immunofluorescence staining and Western blot analysis. After 14 days, the heart weight/body weight ratio and heart weight/tibia length ratio revealed no significant difference between the isoproterenol group and the isoproterenol/evodiamine-treated groups, whereas the increased heart weight was reduced in the isoproterenol/evodiamine-treated groups. Echocardiography revealed that interventricular septal thickness and left ventricular posterior wall thickness at the end diastole decreased in the evodiamine-treated groups. Evodiamine reduced isoproterenol-induced cardiac fibrosis as accessed by normalization in collagen deposition and gene expression of hypertrophic and fibrotic markers. Evodiamine also prevented endothelial-to-mesenchymal transition as evidenced by the increased expression levels of CD31 and CD34, decreased expression levels of α-smooth muscle actin and vimentin, and increased microvascular density in the isoproterenol/evodiamine-treated mice hearts. Furthermore, isoproterenol-induced activation of transforming growth factor-β1/Smad signal was also blunted by evodiamine. Therefore, evodiamine may prevent isoproterenol-induced cardiac fibrosis by regulating endothelial-to-mesenchymal transition, which is probably mediated by the blockage of the transforming growth factor-β1/Smad pathway.

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

Cell Death and Disease ◽

10.1038/s41419-021-03750-4 ◽

2021 ◽

Vol 12 (5) ◽

Author(s):

Lu Zhang ◽

Jiaqi He ◽

Junyan Wang ◽

Jing Liu ◽

Zixin Chen ◽

...

Keyword(s):

Therapeutic Strategy ◽

Cardiac Fibrosis ◽

Smooth Muscle Actin ◽

Heart Tissue ◽

Mesenchymal Transition ◽

Glycation End Products ◽

Endothelial To Mesenchymal Transition ◽

Related Proteins

AbstractEndothelial-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.

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Endothelial-to-Mesenchymal Transition in Human Adipose Tissue Vasculature Alters the Particulate Secretome and Induces Endothelial Dysfunction

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.119.312826 ◽

2019 ◽

Vol 39 (10) ◽

pp. 2168-2191 ◽

Cited By ~ 3

Author(s):

Bronson A. Haynes ◽

Li Fang Yang ◽

Ryan W. Huyck ◽

Eric J. Lehrer ◽

Joshua M. Turner ◽

...

Keyword(s):

Adipose Tissue ◽

Endothelial Dysfunction ◽

Smooth Muscle Actin ◽

Phenotypic Change ◽

Alpha Smooth Muscle Actin ◽

Mesenchymal Transition ◽

Molecular Features ◽

Endothelial To Mesenchymal Transition

Objective: Endothelial cells (EC) in obese adipose tissue (AT) are exposed to a chronic proinflammatory environment that may induce a mesenchymal-like phenotype and altered function. The objective of this study was to establish whether endothelial-to-mesenchymal transition (EndoMT) is present in human AT in obesity and to investigate the effect of such transition on endothelial function and the endothelial particulate secretome represented by extracellular vesicles (EV). Approach and Results: We identified EndoMT in obese human AT depots by immunohistochemical co-localization of CD31 or vWF and α-SMA (alpha-smooth muscle actin). We showed that AT EC exposed in vitro to TGF-β (tumor growth factor-β), TNF-α (tumor necrosis factor-α), and IFN-γ (interferon-γ) undergo EndoMT with progressive loss of endothelial markers. The phenotypic change results in failure to maintain a tight barrier in culture, increased migration, and reduced angiogenesis. EndoMT also reduced mitochondrial oxidative phosphorylation and glycolytic capacity of EC. EVs produced by EC that underwent EndoMT dramatically reduced angiogenic capacity of the recipient naïve ECs without affecting their migration or proliferation. Proteomic analysis of EV produced by EC in the proinflammatory conditions showed presence of several pro-inflammatory and immune proteins along with an enrichment in angiogenic receptors. Conclusions: We demonstrated the presence of EndoMT in human AT in obesity. EndoMT in vitro resulted in production of EV that transferred some of the functional and metabolic features to recipient naïve EC. This result suggests that functional and molecular features of EC that underwent EndoMT in vivo can be disseminated in a paracrine or endocrine fashion and may induce endothelial dysfunction in distant vascular beds.

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Role of endothelial cells in regulating hemoglobin-induced changes in lymphatic pumping

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1992.263.6.h1880 ◽

1992 ◽

Vol 263 (6) ◽

pp. H1880-H1887 ◽

Cited By ~ 1

Author(s):

R. M. Elias ◽

J. Eisenhoffer ◽

M. G. Johnston

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Inhibitory Effect ◽

Direct Inhibitory Effect ◽

Induced Changes ◽

Lymphatic Pumping ◽

Pumping Activity

Studies with a sheep isolated duct preparation in vivo demonstrated that the route of administration of hemoglobin was important in demonstrating its inhibitory effect on lymphatic pumping. With autologous oxyhemoglobin administered intravenously (final plasma concentration 5 x 10(-5) M), pumping was not inhibited. However, the addition of oxyhemoglobin (5 x 10(-5) M) into the reservoir (lumen of the duct) resulted in > 95% inhibition of pumping. The extraluminal administration of oxyhemoglobin (10(-5) M) to bovine mesenteric lymphatics in vitro resulted in a 40% inhibition of pumping, whereas the introduction of oxyhemoglobin (10(-5) M) into the lumen of the vessels suppressed pumping 95%. In vessels mechanically denuded of endothelium, intraluminal oxyhemoglobin inhibited pumping 50%. These results suggested that oxyhemoglobin depressed pumping through an effect on both smooth muscle and endothelium. Once pumping was inhibited with oxyhemoglobin administration, stimulation of the duct with elevations in transmural pressure restored pumping activity when endothelial cells were present. However, in the absence of endothelium, pumping decreased with increases in distending pressures. We conclude that oxyhemoglobin has a direct inhibitory effect on lymphatic smooth muscle. The ability of oxyhemoglobin to alter the pressure range over which the lymph pump operates appears to be dependent on an intact endothelium.

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Endothelial ERK1/2 signaling maintains integrity of the quiescent endothelium

Journal of Experimental Medicine ◽

10.1084/jem.20182151 ◽

2019 ◽

Vol 216 (8) ◽

pp. 1874-1890 ◽

Cited By ~ 13

Author(s):

Nicolas Ricard ◽

Rizaldy P. Scott ◽

Carmen J. Booth ◽

Heino Velazquez ◽

Nicholas A. Cilfone ◽

...

Keyword(s):

Rapid Onset ◽

Cardiac Conduction ◽

Tgfβ Signaling ◽

Mesenchymal Transition ◽

Endothelin 1 ◽

Endocrine Organs ◽

Endothelial To Mesenchymal Transition

To define the role of ERK1/2 signaling in the quiescent endothelium, we induced endothelial Erk2 knockout in adult Erk1−/− mice. This resulted in a rapid onset of hypertension, a decrease in eNOS expression, and an increase in endothelin-1 plasma levels, with all mice dying within 5 wk. Immunostaining and endothelial fate mapping showed a robust increase in TGFβ signaling leading to widespread endothelial-to-mesenchymal transition (EndMT). Fibrosis affecting the cardiac conduction system was responsible for the universal lethality in these mice. Other findings included renal endotheliosis, loss of fenestrated endothelia in endocrine organs, and hemorrhages. An ensemble computational intelligence strategy, comprising deep learning and probabilistic programing of RNA-seq data, causally linked the loss of ERK1/2 in HUVECs in vitro to activation of TGFβ signaling, EndMT, suppression of eNOS, and induction of endothelin-1 expression. All in silico predictions were verified in vitro and in vivo. In summary, these data establish the key role played by ERK1/2 signaling in the maintenance of vascular normalcy.

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Transcriptional regulation of endothelial-to-mesenchymal transition in cardiac fibrosis: role of myocardin-related transcription factor A and activating transcription factor 3

Canadian Journal of Physiology and Pharmacology ◽

10.1139/cjpp-2016-0634 ◽

2017 ◽

Vol 95 (10) ◽

pp. 1263-1270 ◽

Cited By ~ 9

Author(s):

Vibhuti Sharma ◽

Nilambra Dogra ◽

Uma Nahar Saikia ◽

Madhu Khullar

Keyword(s):

Endothelial Cells ◽

Transcription Factor ◽

Cardiac Fibrosis ◽

Activating Transcription Factor 3 ◽

Mesenchymal Transition ◽

Related Transcription Factor ◽

Endothelial To Mesenchymal Transition ◽

Activating Transcription Factor ◽

Transcription Factor 3

The etiology of cardiac fibrogenesis is quite diverse, but a common feature is the presence of activated fibroblasts. Experimental evidence suggests that a subset of cardiac fibroblasts is derived via transition of vascular endothelial cells into fibroblasts by endothelial-to-mesenchymal transition (EndMT). During EndMT, endothelial cells lose their endothelial characteristics and acquire a mesenchymal phenotype. Molecular mechanisms and the transcriptional mediators controlling EndMT in heart during development or disease remain relatively undefined. Myocardin-related transcription factor A facilitates the transcription of cytoskeletal genes by serum response factor during fibrosis; therefore, its specific role in cardiac EndMT might be of importance. Activation of activating transcription factor 3 (ATF-3) during cardiac EndMT is speculative, since ATF-3 responds to a transforming growth factor β (TGF-β) stimulus and controls the expression of the primary epithelial-to-mesenchymal transition markers Snail, Slug, and Twist. Although the role of TGF-β in EndMT-mediated cardiac fibrosis has been established, targeting of the TGF-β ligand has not proven to be a viable anti-fibrotic strategy owing to the broad functional importance of this ligand. Thus, targeting of downstream transcriptional mediators may be a useful therapeutic approach in attenuating cardiac fibrosis. Here, we discuss some of the transcription factors that may regulate EndMT-mediated cardiac fibrosis and their involvement in type 2 diabetes.

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Combination of 13-Cis Retinoic Acid and Lovastatin: Marked Antitumor Potential In Vivo in a Pheochromocytoma Allograft Model in Female Athymic Nude Mice

Endocrinology ◽

10.1210/en.2014-1027 ◽

2014 ◽

Vol 155 (7) ◽

pp. 2377-2390 ◽

Cited By ~ 8

Author(s):

Svenja Nölting ◽

Alessio Giubellino ◽

Yasin Tayem ◽

Karen Young ◽

Michael Lauseker ◽

...

Keyword(s):

Smooth Muscle ◽

Retinoic Acid ◽

Smooth Muscle Actin ◽

Treated Group ◽

Muscle Actin ◽

Tumor Mass ◽

Viable Tumor ◽

Over Time

Currently, there are no reliably effective therapeutic options for metastatic pheochromocytoma (PCC) and paraganglioma. Moreover, there are no therapies that may prevent the onset or progression of tumors in patients with succinate dehydrogenase type B mutations, which are associated with very aggressive tumors. Therefore, we tested the approved and well-tolerated drugs lovastatin and 13-cis-retinoic acid (13cRA) in vitro in an aggressive PCC mouse cell line, mouse tumor tissue-derived (MTT) cells, and in vivo in a PCC allograft nude mouse model, in therapeutically relevant doses. Treatment was started 24 hours before sc tumor cell injection and continued for 30 more days. Tumor sizes were measured from outside by caliper and sizes of viable tumor mass by bioluminescence imaging. Lovastatin showed antiproliferative effects in vitro and led to significantly smaller tumor sizes in vivo compared with vehicle treatment. 13cRA promoted tumor cell growth in vitro and led to significantly larger viable tumor mass and significantly faster increase of viable tumor mass in vivo over time compared with vehicle, lovastatin, and combination treatment. However, when combined with lovastatin, 13cRA enhanced the antiproliferative effect of lovastatin in vivo. The combination-treated mice showed slowest tumor growth of all groups with significantly slower tumor growth compared with the vehicle-treated mice and significantly smaller tumor sizes. Moreover, the combination-treated group displayed the smallest size of viable tumor mass and the slowest increase in viable tumor mass over time of all groups, with a significant difference compared with the vehicle- and 13cRA-treated group. The combination-treated tumors showed highest extent of necrosis, lowest median microvessel density and highest expression of α-smooth muscle actin. The combination of high microvessel density and low α-smooth muscle actin is a predictor of poor prognosis in other tumor entities. Therefore, this drug combination may be a well-tolerated novel therapeutic or preventive option for malignant PCC.

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MIR503HG Loss Promotes Endothelial-to-Mesenchymal Transition in Vascular Disease

Circulation Research ◽

10.1161/circresaha.120.318124 ◽

2021 ◽

Author(s):

João P. Monteiro ◽

Julie Rodor ◽

Axelle Caudrillier ◽

Jessica P Scanlon ◽

Ana-Mishel Spiroski ◽

...

Keyword(s):

Endothelial Cells ◽

Pulmonary Hypertension ◽

Molecular Mechanisms ◽

Clinical Samples ◽

Vascular Remodelling ◽

Mesenchymal Transition ◽

Polypyrimidine Tract Binding Protein ◽

Endothelial To Mesenchymal Transition

Rationale: Endothelial-to-mesenchymal transition (EndMT) is a dynamic biological process involved in pathological vascular remodelling. However, the molecular mechanisms that govern this transition remain largely unknown, including the contribution of long non-coding RNAs (lncRNAs). Objective: To investigate the role of lncRNAs in EndMT and their relevance to vascular remodelling. Methods and Results: To study EndMT in vitro, primary endothelial cells (EC) were treated with transforming growth factor-β2 and interleukin-1β. Single-cell and bulk RNA-sequencing were performed to investigate the transcriptional architecture of EndMT and identify regulated lncRNAs. The functional contribution of seven lncRNAs during EndMT was investigated based on a DsiRNA screening assay. The loss of lncRNA MIR503HG was identified as a common signature across multiple human EC types undergoing EndMT in vitro. MIR503HG depletion induced a spontaneous EndMT phenotype, while its overexpression repressed hallmark EndMT changes, regulating 29% of its transcriptome signature. Importantly, the phenotypic changes induced by MIR503HG were independent of miR-424 and miR-503, which overlap the lncRNA locus. The pathological relevance of MIR503HG down-regulation was confirmed in vivo using Sugen/Hypoxia (SuHx)-induced pulmonary hypertension (PH) in mouse, as well as in human clinical samples, in lung sections and blood outgrowth endothelial cells (BOECs) from pulmonary arterial hypertension (PAH) patients. Overexpression of human MIR503HG in SuHx mice led to reduced mesenchymal marker expression, suggesting MIR503HG therapeutic potential. We also revealed that MIR503HG interacts with the Polypyrimidine Tract Binding Protein 1 (PTB1) and regulates its protein level. PTBP1 regulation of EndMT markers suggests that the role of MIR503HG in EndMT might be mediated in part by PTBP1. Conclusions: This study reports a novel lncRNA transcriptional profile associated with EndMT and reveals the crucial role of the loss of MIR503HG in EndMT and its relevance to pulmonary hypertension.

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