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MS20 NATIVE AND GLYCOXIDIZED HIGH DENSITY LIPOPROTEIN (HDL) MODULATE ADRENAL STEROIDOGENESIS VIA SCAVENGER RECEPTOR CLASS B, TYPE I (SR-BI)
78th EAS Congress
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
Methods: 70 patients were included in research (19 patients with combined hyperlipidemia (HLP), 20 patients with hypertriglyceridemia (HTG), 31 patientscontrol group). 5 normal subjects and 5 patients with HTG were studied for their response to oral fat load. We assessed clinical and laboratory data. We studied ASP concentration in plasma. The lipid meal consisted 200ml of 20% cream mixed with 2 eggs. In experiments in vitro ASP was added to human and mouse fibroblasts in the presence 100 mkM [3 H] oleate. The triacylglycerol synthesis was determined over a concentration range of ASP (10−30 mkg/ml). Results: The subjects with combined HLP and HTG had increased TG level (3.21±0.81 mmol/l, P < 0.05; 3.94±3.16 mmol/l, P < 0.05 v.s. 1.57±0.41 mmol/l). There was no difference of ASP concentration between groups. Patients with c-DLP had increased total cholesterol (TC) level (7.48±0.94 mmol/l, P < 0.05; v.s. 5.26±0.82 mmol/l). There was no correlation between ASP and TG, TC, body mass index in all groups. After oral fat load the plasma triglyceride increased, but ASP level didn’t change significantly. In experiments in vitro ASP induced increase in triacylglycerol synthesis (14−36%, P < 0.05). Conclusions: ASP stimulates triacylglycerol synthesis in fibroblasts. But there is no difference in ASP level between groups of patients. We suppose, the further researches will explain the role of ASP in metabolism of TG. MS18 C-REACTIVE PROTEIN LEVEL AND CAROTID ARTERIES ATHEROSCLEROSIS IN ABDOMINAL OBESITY PATIENTS O. Belyaeva1 , T. Karonova1 , E. Bazhenova1 , E. Baranova1 , O. Berkovich1 , A. Berezina2 , N. Ananieva3 , V. Mandal3 , E. Shlyakhto1 . 1 Saint-Petersburg State Medical University n.a. I.P. Pavlov, 2 Federal Centre of Heart, Blood and Endocrinology n.a. V.A. Almazov, 3 Saint-Petersburg Medical Institute n.a. V.M. Bechterev, Saint Petersburg, Russia Objective: To assess the relationship of C-reactive protein (CRP) level and carotid arteries intima-media complex in the patients with abdominal obesity. Methods: We evaluated severity of atherosclerosis in 203 patients (30 to 55 years of age) with abdominal obesity (according to IDF criteria, 2005). The mean age was 45.9±0.5 years, waist circumference (WC) in men − 107.8±1.3 sm and in women − 98.7±0.9 sm. We used duplex scanning ALOKA SSD3500 (Russia) of common carotid arteries and measurement of serum lipids and CRP levels by standard methodology. Results: Mean intima-media complex was 1.28±0.42 mm with no significant difference between genders. Atherosclerotic plaques in common or/and internal carotid artery were revealed in 39.8%. The mean CRP level was 7.31±0.66 mg/L. It was found that WC correlated with early development of atherosclerosis in carotid arteries (r = 0.16, p = 0.009) as well as with the level of CRP (r = 0.32, p = 0.0001). We did not find correlations of atherosclerosis in carotid arteries with BMI. Positive correlations were revealed between CRP and intima-media complex thickness (r = 0.16, p = 0.02). Negative correlation was found between CRP and triglycerides serum level (r = 0.19, p = 0.0003). Conclusion: We recommend test of duplex scanning of carotid arteries in patients with abdominal obesity older than 30 years for evaluation of early signs of atherosclerosis. MS19 FUNCTIONAL ANALYSIS OF LDLR PROMOTER MUTATIONS ASSOCIATED WITH FAMILIAL HYPERCHOLESTEROLEMIA I. De Castro-Oros ´ 1 , L. Palacios2 , S. Pamp´ın3 , N. Plana4 , L. Masana4 , M. Stef2 , 3 ´ , M. Pocov´ı1 . 1 Dpto. Bioqu´ımica y Biolog´ıa F. Civeira5 , J.C. Rodriguez-Rey ´ de Ciencias Molecular y Celular, Universidad de Zaragoza. Instituto Aragones de la Salud (I+CS), Zaragoza, 2 Progenika Biopharma, S.A., Derio, Vizcaya, 3 Dpto. Biolog´ıa Molecular, Universidad de Cantabria, Santander, 4 Unitat de Medicina Vascular i Metabolisme, Universitat Rovira i Virgili, CIBERDEM, ´ Molecular, Hospital Reus, 5 Unidad de L´ıpidos. Laboratorio de Investigacion Universitario Miguel Servet, Zaragoza, Spain Introduction: Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in LDLR gene affecting the protein functionality. Several mutations have been described in the response element within LDLR promoter, and associated with FH phenotype. Material and Methods: The analysis by Lipochip® platform (Progenika Biopharma) for searching PCSK9, APOB and LDLR mutations showed that 4 patients affected of FH, were carriers as heterozygous of non-described mutations in LDLR promoter: c.(−208)A>T, c.(−140)C>T, c.(−136)C>G and c.(−155)_(−150)delACCCC(−155)_(−154)insTTCTGCAAACTCCTCCC. In silico assays with MatInspector (Genomatix) and Electrophoretic Mobility Assays (EMSA) were accomplished to determine the molecular effect of these mutations in LDLR transcriptional activity. Furthermore, a fragment of 278 bp from RLDL promoter, from −227 to +51, was amplified from DNA of individuals with the subsequent mutations in heterozygosis. Each PCR product, carrier of one mutation, was cloned into a luciferase reporter gene vector pGL3-Basic and transfected into HepG2 using Lipofectamine 2000™ (Invitrogen). Luciferase assays were performed using Dual-Luciferase® Reporter Assay (Promega) in triplicate.
113
Results: In silico and EMSA experiments showed a lower transcription factor binding when c.(−140)C>T, c.(−136)C>G, and c.(−155)_(−150)delACCCC(−155) _(−154)insTTCTGCAAACTCCTCCC mutations were present. Moreover, luciferase assays prove a significant reduction of 94%, 95% and 78%, respectively in LDLR transcriptional activity. These mutations are located in the response elements named as repeat 1 and 2 in the LDLR promoter. No significant differences were observed for the c.(−208)A>T mutation. Conclusion: It is important to analyze the functionality for mutations being in LDLR promoter to perform a correct diagnosis of FH. MS20 NATIVE AND GLYCOXIDIZED HIGH DENSITY LIPOPROTEIN (HDL) MODULATE ADRENAL STEROIDOGENESIS VIA SCAVENGER RECEPTOR CLASS B, TYPE I (SR-BI) S. Kopprasch1 , S. Saha1 , J. Graessler1 , J. Pietzsch2 , S.R. Bornstein1 . 1 Department of Internal Medicine 3, C.G.Carus Medical School, University of Technology Dresden, 2 Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Dresden-Rossendorf, Dresden, Germany Objective: Overactivity of the renin–angiotensin–aldosterone system (RAAS) has been causally related to the pathogenesis of type 2 diabetes and its adverse cardiovascular consequences. Since HDL-derived cholesteryl ester serves as a major source of adrenal hormone synthesis and, moreover, systemic high glucose level may affect lipoprotein structure and function, we investigated the effect of diabetic HDL modification on adrenocortical hormone synthesis. Methods: HDL isolated from blood of healthy volunteers were glycoxidized for 6 days in the presence of 200 mmoL/L glucose. Human adrenocortical cells (H295R) were cultured in DMEM/F12 medium and incubated with native (natHDL) or modified HDL (glycoxHDL). Aldosterone, released in the medium, was measured by RIA. Adrenal SR-BI expression was determined by quantitative RT-PCR and densitometric analysis of Western blotting. Results: HDL glycoxidation resulted in a 6.4-fold increase of TBARS content (p < 0.001) with only minor changes of the protein moiety as estimated by protein carbonyl levels and relative fluorescence (365/430 nm). Incubation of H295R cells with both natHDL and glycoxHDL (1 to 100 mg/mL) was followed by a significant dose-dependent enhancement of aldosterone secretion. GlycoxHDL-induced aldosterone release was blocked to 50% by the highly specific SR-BI inhibitor BLT-1 (p < 0.001). Native and modified HDL induced an increase in adrenocortical SR-BI mRNA and protein expression levels with glycoxHDL having greater impact. Conclusion: HDL-mediated increase in adrenal aldosterone production is at least partially mediated by SR-BI. MS21 COPPER AND MYELOPEROXIDASE-MODIFIED LDLS ACTIVATE NRF2 THROUGH DIFFERENT PATHWAYS OF ROS PRODUCTION IN MACROPHAGES D. Calay1 , A. Rousseau2 , L. Mattart1 , V. Nuyens2 , P. Van Antwerpen3 , N. Moguilevsky4 , K. Zouaoui Boudjeltia2 , M. Raes1 . 1 Research Unit in Cellular Biology (URBC), University of Namur (FUNDP), Namur, 2 Experimental Medicine Laboratory, ULB, CHU Charleroi, Montigny-le-Tilleul, 3 Laboratory of Pharmaceutical Chemistry, Universite Libre de Bruxelles, Brussel, 4 Technology Transfer Office, University of Namur (FUNDP), Namur, Belgium Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2. Interestingly, MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROSproducing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cPLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggests that results obtained until now with copper oxidized-LDLs should be carefully reevaluated taking into consideration physiologically more relevant oxidized LDLs.
Atherosclerosis Supplements 11, no. 2 (2010) 109–222
Methods: 70 patients were included in research (19 patients with combined hyperlipidemia (HLP), 20 patients with hypertriglyceridemia (HTG), 31 patientscontrol group). 5 normal subjects and 5 patients with HTG were studied for their response to oral fat load. We assessed clinical and laboratory data. We studied ASP concentration in plasma. The lipid meal consisted 200ml of 20% cream mixed with 2 eggs. In experiments in vitro ASP was added to human and mouse fibroblasts in the presence 100 mkM [3 H] oleate. The triacylglycerol synthesis was determined over a concentration range of ASP (10−30 mkg/ml). Results: The subjects with combined HLP and HTG had increased TG level (3.21±0.81 mmol/l, P < 0.05; 3.94±3.16 mmol/l, P < 0.05 v.s. 1.57±0.41 mmol/l). There was no difference of ASP concentration between groups. Patients with c-DLP had increased total cholesterol (TC) level (7.48±0.94 mmol/l, P < 0.05; v.s. 5.26±0.82 mmol/l). There was no correlation between ASP and TG, TC, body mass index in all groups. After oral fat load the plasma triglyceride increased, but ASP level didn’t change significantly. In experiments in vitro ASP induced increase in triacylglycerol synthesis (14−36%, P < 0.05). Conclusions: ASP stimulates triacylglycerol synthesis in fibroblasts. But there is no difference in ASP level between groups of patients. We suppose, the further researches will explain the role of ASP in metabolism of TG. MS18 C-REACTIVE PROTEIN LEVEL AND CAROTID ARTERIES ATHEROSCLEROSIS IN ABDOMINAL OBESITY PATIENTS O. Belyaeva1 , T. Karonova1 , E. Bazhenova1 , E. Baranova1 , O. Berkovich1 , A. Berezina2 , N. Ananieva3 , V. Mandal3 , E. Shlyakhto1 . 1 Saint-Petersburg State Medical University n.a. I.P. Pavlov, 2 Federal Centre of Heart, Blood and Endocrinology n.a. V.A. Almazov, 3 Saint-Petersburg Medical Institute n.a. V.M. Bechterev, Saint Petersburg, Russia Objective: To assess the relationship of C-reactive protein (CRP) level and carotid arteries intima-media complex in the patients with abdominal obesity. Methods: We evaluated severity of atherosclerosis in 203 patients (30 to 55 years of age) with abdominal obesity (according to IDF criteria, 2005). The mean age was 45.9±0.5 years, waist circumference (WC) in men − 107.8±1.3 sm and in women − 98.7±0.9 sm. We used duplex scanning ALOKA SSD3500 (Russia) of common carotid arteries and measurement of serum lipids and CRP levels by standard methodology. Results: Mean intima-media complex was 1.28±0.42 mm with no significant difference between genders. Atherosclerotic plaques in common or/and internal carotid artery were revealed in 39.8%. The mean CRP level was 7.31±0.66 mg/L. It was found that WC correlated with early development of atherosclerosis in carotid arteries (r = 0.16, p = 0.009) as well as with the level of CRP (r = 0.32, p = 0.0001). We did not find correlations of atherosclerosis in carotid arteries with BMI. Positive correlations were revealed between CRP and intima-media complex thickness (r = 0.16, p = 0.02). Negative correlation was found between CRP and triglycerides serum level (r = 0.19, p = 0.0003). Conclusion: We recommend test of duplex scanning of carotid arteries in patients with abdominal obesity older than 30 years for evaluation of early signs of atherosclerosis. MS19 FUNCTIONAL ANALYSIS OF LDLR PROMOTER MUTATIONS ASSOCIATED WITH FAMILIAL HYPERCHOLESTEROLEMIA I. De Castro-Oros ´ 1 , L. Palacios2 , S. Pamp´ın3 , N. Plana4 , L. Masana4 , M. Stef2 , 3 ´ , M. Pocov´ı1 . 1 Dpto. Bioqu´ımica y Biolog´ıa F. Civeira5 , J.C. Rodriguez-Rey ´ de Ciencias Molecular y Celular, Universidad de Zaragoza. Instituto Aragones de la Salud (I+CS), Zaragoza, 2 Progenika Biopharma, S.A., Derio, Vizcaya, 3 Dpto. Biolog´ıa Molecular, Universidad de Cantabria, Santander, 4 Unitat de Medicina Vascular i Metabolisme, Universitat Rovira i Virgili, CIBERDEM, ´ Molecular, Hospital Reus, 5 Unidad de L´ıpidos. Laboratorio de Investigacion Universitario Miguel Servet, Zaragoza, Spain Introduction: Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in LDLR gene affecting the protein functionality. Several mutations have been described in the response element within LDLR promoter, and associated with FH phenotype. Material and Methods: The analysis by Lipochip® platform (Progenika Biopharma) for searching PCSK9, APOB and LDLR mutations showed that 4 patients affected of FH, were carriers as heterozygous of non-described mutations in LDLR promoter: c.(−208)A>T, c.(−140)C>T, c.(−136)C>G and c.(−155)_(−150)delACCCC(−155)_(−154)insTTCTGCAAACTCCTCCC. In silico assays with MatInspector (Genomatix) and Electrophoretic Mobility Assays (EMSA) were accomplished to determine the molecular effect of these mutations in LDLR transcriptional activity. Furthermore, a fragment of 278 bp from RLDL promoter, from −227 to +51, was amplified from DNA of individuals with the subsequent mutations in heterozygosis. Each PCR product, carrier of one mutation, was cloned into a luciferase reporter gene vector pGL3-Basic and transfected into HepG2 using Lipofectamine 2000™ (Invitrogen). Luciferase assays were performed using Dual-Luciferase® Reporter Assay (Promega) in triplicate.
113
Results: In silico and EMSA experiments showed a lower transcription factor binding when c.(−140)C>T, c.(−136)C>G, and c.(−155)_(−150)delACCCC(−155) _(−154)insTTCTGCAAACTCCTCCC mutations were present. Moreover, luciferase assays prove a significant reduction of 94%, 95% and 78%, respectively in LDLR transcriptional activity. These mutations are located in the response elements named as repeat 1 and 2 in the LDLR promoter. No significant differences were observed for the c.(−208)A>T mutation. Conclusion: It is important to analyze the functionality for mutations being in LDLR promoter to perform a correct diagnosis of FH. MS20 NATIVE AND GLYCOXIDIZED HIGH DENSITY LIPOPROTEIN (HDL) MODULATE ADRENAL STEROIDOGENESIS VIA SCAVENGER RECEPTOR CLASS B, TYPE I (SR-BI) S. Kopprasch1 , S. Saha1 , J. Graessler1 , J. Pietzsch2 , S.R. Bornstein1 . 1 Department of Internal Medicine 3, C.G.Carus Medical School, University of Technology Dresden, 2 Department of Radiopharmaceutical Biology, Institute of Radiopharmacy, Research Center Dresden-Rossendorf, Dresden, Germany Objective: Overactivity of the renin–angiotensin–aldosterone system (RAAS) has been causally related to the pathogenesis of type 2 diabetes and its adverse cardiovascular consequences. Since HDL-derived cholesteryl ester serves as a major source of adrenal hormone synthesis and, moreover, systemic high glucose level may affect lipoprotein structure and function, we investigated the effect of diabetic HDL modification on adrenocortical hormone synthesis. Methods: HDL isolated from blood of healthy volunteers were glycoxidized for 6 days in the presence of 200 mmoL/L glucose. Human adrenocortical cells (H295R) were cultured in DMEM/F12 medium and incubated with native (natHDL) or modified HDL (glycoxHDL). Aldosterone, released in the medium, was measured by RIA. Adrenal SR-BI expression was determined by quantitative RT-PCR and densitometric analysis of Western blotting. Results: HDL glycoxidation resulted in a 6.4-fold increase of TBARS content (p < 0.001) with only minor changes of the protein moiety as estimated by protein carbonyl levels and relative fluorescence (365/430 nm). Incubation of H295R cells with both natHDL and glycoxHDL (1 to 100 mg/mL) was followed by a significant dose-dependent enhancement of aldosterone secretion. GlycoxHDL-induced aldosterone release was blocked to 50% by the highly specific SR-BI inhibitor BLT-1 (p < 0.001). Native and modified HDL induced an increase in adrenocortical SR-BI mRNA and protein expression levels with glycoxHDL having greater impact. Conclusion: HDL-mediated increase in adrenal aldosterone production is at least partially mediated by SR-BI. MS21 COPPER AND MYELOPEROXIDASE-MODIFIED LDLS ACTIVATE NRF2 THROUGH DIFFERENT PATHWAYS OF ROS PRODUCTION IN MACROPHAGES D. Calay1 , A. Rousseau2 , L. Mattart1 , V. Nuyens2 , P. Van Antwerpen3 , N. Moguilevsky4 , K. Zouaoui Boudjeltia2 , M. Raes1 . 1 Research Unit in Cellular Biology (URBC), University of Namur (FUNDP), Namur, 2 Experimental Medicine Laboratory, ULB, CHU Charleroi, Montigny-le-Tilleul, 3 Laboratory of Pharmaceutical Chemistry, Universite Libre de Bruxelles, Brussel, 4 Technology Transfer Office, University of Namur (FUNDP), Namur, Belgium Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages. Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2. Interestingly, MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROSproducing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cPLA2. This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggests that results obtained until now with copper oxidized-LDLs should be carefully reevaluated taking into consideration physiologically more relevant oxidized LDLs.