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Soluble endoglin effects on endothelial cells in vitro
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Abstracts / Atherosclerosis 252 (2016) e1ee196
Objectives: Cocaine use is a risk factor for ischemic vascular complications. In vivo and in vitro studies indicate that cocaine induces endothelial dysfunction and accelerated atherosclerosis. In the brain, a mechanism by which cocaine exerts its effects is through the Sigma-1 receptor (Sigma1R), but the role of this receptor in systemic endothelial cells is unknown. Objective: to determine the participation of Sigma-1R in the pathogenesis of cocaine-associated cell damage in human umbilical cord endothelial cells (HUVEC). Methods: HUVEC were exposed to cocaine (10 mM) in the presence or absence of the Sigma-1R antagonist BD1047. Activation of RhoA/ROCK was determined by quantifying pMYPT/MYPT levels. Nitric Oxide (NO) was measured based on Griess reaction and procoagulant activity (PCA) by FXa generation. Exposure of von Willebrand factor (VWF) and number of platelets (PLTs) adhered to HUVEC were evaluated by immunofluorescence. Results: Cocaine increased RhoA/ROCK activity compared to controls and Sigma-1R inhibition prevented this activation (P<0.05). Cocaine decreased NO levels and BD1047 hampered this reduction (P<0.05). PCA in HUVEC increased in the presence of cocaine and PLTs, and BD1047 prevented this effect. Cocaine induced the exposure of VWF fibers on the surface of HUVEC, resulting in an increased number of adhered PLTs as compared to controls. Inhibition of Sigma-1R diminished both phenomena. Under flow, inhibition of Sigma-1R prevented adhesion of PLTs to human microvascular endothelial cells stimulated with cocaine. Conclusions: In systemic endothelial cells in vitro, Sigma-1R seems to play a role in cocaine-induced activation of RhoA/ROCK, VWF exposure, increased platelet adhesion, PCA and NO biodisponibility.
EAS16-0230, VASCULAR BIOLOGY: ENDOTHELIUM AND SMOOTH MUSCLE CELLS. P38 MITOGEN-ACTIVATED PROTEIN KINASE IS INVOVLED IN ARGINASEII-MEDIATED ENOS-UNCOUPLING IN AGING AND OBESITY Y. Yu 1, J.P. Montani 1, X. Ming 1, Z. Yang 1. 1 Fribourg University, Department of Medicine, Fribourg, Switzerland Objectives: Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and aging to the increased cardiovascular disease. Given that arginase-II (Arg-II) and p38 mitogen-activated protein kinase (p38mapk) play important roles in vascular system, we investigated here whether p38mapk is involved in Arg-II-mediated eNOSuncoupling in aging and high-fat diet induced obesity model. Methods: Human umbilical vein endothelial cells were transduced with adenovirus overexpressing Arg-II or silencing p38a and Arg-II to detect eNOS-uncoupling and cytokines production. For the animal model, obesity was induced by high-fat diet (HFD, 55% fat) for 14 weeks. The female mice with 2-3 months old and 20- 24 months old were taken as young and old mice. Superoxide and NO production in aortas were detected by en face staining. Results: We demonstrate eNOS-uncoupling, augmented secretion of IL-6 and IL-8, elevation of p38mapk activation and Arg-II levels in senescent endothelial cells. Silencing Arg-II or p38a in senescent cells recouples eNOS and inhibits IL-6 and IL-8 secretion. Arg-II overexpression in young endothelial cells causes eNOS-uncoupling and enhances IL-6 and IL-8 expression, which is prevented by p38mapk inhibition. In mice, aging and HFD both enhanced Arg-II expression and p38mapk activation, which was associated with eNOS-uncoupling. In accordance, aging and obese mice revealed decreased endothelium-dependent relaxations to acetylcholine, whereas Arg-II-/- mice were protected from eNOS-uncoupling and endothelial dysfunction induced by aging or HFD, which was associated with reduced p38mapk activation. Furthermore, inhibition of p38mapk recouples eNOS in aortas from aging and obese mice. Conclusions: Arg-II causes eNOS-uncoupling through activation of p38mapk in aging and HFD-induced obesity.
EAS16-0923, VASCULAR BIOLOGY: ENDOTHELIUM AND SMOOTH MUSCLE CELLS. SOLUBLE ENDOGLIN EFFECTS ON ENDOTHELIAL CELLS IN VITRO M. Varejckova, I. Nemeckova, P. Fikrova, E. Dolezelova, B. Vitverova, P. Nachtigal. Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, Hradec Kralove, Czech Republic Objectives: Endoglin (Eng) is a transmembrane accessory type III receptor for the transforming growth factor-b (TGF-b) and its expression is upregulated in proliferating endothelial cells. Soluble form of endoglin (sEng) has been identified in plasma of patients with preeclampsia, atherosclerosis and other cardiovascular diseases. Hence, endothelial dysfunction plays important role in many cardiovascular pathologies, we decided to test, whether high soluble endoglin induces changes in the expression of markers of endothelial dysfunction, inflammation, oxidative stress and weather is sEng able to affect TGF-b signaling in human umbilical vein endothelial cells (HUVEC) in vitro. In addition, our plan was to test a possible effect of mutual exposure of sEng and inflammation (TNF-a). Methods: HUVEC were obtained from Lonza from CloneticsTM Laboratories. Cells were cultured in gelatin-coated flasks in EGM-2 medium. HUVEC were exposed to recombinant human sEng (50ng/ml or 500ng/ml) for 3 and 16 hours and/or TNF-a (10ng/ml). To see changes in the expression of mRNA of selected markers we used qRT-PCR. Results: Our results showed that only 500ng/ml concentration of sEng after 16 hours induced significant increase in mRNA expression of proinflammatory markers VCAM-1, ICAM-1, COX-2 and TGF-b family members Eng and BMPR2. Moreover, this effect was observed also after sEng and TNF-a co-treatment. Conclusions: In conclusion, our results showed that high concentration of soluble endoglin alone or in combination with inflammation affect endothelial cells with respect to pro-inflammatory markers. However, further study are needed, particularly on the protein level. This work was supported by grants GAUK 1158413C, GACR 15-24015S and SVV/2015/260185.
EAS16-0892, VASCULAR BIOLOGY: ENDOTHELIUM AND SMOOTH MUSCLE CELLS. THE RHOGEF TRIO REGULATES ANGIOGENIC SPROUTING THROUGH VECADHERIN J. Kroon 1, 2, I. Timmerman 2, M. Hoogenboezem 2, S.A. van de Pavert 3, E.M. Weijers 4, P. Koolwijk 4, J.D. van Buul 2. 1 Academic Medical CenterUniversity of Amsterdam, Vascular Medicine, Amsterdam, Netherlands; 2 Sanquin Research, Molecular Cell Biology, Amsterdam, Netherlands; 3 VU University Medical Center, Molecular Cell Biology and Immunology, Amsterdam, Netherlands; 4 VU University Medical Center, Physiology, Amsterdam, Netherlands Objectives: Blood vessels form tight networks that transport cells, nutrients, and fluids to all the organs of the body. Upon damage or occlusion, newly formed vessels sprout from pre-existing ones, a process known as angiogenesis. Also upon a growing plaque, new vessels are found in the intima, supporting further growth and ultimately leading to plaque rupture. Thus, understanding how these novel vessels grow is essential to target such unwanted sprouting. Endothelial cells that line the vessel wall are instrumental in angiogenesis. They start proliferating and migrating out of pre-existing vessels by remodelling their actin cytoskeleton. However, how endothelial cells coordinate this is not fully understood. Methods: Depleting Trio in in vitro sprouting assays or inhibiting Trio in developing intersegmental vessels of zebrafish in vivo showed the requirement of Trio for angiogenesis. Results: Interestingly, rescue experiments showed that global activation of Rac1 and RhoG by the N-terminal GEF domain of Trio was not sufficient to
Abstracts / Atherosclerosis 252 (2016) e1ee196
Objectives: Cocaine use is a risk factor for ischemic vascular complications. In vivo and in vitro studies indicate that cocaine induces endothelial dysfunction and accelerated atherosclerosis. In the brain, a mechanism by which cocaine exerts its effects is through the Sigma-1 receptor (Sigma1R), but the role of this receptor in systemic endothelial cells is unknown. Objective: to determine the participation of Sigma-1R in the pathogenesis of cocaine-associated cell damage in human umbilical cord endothelial cells (HUVEC). Methods: HUVEC were exposed to cocaine (10 mM) in the presence or absence of the Sigma-1R antagonist BD1047. Activation of RhoA/ROCK was determined by quantifying pMYPT/MYPT levels. Nitric Oxide (NO) was measured based on Griess reaction and procoagulant activity (PCA) by FXa generation. Exposure of von Willebrand factor (VWF) and number of platelets (PLTs) adhered to HUVEC were evaluated by immunofluorescence. Results: Cocaine increased RhoA/ROCK activity compared to controls and Sigma-1R inhibition prevented this activation (P<0.05). Cocaine decreased NO levels and BD1047 hampered this reduction (P<0.05). PCA in HUVEC increased in the presence of cocaine and PLTs, and BD1047 prevented this effect. Cocaine induced the exposure of VWF fibers on the surface of HUVEC, resulting in an increased number of adhered PLTs as compared to controls. Inhibition of Sigma-1R diminished both phenomena. Under flow, inhibition of Sigma-1R prevented adhesion of PLTs to human microvascular endothelial cells stimulated with cocaine. Conclusions: In systemic endothelial cells in vitro, Sigma-1R seems to play a role in cocaine-induced activation of RhoA/ROCK, VWF exposure, increased platelet adhesion, PCA and NO biodisponibility.
EAS16-0230, VASCULAR BIOLOGY: ENDOTHELIUM AND SMOOTH MUSCLE CELLS. P38 MITOGEN-ACTIVATED PROTEIN KINASE IS INVOVLED IN ARGINASEII-MEDIATED ENOS-UNCOUPLING IN AGING AND OBESITY Y. Yu 1, J.P. Montani 1, X. Ming 1, Z. Yang 1. 1 Fribourg University, Department of Medicine, Fribourg, Switzerland Objectives: Endothelial nitric oxide synthase (eNOS)-uncoupling links obesity-associated insulin resistance and aging to the increased cardiovascular disease. Given that arginase-II (Arg-II) and p38 mitogen-activated protein kinase (p38mapk) play important roles in vascular system, we investigated here whether p38mapk is involved in Arg-II-mediated eNOSuncoupling in aging and high-fat diet induced obesity model. Methods: Human umbilical vein endothelial cells were transduced with adenovirus overexpressing Arg-II or silencing p38a and Arg-II to detect eNOS-uncoupling and cytokines production. For the animal model, obesity was induced by high-fat diet (HFD, 55% fat) for 14 weeks. The female mice with 2-3 months old and 20- 24 months old were taken as young and old mice. Superoxide and NO production in aortas were detected by en face staining. Results: We demonstrate eNOS-uncoupling, augmented secretion of IL-6 and IL-8, elevation of p38mapk activation and Arg-II levels in senescent endothelial cells. Silencing Arg-II or p38a in senescent cells recouples eNOS and inhibits IL-6 and IL-8 secretion. Arg-II overexpression in young endothelial cells causes eNOS-uncoupling and enhances IL-6 and IL-8 expression, which is prevented by p38mapk inhibition. In mice, aging and HFD both enhanced Arg-II expression and p38mapk activation, which was associated with eNOS-uncoupling. In accordance, aging and obese mice revealed decreased endothelium-dependent relaxations to acetylcholine, whereas Arg-II-/- mice were protected from eNOS-uncoupling and endothelial dysfunction induced by aging or HFD, which was associated with reduced p38mapk activation. Furthermore, inhibition of p38mapk recouples eNOS in aortas from aging and obese mice. Conclusions: Arg-II causes eNOS-uncoupling through activation of p38mapk in aging and HFD-induced obesity.
EAS16-0923, VASCULAR BIOLOGY: ENDOTHELIUM AND SMOOTH MUSCLE CELLS. SOLUBLE ENDOGLIN EFFECTS ON ENDOTHELIAL CELLS IN VITRO M. Varejckova, I. Nemeckova, P. Fikrova, E. Dolezelova, B. Vitverova, P. Nachtigal. Faculty of Pharmacy in Hradec Kralove, Department of Biological and Medical Sciences, Hradec Kralove, Czech Republic Objectives: Endoglin (Eng) is a transmembrane accessory type III receptor for the transforming growth factor-b (TGF-b) and its expression is upregulated in proliferating endothelial cells. Soluble form of endoglin (sEng) has been identified in plasma of patients with preeclampsia, atherosclerosis and other cardiovascular diseases. Hence, endothelial dysfunction plays important role in many cardiovascular pathologies, we decided to test, whether high soluble endoglin induces changes in the expression of markers of endothelial dysfunction, inflammation, oxidative stress and weather is sEng able to affect TGF-b signaling in human umbilical vein endothelial cells (HUVEC) in vitro. In addition, our plan was to test a possible effect of mutual exposure of sEng and inflammation (TNF-a). Methods: HUVEC were obtained from Lonza from CloneticsTM Laboratories. Cells were cultured in gelatin-coated flasks in EGM-2 medium. HUVEC were exposed to recombinant human sEng (50ng/ml or 500ng/ml) for 3 and 16 hours and/or TNF-a (10ng/ml). To see changes in the expression of mRNA of selected markers we used qRT-PCR. Results: Our results showed that only 500ng/ml concentration of sEng after 16 hours induced significant increase in mRNA expression of proinflammatory markers VCAM-1, ICAM-1, COX-2 and TGF-b family members Eng and BMPR2. Moreover, this effect was observed also after sEng and TNF-a co-treatment. Conclusions: In conclusion, our results showed that high concentration of soluble endoglin alone or in combination with inflammation affect endothelial cells with respect to pro-inflammatory markers. However, further study are needed, particularly on the protein level. This work was supported by grants GAUK 1158413C, GACR 15-24015S and SVV/2015/260185.
EAS16-0892, VASCULAR BIOLOGY: ENDOTHELIUM AND SMOOTH MUSCLE CELLS. THE RHOGEF TRIO REGULATES ANGIOGENIC SPROUTING THROUGH VECADHERIN J. Kroon 1, 2, I. Timmerman 2, M. Hoogenboezem 2, S.A. van de Pavert 3, E.M. Weijers 4, P. Koolwijk 4, J.D. van Buul 2. 1 Academic Medical CenterUniversity of Amsterdam, Vascular Medicine, Amsterdam, Netherlands; 2 Sanquin Research, Molecular Cell Biology, Amsterdam, Netherlands; 3 VU University Medical Center, Molecular Cell Biology and Immunology, Amsterdam, Netherlands; 4 VU University Medical Center, Physiology, Amsterdam, Netherlands Objectives: Blood vessels form tight networks that transport cells, nutrients, and fluids to all the organs of the body. Upon damage or occlusion, newly formed vessels sprout from pre-existing ones, a process known as angiogenesis. Also upon a growing plaque, new vessels are found in the intima, supporting further growth and ultimately leading to plaque rupture. Thus, understanding how these novel vessels grow is essential to target such unwanted sprouting. Endothelial cells that line the vessel wall are instrumental in angiogenesis. They start proliferating and migrating out of pre-existing vessels by remodelling their actin cytoskeleton. However, how endothelial cells coordinate this is not fully understood. Methods: Depleting Trio in in vitro sprouting assays or inhibiting Trio in developing intersegmental vessels of zebrafish in vivo showed the requirement of Trio for angiogenesis. Results: Interestingly, rescue experiments showed that global activation of Rac1 and RhoG by the N-terminal GEF domain of Trio was not sufficient to