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CD16positive monocytes exhibit differences in the ability to activate endothelial cells
Abstracts / Atherosclerosis 213 (2010) e6–e19
has not been studied in depth. Therefore we used transcriptomics and related functional analyses to compare cells exposed to laminar shear stress at 15 or 75 dynes/cm2 for 24 h (LSS15-normal or LSS75-high shear stress). Methods and results: LSS75 increased 145 genes and decreased 158 genes in expression more than two-fold relative to LSS15. LSS75 most commonly amplified the effects of LSS15 compared to oscillatory shear stress (OSS ± 5 dynes/cm2 ). Modulation of the metallothioneins (MT1-G, -M, -X) and subunits of NADPH oxidase (NOX2, NOX4, NOX5 and p67phox) accompanied suppression of reactive oxygen species production at LSS75. Modulation of dual specificity phosphatases (DUSPs 1, 5, 8 and 16 increasing and DUSPs 6 and 23 decreasing) accompanied reduced ERK1/2 but increased p38 MAP kinase activity. Amongst vasoactive substances, endothelin-1 expression decreased whereas vasoactive intestinal peptide (VIP) and prostacyclin production increased, despite which cAMP levels declined. Promoter analysis by rVISTA identified a significant over representation of ATF and Nrf2 transcription factor binding sites in genes upregulated by LSS75 compared to LSS15, consistent with previous data on LSS15 compared to OSS. Conclusions: LSS75 in most cases amplifies the protective effect of LSS15, as directly demonstrated for ROS production. However, activation of p38 MAP kinase and reduction of cAMP concentration are stressors that could lead to adverse physiological consequences. doi:10.1016/j.atherosclerosis.2010.08.010 10 Foam cells differentiated from CD14positive/CD16negative or CD14LO/CD16positive monocytes exhibit differences in the ability to activate endothelial cells C.M. Yates, G.B. Nash, G.E. Rainger Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, University of Birmingham, B15 2TT, United Kingdom It is well established that monocytes play a key role in the initiation and progression of atherosclerosis, however the relative contribution of monocyte subsets is largely unknown. We used a monocyte:endothelial cell co-culture model to investigate the capacity of monocyte subsets to differentiate into foam cells and their subsequent ability to activate endothelial cells in a flow based adhesion assay. Using magnetic bead separation CD14positiveCD16negative and CD14loCD16positive monocytes were isolated and cultured on 0.4micrometre pore inserts for 3 days or 6 days in the presence or absence of 50 g/ml oxidised-low density lipoprotein (Ox-LDL). Human umbilical vein endothelial cells (HUVEC) were then co-cultured on the apical side of the filter for 24 h in the absence of Ox-LDL. The ability of the HUVEC to recruit peripheral blood mononuclear cells (PBMCs) was then tested in a flow based adhesion assay. We found that in the absence of OxLDL both subsets stimulated HUVEC so they recruited low levels of PBMCs. However, when cultured in the presence of Ox-LDL, foam cells generated from CD14positiveCD16negative monocytes caused increased EC activation resulting in higher levels of PBMC recruitment compared to CD14loCD16positive derived foam cells at both 4 and 7 days. Thus, we have shown that Ox-LDL is able to generate a proinflammatory phenotype in a specific subset of monocytes. doi:10.1016/j.atherosclerosis.2010.08.011
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13 Necrotic vascular smooth muscle cells are the main cell type responsible for interleukin-1 alpha driven sterile inflammation in atherosclerosis Y. Zheng ∗ , M. Humphry, S. Talib, M.R. Bennett MCH Clarke Cardiovascular Division, Department of Medicine, University of Cambridge, UK CB2 0QQ, United Kingdom Rationale: Vascular smooth muscle cells (VSMCs), macrophages and lymphocytes are the major cell types present within atherosclerotic plaques, and undergo apoptosis and necrosis in late stages of the disease. Cell death reduces plaque stability and consequently may trigger myocardial infarction. IL-1␣ has recently been recognized as a ‘danger signal’ released from necrotic cells that is able to induce inflammation. This study investigated the role of IL-1␣ from necrotic cell types present in atherosclerotic plaques. Methodology and results: VSMCs, macrophages and lymphocytes were made necrotic and their ability to induce inflammatory responses was examined. (1) We find that only necrotic VSMCs induce the release of pro-inflammatory cytokines from healthy VSMCs in vitro. Mature IL-1␣, which was cleaved from its precursor during necrosis, was then identified to be solely responsible for this inflammatory response generated by necrotic VSMCs. (2) Necrotic macrophages and lymphocytes do not process pro-IL-1␣ to the mature cytokine, and macrophages and lymphocytes were not responsive towards recombinant IL-1␣ nor IL-1␣ released from necrotic VSMCs. Conclusions: We reinforce previous findings that IL-1␣ acts as a danger signal released from necrotic cells and further demonstrated that IL-1␣’s inflammatory property is cell type dependent and possibly structurally dependent. Thus, interference with the maturation of IL-1␣ in VSMCs may provide a novel strategy toward dampening sterile inflammation in atherosclerosis and thus improving plaque stability. doi:10.1016/j.atherosclerosis.2010.08.014 14 Apolipoprotein-E regulates the senescence of mouse vascular smooth muscle cells in vitro J.C. Wang ∗ , M.R. Bennett Division of Cardiovascular Medicine, University of Cambridge, Cambridge, CB2 2QQ, United Kingdom Background: Vascular smooth muscle cells (VSMCs) from human atherosclerotic plaques undergo apoptosis and senescence in vitro and in vivo, due to telomere shortening and non-telomeric ‘stressinduced premature senescence’ (SIPS). Apolipoprotein-E-deficient (ApoE−/−) mouse is a model for advanced atherosclerosis. To accurately interpret animal studies, investigating if ApoE−/− VSMCs reproduce the kinetics and mechanisms of human VSMC senescence is crucial. Methods: Aortic VSMCs from wild-type and ApoE−/− mice were analysed by time-lapse videomicroscopy, immunocytochemistry and western-blotting to evaluate cell behaviour, DNA damage and protein expression, respectively. Results: Control VSMCs became senescent within 2–3 passages, consistent with SIPS. ApoE−/− VSMCs senesced at P12–15. A sub-population then transformed spontaneously and resumed proliferation in 10–14 days. This pattern of proliferation followed by transient growth arrest occurred repetitively approximately every 15 passages. The proportion of apoptotic and non-dividing ApoE−/− VSMCs increased dramatically by P14 prior to proliferative arrest, coinciding with extensive DNA damage indicated by
has not been studied in depth. Therefore we used transcriptomics and related functional analyses to compare cells exposed to laminar shear stress at 15 or 75 dynes/cm2 for 24 h (LSS15-normal or LSS75-high shear stress). Methods and results: LSS75 increased 145 genes and decreased 158 genes in expression more than two-fold relative to LSS15. LSS75 most commonly amplified the effects of LSS15 compared to oscillatory shear stress (OSS ± 5 dynes/cm2 ). Modulation of the metallothioneins (MT1-G, -M, -X) and subunits of NADPH oxidase (NOX2, NOX4, NOX5 and p67phox) accompanied suppression of reactive oxygen species production at LSS75. Modulation of dual specificity phosphatases (DUSPs 1, 5, 8 and 16 increasing and DUSPs 6 and 23 decreasing) accompanied reduced ERK1/2 but increased p38 MAP kinase activity. Amongst vasoactive substances, endothelin-1 expression decreased whereas vasoactive intestinal peptide (VIP) and prostacyclin production increased, despite which cAMP levels declined. Promoter analysis by rVISTA identified a significant over representation of ATF and Nrf2 transcription factor binding sites in genes upregulated by LSS75 compared to LSS15, consistent with previous data on LSS15 compared to OSS. Conclusions: LSS75 in most cases amplifies the protective effect of LSS15, as directly demonstrated for ROS production. However, activation of p38 MAP kinase and reduction of cAMP concentration are stressors that could lead to adverse physiological consequences. doi:10.1016/j.atherosclerosis.2010.08.010 10 Foam cells differentiated from CD14positive/CD16negative or CD14LO/CD16positive monocytes exhibit differences in the ability to activate endothelial cells C.M. Yates, G.B. Nash, G.E. Rainger Centre for Cardiovascular Sciences, School of Clinical and Experimental Medicine, University of Birmingham, B15 2TT, United Kingdom It is well established that monocytes play a key role in the initiation and progression of atherosclerosis, however the relative contribution of monocyte subsets is largely unknown. We used a monocyte:endothelial cell co-culture model to investigate the capacity of monocyte subsets to differentiate into foam cells and their subsequent ability to activate endothelial cells in a flow based adhesion assay. Using magnetic bead separation CD14positiveCD16negative and CD14loCD16positive monocytes were isolated and cultured on 0.4micrometre pore inserts for 3 days or 6 days in the presence or absence of 50 g/ml oxidised-low density lipoprotein (Ox-LDL). Human umbilical vein endothelial cells (HUVEC) were then co-cultured on the apical side of the filter for 24 h in the absence of Ox-LDL. The ability of the HUVEC to recruit peripheral blood mononuclear cells (PBMCs) was then tested in a flow based adhesion assay. We found that in the absence of OxLDL both subsets stimulated HUVEC so they recruited low levels of PBMCs. However, when cultured in the presence of Ox-LDL, foam cells generated from CD14positiveCD16negative monocytes caused increased EC activation resulting in higher levels of PBMC recruitment compared to CD14loCD16positive derived foam cells at both 4 and 7 days. Thus, we have shown that Ox-LDL is able to generate a proinflammatory phenotype in a specific subset of monocytes. doi:10.1016/j.atherosclerosis.2010.08.011
e9
13 Necrotic vascular smooth muscle cells are the main cell type responsible for interleukin-1 alpha driven sterile inflammation in atherosclerosis Y. Zheng ∗ , M. Humphry, S. Talib, M.R. Bennett MCH Clarke Cardiovascular Division, Department of Medicine, University of Cambridge, UK CB2 0QQ, United Kingdom Rationale: Vascular smooth muscle cells (VSMCs), macrophages and lymphocytes are the major cell types present within atherosclerotic plaques, and undergo apoptosis and necrosis in late stages of the disease. Cell death reduces plaque stability and consequently may trigger myocardial infarction. IL-1␣ has recently been recognized as a ‘danger signal’ released from necrotic cells that is able to induce inflammation. This study investigated the role of IL-1␣ from necrotic cell types present in atherosclerotic plaques. Methodology and results: VSMCs, macrophages and lymphocytes were made necrotic and their ability to induce inflammatory responses was examined. (1) We find that only necrotic VSMCs induce the release of pro-inflammatory cytokines from healthy VSMCs in vitro. Mature IL-1␣, which was cleaved from its precursor during necrosis, was then identified to be solely responsible for this inflammatory response generated by necrotic VSMCs. (2) Necrotic macrophages and lymphocytes do not process pro-IL-1␣ to the mature cytokine, and macrophages and lymphocytes were not responsive towards recombinant IL-1␣ nor IL-1␣ released from necrotic VSMCs. Conclusions: We reinforce previous findings that IL-1␣ acts as a danger signal released from necrotic cells and further demonstrated that IL-1␣’s inflammatory property is cell type dependent and possibly structurally dependent. Thus, interference with the maturation of IL-1␣ in VSMCs may provide a novel strategy toward dampening sterile inflammation in atherosclerosis and thus improving plaque stability. doi:10.1016/j.atherosclerosis.2010.08.014 14 Apolipoprotein-E regulates the senescence of mouse vascular smooth muscle cells in vitro J.C. Wang ∗ , M.R. Bennett Division of Cardiovascular Medicine, University of Cambridge, Cambridge, CB2 2QQ, United Kingdom Background: Vascular smooth muscle cells (VSMCs) from human atherosclerotic plaques undergo apoptosis and senescence in vitro and in vivo, due to telomere shortening and non-telomeric ‘stressinduced premature senescence’ (SIPS). Apolipoprotein-E-deficient (ApoE−/−) mouse is a model for advanced atherosclerosis. To accurately interpret animal studies, investigating if ApoE−/− VSMCs reproduce the kinetics and mechanisms of human VSMC senescence is crucial. Methods: Aortic VSMCs from wild-type and ApoE−/− mice were analysed by time-lapse videomicroscopy, immunocytochemistry and western-blotting to evaluate cell behaviour, DNA damage and protein expression, respectively. Results: Control VSMCs became senescent within 2–3 passages, consistent with SIPS. ApoE−/− VSMCs senesced at P12–15. A sub-population then transformed spontaneously and resumed proliferation in 10–14 days. This pattern of proliferation followed by transient growth arrest occurred repetitively approximately every 15 passages. The proportion of apoptotic and non-dividing ApoE−/− VSMCs increased dramatically by P14 prior to proliferative arrest, coinciding with extensive DNA damage indicated by