Endothelial and vascular function in mice overexpressing human soluble endoglin

1 p. Nemeckova, Ivana et al.This work was supported by grants from Czech Science foundation GACR number 15-24015S, the Grant Agency of Charles University in Prague (1284214/C and 1158413/C), Charles University in Prague (SVV/2014/260064), European Regional Development Fund under the Innovative Economy Program of the European Union (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09), Ministerio de Economia y Competitividad of Spain (SAF2010-19222 and SAF2013-43421-R and SAF2010-1588), Junta de Castilla y Leon (GR100), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER) and Red de Investigacion Cooperativa en Enfermedades Renales (RD12/0021/0032; REDINREN). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. The Cardiovascular Phenotyping Unit of the University of Salamanca, including the telemetry equipment, was acquired with the support of the European Regional Development Funds (FEDER). Ministerio de Economia y Competitividad (BES-2008-005550). This work has been co-financed by the European Social Fund and the state budget of the Czech Republic (Project No. CZ.1.07/2.3.00/30.0061).Peer reviewe

An animal model for the juvenile non-alcoholic fatty liver disease and non-alcoholic steatohepatitis

11Non Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH) are the hepatic manifestations of the metabolic syndrome; worrisome is the booming increase in pediatric age. To recreate the full spectrum of juvenile liver pathology and investigate the gender impact, male and female C57Bl/6 mice were fed with high fat diet plus fructose in the drinking water (HFHC) immediately after weaning (equal to 3-years old human), and disease progression followed for 16 weeks, until adults (equal to 30-years old human). 100% of subjects of both genders on HFHC diet developed steatosis in 4weeks, and some degree of fibrosis in 8weeks, with the 86% of males and 15% of females presenting a stage 2 fibrosis at 16weeks. Despite a similar final liver damage both groups, a sex difference in the pathology progression was observed. Alterations in glucose homeostasis, dyslipidemia, hepatomegaly and obese phenotype were evident from the very beginning in males with an increased hepatic inflammatory activity. Conversely, such alterations were present in females only at the end of the HFHC diet (with the exception of insulin resistance and the hepatic inflammatory state). Interestingly, only females showed an altered hepatic redox state. This juvenile model appears a good platform to unravel the underlying gender dependent mechanisms in the progression from NAFLD to NASH, and to characterize novel therapeutic approaches.openopenMarin, Veronica; Rosso, Natalia; Dal Ben, Matteo; Raseni, Alan; Boschelle, Manuela; Degrassi, Cristina; Nemeckova, Ivana; Nachtigal, Petr; Avellini, Claudio; Tiribelli, Claudio; Gazzin, SilviaMarin, Veronica; Rosso, NATALIA CAROLINA; DAL BEN, Matteo; Raseni, Alan; Boschelle, Manuela; Degrassi, Cristina; Nemeckova, Ivana; Nachtigal, Petr; Avellini, Claudio; Tiribelli, Claudio; Gazzin, Silvi

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

Monoclonal anti-endoglin antibody TRC105 (carotuximab) prevents hypercholesterolemia and hyperglycemia-induced endothelial dysfunction in human aortic endothelial cells

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

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

32 p.-5 fig.-1 fig. supl. Vitverova, Barbora et al.BACKGROUND AND AIMS:Increased plasma levels of soluble endoglin (sEng) were detected in patients with endothelial dysfunction-related disorders and hypercholesterolemia. In this study, we hypothesized that high levels of sEng accompanied by mild hypercholesterolemia could aggravate endothelial and vessel wall dysfunction and affect endoglin/eNOS signaling in mouse aorta.METHODS:Three-month-old female transgenic mice on CBAxC57BL/6J background, with high levels of sEng (Sol-Eng+high HFD), and their littermates with low levels of sEng (Sol-Eng+low HFD), were fed a high fat diet for six months. Plasma samples were used for biochemical, ELISA and Luminex analyses of total cholesterol, sEng and inflammatory markers. Functional parameters of aorta were assessed with wire myograph 620M. Western blot analyses of membrane endoglin/eNOS signaling and endothelial dysfunction/inflammation markers in aorta were performed.RESULTS:Functional analysis of aorta showed impaired KCl induced vasoconstriction, endothelial-dependent relaxation after the administration of acetylcholine as well as endothelial-independent relaxation induced by sodium nitroprusside in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. Ach-induced vasodilation after administration of l-NAME was significantly higher in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group. The expression of endoglin, p-eNOS/eNOS, pSmad2/3/Smad2/3 signaling pathway was significantly lower in the Sol-Eng+high HFD group compared to the Sol-Eng+low HFD group.CONCLUSIONS:The results indicate that long-term hypercholesterolemia combined with high levels of sEng leads to the aggravation of endothelial and vessel wall dysfunction in aorta, with possible alterations of the membrane endoglin/eNOS signaling, suggesting that high levels of soluble endoglin might be considered as a risk factor of cardiovascular diseases.The work was supported by grants from Czech Science foundation (GACR number GA15-24015S), Czech Health Research Council (AZV CR number 17-31754A), the Grant Agency of Charles University in Prague (number 1284214/C, 1158413C and grant SVV/2016/260293), Ministerio de Economia y Competitividad of Spain (SAF2013-43421-R to CB Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; to CB), and Instituto de Salud Carlos III (PI16/00460 and Retic RD16/0009/0025, REDINREN-FEDER). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds from the European CommissionPeer reviewe

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

Petr Nachtigal et alt. 13 p.-7 fig.Increased levels of a soluble form of endoglin (sEng) circulating in plasma have been detected in various pathological conditions related to cardiovascular system. High concentration of sEng was also proposed to contribute to the development of endothelial dysfunction,but there is no direct evidence to support this hypothesis. Therefore, in the present work we analyzed whether high sEng levels induce endothelial dysfunction in aorta by using transgenic mice with high expression of human sEng. Transgenic mice with high expression of human sEng on CBAxC57Bl/6J background (Sol-Eng+) and age-matched transgenic littermates that do not develop high levels of human soluble endoglin (control animals in this study) on chow diet were used. As expected, male and female Sol-Eng+ transgenic mice showed higher levels of plasma concentrations of human sEng as well as increased blood arterial pressure, as compared to control animals. Functional analysis either in vivo or ex vivo in isolated aorta demonstrated that the endothelium-dependent vascular function was similar in Sol-Eng+ and control mice. In addition, Western blot analysis showed no differences between Sol-Eng+ and control mice in the protein expression levels of endoglin, endothelial NO-synthase (eNOS) and pro-inflammatory ICAM-1 and VCAM-1 from aorta. Our results demonstrate that high levels of soluble endoglin alone do not induce endothelial dysfunction in Sol-Eng+ mice. However, these data do not rule out the possibility that soluble endoglin might contribute to alteration of endothelial function in combination with other risk factors related to cardiovascular disorders.This work was supported by grants from the Grant Agency of Charles University in Prague (300811/C and 1284214/C), Charles University in Prague (SVV/2014/260064), European Regional Development Fund under the Innovative Economy Program of the European Union (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09),Ministerio de Economia y Competitividad of Spain (SAF2010-19222 and SAF2013-43421-R to CB and SAF2010-15881 to JML-N), Junta de Castilla y León (GR100 to JML-N), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER, CB), and Red de Investigación Cooperativa en Enfermedades Renales (RD12/0021/0032; REDINREN, JML-N). CIBERER and REDINREN are initiatives of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDERfunds. The Cardiovascular Phenotyping Unit of the University of Salamanca, including the telemetry equipment, was acquired with the support of the European Regional Development Funds (FEDER).BO is supported by a fellowship from Ministerio de Economia y Competitividad (BES-2008-005550). The publication is co-financed by the European Social Fund and the state budget of the Czech Republic(Project No. CZ.1.07/2.3.00/30.0061).Peer reviewe

Pharmacy Practice and Education in the Czech Republic

The PHARMINE (“Pharmacy Education in Europe”) project studied the organisation of pharmacy education, practice and legislation in the European Union (EU) with the objectives of evaluating to what degree harmonisation had taken place with the EU, and producing documents on each individual EU member state. Part of this work was in the form of a survey of pharmacy education, practice, and legislation in the various member states. We will publish the individual member state surveys as reference documents. This paper presents the results of the PHARMINE survey on pharmacy education, training, and practice in the Czech Republic. Czech community pharmacies sell and provide advice on Rx and Over-the-counter (OTC) medicines; they also provide diagnostic services (e.g., blood pressure measurement). Pharmacists (lékárník in Czech) study for five years and graduate with a Magister (Mgr., equivalent to M.Pharm.) degree. The Mgr. diploma is the only requirement for registration as a pharmacist. Pharmacists can own and manage community pharmacies, or work as responsible pharmacists in pharmacies. All practising pharmacists must be registered with the Czech Chamber of Pharmacists. The ownership of a community pharmacy is not restricted to members of the pharmacy profession; the majority of pharmacies are organised into various pharmacy chains. There are two universities providing higher education in pharmacy in the Czech Republic: the Faculty of Pharmacy in Hradec Kralove, Charles University, which was established in 1969, and the Faculty of Pharmacy of the University of Veterinary and Pharmaceutical Sciences in Brno, which was established in 1991. The pharmacy curriculum is organized as a seamless, fully integrated, five-year master degree course. There is a six-month traineeship supervised by the university, which usually takes place during the fifth year. Thus, the pharmacy curriculum is organised in accordance with the EU directive on sectoral professions that lays down the imperatives for pharmacy education, training, and practice in the various member states of the EU. Currently, no specialisation courses are available at the university level. Specialisation is organised in the form of postgraduate, continuing professional development by the Czech Chamber of Pharmacists, and delivered by the Institute of Postgraduate Education for Health Professions