Publications by authors named "Carmelo Bernabeu"

107 Publications

Pregnancy-Induced High Plasma Levels of Soluble Endoglin in Mice Lead to Preeclampsia Symptoms and Placental Abnormalities.

Authors:
Lucía Pérez-Roque Elena Núñez-Gómez Alicia Rodríguez-Barbero Carmelo Bernabéu José M López-Novoa Miguel Pericacho

Int J Mol Sci 2020 Dec 26;22(1). Epub 2020 Dec 26.

Institute of Biomedical Research of Salamanca (IBSAL), 37007 Salamanca, Spain.

Preeclampsia is a pregnancy-specific disease of high prevalence characterized by the onset of hypertension, among other maternal or fetal signs. Its etiopathogenesis remains elusive, but it is widely accepted that abnormal placentation results in the release of soluble factors that cause the clinical manifestations of the disease. An increased level of soluble endoglin (sEng) in plasma has been proposed to be an early diagnostic and prognostic biomarker of this disease. A pathogenic function of sEng involving hypertension has also been reported in several animal models with high levels of plasma sEng not directly dependent on pregnancy. The aim of this work was to study the functional effect of high plasma levels of sEng in the pathophysiology of preeclampsia in a model of pregnant mice, in which the levels of sEng in the maternal blood during pregnancy replicate the conditions of human preeclampsia. Our results show that wild type pregnant mice carrying human sEng-expressing transgenic fetuses (f()) present high plasma levels of sEng with a timing profile similar to that of human preeclampsia. High plasma levels of human sEng (hsEng) are associated with hypertension, proteinuria, fetal growth restriction, and the release of soluble factors to maternal plasma. In addition, f() mice also present placental alterations comparable to those caused by the poor remodeling of the spiral arteries characteristic of preeclampsia. In vitro and ex vivo experiments, performed in a human trophoblast cell line and human placental explants, show that sEng interferes with trophoblast invasion and the associated pseudovasculogenesis, a process by which cytotrophoblasts switch from an epithelial to an endothelial phenotype, both events being related to remodeling of the spiral arteries. Our findings provide a novel and useful animal model for future research in preeclampsia and reveal a much more relevant role of sEng in preeclampsia than initially proposed.
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http://dx.doi.org/10.3390/ijms22010165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795873PMC
December 2020

Potential Second-Hits in Hereditary Hemorrhagic Telangiectasia.

Authors:
Carmelo Bernabeu Pinar Bayrak-Toydemir Jamie McDonald Michelle Letarte

J Clin Med 2020 Nov 5;9(11). Epub 2020 Nov 5.

Molecular Medicine, Hospital for Sick Children, and Department of Immunology, University of Toronto, Toronto, ON M5G 0A4, Canada.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant genetic disorder that presents with telangiectases in skin and mucosae, and arteriovenous malformations (AVMs) in internal organs such as lungs, liver, and brain. Mutations in (endoglin), (ALK1), and (Smad4) genes account for over 95% of HHT. Localized telangiectases and AVMs are present in different organs, with frequencies which differ among affected individuals. By itself, HHT gene heterozygosity does not account for the focal nature and varying presentation of the vascular lesions leading to the hypothesis of a "second-hit" that triggers the lesions. Accumulating research has identified a variety of triggers that may synergize with HHT gene heterozygosity to generate the vascular lesions. Among the postulated second-hits are: mechanical trauma, light, inflammation, vascular injury, angiogenic stimuli, shear stress, modifier genes, and somatic mutations in the wildtype HHT gene allele. The aim of this review is to summarize these triggers, as well as the functional mechanisms involved.
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http://dx.doi.org/10.3390/jcm9113571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7694477PMC
November 2020

Differential Expression of Circulating Plasma miRNA-370 and miRNA-10a from Patients with Hereditary Hemorrhagic Telangiectasia.

Authors:
Lidia Ruiz-Llorente Virginia Albiñana Luisa M Botella Carmelo Bernabeu

J Clin Med 2020 Sep 3;9(9). Epub 2020 Sep 3.

Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant, vascular disorder that presents with telangiectases and arteriovenous malformations. HHT is a genetically heterogeneous disorder, involving mutations in endoglin (; HHT1) and activin receptor-like kinase 1 (/; HHT2) genes that account for over 85% of all HHT patients. The current diagnosis of HHT patients remains at the clinical level, but many suspected patients do not have a clear HHT diagnosis or do not show pathogenic mutations in HHT genes. This situation has prompted the search for biomarkers to help in the early diagnosis of the disease. We have analyzed the plasma levels in HHT patients of selected micro-RNAs (miRNAs), small single-stranded RNAs that regulate gene expression at the transcriptional level by interacting with specific RNA targets. A total of 16 HHT1 and 17 HHT2 plasma samples from clinically confirmed patients and 16 controls were analyzed in this study. Total RNA was purified from plasma, and three selected miRNAs (miRNA-10a, miRNA-214, and miRNA-370), related to the pathobiology of cardiovascular diseases and potentially targeting or , were measured by quantitative polymerase chain reaction. Compared with controls, levels of miRNA-370, whose putative target is , were significantly downregulated in HHT1, but not in HHT2, whereas the levels of miRNA-10a, whose putative target is , were significantly upregulated in HHT2, but not in HHT1. In addition, the levels of miRNA-214, potentially targeting and did not change in either HHT1 or HHT2 patients versus control samples. While further studies are warranted, these results suggest that dysregulated plasma levels of miRNA-370 or miRNA-10a could help to identify undiagnosed HHT1 or HHT2 patients, respectively.
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http://dx.doi.org/10.3390/jcm9092855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565099PMC
September 2020

Review of Pharmacological Strategies with Repurposed Drugs for Hereditary Hemorrhagic Telangiectasia Related Bleeding.

Authors:
Virginia Albiñana Angel M Cuesta Isabel de Rojas-P Eunate Gallardo-Vara Lucía Recio-Poveda Carmelo Bernabéu Luisa María Botella

J Clin Med 2020 Jun 6;9(6). Epub 2020 Jun 6.

Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 9 Ramiro de Maeztu Street, 28040 Madrid, Spain.

The diagnosis of hereditary hemorrhagic telangiectasia (HHT) is based on the Curaçao criteria: epistaxis, telangiectases, arteriovenous malformations in internal organs, and family history. Genetically speaking, more than 90% of HHT patients show mutations in or genes, both belonging to the TGF-β/BMP9 signaling pathway. Despite clear knowledge of the symptoms and genes of the disease, we still lack a definite cure for HHT, having just palliative measures and pharmacological trials. Among the former, two strategies are: intervention at "ground zero" to minimize by iron and blood transfusions in order to counteract anemia. Among the later, along the last 15 years, three different strategies have been tested: (1) To favor coagulation with antifibrinolytic agents (tranexamic acid); (2) to increase transcription of and with specific estrogen-receptor modulators (bazedoxifene or raloxifene), antioxidants (N-acetylcysteine, resveratrol), or immunosuppressants (tacrolimus); and (3) to impair the abnormal angiogenic process with antibodies (bevacizumab) or blocking drugs like etamsylate, and propranolol. This manuscript reviews the main strategies and sums up the clinical trials developed with drugs alleviating HHT.
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http://dx.doi.org/10.3390/jcm9061766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356836PMC
June 2020

Potential Role of Circulating Endoglin in Hypertension via the Upregulated Expression of BMP4.

Authors:
Eunate Gallardo-Vara Luis Gamella-Pozuelo Lucía Perez-Roque José L Bartha Irene Garcia-Palmero J Ignacio Casal José M López-Novoa Miguel Pericacho Carmelo Bernabeu

Cells 2020 04 16;9(4). Epub 2020 Apr 16.

Centro de Investigaciones Biológicas Margarita Salas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, Spain.

Endoglin is a membrane glycoprotein primarily expressed by the vascular endothelium and involved in cardiovascular diseases. Upon the proteolytic processing of the membrane-bound protein, a circulating form of endoglin (soluble endoglin, sEng) can be released, and high levels of sEng have been observed in several endothelial-related pathological conditions, where it appears to contribute to endothelial dysfunction. Preeclampsia is a multisystem disorder of high prevalence in pregnant women characterized by the onset of high blood pressure and associated with increased levels of sEng. Although a pathogenic role for sEng involving hypertension has been reported in several animal models of preeclampsia, the exact molecular mechanisms implicated remain to be identified. To search for sEng-induced mediators of hypertension, we analyzed the protein secretome of human endothelial cells in the presence of sEng. We found that sEng induces the expression of BMP4 in endothelial cells, as evidenced by their proteomic signature, gene transcript levels, and BMP4 promoter activity. A mouse model of preeclampsia with high sEng plasma levels () showed increased transcript levels of BMP4 in lungs, stomach, and duodenum, and increased circulating levels of BMP4, compared to those of control animals. In addition, after crossing female wild type with male mice, hypertension appeared 18 days after mating, coinciding with the appearance of high plasma levels of BMP4. Also, serum levels of sEng and BMP4 were positively correlated in pregnant women with and without preeclampsia. Interestingly, sEng-induced arterial pressure elevation in mice was abolished in the presence of the BMP4 inhibitor noggin, suggesting that BMP4 is a downstream mediator of sEng. These results provide a better understanding on the role of sEng in the physiopathology of preeclampsia and other cardiovascular diseases, where sEng levels are increased.
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http://dx.doi.org/10.3390/cells9040988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7226995PMC
April 2020

Endoglin Protein Interactome Profiling Identifies TRIM21 and Galectin-3 as New Binding Partners.

Authors:
Eunate Gallardo-Vara Lidia Ruiz-Llorente Juan Casado-Vela María J Ruiz-Rodríguez Natalia López-Andrés Asit K Pattnaik Miguel Quintanilla Carmelo Bernabeu

Cells 2019 09 13;8(9). Epub 2019 Sep 13.

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain.

Endoglin is a 180-kDa glycoprotein receptor primarily expressed by the vascular endothelium and involved in cardiovascular disease and cancer. Heterozygous mutations in the endoglin gene (ENG) cause hereditary hemorrhagic telangiectasia type 1, a vascular disease that presents with nasal and gastrointestinal bleeding, skin and mucosa telangiectases, and arteriovenous malformations in internal organs. A circulating form of endoglin (alias soluble endoglin, sEng), proteolytically released from the membrane-bound protein, has been observed in several inflammation-related pathological conditions and appears to contribute to endothelial dysfunction and cancer development through unknown mechanisms. Membrane-bound endoglin is an auxiliary component of the TGF-β receptor complex and the extracellular region of endoglin has been shown to interact with types I and II TGF-β receptors, as well as with BMP9 and BMP10 ligands, both members of the TGF-β family. To search for novel protein interactors, we screened a microarray containing over 9000 unique human proteins using recombinant sEng as bait. We find that sEng binds with high affinity, at least, to 22 new proteins. Among these, we validated the interaction of endoglin with galectin-3, a secreted member of the lectin family with capacity to bind membrane glycoproteins, and with tripartite motif-containing protein 21 (TRIM21), an E3 ubiquitin-protein ligase. Using human endothelial cells and Chinese hamster ovary cells, we showed that endoglin co-immunoprecipitates and co-localizes with galectin-3 or TRIM21. These results open new research avenues on endoglin function and regulation.
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http://dx.doi.org/10.3390/cells8091082DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769930PMC
September 2019

MMP-12, Secreted by Pro-Inflammatory Macrophages, Targets Endoglin in Human Macrophages and Endothelial Cells.

Authors:
Mikel Aristorena Eunate Gallardo-Vara Matej Vicen Mateo de Las Casas-Engel Luisa Ojeda-Fernandez Concepción Nieto Francisco J Blanco Ana C Valbuena-Diez Luisa M Botella Petr Nachtigal Angel L Corbi María Colmenares Carmelo Bernabeu

Int J Mol Sci 2019 Jun 25;20(12). Epub 2019 Jun 25.

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), 28040 Madrid, Spain.

Upon inflammation, monocyte-derived macrophages (MΦ) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and efficient initial response (GM-MΦ) and a good resolution (M-MΦ) of the inflammatory process. The functional activity of polarized MΦ is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MΦ that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MΦ secretome involved in the shedding of soluble endoglin. We find that the GM-MΦ secretome contains metalloprotease 12 (MMP-12), a GM-MΦ specific marker that may account for the anti-angiogenic activity of the GM-MΦ secretome. Cell surface endoglin is present in both GM-MΦ and M-MΦ, but soluble endoglin is only detected in GM-MΦ culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MΦ and endothelial cells. These data demonstrate a direct correlation between GM-MΦ polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.
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http://dx.doi.org/10.3390/ijms20123107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627183PMC
June 2019

Characterization of a mutation in the zona pellucida module of Endoglin that causes Hereditary Hemorrhagic Telangiectasia.

Authors:
Lidia Ruiz-Llorente Elisa Chiapparino Sara Plumitallo Cesare Danesino Pinar Bayrak-Toydemir Fabio Pagella Guido Manfredi Carmelo Bernabeu Luca Jovine Carla Olivieri

Gene 2019 May 11;696:33-39. Epub 2019 Feb 11.

Molecular Medicine Department, General Biology and Medical Genetics Unit, University of Pavia, Via Forlanini 14, 27100 Pavia, Italy. Electronic address:

Hereditary hemorrhagic telangiectasia (HHT) is a vascular rare disease characterized by nose and gastrointestinal bleeding, skin and mucosa telangiectasias, and arteriovenous malformations in internal organs. HHT shows an autosomal dominant inheritance and a worldwide prevalence of approximately 1:5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG (HHT1) or ACVRL1 (HHT2) genes, which code for the membrane proteins Endoglin and Activin A Receptor Type II-Like Kinase 1 (ALK1), respectively, both belonging to the TGF-β/BMP signaling pathway. In this work, we describe a novel mutation in exon 9 of ENG (c.1145 G > A) found in five affected members of a family, all of them with characteristic symptoms of HHT. This mutation involves Cys382 residue of the Endoglin protein (p.Cys382 > Tyr) in the zona pellucida (ZP) module of its extracellular region. This is a critical residue involved in a conserved intrachain disulphide bond and in the correct folding of the protein. In fact, transfection studies in human cells using Endoglin expression vectors demonstrated that the p.Cys382 > Tyr mutation results in a marked reduction in the levels of the Endoglin protein. These results demonstrate the pathogenic role for this variant in HHT1 and confirm the key function of Cys382 in Endoglin expression.
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http://dx.doi.org/10.1016/j.gene.2019.02.016DOI Listing
May 2019

Endoglin as an Adhesion Molecule in Mature and Progenitor Endothelial Cells: A Function Beyond TGF-β.

Authors:
Elisa Rossi Carmelo Bernabeu David M Smadja

Front Med (Lausanne) 2019 30;6:10. Epub 2019 Jan 30.

Université Paris Descartes, Sorbonne Paris Cité, Paris, France.

Endoglin (ENG) is a transmembrane glycoprotein expressed on endothelial cells that functions as a co-receptor for several ligands of the transforming growth factor beta (TGF-β) family. ENG is also a recognized marker of angiogenesis and mutations in the endoglin gene are responsible for Hereditary Hemorrhagic Telangiectasia (HHT) type 1, a vascular disease characterized by defective angiogenesis, arteriovenous malformations, telangiectasia, and epistaxis. In addition to its involvement in the TGF-β family signaling pathways, several lines of evidence suggest that the extracellular domain of ENG has a role in integrin-mediated cell adhesion via its RGD motif. Indeed, we have described a role for endothelial ENG in leukocyte trafficking and extravasation its binding to leukocyte integrins. We have also found that ENG is involved in vasculogenic properties of endothelial progenitor cells known as endothelial colony forming cells (ECFCs). Moreover, the binding of endothelial ENG to platelet integrins regulate the resistance to shear during platelet-endothelium interactions under inflammatory conditions. Because of the need for more effective treatments in HHT and the involvement of ENG in angiogenesis, current studies are aimed at identifying novel biological functions of ENG which could serve as a therapeutic target. This review focuses on the interaction between ENG and integrins with the aim to better understand the role of this protein in blood vessel formation driven by progenitor and mature endothelial cells.
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http://dx.doi.org/10.3389/fmed.2019.00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363663PMC
January 2019

Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction and .

Authors:
Matej Vicen Barbora Vitverova Radim Havelek Katerina Blazickova Miloslav Machacek Jana Rathouska Iveta Najmanová Eva Dolezelova Alena Prasnicka Magdalena Sternak Carmelo Bernabeu Petr Nachtigal

FASEB J 2019 05 12;33(5):6099-6114. Epub 2019 Feb 12.

Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University, Hradec Kralove, Czech Republic.

Our objective was to investigate the effect of cholesterol [hypercholesterolemia and 7-ketocholesterol (7K)] on endoglin (Eng) expression and regulation with respect to endothelial or vascular dysfunction and . experiments were performed in 2-mo-old atherosclerosis-prone apolipoprotein E-deficient/LDL receptor-deficient (ApoE/LDLR) female mice and their wild-type C57BL/6J littermates. In experiments, human aortic endothelial cells (HAECs) were treated with 7K. ApoE/LDLR mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and Eng and a disruption of NO metabolism. Functional analysis of the aorta demonstrated impaired vascular reactivity, and Western blot analysis revealed down-regulation of membrane Eng/Smad2/3/eNOS signaling in ApoE/LDLR mice. 7K increased Eng expression Krüppel-like factor 6 (KLF6), liver X nuclear receptor, and NF-κB in HAECs. 7K-induced Eng expression was prevented by the treatment with 2-hydroxypropyl-β-cyclodextrin; 8-{[5-chloro-2-(4-methylpiperazin-1-) pyridine-4-carbonyl] amino}-1-(4-fluorophenyl)-4, 5-dihydrobenzo[g]indazole-3-carboxamide; or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic human leukemia promonocytic cell line cells and was prevented by Eng silencing. We concluded that hypercholesterolemia altered Eng expression and signaling, followed by endothelial or vascular dysfunction before formation of atherosclerotic lesions in ApoE/LDLR mice. By contrast, 7K increased Eng expression and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes endothelium, which was prevented by Eng inhibition. Thus, we propose a relevant role for Eng in endothelial or vascular dysfunction or inflammation when exposed to cholesterol.-Vicen, M., Vitverova, B., Havelek, R., Blazickova, K., Machacek, M., Rathouska, J., Najmanová, I., Dolezelova, E., Prasnicka, A., Sternak, M., Bernabeu, C., Nachtigal, P. Regulation and role of endoglin in cholesterol-induced endothelial and vascular dysfunction and .
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http://dx.doi.org/10.1096/fj.201802245RDOI Listing
May 2019

Characterization of a family mutation in the 5' untranslated region of the endoglin gene causative of hereditary hemorrhagic telangiectasia.

Authors:
Lidia Ruiz-Llorente Jamie McDonald Whitney Wooderchak-Donahue Eric Briggs Mark Chesnutt Pinar Bayrak-Toydemir Carmelo Bernabeu

J Hum Genet 2019 Apr 6;64(4):333-339. Epub 2019 Feb 6.

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040, Madrid, Spain.

Hereditary hemorrhagic telangiectasia (HHT) is a vascular disease characterized by nose and gastrointestinal bleeding, telangiectases in skin and mucosa, and arteriovenous malformations in major internal organs. Most patients carry a mutation in the coding region of the endoglin (ENG) or activin A receptor type II-1 (ACVRL1) gene. Nonetheless, in around 15% of patients, sequencing analysis and duplication/deletion tests fail to pinpoint mutations in the coding regions of these genes. In these cases, it has been shown that sequencing of the 5'-untranslated region (5'UTR) of ENG may be useful to identify novel mutations in the ENG non-coding region. Here we report the genetic characterization and functional analysis of the heterozygous mutation c.-142A>T in the 5'UTR region of ENG found in a family with several members affected by HHT. This variant gives rise to a new initiation codon of the protein that involves the change in its open reading frame. Transfection studies in monkey cells using endoglin expression vectors demonstrated that c-142A>T mutation results in a clear reduction in the levels of the endoglin protein. These results support the inclusion of the 5'UTR of ENG in the standard genetic testing for HHT to increase its sensitivity.
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http://dx.doi.org/10.1038/s10038-019-0564-xDOI Listing
April 2019

A role for the Tgf-β/Bmp co-receptor Endoglin in the molecular oscillator that regulates the hair follicle cycle.

Authors:
María I Calvo-Sánchez Sandra Fernández-Martos Elisa Carrasco Gema Moreno-Bueno Carmelo Bernabéu Miguel Quintanilla Jesús Espada

J Mol Cell Biol 2019 01;11(1):39-52

Instituto de Investigaciones Biomédicas 'Alberto Sols', Consejo Superior de Investigaciones Científicas (CSIC)-Departamento de Bioquímica, Universidad Autónoma de Madrid (UAM), Madrid, Spain.

The hair follicle is a biological oscillator that alternates growth, regression, and rest phases driven by the sequential activation of the proliferation/differentiation programs of resident stem cell populations. The activation of hair follicle stem cell niches and subsequent entry into the growing phase is mainly regulated by Wnt/β-catenin signalling, while regression and resting phases are mainly regulated by Tgf-β/Bmp/Smad activity. A major question still unresolved is the nature of the molecular switch that dictates the coordinated transition between both signalling pathways. Here we have focused on the role of Endoglin (Eng), a key co-receptor for members of the Tgf-β/Bmp family of growth factors. Using an Eng haploinsufficient mouse model, we report that Eng is required to maintain a correct follicle cycling pattern and for an adequate stimulation of hair follicle stem cell niches. We further report that β-catenin binds to the Eng promoter depending on Bmp signalling. Moreover, we show that β-catenin interacts with Smad4 in a Bmp/Eng-dependent context and both proteins act synergistically to activate Eng promoter transcription. These observations point to the existence of a growth/rest switching mechanism in the hair follicle that is based on an Eng-dependent feedback cross-talk between Wnt/β-catenin and Bmp/Smad signals.
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http://dx.doi.org/10.1093/jmcb/mjy051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6359924PMC
January 2019

Soluble endoglin regulates expression of angiogenesis-related proteins and induction of arteriovenous malformations in a mouse model of hereditary hemorrhagic telangiectasia.

Authors:
Eunate Gallardo-Vara Simon Tual-Chalot Luisa M Botella Helen M Arthur Carmelo Bernabeu

Dis Model Mech 2018 09 21;11(9). Epub 2018 Sep 21.

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain

Endoglin is a transmembrane glycoprotein expressed in vascular endothelium that plays a key role in angiogenesis. Mutations in the endoglin gene () cause hereditary hemorrhagic telangiectasia type 1 (HHT1), characterized by arteriovenous malformations (AVMs) in different organs. These vascular lesions derive from abnormal processes of angiogenesis, whereby aberrant vascular remodeling leads to focal loss of capillaries. Current treatments for HHT1 include antiangiogenic therapies. Interestingly, a circulating form of endoglin (also known as soluble endoglin, sEng), proteolytically released from the membrane-bound protein and displaying antiangiogenic activity, has been described in several endothelial-related pathological conditions. Using human and mouse endothelial cells, we find that sEng downregulates several pro-angiogenic and pro-migratory proteins involved in angiogenesis. However, this effect is much reduced in endothelial cells that lack endogenous transmembrane endoglin, suggesting that the antiangiogenic activity of sEng is dependent on the presence of endogenous transmembrane endoglin protein. In fact, sEng partially restores the phenotype of endoglin-silenced endothelial cells to that of normal endothelial cells. Moreover, using an established neonatal retinal model of HHT1 with depleted endoglin in the vascular endothelium, sEng treatment decreases the number of AVMs and has a normalizing effect on the vascular phenotype with respect to vessel branching, vascular density and migration of the vascular plexus towards the retinal periphery. Taken together, these data show that circulating sEng can influence vascular development and AVMs by modulating angiogenesis, and that its effect on endothelial cells depends on the expression of endogenous endoglin.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.034397DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176985PMC
September 2018

Soluble endoglin and hypercholesterolemia aggravate endothelial and vessel wall dysfunction in mouse aorta.

Authors:
Barbora Vitverova Katerina Blazickova Iveta Najmanova Matej Vicen Radek Hyšpler Eva Dolezelova Ivana Nemeckova Jurjen Duintjer Tebbens Carmelo Bernabeu Miguel Pericacho Petr Nachtigal

Atherosclerosis 2018 04 9;271:15-25. Epub 2018 Feb 9.

Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University, Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic. Electronic address:

Background And Aims: Increased plasma levels of soluble endoglin (sEng) were detected in patients with endothelial dysfunction-related disorders and hypercholesterolemia. In this study, we hypothesized that high levels of sEng accompanied by mild hypercholesterolemia could aggravate endothelial and vessel wall dysfunction and affect endoglin/eNOS signaling in mouse aorta.

Methods: Three-month-old female transgenic mice on CBAxC57BL/6J background, with high levels of sEng (Sol-Enghigh HFD), and their littermates with low levels of sEng (Sol-Englow HFD), were fed a high fat diet for six months. Plasma samples were used for biochemical, ELISA and Luminex analyses of total cholesterol, sEng and inflammatory markers. Functional parameters of aorta were assessed with wire myograph 620M. Western blot analyses of membrane endoglin/eNOS signaling and endothelial dysfunction/inflammation markers in aorta were performed.

Results: Functional analysis of aorta showed impaired KCl induced vasoconstriction, endothelial-dependent relaxation after the administration of acetylcholine as well as endothelial-independent relaxation induced by sodium nitroprusside in the Sol-Enghigh HFD group compared to the Sol-Englow HFD group. Ach-induced vasodilation after administration of l-NAME was significantly higher in the Sol-Enghigh HFD group compared to the Sol-Englow HFD group. The expression of endoglin, p-eNOS/eNOS, pSmad2/3/Smad2/3 signaling pathway was significantly lower in the Sol-Enghigh HFD group compared to the Sol-Englow HFD group.

Conclusions: The results indicate that long-term hypercholesterolemia combined with high levels of sEng leads to the aggravation of endothelial and vessel wall dysfunction in aorta, with possible alterations of the membrane endoglin/eNOS signaling, suggesting that high levels of soluble endoglin might be considered as a risk factor of cardiovascular diseases.
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http://dx.doi.org/10.1016/j.atherosclerosis.2018.02.008DOI Listing
April 2018

Functional analysis of a novel ENG variant in a patient with hereditary hemorrhagic telangiectasia (HHT) identifies a new Sp1 binding-site.

Authors:
Sara Plumitallo Lidia Ruiz-Llorente Carmen Langa Jacopo Morini Gabriele Babini Donata Cappelletti Laura Scelsi Alessandra Greco Cesare Danesino Carmelo Bernabeu Carla Olivieri

Gene 2018 Mar 4;647:85-92. Epub 2018 Jan 4.

Molecular Medicine Department, General Biology and Medical Genetics Unit, University of Pavia, Via Forlanini 14, 27100 Pavia, Italy. Electronic address:

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare disease, with an autosomal dominant inheritance and a worldwide incidence of about 1: 5000 individuals. In >80% of patients, HHT is caused by mutations in either ENG or ACVRL1, which code for ENDOGLIN and Activin A Receptor Type II-Like Kinase 1 (ALK1), belonging to the TGF-β/BMP signalling pathway. Typical HHT clinical features are mucocutaneous telangiectases, arteriovenous malformations, spontaneous and recurrent epistaxis, as well as gastrointestinal bleedings. An additional, but less frequent, clinical manifestation in some HHT patients is the presence of Pulmonary Arterial Hypertension (PAH). The aim of this work is to describe the functional role of a novel ENG intronic variant found in a patient affected by both HHT and PAH, in order to assess whether it has a pathogenic role. We proved that the variant lies in a novel binding-site for the transcription factor Sp1, known to be involved in the regulation of ENG and ACVRL1 transcription. We confirmed a pathogenic role for this intronic variant, as it significantly reduces ENG transcription by affecting this novel Sp1 binding-site.
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http://dx.doi.org/10.1016/j.gene.2018.01.007DOI Listing
March 2018

Human endoglin as a potential new partner involved in platelet-endothelium interactions.

Authors:
Elisa Rossi Miguel Pericacho Christilla Bachelot-Loza Dominique Pidard Pascale Gaussem Sonia Poirault-Chassac Francisco J Blanco Carmen Langa Consuelo González-Manchón Jose M Lopez Novoa David M Smadja Carmelo Bernabeu

Cell Mol Life Sci 2018 04 28;75(7):1269-1284. Epub 2017 Oct 28.

Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu, 9, 28040, Madrid, Spain.

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann's thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng ) mice compared to Eng animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.
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http://dx.doi.org/10.1007/s00018-017-2694-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5843676PMC
April 2018

Endoglin and alk1 as therapeutic targets for hereditary hemorrhagic telangiectasia.

Authors:
Lidia Ruiz-Llorente Eunate Gallardo-Vara Elisa Rossi David M Smadja Luisa M Botella Carmelo Bernabeu

Expert Opin Ther Targets 2017 10 20;21(10):933-947. Epub 2017 Aug 20.

a Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) , Madrid , Spain.

Introduction: Hereditary Haemorrhagic Telangiectasia (HHT) is as an autosomal dominant trait characterized by frequent nose bleeds, mucocutaneous telangiectases, arteriovenous malformations (AVMs) of the lung, liver and brain, and gastrointestinal bleedings due to telangiectases. HHT is originated by mutations in genes whose encoded proteins are involved in the transforming growth factor β (TGF-β) family signalling of vascular endothelial cells. In spite of the great advances in the diagnosis as well as in the molecular, cellular and animal models of HHT, the current treatments remain just at the palliative level. Areas covered: Pathogenic mutations in genes coding for the TGF-β receptors endoglin (ENG) (HHT1) or the activin receptor-like kinase-1 (ACVRL1 or ALK1) (HHT2), are responsible for more than 80% of patients with HHT. Therefore, ENG and ALK1 are the main potential therapeutic targets for HHT and the focus of this review. The current status of the preclinical and clinical studies, including the anti-angiogenic strategy, have been addressed. Expert opinion: Endoglin and ALK1 are attractive therapeutic targets in HHT. Because haploinsufficiency is the pathogenic mechanism in HHT, several therapeutic approaches able to enhance protein expression and/or function of endoglin and ALK1 are keys to find novel and efficient treatments for the disease.
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http://dx.doi.org/10.1080/14728222.2017.1365839DOI Listing
October 2017

Co-injection of mesenchymal stem cells with endothelial progenitor cells accelerates muscle recovery in hind limb ischemia through an endoglin-dependent mechanism.

Authors:
Elisa Rossi David Smadja Celine Goyard Audrey Cras Blandine Dizier Nour Bacha Anna Lokajczyk Coralie L Guerin Nicolas Gendron Benjamin Planquette Virginie Mignon Carmelo Bernabéu Olivier Sanchez David M Smadja

Thromb Haemost 2017 10 3;117(10):1908-1918. Epub 2017 Aug 3.

Prof. David Smadja, European Hospital Georges Pompidou, Hematology Department and UMR-S1140, 20 rue Leblanc, 75015 Paris, France, Tel.: +31 56093933, Fax: +31 56093393, E-mail:

Endothelial colony-forming cells (ECFCs) are progenitor cells committed to endothelial lineages and have robust vasculogenic properties. Mesenchymal stem cells (MSCs) have been described to support ECFC-mediated angiogenic processes in various matrices. However, MSC-ECFC interactions in hind limb ischemia (HLI) are largely unknown. Here we examined whether co-administration of ECFCs and MSCs bolsters vasculogenic activity in nude mice with HLI. In addition, as we have previously shown that endoglin is a key adhesion molecule, we evaluated its involvement in ECFC/MSC interaction. Foot perfusion increased on day 7 after ECFC injection and was even better at 14 days. Co-administration of MSCs significantly increased vessel density and foot perfusion on day 7 but the differences were no longer significant at day 14. Analysis of mouse and human CD31, and in situ hybridization of the human ALU sequence, showed enhanced capillary density in ECFC+MSC mice. When ECFCs were silenced for endoglin, coinjection with MSCs led to lower vessel density and foot perfusion at both 7 and 14 days (p<0.001). Endoglin silencing in ECFCs did not affect MSC differentiation into perivascular cells or other mesenchymal lineages. Endoglin silencing markedly inhibited ECFC adhesion to MSCs. Thus, MSCs, when combined with ECFCs, accelerate muscle recovery in a mouse model of hind limb ischemia, through an endoglin-dependent mechanism.
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http://dx.doi.org/10.1160/TH17-01-0007DOI Listing
October 2017

Soluble endoglin modulates the pro-inflammatory mediators NF-κB and IL-6 in cultured human endothelial cells.

Authors:
Michala Varejckova Eunate Gallardo-Vara Matej Vicen Barbora Vitverova Petra Fikrova Eva Dolezelova Jana Rathouska Alena Prasnicka Katerina Blazickova Stanislav Micuda Carmelo Bernabeu Ivana Nemeckova Petr Nachtigal

Life Sci 2017 Apr 21;175:52-60. Epub 2017 Mar 21.

Charles University, Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, Hradec Kralove, Czech Republic. Electronic address:

Aims: 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.

Main Methods: 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.

Key Findings: 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, VCAM-1, COX-1, COX-2 and ICAM-1 were detected.

Significance: 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.
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http://dx.doi.org/10.1016/j.lfs.2017.03.014DOI Listing
April 2017

The role of endoglin in post-ischemic revascularization.

Authors:
Elena Núñez-Gómez Miguel Pericacho Claudia Ollauri-Ibáñez Carmelo Bernabéu José M López-Novoa

Angiogenesis 2017 Feb 9;20(1):1-24. Epub 2016 Dec 9.

Renal and Cardiovascular Research Unit, Department of Physiology and Pharmacology, University of Salamanca, Salamanca, Spain.

Following arterial occlusion, blood vessels respond by forming a new network of functional capillaries (angiogenesis), by reorganizing preexisting capillaries through the recruitment of smooth muscle cells to generate new arteries (arteriogenesis) and by growing and remodeling preexisting collateral arterioles into physiologically relevant arteries (collateral development). All these processes result in the recovery of organ perfusion. The importance of endoglin in post-occlusion reperfusion is sustained by several observations: (1) endoglin expression is increased in vessels showing active angiogenesis/remodeling; (2) genetic endoglin haploinsufficiency in humans causes deficient angiogenesis; and (3) the reduction of endoglin expression by gene disruption or the administration of endoglin-neutralizing antibodies reduces angiogenesis and revascularization. However, the precise role of endoglin in the several processes associated with revascularization has not been completely elucidated and, in some cases, the function ascribed to endoglin by different authors is controversial. The purpose of this review is to organize in a critical way the information available for the role of endoglin in several phenomena (angiogenesis, arteriogenesis and collateral development) associated with post-ischemic revascularization.
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http://dx.doi.org/10.1007/s10456-016-9535-4DOI Listing
February 2017

High Levels of Soluble Endoglin Induce a Proinflammatory and Oxidative-Stress Phenotype Associated with Preserved NO-Dependent Vasodilatation in Aortas from Mice Fed a High-Fat Diet.

Authors:
Katerina Jezkova Jana Rathouska Ivana Nemeckova Petra Fikrova Eva Dolezelova Michala Varejckova Barbora Vitverova Kristyna Tysonova Agnieszka Serwadczak Elzbieta Buczek Carmelo Bernabeu Jose M Lopez-Novoa Stefan Chlopicki Petr Nachtigal

J Vasc Res 2016 8;53(3-4):149-162. Epub 2016 Oct 8.

Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University in Prague, Hradec Kralove, Czech Republic.

Aims: A soluble form of endoglin (sEng) was proposed to participate in the induction of endothelial dysfunction in small blood vessels. Here, we tested the hypothesis that high levels of sEng combined with a high-fat diet induce endothelial dysfunction in an atherosclerosis-prone aorta.

Methods And Results: Six-month-old female and male transgenic mice overexpressing human sEng (Sol-Eng+) with low (Sol-Eng+low) or high (Sol-Eng+high) levels of plasma sEng were fed a high-fat rodent diet containing 1.25% cholesterol and 40% fat for 3 months. The plasma cholesterol and mouse sEng levels did not differ in the Sol-Eng+high and Sol-Eng+low mice. The expression of proinflammatory (P-selectin, ICAM-1, pNFκB and COX-2) and oxidative-stress-related markers (HO-1, NOX-1 and NOX-2) in the aortas of Sol-Eng+high female mice was significantly higher than in Sol-Eng+low female mice. Endothelium-dependent vasodilatation induced by acetylcholine was preserved better in the Sol-Eng+ high female mice than in the Sol-Eng+low female mice.

Conclusion: These results suggest that high concentrations of sEng in plasma in combination with a high-fat diet induce the simultaneous activation of proinflammatory, pro-oxidative and vasoprotective mechanisms in mice aorta and the balance of these biological processes determines whether the final endothelial phenotype is adaptive or maladaptive.
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http://dx.doi.org/10.1159/000448996DOI Listing
May 2017

Overexpression of the short endoglin isoform reduces renal fibrosis and inflammation after unilateral ureteral obstruction.

Authors:
José M Muñoz-Félix Lucía Pérez-Roque Elena Núñez-Gómez Bárbara Oujo Miguel Arévalo Laura Ruiz-Remolina Cristina Cuesta Carmen Langa Fernando Pérez-Barriocanal Carmelo Bernabeu José M Lopez-Novoa

Biochim Biophys Acta 2016 09 15;1862(9):1801-14. Epub 2016 Jun 15.

Renal and Cardiovascular Research Unit, Department of Physiology and Pharmacology, University of Salamanca, 37007 Salamanca, Spain; Biomedical Research Institute of Salamanca (IBSAL), 37007 Salamanca, Spain.

Transforming growth factor beta 1 (TGF-β1) is one of the most studied cytokines involved in renal tubulo-interstitial fibrosis, which is characterized by myofibroblast abundance and proliferation, and high buildup of extracellular matrix in the tubular interstitium leading to organ failure. Endoglin (Eng) is a 180-kDa homodimeric transmembrane protein that regulates a great number of TGF-β1 actions in different biological processes, including ECM synthesis. High levels of Eng have been observed in experimental models of renal fibrosis or in biopsies from patients with chronic kidney disease. In humans and mice, two Eng isoforms are generated by alternative splicing, L-Eng and S-Eng that differ in the length and composition of their cytoplasmic domains. We have previously described that L-Eng overexpression promotes renal fibrosis after unilateral ureteral obstruction (UUO). However, the role of S-Eng in renal fibrosis is unknown and its study would let us analyze the possible function of the cytoplasmic domain of Eng in this process. For this purpose, we have generated a mice strain that overexpresses S-Eng (S-ENG(+)) and we have performed an UUO in S-ENG(+) and their wild type (WT) control mice. Our results indicate that obstructed kidney of S-ENG(+) mice shows lower levels of tubulo-interstitial fibrosis, less inflammation and less interstitial cell proliferation than WT littermates. Moreover, S-ENG(+) mice show less activation of Smad1 and Smad2/3 pathways. Thus, S-Eng overexpression reduces UUO-induced renal fibrosis and some associated mechanisms. As L-Eng overexpression provokes renal fibrosis we conclude that Eng-mediated induction of renal fibrosis in this model is dependent on its cytoplasmic domain.
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http://dx.doi.org/10.1016/j.bbadis.2016.06.010DOI Listing
September 2016

Screening pulmonary arteriovenous malformations in a large cohort of Spanish patients with hemorrhagic hereditary telangiectasia.

Authors:
José Antonio Parra José María Cuesta Roberto Zarrabeitia Concepción Fariñas-Álvarez Jesús Bueno Sarah Marqués Carmen Parra-Fariñas María Luisa Botella Carmelo Bernabéu Jesús Zarauza

Int J Cardiol 2016 Sep 14;218:240-245. Epub 2016 May 14.

Department of Cardiology, Hospital Universitario Marqués de Valdecilla, Santander, Cantabria, Spain. Electronic address:

Background And Objectives: Because of the serious nature of potential complications, screening for pulmonary arteriovenous malformations is required in patients with hereditary hemorrhagic telangiectasia. The aim of this study was to evaluate the utility of contrast echocardiography and compare the performance of two contrast agents: agitated saline and Gelofusine.

Material And Methods: Two hundred and five patients screened for PAVMs using TTCE and computed tomography (CT) performed with an interval of less than 180days. Contrast echocardiography studies were graded on a 4-point semiquantitative scale based on the amount of microbubbles seen in left heart chambers.

Results: Positive TTCE findings were seen in 137 (66.8%) patients, whereas CT confirmed PAVMs in 59 (43.1%). Two of 67 grade 1 patients; 18 of 42 grade 2; 17 of 22 grade 3 and all grade 4 had PAVMs on CT. Embolotherapy was feasible in 38.9% patients in grade 2 and 82.3% and 95.2% in grades 3-4. No patients in grade 1 were embolized. The mean cardiac cycle in which bubbles were first seen in the left heart in patients without and with PAVMs on CT was 6.1 and 3.9 (p<0.0001). Compared to saline, Gelofusine produced an overall increase in grade.

Conclusions: No grade 1 patients had treatable PAVMs. There is a need for improvement in the selection of patients for CT in grade 2, where less than half have PAVMs on CT. The cardiac cycle may help to differentiate between patients with and without PAVMs. Gelofusine was not better than saline for PAVM screening.
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http://dx.doi.org/10.1016/j.ijcard.2016.05.065DOI Listing
September 2016

Mice Lacking Endoglin in Macrophages Show an Impaired Immune Response.

Authors:
Luisa Ojeda-Fernández Lucía Recio-Poveda Mikel Aristorena Pedro Lastres Francisco J Blanco Francisco Sanz-Rodríguez Eunate Gallardo-Vara Mateo de las Casas-Engel Ángel Corbí Helen M Arthur Carmelo Bernabeu Luisa M Botella

PLoS Genet 2016 Mar 24;12(3):e1005935. Epub 2016 Mar 24.

Centro de Investigación en Red de Enfermedades Raras (CIBERER), Valencia, Spain.

Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-Osler-Weber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Eng(fl/fl)LysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Eng(fl/fl)LysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.
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http://dx.doi.org/10.1371/journal.pgen.1005935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4806930PMC
March 2016

Transcription factor KLF6 upregulates expression of metalloprotease MMP14 and subsequent release of soluble endoglin during vascular injury.

Authors:
Eunate Gallardo-Vara Francisco J Blanco Mercè Roqué Scott L Friedman Toru Suzuki Luisa M Botella Carmelo Bernabeu

Angiogenesis 2016 Apr 5;19(2):155-71. Epub 2016 Feb 5.

Centro de Investigaciones Biológicas, CSIC, c/Ramiro de Maeztu 9, Madrid, 28040, Spain.

After endothelial injury, the transcription factor Krüppel-like factor 6 (KLF6) translocates into the cell nucleus to regulate a variety of target genes involved in angiogenesis, vascular repair and remodeling, including components of the membrane transforming growth factor beta (TGF-β) receptor complex such as endoglin and activin receptor-like kinase 1. The membrane metalloproteinase 14 (MMP14 or MT1-MMP) targets endoglin to release soluble endoglin and is involved in vascular inflammation and endothelial tubulogenesis. However, little is known about the regulation of MMP14 expression during vascular wounding. In vitro denudation of monolayers of human endothelial cell monolayers leads to an increase in the KLF6 gene transcriptional rate, followed by an upregulation of MMP14 and release of soluble endoglin. Concomitant with this process, MMP14 co-localizes with endoglin in the sprouting endothelial cells surrounding the wound border. MMP14 expression at mRNA and protein levels is increased by ectopic KLF6 and downregulated by KLF6 suppression in cultured endothelial cells. Moreover, after wire-induced endothelial denudation, Klf6 (+/-) mice show lower levels of MMP14 in their vasculature compared with their wild-type siblings. Ectopic cellular expression of KLF6 results in an increased transcription rate of MMP14, and chromatin immunoprecipitation assays show that KLF6 interacts with MMP14 promoter in ECs, this interaction being enhanced during wound healing. Furthermore, KLF6 markedly increases the transcriptional activity of different reporter constructs of MMP14 gene promoter. These results suggest that KLF6 regulates MMP14 transcription and is a critical player of the gene expression network triggered during endothelial repair.
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http://dx.doi.org/10.1007/s10456-016-9495-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4819519PMC
April 2016

Bazedoxifene, a new orphan drug for the treatment of bleeding in hereditary haemorrhagic telangiectasia.

Authors:
Roberto Zarrabeitia Luisa Ojeda-Fernandez Lucia Recio Carmelo Bernabéu Jose A Parra Virginia Albiñana Luisa M Botella

Thromb Haemost 2016 06 28;115(6):1167-77. Epub 2016 Jan 28.

Luisa M. Botella, Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu, 9, Madrid, 28040 Spain, E-mail:

Hereditary haemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is a dominant genetic vascular disorder. In HHT, blood vessels are weak and prone to bleeding, leading to epistaxis and anaemia, severely affecting patients' quality of life. Development of vascular malformations in HHT patients is originated mainly by mutations in ACVRL1/ALK1 (activin receptor-like kinase type I) or Endoglin (ENG) genes. These genes encode proteins of the TGF-β signalling pathway in endothelial cells, controlling angiogenesis. Haploinsufficiency of these proteins is the basis of HHT pathogenicity. It was our objective to study the efficiency of Bazedoxifene, a selective estrogen receptor modulator (SERM) in HHT, looking for a decrease in epistaxis, and understanding the underlying molecular mechanism. Plasma samples of five HHT patients were collected before, and after 1 and 3 months of Bazedoxifene treatment. ENG and ALK1 expression in activated mononuclear cells derived from blood, as well as VEGF plasma levels, were measured. Quantification of Endoglin and ALK1 mRNA was done in endothelial cells derived from HHT and healthy donors, after in vitro treatment with Bazedoxifene. Angiogenesis was also measured by tubulogenesis and wound healing assays. Upon Bazedoxifene treatment, haemoglobin levels of HHT patients increased and the quantity and frequency of epistaxis decreased. Bazedoxifene increased Endoglin and ALK1 mRNA levels, in cells derived from blood samples and in cultured endothelial cells, promoting tube formation. In conclusion, Bazedoxifene seems to decrease bleeding in HHT by partial compensation of haploinsufficiency. The results shown here are the basis of a new orphan drug designation for HHT by the European Medicine Agency (EMA).
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http://dx.doi.org/10.1160/TH15-03-0239DOI Listing
June 2016

Endothelium-derived microparticles from chronically thromboembolic pulmonary hypertensive patients facilitate endothelial angiogenesis.

Authors:
Daria Belik Hilda Tsang John Wharton Luke Howard Carmelo Bernabeu Beata Wojciak-Stothard

J Biomed Sci 2016 Jan 19;23. Epub 2016 Jan 19.

Centre for Pharmacology and Therapeutics, Department of Medicine, Imperial College London, London, UK.

Background: Increased circulating levels of endoglin(+) endothelial microparticles (EMPs) have been identified in several cardiovascular disorders, related to severity. Endoglin is an auxilary receptor for transforming growth factor β (TGF-β) important in the regulation of vascular structure.

Results: We quantified the number of microparticles in plasma of six patients with chronic thromboembolic pulmonary hypertension (CTEPH) and age- and sex-matched pulmonary embolic (PE) and healthy controls and investigated the role of microparticle endoglin in the regulation of pulmonary endothelial function in vitro. Results show significantly increased levels of endoglin(+) EMPs in CTEPH plasma, compared to healthy and disease controls. Co-culture of human pulmonary endothelial cells with CTEPH microparticles increased intracellular levels of endoglin and enhanced TGF-β-induced angiogenesis and Smad1,5,8 phosphorylation in cells, without affecting BMPRII expression. In an in vitro model, we generated endothelium-derived MPs with enforced membrane localization of endoglin. Co-culture of these MPs with endothelial cells increased cellular endoglin content, improved cell survival and stimulated angiogenesis in a manner similar to the effects induced by overexpressed protein.

Conclusions: Increased generation of endoglin(+) EMPs in CTEPH is likely to represent a protective mechanism supporting endothelial cell survival and angiogenesis, set to counteract the effects of vascular occlusion and endothelial damage.
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http://dx.doi.org/10.1186/s12929-016-0224-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717540PMC
January 2016

Endoglin regulates mural cell adhesion in the circulatory system.

Authors:
Elisa Rossi David M Smadja Elisa Boscolo Carmen Langa Miguel A Arevalo Miguel Pericacho Luis Gamella-Pozuelo Alexandre Kauskot Luisa M Botella Pascale Gaussem Joyce Bischoff José M Lopez-Novoa Carmelo Bernabeu

Cell Mol Life Sci 2016 Apr 8;73(8):1715-39. Epub 2015 Dec 8.

Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas (CSIC), c/Ramiro de Maeztu 9, 28040, Madrid, Spain.

The circulatory system is walled off by different cell types, including vascular mural cells and podocytes. The interaction and interplay between endothelial cells (ECs) and mural cells, such as vascular smooth muscle cells or pericytes, play a pivotal role in vascular biology. Endoglin is an RGD-containing counter-receptor for β1 integrins and is highly expressed by ECs during angiogenesis. We find that the adhesion between vascular ECs and mural cells is enhanced by integrin activators and inhibited upon suppression of membrane endoglin or β1-integrin, as well as by addition of soluble endoglin (SolEng), anti-integrin α5β1 antibody or an RGD peptide. Analysis of different endoglin mutants, allowed the mapping of the endoglin RGD motif as involved in the adhesion process. In Eng (+/-) mice, a model for hereditary hemorrhagic telangectasia type 1, endoglin haploinsufficiency induces a pericyte-dependent increase in vascular permeability. Also, transgenic mice overexpressing SolEng, an animal model for preeclampsia, show podocyturia, suggesting that SolEng is responsible for podocytes detachment from glomerular capillaries. These results suggest a critical role for endoglin in integrin-mediated adhesion of mural cells and provide a better understanding on the mechanisms of vessel maturation in normal physiology as well as in pathologies such as preeclampsia or hereditary hemorrhagic telangiectasia.
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http://dx.doi.org/10.1007/s00018-015-2099-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4805714PMC
April 2016

Research on potential biomarkers in hereditary hemorrhagic telangiectasia.

Authors:
Luisa-María Botella Virginia Albiñana Luisa Ojeda-Fernandez Lucia Recio-Poveda Carmelo Bernabéu

Front Genet 2015 31;6:115. Epub 2015 Mar 31.

Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Cientificas , Madrid, Spain ; Centro de Investigación Biomédica en Red de Enfermedades Raras , Madrid, Spain.

Hereditary hemorrhagic telangiectasia (HHT) is a genetically heterogeneous disorder, involving mutations in two predominant genes known as Endoglin (ENG; HHT1) and activin receptor-like kinase 1 (ACVRL1/ALK1; HHT2), as well as in some less frequent genes, such as MADH4/SMAD4 (JP-HHT) or BMP9/GDF2 (HHT5). The diagnosis of HHT patients currently remains at the clinical level, according to the "Curaçao criteria," whereas the molecular diagnosis is used to confirm or rule out suspected HHT cases, especially when a well characterized index case is present in the family or in an isolated population. Unfortunately, many suspected patients do not present a clear HHT diagnosis or do not show pathogenic mutations in HHT genes, prompting the need to investigate additional biomarkers of the disease. Here, several HHT biomarkers and novel methodological approaches developed during the last years will be reviewed. On one hand, products detected in plasma or serum samples: soluble proteins (vascular endothelial growth factor, transforming growth factor β1, soluble endoglin, angiopoietin-2) and microRNA variants (miR-27a, miR-205, miR-210). On the other hand, differential HHT gene expression fingerprinting, next generation sequencing of a panel of genes involved in HHT, and infrared spectroscopy combined with artificial neural network patterns will also be reviewed. All these biomarkers might help to improve and refine HHT diagnosis by distinguishing from the non-HHT population.
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http://dx.doi.org/10.3389/fgene.2015.00115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4379940PMC
April 2015

High soluble endoglin levels do not induce endothelial dysfunction in mouse aorta.

Authors:
Ivana Nemeckova Agnieszka Serwadczak Barbara Oujo Katerina Jezkova Jana Rathouska Petra Fikrova Michala Varejckova Carmelo Bernabeu Jose M Lopez-Novoa Stefan Chlopicki Petr Nachtigal

PLoS One 2015 13;10(3):e0119665. Epub 2015 Mar 13.

Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, Hradec Kralove, 500 05, Czech Republic.

Increased levels of a soluble form of endoglin (sEng) circulating in plasma have been detected in various pathological conditions related to cardiovascular system. High concentration of sEng was also proposed to contribute to the development of endothelial dysfunction, but there is no direct evidence to support this hypothesis. Therefore, in the present work we analyzed whether high sEng levels induce endothelial dysfunction in aorta by using transgenic mice with high expression of human sEng. Transgenic mice with high expression of human sEng on CBAxC57Bl/6J background (Sol-Eng+) and age-matched transgenic littermates that do not develop high levels of human soluble endoglin (control animals in this study) on chow diet were used. As expected, male and female Sol-Eng+ transgenic mice showed higher levels of plasma concentrations of human sEng as well as increased blood arterial pressure, as compared to control animals. Functional analysis either in vivo or ex vivo in isolated aorta demonstrated that the endothelium-dependent vascular function was similar in Sol-Eng+ and control mice. In addition, Western blot analysis showed no differences between Sol-Eng+ and control mice in the protein expression levels of endoglin, endothelial NO-synthase (eNOS) and pro-inflammatory ICAM-1 and VCAM-1 from aorta. Our results demonstrate that high levels of soluble endoglin alone do not induce endothelial dysfunction in Sol-Eng+ mice. However, these data do not rule out the possibility that soluble endoglin might contribute to alteration of endothelial function in combination with other risk factors related to cardiovascular disorders.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0119665PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4359129PMC
January 2016