Endoglin is crucial for atorvastatin induced eNOS expression in endothelial cells in vitro

1p. Varejckova, Michala et al.This work was supported by Grant from Czech Science foundation GACR number 15-24015S, the Grant Agency of Charles University in Prague (300811/C and 1158413/C), Charles University in Prague (SVV/2014/260064), Ministerio de Economia y Competitividad of Spain (SAF2010-19222 and SAF2013-42421-R to CB), and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER to CB). CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. The 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

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

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

Atorvastatin-induced endothelial nitric oxide synthase expression in endothelial cells is mediated by endoglin

11 p.-6 fig.Endoglin, a transforming growth factor β (TGF-β) receptor type III, is co-expressed with endothelial nitric oxide synthase (eNOS) in aortic endothelium in atherosclerotic plaques of mice. Interestingly, atorvastatin (ATV) is able to increase both endoglin and eNOS expression and reduce plaque size beyond its lipid lowering effects but by unknown mechanisms. We hypothesized whether inflammation modulates ATV-dependent induction of endoglin and eNOS expression in vitro in endothelial cells and whether ATV-induced eNOS expression is regulated via endoglin. After treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α, endoglin and eNOS protein expression was reduced, concomitantly with increased levels of cell surface VCAM-1 and soluble endoglin, as determined by flow cytometry, Western blot and ELISA analyses. By contrast, ATV treatment increased endoglin and eNOS protein expression, while preventing TNF-α-mediated downregulation of endoglin and eNOS protein levels. Moreover, suppression of endoglin using small interfering RNA (siRNA), but not inhibition of TGF-β signaling with SB431542, abrogated ATV-induced eNOS expression. These results suggest that ATV treatment prevents inflammation-reduced endoglin and eNOS expression in endothelial cells and that ATV-induced eNOS expression strongly depends on the proper expression of endoglin in HUVECs. Possible implications of these findings might be reflected in pathological conditions characterized by reduced expression of endoglin and eNOS as for example in hereditary hemorrhagic telangiectasia or in other endothelial dysfunctions.This work was supported by The Grant Agency of Charles University in Prague (300811/C and 1158413/C), Charles University in Prague (SVV/2014/260064), Ministerio de Economía y Competitividad of Spain Raras ( (SAF2010-19222 and SAF2013-42421-R to CB), and Centro de Investigación Biomédica en Red de Enfermedades CIBERER to CB).CIBERER is an initiative of the Instituto de Salud Carlos III (ISCIII) of Spain supported by FEDER funds. 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

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

Urinary excretion of nitrites in <i>Sol-Eng</i>^<i>+</i> and control mice.

<p>Urinary excretion of nitrites was measured in urine from <i>Sol-Eng</i>^<i>+</i> and control mice collected in metabolic cages, and corrected by creatinine concentration. Data are shown as mean ± S.E.M. Unpaired t-test.</p

Pressure responses to agonists or antagonists of the NO-cGMP-system in <i>Sol-Eng</i>^<i>+</i> and control mice.

<p>Maximal hypotensive effect of sodium nitroprusside (SNP; 2 mg/Kg b.w.) in <i>Sol-Eng</i>^<i>+</i> and control mice (A). Maximal hypertensive effect of L-NAME (50 mg/Kg b.w.) in <i>Sol-Eng</i>^<i>+</i> and control mice (B). Effects were measured by telemetry. SABP: Systolic blood arterial pressure. Data are shown as mean ± S.E.M. ANOVA and unpaired t-test with respect to control, *p≤0.05; ANOVA and paired t-test with respect to basal conditions, ^#p≤0.05.</p

Impaired vascular contractility in female <i>Sol-Eng</i>^<i>+</i> mice as compared to control mice.

<p>Maximal contraction to KCl (30 mM) (A). Maximal contraction to PGF2α (10μM) (B) and to PHE (1 μM) (C) in <i>Sol-Eng</i>^<i>+</i> and control mice. Comparison of dose-response to PGF2α (D) and PHE (E) in <i>Sol-Eng</i>^<i>+</i> as compared to control mice. Data are shown as mean ± S.E.M. Unpaired t-test, **p≤0.01, ***p≤0.001.</p