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Instability of LDL particles predicts future cardiovascular deaths
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Abstracts / Atherosclerosis 263 (2017) e1ee28
Results: Human macrophages carrying the minor Pro251 variant showed decreased cholesterol content after oxLDL treatment compared to macrophages with the Ser251 variant. Increased cholesterol efflux was observed in oxLDL treated macrophages carrying the minor Pro251 variant as compared to macrophages with the major Ser251 variant. Expression analyses showed increased levels of genes involved in cholesterol efflux and decreased levels of genes involved in lipid synthesis in macrophages treated with oxLDL. Conclusions: These results suggest that the minor Pro251 allele in PLIN2 exert beneficial effects during foam cell formation and atherosclerosis development due to decreased cholesterol accumulation and increased cholesterol efflux in oxLDL treated primary macrophages as compared to macrophages carrying the major Ser251 allele. Late Breaking Session: Late Breaking News In Experimental Sciences LB2:1. INSTABILITY OF LDL PARTICLES PREDICTS FUTURE CARDIOVASCULAR DEATHS Maija Ruuth1, Su Duy Nguyen1, Terhi Vihervaara2, Mika Hilvo2, Teemu Daniel Laajala3, Pradeep Kumar Kondadi4, Gisterå Anton5, Jenni Huusko6, Matti Uusitupa6, Ursula Schwab6,7, Rudel Lawrence8, Daniel Ketelhuth5, Reijo Laaksonen2, Marc Baumann9, Tero Aittokallio3, Matti n Bore Jan4, Kevin Jon Williams4, Petri Jauhiainen10, €o € rni1. 1 Wihuri Research Institute, Helsinki, Finland; Kovanen1, Katariina O 2 Zora Biosciences, Espoo, Finland; 3 Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland; 4 University of Gothenburg, Gothenburg, Sweden; 5 Karolinska Institutet, Stockholm, Sweden; 6 University of Eastern Finland, Kuopio, Finland; 7 Kuopio University Hospital, Kuopio, Finland; 8 Wake Forest School of Medicine, Winston-Salem, NC, USA; 9 University of Helsinki, Helsinki, Finland; 10 National Institute for Health and Welfare, Helsinki, Finland Aim: Aggregation of LDL increases their retention within the arterial intima and induces foam cell formation thereby accelerating atherosclerosis. Nevertheless, the intrinsic susceptibility of human LDL to aggregate, defined here as LDL ‘instability’, has not been systematically evaluated. Methods: We developed a novel, reproducible method to assess LDL instability. LDL phospholipid composition and apoB-100 conformation were determined with mass spectrometry and circular dichroism. Results: Using banked plasma from patients with angiographically defined coronary artery stenosis (Corogene study, n¼48), LDL instability was associated with future cardiovascular deaths independent of conventional cardiovascular risk factors. Aggregated LDL activated macrophages and T cells, two cell types involved in plaque rupture. Unstable LDL contained proportionately more sphingomyelin and less phosphatidylcholine when compared with aggregation-resistant LDL. These compositional findings were verified in healthy human subjects in two independent studies (Health 2000, n¼100; Sysdiet study, n¼58). A causative role of LDL lipid composition in LDL instability was established by lowering the proportion of sphingomyelins in LDL in vitro and in three mouse models in vivo. Using limited proteolysis, we identified a specific domain, HLIDSLIDFLNFPR (4188-4201), in the C-terminus of apoB-100 required in LDL aggregation. Conclusions: Our results implicate a clinically significant role for LDL instability in human atherosclerotic cardiovascular disease. Moreover, measurement of LDL instability may serve as a predictive biomarker for the identification of patients at unrecognized risk of cardiovascular death. LB2:2. C1Q/TNF-RELATED PROTEIN 1 PROMOTES VASODILATORY DYSFUNCTIONS BY INCREASING ARGINASE 1 ACTIVITY AND UNCOUPLING OF ENDOTHELIAL NITRIC OXIDE SYNTHASE Xiaoqun Wang, Chang Li, Zhuhui Liu, Fenghua Ding, Jie Gao, Jiawei Chen, Weifeng Shen, Ruiyan Zhang, Lin Lu. Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Aim: C1q/TNF-related protein (CTRP) 1 was initially identified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.
Previously, we showed that CTRP1 promotes the development of atherosclerosis by increasing endothelial adhesiveness. Here, we sought to investigate whether CTRP1 also influences vascular dilatory functions. Methods: Vascular dilatory responsiveness was compared by intravital microscopy of cremaster arterioles between CTRP1 transgenic (Tg-CTRP1), CTRP1 knockout (CTRP1 KO) and C57 wild type (WT) control mice. In situ production of reactive oxygen species (ROS) was determined by dihydroethidium (DHE) staining. Results: We found a dramatic impairment of endothelium-dependent arteriolar dilation in Tg-CTRP1 mice, whereas vasodilation was markedly enhanced in CTRP1 KO mice as compared to WT controls. Meanwhile, elevated production of reactive oxygen species (ROS) was detected in the vascular wall of Tg-CTRP1 animals. In cultured endothelial cells, CTRP1 stimulation resulted in reduced nitric oxide (NO) bioavailability both in the cell lysates and conditioned media. Furthermore, we found that arginase 1 was significantly increased by CTRP1 in a dose-dependent fashion, thereby leading to endothelial nitric oxide synthase (eNOS)-uncoupling and reactive oxygen species generation. Inhibition of arginase activity by synthetic chemicals markedly improved CTRP1-dependent vasodilatory dysfunctions. Conclusions: These data define the essential role of CTRP1 in mediating vasodilatory dysfunctions, as well as propose a novel mechanism that increased arginase activity by CTRP1 leads to uncoupling of eNOS homodimers, thereby limiting NO biosynthesis and amplifying ROS production.
LB2:3. MARGINAL ZONE B CELLS CONTROL FOLLICULAR HELPER T CELL RESPONSE TO HIGH CHOLESTEROL DIET Meritxell Nus1, Andy P. Sage1, Yuning Lu1, Leanne Masters1, Brian Yh Stephen Newland1, Sandra Weller3, Dimitrios Lam2, Raffort1, Damienne Marcus1, Alison Tsiantoulas4, Juliette Finigan1, Lauren Kitt1, Nichola Figg1, Reinhold Schirmbeck5, Manfred Yeo2, Christoph J. Binder4, Jose L. De La Kneilling6, Giles Sh Pompa7, Ziad Mallat1, 8. 1 Division of Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom; 2 Metabolic Research Laboratories, University of Cambridge and MRC Metabolic Diseases Unit, Wellcome-MRC Institute of Met, Cambridge, United Kingdom; 3 Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Sorbonne Paris Cit e, Universit e Paris Descartes, Paris, France; 4 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 5 Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany; 6 Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Eberhard Karls University, Tubingen, Germany; 7 Intercellular Signalling in Cardiovascular Development & Disease Lab, Centro Nacional de Investigaciones Cardiovascular, Madrid, Spain; 8 Institut National de la Sant e et de la Recherche M edicale (Inserm), Unit 970, Paris Cardiovascular Research Center, Paris, France Aim: The specific role of MZB on atherosclerosis has never been explored and it cannot be anticipated because they participate in both innate and adaptive immune response Methods: We generated mice with B cell lineage-specific (Cd79aCre/+) conditional deletion of Rbpjk (Rbpjkflox/flox) that do not have MZB cells. Lethally irradiated LdlrKO mice were then reconstituted with bone marrow (BM) from Cd79aCre/+;Rbpjkflox/flox or Cd79a+/+;Rbpjkflox/flox control mice and fed with high cholesterol diet (HCD). Mice were also treated with anti-CD4 (for T cell depletion) or anti-Icosl (for follicular helper T (Tfh) depletion) antibodies. Sorted MZB and Tfh cells were analysed by RNAseq. Results: MZB cells selective deletion substantially increased Tfh and germinal center (GC) B cells accumulation in response to HCD, leading to T cell-dependent acceleration of atherosclerosis. MZB cells activate a homeostatic program in response to HCD that regulate Tfh differentiation and accumulation. Upregulation of the transcription factor Atf3 in MZB cells plays a determinant regulatory role in this. HCD promotes increased interaction between MZB and (pre-)Tfh cells outside the follicle, and upregulates MZB cell expression of Pdl1 in an Atf3-dependent manner. Interaction between MZB and Tfh cells leads to Pdl1-mediated suppression of Tfh cell motility, differentiation and accumulation, suppresing Tfh proatherogenic response.
Abstracts / Atherosclerosis 263 (2017) e1ee28
Results: Human macrophages carrying the minor Pro251 variant showed decreased cholesterol content after oxLDL treatment compared to macrophages with the Ser251 variant. Increased cholesterol efflux was observed in oxLDL treated macrophages carrying the minor Pro251 variant as compared to macrophages with the major Ser251 variant. Expression analyses showed increased levels of genes involved in cholesterol efflux and decreased levels of genes involved in lipid synthesis in macrophages treated with oxLDL. Conclusions: These results suggest that the minor Pro251 allele in PLIN2 exert beneficial effects during foam cell formation and atherosclerosis development due to decreased cholesterol accumulation and increased cholesterol efflux in oxLDL treated primary macrophages as compared to macrophages carrying the major Ser251 allele. Late Breaking Session: Late Breaking News In Experimental Sciences LB2:1. INSTABILITY OF LDL PARTICLES PREDICTS FUTURE CARDIOVASCULAR DEATHS Maija Ruuth1, Su Duy Nguyen1, Terhi Vihervaara2, Mika Hilvo2, Teemu Daniel Laajala3, Pradeep Kumar Kondadi4, Gisterå Anton5, Jenni Huusko6, Matti Uusitupa6, Ursula Schwab6,7, Rudel Lawrence8, Daniel Ketelhuth5, Reijo Laaksonen2, Marc Baumann9, Tero Aittokallio3, Matti n Bore Jan4, Kevin Jon Williams4, Petri Jauhiainen10, €o € rni1. 1 Wihuri Research Institute, Helsinki, Finland; Kovanen1, Katariina O 2 Zora Biosciences, Espoo, Finland; 3 Institute for Molecular Medicine Finland (FIMM), Helsinki, Finland; 4 University of Gothenburg, Gothenburg, Sweden; 5 Karolinska Institutet, Stockholm, Sweden; 6 University of Eastern Finland, Kuopio, Finland; 7 Kuopio University Hospital, Kuopio, Finland; 8 Wake Forest School of Medicine, Winston-Salem, NC, USA; 9 University of Helsinki, Helsinki, Finland; 10 National Institute for Health and Welfare, Helsinki, Finland Aim: Aggregation of LDL increases their retention within the arterial intima and induces foam cell formation thereby accelerating atherosclerosis. Nevertheless, the intrinsic susceptibility of human LDL to aggregate, defined here as LDL ‘instability’, has not been systematically evaluated. Methods: We developed a novel, reproducible method to assess LDL instability. LDL phospholipid composition and apoB-100 conformation were determined with mass spectrometry and circular dichroism. Results: Using banked plasma from patients with angiographically defined coronary artery stenosis (Corogene study, n¼48), LDL instability was associated with future cardiovascular deaths independent of conventional cardiovascular risk factors. Aggregated LDL activated macrophages and T cells, two cell types involved in plaque rupture. Unstable LDL contained proportionately more sphingomyelin and less phosphatidylcholine when compared with aggregation-resistant LDL. These compositional findings were verified in healthy human subjects in two independent studies (Health 2000, n¼100; Sysdiet study, n¼58). A causative role of LDL lipid composition in LDL instability was established by lowering the proportion of sphingomyelins in LDL in vitro and in three mouse models in vivo. Using limited proteolysis, we identified a specific domain, HLIDSLIDFLNFPR (4188-4201), in the C-terminus of apoB-100 required in LDL aggregation. Conclusions: Our results implicate a clinically significant role for LDL instability in human atherosclerotic cardiovascular disease. Moreover, measurement of LDL instability may serve as a predictive biomarker for the identification of patients at unrecognized risk of cardiovascular death. LB2:2. C1Q/TNF-RELATED PROTEIN 1 PROMOTES VASODILATORY DYSFUNCTIONS BY INCREASING ARGINASE 1 ACTIVITY AND UNCOUPLING OF ENDOTHELIAL NITRIC OXIDE SYNTHASE Xiaoqun Wang, Chang Li, Zhuhui Liu, Fenghua Ding, Jie Gao, Jiawei Chen, Weifeng Shen, Ruiyan Zhang, Lin Lu. Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China Aim: C1q/TNF-related protein (CTRP) 1 was initially identified as a paralog of adiponectin based on the similarity in C1q domain of these two proteins.
Previously, we showed that CTRP1 promotes the development of atherosclerosis by increasing endothelial adhesiveness. Here, we sought to investigate whether CTRP1 also influences vascular dilatory functions. Methods: Vascular dilatory responsiveness was compared by intravital microscopy of cremaster arterioles between CTRP1 transgenic (Tg-CTRP1), CTRP1 knockout (CTRP1 KO) and C57 wild type (WT) control mice. In situ production of reactive oxygen species (ROS) was determined by dihydroethidium (DHE) staining. Results: We found a dramatic impairment of endothelium-dependent arteriolar dilation in Tg-CTRP1 mice, whereas vasodilation was markedly enhanced in CTRP1 KO mice as compared to WT controls. Meanwhile, elevated production of reactive oxygen species (ROS) was detected in the vascular wall of Tg-CTRP1 animals. In cultured endothelial cells, CTRP1 stimulation resulted in reduced nitric oxide (NO) bioavailability both in the cell lysates and conditioned media. Furthermore, we found that arginase 1 was significantly increased by CTRP1 in a dose-dependent fashion, thereby leading to endothelial nitric oxide synthase (eNOS)-uncoupling and reactive oxygen species generation. Inhibition of arginase activity by synthetic chemicals markedly improved CTRP1-dependent vasodilatory dysfunctions. Conclusions: These data define the essential role of CTRP1 in mediating vasodilatory dysfunctions, as well as propose a novel mechanism that increased arginase activity by CTRP1 leads to uncoupling of eNOS homodimers, thereby limiting NO biosynthesis and amplifying ROS production.
LB2:3. MARGINAL ZONE B CELLS CONTROL FOLLICULAR HELPER T CELL RESPONSE TO HIGH CHOLESTEROL DIET Meritxell Nus1, Andy P. Sage1, Yuning Lu1, Leanne Masters1, Brian Yh Stephen Newland1, Sandra Weller3, Dimitrios Lam2, Raffort1, Damienne Marcus1, Alison Tsiantoulas4, Juliette Finigan1, Lauren Kitt1, Nichola Figg1, Reinhold Schirmbeck5, Manfred Yeo2, Christoph J. Binder4, Jose L. De La Kneilling6, Giles Sh Pompa7, Ziad Mallat1, 8. 1 Division of Cardiovascular Medicine, University of Cambridge, Cambridge, United Kingdom; 2 Metabolic Research Laboratories, University of Cambridge and MRC Metabolic Diseases Unit, Wellcome-MRC Institute of Met, Cambridge, United Kingdom; 3 Institut Necker-Enfants Malades, INSERM U1151-CNRS UMR 8253, Sorbonne Paris Cit e, Universit e Paris Descartes, Paris, France; 4 Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria; 5 Department of Internal Medicine I, Ulm University Hospital, Ulm, Germany; 6 Department of Preclinical Imaging and Radiopharmacy, Werner Siemens Imaging Center, Eberhard Karls University, Tubingen, Germany; 7 Intercellular Signalling in Cardiovascular Development & Disease Lab, Centro Nacional de Investigaciones Cardiovascular, Madrid, Spain; 8 Institut National de la Sant e et de la Recherche M edicale (Inserm), Unit 970, Paris Cardiovascular Research Center, Paris, France Aim: The specific role of MZB on atherosclerosis has never been explored and it cannot be anticipated because they participate in both innate and adaptive immune response Methods: We generated mice with B cell lineage-specific (Cd79aCre/+) conditional deletion of Rbpjk (Rbpjkflox/flox) that do not have MZB cells. Lethally irradiated LdlrKO mice were then reconstituted with bone marrow (BM) from Cd79aCre/+;Rbpjkflox/flox or Cd79a+/+;Rbpjkflox/flox control mice and fed with high cholesterol diet (HCD). Mice were also treated with anti-CD4 (for T cell depletion) or anti-Icosl (for follicular helper T (Tfh) depletion) antibodies. Sorted MZB and Tfh cells were analysed by RNAseq. Results: MZB cells selective deletion substantially increased Tfh and germinal center (GC) B cells accumulation in response to HCD, leading to T cell-dependent acceleration of atherosclerosis. MZB cells activate a homeostatic program in response to HCD that regulate Tfh differentiation and accumulation. Upregulation of the transcription factor Atf3 in MZB cells plays a determinant regulatory role in this. HCD promotes increased interaction between MZB and (pre-)Tfh cells outside the follicle, and upregulates MZB cell expression of Pdl1 in an Atf3-dependent manner. Interaction between MZB and Tfh cells leads to Pdl1-mediated suppression of Tfh cell motility, differentiation and accumulation, suppresing Tfh proatherogenic response.