- Email: [email protected]
Early platelet activation by low density lipoprotein
Thursday June 29, 2000: Poster Abstracts P: W35 Monogenic Hyperlipidemia that to non-transfected cells. Furthermore, both monoclonal antibody EO6, which binds to the oxidized phospholipid POVPC, as well as a POVPC-BSA adduct were able to inhibit the binding of OxLDL to SR-B 1. Conclusions: In addition to its well-described "selective uptake" function, SR-B1 has the ability to "scavenge" OxLDL and, potentially, apoptotic cells as well. The interaction of OxLDL with SR-B1 is apparently mediated to some extent by both the protein moieties and the lipid moieties of the intact particle. In addition, the oxidized pbospholipid POVPC is an important ligand on OxLDL that mediates recognition by SR-B1, ThP21 "VV34] Very low density lipoprotein (VLDL) receptor expression
is activated by dexamethasone in a glncocorticoid receptor-dependent manner in adipocytic 3T3-L1 cells K. Ensler, B. Angelin, M. GMvels. Center for Metabolism and Endocrinology, Department of Medicine, Karolinska Institute at Huddinge Hospital, Sweden LRP and LDL receptors have previously been postulated to be important for the homeostasis of apoE-containing lipoproteins. VLDL receptors belong to the same gene family and binds multiple ligands, some of them related to lipoprotein metabolism. Due to a high expression of VLDL receptors in adipose tissue and muscle the VLDL receptor has been proposed to contribute to lipoprotein metabolism in extrahepatic tissues. These hypotheses are controversial and recent studies link the VLDL receptor to neuronal migration in mice possibly by interaction with non-lipoprotein ligands. Since VLDL receptor knockout mice demonstarted a relatively slower rate of adipose tissue accumulation compared to intact mice we investigated the expression and regulation of VLDL receptors in 3T3-L1 cells, a cell line able to transform from fibroblast to adipocyte-[ike phenotype by addition of dexamethasonc, insulin and isobutylmethylxanthine in the culture medium. During the first 1-3 days of adipocytic formation 3T3-L1 cells showed an increased expression of VLDL receptors. This stimulation could be mimicked by dexamethasone (1 mM) alone in a time- and dosedependent manner. Inclusion of the GR blocker RU-486 (10 raM) inhibited the response. The mouse VLDL receptor promoter was isolated and sequenced. No classical DR-3cis- acting element (GGTACANNNTGTFCT) could be demonstrated. Transfection of 3T3-LI cells with reporter genes containing 1.6, 2.6 and 3.6 kb of upstream promoter sequence showed stimulation by dexamethasone and abrogation by inclusion of RU-486. We demonstrate a hormonal regulation by dexamethasone on the VLDL receptor that involves indirect or direct GR-mediated on the VLDL receptor promoter. Studies adressing the issue of dexamethasone regulation in rive is ongoing.
I ThP22:W34 [ Lipoprotein receptors in extraembryonic tissues of the chicken M. Hermann, M.G. Mahon, K.A. Lindstedt ] , J. Nimpf, W.J. Schneider. Department of Molecular Genetics, A-1030 Vienna, Austria; 2Wihuri Research Institute, SF-O0140 Helsinki, Finland Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via the LDL receptor family member, LR8 (EMBO J. 13, 5165-5175). Endocytosis is accompanied by partial degradation of the yolk precursors' protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. This receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed, at ratios that vary with embryonic age, in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allamois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labelled VLDL in cultured EEC. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Since the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which effects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo. (Supported by the Austrian Science Foundation FWF F-606, F-608).
299
ThP23:W34 ] Early platelet activation by low density lipoprotein I.A.M. Relou 1'2, C.M. Hackeng 1'2 , G. Gorter I , H.A.M. Voorbij 2, H.J.M. van Rijn2, J.-W.N. Akkerman i . t Laboratory for Thrombosis and Haemostasis, Department of Haematology; 2Department of Clinical Chemistry, University Medical Center, Utrecht, The Netherlands Low density lipoprotein (LDL) is known to increase the sensitivity of human platelets for agonists. Little is known about the receptor and the signalling pathways involved. The platelet LDL receptor is different from the classical LDL receptor, described by Brown and Goldstein. We have earlier reported how LDL might sensitize platelets. A first step is the activation of the enzyme p38 MAPK which is activated within 10 sec (at 1 g apoB100/L) after addition of LDL and at concentrations as low as 0.1 g apoB100/L (within 10 rain). A second, equally rapid step is the activation of focal adhesion kinase (pI25FAK), which is an essential step in the formation of focal adhesion plaques, p38 MAPKand p125FAK-activation are both upstream of most platelet signalling pathways and therefore close to the platelet LDL-receptor. It is still unclear which component of LDL is responsible for platelet sensitization. Modification of lysine residues of apt B 100 via carbamylation reduces p38 MAPK activation by 80%, indicating that apoBl00 is required for binding to the platelet surface and subsequent signal generation. Anti-human apoB 100 monoclonal antibodies, 4G3 and 2D8 are both directed against regions of apoB 100 which are susceptible for carbamylation. The antibody 4G3, directed against aa 2980-3084, which is described to inhibit LDL binding to the 'classical' LDL receptor, reduced LDL-platelet binding and subsequent p38 MAPK phosphorylation by 50%. In contrast, 2D8, directed against aa 1438-1481, showed no reduction. After carbamylation of apoBl00, LDL is still able to phosphorylate p38 MAPK after extended incubation time (1-2 hrs), suggesting that the lipid moiety of LDL might also contribute to platelet activation. These observations imply that specific domains of apoB100 are essential for targeting LDL to the platelet membrane, whereafter other signal transduction pathways can be initiated by the lipid moiety.
l ThP24:W34 ] 1
Identification of an HDL and apoAl binding site on baculovirus expressed extraceHular domain of HB2
N. Fidge, Y. Fu. Baker Medical Research Institute Melbourne, Australia
Objective: To confirm the function of HB2 as a candidate HDL receptor. Methods: Constructs of the extracellular domain of HB2 were engineered that coded for residues Ex (extracellular) 1-144, Ex 1-247, Ex 1-334, Ex 1--415 and Ex 1-527, the latter residue terminating at the membrane domain. After transfer to the vector (pBac PAK8) expression plasmids were cotransfected into Spodoptera frugiperda with BacPAK6 viral DNA and bacfectin to generate recombinant baculovirus proteins with yields ranging from 7-12 mg/l culture medium. Ligand blots were performed using HDL, apoAI or LDL as ligands and binding was detected with specific antibodies. Results: Mapping of HDL binding site. Truncated peptides 1-144, 1-247, 1-334 and 1-415 did not bind apoAI or HDL; however HDL binding to peptide 1-527 was similar in strength as binding to full length HB2. Thus a specific binding domain is located proximal (within 144 residues) of the membrane region. Additional experiments showed that the extracellular domain bound apoAI and apoAII but not LDL, confirming the specificity reported previously for full length HB2. Conclusions: Together with receni data demonstrating a connection between HB2 and sterol synthesis, this designation of a precise extracellular binding domain is compelling evidence for a role of HB2 in lipid metabolism, influenced by HDL apoprotein.
P:W35
MONOGENIC HYPERLIPIDEMIA
ThP1 :W35 ] Impaired TG removal in well-controlled type III
hyperlipoproteinemia Kazuo Ishiwata., Yasuhiko Homma, Koichiro Homma, Hideki Ozawa, Shunnosuke Handa. Department of lnternal Medicine, Tokai University School of Medicine, lsehara, 259-1193, Japan Objective: TG removal activity in patients with well-controlled type HI hyperlipoproteinemia was assessed by IV-FrT. Methods: The study was performed in ten normal subjects, and two brothers with type III hyperlipoproteinemia. The older brother showed mild renal
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
"1" C
0
is activated by dexamethasone in a glncocorticoid receptor-dependent manner in adipocytic 3T3-L1 cells K. Ensler, B. Angelin, M. GMvels. Center for Metabolism and Endocrinology, Department of Medicine, Karolinska Institute at Huddinge Hospital, Sweden LRP and LDL receptors have previously been postulated to be important for the homeostasis of apoE-containing lipoproteins. VLDL receptors belong to the same gene family and binds multiple ligands, some of them related to lipoprotein metabolism. Due to a high expression of VLDL receptors in adipose tissue and muscle the VLDL receptor has been proposed to contribute to lipoprotein metabolism in extrahepatic tissues. These hypotheses are controversial and recent studies link the VLDL receptor to neuronal migration in mice possibly by interaction with non-lipoprotein ligands. Since VLDL receptor knockout mice demonstarted a relatively slower rate of adipose tissue accumulation compared to intact mice we investigated the expression and regulation of VLDL receptors in 3T3-L1 cells, a cell line able to transform from fibroblast to adipocyte-[ike phenotype by addition of dexamethasonc, insulin and isobutylmethylxanthine in the culture medium. During the first 1-3 days of adipocytic formation 3T3-L1 cells showed an increased expression of VLDL receptors. This stimulation could be mimicked by dexamethasone (1 mM) alone in a time- and dosedependent manner. Inclusion of the GR blocker RU-486 (10 raM) inhibited the response. The mouse VLDL receptor promoter was isolated and sequenced. No classical DR-3cis- acting element (GGTACANNNTGTFCT) could be demonstrated. Transfection of 3T3-LI cells with reporter genes containing 1.6, 2.6 and 3.6 kb of upstream promoter sequence showed stimulation by dexamethasone and abrogation by inclusion of RU-486. We demonstrate a hormonal regulation by dexamethasone on the VLDL receptor that involves indirect or direct GR-mediated on the VLDL receptor promoter. Studies adressing the issue of dexamethasone regulation in rive is ongoing.
I ThP22:W34 [ Lipoprotein receptors in extraembryonic tissues of the chicken M. Hermann, M.G. Mahon, K.A. Lindstedt ] , J. Nimpf, W.J. Schneider. Department of Molecular Genetics, A-1030 Vienna, Austria; 2Wihuri Research Institute, SF-O0140 Helsinki, Finland Yolk is the major source of nutrients for the developing chicken embryo, but molecular details of the delivery mechanisms are largely unknown. During oogenesis in the chicken, the yolk components vitellogenin and very low density lipoprotein (VLDL) are taken up into the oocytes via the LDL receptor family member, LR8 (EMBO J. 13, 5165-5175). Endocytosis is accompanied by partial degradation of the yolk precursors' protein moieties; however, fragmentation does not abolish binding of VLDL to LR8. This receptor exists in two isoforms that differ by a so-called O-linked sugar domain; the shorter form (LR8-) is the major form in oocytes, and the longer protein (LR8+) predominates in somatic cells. Here we show that both LR8 isoforms are expressed, at ratios that vary with embryonic age, in the extraembryonic yolk sac, which mobilizes yolk for utilization by the embryo, and in the allamois, the embryo's catabolic sink. Stored yolk VLDL interacts with LR8 localized on the surface of the yolk sac endodermal endothelial cells (EEC), is internalized, and degraded, as demonstrated by the catabolism of fluorescently labelled VLDL in cultured EEC. Importantly, EEC express significant levels of microsomal triglyceride transfer protein and protein disulfide isomerase, key components required for lipoprotein synthesis. Since the apolipoprotein pattern of VLDL isolated from the yolk sac-efferent omphalomesenteric vein is very different from that of yolk VLDL, these data strongly suggest that embryo plasma VLDL is resynthesized in the EEC. LR8 is a key mediator of a two-step pathway, which effects the uptake of VLDL from the yolk sac and the subsequent delivery of its components to the growing embryo. (Supported by the Austrian Science Foundation FWF F-606, F-608).
299
ThP23:W34 ] Early platelet activation by low density lipoprotein I.A.M. Relou 1'2, C.M. Hackeng 1'2 , G. Gorter I , H.A.M. Voorbij 2, H.J.M. van Rijn2, J.-W.N. Akkerman i . t Laboratory for Thrombosis and Haemostasis, Department of Haematology; 2Department of Clinical Chemistry, University Medical Center, Utrecht, The Netherlands Low density lipoprotein (LDL) is known to increase the sensitivity of human platelets for agonists. Little is known about the receptor and the signalling pathways involved. The platelet LDL receptor is different from the classical LDL receptor, described by Brown and Goldstein. We have earlier reported how LDL might sensitize platelets. A first step is the activation of the enzyme p38 MAPK which is activated within 10 sec (at 1 g apoB100/L) after addition of LDL and at concentrations as low as 0.1 g apoB100/L (within 10 rain). A second, equally rapid step is the activation of focal adhesion kinase (pI25FAK), which is an essential step in the formation of focal adhesion plaques, p38 MAPKand p125FAK-activation are both upstream of most platelet signalling pathways and therefore close to the platelet LDL-receptor. It is still unclear which component of LDL is responsible for platelet sensitization. Modification of lysine residues of apt B 100 via carbamylation reduces p38 MAPK activation by 80%, indicating that apoBl00 is required for binding to the platelet surface and subsequent signal generation. Anti-human apoB 100 monoclonal antibodies, 4G3 and 2D8 are both directed against regions of apoB 100 which are susceptible for carbamylation. The antibody 4G3, directed against aa 2980-3084, which is described to inhibit LDL binding to the 'classical' LDL receptor, reduced LDL-platelet binding and subsequent p38 MAPK phosphorylation by 50%. In contrast, 2D8, directed against aa 1438-1481, showed no reduction. After carbamylation of apoBl00, LDL is still able to phosphorylate p38 MAPK after extended incubation time (1-2 hrs), suggesting that the lipid moiety of LDL might also contribute to platelet activation. These observations imply that specific domains of apoB100 are essential for targeting LDL to the platelet membrane, whereafter other signal transduction pathways can be initiated by the lipid moiety.
l ThP24:W34 ] 1
Identification of an HDL and apoAl binding site on baculovirus expressed extraceHular domain of HB2
N. Fidge, Y. Fu. Baker Medical Research Institute Melbourne, Australia
Objective: To confirm the function of HB2 as a candidate HDL receptor. Methods: Constructs of the extracellular domain of HB2 were engineered that coded for residues Ex (extracellular) 1-144, Ex 1-247, Ex 1-334, Ex 1--415 and Ex 1-527, the latter residue terminating at the membrane domain. After transfer to the vector (pBac PAK8) expression plasmids were cotransfected into Spodoptera frugiperda with BacPAK6 viral DNA and bacfectin to generate recombinant baculovirus proteins with yields ranging from 7-12 mg/l culture medium. Ligand blots were performed using HDL, apoAI or LDL as ligands and binding was detected with specific antibodies. Results: Mapping of HDL binding site. Truncated peptides 1-144, 1-247, 1-334 and 1-415 did not bind apoAI or HDL; however HDL binding to peptide 1-527 was similar in strength as binding to full length HB2. Thus a specific binding domain is located proximal (within 144 residues) of the membrane region. Additional experiments showed that the extracellular domain bound apoAI and apoAII but not LDL, confirming the specificity reported previously for full length HB2. Conclusions: Together with receni data demonstrating a connection between HB2 and sterol synthesis, this designation of a precise extracellular binding domain is compelling evidence for a role of HB2 in lipid metabolism, influenced by HDL apoprotein.
P:W35
MONOGENIC HYPERLIPIDEMIA
ThP1 :W35 ] Impaired TG removal in well-controlled type III
hyperlipoproteinemia Kazuo Ishiwata., Yasuhiko Homma, Koichiro Homma, Hideki Ozawa, Shunnosuke Handa. Department of lnternal Medicine, Tokai University School of Medicine, lsehara, 259-1193, Japan Objective: TG removal activity in patients with well-controlled type HI hyperlipoproteinemia was assessed by IV-FrT. Methods: The study was performed in ten normal subjects, and two brothers with type III hyperlipoproteinemia. The older brother showed mild renal
Xllth International Symposium on Atherosclerosis, Stockholm, Sweden, June 25-29, 2000
"1" C
0