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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.

16 p.-7 fig. Rossi. E. et al., 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., This work was supported by Grants from Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to C. B., SAF2013-45784-R to J. M. L.-N., and BFU2010-15237 to C. G.-M.), Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 and ER16PIAC707 to C. B.), Red de Investigacion Cooperativa en Enfermedades Renales (REDINREN to J. M. L.-N.), The Conny-Maeva Charitable Foundation (to D. M. S.), and Fundación Renal Iñigo Alvarez de Toledo to J. M. L.-N. CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

19 p.-6 fig. Plumitallo. Sara et al., 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., This work was supported by grants from the Ministerio de Industria, Economia y Competitividad (SAF2013-43421-R to CB) and the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 and ER16PIAC707 to CB) in Spain, both supported by European Regional Development Funds (FEDER). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) in Spain. This work is part of the results of research activity by SP, who holds a research fellowship (2/R.C./2016, n.20160028774) from the IRCCS Fondazione Policlinico San Matteo, Pavia, Italy.

Endoglin is an auxiliary cell surface receptor for TGF-β 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 α1 (CD49a, ITGA1 gene), αL (CD11a, ITGAL gene), αM (CD11b, ITGAM gene) and β2 (CD18, ITGB2 gene) and the chemokine receptor CCR2 (CD192, CCR2 gene), which are downregulated in endoglin transfectants. Moreover, activin A (INHBA gene), a TGF-β 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.

Human endoglin is an RGD-containing transmembrane glycoprotein identified in vascular endothelial cells. Although endoglin is essential for angiogenesis and its expression is up-regulated in inflammation and at sites of leukocyte extravasation, its role in leukocyte trafficking is unknown. This function was tested in endoglin heterozygous mice (Eng+/-) and their wild-type siblings Eng+/+ treated with carrageenan or LPS as inflammatory agents. Both stimuli showed that inflammation-induced leukocyte transendothelial migration to peritoneum or lungs was significantly lower in Eng+/- than in Eng+/+ mice. Leukocyte transmigration through cell monolayers of endoglin transfectants was clearly enhanced in the presence of endoglin. Coating transwells with the RGD-containing extracellular domain of endoglin, enhanced leukocyte transmigration, and this increased motility was inhibited by soluble endoglin. Leukocytes stimulated with CXCL12, a chemokine involved in inflammation, strongly adhered to endoglincoated plates and to endoglin-expressing endothelial cells. This endoglin-dependent adhesion was abolished by soluble endoglin, RGD peptides, the anti-integrin α5β1 inhibitory antibody LIA1/2 and the chemokine receptor inhibitor AMD3100. These results demonstrate for the first time that endothelial endoglin interacts with leukocyte integrin α5β1 via its RGD motif, and this adhesion process is stimulated by the inflammatory chemokine CXCL12, suggesting a regulatory role for endoglin in transendothelial leukocyte trafficking. © 2013 by The American Society of Hematology., Ministerio de Economia y Competitividad of Spain (SAF2010-61 827, SAF2010-15 881); Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER); Red de Investigacion Cooperativa en Enfermedades Renales (REDINDEN)

33 p.-9 fig.-2 tab. Muñoz-Felix. J. M. et al., 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, includ¬ing 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., This study has been supported by grants from Ministerio de Economía y Competitividad of Spain (SAF2013-43421-R to CB; and SAF2013-45784-R to JML-N), Junta de Castilla y León (GR100, JML-N), Institute Queen Sophie for Renal Research, Fundación Renal Íñigo Álvarez de Toledo, Madrid, Spain (0016¬002), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, CB) (ISCIII-CB06/07/0038) and Red de Investigación Cooperativa en Enfermedades Renales (REDINREN, JML-N) (R12/0021/ 0032). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. BO and ENG are supported by fellowships from Ministerio de Economía y Competitividad (BES-2011-048968 and BES-2008-005550). JMMF, LPR and CC are supported by fellowships from Junta de Castilla y León and Fondo Social Europeo (EDU/1204/2010 and EDU/1083/2013).

The interaction and interplay between endothelial cells (ECs) and mural cells (such as vascular smooth muscle cells -VSMCs- and pericytes) play a pivotal role in vascular biology. Endoglin is an RGD-containing ligand of β1 integrins highly expressed by ECs during angiogenesis. Our working hypothesis is that endothelial endoglin acts as an adhesion molecule via integrin recognition motifs, allowing the interaction between ECs and mural cells. We find that suppression of endoglin expression or addition of soluble endoglin inhibits the adhesion between ECs and VSMCs as shown by tubulogenesis assays on matrigel. The EC-VSMC adhesion was also abolished by an antiintegrin α5β1 inhibitory antibody, whereas it was markabsedly enhanced by the integrin activators MnCl2 or CXCL12. The CXCL12-dependent cell adhesion was abolished in the presence of soluble endoglin or a derived pentapeptide containing the RGD motif. Adhesion of cells overexpressing different endoglin mutant constructs, allowed the specific mapping of the endoglin RGD motif as involved in adhesion to VSMCs. Binding of soluble endoglin to VSMCs was markedly enhanced by MnCl2 and CXCL12 and this increase was inhibited by the RGD peptide. Moreover, transgenic mice overexpressing soluble endoglin show podocyturia and lower number of glomerular podocytes, suggesting that soluble endoglin induced the detachment of podocytes 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 development and maturation in normal physiology as well as in pathologies such as preeclampsia, cancer or hereditary hemorrhagic telangiectasia.

This work is licensed under a Creative Commons Attribution 4.0 International License., Elevated levels of a circulating form of the transforming growth factor-β (TGF-β) coreceptor endoglin correlate with poor clinical outcome in different types of cancer. Soluble endoglin (Sol-Eng) is primarily produced by cleavage of cell-surface endoglin by the transmembrane metalloprotease MMP14 that releases most of its extracellular domain. Sol-Eng has been found to contribute to different cardiovascular pathologies, including preeclampsia, a severe hypertensive syndrome of pregnancy. While the anti-angiogenic and pro-hypertensive functions of Sol-Eng appear well established, its role in cancer has not been fully investigated. Recently, we reported that Sol-Eng strongly inhibits signaling through the hepatocyte growth factor (HGF) tyrosine kinase receptor Met in mouse skin spindle carcinoma cells. Sol-Eng also blocked basal and HGF-mediated stimulation of carcinoma cell proliferation, migration and invasion. Taken together, the above results and the anti-angiogenic function exerted by Sol-Eng suggest a suppressor role for Sol-Eng in cancer. This conclusion is discussed in the paradoxical context of Sol-Eng as a marker of poor prognosis and as a potential contributor to the decreased risk of preeclamptic mothers to develop breast cancer later in life., Our research is supported by grants from the Ministerio de Economía y Competitividad (SAF2013-46183-R to MQ, and SAF2013-43421-R to CB), and Comunidad Autónoma de Madrid (S2010/BMD-2359, SkinModel, to MQ). CIBERER is an initiative of the Instituto de Salud Carlos III. GdC was the recipient of a Juan de la Cierva postdoctoral research contract. EP-G and EM-V are the recipients of a postdoctoral research contract from the scientific foundation of Asociación Española Contra el Cáncer (AECC).

Increased levels of soluble endoglin (Sol-Eng) correlate with poor outcome in human cancer. We have previously shown that shedding of membrane endoglin, and concomitant release of Sol-Eng is a late event in chemical mouse skin carcinogenesis associated with the development of undifferentiated spindle cell carcinomas (SpCCs). In this report, we show that mouse skin SpCCs exhibit a high expression of hepatocyte growth factor (HGF) and an elevated ratio of its active tyrosine kinase receptor Met versus total Met levels. We have evaluated the effect of Sol-Eng in spindle carcinoma cells by transfection of a cDNA encoding most of the endoglin ectodomain or by using purified recombinant Sol-Eng. We found that Sol-Eng inhibited both mitogen-activated protein kinase (MAPK) activity and cell growth in vitro and in vivo. Sol-Eng also blocked MAPK activation by transforming growth factor-β1 (TGF-β1) and impaired both basal and HGF-induced activation of Met and downstream MAPK. Moreover, Sol-Eng strongly reduced basal and HGF-stimulated spindle cell migration and invasion. Both Sol-Eng and full-length endoglin were shown to interact with Met by coimmunoprecipitation experiments. However, full-length endoglin expressed at the plasma membrane of spindle carcinoma cells had no effect on Met signaling activity, and was unable to inhibit HGF-induced cell migration/invasion. These results point to a paradoxical suppressor role for Sol-Eng in carcinogenesis., Ministerio de Economía y Competitividad (SAF2010-19152, SAF2013-46183-R to M.Q., and SAF2010-19222, SAF2013-43421-R to C.B.), Comunidad Autónoma de Madrid (S2010/BMD-2359, SkinModel, to M.Q.). GdC was the recipient of a Juan de la Cierva postdoctoral research contract. EP-G and EM-V are the recipients of a postdoctoral research contract from the scientific foundation of Asociación Española Contra el Cáncer (AECC).

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).

Endoglin (CD105) is a cell surface component of the transforming growth factor-β (TGF-β) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5′-flanking sequence of the human endoglin gene has been isolated. The 5′-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFκB, and Mad, as well as TGF-β–, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5′ RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream −400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the −81/+350 fragment as well as major transcriptional regulatory elements within the −400/−141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position −68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-β1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Endoglin (CD105) is a cell surface component of the transforming growth factor-β (TGF-β) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5′-flanking sequence of the human endoglin gene has been isolated. The 5′-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFκB, and Mad, as well as TGF-β–, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5′ RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream −400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the −81/+350 fragment as well as major transcriptional regulatory elements within the −400/−141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position −68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-β1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Endoglin (CD105) is the target gene for the hereditary hemorrhagic telangiectasia type I (HHT1), a dominantly inherited vascular disorder. It shares with betaglycan a limited amino acid sequence homology and being components of the membrane transforming growth factor-beta (TGF-beta) receptor complex. Using rat myoblasts as a model system, we found that overexpression of endoglin led to a decreased TGF-beta response to cellular growth inhibition and plasminogen activator inhibitor-1 synthesis, whereas overexpression of betaglycan resulted in an enhanced response to inhibition of cellular proliferation and plasminogen activator inhibitor-1 induced expression in the presence of TGF-beta. The regulation by endoglin of TGF-beta responses seems to reside on the extracellular domain, as evidenced by the functional analysis of two chimeric proteins containing different combinations of endoglin and betaglycan domains. Binding followed by cross-linking with 125I-TGF-beta1 demonstrated that betaglycan expressing cells displayed a clear increase (about 3. 5-fold), whereas endoglin expressing cells only displayed an slight increment (about 1.6-fold) in ligand binding with respect to mock transfectants. SDS-polyacrylamide gel electrophoresis analysis of radiolabeled receptors demonstrated that expression of endoglin or betaglycan is associated with an increased TGF-beta binding to the signaling receptor complex; however, while endoglin increased binding to types I and II receptors, betaglycan increased the binding to the type II receptor. Conversely, we found that TGF-beta binding to endoglin required the presence of receptor type II as evidenced by transient transfections experiments in COS cells. These findings suggest a role for endoglin in TGF-beta responses distinct from that of betaglycan.

Endoglin is a transmembrane auxiliary receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells. It plays a wide range of physiological roles but its importance on energy balance or insulin sensitivity has been unexplored. Endoglin deficient mice die during midgestation due to cardiovascular defects. Here we report for first time that heterozygous endoglin deficiency in mice decreases high fat diet-induced hepatic triglyceride content and insulin levels. Importantly, these effects are independent of changes in body weight or adiposity. At molecular level, we failed to detect relevant changes in the insulin signalling pathway at basal levels in liver, muscle or adipose tissues that could explain the insulin-dependent effect. However, we found decreased triglyceride content in the liver of endoglin heterozygous mice fed a high fat diet in comparison to their wild type littermates. Overall, our findings indicate that endoglin is a potentially important physiological mediator of insulin levels and hepatic lipid metabolism, This work has been supported by grants from Ministerio de Educacion y Ciencia (CD: BFU2011; ML: RyC-2007-00211; RN: RYC-2008-02219 and SAF2009- 07049; GS: RYC-2009-04972, SAF2010-15881 and SAF2010-19347; JML-N: SAF2010-15881; CB: SAF2010-61827), Xunta de Galicia (ML: 10PXIB208164PR and RN:2010/14), Junta de Castilla y Leo´n (Excellence Group GR100) Fondo Investigationes Sanitarias (ML: PS09/01880), Centro de Investigacio´n Biome´dica en Red (CIBER)de Fisiopatologı´a de la Obesidad y Nutricio´n (CIBERobn), CIBER de Enfermedades Raras (CIBERER) and Red de Investigacio´n Cooperativa en Enfermedades Renales(REDinREN). CIBERobn, CIBERER and RETIC REDinREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. The research leading to these results has also received funding from the European Community’s Seventh Framework Programme under grant agreements (CD, ML and RN: FP7/2007-2013: nu 245009: NeuroFAST, and RN: ERC-2011-StG-OBESITY53-281408)

Hereditary Hemorragic Telangectasia (HHT) is characterized by a bleeding tendency that is postulated to be a consequence of microvessel fragility. HHT type 1 patients present heterozygous mutations in the endoglin gene resulting in a loss of expression of membrane endoglin in their endothelial cells (EC), while platelets in these patients display normal functions in vitro. We have reported previously that endothelial endoglin is involved, via its RGD motif, in inflammation though integrin-mediated leukocyte adhesion and transmigration. We recently found that endoglin-haplodeficient mice (Eng+/–) have significantly higher bleeding time than Eng+/+ animals, therefore, we haypothesized that endoglin may also act as an adhesion molecule via integrin recognition in the interaction between EC and platelets during hemostasis. Coating plastic wells with the extracellular domain of endoglin enhanced platelets adhesion to the wells under static condition and this interaction persist under flow condition. Similarly, platelets could adhere on confluent EC in an endoglin-mediated process, while the silencing of endoglin expression in EC, and the addition of soluble endoglin or of anti-endoglin antibodies, inhibited stable platelet adhesion to EC. Remarkably, platelets from Glanzmann's thrombasthenia patients lacking the€αIIbβ3 integrin were defective for adhesion on EC, wheras CHO cells, ectopically expressing the human αIIbβ3 integrin acquired the capacity to adhere on rat myoblast transfectants expressing human endoglin (L6E9-E), thus confirming the interaction between endoglin and the αIIbβ3 integrin. These results establish a new critical role for endoglin in integrin-mediated adhesion of platelets to endothelium and provide a better understanding on the cellular mechanisms involved in bleeding events occurring in HHT. The author hereby declares no conflict of interest

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.

Endoglin is a TGF-β co-receptor expressed in endothelial cells, where it plays a crucial role in angiogenesis, cardiovascular development and vascular remodeling. In humans, mutations in the endoglin gene give rise to Hereditary Hemorrhagic Telangiectasia type 1 (HHT1), an autosomal dominant disorder associated with vascular lesions in skin, mucosa and internal organs. So far, endoglin cDNA has been sequenced in several species from mammals, amphibians and birds. While in mammals the characterization of endoglin protein expression and function is well documented, little is known about the protein homologue in birds. In silico analysis by multiple sequences alignment showed a low homology score of 30-33 between the full length chicken endoglin protein and several mammalian homologues. However, a high homology score (80-85) was observed with the cytoplasmic and transmembrane regions and the overall structure of the zona pellucida (ZP) and orphan domains of the extracellular region appear to be conserved. Transient expression of chicken endoglin allowed the identification of a 180-kDa disulfide linked homodimer similar to the mammalian homologues. To further characterize its tissue expression, the novel specific monoclonal antibody (mAb) 7H5A8 was generated against chicken endoglin transfectant cells. The mAb 7H5A8 specifically recognized chicken endoglin by western blot, immunoprecipitation, immunofluorescence flow cytometry as well as immunofluorescence microscopy assays and displayed a positive staining of the endothelium in veins and arteries from frozen tissue sections of lung and bursa of Fabricius. These results may help to further understand the endoglin expression in vertebrates. © 2011 Elsevier B.V., Ministerio de Ciencia e Innovación of Spain (SAF2010-19222 to CB and BFU2010-19144 to CC); Genoma España (MEICA); Centro de Investigación Biomédica en Red de Enfermedades Raras; Fondo de Investigaciones Sanitarias; European Union (PI081813)

[Background]: Ischemia in the placenta is considered the base of the pathogenesis of preeclampsia, a pregnancy-specific syndrome in which soluble endoglin (sEng) is a prognostic marker and plays a pathogenic role. Here, we investigated the effects of hypoxia and the downstream pathways in the release of sEng. [Methods and Results]: Under hypoxic conditions, the trophoblast-like cell line JAR showed an increase in sEng parallel to an elevated formation of reactive oxygen species. Because reactive oxygen species are related to the formation of oxysterols, we assessed the effect of 22-(R)-hydroxycholesterol, a natural ligand of the liver X receptor (LXR), and the LXR synthetic agonist T0901317. Treatment of JAR cells or human placental explants with 22-(R)-hydroxycholesterol or T0901317 resulted in a clear increase in sEng that was dependent on LXR. These LXR agonists induced an increased matrix metalloproteinase-14 expression and activity and a significant reduction of its endogenous inhibitor, tissue inhibitor of metalloproteinase-3. In addition, mice treated with LXR agonists underwent an increase in the plasma sEng levels, concomitant with an increase in arterial pressure. Moreover, transgenic mice overexpressing sEng displayed high blood pressure. Finally, administration of an endoglin peptide containing the consensus matrix metalloproteinase-14 cleavage site G-L prevented the oxysterol-dependent increase in arterial pressure and sEng levels in mice. [Conclusions]: These studies provide a clue to the involvement of the LXR pathway in sEng release and its pathogenic role in vascular disorders such as preeclampsia. © 2012 American Heart Association, Inc., This work was supported by grants from the Ministerio de Ciencia e Innovación of Spain (SAF2010-61827 to Dr Bernabeu, SAF2011-29244 to Dr Castrillo, and SAF2010-15881 to Dr Lopez-Novoa), Genoma España (MEICA; Dr Bernabeu), Instituto Reina Sofía de Investigación Nefrológica (FRIAT; Dr Lopez-Novoa), Junta de Castilla and Leon (Excellence Group Grant GR-100 to Dr Lopez-Novoa), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; Dr Bernabeu), and Red de Investigación Cooperativa en Enfermedades Renales (REDINREN; Dr Lopez-Novoa). 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, including the telemetry equipment, has been acquired with the support of FEDER. Dr Llano is recipient of a Ramón y Cajal Research contract.

Background Hereditary hemorrhagic telangiectasia (HHT) is a vascular disorder characterized by epistaxis, arteriovenous malformations, and telangiectases. The majority of the patients have a mutation in the coding region of the activin A receptor type II-like 1 (ACVRL1) or Endoglin (ENG) gene. However, in approximately 15% of cases, sequencing analysis and deletion/duplication testing fail to identify mutations in the coding regions of these genes. Knowing its vital role in transcription and translation control, we were prompted to investigate the 5'untranslated region (UTR) of ENG. Methods and Results We sequenced the 5'UTR of ENG for 154 HHT patients without mutations in ENG or ACVRL1 coding regions. We found a mutation (c.-127C > T), which is predicted to affect translation initiation and alter the reading frame of endoglin. This mutation was found in a family with linkage to the ENG, as well as in three other patients, one of which had an affected sibling with the same mutation. In vitro expression studies showed that a construct with the c.-127C > T mutation alters the translation and decreases the level of the endoglin protein. In addition, a c.-9G > A mutation was found in three patients, one of whom was homozygous for this mutation. Expression studies showed decreased protein levels suggesting that the c.-9G > A is a hypomorphic mutation. Conclusions Our results emphasize the need for the inclusion of the 5'UTR region of ENG in clinical testing for HHT., This work was supported by grants from Ministerio de Ciencia e Innovación of Spain (SAF2007-61827 to CB; SAF08-01218 to LMB), and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER). The CIBER de Enfermedades Raras is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain. This work was also supported by a grant from the International HHT Foundation

9 páginas, 2 figuras, 1 tabla -- PAGS nros. 169-177 Hereditary haemorrhagic telangiectasia (HHT) is a vascular hereditary autosomic dominant disease associatedwith epistaxis, telangiectases, gastrointestinal haemorrhages and arteriovenous malformations in lung, liverand brain. It affects 1–2 in 10,000 people. There are at least three different genes mutated in HHT, ENG,ACVRL1 and MADH4 that encode endoglin, activin receptor-like kinase (ALK1) and Smad4 proteins,respectively. These proteins are involved in the transforming growth factor (TGF)-β superfamily signallingpathway of vascular endothelial cells. Mutations in ENG (HHT1) and ACVRL1 (HHT2) account for more than90% of all HHT mutations. In this article, we review the underlying molecular and cellular bases and thetherapeutic approaches that have been addressed in our laboratory in recent years

Antibodies in serum from some patients with rheumatoid arthritis, recognize bovine albumin present in the milk, as determined by immunoprecipitation analysis from 125I-milk extracts. This antigen was also immunoprecipitated from bovine sera. These and ELISA studies showed that BSA is preferentially recognized over other proteins present in the milk. Panel studies demonstrated that although the average reactivity for BSA was high, only one third of the sera tested displayed a reactivity above the mean. The possibility of a molecular mimicry mechanism in RA between this food antigen and other human antigens was investigated. A sequence alignment analysis showed that the residues 141-157 of bovine albumin significantly differed from the corresponding fragment of human albumin, but were highly homologous with human collagen type I, C1q and vitamin D binding protein. In support of the immunogenicity of this fragment, we found that representative RA sera displayed a specific reactivity for a synthetic peptide containing the BSA residues responsible for the homology. Furthermore, most of the epitopes recognized on BSA by the RA sera seem to be conformationally dependent as heat denaturation or reduction followed by alkylation lead to a diminished recognition.

Senescence of endothelial cells (ECs) may contribute to age-associated cardiovascular diseases, including atherosclerosis and hypertension. The functional and gene expression changes associated with cellular senescence are poorly understood. Here, we have analyzed the expression, during EC senescence, of 2 different isoforms (L, long; S, short) of endoglin, an auxiliary transforming growth factor (TGF)-beta receptor involved in vascular remodeling and angiogenesis. As evidenced by RT-PCR, the S/L ratio of endoglin isoforms was increased during senescence of human ECs in vitro, as well as during aging of mice in vascularized tissues. Next, the effect of S-endoglin protein on the TGF-beta receptor complex was studied. As revealed by coimmunoprecipitation assays, S-endoglin was able to interact with both TGF-beta type I receptors, ALK5 and ALK1, although the interaction with ALK5 was stronger than with ALK1. S-endoglin conferred a lower proliferation rate to ECs and behaved differently from L-endoglin in relation to TGF-beta-responsive reporters with ALK1 or ALK5 specificities, mimicking the behavior of the endothelial senescence markers Id1 and plasminogen activator inhibitor-1. In situ hybridization studies demonstrated the expression of S-endoglin in the endothelium from human arteries. Transgenic mice overexpressing S-endoglin in ECs showed hypertension, decreased hypertensive response to NO inhibition, decreased vasodilatory response to TGF-beta(1) administration, and decreased endothelial nitric oxide synthase expression in lungs and kidneys, supporting the involvement of S-endoglin in the NO-dependent vascular homeostasis. Taken together, these results suggest that S-endoglin is induced during endothelial senescence and may contribute to age-dependent vascular pathology.

11 páginas, 8 figuras -- PAGS nros. 574-584, Transforming growth factor-β (TGF-β) signaling in endothelial cells is able to modulate angiogenesis and vascular remodeling, although the underlying molecular mechanisms remain poorly understood. Endoglin and ALK-1 are components of the TGF-β receptor complex, predominantly expressed in endothelial cells, and mutations in either endoglin or ALK-1 genes are responsible for the vascular dysplasia known as hereditary hemorrhagic telangiectasia. Here we find that the extracellular and cytoplasmic domains of the auxiliary TGF-β receptor endoglin interact with ALK-1 (a type I TGF-β receptor). In addition, endoglin potentiates TGF-β/ALK1 signaling, with the extracellular domain of endoglin contributing to this functional cooperation between endoglin and ALK-1. By contrast, endoglin appears to interfere with TGF-β/ALK-5 signaling. These results suggest that the functional association of endoglin with ALK-1 is critical for the endothelial responses to TGF-β. © 2005 Wiley-Liss, Inc. ALK, activin receptor-like kinase; Dox, doxycycline; HA, hemagglutinin epitope; HHT, hereditary hemorrhagic telangiectasia; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; siRNA, small interference RNA; TGF-β, transforming growth factor-β; TβRI, TGF-β receptor type I; TβRII, TGF-β receptor type II. Transforming growth factor-β (TGF-β) receptors are essential for angiogenesis in development and vascular remodeling in the adult (Oshima et al., 1996; Bourdeau et al., 1999; Li et al., 1999; Arthur et al., 2000; Oh et al., 2000; Larsson et al., 2001; Srinivasan et al., 2003). The TGF-β superfamily signals through binding to a heteromeric complex containing two different transmembrane serine/threonine kinases known as type I (TβRI) and type II (TβRII) signaling receptors (Miyazono et al., 2000; Shi and Massague, 2003). Upon ligand binding to the TβRI/TβRII complex, activated TβRI propagates intracellular signal to the nucleus by phosphorylating members of the Smad family of proteins (Mehra and Wrana, 2002; Derynck and Zhang, 2003). In endothelial cells, the existence of two different types of TβRI, named ALK-1 and ALK-5, has been proposed to alternatively regulate the switch between proliferation and quiescence, respectively (Goumans et al., 2002, 2003a), and to explain the pro- or anti-angiogenic effects of TGF-β (Pepper, 1997; Goumans et al., 2003b). ALK-1 is specifically expressed in endothelial cells where the TGF-β receptor complex also contains an auxiliary co-receptor named endoglin (Gougos and Letarte, 1990; Duff et al., 2003). Endoglin binds different members of the TGF-β superfamily in the presence of the signaling receptors types I and II (Cheifetz et al., 1992; Yamashita et al., 1994; Letamendia et al., 1998; Barbara et al., 1999), and modulates TGF-β1-dependent cellular responses (Lastres et al., 1996; Letamendia et al., 1998; Li et al., 2000; Guerrero-Esteo et al., 2002). Ectopic expression of endoglin counteracts the TGF-β-induced growth inhibition in monocytes (Lastres et al., 1996), myoblasts (Letamendia et al., 1998), and endothelial cells (Li et al., 2000), in agreement with the high endoglin levels found in proliferating versus resting endothelial cells (Miller et al., 1999; Duff et al., 2003). Interestingly, adenoviral expression of a constitutively active form of ALK-1 leads to increased endothelial cell proliferation (Goumans et al., 2002). Both endoglin and ALK-1 are expressed (Gougos and Letarte, 1990; Duff et al., 2003, Seki et al., 2003) and can be co-immunoprecipitated (Abdalla et al., 2000) on endothelial cells, and play important roles in cardiovascular development and vascular remodeling. Genetic inactivation of endoglin or ALK-1 in the mouse shows that embryos homozygous for these genes die at 10–10.5 days postcoitum due to vascular and cardiac anomalies (Bourdeau et al., 1999; Li et al., 1999; Arthur et al., 2000; Oh et al., 2000). Furthermore, genes encoding endoglin and ALK-1 are targets for the autosomal dominant disorder known as hereditary hemorrhagic telangiectasia (McAllister et al., 1994; Johnson et al., 1996). In spite of the above data suggesting the physical interaction and functional collaboration between endoglin and ALK-1 within the endothelial TGF-β receptor pathway, the molecular basis for this association is not understood. In this study, we have analyzed the interaction between endoglin and ALK-1, as well as the role of endoglin in the ALK-1 mediated TGF-β signaling, This work was supported by Fondo de Investigacion Sanitaria grant PI020200, Ministerio de Educacion y Ciencia grant SAF2004-01390 (to C.B.), National Center for Research Resources Grant P20 15555 from the National Institutes of Health (to C.P.H.V.), and Ministerio de Educacion Cultura y Deporte postdoctoral fellowship to J.F.S.

12 páginas, 6 figuras, 1 tabla -- PAGS nros. 561-572, Endoglin (CD105) is a homodimeric membrane glycoprotein, which acts as a TGF-β coreceptor in the vasculature and plays an important role in cardiovascular development and vascular remodelling. To isolate putative genes regulated by endoglin expression, a PCR-based RNA fingerprinting technique was carried out. Myoblasts stably transfected with endoglin showed a decrease in the expression of lumican both at the RNA and protein levels. Lumican is a proteoglycan of the extracellular matrix, belonging to the SLRP (Small Leucine-Rich Repeat Proteoglycans) family. Lumican down-regulation by endoglin appeared to be controlled, at least in part, at the transcriptional level, as indicated by RT-PCR, and transient transfection experiments using a lumican promoter reporter based vector. This inverse correlation between endoglin and lumican expression was substantiated by immunohistochemical staining of vessels from human tissues. Thus, cells belonging to the high endothelia, such as tonsil, express a large amount of endoglin, and the lumican content of their matrix is considerably reduced. Conversely, in resting endothelia, such as that of large vessels, the expression of endoglin is reduced whereas the amount of lumican is greatly increased. The inverse regulation in the expression of endoglin and lumican was also evident after TGF-β treatments since endoglin was up-regulated, whereas lumican was down-regulated by this cytokine. This report describes for the first time a relationship between endoglin and lumican expression, This work was supported by grants from Ministerio de Ciencia y Tecnologı́a (SAF2000-0132), Fondo de Investigacion Sanitaria (PI020200), and Comunidad Autónoma de Madrid (CAM) to C.B. FS-R is recipient of a postdoctoral fellowship from CAM

The vascular endothelium participates in angiogenesis, inflammation and the immune response, which are modulated by vasoactive cytokines such as tumour necrosis factor-alpha (TNF alpha) and transforming growth factor-beta 1 (TGF beta 1). CD105 is a component of the TGF beta receptor complex and is abundantly expressed in activated/injured endothelium where it is implicated in multiple cellular processes. Up-regulation of CD105 in synovial cells of rheumatoid arthritis and psoriatic lesions implies a possible role in the pathogenesis of such inflammatory disorders. The pro-inflammatory cytokine, TNF alpha, and anti-inflammatory cytokine, TGF beta 1, regulate multiple cellular processes such as proliferation, differentiation and apoptosis. Our hypothesis is that CD105 gene expression in endothelial cells is regulated by the multifunctional cytokines TNF alpha and TGF beta 1. By using human dermal microvascular endothelial cells the present study has shown that long-term treatment with TNF alpha (0.1-5 ng/ml) elicited a concentration- and time-dependent significant suppression (over 50% reduction) in CD105 protein levels. The observations that no significant alterations in the CD105 mRNA levels or in the CD105 promoter activity were found and that the potent inhibitor of NF kappa B, PDTC, did not affect the TNF alpha action suggest that CD105 down-regulation by TNF alpha is not at the transcriptional level. In contrast to TNF alpha, TGF beta 1 significantly elevated CD105 protein and mRNA expression (approximately 2-fold increase) through activation of its promoter activity. From these data we conclude that TNF alpha and TGF beta 1 exert opposing effects on CD105 expression in human vascular endothelial cells and that CD105 is enmeshed in the network of signal pathways modulating multiple cellular functions.

Endoglin is an endothelial membrane glycoprotein involved in cardiovascular morphogenesis and vascular remodeling. It associates with transforming growth factor-beta (TGF-beta) signaling receptors to bind TGF-beta family members, forming a functional receptor complex. Arterial injury leads to up-regulation of endoglin, but the underlying regulatory events are unknown. The transcription factor KLF6, an immediate-early response gene induced in endothelial cells during vascular injury, transactivates TGF-beta, TGF-beta signaling receptors, and TGF-beta-stimulated genes. KLF6 and, subsequently, endoglin were colocalized to vascular endothelium (ie, expressed in the same cell type) following carotid balloon injury in rats. After endothelial denudation, KLF6 was induced and translocated to the nucleus; this was followed 6 hours later by increased endoglin expression. Transient overexpression of KLF6, but not Egr-1, stimulated endogenous endoglin mRNA and transactivated the endoglin promoter. This transactivation was dependent on a GC-rich tract required for basal activity of the endoglin promoter driven by the related GC box binding protein, Sp1. In cells lacking Sp1 and KLF6, transfected KLF6 and Sp1 cooperatively transactivated the endoglin promoter and those of collagen alpha1(I), urokinase-type plasminogen activator, TGF-beta1, and TGF-beta receptor type 1. Direct physical interaction between Sp1 and KLF6 was documented by coimmunoprecipitation, pull-down experiments, and the GAL4 one-hybrid system, mapping the KLF6 interaction to the C-terminal domain of Sp1. These data provide evidence that injury-induced KLF6 and preexisting Sp1 may cooperate in regulating the expression of endoglin and related members of the TGF-beta signaling complex in vascular repair.

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 pages, 6 figures., Endoglin is a transmembrane glycoprotein 633 residues in length expressed at the surface of endothelial cells as a disulphide-linked homodimer; the specific cysteine residues involved in endoglin dimerization are unknown. Mutations in the coding region of the endoglin gene are responsible for hereditary haemorrhagic telangiectasia type 1 (HHT1), a dominantly inherited vascular disorder. Many of these mutations, if translated, would lead to truncated forms of the protein. It is therefore of interest to assess the protein expression of different truncated forms of endoglin. Infections in vitro or in vivo with recombinant vaccinia virus, as well as transient transfections with expression vectors, were used to express normal and truncated forms of endoglin. Truncated mutants could be classified into three different groups: (1) those that did not produce stable transcripts; (2) those that produced stable transcripts but did not secrete the protein; and (3) those that secreted a soluble dimeric protein. This is the first time that a recombinant truncated form of endoglin has been found to be expressed in a soluble form. Because a chimaeric construct encoding the N-terminal sequence of platelet/endothelial cell adhesion molecule (PECAM-1) antigen fused to residues Ile281–Ala658 of endoglin also yielded a dimeric surface protein, these results suggest that cysteine residues contained within the fragment Cys330–Cys412 are involved in disulphide bond formation. Infection with vaccinia recombinants encoding an HHT1 mutation did not affect the expression of the normal endoglin, and did not reveal an association of the recombinant soluble form with the transmembrane endoglin, supporting a haploinsufficiency model for HHT1., This work has been supported by grants from Comisión Interministerial de Ciencia y Tecnología (CICYT-SAF97-0034 and CICYT-BIO94-0118), Comunidad Autónoma de Madrid (CAM) and Biomed Program of the European Community (BMH4-T95-0995).

Human immunoglobulins treated at 55 degrees C in vitro are able to interact with maleylated bovine serum albumin (mBSA), but not with unmodified BSA. Gel filtration experiments demonstrated that the mBSA binding is associated with a high molecular weight complex of aggregated IgG. This aggregated IgG with binding capacity for mBSA could also be generated in vitro by treatment of human IgG at 37 degrees C or 40 degrees C and by incubation with human neutrophils. Furthermore, IgG aggregates with binding activity for mBSA could be detected in untreated synovial fluids from rheumatoid arthritis patients, indicating that these complexes occur in vivo. The phenomenon of binding to aggregated IgG was extended to other modified proteins such as maleylated human serum albumin (mHSA), acetyl low density lipoprotein (Ac-LDL) and BSA reacted with oxidized linolenic acid. Soluble forms of these modified proteins were able to compete for the interaction between aggregated IgG and surface-bound mBSA. We also found that aggregated IgG enhanced the Ac-LDL-dependent foam cell formation. These findings suggest a role for aggregated IgG in the metabolism of oxidized proteins.

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.).

25 p.-10 fig. Rossi, Elisa et al., This study has been supported by grants from Ministerio de Economia y Competitividad of Spain (SAF2010-19222 and SAF2013-43421-R to CB and SAF2010-15881 and SAF2013-45784-R to JML-N), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to CB), Red de Investigacion Cooperativa en Enfermedades Renales (REDINREN to JML-N), Fundación Renal Íñigo Alvarez de Toledo (to JML-N and CB), Consejo Superior den Investigaciones Cientificas (PA1003298 to ER) and The Conny-Maeva Charitable Foundation (to DMS and ER). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.

4 pages, 2 figures., Endoglin is an homodimeric membrane antigen with capacity to bind transforming growth factor-beta (TGF-beta). Phosphorylation of human endoglin was demonstrated in endothelial cells as well as in mouse fibroblast transfectants expressing two isoforms, L-endoglin or S-endoglin, with distinct cytoplasmic domains. The extent of L-endoglin phosphorylation was found to be 8-fold higher than that of S-endoglin, and phosphopeptide analyses revealed at least three different phosphorylation sites for L-endoglin, whereas S-endoglin produces only one phosphopeptide. The immunoprecipitated L-endoglin was found to be phosphorylated mainly on serine, and, to a minor extent, on threonine, residues. Treatment of the cells with TGF-beta 1 or the protein kinase C inhibitor H-7 resulted in a reduction of the levels of endoglin phosphorylation., This work was supported by grants from CICYT (SAL91-0507 to C. B.), CAM (151/92 to C. B. and C263/91 A to J. M.-P.) and DGICYT (PB92-0116 to J.M.-P) of Spain.

The presence of antibodies in rheumatoid arthritis (RA) patients to antigens on the synoviocyte surface has recently been reported (Scand. J. Immunol. 27, 295, 1988). Here we have further characterized these antigens and found that they are exogenous proteins acquired from the bovine serum used in the culture medium. By immunoprecipitation and ELISA studies, we have identified bovine albumin and transferrin as the antigens recognized by the RA antibodies. These specificities were found not only in the sera but also in the synovial fluid from RA patients. A comparative study with a large panel of RA sera did not show a correlation in the antibody specificities for bovine albumin, bovine transferrin, or the 65-kDa heat shock protein from Mycobacterium bovis. Similar experiments using rabbit and monkey sera as well as human synovial fluid and serum as a source of antigen did not reveal any reactivity with a highly positive RA serum. By sequence alignment, a high degree of homology between residues 142-156 from bovine albumin and residues 65-78 from human pro-collagen alpha 1 (I) was found. The capacity of the synoviocytes to bind exogenous antigens and the presence of antibodies to bovine proteins, normally present in the diet, suggest a role for these type A synoviocytes as well as a possible involvement of food antigens in the pathogenesis of RA.

22 páginas, 9 figuras, 2 tablas.-- et al., [Background]: Activin receptor-like kinase 1 (ALK1) is a Transforming Growth Factor-β (TGF-β) receptor type I, mainly expressed in endothelial cells that plays a pivotal role in vascular remodelling and angiogenesis. Mutations in the ALK1 gene (ACVRL1) give rise to Hereditary Haemorrhagic Telangiectasia, a dominant autosomal vascular dysplasia caused by a haploinsufficiency mechanism. In spite of its patho-physiological relevance, little is known about the transcriptional regulation of ACVRL1. Here, we have studied the different origins of ACVRL1 transcription, we have analyzed in silico its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of ACVRL1. [Results]: We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of ACVRL1 transcripts, finding two new transcriptional origins, upstream of the one previously described, that give rise to a new exon undiscovered to date. The 5'-proximal promoter region of ACVRL1 (-1,035/+210) was analyzed in silico, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, ACVRL1 promoter reporters did not present any significant transcriptional activity, whereas increasing concentrations of Sp1 triggered a dose-dependent stimulation of its transcription. Moreover, silencing Sp1 in HEK293T cells resulted in a marked decrease of ACVRL1 transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of ACVRL1 in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in ACVRL1 transcription, whereas in vitro hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of ACVRL1. [Conclusions]: Our results describe two new transcriptional start sites in ACVRL1 gene, and indicate that Sp1 is a key regulator of ACVRL1 transcription, providing new insights into the molecular mechanisms that contribute to the expression of ACVRL1 gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of ACVRL1 gene transcriptional activity., This work was supported by grants from Ministerio de Ciencia e Innovación of Spain (SAF2007-61827 to CB; SAF08-01218 to LMB, and predoctoral fellowship BES-2005-7974 to EMG-M), National Institutes of Health & National Center for Research Resources (P20-RR-15555 and HL083151 to CPV; DK56621 and DK37340 to SLF) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER).We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).