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The efflux of cholesterol from ABC1-expressing J774 macrophages is highest when exposed to serum from human apo AII transgenic mice
Posters 7. HDL
72
0.0001) plasma values. More, the regression analyses shows a significative inverse correlation of CRP versus HDL (r = -0.41, p = 0.0196) and apoprotein A-1 (r = -0.55, p = 0.0006). Atherosclerosis produced by hypoalpha might in part be explained by an upregulation of inflammatory mechanisms. ~--~
THE ASSOCIATION OF HEPATIC LIPASE ACTIVITY WITH CORONARY ARTERY DISEASE DEPENDS ON THE HDL CHOLESTEROL LEVEL
(r = 0.78, P < 0.0001) and hapo A-II (r = 0.71, P < 0.0001). Considering that C accumulation in macrophages may lead to fatty streak formation and ultimately to aortic lesions, we propose that stimulation of ABC1 mediated C effiux from J774 cells by the plasma of mice overexpressing hapo A-II may protect against atherogenesis. References
[1] Boisfer et al., J. Biol. Chem. 1999, 274." 11564-11572. [2] Fournier et al., Arterioseler. Thromb. Vase. Biol. 1997, 17_5."2685-2691. [3] Bortnick et al., J. Biol. Chem. 2000, 275: 28634--28640.
K.A. Dugi, K. Brandauer, N. Schrnidt, W. Fiehn, J. Kreuzer. Dept.
Medicine I (Endocrinology and Metabolism), Heidelberg University Hospital, Bergheimer S~ 58, 69115 Heidelberg, Germany
[ffi--0~ HUMAN APOLIPOPROTEIN A-II EXPRESSION IN MICE DECREASES ANTIOXYDANT PROPERTIES OF HDL
Although the fundamental role of hepatic lipase (HL) in lipid metabolism is well established, it remains disputed whether it functions in a pro- or anti-atherogenic fashion. We have recently demonstrated in 200 men with coronary artery disease (CAD) that low HL activity in post heparin plasma is inversely correlated with CAD extent, despite the fact that low HL is also associated with higher plasma levels of antiatherogenic HDL cholesterol. To better understand the relation of HL activity with HDL and CAD, we grouped the patients according to factors known to be associated with HL such as body mass index (BMI), insulin levels, and HDL. The inverse association of HL activity with CAD extent was similar in patients with a BMI of more or less than the median of 27.4 kg/m2 and in patients with low or high insulin levels. We found a striking difference, however, between the patients with an HDL cholesterol of more or less than the median of 37 mg/dl, in patients with lower HDL cholesterol, no significant association of HL activity with CAD extent was detected. In the 100 patients with HDL cholesterol above 37 mg/dl, however, there was a highly significant inverse association of HL activity with CAD extent (r = -0.30, p < 0.005). This finding was confirmed with a newer, fluorometric HL assay (r = -0.32, p < 0.005). Finally, in the high I-IDL group, the -514C/T polymorphism in the HL promoter was associated with lower HL activity (r = -0.40, p < 0.001) and greater CAD extent (r = 0.20, p = 0.05). In summary: 1) The association of low HL activity with greater CAD extent is independent of BMI and insulin levels. 2) The association of HL with CAD is mainly due to patients with higher HDL cholesterol. 3) In patients with higher HDL cholesterol levels determination of hepatic lipase activity may help to define CAD risk.
E. Boisfer1, D. Stengel2, D. Pastier 1, E. Dugu 61 , P.M. Laplaud 2, N. Dousset3, E. Ninio 2, A.-D. Kalopissis 1. 1U505 luserm, Centre
•0-•
THE EFFLUX OFCHOLESTEROL FROM ABC1-EXPRESSING J774 MACROPHAGES IS HIGHEST WHEN EXPOSED TO SERUM FROM HUMAN apo AII TRANSGENIC MICE
A.D. Kalopissis I , N. Fournier2, A. Cogny2, D. Pastier 1, D. Goudounecbe2, J. Chambaz 1, J.L. Paul 2, N. Moatti 2. 1 U505 Inserm, Paris; 2Laboratoire
de Biochimie, Facult~ des Sciences Pharmaceutiques, Chdtenay-Malabry, France The regulation of cholesterol (C) efltux from tissues is linked both to the nature of the acceptor particles and to specific receptors. The C effinx by aqueous diffusion to phospholipid (PL)-rich acceptors such as HDL can be assessed in Fu5AH rat hepatoma ceils, whereas the C efflux mediated by lipid-poor apolipoproteins and regulated by ATP binding cassette transporter 1 (ABC1) can be measured in macrophage-like cells. We previously generated transgenic mice overexpressing human apo A-II (hAIltg mice), which display a marked hypertriglyceridemia and low plasma HDL levels (1). We presently addressed the ability of whole plasma and HDL from these mice to promote C eftlux from Fu5AH cells and J774 macrophages. Plasma from hAlltg mice had a markedly reduced capacity to induce C efltux from Fu5AH cells, compared with control plasma. This concords with the well established dependance of C efflux from the SR-Bl-rich Fu5AH cells on the HDL-PL concentration (2). Moreover, when tested at the same HDL-PL concentrations, HDL from hA/Itg mice induced less C effiux than control HDL. In the absence of cAMP, J774 macrophages are negative for ABC1, whereas pretreatment with cAMP upregulates this receptor (3). Thus, the difference in effinx between ABC1 negative and positive J774 cells reflects the effect of ABC1. All groups showed similar efltux abilities from ABC1 negative cells. Pretreatment with cAMP had no effect on the C efflux to control plasma, whereas it induced a markedly increased effinx to hAIItg plasma, in proportion to hapo A-II expression (up to 70% stimulation). Strong positive correlations were established between percent cAMP stimulation of effhix and plasma concentrations of triglyceride
de Recherche des Cordeliers, Paris; 2 U525 Inserm, Htpital de la Pitid-Salp~tridre, Paris," 3Laboratoire de Biochimie 1E, Htpital de Rangeuil, Toulouse, France Transgenic mice overexpressing human apolipoprotein A-II (hapo A-II) display high VLDL and low HDL levels (1). Because in mice HDL carry two enzymes with antioxidant properties, paraoxonase (PON) and plateletactivating factor acetylhydrolase (PAF-AH), we measured their activities and the susceptibility to oxidation of apo B-containing lipoproteins (LPB) and HDL. PON and PAF-AH activities decreased up to 43% in the serum of transgenic mice compared with controls, under ad libitum feeding conditions and after an overnight fast. Interestingly, the activities o f both enzymes increased 1.6 to 2-fold in fasted, compared with fed, animals oftbe same genotype. The variations of PON and PAF-AH activities occurred in parallel to the variations in plasma HDL levels irrespective of the nutritional state. The major part of PON and PAF-AH activities was associated with HDL, except in the higher-expressing X mice fed ad libimm, in which 13% of PAF-AH activity was associated with VLDL, which accumulate in the circulation of these mice. The production of thiobarbituric acid reactive substances (TBARS) and that of hydroperoxides increased in LP-B of transgenic mice as compared with LP-B of control mice, whereas HDL susceptibility to oxidation was similar irrespective of genotype. PON mRNA levels in the liver, its major site of synthesis, were similar in transgenie and control animals, either fed or fasted overnight. Our results suggest that the lower PON and PAF-AH activities in transgenic mice were related to the decrease in plasma HDL levels and not to a decreased transcription rate of the enzymes. The greater susceptibility to oxidation of LP-B in mice overexpressing hapo A-II may be associated with increased risk of developing aortic lesions. References
[1] Boisfer et al., J. Biol. Chem. 1999, 274: 11564-11572. ~'~
DECREASED EXPRESSION OF SCAVENGER RECEPTOR CLASS B TYPE I BY CHOLESTERYL ESTER TRANSFER PROTEIN ANTISENSE INHIBITION IN HepG2 CELLS
Z.P. Huang, A. Inazu, A. Nohara, T. Higashikata, H. Mabuchi. The
Second Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan Both scavenger receptor class B type I (SR-BI), a HDL receptor, and cholesteryl ester transfer protein (CETP) can return plasma HDL-CE to liver for catabolism. It is not yet known how these two pathways act synergistically on transfering HDL-CE. We focused on changes in SR-BI during inhibition of CETP expression and used CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells to identify potential interaction between CETP and hepatic lipoprotein receptors. Twenty hours after ODNs against CETP were delivered to HepG2 ceils, CETP expression markedly decreased. RT-PCR and EUSA protein quantitation analysis showed that the CETP levels were reduced by 70% and 51% in cells treated with antisense ODNs compared with cells treated with sense ODNs, respectively. RT-PCR and Western blotting showed a significant 50% reduction in SR-BI expression during down-regulatiun of CETP. Moreover, the CE levels in cells used for antisense treatment increased progressively, mRNA levels of sterol regulatory element binding protein-la (SREBP-la), a regulatory factor of SR-BI, were found to have decreased by 40%. These findings suggest that SR-BI expression is down-regulated in response to an increase in cellular cholesterol stores and a decrease in sterol signal transduction. Suppression of CETP expression
72nd EAS Congress
72
0.0001) plasma values. More, the regression analyses shows a significative inverse correlation of CRP versus HDL (r = -0.41, p = 0.0196) and apoprotein A-1 (r = -0.55, p = 0.0006). Atherosclerosis produced by hypoalpha might in part be explained by an upregulation of inflammatory mechanisms. ~--~
THE ASSOCIATION OF HEPATIC LIPASE ACTIVITY WITH CORONARY ARTERY DISEASE DEPENDS ON THE HDL CHOLESTEROL LEVEL
(r = 0.78, P < 0.0001) and hapo A-II (r = 0.71, P < 0.0001). Considering that C accumulation in macrophages may lead to fatty streak formation and ultimately to aortic lesions, we propose that stimulation of ABC1 mediated C effiux from J774 cells by the plasma of mice overexpressing hapo A-II may protect against atherogenesis. References
[1] Boisfer et al., J. Biol. Chem. 1999, 274." 11564-11572. [2] Fournier et al., Arterioseler. Thromb. Vase. Biol. 1997, 17_5."2685-2691. [3] Bortnick et al., J. Biol. Chem. 2000, 275: 28634--28640.
K.A. Dugi, K. Brandauer, N. Schrnidt, W. Fiehn, J. Kreuzer. Dept.
Medicine I (Endocrinology and Metabolism), Heidelberg University Hospital, Bergheimer S~ 58, 69115 Heidelberg, Germany
[ffi--0~ HUMAN APOLIPOPROTEIN A-II EXPRESSION IN MICE DECREASES ANTIOXYDANT PROPERTIES OF HDL
Although the fundamental role of hepatic lipase (HL) in lipid metabolism is well established, it remains disputed whether it functions in a pro- or anti-atherogenic fashion. We have recently demonstrated in 200 men with coronary artery disease (CAD) that low HL activity in post heparin plasma is inversely correlated with CAD extent, despite the fact that low HL is also associated with higher plasma levels of antiatherogenic HDL cholesterol. To better understand the relation of HL activity with HDL and CAD, we grouped the patients according to factors known to be associated with HL such as body mass index (BMI), insulin levels, and HDL. The inverse association of HL activity with CAD extent was similar in patients with a BMI of more or less than the median of 27.4 kg/m2 and in patients with low or high insulin levels. We found a striking difference, however, between the patients with an HDL cholesterol of more or less than the median of 37 mg/dl, in patients with lower HDL cholesterol, no significant association of HL activity with CAD extent was detected. In the 100 patients with HDL cholesterol above 37 mg/dl, however, there was a highly significant inverse association of HL activity with CAD extent (r = -0.30, p < 0.005). This finding was confirmed with a newer, fluorometric HL assay (r = -0.32, p < 0.005). Finally, in the high I-IDL group, the -514C/T polymorphism in the HL promoter was associated with lower HL activity (r = -0.40, p < 0.001) and greater CAD extent (r = 0.20, p = 0.05). In summary: 1) The association of low HL activity with greater CAD extent is independent of BMI and insulin levels. 2) The association of HL with CAD is mainly due to patients with higher HDL cholesterol. 3) In patients with higher HDL cholesterol levels determination of hepatic lipase activity may help to define CAD risk.
E. Boisfer1, D. Stengel2, D. Pastier 1, E. Dugu 61 , P.M. Laplaud 2, N. Dousset3, E. Ninio 2, A.-D. Kalopissis 1. 1U505 luserm, Centre
•0-•
THE EFFLUX OFCHOLESTEROL FROM ABC1-EXPRESSING J774 MACROPHAGES IS HIGHEST WHEN EXPOSED TO SERUM FROM HUMAN apo AII TRANSGENIC MICE
A.D. Kalopissis I , N. Fournier2, A. Cogny2, D. Pastier 1, D. Goudounecbe2, J. Chambaz 1, J.L. Paul 2, N. Moatti 2. 1 U505 Inserm, Paris; 2Laboratoire
de Biochimie, Facult~ des Sciences Pharmaceutiques, Chdtenay-Malabry, France The regulation of cholesterol (C) efltux from tissues is linked both to the nature of the acceptor particles and to specific receptors. The C effinx by aqueous diffusion to phospholipid (PL)-rich acceptors such as HDL can be assessed in Fu5AH rat hepatoma ceils, whereas the C efflux mediated by lipid-poor apolipoproteins and regulated by ATP binding cassette transporter 1 (ABC1) can be measured in macrophage-like cells. We previously generated transgenic mice overexpressing human apo A-II (hAIltg mice), which display a marked hypertriglyceridemia and low plasma HDL levels (1). We presently addressed the ability of whole plasma and HDL from these mice to promote C eftlux from Fu5AH cells and J774 macrophages. Plasma from hAlltg mice had a markedly reduced capacity to induce C efltux from Fu5AH cells, compared with control plasma. This concords with the well established dependance of C efflux from the SR-Bl-rich Fu5AH cells on the HDL-PL concentration (2). Moreover, when tested at the same HDL-PL concentrations, HDL from hA/Itg mice induced less C effiux than control HDL. In the absence of cAMP, J774 macrophages are negative for ABC1, whereas pretreatment with cAMP upregulates this receptor (3). Thus, the difference in effinx between ABC1 negative and positive J774 cells reflects the effect of ABC1. All groups showed similar efltux abilities from ABC1 negative cells. Pretreatment with cAMP had no effect on the C efflux to control plasma, whereas it induced a markedly increased effinx to hAIItg plasma, in proportion to hapo A-II expression (up to 70% stimulation). Strong positive correlations were established between percent cAMP stimulation of effhix and plasma concentrations of triglyceride
de Recherche des Cordeliers, Paris; 2 U525 Inserm, Htpital de la Pitid-Salp~tridre, Paris," 3Laboratoire de Biochimie 1E, Htpital de Rangeuil, Toulouse, France Transgenic mice overexpressing human apolipoprotein A-II (hapo A-II) display high VLDL and low HDL levels (1). Because in mice HDL carry two enzymes with antioxidant properties, paraoxonase (PON) and plateletactivating factor acetylhydrolase (PAF-AH), we measured their activities and the susceptibility to oxidation of apo B-containing lipoproteins (LPB) and HDL. PON and PAF-AH activities decreased up to 43% in the serum of transgenic mice compared with controls, under ad libitum feeding conditions and after an overnight fast. Interestingly, the activities o f both enzymes increased 1.6 to 2-fold in fasted, compared with fed, animals oftbe same genotype. The variations of PON and PAF-AH activities occurred in parallel to the variations in plasma HDL levels irrespective of the nutritional state. The major part of PON and PAF-AH activities was associated with HDL, except in the higher-expressing X mice fed ad libimm, in which 13% of PAF-AH activity was associated with VLDL, which accumulate in the circulation of these mice. The production of thiobarbituric acid reactive substances (TBARS) and that of hydroperoxides increased in LP-B of transgenic mice as compared with LP-B of control mice, whereas HDL susceptibility to oxidation was similar irrespective of genotype. PON mRNA levels in the liver, its major site of synthesis, were similar in transgenie and control animals, either fed or fasted overnight. Our results suggest that the lower PON and PAF-AH activities in transgenic mice were related to the decrease in plasma HDL levels and not to a decreased transcription rate of the enzymes. The greater susceptibility to oxidation of LP-B in mice overexpressing hapo A-II may be associated with increased risk of developing aortic lesions. References
[1] Boisfer et al., J. Biol. Chem. 1999, 274: 11564-11572. ~'~
DECREASED EXPRESSION OF SCAVENGER RECEPTOR CLASS B TYPE I BY CHOLESTERYL ESTER TRANSFER PROTEIN ANTISENSE INHIBITION IN HepG2 CELLS
Z.P. Huang, A. Inazu, A. Nohara, T. Higashikata, H. Mabuchi. The
Second Department of Internal Medicine, School of Medicine, Kanazawa University, Kanazawa, Japan Both scavenger receptor class B type I (SR-BI), a HDL receptor, and cholesteryl ester transfer protein (CETP) can return plasma HDL-CE to liver for catabolism. It is not yet known how these two pathways act synergistically on transfering HDL-CE. We focused on changes in SR-BI during inhibition of CETP expression and used CETP antisense oligodeoxynucleotides (ODNs) in HepG2 cells to identify potential interaction between CETP and hepatic lipoprotein receptors. Twenty hours after ODNs against CETP were delivered to HepG2 ceils, CETP expression markedly decreased. RT-PCR and EUSA protein quantitation analysis showed that the CETP levels were reduced by 70% and 51% in cells treated with antisense ODNs compared with cells treated with sense ODNs, respectively. RT-PCR and Western blotting showed a significant 50% reduction in SR-BI expression during down-regulatiun of CETP. Moreover, the CE levels in cells used for antisense treatment increased progressively, mRNA levels of sterol regulatory element binding protein-la (SREBP-la), a regulatory factor of SR-BI, were found to have decreased by 40%. These findings suggest that SR-BI expression is down-regulated in response to an increase in cellular cholesterol stores and a decrease in sterol signal transduction. Suppression of CETP expression
72nd EAS Congress