Papel de las isomorfas de endoglina en el linaje mieloide

175 p.-40 fig.-4 tab.-1 tab suppl.Endoglin (also known as CD105) is a type I homodimeric transmembrane glycoprotein that can act as an auxiliary receptor for members of the TGF-β superfamily (Cheifetz et al., 1992; Bellon et al., 1993). Structurally, endoglin belongs to the zona pellucida (ZP) family of proteins that share a ZP domain of ∼260 amino acid residues at their yuxtamembrane extracellular region (Llorca et al., 2007). Endoglin also contains at its N-terminal extracellular region an Orphan domain (OD), comprising ∼325 amino acids, whose sequence shows no homology to any other protein, but it was recently identified as the minimal active endoglin domain needed for partner recognition (Llorca et al., 2007; Castonguay et al., 2011; Alt et al., 2012). Endoglin expression and function has been widely described in the context of endothelial cells and vascular physiology (Bernabeu et al., 2007; Lopez-Novoa and Bernabeu, 2010), playing a key role in many pathological processes, including Hereditary Hemorrhagic Telangiectasia (HHT), cancer angiogenesis, preeclampsia or hypertension (Bernabeu et al., 2009; Shovlin, 2010; Kapur et al., 2012; Rana et al., 2012; Valbuena-Diez et al., 2012). In addition, increased levels of membrane and soluble forms of endoglin have been linked to inflammatory processes such as wound healing, atherosclerosis, psoriasis or rheumatoid arthritis (Rulo et al., 1995; Conley et al., 2000; Torsney et al., 2002; Lopez-Novoa and Bernabeu, 2010). There are two different isoforms of endoglin that share an identical extracellular domain, but differ from each other in the length and composition of their cytoplasmic tail (Cheifetz et al., 1992; Bellon et al., 1993). The endoglin gene (ENG) is predominantly expressed as a long isoform (L-endoglin), but its pre-mRNA can be alternatively spliced by a mechanism of intron retention, yielding a less abundant form, known as short endoglin (S-endoglin). This intron retention is driven by the SRSF1 splicing factor, that physically competes with the minor spliceosome for the elimination of the last intron between exons #13 and #14 (Blanco and Bernabeu, 2012). When transcribed, the last intron of ENG bears an early stop codon that affects the open reading frame and truncates the mature protein at the cytoplasmic region (Blanco et al., 2008). So far, most studies published about endoglin have focused on L-endoglin. The expression of the short variant (S-endoglin) was first described in humans (Bellon et al., 1993) and later in mice (Perez-Gomez et al., 2005). The cytoplasmic region of human L-endoglin is composed by 47 amino acids with a high content of serine and threonine residues susceptible to be phosphorylated (Lastres et al., 1994). Also, the sequence Ser-Met-Ala (SMA) in the C-terminal end of L-endoglin is a docking site for proteins with a PDZ domain and is involved in cytoskeleton organization (Koleva et al., 2006). By contrast, the sequence of the S-endoglin cytoplasmic tail is only 14 amino acids long, the last 7 residues being specific for this isoform. These structural differences may account for the distinct functional effects of L-endoglin and S-endoglin (Blanco et al., 2008; Velasco et al., 2008). Up to date the tridimensional structure of the cytoplasmic domain of both long and short isoforms of endoglin has not been solved.. Interestingly, a role for S-endoglin during endothelial senescence has been described (Blanco et al., 2008). Thus, the S-endoglin/L-endoglin ratio is increased during senescence of human endothelial cells in vitro, as well as during aging of mice in vascularized tissues. The switch between L-endoglin and S-endoglin affects the TGF-β- mediated cell signaling promoting the ALK5/Smad3 pathway instead of the ALK1/Smad1 route. This is due to the preferential affinity of the short cytoplasmic domain for ALK5 that promotes the expression of target genes such as SERPINE-1 (PAI-1) and PTGS2 (COX-2), and inhibits NOS3 (eNOS). Furthermore, transgenic mice overexpressing S-endoglin showed hypertension, decreased hypertensive response to NO inhibition, decreased vasodilatory response to TGF-β1 administration, and decreased endothelial nitric oxide synthase expression in lungs and kidneys, supporting the involvement of S-endoglin in the NO-dependent vascular homeostasis (Blanco et al., 2008). Taken together, these results suggest that S-endoglin is induced during endothelial senescence and may contribute to age-dependent vascular pathology. During aging, immune and inflammatory responses are impaired in a general immunosenescence process where macrophage functions are compromised (Cassado Ados et al., 2011; Mahbub et al., 2012). Aged macrophages show reduced functional activity, leading to accumulation of unphagocytosed cellular debris, chronic inflammation and exacerbation of tissue damage and aging (Shaw et al., 2010; Li, 2013). Macrophages participate in the immune response according to a preferential M1 or M2 polarization, which is involved not only in the response to pathogens, tumor progression or angiogenesis, but also in tissue homeostasis, autoimmunity or fibrosis (Sunderkotter et al., 1994; Werner and Alzheimer, 2006; Benoit et al., 2008). Although many studies on endoglin have focused on endothelial cells, endoglin expression has been also detected in some myeloid precursors and macrophages. Thus, endoglin acts as a critical regulator of the hematopoietic development, in part by modulating the signaling involving members of the TGF-β superfamily (Borges et al., 2012). Later in development, endoglin is present at low levels on monocytes, but it is markedly upregulated during the monocyte-macrophage transition (Lastres et al., 1992; O'Connell et al., 1992). Moreover, in the human monocytic line U937, ectopic expression of endoglin counteracts TGF-β1-dependent cellular responses (Lastres et al., 1996). Overall, the role of endoglin in the myeloid lineage is poorly understood, especially regarding the individual function of each isoform. Matrix metalloproteinases are a family of metalloproteases that contain a zinc atom at their active site and are able to degrade matrix macromolecules including collagen, laminin, and elastin (Shapiro, et al. 1992; Shapiro, et al. 1993). In addition to their ability to degrade extracellular matrix components, some MMPs also cleave cytokines and antiproteolytic molecules. Up to date the only described protease able to cut membrane endoglin and release the soluble form of the protein is the membrane MMP14 (Hawinkels, et al. 2010; Valbuena-Diez, et al. 2012). Human macrophage elastase (MMP12) was first identified as an elastolytic metalloproteinase secreted by inflammatory macrophages (Banda and Werb 1981). Like other MMPs, MMP12 is a zinc-dependent neutral endopeptidase; it is secreted as a 54-kDa proform protein that undergoes self-activation through autolytic processing to produce several active forms of the enzyme (Shapiro, et al. 1993). In addition to elastolytic activity, MMP12 has been shown to be capable of degrading a broad spectrum of extracellular matrix components, including type IV collagen, fibronectin, laminin, vitronectin, proteoglycans, chondroitin sulfate, and myelin basic protein (Gronski, et al. 1997) .One apparently important function of catalytic MMP12 in vivo is its ability to activate other MMPs such as MMP2 and MMP3, by which MMP12 exacerbates the cascade of proteolytic processes (Matsumoto, et al. 1998). Moreover, the role of MMP12 as an anti-angiogenic factor has been described (Houghton, et al. 2006; Nocito, et al. 2008). Interestingly, MMP12 is considered as an M1 phenotype pro-inflammatory macrophage marker and its expression is induced by the cytokine GM-CSF (de las Casas-Engel, et al. 2013). Additionally, MMP12 is reported as a pro-inflammatory protease and in extension is a marker of M1 phenotype effectors (de las Casas-Engel, et al. 2013; Nenan, et al. 2005). The presence of this protease has been already reported in inflammation-associated pathologies like atherosclerosis, rheumatoid arthritis, systemic sclerosis and others like psoriasis or chronic obstructive pulmonary disease (COPD) (Chaudhuri, et al. 2012; Johnson, et al. 2011; Liu, et al. 2004; Suomela, et al. 2001). Because the exacerbated basal inflammatory response is associated with increased plasma levels of both soluble endoglin and MMP12 in those pathologies, it is likely that the macrophage metalloelastase (MMP12) is involved in the shedding of membrane endoglin in inflammatory associated processes. 2. AIMS • Study the tridimensional structure at high resolution of the cytoplasmic region of the two existing endoglin isoforms (Long endoglin and Short endoglin). • Analyze the role of both endoglin isoforms in the context of the myeloid lineage and their possible implication in macrophage senescence. • Examine the relationship between the soluble endoglin release and the macrophage polarization into M1 or M2 phenotypes. 3. RESULTS The results obtained by RMN analysis of the peptide corresponding to the cytoplasmic tail of the short isoform of endoglin showed that, due to its small size, the peptide does not have a defined secondary structure. The experimental approaches to solve the structure of the cytoplasmic domain of the long isoform by X-Ray crystallography have not been successful. The crystallization assays performed with the synthesized recombinant proteins (cytoplasmic domain of EngL and LIM domain) did not yield crystals for the subsequent structural analysis. Because S-endoglin is a marker of endothelial senescence, we first assessed the expression of both endoglin isoforms in a senescence-like stage of monocytes/macrophages subjected to oxidative stress. First, macrophages were obtained from the peritoneal cavity of 2 months old mice. Most of these cells correspond to the myeloid lineage, mainly to tissue resident large peritoneal macrophages (LPM) (Cassado Ados et al., 2011). A large proportion of these LPMs were positive for the senescence-associated β-Galactosidase staining and there was a marked increase in the percentage of senescent macrophages after hydrogen peroxide (H2O2) treatment. In addition, increased levels of S-endoglin (a marker of cellular senescence), were observed upon oxidative stress, as evidenced by semiquantitative RT-PCR analysis of endoglin transcripts. Besides, oxidative stress-induced senescence in human activated monocytes from four different donors demonstrated a clear increase in the transcript levels of the senescence markers S-endoglin and PAI-1, whereas L-endoglin levels underwent a much lower increase. To assess the effect of in vivo aging, LPMs and recruited small peritoneal macrophages (SPM) were analyzed upon zymosan treatment. S-endoglin levels were clearly increased in both LPM and SPM at 6 and 12 months of age mice (up to 16-fold), relative to 2 months old animals.Mo Moreover, the senescent marker Pai-1 showed a statistically significant increase in LPM (12 m), whereas L-endoglin transcript levels were augmented in LPM (6 m and 12 m) and SPM (12 m), although at lower levels than S-endoglin. Of note, the upregulation of S-endoglin preceded that of Pai-1 in LPM and no significant changes of Pai-1 levels in infiltrating SPM during aging were observed. Taken together, these results suggest that S-endoglin is a marker of macrophage senescence that is upregulated during in vitro oxidative stress-induced senescence and in vivo aging. To investigate the effect of both endoglin isoforms on protein expression, a proteomic analysis using SILAC technology was applied to quantify differences in protein expression level among U937 endoglin transfected cells. In this manner, we were able to identify and quantify a set of 890 differentially expressed proteins in the three subcellular fractions from endoglin transfectants. The Venn diagram representation showed a common effect of both endoglin isoforms in some of the regulated proteins, suggesting that this effect was mediated by their shared extracellular domain. In fact, the same regulatory effect was observed for 166 proteins (26 upregulated and 140 downregulated). By contrast, six proteins showed an opposite regulation between Lendoglin and S-endoglin transfectants (upregulated in L-endoglin and downregulated in S-endoglin cells). Of note, a large number of proteins (546) with an independent behaviour in either L-endoglin (106 upregulated and 143 downregulated) or S-endoglin (36 upregulated and 261 downregulated) transfectants were identified, suggesting that this effect depends on the endoglin cytoplasmic region. To confirm the results from the SILAC analysis, we decided to validate some of the differentially expressed proteins. For this purpose, proteins like Smad3, Calmodulin or CD13 were analysed by western blot or flow cytometry techniques. Next, we investigated the possible biological functions affected by endoglin overexpression. The SILAC protein list was subjected to the Bio-informatic Ingenuity Pathways Analysis (IPA) using the Ingenuity Knowledge Base (Genes Only) as a reference set. To assess whether these cellular functions were indeed affected in endoglin transfectants, specific functional assays were carried out. Both endoglin transfectants showed an altered “cell trafficking” profile in the bioinformatic analysis, most likely due to the marked reduction of integrin levels, including those of β1, α5 and β2 subunits. Accordingly, cell adhesion assays clearly demonstrated an impaired binding of both endoglin transfectants to fibronectin compared to mock controls. The IPA results also yielded that "cell cycle" was one of the compromised biological functions, although the statistical significance was much higher in S-endoglin (lowest p value, 4.76x10-6) than L-endoglin (lowest p-value, 3.99x10-4) transfectants. Indeed, the growth rate is significantly slower in S-endoglin transfectants compared to mock controls or Lendoglin transfectants. According to the IPA analysis, cell death and survival are affected in both endoglin transfectants, although in opposite ways regarding apoptosis. To test this hypothesis, the GM-CSF-induced pro-apoptotic effect in U937 cells (Okuma et al., 2000) was assessed. S-endoglin transfectants showed a decreased survival rate in response to three different doses of the pro-apoptotic cytokine, whereas L-endoglin transfectants showed a slightly higher, but not significant, survival rate than control cells.The SILAC analysis also suggested that the metabolism of reactive oxygen species (ROS) was affected in both endoglin transfectants. Thus, redox homeostasis maintaining protein AIFM1 and antioxidant protein PRDX6 are down-expressed, while the NADPH oxidase CYBB is upregulated in endoglin transfectants. Based on these data, it can be postulated that L-endoglin and S-endoglin transfectants might display an enhanced production of ROS levels. However, this production might be compensated in L-endoglin, but not in S-endoglin transfectants due to an increased protein levels of superoxide dismutase 2 (SOD2), which may contribute to free radical scavenging. This interpretation is compatible with the actual measurement of ROS production in U937 cells, showing that S-endoglin expressing cells display levels of ROS higher than those of L-endoglin or mock transfectants. It is well known that aging impairs macrophage polarization into M1 and M2 subtypes (Mahbub et al., 2012). Because S-endoglin is upregulated upon macrophage senescence in vitro and during in vivo aging, we assessed the role of both endoglin isoforms in the phorbol myristate acetate (PMA)-induced macrophage differentiation of U937 cells (Cabanas et al., 1990). Thus, PMA-treated L-endoglin, S-endoglin and mock U937 transfectants were analyzed by quantitative PCR to assess the transcript levels of different M1 and M2 marker genes. The expression of classical M1 genes like IL-6, IL23A or IL12A was higher in mock and L-endoglin transfectants than in S-endoglintransfected cells. By contrast, typical M2 genes like MAFB, STAB1 or SERPINB2 were more highly expressed in S-endoglin transfectants than in mock transfected or Lendoglin transfectants. Thus, at the transcriptomic level, the presence of S-endoglin skews myeloid cell polarization towards M2, whereas the expression of L-endoglin does not have an overt influence of the expression of polarization markers, further emphasizing the different signalling capabilities of both endoglin isoforms. The PMA-dependent differentiation of U937 cells constitutes a well-established model for macrophage differentiation (Cabanas et al., 1990). Therefore, we decided to assess the potential influence of both endoglin isoforms on the functional polarization of myeloid cells using this experimental system. To that end, the responsiveness of endoglin transfectants to LPS, that favours the acquisition of an M1 phenotype, was evaluated. Analysis of the secreted cytokine profile showed the release of proinflammatory cytokines to the supernatant in mock, L-endoglin and S-endoglin populations. Overall, endoglin transfectants displayed lower levels of LPS-induced pro inflammatory cytokines than mock controls. However, S-endoglin transfectants showed the lowest inflammatory profile among the three cell types, in agreement with their M2-like phenotype. Next, we investigated the underlying molecular basis for the endoglin role in macrophage polarization. It is noteworthy that activin-A is a key regulator of macrophage polarization that promotes a pro-inflammatory phenotype and inhibits the acquisition of an anti-inflammatory status (Sierra-Filardi et al., 2011). Because gene expression experiments showed an altered regulation of activin-A in endoglin transfectants, we assessed the possible deregulated protein expression of this cytokine in U937 transfectants. The secretion of activin-A was markedly increased (more than two-fold) in L-endoglin transfectants compared to mock controls. By contrast, activin-A levels in culture supernatants of S-endoglin transfectants were clearly reduced relative to controls. Taken together, the polarization studies suggest that in monocytic cells L-endoglin promotes an M1-like phenotype, whereas Sendoglin favours the expression of M2 markers and functions.Those data suggest that the cutting points for MMP12 could be the same as the ones described for MMP14. In addition, the MMP408 compound seems to be a good specific inhibitor for MMP12. A significant increase in the level of soluble endoglin was observed when COS7 cells were transfected with endoglin and MMP12 expression vectors. The background of untreated COS7 cells does not include membrane endoglin, nor MMP12 or MMP14. Therefore, we confirmed the ability of MMP12 as an endoglin cutting protease, making stronger the relationship of both proteins as M1 phenotype markers. Supporting this hypothesis we were able to counteract the increase of soluble endoglin in M1-GM-CSF polarization just by adding the MMP408 inhibitor during GM-CSF differentiation. The incubation of primary cultures from human endothelial cells (HUVECS) with the conditioned media of M1-M2 macrophages showed an increase of soluble endoglin levels in the resultant supernatant. Parallel experiments showed that the inhibitor MMP408 decreased the release of soluble endoglin in HUVECS cultured with the M1 conditioned media, similarly as we observed in macrophage cultures. The MMP12 activity in endothelial cells is an important issue due to the exponential increase of the membrane endoglin susceptible to be released in an inflammatory context. Finally, using an in vivo model, the injection of LPS in mice resulted in a significant increase of soluble endoglin levels in plasma after 48 hours of incubation. As controls, the gavage of the MMP408 or vehicle and the injection of PBS instead of LPS had no effect on the basal or induced levels of soluble endoglin. Moreover, the LPS-induced soluble endoglin levels were markedly decreased upon treatment with the MMP12 inhibitor (MMP408). Thus, the effect of the inhibitor in both macrophage and endothelial in vitro models can be reproduced in an in vivo inflammatory scenario.4. CONCLUSIONS • The cytoplasmic domain of the short isoform of endoglin does not have a defined secondary structure, as shown by the RMN analysis. Due to the methodological difficulties in the resolution of the cytoplasmic domain of the long isoform by X-Ray Crystallography, further studies are necessary using RMN with a previous isotopic labeling to increase the sensitivity and complete the comparative structural study of both endoglin isoforms. • We have established the short isoform of endoglin (EngS) as a potential marker of senescence in the myeloid lineage. The disequilibrium between the EngL/EngS ratio, which is closely related to aging, can potentiate the inefficient innate immune response in the elderly. • The proteomic analysis using SILAC has demonstrated that both endoglin isoforms modulate key biological processes in myeloid cells, such as the adhesion, proliferation, survival or ROS accumulation. In addition, EngS seems to promote an elderly-related phenotype in the previously mentioned processes. • The over-expression of the short isoform of endoglin, favors the polarization of the macrophages into an anti-inflammatory phenotype (M2). The observed switch of expression from EngL to EngS can be included in the immunosenescence process. • Membrane endoglin is a substrate for the macrophage elastase MMP12, a protein associated to a pro-inflammatory M1 respons

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

Upon inflammation, monocyte-derived macrophages (MF) infiltrate blood vessels to regulate several processes involved in vascular pathophysiology. However, little is known about the mediators involved. Macrophage polarization is crucial for a fast and e cient initial response (GM-MF) and a good resolution (M-MF) of the inflammatory process. The functional activity of polarized MF is exerted mainly through their secretome, which can target other cell types, including endothelial cells. Endoglin (CD105) is a cell surface receptor expressed by endothelial cells and MF that is markedly upregulated in inflammation and critically involved in angiogenesis. In addition, a soluble form of endoglin with anti-angiogenic activity has been described in inflammation-associated pathologies. The aim of this work was to identify components of the MF secretome involved in the shedding of soluble endoglin. We find that the GM-MF secretome contains metalloprotease 12 (MMP-12), a GM-MF specific marker that may account for the anti-angiogenic activity of the GM-MF secretome. Cell surface endoglin is present in both GM-MF and M-MF, but soluble endoglin is only detected in GM-MF culture supernatants. Moreover, MMP-12 is responsible for the shedding of soluble endoglin in vitro and in vivo by targeting membrane-bound endoglin in both MF and endothelial cells. These data demonstrate a direct correlation between GM-MF polarization, MMP-12, and soluble endoglin expression and function. By targeting endothelial cells, MMP-12 may represent a novel mediator involved in vascular homeostasis.Ministerio de Ciencia, Innovación y Universidades of Spain (SAF2013-43421-R to C.B.; SAF2017-83785-R and SAF2014-23801 to A.L.C.)Consejo Superior de Investigaciones Cientificas (201920E022 to C.B.)Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIII-CB06/07/0038 to C.B.)Czech Republic Specific University Research (SVV-260414 to P.N.)CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER fundsM.A. was funded with a fellowship from Ministerio de Ciencia e Innovación (BES-2008-003888)M.V. was supported by a short-term mobility fellowship from the European Erasmus Programm

PI3K-regulated Glycine N-methyltransferase is required for the development of prostate cancer

[EN] Glycine N-Methyltransferase (GNMT) is a metabolic enzyme that integrates metabolism and epigenetic regulation. The product of GNMT, sarcosine, has been proposed as a prostate cancer biomarker. This enzyme is predominantly expressed in the liver, brain, pancreas, and prostate tissue, where it exhibits distinct regulation. Whereas genetic alterations in GNMT have been associated to prostate cancer risk, its causal contribution to the development of this disease is limited to cell line-based studies and correlative human analyses. Here we integrate human studies, genetic mouse modeling, and cellular systems to characterize the regulation and function of GNMT in prostate cancer. We report that this enzyme is repressed upon activation of the oncogenic Phosphoinositide-3-kinase (PI3K) pathway, which adds complexity to its reported dependency on androgen signaling. Importantly, we demonstrate that expression of GNMT is required for the onset of invasive prostate cancer in a genetic mouse model. Altogether, our results provide further support of the heavy oncogenic signal-dependent regulation of GNMT in prostate cancer.We are grateful to the Carracedo lab for valuable input, to Drs. Ana M. Aransay, James D. Sutherland and F. Elortza for technical advice, and Drs. Michelle Clasquin, Katie Sellers and Katya Marjon at Agios Pharmaceuticals for performing, processing and analyzing the metabolomics experiments. We thank the Basque Biobank for Research (BIOEF) for the support with prostate specimen acquisition and management. A.A-A. was funded by the Basque Government (predoctoral fellowship). V.T. is funded by Fundación Vasca de Innovación e Investigación Sanitarias, BIOEF (BIO15/CA/052), the AECC J.P. Bizkaia, the Basque Department of Health (2016111109) and the MICINN RTI2018-097267-B-I00. I.M. is supported by Fundación Cris Contra el Cáncer (PR_TPD_2020-19). The work of A. Carracedo is supported by the Basque Department of Industry, Tourism and Trade (Elkartek), the department of education (IKERTALDE IT1106-16) and health (RIS3), the BBVA foundation, the MICINN (SAF2016-79381-R; PID2019-108787RB-I00 (FEDER/EU); Severo Ochoa Excellence Accreditation SEV-2016-0644; Excellence Networks RED2018-102769-T), the AECC (GCTRA18006CARR), Vencer el Cáncer Foundation, La Caixa Foundation (ID 100010434), under the agreement LCF/PR/HR17/ and the European Research Council (Starting Grant 336343, PoC 754627, Consolidator Grant 819242). CIBERONC was co-funded with FEDER funds and funded by ISCIII. We are grateful for the support of Mondravember and Movembergara. A.E. was supported by MCIN/AEI/10.13039/501100011033 and the EU programme NextGenerationEU/PRTR (IJC2020-043583-I). The work of JM Mato was supported by NIH grant R01CA172086 and SAF2017-88041-R. EB is funded by the MICINN (BFU2016-76872-R (FEDER/EU), PID2019-108112RB-I00, and Excellence Networks SAF2017-90794-REDT)

Mice lacking endoglin in macrophages show an impaired immune response

24 p.-9 fig.-1 tab. Ojeda Fernández, Luisa et al.Endoglin is an auxiliary receptor for members of the TGF-β superfamily and plays an important role in the homeostasis of the vessel wall. Mutations in endoglin gene (ENG) or in the closely related TGF-β receptor type I ACVRL1/ALK1 are responsible for a rare dominant vascular dysplasia, the Hereditary Hemorrhagic Telangiectasia (HHT), or Rendu-OslerWeber syndrome. Endoglin is also expressed in human macrophages, but its role in macrophage function remains unknown. In this work, we show that endoglin expression is triggered during the monocyte-macrophage differentiation process, both in vitro and during the in vivo differentiation of blood monocytes recruited to foci of inflammation in wild-type C57BL/6 mice. To analyze the role of endoglin in macrophages in vivo, an endoglin myeloid lineage specific knock-out mouse line (Engfl/flLysMCre) was generated. These mice show a predisposition to develop spontaneous infections by opportunistic bacteria. Engfl/flLysMCre mice also display increased survival following LPS-induced peritonitis, suggesting a delayed immune response. Phagocytic activity is impaired in peritoneal macrophages, altering one of the main functions of macrophages which contributes to the initiation of the immune response. We also observed altered expression of TGF-β1 target genes in endoglin deficient peritoneal macrophages. Overall, the altered immune activity of endoglin deficient macrophages could help to explain the higher rate of infectious diseases seen in HHT1 patients.This work was funded by: Ministerio de Economía y Competitividad of Spain (SAF2011-23475 to LMB; SAF2013-43421-R and SAF2010- 19222 to CB.Peer reviewe

Structural and Functional Insights into Endoglin Ligand Recognition and Binding

Endoglin, a type I membrane glycoprotein expressed as a disulfide-linked homodimer on human vascular endothelial cells, is a component of the transforming growth factor (TGF)-β receptor complex and is implicated in a dominant vascular dysplasia known as hereditary hemorrhagic telangiectasia as well as in preeclampsia. It interacts with the type I TGF-β signaling receptor activin receptor-like kinase (ALK)1 and modulates cellular responses to Bone Morphogenetic Protein (BMP)-9 and BMP-10. Structurally, besides carrying a zona pellucida (ZP) domain, endoglin contains at its N-terminal extracellular region a domain of unknown function and without homology to any other known protein, therefore called the orphan domain (OD). In this study, we have determined the recognition and binding ability of full length ALK1, endoglin and constructs encompassing the OD to BMP-9 using combined methods, consisting of surface plasmon resonance and cellular assays. ALK1 and endoglin ectodomains bind, independently of their glycosylation state and without cooperativity, to different sites of BMP-9. The OD comprising residues 22 to 337 was identified among the present constructs as the minimal active endoglin domain needed for partner recognition. These studies also pinpointed to Cys350 as being responsible for the dimerization of endoglin. In contrast to the complete endoglin ectodomain, the OD is a monomer and its small angle X-ray scattering characterization revealed a compact conformation in solution into which a de novo model was fitted

Papel de las isomorfas de endoglina en el linaje mieloide

Endoglina es una glicoproteína de membrana con un papel crucial en el remodelado vascular y la angiogénesis. Habiéndose descrito su función e implicaciones fisiopatológicas principalmente en el ámbito de la célula endotelial, su papel en el linaje mieloide, donde también se expresa, está todavía por elucidar. En la presente memoria se aborda el estudio de las dos isoformas de endoglina en la línea promonocítica U937, mediante análisis proteómico SILAC. Al analizar las proteínas alteradas debido a la acción de las isoformas de endoglina, se ha identificado a la isoforma corta de endoglina (EngS) como un marcador de senescencia en macrófagos, observándose alteraciones en adhesión, supervivencia, proliferación o acumulación de especies reactivas de oxígeno. Los análisis in vivo e in vitro realizados, confirman la validez del desequilibrio entre la expresión de la isoforma corta frente a la mayoritaria (isoforma larga, EngL) como un marcador del estado envejecido en estas células. Además, se ha observado una polarización preferencial de los transfectantes de endoglina corta hacia el fenotipo macrofágico anti-inflamatorio-M2 y una peor respuesta generalizada a estímulos pro-inflamatorios-M1. Este comportamiento se puede englobar dentro del deterioro en la respuesta inmune innata asociada a la inmunosenescencia. Por otra parte se ha analizado el perfil liberación de una tercera forma de endoglina, la endoglina soluble. Se ha identificado por primera vez esta forma soluble de endoglina como un marcador de polarización de macrófagos M1. Además se ha descrito un nuevo mecanismo proteolítico de liberación de endoglina de membrana por medio de la metaloproteasa de macrófagos MMP12. Se ha establecido por tanto una relación directa entre endoglina soluble y MMP12 como marcadores de respuesta inflamatoria en macrófagos, un hallazgo que está apoyado por el hecho de que ambos factores solubles están aumentados en enfermedades que tienen asociado un proceso inflamatorio

Expression of endoglin isoforms in the myeloid lineage and their role during aging and macrophage polarization

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].Peer reviewe

Experimental gene therapies for the NCLs

The neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease, are a group of rare monogenic neurodegenerative diseases predominantly affecting children. All NCLs are lethal and incurable and only one has an approved treatment available. To date, 13 NCL subtypes (CLN1-8, CLN10-14) have been identified, based on the particular disease-causing defective gene. The exact functions of NCL proteins and the pathological mechanisms underlying the diseases are still unclear. However, gene therapy has emerged as an attractive therapeutic strategy for this group of conditions. Here we provide a short review discussing updates on the current gene therapy studies for the NCLs

Gene Therapy Targeting the Inner Retina Rescues the Retinal Phenotype in a Mouse Model of CLN3 Batten Disease

The neuronal ceroid lipofuscinoses (NCLs), often referred to as Batten disease, are inherited lysosomal storage disorders that represent the most common neurodegeneration during childhood. Symptoms include seizures, vision loss, motor and cognitive decline, and premature death. The development of brain-directed treatments for NCLs has made noteworthy progress in recent years. Clinical trials are currently ongoing or planned for different forms of the disease. Despite these promising advances, it is unlikely that therapeutic interventions targeting the brain will prevent loss of vision in patients as retinal cells remain untreated and will continue to degenerate. Here, we demonstrate that Cln3(Δex7/8) mice, a mouse model of CLN3 Batten disease with juvenile onset, suffer from a decline in inner retinal function resulting from the death of rod bipolar cells, interneurons vital for signal transmission from photoreceptors to ganglion cells in the retina. We also show that this ocular phenotype can be treated by adeno-associated virus (AAV)-mediated expression of CLN3 in cells of the inner retina, leading to significant survival of bipolar cells and preserved retinal function. In contrast, the treatment of photoreceptors, which are lost in patients at late disease stages, was not therapeutic in Cln3(Δex7/8) mice, underlining the notion that CLN3 disease is primarily a disease of the inner retina with secondary changes in the outer retina. These data indicate that bipolar cells play a central role in this disease and identify this cell type as an important target for ocular AAV-based gene therapies for CLN3 disease

Experimental and modelling SAXS parameters.

a<p>Values for I(0) have been extrapolated by the Guinier approximation from the experimental scattering profiles.</p>b<p>Concentration of the protein used for the calculation of the estimated Mw.</p>c<p>Relative molecular mass estimated from I(0) and the concentration of the protein through BSA calibration.</p>d<p>The Porod volume was calculated using PRIMUS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029948#pone.0029948-Mertens1" target="_blank">[41]</a>.</p>e<p>Expected molecular mass predicted from the sequence and assuming full occupation of the glycosylation sites.</p>f<p>Rg (Guinier), Rg (GNOM), radius of gyration given by the Guinier approximation, and calculated by the program GNOM, respectively, given in nm.</p>g<p>Maximum dimension of the macromolecules. χ²^(over) Discrepancy between the SAXS profile and its fit by the overall shapes-models calculated by DAMMIF, and χ^{2 (sasref)} the average discrepancy of the best atomic models estimated with the program CRYSOL. ND, not determined.</p