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Electronegative LDL (LDL(-)) induces IL-1b release in monocytes and macrophages through caspase-1 activation
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Abstracts / Atherosclerosis 241 (2015) e72ee148
From 749 differentially expressed transcripts, Gene Ontology analysis identified the LXR pathway as the most overrepresented; and up-regulation of LXRa,b and RXRa,b and downstream genes was confirmed by qRTPCR. Moreover, we demonstrated increased nuclear LXR translocation in FCMs from sponges or atherosclerotic plaques, consistent with recent data by (Spann NJ, Cell. 2012;151:138-52). However, we found no change markers for macrophage M1/M2 polarization, implying that foam cell formation in itself is neither pro- nor anti-inflammatory. The second most over-represented pathway was Fibrosis. Most notably, the pro-collagen peptidase, BMP1, and several collagen polypeptide and proteoglycan genes were increased in FCMs, as were connective tissue growth factor and pro-fibrotic signalling molecules of the Fos and Jun families. All the changes were confirmed by qRT-PCR. Moreover, we showed increased production of matrix proteins, CTGF, and phosphorylation of SMAD2, a major transcription factor mediating fibrosis, in FCMs from sponges and plaques. Our results indicate, somewhat paradoxically, that foam cell formation in vivo is pro-fibrotic, implying that it helps to stabilize plaques. EAS-0680. ELECTRONEGATIVE LDL (LDL(-)) INDUCES IL-1B RELEASE IN MONOCYTES AND MACROPHAGES THROUGH CASPASE-1 ACTIVATION €ki 2, M. Moya 1, J. Sa nchez-Quesada 1, J. M. Estruch-Alrich 1, *, K. Rajama €o € rni 2, S. Benitez 1. 1 Biochemistry, n ~ ez-Llanos 1, P.T. Kovanen 2, K. O Ordo Institut de Recerca Biom edica e Hospital de Sant Pau, Barcelona, Spain; 2 Lipoprotein Particles and their modification, Wihuri Research Institute, Helsinki, Finland
Aim: The role of several heat shock proteins (Hsps) in atherosclerosis is well established. This is less clear for heat shock protein 27 (Hsp27) and its phosphorylated forms. Phosphorylation of Hsp27 may modulate several processes including: cell survival and inflammation. We aimed to assess the expression and distribution of Hsp27, pHsp27, p38MAPK and pp38MAPK in advanced atherosclerotic lesions in the aortic arch and myocardium of hyper- and normo-cholesterolaemic rabbits. Methods: Atherosclerotic lesions were induced in male New Zealand White rabbits by feeding a high cholesterol-diet (0.3-2%) for 18 weeks; maintaining serum cholesterol of approximately 20 mmol/l over this period. The aortic arch and myocardial tissues were analysed by Western blot, immunohistochemistry and double-immunofluorescence. Results: Western blot analysis showed a significant increase in both the monomeric and dimeric forms of Hsp27 and pHsp27, p38MAPK and pp38MAPK in advanced atherosclerotic lesions and in myocardial tissues from cholesterol-fed rabbits when compared to the tissues from control rabbits. These results were confirmed by immunohistochemical analysis, where increases in Hsp27 and pHsp27 immunoreactivity were observed in the advanced atherosclerotic lesions. Furthermore, double-labeling showed intense expression of pHsp27 and p-p38MAPK in the regions that were also rich in macrophages. Conclusion: High levels of pHsp27 expression within advanced atherosclerotic lesions were spatially associated with increased p-p38MAPK, and co-localised with regions rich in macrophages, suggesting that this coexpression may have a profound effect on the cellular events (proliferation, apoptosis or necrosis) and inflammation leading to the development of advanced atherosclerotic lesions.
* Corresponding author. Aim: LDL(-) induces IL-1b release in monocytes by promoting NLRP3 and pro-IL-1b expression. However, its effect on caspase-1 activation was not known. Caspase-1 mediates the processing of pro-IL-1b into its activated secreted form. It was assessed whether LDL(-) induces caspase-1 activation in monocytes and monocyte-derived macrophages (MDM), thereby contributing to IL-1b release in both cell types. Methods: LDL(-) was isolated from total LDL by anion exchange chromatography. Monocytes were isolated by density gradient centrifugation. The differentiation of monocytes into MDM was made by maintaining cells for 7 days in Macrophage Serum-Free Medium with macrophage-colony stimulating factor. After incubation with stimuli, IL-1b was measured in the cell supernatant by ELISA. Cells were pretreated with increasing concentrations of the Kþ channel inhibitors TEA and BaCl2, or with the caspase-1-inhibitor ZYVAD. To evaluate intracellular activated caspase-1, cells were incubated with FAM-fluorescent labelled inhibitors (FLICA) and intracellular fluorescence was measured. Results: It was found that Kþ channel inhibitors decreased IL-1b release promoted by LDL(-) in a dose-dependent manner. Kþ efflux is an upstream step for caspase-1 activation. Data show that cells incubated with LDL(-) had around two-fold increase in active caspase-1 versus native LDL. In the presence of the caspase-1 inhibitor ZYVAD, LDL(-)-induced IL-1b release was disminished in a dosedependent manner. IL-1b release promoted by LDL(-) in MDM in the absence of ZYVAD was almost two-fold compared to that promoted by LDL(þ). Conclusion: The data confirms the role of caspase-1 activation in the IL-1b release promoted by LDL(-) in monocytes and MDM.
EAS-0721. INCREASED EXPRESSION OF PHOSPHORYLATED FORMS OF HEAT SHOCK PROTEIN-27 AND P38MAPK IN MACROPHAGES WITHIN RABBIT ADVANCED ATHEROSCLEROTIC LESIONS S. Shafi 1, *, R. Codrington 1, L. Gideon 1, G.A. Ferns 2. 1 Biochemistry and Physiology, University of Surrey, Guildford, United Kingdom; 2 Brighton and Sussex Medical School Division of Medical Education University of Brighton, University of Surrey, Brighton, United Kingdom
* Corresponding author.
EAS-0789. LONG-CHAIN METABOLITES OF VITAMIN E: A NEW CLASS OF REGULATORY METABOLITES MODULATING ATHEROGENIC PROCESSES? € lz 1, S. Ciffolilli 2, R.M. Pellegrino 3, G. Cruciani 3, M. M. Wallert 1, *, L. Schmo Glei 4, O. Werz 5, A. Mosig 6, K. Rennert 6, F. Galli 2, M. Birringer 7, S. Lorkowski 1. 1 Department of Nutritional Biochemistry, Institute of Nutrition University of Jena, Jena, Germany; 2 Lab of Biochemistry and Nutrition, Department of Pharmaceutical Sciences University of Perugia, Perugia, Italy; 3 Lab of Molecular Modeling and Chemoinformatics, Department of Chemistry University of Perugia, Perugia, Italy; 4 Department of Nutritional Toxicology, Institute of Nutrition University of Jena, Jena, Germany; 5 Department of Pharmaceutical Chemistry, Institute of Pharmacy University of Jena, Jena, Germany; 6 Department of Molecular Hemostaseology, University Hospital Jena, Jena, Germany; 7 Department of Nutritional Food and Consumer Studies, University of Applied Sciences Fulda, Fulda, Germany
* Corresponding author. Vitamin E, particularly a-tocopherol (a-TOH), is still widely used to prevent age-related diseases such as cardiovascular disease although it failed to be atheroprotective in clinical trials. Aside from hepatic a-TOH metabolism, which forms the long-chain metabolites (a-LCM) a-13’-OH and a-13’COOH by CYP4F2/3A4-dependent u-hydroxylation followed by a-oxidation, physiological role and molecular mechanisms of action of a-LCM are poorly understood. We propose that a-LCM have biological functions that need further exploration. Using GC/MS and LC/MS we detected a-LCM in human serum before and after supplementation with RRR-a-TOH and provide evidence for their systemic bioavailability. In macrophages we found induced expression of CD36, the oxLDL scavenger receptor. Simultaneously a-LCM significantly decreased uptake and accumulation of oxLDL by macrophages, likely due to reduced phagocytosis. Screening inflammatory genes in LPS-stimulated macrophages showed significant blocking of proinflammatory pathways by a-LCM. Reduction of Cox2 and iNos expression resulted in decreased production of respective signaling molecules. Modulation of macrophage foam cell formation and inflammatory response was structure-functiondependent. Interestingly, a-LCM acted at lower concentrations and different mechanisms compared to a-TOH. Microarray analyses provided
Abstracts / Atherosclerosis 241 (2015) e72ee148
From 749 differentially expressed transcripts, Gene Ontology analysis identified the LXR pathway as the most overrepresented; and up-regulation of LXRa,b and RXRa,b and downstream genes was confirmed by qRTPCR. Moreover, we demonstrated increased nuclear LXR translocation in FCMs from sponges or atherosclerotic plaques, consistent with recent data by (Spann NJ, Cell. 2012;151:138-52). However, we found no change markers for macrophage M1/M2 polarization, implying that foam cell formation in itself is neither pro- nor anti-inflammatory. The second most over-represented pathway was Fibrosis. Most notably, the pro-collagen peptidase, BMP1, and several collagen polypeptide and proteoglycan genes were increased in FCMs, as were connective tissue growth factor and pro-fibrotic signalling molecules of the Fos and Jun families. All the changes were confirmed by qRT-PCR. Moreover, we showed increased production of matrix proteins, CTGF, and phosphorylation of SMAD2, a major transcription factor mediating fibrosis, in FCMs from sponges and plaques. Our results indicate, somewhat paradoxically, that foam cell formation in vivo is pro-fibrotic, implying that it helps to stabilize plaques. EAS-0680. ELECTRONEGATIVE LDL (LDL(-)) INDUCES IL-1B RELEASE IN MONOCYTES AND MACROPHAGES THROUGH CASPASE-1 ACTIVATION €ki 2, M. Moya 1, J. Sa nchez-Quesada 1, J. M. Estruch-Alrich 1, *, K. Rajama €o € rni 2, S. Benitez 1. 1 Biochemistry, n ~ ez-Llanos 1, P.T. Kovanen 2, K. O Ordo Institut de Recerca Biom edica e Hospital de Sant Pau, Barcelona, Spain; 2 Lipoprotein Particles and their modification, Wihuri Research Institute, Helsinki, Finland
Aim: The role of several heat shock proteins (Hsps) in atherosclerosis is well established. This is less clear for heat shock protein 27 (Hsp27) and its phosphorylated forms. Phosphorylation of Hsp27 may modulate several processes including: cell survival and inflammation. We aimed to assess the expression and distribution of Hsp27, pHsp27, p38MAPK and pp38MAPK in advanced atherosclerotic lesions in the aortic arch and myocardium of hyper- and normo-cholesterolaemic rabbits. Methods: Atherosclerotic lesions were induced in male New Zealand White rabbits by feeding a high cholesterol-diet (0.3-2%) for 18 weeks; maintaining serum cholesterol of approximately 20 mmol/l over this period. The aortic arch and myocardial tissues were analysed by Western blot, immunohistochemistry and double-immunofluorescence. Results: Western blot analysis showed a significant increase in both the monomeric and dimeric forms of Hsp27 and pHsp27, p38MAPK and pp38MAPK in advanced atherosclerotic lesions and in myocardial tissues from cholesterol-fed rabbits when compared to the tissues from control rabbits. These results were confirmed by immunohistochemical analysis, where increases in Hsp27 and pHsp27 immunoreactivity were observed in the advanced atherosclerotic lesions. Furthermore, double-labeling showed intense expression of pHsp27 and p-p38MAPK in the regions that were also rich in macrophages. Conclusion: High levels of pHsp27 expression within advanced atherosclerotic lesions were spatially associated with increased p-p38MAPK, and co-localised with regions rich in macrophages, suggesting that this coexpression may have a profound effect on the cellular events (proliferation, apoptosis or necrosis) and inflammation leading to the development of advanced atherosclerotic lesions.
* Corresponding author. Aim: LDL(-) induces IL-1b release in monocytes by promoting NLRP3 and pro-IL-1b expression. However, its effect on caspase-1 activation was not known. Caspase-1 mediates the processing of pro-IL-1b into its activated secreted form. It was assessed whether LDL(-) induces caspase-1 activation in monocytes and monocyte-derived macrophages (MDM), thereby contributing to IL-1b release in both cell types. Methods: LDL(-) was isolated from total LDL by anion exchange chromatography. Monocytes were isolated by density gradient centrifugation. The differentiation of monocytes into MDM was made by maintaining cells for 7 days in Macrophage Serum-Free Medium with macrophage-colony stimulating factor. After incubation with stimuli, IL-1b was measured in the cell supernatant by ELISA. Cells were pretreated with increasing concentrations of the Kþ channel inhibitors TEA and BaCl2, or with the caspase-1-inhibitor ZYVAD. To evaluate intracellular activated caspase-1, cells were incubated with FAM-fluorescent labelled inhibitors (FLICA) and intracellular fluorescence was measured. Results: It was found that Kþ channel inhibitors decreased IL-1b release promoted by LDL(-) in a dose-dependent manner. Kþ efflux is an upstream step for caspase-1 activation. Data show that cells incubated with LDL(-) had around two-fold increase in active caspase-1 versus native LDL. In the presence of the caspase-1 inhibitor ZYVAD, LDL(-)-induced IL-1b release was disminished in a dosedependent manner. IL-1b release promoted by LDL(-) in MDM in the absence of ZYVAD was almost two-fold compared to that promoted by LDL(þ). Conclusion: The data confirms the role of caspase-1 activation in the IL-1b release promoted by LDL(-) in monocytes and MDM.
EAS-0721. INCREASED EXPRESSION OF PHOSPHORYLATED FORMS OF HEAT SHOCK PROTEIN-27 AND P38MAPK IN MACROPHAGES WITHIN RABBIT ADVANCED ATHEROSCLEROTIC LESIONS S. Shafi 1, *, R. Codrington 1, L. Gideon 1, G.A. Ferns 2. 1 Biochemistry and Physiology, University of Surrey, Guildford, United Kingdom; 2 Brighton and Sussex Medical School Division of Medical Education University of Brighton, University of Surrey, Brighton, United Kingdom
* Corresponding author.
EAS-0789. LONG-CHAIN METABOLITES OF VITAMIN E: A NEW CLASS OF REGULATORY METABOLITES MODULATING ATHEROGENIC PROCESSES? € lz 1, S. Ciffolilli 2, R.M. Pellegrino 3, G. Cruciani 3, M. M. Wallert 1, *, L. Schmo Glei 4, O. Werz 5, A. Mosig 6, K. Rennert 6, F. Galli 2, M. Birringer 7, S. Lorkowski 1. 1 Department of Nutritional Biochemistry, Institute of Nutrition University of Jena, Jena, Germany; 2 Lab of Biochemistry and Nutrition, Department of Pharmaceutical Sciences University of Perugia, Perugia, Italy; 3 Lab of Molecular Modeling and Chemoinformatics, Department of Chemistry University of Perugia, Perugia, Italy; 4 Department of Nutritional Toxicology, Institute of Nutrition University of Jena, Jena, Germany; 5 Department of Pharmaceutical Chemistry, Institute of Pharmacy University of Jena, Jena, Germany; 6 Department of Molecular Hemostaseology, University Hospital Jena, Jena, Germany; 7 Department of Nutritional Food and Consumer Studies, University of Applied Sciences Fulda, Fulda, Germany
* Corresponding author. Vitamin E, particularly a-tocopherol (a-TOH), is still widely used to prevent age-related diseases such as cardiovascular disease although it failed to be atheroprotective in clinical trials. Aside from hepatic a-TOH metabolism, which forms the long-chain metabolites (a-LCM) a-13’-OH and a-13’COOH by CYP4F2/3A4-dependent u-hydroxylation followed by a-oxidation, physiological role and molecular mechanisms of action of a-LCM are poorly understood. We propose that a-LCM have biological functions that need further exploration. Using GC/MS and LC/MS we detected a-LCM in human serum before and after supplementation with RRR-a-TOH and provide evidence for their systemic bioavailability. In macrophages we found induced expression of CD36, the oxLDL scavenger receptor. Simultaneously a-LCM significantly decreased uptake and accumulation of oxLDL by macrophages, likely due to reduced phagocytosis. Screening inflammatory genes in LPS-stimulated macrophages showed significant blocking of proinflammatory pathways by a-LCM. Reduction of Cox2 and iNos expression resulted in decreased production of respective signaling molecules. Modulation of macrophage foam cell formation and inflammatory response was structure-functiondependent. Interestingly, a-LCM acted at lower concentrations and different mechanisms compared to a-TOH. Microarray analyses provided