Ivana Nemeckova

Soluble endoglin, hypercholesterolemia and endothelial dysfunction

A soluble form of endoglin (sEng) is known to be an extracellular domain of the full-length membrane endoglin, which is elevated during various pathological conditions related to vascular endothelium. In the current review, we tried to summarize a possible role of soluble endoglin in cardiovascular pathologies, focusing on its relation to endothelial dysfunction and cholesterol levels. We discussed sEng as a proposed biomarker of cardiovascular disease progression, cardiovascular disease treatment and endothelial dysfunction. We also addressed a potential interaction of sEng with TGF-β/eNOS or BMP-9 signaling. We suggest soluble endoglin levels to be monitored, because they reflect the progression/treatment efficacy of cardiovascular diseases related to endothelial dysfunction and hypercholesterolemia. A possible role of soluble endoglin as an inducer of endothelial dysfunction however remains to be elucidated.

Spirulina platensis and phycocyanobilin activate atheroprotective heme oxygenase-1: a possible implication for atherogenesis

Spirulina platensis, a water blue-green alga, has been associated with potent biological effects, which might have important relevance in atheroprotection. We investigated whether S. platensis or phycocyanobilin (PCB), its tetrapyrrolic chromophore, can activate atheroprotective heme oxygenase-1 (Hmox1), a key enzyme in the heme catabolic pathway responsible for generation of a potent antioxidant bilirubin, in endothelial cells and in a mouse model of atherosclerosis. In vitro experiments were performed on EA.hy926 endothelial cells exposed to extracts of S. platensis or PCB. In vivo studies were performed on ApoE-deficient mice fed a cholesterol diet and S. platensis. The effect of these treatments on Hmox1, as well as other markers of oxidative stress and endothelial dysfunction, was then investigated. Both S. platensis and PCB markedly upregulated Hmox1 in vitro, and a substantial overexpression of Hmox1 was found in aortic atherosclerotic lesions of ApoE-deficient mice fed S. platensis. In addition, S. platensis treatment led to a significant increase in Hmox1 promoter activity in the spleens of Hmox-luc transgenic mice. Furthermore, both S. platensis and PCB were able to modulate important markers of oxidative stress and endothelial dysfunction, such as eNOS, p22 NADPH oxidase subunit, and/or VCAM-1. Both S. platensis and PCB activate atheroprotective HMOX1 in endothelial cells and S. platensis increased the expression of Hmox1 in aortic atherosclerotic lesions in ApoE-deficient mice, and also in Hmox-luc transgenic mice beyond the lipid lowering effect. Therefore, activation of HMOX1 and the heme catabolic pathway may represent an important mechanism of this food supplement for the reduction of atherosclerotic disease.

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

Non 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.

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, pNFκB 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.

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

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‐&bgr; 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‐&bgr; signaling. IL6 and NF&kgr;B 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‐&kgr;B/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‐&kgr;B, 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.

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

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.

Endothelial and vascular function in mice overexpressing human soluble endoglin

Introduction and aims: Endoglin, a 180 kDa membrane glycoprotein, is a TGF-beta co-receptor which is overexpressed in several models of chronic kidney disease but its specific function in renal fibrosis remains still undefined. Two membrane isoforms generated by alternative splicing have been described, full length Endoglin (L-Eng, the most abundant isoform) and S-Endoglin (short) that differ from L-Eng in the absence of the cytoplasmic tail. We have recently demonstrated that L-Eng overexpression enhances fibrosis in the unilateral ureteral obstruction (UUO) model of kidney fibrosis. The aim of the present study was to assess the effect of S-Endoglin overexpression in renal tubulo-interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Methods: For this purpose, a transgenic mouse which ubiquitously overexpresses human S-Endoglin (SENG+) was generated. UUO was performed in S-ENG+ mice and their wild type littermates. Kidney Fibrosis was determined by morphometric techniques and by the expression of fibrosis-related molecules (collagen I, fibronectin) by western blot. Results: Obstructed kidneys from S-ENG+ mice showed reduced tubulo-interstitial fibrotic area and lower amounts of collagen I and fibronectin than obstructed kidneys from WT mice. Moreover, western blot analysis showed that levels of p-Smad1 and p-Smad3 were higher in obstructed kidneys, being these increase significantly lower in obstructed kidneys from S-ENG+ than in those from WT animals. Conclusions: The overexpression of S-ENG reduces kidney fibrosis. These results are exactly the opposite of those obtained in L-ENG mice. Therefore, we conclude that the intracellular domain of endoglin plays a major role in regulating its fibrotic effects.1Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic; 3Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Madrid, Spainendoglin release upon vascular injury Bernabeu C1; Gallardo-Vara E1; Blanco F1; Roque M2; Friedman SL3; Suzuki T4; Botella LM1 1Centro de Investigaciones Biológicas, CSIC, and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain; 2Servei de Cardiología, Institut del Torax, Hospital Clinic, Barcelona, Spain; 3Division of Liver Disease, Mount Sinai School of Medicine, New York, NY, USA; 4Department of Cardiovascular Medicine, The University of Tokyo, Tokyo, Japan11th International HHT Scientific Conference June 11–14, 2015 Captiva, Florida USA Springer Science+Business Media Dordrecht 2015 SCIENTIFIC PROGRAM COMMITTEE

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

Introduction and aims: Endoglin, a 180 kDa membrane glycoprotein, is a TGF-beta co-receptor which is overexpressed in several models of chronic kidney disease but its specific function in renal fibrosis remains still undefined. Two membrane isoforms generated by alternative splicing have been described, full length Endoglin (L-Eng, the most abundant isoform) and S-Endoglin (short) that differ from L-Eng in the absence of the cytoplasmic tail. We have recently demonstrated that L-Eng overexpression enhances fibrosis in the unilateral ureteral obstruction (UUO) model of kidney fibrosis. The aim of the present study was to assess the effect of S-Endoglin overexpression in renal tubulo-interstitial fibrosis induced by unilateral ureteral obstruction (UUO). Methods: For this purpose, a transgenic mouse which ubiquitously overexpresses human S-Endoglin (SENG+) was generated. UUO was performed in S-ENG+ mice and their wild type littermates. Kidney Fibrosis was determined by morphometric techniques and by the expression of fibrosis-related molecules (collagen I, fibronectin) by western blot. Results: Obstructed kidneys from S-ENG+ mice showed reduced tubulo-interstitial fibrotic area and lower amounts of collagen I and fibronectin than obstructed kidneys from WT mice. Moreover, western blot analysis showed that levels of p-Smad1 and p-Smad3 were higher in obstructed kidneys, being these increase significantly lower in obstructed kidneys from S-ENG+ than in those from WT animals. Conclusions: The overexpression of S-ENG reduces kidney fibrosis. These results are exactly the opposite of those obtained in L-ENG mice. Therefore, we conclude that the intracellular domain of endoglin plays a major role in regulating its fibrotic effects.1Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Hradec Kralove, Czech Republic; 3Centro de Investigaciones Biologicas, Consejo Superior de Investigaciones Científicas (CSIC), and Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Madrid, Spainendoglin release upon vascular injury Bernabeu C1; Gallardo-Vara E1; Blanco F1; Roque M2; Friedman SL3; Suzuki T4; Botella LM1 1Centro de Investigaciones Biológicas, CSIC, and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), 28040 Madrid, Spain; 2Servei de Cardiología, Institut del Torax, Hospital Clinic, Barcelona, Spain; 3Division of Liver Disease, Mount Sinai School of Medicine, New York, NY, USA; 4Department of Cardiovascular Medicine, The University of Tokyo, Tokyo, Japan11th International HHT Scientific Conference June 11–14, 2015 Captiva, Florida USA Springer Science+Business Media Dordrecht 2015 SCIENTIFIC PROGRAM COMMITTEE

CARDIOTOXICITY OF CHRONIC SUNITINIB TREATMENT IN NORMOTENSIVE AND SPONTANEOUSLY HYPERTENSIVE RATS

Aims Sunitinib is a modern multi-targeted tyrosine kinase inhibitor, which showed great promise in the treatment of human cancer. However, its chronic use may be associated with cardiotoxicity, which is poorly understood. Hence, the aim of the present study was to investigate potential cardiotoxicity of chronic sunitinib treatment in normotensive and spontaneously hypertensive rats. Methods Spontaneously hypertensive rats (SHR) and their normotensive littermates (Wistar Kyoto, WKY) were used in the study (n=32). Sunitinib (10 mg/kg) was administered daily for 8 weeks and after a wash out period (5 days) a re-challenge for 2 and 8 weeks followed in WKY and SHR rats, respectively. Results Sunitinib treatment induced a significant left ventricular (LV) dysfunction and decline in heart rate. This was accompanied by increased LV BNP expression, increased BNP plasma concentrations and increased lung to body weight ratio. Remarkably, no such change was found in SHR despite significantly longer duration of treatment. Moreover, sunitinib-induced cardiac dysfunction in WKY was accompanied by marked expression of numerous hypoxia-regulated genes and inflammatory molecules. Conclusions In conclusion, our findings show different propensity of WKY and SHR animals towards sunitinib cardiotoxicity development and suggest an important role of hypoxic signaling in this process. Furthermore, possible involvement of “hibernating myocardium” in sunitinib-induced cardiac dysfunction deserves further study, particularly with respect to recently reported impairment of cardiac microvasculature. 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. This work was supported by project UNCE 33/2012.