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THE EFFECTS OF LOW-DENSITY LIPOPROTEIN ON THE ADHESION OF LEUKOCYTES ON ENDOTHELIAL CELLS
Poster Abstracts / Cardiovascular Pathology 13 (2004) S17–S79
P036 THE EFFECTS OF LOW-DENSITY LIPOPROTEIN ON THE ADHESION OF LEUKOCYTES ON ENDOTHELIAL CELLS. Chauying Jen, Chih-Yung Huang, Hsiun-ing Chen. Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 701, TAIWAN. Previous studies have shown that the endothelium overlying atherosclerotic lesions expresses various adhesion molecules. Both native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) have been reported to increase the expression of adhesion molecules in endothelial cells. Some of these adhesion molecules may be involved in the progression of rolling/ adhesion of leukocytes. Therefore, LDL may contribute to the development of arterial atherosclerosis by altering the function of vascular endothelium, promoting the adhesion of leukocytes and their migration from vascular lumen to intima. This study was undertaken to quantify the adhesiveness between various leukocytes and endothelial cells pretreated with nLDL or oxLDL. To address this subject, we used a tapered flow chamber that has a linear variation of shear stress across it flow channel, starting from a predetermined maximum value at the entrance and falling to zero at the exit. Cultured human umbilical vein endothelial cells (HUVECs) on a glass slide served as the testing surface of the chamber to allow sedimentation and adhesion of leukocytes. The nLDL was prepared from human serum by density gradient ultracentrifugation, and subsequently oxidized to different extent by copper-ion-catalysis. The degree of oxidation of oxLDL was characterized by agarose gel electrophoresis, by detecting the TBAR values and the production of conjugated-diene. Peripheral leukocytes collected from normal male subjects were loaded into this chamber and their adhesiveness to HUVECs was accessed by flushing them with a buffer under specific local shear stresses. Our preliminary data showed that the mobility of oxLDL on agarose gel increased with oxidation time. When oxLDL was oxidized with copper for 12 hours, it had the highest TBAR value. The pretreatment of oxLDL (0.1 mg/ml) for more than 16 hours would change cell morphology of HUVECs, but these cells survived longer than 24 hours. Leukocytes adhered more readily to oxLDL-pretreated HUVECs than to the control group. In the future, we well apply specific antibodies to verify which adhesion molecules are involved in these adhesive interactions. This study should provide valuable information about not only the adhesion between leukocytes and endothelial cells, but also the roles of LDL in modulating this important cell-cell interaction. Supported by National Sciences Council, Taiwan (Grant No. NSC92-2320B-006-040, NSC91-2320-B-006-044, and NSC91-2320-B-006-045)
P037 POTENTIAL ROLE FOR GLYCOGEN SYNTHASE KINASE (GSK)-3 IN ENDOPLASMIC RETICULUM (ER) STRESS-INDUCED ATHEROSCLEROSIS. Anna J Kim, Yuan Y Shi, Ji Zhou, Richard C Austin, Geoff H Werstuck. McMaster University and the Henderson Research Centre, Hamilton, Ontario. Diabetes mellitus is a major independent risk factor for cardiovascular disease and stroke however the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway leading to the production of glucosamine-6-phosphate. We have shown that elevated levels of intracellular glucosamine interfere with protein folding in the endoplasmic reticulum (ER) causing ER stress in cell types relevant to the development of atherosclerosis, including human aortic smooth muscle cells, endothelial cells, monocytes and hepatocytes. We have demonstrated that glucosamine and other ER stress-inducing agents promote lipid accumulation, activate inflammatory pathways and cause apoptosis – the hallmark features of atherosclerosis. We hypothesize that the accumulation of intracellular glucosamine observed in conditions of hyperglycemia may promote
S29
atherogenesis via a mechanism that involves ER stress. To examine the relevance of the ER stress pathway in vivo, we have developed a streptozotocin-induced diabetic, apoE-deficient mouse model of atherosclerosis. Using molecular biological and histological techniques we have shown that hyperglycemia is associated with accelerated atherosclerosis, tissue specific ER stress and disrupted lipid metabolism. The identification of this novel atherogenic pathway has provided us with new targets for the development of potential anti-atherosclerotic drugs. Our results suggest that glycogen synthase kinase (GSK) – 3 activity plays a role in ER stress-induced cellular dysfunction. Furthermore, we have demonstrated that by inhibiting GSK-3 we can protect cells from ER stress-induced lipid accumulation and apoptosis. We are currently investigating the anti-atherogenic effects of novel GSK-3 inhibitors in our animal model systems. Canadian Institutes for Health Research Ontario Research and Development Challenge Fund Hamilton Health Science Foundation
P038 ATHERIN: A NEWLY IDENTIFIED LDL-BINDING PROTEIN IN HUMAN ATHEROSCLEROTIC LESIONS. Ann M. Lees, Anne E. Deconinck, Bruce D. Campbell, Robert S. Lees. Boston Heart Foundation, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, Atherex, Inc., Cambridge, MA. Focal retention of LDL in the artery wall is one of the earliest events in atherogenesis. It is a critical step because macrophages become foam cells, the precursors of fatty streaks and atherosclerotic plaques, by accumulating LDL that has been retained in the artery long enough to become extensively oxidized. In earlier experiments with the healing balloon-catheter deendothelialized rabbit aorta, a model for very early arterial lesions, we found prolonged focal LDL accumulation localized to the edges of regenerating endothelial islands. To identify previously unrecognized LDL-binding proteins specific for atherosclerotic lesions, we generated a cDNA expression library from healing deendothelialized rabbit aorta. In addition to native LDL, we screened the library with methylated LDL, which is not recognized by cell-surface receptors, to avoid selecting known LDLbinding proteins. Screening led to the identification of a new, 56 kDa LDL-binding protein that we call atherin. It binds LDL with a Kd of 15 nM, and is highly conserved across species, with over 90% amino-acid sequence identity between human, rabbit, rat, and mouse. We performed immunofluorescent and confocal microscopic studies of human atherosclerotic lesions freshly obtained at autopsy, using antibodies against LDL, atherin, and CD68, a marker for macrophages. We found consistent co-localization of atherin and LDL in very early lesions represented by minimal intimal thickening, as well as in advanced lesions. Atherin was not present in normal artery. In lesions, it appears to be made by smooth muscle cells, which export it to the extracellular compartment where it binds LDL. Colocalization also occurs within foam cells, which appear to ingest a tightly bound complex of LDL and atherin. We conclude that focal accumulation of LDL in arterial lesions is initiated by binding of LDL to atherin, and thus, that atherin plays a primary role in atherogenesis through its ability to immobilize LDL long enough for it to become extensively oxidized and internalized by macrophages.
P039 HIGH-DENSITY LIPOPROTEIN SUBFRACTION 3 DECREASES ADAMTS-1 EXPRESSION INDUCED. Giuseppe Danilo Norata, Hanna Bjo¨rk, Anders Hamsten, Alberico Luigi Catapano, Per Eriksson. Department of Pharmacological Sciences, University of Milan, Italy, Atherosclerosis Research Unit, King Gustaf V Research Institute, Department of Medicine, Karolinska Hospital. Endothelial expression of matrix metalloproteinases has been implicated in angiogenesis and endothelial cell proliferation. Recently, it has been
P036 THE EFFECTS OF LOW-DENSITY LIPOPROTEIN ON THE ADHESION OF LEUKOCYTES ON ENDOTHELIAL CELLS. Chauying Jen, Chih-Yung Huang, Hsiun-ing Chen. Department of Physiology, College of Medicine, National Cheng Kung University, Tainan 701, TAIWAN. Previous studies have shown that the endothelium overlying atherosclerotic lesions expresses various adhesion molecules. Both native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) have been reported to increase the expression of adhesion molecules in endothelial cells. Some of these adhesion molecules may be involved in the progression of rolling/ adhesion of leukocytes. Therefore, LDL may contribute to the development of arterial atherosclerosis by altering the function of vascular endothelium, promoting the adhesion of leukocytes and their migration from vascular lumen to intima. This study was undertaken to quantify the adhesiveness between various leukocytes and endothelial cells pretreated with nLDL or oxLDL. To address this subject, we used a tapered flow chamber that has a linear variation of shear stress across it flow channel, starting from a predetermined maximum value at the entrance and falling to zero at the exit. Cultured human umbilical vein endothelial cells (HUVECs) on a glass slide served as the testing surface of the chamber to allow sedimentation and adhesion of leukocytes. The nLDL was prepared from human serum by density gradient ultracentrifugation, and subsequently oxidized to different extent by copper-ion-catalysis. The degree of oxidation of oxLDL was characterized by agarose gel electrophoresis, by detecting the TBAR values and the production of conjugated-diene. Peripheral leukocytes collected from normal male subjects were loaded into this chamber and their adhesiveness to HUVECs was accessed by flushing them with a buffer under specific local shear stresses. Our preliminary data showed that the mobility of oxLDL on agarose gel increased with oxidation time. When oxLDL was oxidized with copper for 12 hours, it had the highest TBAR value. The pretreatment of oxLDL (0.1 mg/ml) for more than 16 hours would change cell morphology of HUVECs, but these cells survived longer than 24 hours. Leukocytes adhered more readily to oxLDL-pretreated HUVECs than to the control group. In the future, we well apply specific antibodies to verify which adhesion molecules are involved in these adhesive interactions. This study should provide valuable information about not only the adhesion between leukocytes and endothelial cells, but also the roles of LDL in modulating this important cell-cell interaction. Supported by National Sciences Council, Taiwan (Grant No. NSC92-2320B-006-040, NSC91-2320-B-006-044, and NSC91-2320-B-006-045)
P037 POTENTIAL ROLE FOR GLYCOGEN SYNTHASE KINASE (GSK)-3 IN ENDOPLASMIC RETICULUM (ER) STRESS-INDUCED ATHEROSCLEROSIS. Anna J Kim, Yuan Y Shi, Ji Zhou, Richard C Austin, Geoff H Werstuck. McMaster University and the Henderson Research Centre, Hamilton, Ontario. Diabetes mellitus is a major independent risk factor for cardiovascular disease and stroke however the molecular and cellular mechanisms by which diabetes contributes to the development of vascular disease are not fully understood. It is well established that hyperglycemia promotes intracellular glucose flux through the hexosamine pathway leading to the production of glucosamine-6-phosphate. We have shown that elevated levels of intracellular glucosamine interfere with protein folding in the endoplasmic reticulum (ER) causing ER stress in cell types relevant to the development of atherosclerosis, including human aortic smooth muscle cells, endothelial cells, monocytes and hepatocytes. We have demonstrated that glucosamine and other ER stress-inducing agents promote lipid accumulation, activate inflammatory pathways and cause apoptosis – the hallmark features of atherosclerosis. We hypothesize that the accumulation of intracellular glucosamine observed in conditions of hyperglycemia may promote
S29
atherogenesis via a mechanism that involves ER stress. To examine the relevance of the ER stress pathway in vivo, we have developed a streptozotocin-induced diabetic, apoE-deficient mouse model of atherosclerosis. Using molecular biological and histological techniques we have shown that hyperglycemia is associated with accelerated atherosclerosis, tissue specific ER stress and disrupted lipid metabolism. The identification of this novel atherogenic pathway has provided us with new targets for the development of potential anti-atherosclerotic drugs. Our results suggest that glycogen synthase kinase (GSK) – 3 activity plays a role in ER stress-induced cellular dysfunction. Furthermore, we have demonstrated that by inhibiting GSK-3 we can protect cells from ER stress-induced lipid accumulation and apoptosis. We are currently investigating the anti-atherogenic effects of novel GSK-3 inhibitors in our animal model systems. Canadian Institutes for Health Research Ontario Research and Development Challenge Fund Hamilton Health Science Foundation
P038 ATHERIN: A NEWLY IDENTIFIED LDL-BINDING PROTEIN IN HUMAN ATHEROSCLEROTIC LESIONS. Ann M. Lees, Anne E. Deconinck, Bruce D. Campbell, Robert S. Lees. Boston Heart Foundation, Harvard-MIT Division of Health Sciences and Technology, Cambridge, MA, Atherex, Inc., Cambridge, MA. Focal retention of LDL in the artery wall is one of the earliest events in atherogenesis. It is a critical step because macrophages become foam cells, the precursors of fatty streaks and atherosclerotic plaques, by accumulating LDL that has been retained in the artery long enough to become extensively oxidized. In earlier experiments with the healing balloon-catheter deendothelialized rabbit aorta, a model for very early arterial lesions, we found prolonged focal LDL accumulation localized to the edges of regenerating endothelial islands. To identify previously unrecognized LDL-binding proteins specific for atherosclerotic lesions, we generated a cDNA expression library from healing deendothelialized rabbit aorta. In addition to native LDL, we screened the library with methylated LDL, which is not recognized by cell-surface receptors, to avoid selecting known LDLbinding proteins. Screening led to the identification of a new, 56 kDa LDL-binding protein that we call atherin. It binds LDL with a Kd of 15 nM, and is highly conserved across species, with over 90% amino-acid sequence identity between human, rabbit, rat, and mouse. We performed immunofluorescent and confocal microscopic studies of human atherosclerotic lesions freshly obtained at autopsy, using antibodies against LDL, atherin, and CD68, a marker for macrophages. We found consistent co-localization of atherin and LDL in very early lesions represented by minimal intimal thickening, as well as in advanced lesions. Atherin was not present in normal artery. In lesions, it appears to be made by smooth muscle cells, which export it to the extracellular compartment where it binds LDL. Colocalization also occurs within foam cells, which appear to ingest a tightly bound complex of LDL and atherin. We conclude that focal accumulation of LDL in arterial lesions is initiated by binding of LDL to atherin, and thus, that atherin plays a primary role in atherogenesis through its ability to immobilize LDL long enough for it to become extensively oxidized and internalized by macrophages.
P039 HIGH-DENSITY LIPOPROTEIN SUBFRACTION 3 DECREASES ADAMTS-1 EXPRESSION INDUCED. Giuseppe Danilo Norata, Hanna Bjo¨rk, Anders Hamsten, Alberico Luigi Catapano, Per Eriksson. Department of Pharmacological Sciences, University of Milan, Italy, Atherosclerosis Research Unit, King Gustaf V Research Institute, Department of Medicine, Karolinska Hospital. Endothelial expression of matrix metalloproteinases has been implicated in angiogenesis and endothelial cell proliferation. Recently, it has been