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NEUTROPHIL RECRUITMENT IN MURAL THROMBUS OF HUMAN ABDOMINAL AORTIC ANEURYSM
14
Workshops WO14 Chemokine and innate receptors in vascular disease
Methods: Immunofluorescence confocal microscopy, Western blotting, cryothin section-immunogold electron microscopy and SDS-freeze-fracture immunocytochemistry. Results: Our present findings represent a challenge to the prevailing hypothesis of lipid droplet biogenesis. Especially the three-dimensional perspectives provided by freeze-fracture electron microscopy demonstrate unequivocally that at sites of close association the lipid droplet is not situated within the ER membrane; rather, both ER membranes lie external to and follow the contour of the lipid droplet, enclosing it in a manner akin to an egg cup (the ER) holding an egg (the lipid droplet). Freeze-fracture cytochemistry demonstrates that the PAT family protein adipophilin is concentrated in prominent clusters in the cytoplasmic leaflet of the ER membrane closely apposed to the lipid droplet envelope. Conclusion: The obvious associations of lipid droplets, adipophilin clusters and ER membranes indicate that these features play important roles in the transfer of lipids and proteins into lipid droplets. These findings call for a re-evaluation of the prevailing hypothesis of lipid droplet biogenesis. WO13-OR6 INFLAMMATION CAUSES STATIN RESISTANCE: A POTENTIAL MECHANISM FOR INEFFECTIVENESS OF STATIN THERAPY X.Z. Ruan 1 , Y. Chen 2 , A. Huang 2 , J.F. Moorhead 1 , S.H. Powis 1 , Z. Varghese 1 . 1 Lipid Research Unit, Centre for Nephrology, Royal Free and University College Medical School, UCL, London, UK; 2 Centre for Lipid Research, Chongqing Medical University, Chongqing, China Background and aims: Inhibitors of HMGCoA reductase have revolutionized the treatment of high blood cholesterol levels. This leads to a significant reduction in plasma cholesterol level and cardiovascular disease in the general population. However, the 4D trial in dialyzed patients with diabetes-associated inflammatory states failed to show a significant reduction in cardiovascular events. We aimed to test if chronic inflammation causes statin resistance. Methods: Human hepatic cells (HepG2) were cultured with various dose of mevastatin in the absence or presence of pro-inflammatory cytokine IL-1beta. HMGCoA reductase activity was measured by detecting the amount of mevalonate-3-14C. Cholesterol synthesis was observed by measuring incorporation of 14C acetate into non-soaponifiablelipids. The mRNA expression was examined by real time PCR. Results: Mevastatin decreased HMGCoA reductase activity and intracellular cholesterol synthesis, thereby up-regulated LDL receptor mRNA expression in the absence of IL-1beta. However, IL-1beta overrode the suppression of HMGCoA reductase activity and cholesterol synthesis caused by mevastatin. The function of statin was impaired under inflammatory stress. The concentration of mevastatin for 50% inhibition of HMGCoA reductase activity (IC50) was 30 microM, but we observed only 28% inhibition at this concentration in the presence of IL-1beta. The concentration of mevastatin required for 200% up-regulation of LDL receptor (UC200) was 0.4microM, which was significantly increased to 1.7microM in the presence of IL-1beta. Conclusions: Inflammation overrides the suppression of HMGCoA reductase activity by statins and causes statin resistance. Hence a higher concentration of statin may be required to achieve the same biological effects in inflammatory states.
WO14 CHEMOKINE AND INNATE RECEPTORS IN VASCULAR DISEASE WO14-OR3 NEUTROPHIL RECRUITMENT IN MURAL THROMBUS OF HUMAN ABDOMINAL AORTIC ANEURYSM X. Houard, Z. Touat, V. Ollivier, M. Philippe, L. Louedec, J.B. Michel. INSERM U698, Bichat Hospital, Paris, France Background and aims: Mural thrombus plays a major role in the progression of abdominal aortic aneurysm (AAA), especially via its enrichment of neutrophils. This study focused on mediators involved in neutrophil recruitment to AAA mural thrombus. Methods: Luminal, intermediate and abluminal layers of 29 human mural thrombi were dissected and separately incubated in cell culture medium. Conditioned media were then analyzed for neutrophil markers (elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase
and α-defensin peptides), RANTES, platelet factor 4 (PF4) and interleukin8. In parallel, neutrophil chemotaxis induced by conditioned media was assessed in vitro. This study was completed by immunohistochemical localization of neutrophils (CD66b, NGAL, proteinase 3), platelets (CD61) interleukin-8 and RANTES in AAA. Results: Immunohistochemistry for neutrophils and platelets showed their localization in the luminal layer of the thrombus at the interface with the circulating blood. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were mainly released by the luminal pole of the thrombus where their concentrations were significantly correlated. The thrombus luminal layer was also the main source of interleukin-8. This luminal pole of the thrombus displays potent chemotactic activity in vitro for human neutrophils as compared to the abluminal pole. Interestingly, interleukin-8 concentration released by the luminal layer correlates with neutrophil chemotaxis activity. In addition, RANTES and interleukin-8 blocking antibodies inhibit neutrophil chemotaxis induced by conditioned media from the luminal part of the thrombus. Conclusions: Taken together, these results suggest that platelet-derived RANTES and interleukin-8 are involved in neutrophil recruitment to the luminal layer of the mural thrombus of AAA. WO14-OR4 FREE HEMOGLOBIN:A CHEMOTACTIC STIMULUS FOR THE INNATE IMMUNITY B. Buttari 1 , E. Profumo 1 , A. Tagliani 2 , F. Ippoliti 3 , R. Businaro 2 , R. Rigano’ 2 . 1 Dipartimento Di Malattie Infettive, Parassitarie Ed Immunomediate, Istituto Superiore Di Sanità, Rome, Italy; 2 Dipartimento Di Scienze Cardiovascolari, Università Di Roma Sapienza, Rome, Italy; 3 Dipartimento Di Medicina Sperimentale, Università Di Roma Sapienza, Rome, Italy Background and aims: Atherosclerosis is a chronic inflammatory disease in which immune responses are key pathogenetic factors. The inflammatory cell infiltrate within atherosclerotic plaques consists of activated T cells, B cells, monocytes, dendritic cells. Convincing evidence supports a determinant role of autoimmune responses to self antigens in the progression of atherosclerosis. The signals that induce cells infiltration into the atherosclerotic lesions are not well identified. It is known that most autoantigens induce chemokine receptor mediated cell migration. We demonstrated that haemoglobin (Hb) activates innate and adaptive immune responses in patients with carotid atherosclerosis. The aims of this study was to investigate the possible chemotactic activity of human Hb for human immature dendritic cells (iDCs) and to identify the corresponding receptors. Methods: Hb (10-200 ng/ml) with or without the chemokine Mip-1β (100 ng/ml) was placed in the lower wells of microBoyden chemotaxis chamber and 100 µl of monocyte-derived iDCs were placed in the upper wells for 4 hours. Migrated cells were analyzed by flow cytometry. To investigate the involvement of receptor binding, we analyzed ERK1/2 and p38 signal pathways by the use of Face Chemi assays and we monitored calcium mobilization before and after stimulation with Hb by the use of Fura-2. Results: Our findings indicate that Hb chemoattracts iDCs, and actively interacts with membrane DC receptors inducing the phosphorilation of ERK1/2 and p38. Conclusion: We hypothesize that free Hb, abundant in advanced lesions, contributes to DC infiltration into atherosclerotic lesions thus leading to subsequent amplification of the inflammatory response. WO14-OR5 A NOVEL ROLE FOR IGF2/IGF2R SIGNALING IN HOMING OF ENDOTHELIAL PROGENITOR CELLS Y.-G. Kwon, Y.S. Maeng. Dept. of Biochemistry, Yonsei University, Seoul, South Korea Endothelial progenitor cells (EPC) contribute to the formation of vascular networks during embryonic development and also to neovascularization in settings of tissue ischemia or tumors in postnatal life. However, the soluble factors responsible for EPC recruitment to a neovascular zone are rarely identified. In this study, we show that insulin-like growth factor 2 (IGF2), which has a comparable activity with stromal cell-derived factor-1, is a prominent homing factor for EPCs. The IGF2/mannose-6 phosphate receptor (IGF2R) is highly expressed in EPCs isolated from human cord blood and CD34+ mononuclear cells from mouse bone marrow (BMMNCs), whereas IGF2 was not present in these cells. IGF2 enhanced
77th Congress of the European Atherosclerosis Society, April 26–29, 2008, Istanbul, Turkey
Workshops WO14 Chemokine and innate receptors in vascular disease
Methods: Immunofluorescence confocal microscopy, Western blotting, cryothin section-immunogold electron microscopy and SDS-freeze-fracture immunocytochemistry. Results: Our present findings represent a challenge to the prevailing hypothesis of lipid droplet biogenesis. Especially the three-dimensional perspectives provided by freeze-fracture electron microscopy demonstrate unequivocally that at sites of close association the lipid droplet is not situated within the ER membrane; rather, both ER membranes lie external to and follow the contour of the lipid droplet, enclosing it in a manner akin to an egg cup (the ER) holding an egg (the lipid droplet). Freeze-fracture cytochemistry demonstrates that the PAT family protein adipophilin is concentrated in prominent clusters in the cytoplasmic leaflet of the ER membrane closely apposed to the lipid droplet envelope. Conclusion: The obvious associations of lipid droplets, adipophilin clusters and ER membranes indicate that these features play important roles in the transfer of lipids and proteins into lipid droplets. These findings call for a re-evaluation of the prevailing hypothesis of lipid droplet biogenesis. WO13-OR6 INFLAMMATION CAUSES STATIN RESISTANCE: A POTENTIAL MECHANISM FOR INEFFECTIVENESS OF STATIN THERAPY X.Z. Ruan 1 , Y. Chen 2 , A. Huang 2 , J.F. Moorhead 1 , S.H. Powis 1 , Z. Varghese 1 . 1 Lipid Research Unit, Centre for Nephrology, Royal Free and University College Medical School, UCL, London, UK; 2 Centre for Lipid Research, Chongqing Medical University, Chongqing, China Background and aims: Inhibitors of HMGCoA reductase have revolutionized the treatment of high blood cholesterol levels. This leads to a significant reduction in plasma cholesterol level and cardiovascular disease in the general population. However, the 4D trial in dialyzed patients with diabetes-associated inflammatory states failed to show a significant reduction in cardiovascular events. We aimed to test if chronic inflammation causes statin resistance. Methods: Human hepatic cells (HepG2) were cultured with various dose of mevastatin in the absence or presence of pro-inflammatory cytokine IL-1beta. HMGCoA reductase activity was measured by detecting the amount of mevalonate-3-14C. Cholesterol synthesis was observed by measuring incorporation of 14C acetate into non-soaponifiablelipids. The mRNA expression was examined by real time PCR. Results: Mevastatin decreased HMGCoA reductase activity and intracellular cholesterol synthesis, thereby up-regulated LDL receptor mRNA expression in the absence of IL-1beta. However, IL-1beta overrode the suppression of HMGCoA reductase activity and cholesterol synthesis caused by mevastatin. The function of statin was impaired under inflammatory stress. The concentration of mevastatin for 50% inhibition of HMGCoA reductase activity (IC50) was 30 microM, but we observed only 28% inhibition at this concentration in the presence of IL-1beta. The concentration of mevastatin required for 200% up-regulation of LDL receptor (UC200) was 0.4microM, which was significantly increased to 1.7microM in the presence of IL-1beta. Conclusions: Inflammation overrides the suppression of HMGCoA reductase activity by statins and causes statin resistance. Hence a higher concentration of statin may be required to achieve the same biological effects in inflammatory states.
WO14 CHEMOKINE AND INNATE RECEPTORS IN VASCULAR DISEASE WO14-OR3 NEUTROPHIL RECRUITMENT IN MURAL THROMBUS OF HUMAN ABDOMINAL AORTIC ANEURYSM X. Houard, Z. Touat, V. Ollivier, M. Philippe, L. Louedec, J.B. Michel. INSERM U698, Bichat Hospital, Paris, France Background and aims: Mural thrombus plays a major role in the progression of abdominal aortic aneurysm (AAA), especially via its enrichment of neutrophils. This study focused on mediators involved in neutrophil recruitment to AAA mural thrombus. Methods: Luminal, intermediate and abluminal layers of 29 human mural thrombi were dissected and separately incubated in cell culture medium. Conditioned media were then analyzed for neutrophil markers (elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase
and α-defensin peptides), RANTES, platelet factor 4 (PF4) and interleukin8. In parallel, neutrophil chemotaxis induced by conditioned media was assessed in vitro. This study was completed by immunohistochemical localization of neutrophils (CD66b, NGAL, proteinase 3), platelets (CD61) interleukin-8 and RANTES in AAA. Results: Immunohistochemistry for neutrophils and platelets showed their localization in the luminal layer of the thrombus at the interface with the circulating blood. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were mainly released by the luminal pole of the thrombus where their concentrations were significantly correlated. The thrombus luminal layer was also the main source of interleukin-8. This luminal pole of the thrombus displays potent chemotactic activity in vitro for human neutrophils as compared to the abluminal pole. Interestingly, interleukin-8 concentration released by the luminal layer correlates with neutrophil chemotaxis activity. In addition, RANTES and interleukin-8 blocking antibodies inhibit neutrophil chemotaxis induced by conditioned media from the luminal part of the thrombus. Conclusions: Taken together, these results suggest that platelet-derived RANTES and interleukin-8 are involved in neutrophil recruitment to the luminal layer of the mural thrombus of AAA. WO14-OR4 FREE HEMOGLOBIN:A CHEMOTACTIC STIMULUS FOR THE INNATE IMMUNITY B. Buttari 1 , E. Profumo 1 , A. Tagliani 2 , F. Ippoliti 3 , R. Businaro 2 , R. Rigano’ 2 . 1 Dipartimento Di Malattie Infettive, Parassitarie Ed Immunomediate, Istituto Superiore Di Sanità, Rome, Italy; 2 Dipartimento Di Scienze Cardiovascolari, Università Di Roma Sapienza, Rome, Italy; 3 Dipartimento Di Medicina Sperimentale, Università Di Roma Sapienza, Rome, Italy Background and aims: Atherosclerosis is a chronic inflammatory disease in which immune responses are key pathogenetic factors. The inflammatory cell infiltrate within atherosclerotic plaques consists of activated T cells, B cells, monocytes, dendritic cells. Convincing evidence supports a determinant role of autoimmune responses to self antigens in the progression of atherosclerosis. The signals that induce cells infiltration into the atherosclerotic lesions are not well identified. It is known that most autoantigens induce chemokine receptor mediated cell migration. We demonstrated that haemoglobin (Hb) activates innate and adaptive immune responses in patients with carotid atherosclerosis. The aims of this study was to investigate the possible chemotactic activity of human Hb for human immature dendritic cells (iDCs) and to identify the corresponding receptors. Methods: Hb (10-200 ng/ml) with or without the chemokine Mip-1β (100 ng/ml) was placed in the lower wells of microBoyden chemotaxis chamber and 100 µl of monocyte-derived iDCs were placed in the upper wells for 4 hours. Migrated cells were analyzed by flow cytometry. To investigate the involvement of receptor binding, we analyzed ERK1/2 and p38 signal pathways by the use of Face Chemi assays and we monitored calcium mobilization before and after stimulation with Hb by the use of Fura-2. Results: Our findings indicate that Hb chemoattracts iDCs, and actively interacts with membrane DC receptors inducing the phosphorilation of ERK1/2 and p38. Conclusion: We hypothesize that free Hb, abundant in advanced lesions, contributes to DC infiltration into atherosclerotic lesions thus leading to subsequent amplification of the inflammatory response. WO14-OR5 A NOVEL ROLE FOR IGF2/IGF2R SIGNALING IN HOMING OF ENDOTHELIAL PROGENITOR CELLS Y.-G. Kwon, Y.S. Maeng. Dept. of Biochemistry, Yonsei University, Seoul, South Korea Endothelial progenitor cells (EPC) contribute to the formation of vascular networks during embryonic development and also to neovascularization in settings of tissue ischemia or tumors in postnatal life. However, the soluble factors responsible for EPC recruitment to a neovascular zone are rarely identified. In this study, we show that insulin-like growth factor 2 (IGF2), which has a comparable activity with stromal cell-derived factor-1, is a prominent homing factor for EPCs. The IGF2/mannose-6 phosphate receptor (IGF2R) is highly expressed in EPCs isolated from human cord blood and CD34+ mononuclear cells from mouse bone marrow (BMMNCs), whereas IGF2 was not present in these cells. IGF2 enhanced
77th Congress of the European Atherosclerosis Society, April 26–29, 2008, Istanbul, Turkey