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7-ketocholesterol treatment modulates expression of the cell adhesion molecules in human aortic endothelial cells-pilot study
Abstracts / Atherosclerosis 263 (2017) e111ee282
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PO077. 7-KETOCHOLESTEROL TREATMENT MODULATES EXPRESSION OF THE CELL ADHESION MOLECULES IN HUMAN AORTIC ENDOTHELIAL CELLS-PILOT STUDY
vivo and in vitro. On the other hand, not all typical markers of endothelial dysfunction in endothelial cells or functional properties in aorta were changed in the presence of high levels of soluble endoglin. In other words, more studies are necessary to answer the question whether soluble endoglin can induce endothelial dysfunction and promote atherogenesis.
1, Matej Vicen1, Michala Varejckova1, Radim Havelek2, Barbora Vitverova Petra Fikrov a1, Eva Dolezelova1, Jana Rathouska1, Katerina Blazickova1, Ivana Nemeckova1, Petr Nachtigal1. 1 Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Biological and lov Medical Sciences, Hradec Kra e, Czech Republic; 2 Charles University in Prague, Faculty of Medicine in Hradec Kralove, Department of Medical lov Biochemistry, Hradec Kra e, Czech Republic
PO079. CONTRIBUTION OF THE CLASSICAL NF-B PATHWAY TO VENOUS ENDOTHELIAL INFLAMMATION FOLLOWING ACUTE INCREASES IN SHEAR STRESS: IMPLICATIONS FOR VEIN GRAFT FAILURE
Aim: Upregulation of cell adhesion molecules is essential for the development of endothelial dysfunction and atherosclerosis. P/E-selectins, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) mediate adhesion and transmigration of leukocytes through endothelium. In this pilot study, we tested the effect of oxidized cholesterol - 7-ketocholesterol (7-K) and tumor necrosis factor alpha (TNFa) on the expression of P/E selectins, VCAM-1 and ICAM-1 in Human aortic endothelial cells (HAECs). Methods: (HAECs) were exposed to TNF-a (10 ng/mL) for 18 hours or 7-K (10 ug/mL) for 12 and 24 hours. Protein expression and ratio of apoptotic cells was evaluated by immunofluorescence flow cytometry analysis. Results: Significantly stronger basal expression of ICAM-1 was detected when compared with P/E selectin and VCAM-1 expression. TNF-a treatment significantly increased expression of VCAM-1 (P< 0,0001), ICAM-1 (P< 0,0001) and P/E selectins (P< 0,05) in HAECs. 7-K significantly increased expression of ICAM-1 (P< 0,001) and P/E selectins (P? 0,05) after 12h premedication when compared to non-treated cells. After 24h was increased also expression of VCAM-1 (P< 0,01). Conclusion: In this study, we demonstrated that 7-K as well as TNF-a induced pro-inflammatory reaction in endothelial cells. Pro-inflammatory effects of 7-K were less pronounced than TNF-a. These results suggest that this experimental design is suitable for our prospective experiments focusing on endothelial dysfunction and endoglin. This work was supported by grants from Czech Science Foundation (GACR 15-24015S, GAUK 1158413C and SVV/2016/260293).
PO078. SOLUBLE ENDOGLIN PARTICIPATE ON ENDOTHELIAL DYSFUNCTION: OR NOT?
THE
DEVELOPMENT
OF
Blazickova1, Jana Rathouska1, Iveta Petr Nachtigal1, Katerina Najmanova1, Barbora Vitverova1, Matej Vicen1, Eva Dolezelova1, Ivana Nemeckova1, Stefan Chlopicki2. 1 Charles University, Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, Hradec Kralove, Czech Republic; 2 Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland Aim: A soluble form of tissue endoglin (sEng) circulating in plasma has been proposed to be at least partially responsible for the induction of endothelial dysfunction, however in blood vessels not related to atherosclerosis. We provided couple of experiments in order to reveal whether high levels of soluble endoglin might upregulate endothelial dysfunction markers and induce endothelial dysfunction both in vitro and in vivo. Methods: Transgenic mice overexpressing human sEng (high Sol-Eng+) and their and age-matched transgenic littermates that do not develop high levels of human sEng (low Sol-Eng+) were fed high fat diet for 3 months and HUVEC and HEK were exposed to recombinant human endoglin at concentration 40 e 500 ng/mL at different times (16 e 48 hours). Results: High soluble endoglin levels and the presence of mild hypercholesteromia resulted in induction of inflammation in aorta of high SolEng+ mice. On the contrary, functional properties of aorta were not altered. Soluble endoglin treatment in endothelial cells resulted in upregulation of NFkB and IL-6 expression but in increased expression of cell adhesion molecules. Conclusions: In conclusion, current results show that high levels of soluble endoglin induce signs of inflammation in vascular endothelium both in
Alexander Ward, Gianni Angelini, Sarah George, Mustafa Zakkar. University of Bristol, School of Clinical Sciences, Bristol, United Kingdom Aim: The long saphenous vein (LSV) is the most commonly used conduit for coronary artery bypass grafting; however, its use is complicated by the progression of inflammation, intimal hyperplasia and accelerated atherosclerosis. Once implanted, venous ECs in the vein graft are activated in response to acute changes in haemodynamics, although the contribution of inflammatory signalling pathways remains poorly understood. We hypothesised that inhibiting classical NF-kB activation will prevent inflammation in venous ECs in response to acute shear stress (SS). Methods: HUVECs were cultured in static conditions or exposed to laminar SS at 12dyn/cm2 for 0.5-4 hours using parallel-plate flow chambers. Cells were pre-treated prior to SS exposure with either NF-kB inhibitor - BAY117085 (10mM) or DMSO. Alternately, adenovirus-delivered over-expression of WT-IkBa was used prior to SS. Finally, using an ex vivo perfusion model, human LSV resected from patients were exposed to acute SS for 0.5-4 hours. Results: This pro-inflammatory response to acute SS was associated with activation of the classical NF-kB pathway: increased nuclear translocation of p65 (n¼3, 3.04±0.28 fold change±SEM, p<0.01) both in vitro and ex vivo, enhanced p65 DNA binding activity (n¼3, 1.25±0.03, p<0.05) and significantly increased phosphorylation at Ser-276 at both 30 and 90 minutes. Moreover, both adenoviral-delivered overexpression of IkBaand BAY11-7085 significantly reduced MCP-1 mRNA levels following 90 minutes of SS (n¼3, 87% reduction, p<0.05). Conclusions: Our data suggest that inhibition of the classical NF-kB pathway reduces inflammation induced in the venous endothelium following acute high shear stress and may provide a novel pre-treatment option for vein graft failure.
PO080. FICUS DELTOIDEA TRENGGANUENSIS IS THE MOST POTENT VARIANT IN REDUCING ENDOTHELIAL ACTIVATION AND MONOCYTE-ENDOTHELIAL CELL INTERACTION IN STIMULATED HUMAN CORONARY ARTERY ENDOTHELIAL CELLS Suhaila Muid1, 2, Effat Omar1, 2, Radzi Ikhsan Ahmad1, 2, Nor Hadiani Ismail3, Noor Alicezah Mohd Kasim1, 2, Abdul Manaf Ali4, Nurul'ain Abu Bakar1, Hapizah Nawawi1, 2. 1 Institute for Pathology, Laboratory and Forensic Medicine (I-PPerForM), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia; 2 Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia; 3 Atta-ur-Rahman Institute, Universiti Teknologi MARA, Puncak Alam, Malaysia; 4 Faculty of Bioresources & Food Industry, Universiti Sultan Zainal Abidin (UniSZA), Kuala Terengganu, Malaysia Aim: To investigate the effects of four variants of Ficus deltoidea (FD) on endothelial activation and monocyte-endothelial cell interaction in human coronary artery endothelial cells (HCAECs). Methods: Four variants of FD (Trengganuensis, Kunstleri, Deltoidea and Intermedia) were used in this study. HCAECs were cultured in 25 cm2 flasks to confluency and incubated with different concentrations of the FD crude extracts (5-40 mg/ml) together with lipopolysaccharide (LPS) for 16 hours. Protein expression of endothelial activation biomarkers [soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and e-selectin] in the supernatant were
e135
PO077. 7-KETOCHOLESTEROL TREATMENT MODULATES EXPRESSION OF THE CELL ADHESION MOLECULES IN HUMAN AORTIC ENDOTHELIAL CELLS-PILOT STUDY
vivo and in vitro. On the other hand, not all typical markers of endothelial dysfunction in endothelial cells or functional properties in aorta were changed in the presence of high levels of soluble endoglin. In other words, more studies are necessary to answer the question whether soluble endoglin can induce endothelial dysfunction and promote atherogenesis.
1, Matej Vicen1, Michala Varejckova1, Radim Havelek2, Barbora Vitverova Petra Fikrov a1, Eva Dolezelova1, Jana Rathouska1, Katerina Blazickova1, Ivana Nemeckova1, Petr Nachtigal1. 1 Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Biological and lov Medical Sciences, Hradec Kra e, Czech Republic; 2 Charles University in Prague, Faculty of Medicine in Hradec Kralove, Department of Medical lov Biochemistry, Hradec Kra e, Czech Republic
PO079. CONTRIBUTION OF THE CLASSICAL NF-
Aim: Upregulation of cell adhesion molecules is essential for the development of endothelial dysfunction and atherosclerosis. P/E-selectins, vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) mediate adhesion and transmigration of leukocytes through endothelium. In this pilot study, we tested the effect of oxidized cholesterol - 7-ketocholesterol (7-K) and tumor necrosis factor alpha (TNFa) on the expression of P/E selectins, VCAM-1 and ICAM-1 in Human aortic endothelial cells (HAECs). Methods: (HAECs) were exposed to TNF-a (10 ng/mL) for 18 hours or 7-K (10 ug/mL) for 12 and 24 hours. Protein expression and ratio of apoptotic cells was evaluated by immunofluorescence flow cytometry analysis. Results: Significantly stronger basal expression of ICAM-1 was detected when compared with P/E selectin and VCAM-1 expression. TNF-a treatment significantly increased expression of VCAM-1 (P< 0,0001), ICAM-1 (P< 0,0001) and P/E selectins (P< 0,05) in HAECs. 7-K significantly increased expression of ICAM-1 (P< 0,001) and P/E selectins (P? 0,05) after 12h premedication when compared to non-treated cells. After 24h was increased also expression of VCAM-1 (P< 0,01). Conclusion: In this study, we demonstrated that 7-K as well as TNF-a induced pro-inflammatory reaction in endothelial cells. Pro-inflammatory effects of 7-K were less pronounced than TNF-a. These results suggest that this experimental design is suitable for our prospective experiments focusing on endothelial dysfunction and endoglin. This work was supported by grants from Czech Science Foundation (GACR 15-24015S, GAUK 1158413C and SVV/2016/260293).
PO078. SOLUBLE ENDOGLIN PARTICIPATE ON ENDOTHELIAL DYSFUNCTION: OR NOT?
THE
DEVELOPMENT
OF
Blazickova1, Jana Rathouska1, Iveta Petr Nachtigal1, Katerina Najmanova1, Barbora Vitverova1, Matej Vicen1, Eva Dolezelova1, Ivana Nemeckova1, Stefan Chlopicki2. 1 Charles University, Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, Hradec Kralove, Czech Republic; 2 Jagiellonian Centre for Experimental Therapeutics (JCET), Krakow, Poland Aim: A soluble form of tissue endoglin (sEng) circulating in plasma has been proposed to be at least partially responsible for the induction of endothelial dysfunction, however in blood vessels not related to atherosclerosis. We provided couple of experiments in order to reveal whether high levels of soluble endoglin might upregulate endothelial dysfunction markers and induce endothelial dysfunction both in vitro and in vivo. Methods: Transgenic mice overexpressing human sEng (high Sol-Eng+) and their and age-matched transgenic littermates that do not develop high levels of human sEng (low Sol-Eng+) were fed high fat diet for 3 months and HUVEC and HEK were exposed to recombinant human endoglin at concentration 40 e 500 ng/mL at different times (16 e 48 hours). Results: High soluble endoglin levels and the presence of mild hypercholesteromia resulted in induction of inflammation in aorta of high SolEng+ mice. On the contrary, functional properties of aorta were not altered. Soluble endoglin treatment in endothelial cells resulted in upregulation of NFkB and IL-6 expression but in increased expression of cell adhesion molecules. Conclusions: In conclusion, current results show that high levels of soluble endoglin induce signs of inflammation in vascular endothelium both in
Alexander Ward, Gianni Angelini, Sarah George, Mustafa Zakkar. University of Bristol, School of Clinical Sciences, Bristol, United Kingdom Aim: The long saphenous vein (LSV) is the most commonly used conduit for coronary artery bypass grafting; however, its use is complicated by the progression of inflammation, intimal hyperplasia and accelerated atherosclerosis. Once implanted, venous ECs in the vein graft are activated in response to acute changes in haemodynamics, although the contribution of inflammatory signalling pathways remains poorly understood. We hypothesised that inhibiting classical NF-kB activation will prevent inflammation in venous ECs in response to acute shear stress (SS). Methods: HUVECs were cultured in static conditions or exposed to laminar SS at 12dyn/cm2 for 0.5-4 hours using parallel-plate flow chambers. Cells were pre-treated prior to SS exposure with either NF-kB inhibitor - BAY117085 (10mM) or DMSO. Alternately, adenovirus-delivered over-expression of WT-IkBa was used prior to SS. Finally, using an ex vivo perfusion model, human LSV resected from patients were exposed to acute SS for 0.5-4 hours. Results: This pro-inflammatory response to acute SS was associated with activation of the classical NF-kB pathway: increased nuclear translocation of p65 (n¼3, 3.04±0.28 fold change±SEM, p<0.01) both in vitro and ex vivo, enhanced p65 DNA binding activity (n¼3, 1.25±0.03, p<0.05) and significantly increased phosphorylation at Ser-276 at both 30 and 90 minutes. Moreover, both adenoviral-delivered overexpression of IkBaand BAY11-7085 significantly reduced MCP-1 mRNA levels following 90 minutes of SS (n¼3, 87% reduction, p<0.05). Conclusions: Our data suggest that inhibition of the classical NF-kB pathway reduces inflammation induced in the venous endothelium following acute high shear stress and may provide a novel pre-treatment option for vein graft failure.
PO080. FICUS DELTOIDEA TRENGGANUENSIS IS THE MOST POTENT VARIANT IN REDUCING ENDOTHELIAL ACTIVATION AND MONOCYTE-ENDOTHELIAL CELL INTERACTION IN STIMULATED HUMAN CORONARY ARTERY ENDOTHELIAL CELLS Suhaila Muid1, 2, Effat Omar1, 2, Radzi Ikhsan Ahmad1, 2, Nor Hadiani Ismail3, Noor Alicezah Mohd Kasim1, 2, Abdul Manaf Ali4, Nurul'ain Abu Bakar1, Hapizah Nawawi1, 2. 1 Institute for Pathology, Laboratory and Forensic Medicine (I-PPerForM), Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia; 2 Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia; 3 Atta-ur-Rahman Institute, Universiti Teknologi MARA, Puncak Alam, Malaysia; 4 Faculty of Bioresources & Food Industry, Universiti Sultan Zainal Abidin (UniSZA), Kuala Terengganu, Malaysia Aim: To investigate the effects of four variants of Ficus deltoidea (FD) on endothelial activation and monocyte-endothelial cell interaction in human coronary artery endothelial cells (HCAECs). Methods: Four variants of FD (Trengganuensis, Kunstleri, Deltoidea and Intermedia) were used in this study. HCAECs were cultured in 25 cm2 flasks to confluency and incubated with different concentrations of the FD crude extracts (5-40 mg/ml) together with lipopolysaccharide (LPS) for 16 hours. Protein expression of endothelial activation biomarkers [soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and e-selectin] in the supernatant were