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Interest of mox-LDL to initiate resolution of inflammatory process by production of resolvin-D1
Abstracts / Atherosclerosis 263 (2017) e111ee282
intervention group were used to investigate the changes of autophagyrelated proteins such as LC3, beclin-1, p62 and LAMP1. To determine autophagy level, RFP- GFP-LC3 adenovirus transfection was used to detect the activity of autophagy. Foam cell formation was detected by oil red O staining. Total cholesterol content was measured by enzyme method to asses the foam cell formation. AMPK inhibitor compound C was used to inhibit the activity of AMPK, and the expression of Atg7 and LC3 was detected. The pharmacological inhibitor of autophagy 3-MA was also applied to confirm the role of autophagy in PDGF-BB intervention. Results: VSMC foam cell formation were impeded by PDGF-BB,and was associated with the up-regulation of autophagy. PDGF-BB increased the expression of p-AMPK and the AMPK specific inhibitor Compound C abolished PDGF-BB induced autophagy, 3-MA attenuated the inhibition of VSMC foam formation induced by PDGF-BB. Conclusions: This study demonstrates the role of autophagy in VSMC foam formation and highlights PDGF-BB as a protective target for atherosclerosis.
PO063. INTEREST OF MOX-LDL TO INITIATE RESOLUTION OF INFLAMMATORY PROCESS BY PRODUCTION OF RESOLVIN-D1 Damien Dufour1, Alia Khalil2, Alexandre Rousseau2, Caroline Noyon1, dric Delporte1, Jean Ne ve1, Luc Vanhamme3, Melissa Cortese1, Ce Karim Zouaoui Boudjeltia2, Pierre Van Antwerpen1. 1 Laboratory of Pharmaceutical Chemistry and Analytical Platform of the Faculty of Pharmacy, ULB, Bruxelles, Belgium; 2 Laboratory of Experimental Medicine, CHU-Charleroi A V esale, ISPPC, Montigny-le-Tilleul, Belgium; 3 Laboratory of Neurovascular Signaling, ULB, Gosselies, Belgium Aim: Low-density lipoprotein (LDL) oxidation is an important process in establishment and development of atherosclerosis. An important contribution to the LDL oxidation mechanism comes from myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and in monocytes. During inflammatory conditions, MPO can be released into the extracellular space , promoting oxidative stress and producing specifically oxidized LDL called Mox-LDL which exerts a proinflammatory response in the endothelium by activation of endothelial cells, monocytes/macrophages and induces the formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (PUFA) and which promotes resolution of inflammation at nanogramme level. Methods: In the present work, the synthesis of RvD1 and its precursors 17S-HDHA and DHA by endothelial cells were studied by LC/MSMS in the presence of several concentrations of Mox-LDL and were compared to nonphysiologic model of oxidized-LDL produced by copper (Ox-LDL). Results: Results showed an important contribution of Mox-LDL in the synthesis of RvD1 and 17S-HDHA from DHA in dose-dependent manner in comparison to results obtained by incubation of endothelial cells with OxLDL. Conclusions: These results suggest that if Mox-LDL were known to be proinflammatory and deleterious in the context of atherosclerosis, they are also able to induce a pro-resolution effect by induction of RvD1 from DHA by endothelial cells. Transfection of LOX-1 Si-RNA did not modulate RvD1 production from endothelial cells. However, antioxidant Trolox inhibits partially the RvD1 production suggesting an activation of this process by oxidative stress.
PO064. ALPHA-ISO-CUBEBENE ATTENUATES VSMC PROLIFERATION INDUCED BY PDGF VIA INHIBITION OF OSTEOPONTIN EXPRESSION Min A. Jang1, Seung Eun Baek1, Eun Jung Kim1, So Youn Park1, Young Whan Choi2, Chi Dae Kim1. 1 Department of Pharmacology, School of Medicine, Pusan National University, Yangsan, South Korea; 2 College of Natural Resources & Life Sciences, Pusan National University, Gyeongnam, South Korea
e131
Aim: a-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms. Thus, we examined the effect of ICB on vascular smooth muscle cell (VSMC) proliferation, a key feature of proliferative vascular diseases. Methods: VSMCs were primary cultured from rat thoracic aorta, and cell proliferation was measured by cell count and MTT assays. OPN expression in cells was determined by Western blot. Reporter and ChIP assays were used to investigate transcription factors. Results: Cell proliferation and OPN expression were up-regulated in PDGFstimulated VSMCs, which were attenuated by an OPN antibody. In aortic tissues exposed to PDGF, sprouting VSMC numbers increased, which was attenuated in OPN-deficient tissues. Furthermore, VSMC proliferation and OPN expression induced by PDGF were attenuated by ICB. Reporter assays showed that the promoter region 538-234 bp of the transcription start site was responsible for transcriptional activity enhancement by PDGF, which was significantly inhibited by ICB. Putative binding sites for AP-1 and C/ EBPb in the indicated promoter region were suggested by TF Search, and increased binding of AP-1 and C/EBPb in PDGF-treated VSMCs was demonstrated using a ChIP assay. The increased bindings of AP-1 and C/ EBPb into OPN promoter were attenuated by ICB. Moreover, the PDGFinduced expression of OPN was attenuated in VSMCs transfected with siRNA for AP-1 and C/EBPb. Conclusions: The results of present study indicate that ICB inhibit VSMC proliferation by inhibiting the AP-1 and C/EBPb signaling pathways and thus downregulating OPN expression.
PO065. INVOLVEMENT OF ATP-SENSITIVE POTASSIUM CHANNELS IN EFFECT OF PINACIDIL ON SAPHENOUS VEIN OBTAINED FROM THE PATIENTS WITH AND WITHOUT TYPE 2 DIABETES MELLITUS Jovana Rajkovic1, Miodrag Peric2, Jelena Stanisic3, Jelena Rakocevic4, Radmila Novakovic1, Vladimir Djokic1, Milica Labudovic-Borovic4, Snezana Tepavcevic3, Vladimir Zivanovic5, Vladimir Kanjuh6, Helmut Heinle7, Ljiljana Gojkovic-Bukarica1. 1 Institute of Pharmacology, Clinical Pharmacology and Toxicology, Department of Cardiovascular Pharmacology, Belgrade, Serbia; 2 Dedinje Cardiovascular Institute, Department of Cardiac Surgery, Belgrade, Serbia; 3 Vinca Institute of Nuclear Sciences, Department of Molecular Biology and Endocrinology, Belgrade, Serbia; 4 Institute of Histology and Embryology, Department of Cardiovascular Histology, Belgrade, Serbia; 5 University Clinical Hospital Center Dr Dragisa Misovic, Department of Cardiology, Belgrade, Serbia; 6 Serbian Academy of Sciences and Arts, Department of Medical Sciences, Belgrade, Serbia; 7 Institute of Physiology, Department of Physiology, Tuebingen, Germany Aim: Type 2 diabetes mellitus (T2DM) increases cardiovascular complications. It is possible that the diabetic vascular dysfunction is associated with the reduced activity of the different smooth muscle potassium (K+) channels. Thus, the objective of our study was to investigate the differences in the involvement of ATP-sensitive K+ channels (KATP) in the relaxant effect of pinacidil on the human saphenous vein (HSV) obtained from the patients with and without T2DM. Methods: The rings of HSV, without endothelium, obtained from the patients undergoing a coronary bypass surgery, were mounted in an organ bath system and an isometric tension was being recorded. The relaxation of HSV, precontracted with phenylephrine, was produced by pinacidil, a KATP channel opener. The expression of KATP subunits (Kir6.1, Kir6.2 and SUR-2B) was detected by immunohistochemistry and by western blot. Results: Pinacidil produces comparable effects on the HSV from the patients with and without T2DM. Glibenclamide, a selective blocker of KATP channels, antagonized pinacidil effects on the HSV from patients with and without T2DM (P < 0.001 vs. P < 0.05). All three types of KATP subunits are expressed on smooth muscle cells of HSV. While there are no differences in expression of Kir6.1 and Kir6.2, expression of SUR-2B is smaller in HSV from patients with T2DM (P < 0.05). Conclusions: Pinacidil produces comparable relaxation of HSV in patients with/without T2DM. According to the effect of glibenclamide and applied molecular analysis it is possible that diabetes is associated with the reduced expression of SUR-2B subunit in vascular smooth muscle of HSV.
intervention group were used to investigate the changes of autophagyrelated proteins such as LC3, beclin-1, p62 and LAMP1. To determine autophagy level, RFP- GFP-LC3 adenovirus transfection was used to detect the activity of autophagy. Foam cell formation was detected by oil red O staining. Total cholesterol content was measured by enzyme method to asses the foam cell formation. AMPK inhibitor compound C was used to inhibit the activity of AMPK, and the expression of Atg7 and LC3 was detected. The pharmacological inhibitor of autophagy 3-MA was also applied to confirm the role of autophagy in PDGF-BB intervention. Results: VSMC foam cell formation were impeded by PDGF-BB,and was associated with the up-regulation of autophagy. PDGF-BB increased the expression of p-AMPK and the AMPK specific inhibitor Compound C abolished PDGF-BB induced autophagy, 3-MA attenuated the inhibition of VSMC foam formation induced by PDGF-BB. Conclusions: This study demonstrates the role of autophagy in VSMC foam formation and highlights PDGF-BB as a protective target for atherosclerosis.
PO063. INTEREST OF MOX-LDL TO INITIATE RESOLUTION OF INFLAMMATORY PROCESS BY PRODUCTION OF RESOLVIN-D1 Damien Dufour1, Alia Khalil2, Alexandre Rousseau2, Caroline Noyon1, dric Delporte1, Jean Ne ve1, Luc Vanhamme3, Melissa Cortese1, Ce Karim Zouaoui Boudjeltia2, Pierre Van Antwerpen1. 1 Laboratory of Pharmaceutical Chemistry and Analytical Platform of the Faculty of Pharmacy, ULB, Bruxelles, Belgium; 2 Laboratory of Experimental Medicine, CHU-Charleroi A V esale, ISPPC, Montigny-le-Tilleul, Belgium; 3 Laboratory of Neurovascular Signaling, ULB, Gosselies, Belgium Aim: Low-density lipoprotein (LDL) oxidation is an important process in establishment and development of atherosclerosis. An important contribution to the LDL oxidation mechanism comes from myeloperoxidase (MPO), a heme enzyme present in azurophil granules of neutrophils and in monocytes. During inflammatory conditions, MPO can be released into the extracellular space , promoting oxidative stress and producing specifically oxidized LDL called Mox-LDL which exerts a proinflammatory response in the endothelium by activation of endothelial cells, monocytes/macrophages and induces the formation of foam cells. Resolvin D1 (RvD1) is a compound derived from the metabolism of docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid (PUFA) and which promotes resolution of inflammation at nanogramme level. Methods: In the present work, the synthesis of RvD1 and its precursors 17S-HDHA and DHA by endothelial cells were studied by LC/MSMS in the presence of several concentrations of Mox-LDL and were compared to nonphysiologic model of oxidized-LDL produced by copper (Ox-LDL). Results: Results showed an important contribution of Mox-LDL in the synthesis of RvD1 and 17S-HDHA from DHA in dose-dependent manner in comparison to results obtained by incubation of endothelial cells with OxLDL. Conclusions: These results suggest that if Mox-LDL were known to be proinflammatory and deleterious in the context of atherosclerosis, they are also able to induce a pro-resolution effect by induction of RvD1 from DHA by endothelial cells. Transfection of LOX-1 Si-RNA did not modulate RvD1 production from endothelial cells. However, antioxidant Trolox inhibits partially the RvD1 production suggesting an activation of this process by oxidative stress.
PO064. ALPHA-ISO-CUBEBENE ATTENUATES VSMC PROLIFERATION INDUCED BY PDGF VIA INHIBITION OF OSTEOPONTIN EXPRESSION Min A. Jang1, Seung Eun Baek1, Eun Jung Kim1, So Youn Park1, Young Whan Choi2, Chi Dae Kim1. 1 Department of Pharmacology, School of Medicine, Pusan National University, Yangsan, South Korea; 2 College of Natural Resources & Life Sciences, Pusan National University, Gyeongnam, South Korea
e131
Aim: a-Iso-cubebene (ICB) is a dibenzocyclooctadiene lignin contained in Schisandra chinensis (SC), a well-known medicinal herb that ameliorates cardiovascular symptoms. Thus, we examined the effect of ICB on vascular smooth muscle cell (VSMC) proliferation, a key feature of proliferative vascular diseases. Methods: VSMCs were primary cultured from rat thoracic aorta, and cell proliferation was measured by cell count and MTT assays. OPN expression in cells was determined by Western blot. Reporter and ChIP assays were used to investigate transcription factors. Results: Cell proliferation and OPN expression were up-regulated in PDGFstimulated VSMCs, which were attenuated by an OPN antibody. In aortic tissues exposed to PDGF, sprouting VSMC numbers increased, which was attenuated in OPN-deficient tissues. Furthermore, VSMC proliferation and OPN expression induced by PDGF were attenuated by ICB. Reporter assays showed that the promoter region 538-234 bp of the transcription start site was responsible for transcriptional activity enhancement by PDGF, which was significantly inhibited by ICB. Putative binding sites for AP-1 and C/ EBPb in the indicated promoter region were suggested by TF Search, and increased binding of AP-1 and C/EBPb in PDGF-treated VSMCs was demonstrated using a ChIP assay. The increased bindings of AP-1 and C/ EBPb into OPN promoter were attenuated by ICB. Moreover, the PDGFinduced expression of OPN was attenuated in VSMCs transfected with siRNA for AP-1 and C/EBPb. Conclusions: The results of present study indicate that ICB inhibit VSMC proliferation by inhibiting the AP-1 and C/EBPb signaling pathways and thus downregulating OPN expression.
PO065. INVOLVEMENT OF ATP-SENSITIVE POTASSIUM CHANNELS IN EFFECT OF PINACIDIL ON SAPHENOUS VEIN OBTAINED FROM THE PATIENTS WITH AND WITHOUT TYPE 2 DIABETES MELLITUS Jovana Rajkovic1, Miodrag Peric2, Jelena Stanisic3, Jelena Rakocevic4, Radmila Novakovic1, Vladimir Djokic1, Milica Labudovic-Borovic4, Snezana Tepavcevic3, Vladimir Zivanovic5, Vladimir Kanjuh6, Helmut Heinle7, Ljiljana Gojkovic-Bukarica1. 1 Institute of Pharmacology, Clinical Pharmacology and Toxicology, Department of Cardiovascular Pharmacology, Belgrade, Serbia; 2 Dedinje Cardiovascular Institute, Department of Cardiac Surgery, Belgrade, Serbia; 3 Vinca Institute of Nuclear Sciences, Department of Molecular Biology and Endocrinology, Belgrade, Serbia; 4 Institute of Histology and Embryology, Department of Cardiovascular Histology, Belgrade, Serbia; 5 University Clinical Hospital Center Dr Dragisa Misovic, Department of Cardiology, Belgrade, Serbia; 6 Serbian Academy of Sciences and Arts, Department of Medical Sciences, Belgrade, Serbia; 7 Institute of Physiology, Department of Physiology, Tuebingen, Germany Aim: Type 2 diabetes mellitus (T2DM) increases cardiovascular complications. It is possible that the diabetic vascular dysfunction is associated with the reduced activity of the different smooth muscle potassium (K+) channels. Thus, the objective of our study was to investigate the differences in the involvement of ATP-sensitive K+ channels (KATP) in the relaxant effect of pinacidil on the human saphenous vein (HSV) obtained from the patients with and without T2DM. Methods: The rings of HSV, without endothelium, obtained from the patients undergoing a coronary bypass surgery, were mounted in an organ bath system and an isometric tension was being recorded. The relaxation of HSV, precontracted with phenylephrine, was produced by pinacidil, a KATP channel opener. The expression of KATP subunits (Kir6.1, Kir6.2 and SUR-2B) was detected by immunohistochemistry and by western blot. Results: Pinacidil produces comparable effects on the HSV from the patients with and without T2DM. Glibenclamide, a selective blocker of KATP channels, antagonized pinacidil effects on the HSV from patients with and without T2DM (P < 0.001 vs. P < 0.05). All three types of KATP subunits are expressed on smooth muscle cells of HSV. While there are no differences in expression of Kir6.1 and Kir6.2, expression of SUR-2B is smaller in HSV from patients with T2DM (P < 0.05). Conclusions: Pinacidil produces comparable relaxation of HSV in patients with/without T2DM. According to the effect of glibenclamide and applied molecular analysis it is possible that diabetes is associated with the reduced expression of SUR-2B subunit in vascular smooth muscle of HSV.