- Email: [email protected]
3.P.169 Expression of tissue factor and tissue factor pathway inhibitor in human macrophages
Wednesday 8 October 1997 : Posters Macrophages and atherosclerosis of acyl CoA : cholesterol acyl transferase (ACAT) results in the accumulation of free cholesterol (FC) . In the absence of an extracellular FC acceptor, deposition of FC is accompanied by a stimulation in phospholipid (PL) synthesis and cell toxicity . In the present study we observed that the metabolic changes that are associated with FC accumulation were blocked if the ACAT-treated cells were simultaneously exposed to the cholesterol intracellular transport inhibitor U18666A (2 sg/ml) . Furthermore, these effects were not due to differences in free cholesterol accumulation . Phospholipid synthesis and cell toxicity were reduced or completely eliminated with U18666A co-treatment . This implies that the FC in these cells is in a different subcellular location than the FC in cells treated with an ACAT inhibitor alone . To address this we investigated FC efflux. FC efflux from untreated CE-loaded macrophages to cyclodextrins exhibits kinetics indicative of two pools of cholesterol ; a fast and a slow pool. The addition of the ACAT inhibitor CP- 113, 818 (2 zg/ml) significantly decreases the t1/2 for cholesterol efflux from the kinetically slow pool of cholesterol . ACAT inhibition had no other effect on cholesterol efflux . The cells co-treated with U18666A had kinetics of efflux similar to those of control cells. Thus, it appears that U18666A sequesters excess FC in an intracellular pool that does not trigger metabolic responses associated with FC toxicity and is not readily available for efflux to cyclodextrins .
3 .P.165
Adhesion of monocytes in a coculture model of the arterial wall . Influence of LDL and LPC
F. Kinardl, F. Verhoeyel, K. Jaworskil , Th . Sergent-Ennelen 2 , P. Holvoet 3 , D . Collen3 , Y.J . Schneider2 , A. Trouet', C . Remaclet . Laboratory of Cell Biology; 2 Laboratory of Cell Biochemistry; University of Louvain; 3 Center for Molecular and Vascular Biology, University of Leuven, Belgium One of the first events occurring in the process of atherogenesis is the adhesion of monocytes to the endothelial cells. The model we used to study these interactions is a compartmentalized coculture of arterial endothelial and smooth muscle cells. These cells were harvested from porcine arteries and grown to two passages from primary culture, in serum-containing medium . Thereafter, the cells were plated on the opposite sides of a poly-(ethylene terephtalate) membrane with pores of 1 ttm or 3 ttm diameter, and cultivated in serum-free medium . Low density lipoproteins (LDL) were isolated from human plasma by ultracentrifugation . Endothelial cells inoculated alone (monoculture) or in coculture with smooth muscle cells were preincubated with native LDL, copper oxidized LDL, malondialdehyde modified LDL (MDA-LDL), or lyso-phosphatidylcholine (LPC), for 24 hours . The culture media were then removed and THP-1 monocytic cells labelled with Dil were inoculated on the endothelial cells . After 6 hours, cultures were fixed, counterstained with DiO and THP-I cells were counted . Whereas no influence of native LDL, oxidized LDL and MDA-LDL was observed, the number of THP-1 that adhered to endothelial cells preincubated with LPC was 2 times higher in monoculture and 1 .6 times higher in coculture, than in the control group without LDL . As shown in a preliminary experiment, the presence of serum factors was necessary to induce an increase of THP-1 adhesion to endothelial cells preincubated with oxidized LDL . These experiments will also permit to study the successive steps of the interactions of monocytes with the in vitro model of the arterial wall : migration under the endothelium, and then to the medial layer, followed by the lipid accumulation .
3.P.166
Proteolytic enzyme release by macrophages in the destabilisation process of atherosclerotic plaques
H .J . Knieriem', Z. Jurukova 2 .'Dept. Path, Bethesda Hosp., Duisburg, Germany; 2 Dept. of Path., Med. Academy, Sofia, Bulgaria Metallopreoteinases (MMPs)-a group of enzymes that degrade extracellular matrix (ECM) components produced by macrophages (Ma) are involved in localized fragilization and weakening of the collagen and elastin framework of the atherosclerotic plaque . We investigated human atheroscl . plaques removed by endarterectomy (n = 34) . Immunohistochemically the cellular composition of the plaque (SMC, Ma) was analysed and the cellular expression of MMPs and the endogenous inhibitors of MMPs - TIMPs were identified . In fibrolipid plaques with a thick fibrocellular cap overlying a limited lipid core (n = 16) SMCs of the lesion stained for gelatinase-A and TIMP-1 and TIMP-2 . Groups of Ma-foam cells were positive for stromelysin, TIMP-1 and TIMP-2 . In contrast, in plaques with a large atheroma, covered by a distinct fibrous cap, large Ma- derived foam cell accumulations in the fibrous cap and around the lipid core exhibited an intensive expression of gelatinase A, stromelysin and interstitial collagenase, and a very low, or even lacking expression of TIMPs . Our results demonstrate an enhanced production of MMPs by Ma's in
233
unstable plaques with a large atheroma, correlated with a suppressed secretion of TIMPs and promoting ECM-catabolism . These data reveal the role of MMPs in the destruction of the fibrous cap and therefore supporting the destabilization process of atherosclerotic plaques .
3 .P.167
Cholesterol metabolism and efflux in human THP-1 macrophages
L . Kritharides 12 , A . Christian, G. Stoudt2 , D. Morel', G . RothblaO . 'The Heart Research Institute, Sydney, Australia ; 'Medical College of Pennsylvania, Philadelphia, USA Lipid metabolism and cholesterol efflux in human THP-1 monocytemacrophages have been investigated to clarify the behavior of macrophages in human atherosclerosis . THP-1 macrophages were loaded with acetylated low density lipoprotein (AcLDL) together with 3H-cholesterol (3H-FC) . Free (FC) and esterified (CE) cholesterol mass were determined by gas chromatography and radiolabelled FC and CE were separated by thin layer chromatography . After loading, THP- 1 macrophages accumulated FC and CE, assessed by both mass and radioactivity. Inhibition of acyl CoA acyl transferase (ACAT) during loading decreased cell 3H-CE by 95 f 1 .4% and cell CE mass by 66.0 ± 4 .0%, indicating some cell CE mass represented undegraded AcLDL-derived CE . In AcLDL-loaded cells pulsed with 3H-oleate, CE, triglyceride (TG) and phospholipid fractions respectively contained 1 .0 f 0 .07%, 80.0 ± 0 .5%, and 18 .9 plusmn ; 0 .3% of cell 3H-oleate . Cell TG mass determined enzymatically ranged front 74 .8 ± 10.0 to 142.7 ± 10 .1 µg/mg cell protein over several experiments, and greatly exceeded cell cholesterol ester mass after 24 hour loading with AcLDL . ACAT inhibition after loading established that 37 .1 ± 4 .9% of cellular 3H-CE was hydrolysed over 24 hours, and hydrolysis was unaffected by cholesterol efflux . Efflux kinetics were determined after selectively loading cells with FC . Human apolipoprotein A-I (apo A-I), rHDL-PC (reconstituted HDL) and HDL3 (high density lipoprotein) respectively removed 46.6 ± 3 .7%, 61 .3 ± 3 .4%, and 76 .4 ± 10.1% of 3H-FC from THP-I cells in 24 firs . Of these acceptors, only phospholipid-free apo A-I became saturated with cholesterol by 24 hrs . 97% of effluxed FC derived from a slow pool with a ti/2 ranging from 27 .7 h for HDL to 69 .3 h for Apo A-I. In conclusion, human THP-1 cells contain large quantities of metabolically active triglyceride, demonstrate slow CE hydrolysis, and net FC efflux is substantially enhanced by the presence of phospholipid in acceptor particles .
3 .P.168
Aggregated LDL induces and enters surface-connected compartments of human monocyte-macrophages : A process independent of the LDL receptor
H.S . Kruth, W.Y. Zhang, P.M . Gaynor. Section of Experimental Atherosclerosis, NHLBI, NIH, Bethesda, Maryland Aggregation of LDL stimulates its uptake by macrophages. We have now shown that aggregated LDL produced by vortexing (VxLDL) induces and becomes sequestered in large amounts within surface-connected compartments (SCC) of human monocyte-derived macrophages, through a process different from phagocytosis . Because of their surface connections, trypsin could release VxLDL from these SCC . Formation of SCC and accumulation of VxLDL in SSC were temperature dependent cell-mediated processes blocked by cytochalasin D, but not by nocodazole . The pathways for sequestration of VxLDL within SCC and degradation of VxLDL were different . Degradation of 125 1-VxLDL saturated at 100 Ag/ml while cell-association of 125 I-VxLDL did not saturate up to 400 Ag/ml . Further, uptake of VxLDL into SCC was not mediated by the LDL receptor because sequestration was not decreased by heparin, cholesterol-enrichment of macrophages, or methylation of LDL . This was in contrast to the pathway that mediated most of the degradation of VxLDL (possibly phagocytosis), which did appear to be mediated by the LDL receptor. VxLDL in SCC was degraded only slowly but through a chloroquine-sensitive pathway . This novel endocytosis pathway for uptake of aggregated LDL allows the macrophage to store large amounts of aggregated LDL lipoprotein before it is processed further.
3 .P.169
Expression of tissue factor and tissue factor pathway inhibitor in human macrophages
P. Lesnik, L . Petit, C . Dachet, M. Moreau, J . Chapman. INSERM U-321, La Pitie, 83, Bd. de l'H6pital, 75651 Paris Cedex 13, France Lipid-laden macrophages are a prominent feature of rupture-prone regions of atherosclerotic plaques . These cells express Tissue factor (TF), which after
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Wednesday 8 October 1997 : Posters Macrophages and atherosclerosis
plaque rupture may be exposed to the bloodstream, resulting in activation of the extrinsic coagulation pathway . Since TF is a major determinant of thrombin generation, we have investigated the possibility that macrophages or macrophage foam cells synthesize Tissue Factor Pathway Inhibitor (TFPI), thereby contributing to local regulation of TF activity. Indeed, Northern blot analysis of TFPI mRNA from cultured human monocyte-derived macrophages (HMDM) revealed a single band at 4.2 kb with weak intensity. Moreover, gel filtration of HMDM supernatants showed the presence of an active 100 kDa form of TFPI, which was recovered in SDS-PAGE under non-reducing conditions ; under reducing conditions however, the immunoblot revealed a 40 kDa form of TFPI. The TFPI present in HMDM supernatants possessed heparin binding affinity, suggesting potential interaction of TFPI with heparin sulphate proteoglycans . In addition, stimulation of foam cell formation by incubation of macrophages for 48 hours with exogenous free cholesterol indicated that neither the biological activity nor the de novo synthesis of TFPI protein were affected . In contrast, cholesterol loading induced significant upregulation of TF activity (23 .10 ± 4 .57 vs 9 .70 ± 0 .68 mU/mg cell protein ; cholesterol-treated cells vs control cells, p = 0 .01). Our data indicate that enhancement of the procoagulant activity of TF in macrophage-derived foam cells is not counterbalanced by upregulation of TFPI activity, thereby suggesting that macrophage foam cells are in a procoagulant state ; they may therefore contribute to thrombus generation upon plaque rupture .
3 .R170
Butylated hydroxytoluene and N-acetylcysteine decrease the production of tumor necrosis factor-a from human alveolar macrophages
L . Mattsson Hultdn, H. Lindmark, H . Schersten, G. Riise, F. Nilsson, O . Wiklund. Wallenberg Laboratory and Cardiothoracic Surgery, Sahlgrenska University Hospital, Goteborg, Sweden Tumor necrosis factor-a (TNF-a) is a polypeptide cytokine with a wide range of metabolic, immunologic, and inflammatory activities . The numerous immunomodulatory effects of TNF-a suggest a central role for this mediator in association with atherosclerosis . Antioxidants have been described as inhibitors of LPS stimulated TNF-a production in rabbit alveolar macrophages and in human mononuclear cells but not in mouse peritoneal macrophages . In this study we investigated the possible role of antioxidants, such as butylated hydroxytoluene (BHT) and N-acetylcysteine (NAC) in regulating TNF production in human alveolar macrophages . Macrophages from lung-transplanted patients were cultured with BHT (10 /Lg/ml) or NAC (1 mg/ml), with or without lipopolysaccaride (LPS) activation . TNF-a was analysed using ELISA-kit and quantitative RT PCR was used for the TNF-a mRNA analyses . After a 4 h incubation with BHT, a decreased release of TNF-a was demonstrated (43 pg/mg cell protein and 473 pg/mg cell protein from BHT treated and control cells, respectively) . Incubation of macrophages with NAC resulted in an about 60% reduction of TNF-a secretion and a 50% reduction in TNF-a mRNA . In LPS-stimulated cells the TNF-a secretion was 2704 pg/mg cell protein, and 479 pg/mg cell protein when BHT was present . Similarly, the secretion of TNF-a from LPS-activated cells was decreased by 60% in the presence of NAC . In summary, human alveolar macrophages treated with an antioxidant suppress the secretion of TNF-a . These findings may have important implications in the treatment of atherosclerosis by alter the inflammatory processes of the disease .
3 .P.171
The activation of protein kinase C is involved in oxidized low density lipoprotein-induced macrophage growth
Takeshi Matsumura l , Shozo Kobori l , Masakazu Sakail, Yoshichika Anami l , Takeshi Biwa1 , Tons Takemura l , Seikoh Horiuchi2 , Motoaki Shichiri t . 'Department of Metabolic Medicine ; DDepartment of Biochemistry, Kumamoto University Scool of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan Recent studies from our laboratory have demonstrated that oxidized low density lipoprotein (Ox-LDL) induces macrophage growth in vitro . The present study was undertaken to elucidate the intracellular signaling pathways for macrophage growth . Ox-LDL initiated a rapid and transient rise in intracellular free calcium ion and induced activation of membrane protein kinase C (PKC) . Pertussis toxin completely inhibited Ox-LDL-induced rise in free calcium ion and significantly inhibited macrophage growth by 50% . Moreover, PKC inhibitors, calphostin C and H-7, significantly inhibited Ox-LDL-induced macrophage growth by 80% . On the other hand, phospholipase A2-treated acetylated LDL did not induce rise in calcium, but significantly induced activation of PKC . Moreover, phospholipase A2-treated acetylated LDL-induced macrophage growth was significantly inhibited by calphostin C by 90% .
Our results suggest that the presence of two intracellular signaling pathways for activation of PKC, a rise in calcium which was mediated by pertussis toxin sensitive G-protein and the internalization of lysophosphatidylcholine through the scavenger receptor (SR-Al/All) . These two pathways may play an important role in Ox-LDL-induced macrophage growth .
3 .P.172
Embryonic stem cell differentiated macrophages : A model system for the evaluation of macrophage foam cell formation and function
K .J . Moore, L.P. Andersson, M .W. Freeman . Massachusetts General Hospital, Boston MA, USA In the early development of the atherosclerotic plaque, monocytes are recruited to the intima where they bind and internalize modified lipoproteins to form foam cells. Foam cell formation is thought to depend on the uptake of modified LDL by macrophage (MO) scavenger receptors (SR), which include members of the SR-A and SR-B families of these transmembrane proteins . Studies of the consequence of foam cell formation on MO behaviour have been hampered by limitations of the available monocyte/Mo cell lines . In this study, we explored the use of in vitro differentiated embryonic stem (ES) cells for studying M¢ foam cell formation and function . ES differentiated M¢ (ES-MO) were >90% positive for the MO markers F4/80 and CD11b as determined by immunostaining . ES-MO phagocytosed latex beads and produced cytokines in response to inflammatory stimuli . Expression of the modified LDL receptors SR-A and CD36 was demonstrated in ES-MO by RT PCR, and SR-A expression was confirmed by immunostaining . In the presence of acetylated LDL (AcLDL), ES-Mc formed characteristic foam cells as visualized by oil red 0 staining . Furthermore, ES-MO degradation of 125 1-AcLDL exceeded that of the P388D1 cell line commonly exploited in SR degradation studies . In conclusion, these results demonstrate that ES-M¢ express the modified LDL receptors SR-A and CD36, internalize and degrade AcLDL, and thus, are a suitable model system for studying M¢ foam cell formation. The ease of genetic manipulation of ES cells prior to differentiation to MO provides a powerful experimental tool to identify key events in foam cells formation and function .
3 . P.173*
Expression of cholesterol 7a-hydroxylase in cultured RAW macrophages reduces cholesterol loading by acetyl LDL
Gina L. Moore, Neil Sheth, Roger A . Davis . Mammalian Cell and Molecular Biology Lab, San Diego State University, San Diego, California, USA Cholesterol 7a-hydroxylase (7aH) provides the liver with the unique ability to take up cholesterol-rich lipoproteins from plasma and excrete the cholesterol into bile through the formation of bile acids . We examined the possibility that expression of 7aH in macrophages might help prevent the accumulation of cholesterol esters, a hallmark of atherosclerosis . Cholesterol metabolism between RAW mouse macrophages, stably expressing 7aH (7a-RAW) and wild-type RAW cells were compared with and without acetyl-LDL . In the absence of acetyl-LDL, the cellular content of free cholesterol was similar between both cell types (in .sg/mg protein : 8 ± 5 vs . 6 ± 3 for RAW and 7a-RAW cells, respectively) . In contrast, the mass of cholesterol esters in RAW cells was 4-fold greater compared to 7a-RAW cells (in ttg/mg protein : 1 .2 vs . 0.31) . When cultured in the presence of 25 itg protein/ml acetyl-LDI, for 48 hours, RAW cells accumulated more free cholesterol (75 ± 14 µg/mg vs . 48 ± 5 ttg/mg protein) and cholesterol ester (41 .2 ttg/mg vs . 2.9 ttg/mg protein) than that observed in 7a-RAW cells . The decreased accumulation of cholesterol and cholesterol esters in 7a-RAW cells was not associated with decreased levels of synthesis, as determined by 14C-acetate incorporation . 7a-RAW cells displayed a greater rate of excretion of 14C-cholesterol into the cultured medium compared to wild-type cells . This excretion of 14 C-cholesterol was associated with the formation and excretion of a polar 14 C-labeled sterol . Based on these data, we propose that expression of 7aH decreases the accumulation of cholesterol by producing an amphipathic sterol . This amphipathic sterol, like bile acids, may promote the efflux of cholesterol from the macrophage .
11th International Symposium on Atherosclerosis, Paris, October 1997
3 .P.165
Adhesion of monocytes in a coculture model of the arterial wall . Influence of LDL and LPC
F. Kinardl, F. Verhoeyel, K. Jaworskil , Th . Sergent-Ennelen 2 , P. Holvoet 3 , D . Collen3 , Y.J . Schneider2 , A. Trouet', C . Remaclet . Laboratory of Cell Biology; 2 Laboratory of Cell Biochemistry; University of Louvain; 3 Center for Molecular and Vascular Biology, University of Leuven, Belgium One of the first events occurring in the process of atherogenesis is the adhesion of monocytes to the endothelial cells. The model we used to study these interactions is a compartmentalized coculture of arterial endothelial and smooth muscle cells. These cells were harvested from porcine arteries and grown to two passages from primary culture, in serum-containing medium . Thereafter, the cells were plated on the opposite sides of a poly-(ethylene terephtalate) membrane with pores of 1 ttm or 3 ttm diameter, and cultivated in serum-free medium . Low density lipoproteins (LDL) were isolated from human plasma by ultracentrifugation . Endothelial cells inoculated alone (monoculture) or in coculture with smooth muscle cells were preincubated with native LDL, copper oxidized LDL, malondialdehyde modified LDL (MDA-LDL), or lyso-phosphatidylcholine (LPC), for 24 hours . The culture media were then removed and THP-1 monocytic cells labelled with Dil were inoculated on the endothelial cells . After 6 hours, cultures were fixed, counterstained with DiO and THP-I cells were counted . Whereas no influence of native LDL, oxidized LDL and MDA-LDL was observed, the number of THP-1 that adhered to endothelial cells preincubated with LPC was 2 times higher in monoculture and 1 .6 times higher in coculture, than in the control group without LDL . As shown in a preliminary experiment, the presence of serum factors was necessary to induce an increase of THP-1 adhesion to endothelial cells preincubated with oxidized LDL . These experiments will also permit to study the successive steps of the interactions of monocytes with the in vitro model of the arterial wall : migration under the endothelium, and then to the medial layer, followed by the lipid accumulation .
3.P.166
Proteolytic enzyme release by macrophages in the destabilisation process of atherosclerotic plaques
H .J . Knieriem', Z. Jurukova 2 .'Dept. Path, Bethesda Hosp., Duisburg, Germany; 2 Dept. of Path., Med. Academy, Sofia, Bulgaria Metallopreoteinases (MMPs)-a group of enzymes that degrade extracellular matrix (ECM) components produced by macrophages (Ma) are involved in localized fragilization and weakening of the collagen and elastin framework of the atherosclerotic plaque . We investigated human atheroscl . plaques removed by endarterectomy (n = 34) . Immunohistochemically the cellular composition of the plaque (SMC, Ma) was analysed and the cellular expression of MMPs and the endogenous inhibitors of MMPs - TIMPs were identified . In fibrolipid plaques with a thick fibrocellular cap overlying a limited lipid core (n = 16) SMCs of the lesion stained for gelatinase-A and TIMP-1 and TIMP-2 . Groups of Ma-foam cells were positive for stromelysin, TIMP-1 and TIMP-2 . In contrast, in plaques with a large atheroma, covered by a distinct fibrous cap, large Ma- derived foam cell accumulations in the fibrous cap and around the lipid core exhibited an intensive expression of gelatinase A, stromelysin and interstitial collagenase, and a very low, or even lacking expression of TIMPs . Our results demonstrate an enhanced production of MMPs by Ma's in
233
unstable plaques with a large atheroma, correlated with a suppressed secretion of TIMPs and promoting ECM-catabolism . These data reveal the role of MMPs in the destruction of the fibrous cap and therefore supporting the destabilization process of atherosclerotic plaques .
3 .P.167
Cholesterol metabolism and efflux in human THP-1 macrophages
L . Kritharides 12 , A . Christian, G. Stoudt2 , D. Morel', G . RothblaO . 'The Heart Research Institute, Sydney, Australia ; 'Medical College of Pennsylvania, Philadelphia, USA Lipid metabolism and cholesterol efflux in human THP-1 monocytemacrophages have been investigated to clarify the behavior of macrophages in human atherosclerosis . THP-1 macrophages were loaded with acetylated low density lipoprotein (AcLDL) together with 3H-cholesterol (3H-FC) . Free (FC) and esterified (CE) cholesterol mass were determined by gas chromatography and radiolabelled FC and CE were separated by thin layer chromatography . After loading, THP- 1 macrophages accumulated FC and CE, assessed by both mass and radioactivity. Inhibition of acyl CoA acyl transferase (ACAT) during loading decreased cell 3H-CE by 95 f 1 .4% and cell CE mass by 66.0 ± 4 .0%, indicating some cell CE mass represented undegraded AcLDL-derived CE . In AcLDL-loaded cells pulsed with 3H-oleate, CE, triglyceride (TG) and phospholipid fractions respectively contained 1 .0 f 0 .07%, 80.0 ± 0 .5%, and 18 .9 plusmn ; 0 .3% of cell 3H-oleate . Cell TG mass determined enzymatically ranged front 74 .8 ± 10.0 to 142.7 ± 10 .1 µg/mg cell protein over several experiments, and greatly exceeded cell cholesterol ester mass after 24 hour loading with AcLDL . ACAT inhibition after loading established that 37 .1 ± 4 .9% of cellular 3H-CE was hydrolysed over 24 hours, and hydrolysis was unaffected by cholesterol efflux . Efflux kinetics were determined after selectively loading cells with FC . Human apolipoprotein A-I (apo A-I), rHDL-PC (reconstituted HDL) and HDL3 (high density lipoprotein) respectively removed 46.6 ± 3 .7%, 61 .3 ± 3 .4%, and 76 .4 ± 10.1% of 3H-FC from THP-I cells in 24 firs . Of these acceptors, only phospholipid-free apo A-I became saturated with cholesterol by 24 hrs . 97% of effluxed FC derived from a slow pool with a ti/2 ranging from 27 .7 h for HDL to 69 .3 h for Apo A-I. In conclusion, human THP-1 cells contain large quantities of metabolically active triglyceride, demonstrate slow CE hydrolysis, and net FC efflux is substantially enhanced by the presence of phospholipid in acceptor particles .
3 .P.168
Aggregated LDL induces and enters surface-connected compartments of human monocyte-macrophages : A process independent of the LDL receptor
H.S . Kruth, W.Y. Zhang, P.M . Gaynor. Section of Experimental Atherosclerosis, NHLBI, NIH, Bethesda, Maryland Aggregation of LDL stimulates its uptake by macrophages. We have now shown that aggregated LDL produced by vortexing (VxLDL) induces and becomes sequestered in large amounts within surface-connected compartments (SCC) of human monocyte-derived macrophages, through a process different from phagocytosis . Because of their surface connections, trypsin could release VxLDL from these SCC . Formation of SCC and accumulation of VxLDL in SSC were temperature dependent cell-mediated processes blocked by cytochalasin D, but not by nocodazole . The pathways for sequestration of VxLDL within SCC and degradation of VxLDL were different . Degradation of 125 1-VxLDL saturated at 100 Ag/ml while cell-association of 125 I-VxLDL did not saturate up to 400 Ag/ml . Further, uptake of VxLDL into SCC was not mediated by the LDL receptor because sequestration was not decreased by heparin, cholesterol-enrichment of macrophages, or methylation of LDL . This was in contrast to the pathway that mediated most of the degradation of VxLDL (possibly phagocytosis), which did appear to be mediated by the LDL receptor. VxLDL in SCC was degraded only slowly but through a chloroquine-sensitive pathway . This novel endocytosis pathway for uptake of aggregated LDL allows the macrophage to store large amounts of aggregated LDL lipoprotein before it is processed further.
3 .P.169
Expression of tissue factor and tissue factor pathway inhibitor in human macrophages
P. Lesnik, L . Petit, C . Dachet, M. Moreau, J . Chapman. INSERM U-321, La Pitie, 83, Bd. de l'H6pital, 75651 Paris Cedex 13, France Lipid-laden macrophages are a prominent feature of rupture-prone regions of atherosclerotic plaques . These cells express Tissue factor (TF), which after
11th International Symposium on Atherosclerosis, Paris, October 1997
234
Wednesday 8 October 1997 : Posters Macrophages and atherosclerosis
plaque rupture may be exposed to the bloodstream, resulting in activation of the extrinsic coagulation pathway . Since TF is a major determinant of thrombin generation, we have investigated the possibility that macrophages or macrophage foam cells synthesize Tissue Factor Pathway Inhibitor (TFPI), thereby contributing to local regulation of TF activity. Indeed, Northern blot analysis of TFPI mRNA from cultured human monocyte-derived macrophages (HMDM) revealed a single band at 4.2 kb with weak intensity. Moreover, gel filtration of HMDM supernatants showed the presence of an active 100 kDa form of TFPI, which was recovered in SDS-PAGE under non-reducing conditions ; under reducing conditions however, the immunoblot revealed a 40 kDa form of TFPI. The TFPI present in HMDM supernatants possessed heparin binding affinity, suggesting potential interaction of TFPI with heparin sulphate proteoglycans . In addition, stimulation of foam cell formation by incubation of macrophages for 48 hours with exogenous free cholesterol indicated that neither the biological activity nor the de novo synthesis of TFPI protein were affected . In contrast, cholesterol loading induced significant upregulation of TF activity (23 .10 ± 4 .57 vs 9 .70 ± 0 .68 mU/mg cell protein ; cholesterol-treated cells vs control cells, p = 0 .01). Our data indicate that enhancement of the procoagulant activity of TF in macrophage-derived foam cells is not counterbalanced by upregulation of TFPI activity, thereby suggesting that macrophage foam cells are in a procoagulant state ; they may therefore contribute to thrombus generation upon plaque rupture .
3 .R170
Butylated hydroxytoluene and N-acetylcysteine decrease the production of tumor necrosis factor-a from human alveolar macrophages
L . Mattsson Hultdn, H. Lindmark, H . Schersten, G. Riise, F. Nilsson, O . Wiklund. Wallenberg Laboratory and Cardiothoracic Surgery, Sahlgrenska University Hospital, Goteborg, Sweden Tumor necrosis factor-a (TNF-a) is a polypeptide cytokine with a wide range of metabolic, immunologic, and inflammatory activities . The numerous immunomodulatory effects of TNF-a suggest a central role for this mediator in association with atherosclerosis . Antioxidants have been described as inhibitors of LPS stimulated TNF-a production in rabbit alveolar macrophages and in human mononuclear cells but not in mouse peritoneal macrophages . In this study we investigated the possible role of antioxidants, such as butylated hydroxytoluene (BHT) and N-acetylcysteine (NAC) in regulating TNF production in human alveolar macrophages . Macrophages from lung-transplanted patients were cultured with BHT (10 /Lg/ml) or NAC (1 mg/ml), with or without lipopolysaccaride (LPS) activation . TNF-a was analysed using ELISA-kit and quantitative RT PCR was used for the TNF-a mRNA analyses . After a 4 h incubation with BHT, a decreased release of TNF-a was demonstrated (43 pg/mg cell protein and 473 pg/mg cell protein from BHT treated and control cells, respectively) . Incubation of macrophages with NAC resulted in an about 60% reduction of TNF-a secretion and a 50% reduction in TNF-a mRNA . In LPS-stimulated cells the TNF-a secretion was 2704 pg/mg cell protein, and 479 pg/mg cell protein when BHT was present . Similarly, the secretion of TNF-a from LPS-activated cells was decreased by 60% in the presence of NAC . In summary, human alveolar macrophages treated with an antioxidant suppress the secretion of TNF-a . These findings may have important implications in the treatment of atherosclerosis by alter the inflammatory processes of the disease .
3 .P.171
The activation of protein kinase C is involved in oxidized low density lipoprotein-induced macrophage growth
Takeshi Matsumura l , Shozo Kobori l , Masakazu Sakail, Yoshichika Anami l , Takeshi Biwa1 , Tons Takemura l , Seikoh Horiuchi2 , Motoaki Shichiri t . 'Department of Metabolic Medicine ; DDepartment of Biochemistry, Kumamoto University Scool of Medicine, 1-1-1 Honjo, Kumamoto 860, Japan Recent studies from our laboratory have demonstrated that oxidized low density lipoprotein (Ox-LDL) induces macrophage growth in vitro . The present study was undertaken to elucidate the intracellular signaling pathways for macrophage growth . Ox-LDL initiated a rapid and transient rise in intracellular free calcium ion and induced activation of membrane protein kinase C (PKC) . Pertussis toxin completely inhibited Ox-LDL-induced rise in free calcium ion and significantly inhibited macrophage growth by 50% . Moreover, PKC inhibitors, calphostin C and H-7, significantly inhibited Ox-LDL-induced macrophage growth by 80% . On the other hand, phospholipase A2-treated acetylated LDL did not induce rise in calcium, but significantly induced activation of PKC . Moreover, phospholipase A2-treated acetylated LDL-induced macrophage growth was significantly inhibited by calphostin C by 90% .
Our results suggest that the presence of two intracellular signaling pathways for activation of PKC, a rise in calcium which was mediated by pertussis toxin sensitive G-protein and the internalization of lysophosphatidylcholine through the scavenger receptor (SR-Al/All) . These two pathways may play an important role in Ox-LDL-induced macrophage growth .
3 .P.172
Embryonic stem cell differentiated macrophages : A model system for the evaluation of macrophage foam cell formation and function
K .J . Moore, L.P. Andersson, M .W. Freeman . Massachusetts General Hospital, Boston MA, USA In the early development of the atherosclerotic plaque, monocytes are recruited to the intima where they bind and internalize modified lipoproteins to form foam cells. Foam cell formation is thought to depend on the uptake of modified LDL by macrophage (MO) scavenger receptors (SR), which include members of the SR-A and SR-B families of these transmembrane proteins . Studies of the consequence of foam cell formation on MO behaviour have been hampered by limitations of the available monocyte/Mo cell lines . In this study, we explored the use of in vitro differentiated embryonic stem (ES) cells for studying M¢ foam cell formation and function . ES differentiated M¢ (ES-MO) were >90% positive for the MO markers F4/80 and CD11b as determined by immunostaining . ES-MO phagocytosed latex beads and produced cytokines in response to inflammatory stimuli . Expression of the modified LDL receptors SR-A and CD36 was demonstrated in ES-MO by RT PCR, and SR-A expression was confirmed by immunostaining . In the presence of acetylated LDL (AcLDL), ES-Mc formed characteristic foam cells as visualized by oil red 0 staining . Furthermore, ES-MO degradation of 125 1-AcLDL exceeded that of the P388D1 cell line commonly exploited in SR degradation studies . In conclusion, these results demonstrate that ES-M¢ express the modified LDL receptors SR-A and CD36, internalize and degrade AcLDL, and thus, are a suitable model system for studying M¢ foam cell formation. The ease of genetic manipulation of ES cells prior to differentiation to MO provides a powerful experimental tool to identify key events in foam cells formation and function .
3 . P.173*
Expression of cholesterol 7a-hydroxylase in cultured RAW macrophages reduces cholesterol loading by acetyl LDL
Gina L. Moore, Neil Sheth, Roger A . Davis . Mammalian Cell and Molecular Biology Lab, San Diego State University, San Diego, California, USA Cholesterol 7a-hydroxylase (7aH) provides the liver with the unique ability to take up cholesterol-rich lipoproteins from plasma and excrete the cholesterol into bile through the formation of bile acids . We examined the possibility that expression of 7aH in macrophages might help prevent the accumulation of cholesterol esters, a hallmark of atherosclerosis . Cholesterol metabolism between RAW mouse macrophages, stably expressing 7aH (7a-RAW) and wild-type RAW cells were compared with and without acetyl-LDL . In the absence of acetyl-LDL, the cellular content of free cholesterol was similar between both cell types (in .sg/mg protein : 8 ± 5 vs . 6 ± 3 for RAW and 7a-RAW cells, respectively) . In contrast, the mass of cholesterol esters in RAW cells was 4-fold greater compared to 7a-RAW cells (in ttg/mg protein : 1 .2 vs . 0.31) . When cultured in the presence of 25 itg protein/ml acetyl-LDI, for 48 hours, RAW cells accumulated more free cholesterol (75 ± 14 µg/mg vs . 48 ± 5 ttg/mg protein) and cholesterol ester (41 .2 ttg/mg vs . 2.9 ttg/mg protein) than that observed in 7a-RAW cells . The decreased accumulation of cholesterol and cholesterol esters in 7a-RAW cells was not associated with decreased levels of synthesis, as determined by 14C-acetate incorporation . 7a-RAW cells displayed a greater rate of excretion of 14C-cholesterol into the cultured medium compared to wild-type cells . This excretion of 14 C-cholesterol was associated with the formation and excretion of a polar 14 C-labeled sterol . Based on these data, we propose that expression of 7aH decreases the accumulation of cholesterol by producing an amphipathic sterol . This amphipathic sterol, like bile acids, may promote the efflux of cholesterol from the macrophage .
11th International Symposium on Atherosclerosis, Paris, October 1997