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Acute global effects of high-density lipoprotein on activated endothelial cells—Inhibition of CXCL10
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Abstracts / Atherosclerosis 213 (2010) e6–e19
human carotid plaques to map the pro-inflammatory milieu responsible for plaque instability. Methodology: Carotid endarterectomies from symptomatic (n = 35) and asymptomatic (n = 32) patients were enzymatically dissociated producing mixed macrophage-rich, atheroma cell suspensions which were cultured for 24 h [1]. Supernatants were interrogated with a 45-analyte panel on a Luminex 100 platform. Analyte inter-relationships were described statistically via Spearman correlation. Resulting sets were analyzed via ingenuity pathways analysis v7.6. A 1.5-fold cut-off was set to identify proteins whose expression was significantly increased or decreased. Right-tailed Fisher’s exact test determined significance (p-value) of protein over-representation compared to the result expected by a random protein set. Results: Tumour necrosis factor (TNF)-alpha, interleukin (IL)1alpha, IL-1beta, IL-6, IL-10, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), CCL2, CCL5, CCL20, CXCL9, MMP1, MMP3, MMP8 and MMP9 levels were significantly higher in symptomatic plaques (p < 0.05). Analyte levels were not significantly related to clinical parameters. Inter-related analyte ‘clusters’ indentified, in particular GM-CSF correlated significantly with IL-1, IL-6 and TNFalpha (p < 0.05). Top-ranked biological pathways associated with differences between symptomatic and asymptomatic data sets (identified via Ingenuity) focused on the interplay between innate and adaptive immunity, nuclear factor-kappaB and MAPK signalling, and hypercytokinemia in inducing pathology and matrix degradation. Conclusions: The inflammatory milieu within human unstable plaque is in keeping with a predominance of M1-type macrophages, and correlates with MMP production. Reference [1] Monaco, et al. Proc Natl Acad Sci U S A 2004;13(101):5634–9.
doi:10.1016/j.atherosclerosis.2010.08.041 41 Foam cell formation affects MHCI processing and presentation in vitro while hypercholesteremia induces a DC-like phenotype in macrophages in vivo K.L.L. Habets 1,∗ , V. Frodermann 1 , A.C. Foks 1 , G.H.M. Puijvelde 1 , R.E.M. Toes 2 , Th.J.C. van Berkel 1 , J. Kuiper 1
van
1
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands 2 Department of Rheumatology, Leiden University Medical Centre, Leiden, The Netherlands
Background: Recent reports indicate that foam cell formation induces a differentiation of macrophages into a dendritic cell-like phenotype. Because dendritic cells are the most potent antigen presenting cells, these changes could have implications in antigen presentation and subsequent T cell activation. Methods and results: To evaluate the effects of foam cell formation we have cultured bone-marrow derived macrophages in the presence of oxLDL (10 g/ml). We observed a dendritic cell like phenotype with increased CD11c, MHCI, MHCII and costimulatory molecules suggesting an increased capacity to induce a specific T cell response. Consequently, we showed that MHCI antigen presentation and processing of ovalbumin was increased while MHCII induced T cell activation was not altered. Next we evaluated the effects of hypercholesteremia on peripheral macrophages in LDLr−/− mice. Again we observed an increase of CD11chigh and MHCI within the macrophage population. Furthermore, the
activation status of both dendritic cells as macrophages was increased. Conclusions: Both hypercholesteremic conditions as in vitro foam cell formation not only induced a dendritic-cell like phenotype in macrophages, but also provide macrophages with dendritic-cell like capacities. These data support the hypothesis that foam cell macrophages could play an important role in inducing an immune response during the early stages of atherosclerotic lesion development. However, further investigation about the role of extreme foam cell formation and its effect on antigen presentation in vivo needs to be further elucidated. doi:10.1016/j.atherosclerosis.2010.08.042 42 Increased foam cell formation and atherosclerotic plaque apoptosis in LDLr−/− mice lacking macrophage Mcl-1 M.M. Westra 1 , I. Bot 1 , M. Bot 1 , S.C.A. de Jager 1 , I. Dzhagalov 2 , Y.W. He 1 , Th.J.C. van Berkel 1 , E.A.L. Biessen 1,3 1
Biopharmaceutics, LACDR, Leiden, The Netherlands Department of Cardiology, Leiden University Medical Center, The Netherlands 3 Department of Pathology, CARIM, The Netherlands 2
The anti-apoptotic Bcl-1 family member Mcl-1 plays an important, in neutrophils essential, role in leukocyte survival and differentiation. Here, we investigated the impact of Mcl-1 deletion in myeloid cells on atherosclerosis in WTD fed LDLr−/−. First, Mcl-1−/− peritoneal macrophages had an increased sensitivity to Ox-LDL induced cell death and showed altered expression of several pro-apoptotic Bcl-2 family members as compared to WT macrophages. In keeping apoptotic cell content in aortic root lesions of Mcl-1−/− chimeras was elevated by 77% compared to WT controls. Second lipid uptake by peritoneal Mcl-1−/− macrophages was enhanced in vitro as well as in vivo. Third, Mcl-1−/− macrophages showed a clear shift towards a proinflammatory M1 phenotype as apparent from their cytokine expression pattern and reduced phagocytotic capacity. Despite these profound pro-atherogenic effects of Mcl-1 deficiency both plaque development and progression did not differ between Mcl-1−/− and WT BM recipients. This seeming paradox may well be imputed to altered neutrophil numbers and migratory capacity in Mcl-1 deficiency, attenuating lesional neutrophil infiltration. In conclusion, myeloid Mcl-1 deletion enhances Ox-LDL induced foam cell formation and cell death, favors a pro-inflammatory macrophage phenotype and alters neutrophil characteristics, Overall deficiency of this key gene markedly increased plaque cell apoptosis, but did neither affect plaque initiation nor growth. doi:10.1016/j.atherosclerosis.2010.08.043 43 Acute global effects of high-density lipoprotein on activated endothelial cells—Inhibition of CXCL10 A. Shoreim 1,∗ , J. Dennis 2 , K. Laing 2 , S. Petrova 1 , G.W. Cockerill 1 1
Department of Cardiovascular Sciences, St George’s University of London, Cranmer Terrace, SW17 0RE, United Kingdom 2 Department of Infectious Diseases, St George’s University of London, Cranmer Terrace, SW17 0RE, United Kingdom Rationale: Plasma concentration of high-density lipoprotein (HDL) is inversely proportional to the incidence of coronary artery
Abstracts / Atherosclerosis 213 (2010) e6–e19
disease, although the precise mechanism of protection remains unclear. Using activated human umbilical vein endothelial cells (HUVEC) as a model of ‘athero-prone’ endothelium, we investigated the acute transcriptional changes in response to treatment with reconstituted discoidal HDL (rHDL), to identify genes/processes important in mediating protection. Method: Confluent HUVECs (n = 3) activated with TNFalpha; (10 ng/ml) for 4 h were treated with rHDL (0.1 mg/ml or 1 mg/ml) for a further 8 h. Transcriptome changes were identified using SentrixTM beadchip microarray HT-12 (n = 3), per treatment. Following normalisation, data was analysed using GenespringTM . Differential expression was validated using quantitative realtime PCR.
e19
Results: In addition to changes in expression of genes involved in lipid metabolism, we have identified changes in CXCL10, a cytokine-induced T-cell chemokine important in innate immunity. Treatment with rHDL (1 mg/ml) results in a significant reduction in cytokine-induced expression: 2.25 ± 0.27 (TNFalpha) vs. 0.98 ± 0.12 (rHDL), n-3, p ≤0.05. Conclusion: Our data supports a role for HDL in reducing the innate immune response. Further studies will investigate the effect of HDL on the protein expression and time course of endothelial CXCL10. doi:10.1016/j.atherosclerosis.2010.08.044
Abstracts / Atherosclerosis 213 (2010) e6–e19
human carotid plaques to map the pro-inflammatory milieu responsible for plaque instability. Methodology: Carotid endarterectomies from symptomatic (n = 35) and asymptomatic (n = 32) patients were enzymatically dissociated producing mixed macrophage-rich, atheroma cell suspensions which were cultured for 24 h [1]. Supernatants were interrogated with a 45-analyte panel on a Luminex 100 platform. Analyte inter-relationships were described statistically via Spearman correlation. Resulting sets were analyzed via ingenuity pathways analysis v7.6. A 1.5-fold cut-off was set to identify proteins whose expression was significantly increased or decreased. Right-tailed Fisher’s exact test determined significance (p-value) of protein over-representation compared to the result expected by a random protein set. Results: Tumour necrosis factor (TNF)-alpha, interleukin (IL)1alpha, IL-1beta, IL-6, IL-10, granulocyte-macrophage colonystimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), CCL2, CCL5, CCL20, CXCL9, MMP1, MMP3, MMP8 and MMP9 levels were significantly higher in symptomatic plaques (p < 0.05). Analyte levels were not significantly related to clinical parameters. Inter-related analyte ‘clusters’ indentified, in particular GM-CSF correlated significantly with IL-1, IL-6 and TNFalpha (p < 0.05). Top-ranked biological pathways associated with differences between symptomatic and asymptomatic data sets (identified via Ingenuity) focused on the interplay between innate and adaptive immunity, nuclear factor-kappaB and MAPK signalling, and hypercytokinemia in inducing pathology and matrix degradation. Conclusions: The inflammatory milieu within human unstable plaque is in keeping with a predominance of M1-type macrophages, and correlates with MMP production. Reference [1] Monaco, et al. Proc Natl Acad Sci U S A 2004;13(101):5634–9.
doi:10.1016/j.atherosclerosis.2010.08.041 41 Foam cell formation affects MHCI processing and presentation in vitro while hypercholesteremia induces a DC-like phenotype in macrophages in vivo K.L.L. Habets 1,∗ , V. Frodermann 1 , A.C. Foks 1 , G.H.M. Puijvelde 1 , R.E.M. Toes 2 , Th.J.C. van Berkel 1 , J. Kuiper 1
van
1
Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands 2 Department of Rheumatology, Leiden University Medical Centre, Leiden, The Netherlands
Background: Recent reports indicate that foam cell formation induces a differentiation of macrophages into a dendritic cell-like phenotype. Because dendritic cells are the most potent antigen presenting cells, these changes could have implications in antigen presentation and subsequent T cell activation. Methods and results: To evaluate the effects of foam cell formation we have cultured bone-marrow derived macrophages in the presence of oxLDL (10 g/ml). We observed a dendritic cell like phenotype with increased CD11c, MHCI, MHCII and costimulatory molecules suggesting an increased capacity to induce a specific T cell response. Consequently, we showed that MHCI antigen presentation and processing of ovalbumin was increased while MHCII induced T cell activation was not altered. Next we evaluated the effects of hypercholesteremia on peripheral macrophages in LDLr−/− mice. Again we observed an increase of CD11chigh and MHCI within the macrophage population. Furthermore, the
activation status of both dendritic cells as macrophages was increased. Conclusions: Both hypercholesteremic conditions as in vitro foam cell formation not only induced a dendritic-cell like phenotype in macrophages, but also provide macrophages with dendritic-cell like capacities. These data support the hypothesis that foam cell macrophages could play an important role in inducing an immune response during the early stages of atherosclerotic lesion development. However, further investigation about the role of extreme foam cell formation and its effect on antigen presentation in vivo needs to be further elucidated. doi:10.1016/j.atherosclerosis.2010.08.042 42 Increased foam cell formation and atherosclerotic plaque apoptosis in LDLr−/− mice lacking macrophage Mcl-1 M.M. Westra 1 , I. Bot 1 , M. Bot 1 , S.C.A. de Jager 1 , I. Dzhagalov 2 , Y.W. He 1 , Th.J.C. van Berkel 1 , E.A.L. Biessen 1,3 1
Biopharmaceutics, LACDR, Leiden, The Netherlands Department of Cardiology, Leiden University Medical Center, The Netherlands 3 Department of Pathology, CARIM, The Netherlands 2
The anti-apoptotic Bcl-1 family member Mcl-1 plays an important, in neutrophils essential, role in leukocyte survival and differentiation. Here, we investigated the impact of Mcl-1 deletion in myeloid cells on atherosclerosis in WTD fed LDLr−/−. First, Mcl-1−/− peritoneal macrophages had an increased sensitivity to Ox-LDL induced cell death and showed altered expression of several pro-apoptotic Bcl-2 family members as compared to WT macrophages. In keeping apoptotic cell content in aortic root lesions of Mcl-1−/− chimeras was elevated by 77% compared to WT controls. Second lipid uptake by peritoneal Mcl-1−/− macrophages was enhanced in vitro as well as in vivo. Third, Mcl-1−/− macrophages showed a clear shift towards a proinflammatory M1 phenotype as apparent from their cytokine expression pattern and reduced phagocytotic capacity. Despite these profound pro-atherogenic effects of Mcl-1 deficiency both plaque development and progression did not differ between Mcl-1−/− and WT BM recipients. This seeming paradox may well be imputed to altered neutrophil numbers and migratory capacity in Mcl-1 deficiency, attenuating lesional neutrophil infiltration. In conclusion, myeloid Mcl-1 deletion enhances Ox-LDL induced foam cell formation and cell death, favors a pro-inflammatory macrophage phenotype and alters neutrophil characteristics, Overall deficiency of this key gene markedly increased plaque cell apoptosis, but did neither affect plaque initiation nor growth. doi:10.1016/j.atherosclerosis.2010.08.043 43 Acute global effects of high-density lipoprotein on activated endothelial cells—Inhibition of CXCL10 A. Shoreim 1,∗ , J. Dennis 2 , K. Laing 2 , S. Petrova 1 , G.W. Cockerill 1 1
Department of Cardiovascular Sciences, St George’s University of London, Cranmer Terrace, SW17 0RE, United Kingdom 2 Department of Infectious Diseases, St George’s University of London, Cranmer Terrace, SW17 0RE, United Kingdom Rationale: Plasma concentration of high-density lipoprotein (HDL) is inversely proportional to the incidence of coronary artery
Abstracts / Atherosclerosis 213 (2010) e6–e19
disease, although the precise mechanism of protection remains unclear. Using activated human umbilical vein endothelial cells (HUVEC) as a model of ‘athero-prone’ endothelium, we investigated the acute transcriptional changes in response to treatment with reconstituted discoidal HDL (rHDL), to identify genes/processes important in mediating protection. Method: Confluent HUVECs (n = 3) activated with TNFalpha; (10 ng/ml) for 4 h were treated with rHDL (0.1 mg/ml or 1 mg/ml) for a further 8 h. Transcriptome changes were identified using SentrixTM beadchip microarray HT-12 (n = 3), per treatment. Following normalisation, data was analysed using GenespringTM . Differential expression was validated using quantitative realtime PCR.
e19
Results: In addition to changes in expression of genes involved in lipid metabolism, we have identified changes in CXCL10, a cytokine-induced T-cell chemokine important in innate immunity. Treatment with rHDL (1 mg/ml) results in a significant reduction in cytokine-induced expression: 2.25 ± 0.27 (TNFalpha) vs. 0.98 ± 0.12 (rHDL), n-3, p ≤0.05. Conclusion: Our data supports a role for HDL in reducing the innate immune response. Further studies will investigate the effect of HDL on the protein expression and time course of endothelial CXCL10. doi:10.1016/j.atherosclerosis.2010.08.044