Endothelial cells are activated during hypoxia via endoglin/ALK-1/SMAD1/5 signaling in vivo and in vitro

doi:10.1016/j.bbrc.2009.12.170

Biochemical and Biophysical Research Communications

Volume 392, Issue 3, 12 February 2010, Pages 283-288

https://doi.org/10.1016/j.bbrc.2009.12.170 Get rights and content

Abstract

Endoglin (ENG) promotes angiogenesis by enhancing activation of TGF-β type I receptors ALK-1 and ALK-5. ALK-1 phosphorylates transcription factors SMAD1/5, which bind to BMP-responsive elements (BRE), whereas ALK-5 phosphorylates SMAD3, which binds to CAGA elements. Expression of ENG is increased during myocardial infarction (MI). We investigated which ENG signaling pathway is activated in endothelial cells during hypoxia. Expression of ENG, ALK-1, ALK-5, and phosphorylated SMAD1/3/5 by immunostaining and immunoblotting in a mouse model of myocardial infarction (MI) and in hypoxic human aortic endothelial cells (HAECs) was evaluated. Activation of BRE and CAGA was measured by luciferase assays in cells transfected with plasmids expressing ENG or ALK-1 and the number of cells was quantified. mRNA expression of the target genes of TGF-β signaling, ID1 and BCL-X, was quantified by real-time RT-PCR. Expression of ENG, ALK-1 and phosphorylated SMAD1/5, but not ALK-5 or phosphorylated SMAD3, was significantly increased in hypoxic endothelial cells in vivo and in vitro. Overexpression of both ENG and ALK-1 significantly increased BRE but not CAGA activity, expression of ID1 and BCL-X and the number of HAECs at hypoxia. ENG/ALK-1 signaling is one of the factors that regulate endothelial cell activity during adaptive cardiac angiogenesis.

Introduction

Myocardial infarction (MI) leads to impaired perfusion of the heart resulting in hypoxic injury that, in turn, promotes formation of new vessels by inducing a wide spectrum of angiogenic genes [1]. Newly formed vessels allow increased blood flow, thus restoring the oxygen supply to the affected myocardium to salvage ischemic heart damage. Endothelial cells are the major players in the process of angiogenesis. Studies determining mechanisms that dictate proliferation, migration, and remodeling of differentiated endothelial cells located in peri-infarct areas are of a great interest to clinical cardiology.

Transforming growth factor-β (TGF-β) plays important roles not only in cellular growth and development, but also in angiogenesis by binding to specific serine/threonine kinase receptors [2]. Endoglin (ENG), a homodimeric transmembrane glycoprotein, is an accessory TGF-β receptor and is predominantly expressed by vascular endothelial cells where it regulates endothelial cell proliferation and migration that are crucial for angiogenesis [3]. ENG forms complexes with two different type I receptors expressed by endothelial cells, a restrictly expressed activin receptor-like kinase-1 (ALK-1) and a broadly expressed ALK-5, to modulate angiogenesis by regulating TGF-β/ALK signaling. After activating these receptors, signals are transduced from the membrane to the nucleus via phosphorylation of transcriptional factors (called SMADs) that are translocated into the nucleus to regulate the transcriptional activity of targeted genes. ALK-1 activation induces phosphorylation of SMAD1/5 and has been proposed to stimulate endothelial cell proliferation and migration, whereas ALK-5 activation phosphorylates SMAD2/3, which has been shown to inhibit these processes [4]. Thus, it seems that expression of genes downstream of TGF-β in endothelial cells may be modulated differently under certain circumstances.

Recent reports indicate that hypoxia increases expression of ENG in endothelial cells [5] and in infarcted murine hearts [6]. However, the preferentially activated signaling pathway downstream of ENG in endothelial cells during MI has not yet been determined. Therefore, we conducted the present study to evaluate the expression of ENG and ALK receptors, as well as phosphorylation of SMADs, in infarcted murine hearts and confirmed our findings in hypoxic endothelial cells. Following activation of the ENG/ALK-1 signaling pathway, we studied the activity of SMAD binding elements, expression of some target genes, and cell proliferation in hypoxic endothelial cells. Our studies indicated that increased expression of ENG promotes expression of ALK-1, but not ALK-5, in MI and hypoxic endothelial cells. In addition, activation of BRE elements was induced as a consequence of increased phosphorylation of SMAD1/5. Finally, expression of ENG and ALK-1 regulated proliferation of endothelial cells. Our results from both in vivo and in vitro studies converge to indicate a specific ENG/ALK-1/SMAD1/5 signaling pathway in regulating endothelial cell activity during MI.

Section snippets

Materials and methods

Mouse model of myocardial infarction. Male C57BL/6 mice aged 8–10 weeks (M&B, Ejby, Denmark) were housed in a temperature-controlled room at 21 ± 2 °C under a constant 12:12-h light–dark cycle with access to standard laboratory food and water. Myocardial infarction (MI) was induced by permanent ligation of the left coronary artery as described previously [7]. Age-matched control mice were submitted to the same surgery with the exception of coronary artery ligation and defined as a sham control

Hypoxia increases expression of ENG in vivo and in vitro

Immunohistochemical staining of left ventricle sections with an anti-ENG antibody 1 week after MI showed that ENG was strongly expressed in peri-infarct areas (Fig. 1A and B). Vessels growing into the infarct core from the rim of infarction also strongly expressed ENG (Fig. 1C). In serial sections from left ventricles 1 week after MI, ENG co-localized with the endothelial cell surface marker CD31 (Fig. 1D), demonstrating that ENG was expressed by the vascular endothelium. Expression of ENG was

Discussion

In this study, we show that expression of ENG, ALK-1 and SMAD1/5 increases in infarcted mouse ventricles and in hypoxic endothelial cells in vitro. By contrast, expression of ALK-5 and SMAD3 does not increase in infarcted mouse ventricles. We analyzed promoter activation downstream of ENG signaling in endothelial cells and show that hypoxia or overexpression of ENG increased the activity of BRE but not CAGA. Overexpression of ENG also increases expression of ID1 and BCL-X, target genes of

Acknowledgments

The authors thank Dr. Rosie Perkins for editing the manuscript, Dr. Carmelo Bernabeu for plasmid vector expressing ENG, and Dr. Kohei Miyazono for plasmid vector driving luciferase expression under the control of BRE or CAGA. This work was supported by the Swedish Research Council, the Swedish Heart-Lung Foundation, the Göran Gustafsson Foundation, the Swedish Foundation for Strategic Research, and the Sahlgrenska Hospital Funds.

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In infarcted and failing hearts, TGF-β superfamily members play an important role in regulation of inflammatory, reparative, fibrogenic, and hypertrophic responses through activation of Smad-dependent and Smad-independent cascades. This review manuscript discusses the mechanisms of regulation and role of Smad pathways in myocardial infarction and in heart failure. Cardiomyocyte-specific Smad1 activation exerts protective anti-apoptotic actions following ischemia/reperfusion. In contrast, the role of the Smad1/5/8 cascade in reparative, immune, and vascular cells infiltrating the infarcted heart is unknown. Smad3, but not Smad2 is implicated in repair of the infarcted heart, by activating reparative myofibroblasts and by promoting anti-inflammatory transition in macrophages. However, prolonged activation of Smad3 may promote adverse remodeling and fibrosis. The inhibitory Smad, Smad7 restrains TGF-β-induced fibroblast activation, but also exerts TGF-independent actions through inhibition of receptor tyrosine kinase signaling. Cell-specific approaches targeting Smad pathways may hold therapeutic promise in myocardial infarction and in heart failure.
The role of endothelial miRNAs in myocardial biology and disease
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Evidence provided by overexpression and knockdown studies in mice and zebrafish demonstrate that miR-26a impairs myocardial angiogenesis by targeting the bone morphogenetic protein (BMP) – SMAD1 – inhibitor of DNA binding 1 (Id1) axis [69]. Repression of SMAD1 expression leads to decreased levels of the Id1 transcription factor [70], and accordingly to the inhibition of growth and migration of ECs due to changes in the p21WAF/CIP/p27 cell cycle inhibitor [71–73]. Furthermore, in vitro studies show that miR-26a targets the Nogo B receptor (NgBR), thus, affects NgBR – NO – VEGF axis leading to attenuated VEGF-mediated angiogenesis [74].
The myocardium is a highly structured pluricellular tissue which is governed by an intricate network of intercellular communication. Endothelial cells are the most abundant cell type in the myocardium and exert crucial roles in both healthy myocardium and during myocardial disease. In the last decade, microRNAs have emerged as new actors in the regulation of cellular function in almost every cell type. Here, we review recent evidence on the regulatory function of different microRNAs expressed in endothelial cells, also called endothelial microRNAs, in healthy and diseased myocardium. Endothelial microRNA emerged as modulators of angiogenesis in the myocardium, they are implicated in the paracrine role of endothelial cells in regulating cardiac contractility and homeostasis, and interfere in the crosstalk between endothelial cells and cardiomyocytes.
Soluble endoglin modulates the pro-inflammatory mediators NF-κB and IL-6 in cultured human endothelial cells
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Membrane endoglin can bind TGF-β1 and TGF-β3 isoforms in association with TGF-β receptors II and I (ALKs) and form a functional receptor complex [11]. Moreover, in vitro studies demonstrated that membrane endoglin affects TGF-β signaling by modulating the activity of Smad transcription factors [12,13]. Several studies have revealed an interplay between two signaling pathways involving membrane endoglin.
Endoglin is a transmembrane glycoprotein, that plays an important role in regulating endothelium. Proteolytic cleavage of membrane endoglin releases soluble endoglin (sEng), whose increased plasma levels have been detected in diseases related to the cardiovascular system. It was proposed that sEng might damage vascular endothelium, but detailed information about the potential mechanisms involved is not available. Thus, we hypothesized that sEng contributes to endothelial dysfunction, leading to a pro-inflammatory phenotype by a possible modulation of the TGF-β and/or inflammatory pathways.
Human umbilical vein endothelial cells (HUVECs) and Human embryonic kidney cell line (HEK293T) were treated with different sEng concentration and time in order to reveal possible effect on biomarkers of inflammation and TGF-β signaling. IL6 and NFκB reporter luciferase assays, quantitative real-time PCR analysis, Western blot analysis and immunofluorescence flow cytometry were used.
sEng treatment results in activation of NF-κB/IL-6 expression, increased expression of membrane endoglin and reduced expression of Id-1. On the other hand, no significant effects on other markers of endothelial dysfunction and inflammation, including eNOS, peNOS^S1177, VCAM-1, COX-1, COX-2 and ICAM-1 were detected.
As a conclusion, sEng treatment resulted in an activation of NF-κB, IL-6, suggesting activation of pro-inflammatory phenotype in endothelial cells. The precise mechanism of this activation and its consequence remains to be elucidated. A combined treatment of sEng with other cardiovascular risk factors will be necessary in order to reveal whether sEng is not only a biomarker of cardiovascular diseases, but also a protagonist of endothelial dysfunction.
TGF-β signalling in tumour associated macrophages
2017, Immunobiology
Tumour associated macrophages (TAM) represent an important component of tumour stroma. They develop under the influence of tumour microenvironment where transforming growth factor (TGF)β is frequently present. Activities of TAM regulated by TGFβ stimulate proliferation of tumour cells and lead to tumour immune escape. Despite high importance of TGFβ-induction of TAM activities till now our understanding of the mechanism of this induction is limited. We have previously developed a model of type 2 macrophages (M2) resembling certain properties of TAM. We established that in M2 TGFβRII is regulated on the level of subcellular sorting by glucocorticoids. Further studies revealed that in M2 with high levels of TGFβRII on the surface TGFβ activates not only its canonical Smad2/3-mediated signaling, but also Smad1/5-mediated signaling, what is rather typical for bone morphogenetic protein (BMP) stimulation. Complexity of macrophage populations, however, allows assumption that TGFβ signalling may function in different ways depending on the functional state of the cell. To understand the peculiarities of TGFβ signalling in human TAMs experimental systems using primary cells have to be developed and used together with the modern mathematical modelling approaches.
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Angiogenesis is essential for mammalian development and tissue homeostasis, and is involved in several pathological processes, including tumor growth and dissemination. Many factors within the tissue microenvironment are known to modulate angiogenesis, including cytokines, such as transforming growth factor beta (TGFβ), and oxygen level. TGFβ exists in three different isoforms (1, 2 and 3), all of which (albeit in different contexts) might mediate angiogenesis and are able to induce endothelial–mesenchymal transition (EndoMT), a process involved in heart development, pathologic fibrosis and, as recently reported, in angiogenesis. Low oxygen level, referred to as hypoxia, has been independently shown to induce angiogenesis, modulate TGFβ signalling and promote EndoMT. However, how these phenomena might be interconnected to drive angiogenesis is rather unexplored. To begin addressing the potential contribution of TGFβ-induced EndoMT to angiogenesis, and to explore how microenvironmental hypoxia might influence these processes, we investigated the effect of TGFβ isoforms 1 and 2 on early EndoMT response in cultured adult endothelium under standard (21 %) and hypoxic (1 %) culture conditions. Our data indicates that EndoMT-like changes, such as an increase in expression and nuclear translocation of Snail, Slug and Zeb1, and reduction of VE-cadherin expression, occur in response to TGFβ1 and/or TGFβ2 as early as 6 h after stimulation and might be enhanced by hypoxia in an isoform-specific manner. Further, hypoxia enhances canonical TGFβ signalling, and appears to be a key determinant of Snail's differential involvement in endothelial cell responses to TGFβ1 versus TGFβ2.
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Its expression has been shown to increase during myocardial infarction (MI). Endothelial cells are activated during hypoxia via endoglin/ALK-1/SMAD1/5 signaling in vivo and in vitro, thus regulating endothelial cell activity during adaptive cardiac angiogenesis (Tian et al., 2010). Nrp1 was shown to act as a co-receptor for VEGF protein family (Grunewald et al., 2010).
Various preconditioning strategies influence regeneration properties of stem cells. Preconditioned stem cells generally show better cell survival, increased differentiation, enhanced paracrine effects, and improved homing to the injury site by regulating the expression of tissue-protective cytokines and growth factors. In this study, we analyzed gene expression pattern of growth factors through RT-PCR after treatment of mesenchymal stem cells (MSCs) with a metabolic inhibitor, 2,4 dinitrophenol (DNP) and subsequent re-oxygenation for periods of 2, 6, 12 and 24 h. These growth factors play important roles in cardiomyogenesis, angiogenesis and cell survival. Mixed pattern of gene expression was observed depending on the period of re-oxygenation. Of the 13 genes analyzed, ankyrin repeat domain 1 (Ankrd1) and GATA6 were downregulated after DNP treatment and subsequent re-oxygenations. Ankrd1 expression was, however, increased after 24 h of re-oxygenation. Placental growth factor (Pgf), endoglin (Eng), neuropilin (Nrp1) and jagged 1 (Jag1) were up-regulated after DNP treatment. Gradual increase was observed as re-oxygenation advances and by the end of the re-oxygenation period the expression started to decrease and ultimately regained normal values. Epiregulin (Ereg) was not expressed in normal MSCs but its expression increased gradually from 2 to 24 h after re-oxygenation. No change was observed in the expression level of connective tissue growth factor (Ctgf) at any time period after re-oxygenation. Kindlin3, kinase insert domain receptor (Kdr), myogenin (Myog), Tbx20 and endothelial tyrosine kinase (Tek) were not expressed either in normal cells or cells treated with DNP. It can be concluded from the present study that MSCs adjust their gene expression levels under the influence of DNP induced metabolic stress. Their levels of expression vary with varying re-oxygenation periods. Preconditioning of MSCs with DNP can be used for enhancing the potential of these cells for better regeneration.

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¹: These authors contributed equally to this work.

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Endothelial cells are activated during hypoxia via endoglin/ALK-1/SMAD1/5 signaling in vivo and in vitro

Abstract

Introduction

Section snippets

Materials and methods

Hypoxia increases expression of ENG in vivo and in vitro

Discussion

Acknowledgments

Trends Cardiovasc. Med.

Mech. Dev.

J. Biol. Chem.

Anal. Biochem.

Trends Cell Biol.

Cancer Cell

Therapeutic angiogenesis and vasculogenesis for ischemic disease. Part I: angiogenic cytokines

Circulation

TGF-β receptor function in the endothelium

Cardiovasc. Res.

Interaction and functional interplay between endoglin and ALK-1, two components of the endothelial transforming growth factor-β receptor complex

J. Cell. Physiol.

Endoglin has a crucial role in blood cell-mediated vascular repair

Circulation

Protein disulfide isomerase increases in myocardial endothelial cells in mice exposed to chronic hypoxia: a stimulatory role in angiogenesis

Am. J. Physiol. Heart Circ. Physiol.