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15 p.-11 fig., Endoglin (Eng) is a co-receptor of the transforming growth factor β superfamily playing an important role in endothelial dysfunction. TRC105 (carotuximab) is a monoclonal antibody that blocks Eng and its downstream Smad signaling pathway. Here we have investigated for the first time the effects of TRC105 treatment on the development of endothelial dysfunction induced by 7-ketocholesterol (7K) or high glucose (HG), focusing on Eng expression, signaling, and function. In the hypercholesterolemia study, human aortic endothelial cells (HAoECs) were treated with TRC105 (300 μg/ml) for 1 h, followed by the addition of 7K (10 μg/ml) for another 12 h. In the hyperglycemia study, HAoECs were exposed to HG (45 mM) for 60 h, followed by the addition of TRC105 for another 12 h, and cells treated with 5mM glucose and 40 mM mannitol served as control. Protein levels, adhesion, and transmigration of monocytes were assessed by flow cytometry, mRNA expression was measured by qRT-PCR. 7K and HG treatment increased protein levels of NF-κB and Eng and adhesion and transmigration of monocytes through HAoECs monolayer. TRC105 pretreatment reduced the 7K- or HG-induced Eng protein levels and pSmad1/5 and pSmad2/3 signaling. Despite increased protein levels of P-selectin and VCAM-1, TRC105 mediated blockage of Eng prevented 7K- and HG-induced adhesion and transmigration of monocytes through endothelial monolayers. These results suggest that TRC105-mediated Eng blockage can counteract the hypercholesterolemia- and hyperglycemia-induced endothelial dysfunction in HAoECs, suggesting that Eng might be a potential therapeutic target in disorders associated with elevated cholesterol and glucose levels., This research was supported by grants from the Grant Agency of Charles University (GAUK No. 1130120), Czech Science Foundation (GACR 22-14961S), Specific University Research (SVV 260 549), efficiency and safety improvement of current drugs and nutraceuticals: advanced methods new challenge grant (EFSA-CDN; No. CZ.02.1.01/0.0/0.0/16_019/0000841), and by Consejo Superior de Investigaciones Científicas (CSIC), grant number: 201920E022.

13 p.-3 fig.-1 tab., Background: Disrupted endothelial BMP9/10 signaling may contribute to the pathophysiology of both hereditary hemorrhagic telangiectasia (HHT) and pulmonary arterial hypertension (PAH), yet loss of circulating BMP9 has not been confirmed in individuals with ultra-rare homozygous GDF2 (BMP9 gene) nonsense mutations. We studied two pediatric patients homozygous for GDF2 (BMP9 gene) nonsense mutations: one with PAH (c.[76C>T];[76C>T] or p.[Gln26Ter];[Gln26Ter] and a new individual with pulmonary arteriovenous malformations (PAVMs; c.[835G>T];[835G>T] or p.[Glu279Ter];[Glu279Ter]); both with facial telangiectases., Methods: Plasma samples were assayed for BMP9 and BMP10 by ELISA. In parallel, serum BMP activity was assayed using an endothelial BRE-luciferase reporter cell line (HMEC1-BRE). Proteins were expressed for assessment of secretion and processing., Results: Plasma levels of both BMP9 and BMP10 were undetectable in the two homozygous index cases and this corresponded to low serum-derived endothelial BMP activity in the patients. Measured BMP9 and BMP10 levels were reduced in the asymptomatic heterozygous p.[Glu279Ter] parents, but serum activity was normal. Although expression studies suggested alternate translation can be initiated at Met57 in the p.[Gln26Ter] mutant, this does not result in secretion of functional BMP9., Conclusion: Collectively, these data show that homozygous GDF2 mutations, leading to a loss of circulating BMP9 and BMP10, can cause either pediatric PAH and/or "HHT-like" telangiectases and PAVMs. Although patients reported to date have manifestations that overlap with those of HHT, none meet the Curaçao criteria for HHT and seem distinct from HHT in terms of the location and appearance of telangiectases, and a tendency for tiny, diffuse PAVMs., This study was supported by British Heart Foundation Grant/Award Numbers: FS/15/62/323032, RG/13/4/30107, and RG/19/3/34265; Consejo Superior de Investigaciones Científicas (CSIC) Grant/Award Number: 201920E022; Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) Grant/ Award Numbers: ISCIII-CB06/07/0038 and CNV-234-PRF-360.

13 p.-5 fig., Endoglin (Eng, CD105) is a type I membrane glycoprotein that functions in endothelial cells as an auxiliary receptor for transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) family members and as an integrin ligand, modulating the vascular pathophysiology. Besides the membrane-bound endoglin, there is a soluble form of endoglin (sEng) that can be generated by the action of the matrix metalloproteinase (MMP)-14 or -12 on the juxtamembrane region of its ectodomain. High levels of sEng have been reported in patients with preeclampsia, hypercholesterolemia, atherosclerosis and cancer. In addition, sEng is a marker of cardiovascular damage in patients with hypertension and diabetes, plays a pathogenic role in preeclampsia, and inhibits angiogenesis and tumor proliferation, migration, and invasion in cancer. However, the mechanisms of action of sEng have not yet been elucidated, and new tools and experimental approaches are necessary to advance in this field. To this end, we aimed to obtain a fluorescent form of sEng as a new tool for biological imaging. Thus, we cloned the extracellular domain of endoglin in the pEGFP-N1 plasmid to generate a fusion protein with green fluorescent protein (GFP), giving rise to pEGFP-N1/Eng.EC. The recombinant fusion protein was characterized by transient and stable transfections in CHO-K1 cells using fluorescence microscopy, SDS-PAGE, immunodetection, and ELISA techniques. Upon transfection with pEGFP-N1/Eng.EC, fluorescence was readily detected in cells, indicating that the GFP contained in the recombinant protein was properly folded into the cytosol. Furthermore, as evidenced by Western blot analysis, the secreted fusion protein yielded the expected molecular mass and displayed a specific fluorescent signal. The fusion protein was also able to bind to BMP9 and BMP10 in vitro. Therefore, the construct described here could be used as a tool for functional in vitro studies of the extracellular domain of endoglin., This research was funded by grants from Ministerio de Ciencia, Innovación y Universidades of Spain (SAF2013-43421-R to C.B. and RTI2018-102242-B-I00 to M.C.V.), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B. and contract CNV-234-PRF-360 to L.R.-L.) and Consejo Superior de Investigaciones Científicas (CSIC; 201920E022 to C.B.) and the British Heart Foundation (Programme Grant RG/19/3/34265 to P.D.U. and N.W.M.). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

7 p.-4 fig., 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., This work was supported by the Ministerio de Economía, Industria y Competitividad (Grant SAF2013-43421-R to CB); the Consejo Superior de Investigaciones Científicas (Grant 201420E039 and 201920E022 to CB); and the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; Grant ISCIII-CB06/07/0038 to CB and contract to LR-L) of Spain. CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

Endoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p &lt, 0.05) and an increase in pseudo-tube width (41 ± 4.5%, p &lt, 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p &lt, 0.01), which is associated to a higher Ca2+ mobilization (p &lt, 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p &lt, 0.001, p &lt, 0.01, and p &lt, 0.01, respectively) as well as actin/tubulin distribution (p &lt, 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p &lt, 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway.

228 p. (This book is a printed edition of the Special Issue Hereditary Hemorrhagic Telangiectasia: Recent Advances and Future Challenges that was published in JCM), Hereditary hemorrhagic telangiectasia (HHT) is an autosomal heritable disease, leading to vascular malformations, ranging from mucocutaneous telangiectases to large arteriovenous malformations, which can occur in different organs. HHT is associated with a decreased quality of life and severe complications. If untreated, the disease leads to decreased life expectancy. Recent years have brought advances in diagnosis and treatment, but not a cure for HHT. The exact molecular etiology is still unknown and there is an urgent need for more systematic research. Because understanding the mechanisms of disease is essential for the development of new medicines or therapeutic strategies, this book aims to highlight not only the current knowledge regarding the diagnosis and treatment of HHT, but also the newest insights into the molecular basis of HHT.Among the different contributions, we would like to emphasize: (i) the key role of a systematic screening and treatment of patients at HHT Centers (de Gussem et al. 2020), (ii) the knowledge about genetics, genotype-phenotype correlations, second-hits and circulating biomarkers of HHT (Bernabeu et al. 2020; Cannavicci et al. 2020; Geisthoff et al. 2020; Kilian et al. 2020; Ruiz-Llorente et al. 2020); (iii) advances in imaging and treatment of arteriovenous malformations of the lung,liver and gastrointestinal tract using contrast-enhanced magnetic resonance angiography, computed tomography, endoscopy and embolotherapy (Daniel et al. 2020; Harwin et al. 2020; Mora-Lujan et al. 2020; Majumdar et al. 2020; Van den Heuvel et al. 2020; Schneider et al. 2021), and (iv)pharmacological strategies for HHT-related nose and gastrointestinal bleeding, using well known and repurposed drugs, including bevacizumab, propranolol, tacrolimus, and antithrombotic drugs(Albi ̃nana et al. 2020; Dupuis-Girod et al. 2020; Gaetani et al. 2020; Mei-Zahav et al. 2020;Mora-Lujan et al. 2020).

8 p.-2 fig. This entry is adapted from 10.3390/jcm9113571

21 p.-2 fig., 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 ENG (endoglin), ACVRL1 (ALK1), and MADH4 (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., This research was funded by Consejo Superior de Investigaciones Científicas of Spain (CSIC; grant 201920E022 to C.B.).

8 p.-2 fig., 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-b) 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-b 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 via 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-endotheliuminteractions 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., DS wrote the manuscript and provided funding support.

7 p.-4 fig., 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., This work was supported by grants from Ministerio de Economia, Industria y Competitividad (SAF2013-43421-R to CB), Mobility Program Salvador de Madariaga, Ministerio de Educacion, Cultura y Deporte (PRX17/00142 to CB), Consejo Superior de Investigaciones Cientificas (201420E039 and 201920E022 to CB), and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to CB and contract CNV-234-PRF-360 to LR-L) of Spain.

41 p.-7 fig. Objective: To investigate the effect of cholesterol (hypercholesterolemia/7- ketocholesterol) on endoglin expression and regulation with respect to endothelial/vascular dysfunction in vivo and in vitro. Approach and results: In vivo experiments were performed in two-month-old ApoE-/-/LDLR-/- female mice and their wild type C57BL/6J littermates. In in vitro experiments, Human Aortic Endothelial Cells (HAECs) were treated with 7-ketocholesterol (7K). ApoE-/-/LDLR-/- mice developed hypercholesterolemia accompanied by increased circulating levels of P-selectin and endoglin and a disruption of NO metabolism. Functional analysis of aorta demonstrated impaired vascular reactivity and Western blot analysis revealed downregulation of membrane Eng/Smad2/3/eNOS signaling in ApoE-/-/LDLR-/- mice. 7K increased endoglin expression via KLF6, LXR and NF-κB in HAECs. 7K-induced endoglin expression was prevented by the treatment with 2-hydroxypropyl-β-cyclodextrin, PHA-408 or by KLF6 silencing. 7K induced increased adhesion and transmigration of monocytic THP-1 cells, was prevented by endoglin silencing. Conclusions: Hypercholesterolemia altered endoglin expression and signaling, followed by endothelial/vascular dysfunction before formation of atherosclerotic lesions in ApoE-/-/LDLR- /- mice. By contrast, 7-ketocholesterol increased endoglin expression, and induced inflammation in HAECs, which was followed by an increased adhesion and transmigration of monocytes via endothelium, which was prevented by endoglin inhibition. Thus, we propose a relevant role for endoglin in endothelial/vascular dysfunction/inflammation when exposed to cholesterol This work was supported by project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) cofunded by ERDF, grants of Charles University Grant Agency, GAUK 1158413C, 1216217,Specific University Research SVV/2017/260414 and Czech Health Research Council (AZV CR17-31754A). CB was supported by grants from Consejo Superior de Investigaciones Cientificas(201420E039) and Centro de Investigacion Biomedica en Red de Enfermedades Raras(CIBERER; ISCIII-CB06/07/0038). CIBERER is an initiative of the Instituto de Salud Carlos III(ISCIII) of Spain supported by FEDER funds.

14 p., [EN]Hereditary diseases constitute a major group within the rare diseases that includes those caused by mutations in a single gene (monogenic) or in more than one gene (multigenic). The discovery that a certain gene is associated with the development of a disease has become a paradigm of current biomedical research with huge implications in clinical practice. Hence, molecular diagnosis based on the search for mutations in genes associated with a disease, has not only become essential to support the clinical diagnosis, but often provides diagnostic criteria that allow customized patient management. Although we have become familiar with terms like genes, Human Genome or DNA Sequencing, we often forget that the identification of a gene responsible for a disease is only the starting point of a long process whose main objectives are to establish the molecular mechanisms underlying the pathology and the development of strategies to prevent or cure the disease. Here, we will review recent advances in two rare diseases, one multigenic, the atypical hemolytic uremic syndrome, and the other one monogenic, the hereditary hemorrhagic telangiectasia, to illustrate the model of multidisciplinary research that characterizes current biomedical research and how the knowledge generated in this area has been transferred to clinical practice., [ES]Dentro de las enfermedades raras, las enfermedades hereditarias constituyen un grupo mayoritario que incluye las ocasionadas por mutaciones en un solo gen (monogénicas) o aquellas ocasionadas por más de un gen (poligénicas). Descubrir que un determinado gen se asocia con el desarrollo de una patología se ha convertido en un paradigma de la investigación biomédica actual que tiene enormes repercusiones en la práctica clínica. Así, el diagnóstico molecular, basado en la búsqueda de mutaciones en los genes asociados a una patología, no solo se ha convertido en un refuerzo imprescindible del diagnóstico clínico, sino que frecuentemente aporta criterios a ese diagnóstico que permiten un manejo personalizado de los pacientes. Sin embargo, nos hemos familiarizado tanto con términos como genes, genoma humano o secuenciación de ADN que frecuentemente olvidamos que la identificación de un gen como responsable de una enfermedad es a menudo solo el punto de partida de un largo proceso cuyos objetivos fundamentales son establecer los mecanismos moleculares que determinan la patología y el desarrollo de estrategias que la eviten o la remedien. En este artículo revisaremos los avances recientes en dos patologías raras, una poligénica, el síndrome hemolítico urémico atípico, y otra monogénica, la telangiectasia hemorrágica hereditaria, para ilustrar el modelo de investigación multidisciplinar que caracteriza a la investigación biomédica actual y cómo el conocimiento generado en esta área se ha trasladado a la práctica clínica.

1p., Objective: A soluble form of tissue/membrane endoglin (sEng) circulating in plasma is increased in hypercholesterolemia, atherosclerosis and type II diabetes mellitus patients. Moreover, it has been proposed that sEng might be least partially responsible for the induction of endothelial dysfunction (but not studied in atherosclerosis prone blood vessels so far) and related to the presence of hypercholesterolemia. We provided couple of experiments in order to reveal whether high levels of sEng combined with hypercholesterolemia might affect vascular/endothelial function and inflammation in mice aorta., Methods: Three-month-old female transgenic mice on CBAxC57BL/6J background with high levels of sEng (Sol-Eng+ high HFD) and their littermates with low levels of sEng (Sol-Eng+ low HFD) were fed high fat diet (HFD) for either 3 months or 6 months. Plasma samples were used for biochemical analyses of total cholesterol and ELISA analyses of sEng and inflammatory markers. Functional parameters of aorta were assessed by means of wire myograph 620M. Western Blot analysis endothelial dysfunction/inflammation markers in aorta was performed., Results: 3 months exposure to high sEng levels and HFD resulted in induction of inflammation (increased expression of P-selectin, ICAM-1, pNFκB and COX-2) in aorta of high Sol-Eng+high HFD. Moreover, 6 months exposure to high sEng levels and HFD resulted in impaired KCl induced vasoconstriction, endothelial-dependent relaxation after administration of acetylcholine, endothelial-independent relaxation induced by sodium nitroprusside. In addition, the expressions of endoglin, p-eNOS/eNOS, pSmad2/3/Smad2/3 signaling pathway were significantly lower in Sol-Eng+high HFD group compared to Sol-Eng+low HFD group., Conclusions: Current results show that high levels of soluble endoglin in combination with hypercholesterolemia induce signs of inflammation and endothelial/vascular dysfunction in aorta with possible alteration of membrane endoglin/eNOS signaling. Thus, we might propose that high levels of soluble endoglin presented in patients with hypercholesterolemia, atherosclerosis and type II diabetes mellitus might worsen endothelial dysfunction in combination with hypercholesterolemia, thus soluble endoglin might be considered as a risk factor of cardiovascular diseases., This work was supported by grant from Czech Health Research Council AZV CR number 17-31754A.

14 p.-6 fig., 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., The work was supported by grants from Czech Science Foundation GACR No. GA15-24015S, the Grant Agency of Charles University in Prague No. 1284214/C, No. 1158413C and SVV/2016/260293, the European Regional Development Fund under the Innovative Economy Program of the European Union (grant coordinated by JCET-UJ, No. POIG.01.01.02-00-069/09), the Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to C.B. and SAF2013-45784-R to J.M.L.-N.), the Junta de Castilla y Leon (GR100 to J.M.L.-N.), CIBERER (to C.B.) and Red de Investigación Cooperativa en Enfermedades Renales (REDINREN, RD12/0021/0032 to J.M.L.-N.). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III of Spain supported by European Regional Development Funds (FEDER). The Cardiovascular Phenotyping Unit of the University of Salamanca received the support of FEDER.

22 p.-6 fig.-1 tab.-3 fig. supl.-2 tab.supl., Endoglin is a transmembrane glycoprotein expressed in vascular endothelium that plays a key role in angiogenesis. Mutations in the endoglin gene (ENG) cause Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), characterized by arteriovenous malformations (AVMs) in different organs. These vascular lesions derive from abnormal processes of angiogenesis where aberrant vascular remodeling leads to focal loss of capillaries. Current treatments for HHT1 include anti-angiogenic therapies. Interestingly, a circulating form of endoglin (also known as soluble endoglin, sEng), proteolytically released from the membrane-bound protein and displaying anti-angiogenic 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 anti-angiogenic 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 back 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 expression of endogenous endoglin., This work was supported by grants from Ministerio de Economia, Industria y Competitividad of Spain (SAF2010-19222 and SAF2013-43421-R to CB), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038) and the British Heart Foundation (PG/14/86/31177 to HMA). EG-V was supported by the international mobility program of Ministerio deEconomía, Industria y Competitividad (EEBB-I-14-09020 and EEBB-I-15-10398). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

34 p.-7 fig., 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, peNOSS1177, 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., This work was supported by grants from Czech Science Foundation (GACR 15-24015S,GAUK 1158413C, SVV/2016/260293 and SVV/2017/260414 to Petr Nachtigal), Ministerio de Economía y Competitividad of Spain (SAF2013-43421-R to Carmelo Bernabéu), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIIICB06/07/0038 and ER16PIAC707 to CB). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

11 p.-6 fig., Endoglin, a transforming growth factor β (TGF-β) receptor type III, is co-expressed with endothelial nitric oxide synthase (eNOS) in aortic endothelium in atherosclerotic plaques of mice. Interestingly, atorvastatin (ATV) is able to increase both endoglin and eNOS expression and reduce plaque size beyond its lipid lowering effects but by unknown mechanisms. We hypothesized whether inflammation modulates ATV-dependent induction of endoglin and eNOS expression in vitro in endothelial cells and whether ATV-induced eNOS expression is regulated via endoglin. After treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α, endoglin and eNOS protein expression was reduced, concomitantly with increased levels of cell surface VCAM-1 and soluble endoglin, as determined by flow cytometry, Western blot and ELISA analyses. By contrast, ATV treatment increased endoglin and eNOS protein expression, while preventing TNF-α-mediated downregulation of endoglin and eNOS protein levels. Moreover, suppression of endoglin using small interfering RNA (siRNA), but not inhibition of TGF-β signaling with SB431542, abrogated ATV-induced eNOS expression. These results suggest that ATV treatment prevents inflammation-reduced endoglin and eNOS expression in endothelial cells and that ATV-induced eNOS expression strongly depends on the proper expression of endoglin in HUVECs. Possible implications of these findings might be reflected in pathological conditions characterized by reduced expression of endoglin and eNOS as for example in hereditary hemorrhagic telangiectasia or in other endothelial dysfunctions., This work was supported by The Grant Agency of Charles University in Prague (300811/C and 1158413/C), Charles University in Prague (SVV/2014/260064), Ministerio de Economía y Competitividad of Spain Raras ( (SAF2010-19222 and SAF2013-42421-R to CB), and Centro de Investigación Biomédica en Red de Enfermedades CIBERER to CB).CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. The publication is co-financed by the European Social Fund and the state budget of the Czech Republic (Project No. CZ.1.07/2.3.00/30.0061).

40 p.-7 fig.-1 tab. Endoglin is an auxiliary cell surface receptor for TGF-beta family members. Two different alternatively spliced isoforms, long (L)-endoglin and short (S)-endoglin, have been reported. S-endoglin and L-endoglin proteins vary from each other in their cytoplasmic tails that contain 14 and 47 amino acids, respectively. A critical role for endoglin in vascular development has primarily been studied in endothelial cells. In addition, endoglin expression is upregulated during monocyte-to-macrophage differentiation; however, little is known about its role in this myeloid context. To investigate the function of endoglin in monocytes, stable transfectants expressing the two endoglin isoforms in the promonocytic human cell line U937 were generated. The differential gene expression fingerprinting of these endoglin transfectants using DNA microarrays and further bioinformatics analysis showed a clear alteration in essential biological functions, mainly those related to "Cellular Movement", including cell adhesion and transmigration. Interestingly, these cellular functions are highly dependent on adhesion molecules, including integrins alpha 1 (CD49a, ITGA1 gene), alpha L (CD11a, ITGAL gene), alpha M(CD11b, ITGAM gene) and beta 2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192, CCR2 gene), which are downregulated in endoglin transfectants. Moreover, activin A (INHBA gene), a TGF-beta superfamily member involved in macrophage polarization, was distinctly affected in each endoglin transfectant, and may contribute to the regulated expression of integrins. These data were confirmed by quantitative PCR, flow cytometry and functional tests. Taken together, these results provide new insight into endoglin function in monocytes. This study has been supported by grants from Ministerio de Economia y Competitividad of Spain (SAF2010-19222 to CB), Genoma España (MEICA to CB), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, CB). FJB is a scientific researcher from the CIBERER, an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

14 p.-10 fig., Endoglin is an auxiliary component of the transforming growth factor-beta (TGF-beta) receptor system, able to associate with the signaling receptor types I (TbetaRI) and II (TbetaRII) in the presence of ligand and to modulate the cellular responses to TGF-beta1. Endoglin cannot bind ligand on its own but requires the presence of the signaling receptors, supporting a critical role for the interaction between endoglin and TbetaRI or TbetaRII. This study shows that full-length endoglin interacts with both TbetaRI and TbetaRII, independently of their kinase activation state or the presence of exogenous TGF-beta1. Truncated constructs encoding either the extracellular or the cytoplasmic domains of endoglin demonstrated that the association with the signaling receptors occurs through both extracellular and cytoplasmic domains. However, a more specific mapping revealed that the endoglin/TbetaRI interaction was different from that of endoglin/TbetaRII. TbetaRII interacts with the amino acid region 437-558 of the extracellular domain of endoglin, whereas TbetaRI interacts not only with the region 437-558 but also with the protein region located between amino acid 437 and the N terminus. Both TbetaRI and TbetaRII interact with the cytoplasmic domain of endoglin, but TbetaRI only interacts when the kinase domain is inactive, whereas TbetaRII remains associated in its active and inactive forms. Upon association, TbetaRI and TbetaRII phosphorylate the endoglin cytoplasmic domain, and then TbetaRI, but not TbetaRII, kinase dissociates from the complex. Conversely, endoglin expression results in an altered phosphorylation state of TbetaRII, TbetaRI, and downstream Smad proteins as well as a modulation of TGF-beta signaling, as measured by the reporter gene expression. These results suggest that by interacting through its extracellular and cytoplasmic domains with the signaling receptors, endoglin might affect TGF-beta responses., This work was supported by grants from Ministerio de Ciencia y Tecnologı́a (SAF2000-0132) and Comunidad Autónoma de Madrid (CAM).

20 p.-6 fig.-3 fig. supl.-2 tab. supl., Endoglin plays a crucial role in pathophysiological processes such as hereditary hemorrhagic telangiectasia (HHT), preeclampsia and cancer. Endoglin expression is upregulated during the monocyte-to-macrophage transition, but little is known about its regulation and function in these immune cells. Two different alternatively spliced isoforms of endoglin have been reported, L-endoglin and S-endoglin. Although L-endoglin is the predominant variant, here, we found that there was an increased expression of the S-endoglin isoform during senescence of the myeloid lineage in human and murine models. We performed a stable isotope labelling of amino acids in cell culture (SILAC) analysis of both L-endoglin and S-endoglin transfectants in the human promonocytic cell line U937. Analysis of differentially expressed protein clusters allowed the identification of cellular activities affected during aging. S-endoglin expression led to decreased cellular proliferation and a decreased survival response to granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced apoptosis, as well as increased oxidative stress. Gene expression and functional studies suggested that there was a non-redundant role for each endoglin isoform in monocyte biology. In addition, we found that S-endoglin impairs the monocytic differentiation into the pro-inflammatory M1 phenotype and contributes to the compromised status of macrophage functions during aging., This study was supported by grants from Ministerio de Economía y Competitividad of Spain [grant number SAF2010-19222 to C.B.]; and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) (to C.B.). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. M.A. was supported by a fellowship from Ministerio de Ciencia e Innovación [grant number BES-2008-003888].

10 p.-9 fig.-1 tab., Endoglin is the gene mutated in hereditary hemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Endoglin glycoprotein is a component of the transforming growth factor type ß (TGF-ß) receptor system which is highly expressed by endothelial cells, and at lower levels on fibroblasts and smooth muscle cells, suggesting the involvement of these lineages in the HHT1 vascular dysplasia. Overexpression of endoglin in mouse NCTC929 fibroblasts led to decreased migration in chemotactic and wound healing assays, as well as changes in the cellular morphology. When plated on uncoated surfaces, endoglin transfectants formed intercellular clusters, endoglin being not specifically localized to the cell-cell junctions, but homogenously distributed on the cellular surface. Although the expression of α5ß1 integrin and of an activation epitope of ß1 integrin were unchanged, a polyclonal antibody to α5ß1 integrin was able to inhibit cluster formation, suggesting the involvement of integrin ligand/s. In fact, coating with fibronectin, laminin, or an RGD-containing 80 kDa fragment of fibronectin were able to prevent the cellular clustering. Furthermore, synthesis of plasminogen activator inhibitor 1 (PAI-1), and to a weak extent that of fibronectin, were inhibited in endoglin transfectants. Thus, the presence of endoglin in mouse NCTC929 fibroblasts is associated with reduced production of certain extracellular matrix (ECM) components, which might explain their altered morphology, migration and intercellular cluster formation., This work has been supported by grants from Camisión Interministerial de Ciencia yTecnología (CICYT-SAF97-0034 to C. Bernabéu, and CICYT-SAF97-0064-C03-02 to A. García-Pardo), Comunidad Autónoma de Madrid (CAM) and Biomed Program of the European Community (BMH4-CT95-0995 to C. Bernabéu).

11 p.-6 fig., TGF-beta superfamily co-receptors are emerging as targets for cancer therapy, acting both directly on cells and indirectly on the tumour neovasculature. Endoglin (CD105), an accessory component of the TGF-beta receptor complex, is expressed in certain melanoma cell lines and the endothelial cells of tumour neovessels. Targeting endoglin with immunotoxins is an attractive approach for actively suppressing the blood supply to tumours. Here, we report evidence indicating that endoglin is expressed in mouse melanoma B16MEL4A5 and mouse fibroblast L929 cell lines. We prepared an immunotoxin to target endoglin by coupling the rat anti-mouse MJ7/18 (IgG2a) monoclonal antibody (mAb) to the non-toxic type 2 ribosome-inactivating protein nigrin b (Ngb) with N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP) as a linker with a molar nigrin b at a MJ7/18 stoichiometry of 2:1. The MJ7-Ngb immunotoxin generated killed both cell lines, with IC50 values of 4.2 × 10−9 M for B16MEL4A5 and 7.7 × 10−11 M for L929 cells. For in vivo assays of the immunotoxin, B16MEL4A5 cells were injected subcutaneously into the right flanks of 6-week-old C57BL/6 J mice. When the animals developed palpable solid tumours, they were subjected to treatment with the immunotoxin. While treatment with either MJ7/18 mAb or Ngb did not affect tumour development, treatment with the immunotoxin completely and steadily blocked tumour growth up to 7 days, after which some tumours re-grew. Thus, vascular-targeting therapy with this anti-vascular immunotoxin could promote the destruction of newly created tumour vessels at early stages of B16MEL4A5 tumour development and readily accessible CD105+ B16MEL4A5 melanoma cells., This research was supported by grants from the Junta de Castilla y León (Grupo de Excelencia GR106 and Convenio-Consejería de Sanidad) and UVA-GIR funding to T.G., Fondo de Investigaciones Sanitarias FISPI02/0917 to R.M. and FISPI04/1279 to J.M.F., Ministerio de Ciencia e Innovación of Spain (SAF2010-19222 to C.B.) and Genoma España (MEICA) to C.B.

7 p.-5 fig., CD105 is a receptor for transforming growth factor-beta but it is also considered to be involved in cellular functions such as cell adhesion and migration. Using CD105 transfected rat myoblasts, we have investigated the role of CD105 in cell adhesion, spreading, growth and migration. CD105 transfected myoblasts expressed abundant CD105, which was preferentially located within focal adhesion sites. These cells took on a bipolar morphology whereas mock cells remained polygonal or rounded, and when wounded, CD105 expressing cells realigned their long axis prior to migrating and migrated as a cohort of cells. CD105 expression promoted cellular attachment, spreading, survival and growth in serum-free conditions and each of these parameters could be inhibited by a RGD-containing peptide but not a RAD-containing peptide. Mock-transfected cells could not attach, spread or grow under these conditions. Attachment, spreading and growth in CD105 expressing cells could be promoted by the addition of a monoclonal antibody against CD105. Expression of CD105 resulted in the phosphorylation of JNK1 but had no effect on beta1 integrin expression. From this preliminary study, we conclude that in addition to acting as a transforming growth factor-beta receptor, CD105 has an important role in cell adhesion, migration and survival.

11p.-6 fig., Endoglin is a transforming growth factor-beta (TGF-beta) co-receptor expressed mainly on endothelial cells and involved in cardiovascular development, angiogenesis, and vascular remodeling. This is illustrated by the fact that mutations in the endoglin gene give rise to hereditary hemorrhagic telangiectasia type 1, a dominant vascular disease with clinical manifestations that originate by a mechanism of haploinsufficiency. Thus, studies on the regulated expression of endoglin are crucial to devising therapeutic strategies for hereditary hemorrhagic telangiectasia type 1. Endoglin is highly expressed in the neovasculature associated with hypoxia such as ischemic tissues and tumors, but the molecular mechanism of this up-regulation is unknown. Here, we have investigated the possible regulation of endoglin expression by hypoxia. Surface protein, transcript, and promoter activity levels of endoglin were found to be up-regulated by hypoxia, indicating that the regulation takes place at the transcriptional level. A hypoxia-responsive element downstream of the main transcription start site of the endoglin gene was functionally characterized. Whereas hypoxia alone moderately stimulated endoglin transcription, addition of TGF-beta under bypoxic conditions resulted in transcriptional cooperation between both signaling pathways, leading to marked stimulation of endoglin expression. Because basal endoglin transcription is sustained by Sp1, and TGF-beta and hypoxia signaling pathways are mediated by Smad proteins and hypoxia-inducible factor-1 (HIF-1), respectively, the involvement of these transcription factors was analyzed. Functional and co-immunoprecipitation experiments demonstrated the existence of a multiprotein complex (Sp1.Smad3.HIF-1) on the endoglin promoter, mediating the cooperation between the hypoxia and TGF-beta pathways. Within this multiprotein complex, Smad3 appears to function not only as a coactivator factor, but also as an adaptor between HIF-1 and Sp1. We propose that basal endoglin transcription (highly dependent on Sp1) may switch from a constitutive to an inducible state through Sp1 interaction with HIF-1 and Smad transcription factors, induced by hypoxia and TGF-beta, respectively., This work was supported in part by Ministerio de Ciencia y Tecnologı́a Grant SAF2000-0132 and by the Comunidad Autónoma de Madrid.

10 p.-6 fig., Endoglin, a component of the transforming growth factor-beta (TGF-beta) receptor complex expressed on endothelial cells, is involved in cardiovascular morphogenesis and vascular remodeling, as exemplified by the fact that the endoglin gene is the target for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia type 1. Since haploinsufficiency is the underlying mechanism for hereditary hemorrhagic telangiectasia type 1, understanding the regulation of endoglin gene expression appears to be a crucial step to correct the disease. In this study we have identified an Sp1 site at -37 as a critical element for the basal transcription of the endoglin TATA-less promoter. Since endoglin promoter activity is stimulated by TGF-beta and this stimulation is located at the Sp1-containing proximal region, we have investigated the possible involvement of Sp1 in the TGF-beta-mediated induction. Mutation of the Sp1-binding sequence, or addition of the Sp1 inhibitor WP631, abolished both the basal transcription activity and the TGF-beta responsiveness of the endoglin promoter. Binding of Sp1 and Smad3 to the proximal promoter region -50/-29 was evidenced by electrophoretic mobility shift assays and DNA affinity precipitation studies. Furthermore, synergistic cooperation on the promoter activity between Sp1 and TGF-beta or Smad3 could be demonstrated by co-transfection experiments of reporter promoter constructs. The molecular mechanism underlying this cooperation appears to involve a direct physical interaction between Sp1 and Smad3/Smad4., This work was supported in part by Ministerio de Ciencia y Tecnologı́a Grants SAF2000-0132 and SAF98/0068 and by the Comunidad Autónoma de Madrid.

10 p.-7 fig., Signaling by transforming growth factor (TGF)-beta family members is mediated by Smad proteins that regulate gene transcription through functional cooperativity and association with other DNA-binding proteins. The hypoxia-inducible factor (HIF)-1 is a transcriptional complex that plays a key role in oxygen-regulated gene expression. We demonstrate that hypoxia and TGF-beta cooperate in the induction of the promoter activity of vascular endothelial growth factor (VEGF), which is a major stimulus in the promotion of angiogenesis. This cooperation has been mapped on the human VEGF promoter within a region at -1006 to -954 that contains functional DNA-binding sequences for HIF-1 and Smads. Optimal HIF-1alpha-dependent induction of the VEGF promoter was obtained in the presence of Smad3, suggesting an interaction between these proteins. Consistent with this, co-immunoprecipitation experiments revealed that HIF-1alpha physically associates with Smad3. These results demonstrate that both TGF-beta and hypoxia signaling pathways can synergize in the regulation of VEGF gene expression at the transcriptional level., This work was supported in part by Grants SAF2000-0132 and SAF98/0068 from the Ministerio de Ciencia y Tecnología and Comunidad Autónoma de Madrid.

8 p.-5 fig.-1 tab., Hereditary hemorrhagic telangiectasia (HHT), the most common inherited vascular disorder, is caused by mutations in genes involved in the transforming growth factor beta (TGF-β) signaling pathway (ENG, ACVRL1, and SMAD4). Yet, approximately 15% of individuals with clinical features of HHT do not have mutations in these genes, suggesting that there are undiscovered mutations in other genes for HHT and possibly vascular disorders with overlapping phenotypes. The genetic etiology for 191 unrelated individuals clinically suspected to have HHT was investigated with the use of exome and Sanger sequencing; these individuals had no mutations in ENG, ACVRL1, and SMAD4. Mutations in BMP9 (also known as GDF2) were identified in three unrelated probands. These three individuals had epistaxis and dermal lesions that were described as telangiectases but whose location and appearance resembled lesions described in some individuals with RASA1-related disorders (capillary malformation-arteriovenous malformation syndrome). Analyses of the variant proteins suggested that mutations negatively affect protein processing and/or function, and a bmp9-deficient zebrafish model demonstrated that BMP9 is involved in angiogenesis. These data confirm a genetic cause of a vascular-anomaly syndrome that has phenotypic overlap with HHT., This work was funded by the Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, a National Institutes of Health grant R01 (HL079108 to B.L.R.), a British Heart Foundation Programme grant (RG/08/002/24718 to N.W.M), and the Ministerio de Economia y Competitividad of Spain (SAF2010-19222 to C.B.).