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

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).Peer reviewe

A vision on air pollution in cities and the human effect

[EN] The human effect on air quality in cities and the evolution of air pollution is obvious, due to economic activity, vehicle traffic, etc. The situation created indirectly by COVID-19, has caused many countries to impose during certain periods restriction of movement and stoppage of economic activities, which has allowed us to observe the instant effect that occurs on the air quality in cities. This article discusses what the observed effect has been, focusing on the early moments of the pandemic (January 2020 to March 2020), with an analysis of the situation from its origin in China to its arrival in Europe and more specifically the situation created in Spain. After the analysis of the situation, it can be seen the large reduction of pollutants in the air of different cities, and in particular in Spain, which came to reduce about 80%. All this leads us to the observation of how human activity can greatly influence air pollution.Cárcel Carrasco, FJ.; Peñalvo López, E.; Carnero, MC.; Langa Sanchis, J. (2020). A vision on air pollution in cities and the human effect. VITRUVIO - International Journal of Architectural Technology and Sustainability. 5(2):57-70. https://doi.org/10.4995/vitruvio-ijats.2020.14607OJS57705

Potential of native Trichoderma strains as antagonists for the control of fungal wood pathologies in young grapevine plants

Neofusicoccum parvum and Rhizoctonia solani are fungal pathogens with an increasing incidence in young grapevine plants. In this study, the antagonistic potential of some strains of the genus Trichoderma isolated from grapevine against these pathogens was investigated at the laboratory and greenhouse levels. In-plate confrontation assays showed that the selected Trichoderma strains could inhibit the mycelial growth of both taxa, being more effective against N. parvum. In the in vivo assays, the biocontrol activity of the mentioned strains against the pathogens, when applied either simultaneously or successively, was tested on both grafted plants and seedlings germinated from seed. The effectiveness of the treatments was evaluated by comparing biomass weight and vascular rot lengths data. In seedling trials, successive treatments resulted in higher root development and a lower colonization rate of the pathogens, especially against R. solani. In grafted plants, some disparity was observed against N. parvum: simultaneous treatments resulted in higher aerial biomass, but successive treatments resulted in higher root biomass and lower necrosis. Against R. solani, simultaneous treatments were clearly more effective, with higher root and aerial length values and lower necrosis. The obtained data suggest that the use of Trichoderma spp. isolates can constitute an alternative to conventional fungicides to control certain grapevine wood diseases

Genomic Selection Signatures In Sheep From The Western Pyrenees

Background: The current large spectrum of sheep phenotypic diversity results from the combined product of sheep selection for different production traits such as wool, milk and meat, and its natural adaptation to new environments. In this study, we scanned the genome of 25 Sasi Ardi and 75 Latxa sheep from the Western Pyrenees for three types of regions under selection: (1) regions underlying local adaptation of Sasi Ardi semi-feral sheep, (2) regions related to a long traditional dairy selection pressure in Latxa sheep, and (3) regions experiencing the specific effect of the modern genetic improvement program established for the Latxa breed during the last three decades. Results: Thirty-two selected candidate regions including 147 annotated genes were detected by using three statistical parameters: pooled heterozygosity H, Tajima's D, and Wright's fixation index F-st. For Sasi Ardi sheep, chromosomes Ovis aries (OAR) 4, 6, and 22 showed the strongest signals and harbored several candidate genes related to energy metabolism and morphology (BBS9, ELOVL3 and LDB1), immunity (NFKB2), and reproduction (H2AFZ). The major genomic difference between Sasi Ardi and Latxa sheep was on OAR6, which is known to affect milk production, with highly selected regions around the ABCG2, SPP1, LAP3, NCAPG, LCORL, and MEPE genes in Latxa sheep. The effect of the modern genetic improvement program on Latxa sheep was also evident on OAR15, on which several olfactory genes are located. We also detected several genes involved in reproduction such as ESR1 and ZNF366 that were affected by this selection program. Conclusions: Natural and artificial selection have shaped the genome of both Sasi Ardi and Latxa sheep. Our results suggest that Sasi Ardi traits related to energy metabolism, morphological, reproductive, and immunological features have been under positive selection to adapt this semi-feral sheep to its particular environment. The highly selected Latxa sheep for dairy production showed clear signatures of selection in genomic regions related to milk production. Furthermore, our data indicate that the selection criteria applied in the modern genetic improvement program affect immunity and reproduction traits.The authors gratefully acknowledge support from the University of the Basque Country (UPV/EHU) and the Conservatoire des Races d'Aquitaine (US13/29

Generation of a Soluble Form of Human Endoglin Fused to Green Fluorescent Protein.

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

Characterization of chicken endoglin, a member of the zona pellucida family of proteins, and its tissue expression

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)Peer Reviewe

Soluble endoglin antagonizes Met signaling in spindle carcinoma cells

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).Peer reviewe

5'UTR mutations of ENG cause hereditary hemorrhagic telangiectasia

<p>Abstract</p> <p>Background</p> <p>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 (<it>ACVRL1</it>) or Endoglin (<it>ENG</it>) 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 <it>ENG</it>.</p> <p>Methods and Results</p> <p>We sequenced the 5'UTR of <it>ENG </it>for 154 HHT patients without mutations in <it>ENG </it>or <it>ACVRL1 </it>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 <it>ENG</it>, as well as in three other patients, one of which had an affected sibling with the same mutation. <it>In vitro </it>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.</p> <p>Conclusions</p> <p>Our results emphasize the need for the inclusion of the 5'UTR region of <it>ENG </it>in clinical testing for HHT.</p

Characterization of the human Activin-A receptor type II-like kinase 1 (ACVRL1) promoter and its regulation by Sp1

<p>Abstract</p> <p>Background</p> <p>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 (<it>ACVRL1</it>) 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 <it>ACVRL1</it>. Here, we have studied the different origins of <it>ACVRL1 </it>transcription, we have analyzed <it>in silico </it>its 5'-proximal promoter sequence and we have characterized the role of Sp1 in the transcriptional regulation of <it>ACVRL1</it>.</p> <p>Results</p> <p>We have performed a 5'Rapid Amplification of cDNA Ends (5'RACE) of <it>ACVRL1 </it>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 <it>ACVRL1 </it>(-1,035/+210) was analyzed <it>in silico</it>, finding that it lacks TATA/CAAT boxes, but contains a remarkably high number of GC-rich Sp1 consensus sites. In cells lacking Sp1, <it>ACVRL1 </it>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 <it>ACVRL1 </it>transcriptional activity. Chromatin immunoprecipitation assays demonstrated multiple Sp1 binding sites along the proximal promoter region of <it>ACVRL1 </it>in endothelial cells. Furthermore, demethylation of CpG islands, led to an increase in <it>ACVRL1 </it>transcription, whereas <it>in vitro </it>hypermethylation resulted in the abolishment of Sp1-dependent transcriptional activation of <it>ACVRL1</it>.</p> <p>Conclusions</p> <p>Our results describe two new transcriptional start sites in <it>ACVRL1 </it>gene, and indicate that Sp1 is a key regulator of <it>ACVRL1 </it>transcription, providing new insights into the molecular mechanisms that contribute to the expression of <it>ACVRL1 </it>gene. Moreover, our data show that the methylation status of CpG islands markedly modulates the Sp1 regulation of <it>ACVRL1 </it>gene transcriptional activity.</p