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

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

Endoglin is not expressed with cell adhesion molecules in aorta during atherogenesis in apoE-deficient mice

Endoglin (TGF-β receptor III), has been demonstrated to affect vascular endothelium and atherosclerosis. Moreover, it was also demonstrated that endoglin is involved in inflammation and plays a role in leukocyte adhesion and transmigration in vitro and in vivo but not in atherosclerosis related vessels. In this study, we wanted to evaluate endoglin expression in two different parts of the aorta (heart aortic sinus and ascending aorta) and assess its potential simultaneous expression with cell adhesion molecules in nonatherosclerotic and atherosclerotic aortas of apoEdeficient mice. Ten-week–old female apolipoprotein E-deficient mice on a C57BL/6J background (n=24) were randomly subdivided into three groups and were fed either chow diet (for another two months) or Western type diet (for another two or four months). Immunohistochemical staining of endoglin, VCAM-1 and P-selectin in aortic sinus and ascending aorta was performed. Endoglin expression was detected only in endothelial cells and varied during atherogenic process in aorta but not in aortic sinus. Moreover, its expression seemed to be weaker in aorta when compared to aortic sinus and the positivity was detected only in endothelium covering atherosclerotic lesions but not in non-atherosclerotic endothelium regardless of the plaque size. Endoglin was not expressed with P-selectin and VCAM-1 in aortic endothelium in any studied group. This study shows that endothelial expression of endoglin is related to the atherogenic process predominantly in aorta outside the heart. Moreover, endoglin is not localized with cell adhesion molecules involved in atherosclerosis, suggesting it might not participate in leukocyte accumulation in aorta of apoEdeficient mice during atherogenesis

Transgenic human C-reactive protein affects oxidative stress but not inflammation biomarkers in the aorta of spontaneously hypertensive rats

Abstract Background C-reactive protein (CRP) is an acute inflammatory protein detected in obese patients with metabolic syndrome. Moreover, increased CRP levels have been linked with atherosclerotic disease, congestive heart failure, and ischemic heart disease, suggesting that it is not only a biomarker but also plays an active role in the pathophysiology of cardiovascular diseases. Since endothelial dysfunction plays an essential role in various cardiovascular pathologies and is characterized by increased expression of cell adhesion molecules and inflammatory markers, we aimed to detect specific markers of endothelial dysfunction, inflammation, and oxidative stress in spontaneously hypertensive rats (SHR) expressing human CRP. This model is genetically predisposed to the development of the metabolic syndrome. Methods Transgenic SHR male rats (SHR-CRP) and non-transgenic SHR (SHR) at the age of 8 months were used. Metabolic profile (including serum and tissue triglyceride (TAG), serum insulin concentrations, insulin-stimulated incorporation of glucose, and serum non-esterified fatty acids (NEFA) levels) was measured. In addition, human serum CRP, MCP-1 (monocyte chemoattractant protein-1), and adiponectin were evaluated by means of ELISA, histological analysis was used to study morphological changes in the aorta, and western blot analysis of aortic tissue was performed to detect expression of endothelial, inflammatory, and oxidative stress markers. Results The presence of human CRP was associated with significantly decreased insulin-stimulated glycogenesis in skeletal muscle, increased muscle and hepatic accumulation of TAG and decreased plasmatic cGMP concentrations, reduced adiponectin levels, and increased monocyte chemoattractant protein-1 (MCP-1) levels in the blood, suggesting pro-inflammatory and presence of multiple features of metabolic syndrome in SHR-CRP animals. Histological analysis of aortic sections did not reveal any visible morphological changes in animals from both SHR and SHR-CRP rats. Western blot analysis of the expression of proteins related to the proper function of endothelium demonstrated significant differences in the expression of p-eNOS/eNOS in the aorta, although endoglin (ENG) protein expression remained unaffected. In addition, the presence of human CRP in SHR in this study did not affect the expression of inflammatory markers, namely p-NFkB, P-selectin, and COX2 in the aorta. On the other hand, biomarkers related to oxidative stress, such as HO-1 and SOD3, were significantly changed, indicating the induction of oxidative stress. Conclusions Our findings demonstrate that CRP alone cannot fully induce the expression of endothelial dysfunction biomarkers, suggesting other risk factors of cardiovascular disorders are necessary to be involved to induce endothelial dysfunction with CRP

Soluble endoglin modulates the pro-inflammatory mediators NF-kappa B and IL-6 in cultured human endothelial cells

34 p.-7 fig.Aims: Endoglin is a transmembrane glycoprotein, that plays an important role in regulating endothelium. Proteolytic cleavage of membrane endoglin releases soluble endoglin (sEng), whose increased plasma levels have been detected in diseases related to the cardiovascular system. It was proposed that sEng might damage vascular endothelium, but detailed information about the potential mechanisms involved is not available. Thus, we hypothesized that sEng contributes to endothelial dysfunction, leading to a pro-inflammatory phenotype by a possible modulation of the TGF-β and/or inflammatory pathways.Main methods: Human umbilical vein endothelial cells (HUVECs) and Human embryonic kidney cell line (HEK293T) were treated with different sEng concentration and time in order to reveal possible effect on biomarkers of inflammation and TGF-β signaling. IL6 and NFκB reporter luciferase assays, quantitative real-time PCR analysis, Western blot analysis and immunofluorescence flow cytometry were used.Key findings: sEng treatment results in activation of NF-κB/IL-6 expression, increased expression of membrane endoglin and reduced expression of Id-1. On the other hand, no significant effects on other markers of endothelial dysfunction and inflammation, including eNOS, peNOSS1177, VCAM-1, COX-1, COX-2 and ICAM-1 were detected.Significance: As a conclusion, sEng treatment resulted in an activation of NF-κB, IL-6, suggesting activation of pro-inflammatory phenotype in endothelial cells. The precise mechanism of this activation and its consequence remains to be elucidated. A combined treatment of sEng with other cardiovascular risk factors will be necessary in order to reveal whether sEng is not only a biomarker of cardiovascular diseases, but also a protagonist of endothelial dysfunction.This work was supported by grants from Czech Science Foundation (GACR 15-24015S,GAUK 1158413C, SVV/2016/260293 and SVV/2017/260414 to Petr Nachtigal), Ministerio de Economía y Competitividad of Spain (SAF2013-43421-R to Carmelo Bernabéu), Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER; ISCIIICB06/07/0038 and ER16PIAC707 to CB). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds.Peer reviewe

High soluble endoglin levels and high fat diet effects on mouse aorta

2 p.The study was supported by grant from Czech Science foundation GACR number 15-24015S, The Grant Agency of Charles University in Prague number 1284214/C and grant SVV/2014/260064. 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. European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCET-UJ, No POIG.01.01.02-00-069/09).Peer reviewe

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

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

High levels of soluble endoglin induce a proinflammatory and oxidative-stress phenotype associated with preserved NO-dependent vasodilatation in aortas from mice fed a high-fat diet

Aims: A soluble form of endoglin (sEng) was proposed to participate in the induction of endothelial dysfunction in small blood vessels. Here, we tested the hypothesis that high levels of sEng combined with a high-fat diet induce endothelial dysfunction in an atherosclerosis-prone aorta. Methods and Results: Six-month-old female and male transgenic mice overexpressing human sEng (Sol-Eng(+)) with low (Sol-Eng(+) low) or high (Sol-Eng(+) high) levels of plasma sEng were fed a high-fat rodent diet containing 1.25\% cholesterol and 40% fat for 3 months. The plasma cholesterol and mouse sEng levels did not differ in the Sol-Eng(+) high and Sol-Eng(+) low mice. The expression of proinflammatory (P-selectin, ICAM-1, pNFkB and COX-2) and oxidative-stress-related markers (HO-1, NOX-1 and NOX-2) in the aortas of Sol-Eng(+) high female mice was significantly higher than in Sol-Eng(+) low female mice. Endothelium-dependent vasodilatation induced by acetylcholine was preserved better in the Sol-Eng(+) high female mice than in the Sol-Eng(+) low female mice. Conclusion: These results suggest that high concentrations of sEng in plasma in combination with a high-fat diet induce the simultaneous activation of proinflammatory, pro-oxidative and vasoprotective mechanisms in mice aorta and the balance of these biological processes determines whether the final endothelial phenotype is adaptive or maladaptive