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Mechanisms of apolipoprotein e gene regulation in macrophages
Abstracts / Atherosclerosis 235 (2014) e84–e191
Objectives: Hypertension is one of major risk factors for atherosclerosis, which is characterized by the internalization of lipids into the arteries mediated by receptors such as LRP1. Our objective was to determine the relationship between ITM and HTA with the expression and the polymorphisms of LRP1 gene. Methods: We analyzed 71 subjects (42 controls and 29 with essential hypertension), in all subjects were taken lipid profile and LRP1 expression by monocytes culture through RT- PCR. In addition, 5 polymorphisms of the LRP1 gene (rs11172113, rs1466535, rs1800194, rs1140648 and rs1800164) were analyzed. Results: Our preliminary results shown a significant difference in the LRP1 expression in hypertensive subjects compared to the control group (P¼0.015). Also, a correlation between the LRP1 expression and IMT (P¼0.020) in the hypertensive group. The analysis between the genotype and allele frequencies of our control relating to the hypertensive group shown a significant association with the polymorphism C.67+4469T>C (rs11172113) in the co-dominant model TT vs CC (OR¼8.14; 95%CI 1.31-50.25; P¼0.042), and c.9783 G>A (rs1140648) in GG vs AA (OR ¼ 0.107; 95%CI 0.018-0.604; P¼0.02). Analyzing the frequencies in relation to clinical parameters, in the hypertensive group was found significant differences in c.67+4469T>C (rs11172113) (CT vs TT, P¼ 0.043); and c.3546C>T (rs1800194) (CT vs CC, P¼0.034) with GIM and only was found significant differences in c.191687G>A (rs1466535) with the LRP1 expression (CC vs. CT P¼0.037). Conclusion: In the c.67+4469 T>C (rs11172113) polymorphism, the C allele could be acting as a protector for hypertension, whereas c.9783G>A (rs1140648) polymorphism, the allele A could be associated with an increased risk of hypertension. 17 - Gene expression EAS-0471. MECHANISMS OF MACROPHAGES
APOLIPOPROTEIN
E
GENE
REGULATION
IN
V. Truscaa, D. Kardassisb, M. Simionescua, A. Gafencua a Genomics Transcriptomics and Molecular Therapies, Institute of Cellular Biology and Pathology " N. Simionescu", Bucharest, Romania; b Mammalian Gene Expression Group, Univ. Crete Medical School, Heraklion, Greece
Objectives: The atheroprotective role of apolipoprotein E (apoE) is well established, but during inflammation, the apoE expression in macrophages is reduced. The aim of the study is to determine the signaling pathways involved in the repression of apoE gene in response to lipopolysaccharide (LPS) treatment, and to reveal other regulatory mechanisms that increase the apoE expression in macrophages. Methods: ApoE proximal promoter (-500/+73) or its deletion mutants and the distal multienhancer 2 (ME.2) or its deletion mutants were cloned alone or together in pGL3 basic vector. Transient transfections were performed by calcium phosphate precipitation. Chromosome conformational capture (3C) experiments were carried out on human normal or PMAdifferentiated monocytes, using PstI restriction enzyme. DNA binding assays were performed using biotinylated DNA fragments of the apoE promoter and ME.2 and the nuclear extract obtained from macrophages. Results: The data showed that Tpl-2 and MEKK1 kinases are primarily responsible for the downregulation of apoE promoter activity by LPS. These kinases activate NF-kB and AP-1 that act on the -55/+73apoE promoter as well as on the ME.2. Transient transfections and 3C assays demonstrated that the ME.2 interacts with the apoE promoter, in a macrophage-specific manner. Thus, the transcription factors that bind on the ME.2 can modulate the apoE gene expression in macrophages. Our data showed that STAT1 binds to the 174-182 region of ME.2 and RXRa binds at the 405-420 fragment of ME.2 and both specifically upregulate the apoE gene expression in macrophages. In addition, we revealed that STAT1 can physically interact with RXRa. Conclusion: Taken together, the data show that in macrophages apoE is downregulated by the inflammatory stress, but the expression can be specifically upregulated by some transcription factors that bind on the ME.2. This can be used for the future therapies of atherosclerosis, to avoid hypertriglyceridemia induced by systemic overexpression of apoE.
e133
17 - Gene expression EAS-0659. GENOTYPE PECULIARITIES AND PROGNOSIS DEPENDING ON APOLIPOPROTEIN A LEVEL IN PATIENTS WITH HEART FAILURE WHO SUFFERED ACUTE CORONARY SYNDROME N. Koziolova, E. Polyanskaya Internal diseases, Perm State Medical Academy, Perm, Russia Objectives: to evaluate the course of heart failure (HF) and peculiarities of genotype in CHD patients who suffered acute coronary syndrome (ACS) depending on apolipoprotein A level (ApoA) in debut. Methods: 87 patients with ACS and HF underwent a two-stage examination. A single-step clinical examination was performed at the first stage during the first three days beginning from ACS development. At this very stage medical history, clinical symptoms, ApoB level, lipid spectrum, genotypes of fibrinogen, apoB and protein C genes were evaluated. Also, HF severity was evaluated through determining NTproBNP increase and myocardial collagenolisis was evaluated through determining TIMP-1. The second stage- two-year monitoring with endpoints recording: cardio-vascular death, new ACS, and CHF worsening. Results: patients with a high ApoA level>105mg/dL made up 66.7%(STEMI–35,7%, NSTEMI-55,4%, UA–8,9%). Patients with a low ApoA level made up 33,3%(STEMI–42,9%, NSTEMI–39,3%, UA–17,9%). In patients with low ApoA the ApoB/ApoA index was significantly higher, than in group with high ApoA level: 1,150,15vs0,930,11(?<0,001). Frequency of poor-prognostic ApoB/ApoA index>1.1 was significantly higher in low ApoA:(82.14% vs66.07%,?¼0,002). Myocardial stress by NT-proBNP was more intensive in low ApoA: 943,66362,14pg/mL vs 726,04283,58pg/ mL,?¼0,003. There was more intensive collagenolisis (according to lower TIMP-I) in low ApoA group: 621,805,22 vs 634,22,75(?<0,001).Unfavourable TT allele of ApoB gene isolated and combined with unfavourable A allele of fibrinogen gene and CC allele of protein C gene were found more often in patients with high ApoB (?? allele:35,6%vs11,6%,?¼0,057, combination:42,2%vs11,6%,?¼0,024). Prognosis was almost equal in both groups(14,29%of unfavourable prognosis vs25,00%,?¼0,521). ApoB/ApoA appeared to be more valuable in terms of prognosis: endpoints had76.2% with the level of >1,1and 20.7%with the level of<1,1,?¼0,002. Conclusion: low ApoA in ACS debut in HF patients is associated with more severe HF and presence of unfavourable genes’ alleles in genotype affecting the prognosis after ACS. ApoB/ApoA of>1,1, but not ApoA level appeared to be more valuable in terms of prognosis. 17 - Gene expression EAS-0733. GENE-EXPRESSION PROFILING OF LYMPH NODES REVEALS THAT APOAI DEFICIENCY IN APOE-KO MICE INDUCES A DRAMATIC ACTIVATION OF THE IMMUNE RESPONSE C. Parolinia, S. Manzinia, M. Busnellia, M. Chiarab, F. Delleraa, G.S. Ganzettia, C.R. Sirtoria, D.S. Hornerb, G. Chiesaa a Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy; b Department of Biosciences, Università degli Studi di Milano, Milan, Italy
Objectives: Systems biology approach, integrating data from large-scale measurements, could contribute to decipher the complex interplay among thousands of molecules in multiple interacting cells. Preliminary data showed that apoA-I deficiency in apoE-KO mice worsens atherosclerosis development at least in part, by modulating the inflammatory response mediated by CD4+ T lymphocytes. Based on these results, in the present study, transcriptomic analysis was carried out to identify novel candidate genes/pathways influenced by apoA-I deficiency in lymph nodes from athero-prone apoE-KO mice.
Objectives: Hypertension is one of major risk factors for atherosclerosis, which is characterized by the internalization of lipids into the arteries mediated by receptors such as LRP1. Our objective was to determine the relationship between ITM and HTA with the expression and the polymorphisms of LRP1 gene. Methods: We analyzed 71 subjects (42 controls and 29 with essential hypertension), in all subjects were taken lipid profile and LRP1 expression by monocytes culture through RT- PCR. In addition, 5 polymorphisms of the LRP1 gene (rs11172113, rs1466535, rs1800194, rs1140648 and rs1800164) were analyzed. Results: Our preliminary results shown a significant difference in the LRP1 expression in hypertensive subjects compared to the control group (P¼0.015). Also, a correlation between the LRP1 expression and IMT (P¼0.020) in the hypertensive group. The analysis between the genotype and allele frequencies of our control relating to the hypertensive group shown a significant association with the polymorphism C.67+4469T>C (rs11172113) in the co-dominant model TT vs CC (OR¼8.14; 95%CI 1.31-50.25; P¼0.042), and c.9783 G>A (rs1140648) in GG vs AA (OR ¼ 0.107; 95%CI 0.018-0.604; P¼0.02). Analyzing the frequencies in relation to clinical parameters, in the hypertensive group was found significant differences in c.67+4469T>C (rs11172113) (CT vs TT, P¼ 0.043); and c.3546C>T (rs1800194) (CT vs CC, P¼0.034) with GIM and only was found significant differences in c.191687G>A (rs1466535) with the LRP1 expression (CC vs. CT P¼0.037). Conclusion: In the c.67+4469 T>C (rs11172113) polymorphism, the C allele could be acting as a protector for hypertension, whereas c.9783G>A (rs1140648) polymorphism, the allele A could be associated with an increased risk of hypertension. 17 - Gene expression EAS-0471. MECHANISMS OF MACROPHAGES
APOLIPOPROTEIN
E
GENE
REGULATION
IN
V. Truscaa, D. Kardassisb, M. Simionescua, A. Gafencua a Genomics Transcriptomics and Molecular Therapies, Institute of Cellular Biology and Pathology " N. Simionescu", Bucharest, Romania; b Mammalian Gene Expression Group, Univ. Crete Medical School, Heraklion, Greece
Objectives: The atheroprotective role of apolipoprotein E (apoE) is well established, but during inflammation, the apoE expression in macrophages is reduced. The aim of the study is to determine the signaling pathways involved in the repression of apoE gene in response to lipopolysaccharide (LPS) treatment, and to reveal other regulatory mechanisms that increase the apoE expression in macrophages. Methods: ApoE proximal promoter (-500/+73) or its deletion mutants and the distal multienhancer 2 (ME.2) or its deletion mutants were cloned alone or together in pGL3 basic vector. Transient transfections were performed by calcium phosphate precipitation. Chromosome conformational capture (3C) experiments were carried out on human normal or PMAdifferentiated monocytes, using PstI restriction enzyme. DNA binding assays were performed using biotinylated DNA fragments of the apoE promoter and ME.2 and the nuclear extract obtained from macrophages. Results: The data showed that Tpl-2 and MEKK1 kinases are primarily responsible for the downregulation of apoE promoter activity by LPS. These kinases activate NF-kB and AP-1 that act on the -55/+73apoE promoter as well as on the ME.2. Transient transfections and 3C assays demonstrated that the ME.2 interacts with the apoE promoter, in a macrophage-specific manner. Thus, the transcription factors that bind on the ME.2 can modulate the apoE gene expression in macrophages. Our data showed that STAT1 binds to the 174-182 region of ME.2 and RXRa binds at the 405-420 fragment of ME.2 and both specifically upregulate the apoE gene expression in macrophages. In addition, we revealed that STAT1 can physically interact with RXRa. Conclusion: Taken together, the data show that in macrophages apoE is downregulated by the inflammatory stress, but the expression can be specifically upregulated by some transcription factors that bind on the ME.2. This can be used for the future therapies of atherosclerosis, to avoid hypertriglyceridemia induced by systemic overexpression of apoE.
e133
17 - Gene expression EAS-0659. GENOTYPE PECULIARITIES AND PROGNOSIS DEPENDING ON APOLIPOPROTEIN A LEVEL IN PATIENTS WITH HEART FAILURE WHO SUFFERED ACUTE CORONARY SYNDROME N. Koziolova, E. Polyanskaya Internal diseases, Perm State Medical Academy, Perm, Russia Objectives: to evaluate the course of heart failure (HF) and peculiarities of genotype in CHD patients who suffered acute coronary syndrome (ACS) depending on apolipoprotein A level (ApoA) in debut. Methods: 87 patients with ACS and HF underwent a two-stage examination. A single-step clinical examination was performed at the first stage during the first three days beginning from ACS development. At this very stage medical history, clinical symptoms, ApoB level, lipid spectrum, genotypes of fibrinogen, apoB and protein C genes were evaluated. Also, HF severity was evaluated through determining NTproBNP increase and myocardial collagenolisis was evaluated through determining TIMP-1. The second stage- two-year monitoring with endpoints recording: cardio-vascular death, new ACS, and CHF worsening. Results: patients with a high ApoA level>105mg/dL made up 66.7%(STEMI–35,7%, NSTEMI-55,4%, UA–8,9%). Patients with a low ApoA level made up 33,3%(STEMI–42,9%, NSTEMI–39,3%, UA–17,9%). In patients with low ApoA the ApoB/ApoA index was significantly higher, than in group with high ApoA level: 1,150,15vs0,930,11(?<0,001). Frequency of poor-prognostic ApoB/ApoA index>1.1 was significantly higher in low ApoA:(82.14% vs66.07%,?¼0,002). Myocardial stress by NT-proBNP was more intensive in low ApoA: 943,66362,14pg/mL vs 726,04283,58pg/ mL,?¼0,003. There was more intensive collagenolisis (according to lower TIMP-I) in low ApoA group: 621,805,22 vs 634,22,75(?<0,001).Unfavourable TT allele of ApoB gene isolated and combined with unfavourable A allele of fibrinogen gene and CC allele of protein C gene were found more often in patients with high ApoB (?? allele:35,6%vs11,6%,?¼0,057, combination:42,2%vs11,6%,?¼0,024). Prognosis was almost equal in both groups(14,29%of unfavourable prognosis vs25,00%,?¼0,521). ApoB/ApoA appeared to be more valuable in terms of prognosis: endpoints had76.2% with the level of >1,1and 20.7%with the level of<1,1,?¼0,002. Conclusion: low ApoA in ACS debut in HF patients is associated with more severe HF and presence of unfavourable genes’ alleles in genotype affecting the prognosis after ACS. ApoB/ApoA of>1,1, but not ApoA level appeared to be more valuable in terms of prognosis. 17 - Gene expression EAS-0733. GENE-EXPRESSION PROFILING OF LYMPH NODES REVEALS THAT APOAI DEFICIENCY IN APOE-KO MICE INDUCES A DRAMATIC ACTIVATION OF THE IMMUNE RESPONSE C. Parolinia, S. Manzinia, M. Busnellia, M. Chiarab, F. Delleraa, G.S. Ganzettia, C.R. Sirtoria, D.S. Hornerb, G. Chiesaa a Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Milan, Italy; b Department of Biosciences, Università degli Studi di Milano, Milan, Italy
Objectives: Systems biology approach, integrating data from large-scale measurements, could contribute to decipher the complex interplay among thousands of molecules in multiple interacting cells. Preliminary data showed that apoA-I deficiency in apoE-KO mice worsens atherosclerosis development at least in part, by modulating the inflammatory response mediated by CD4+ T lymphocytes. Based on these results, in the present study, transcriptomic analysis was carried out to identify novel candidate genes/pathways influenced by apoA-I deficiency in lymph nodes from athero-prone apoE-KO mice.