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Th-P15:106 Advanced glycation end products (AGE), in contrast to OXLDL, do not stimulate macrophage gene expression VIA CD36
P15
516
Thurs&ty, June 22, 2006: Poster Session Pathophysiology of lipids arm lipoproteins
M e t h o d s : 9 normolipidemic men received two different test meals enriched with POO or ROO. Postprandial blood samples were collected at 2, 4 and 6 hours and TRL isolated by ultracentrifugation. THP-1 monocytes were differentiated into macrophages and incubated for 24h with TRL. mRNA levels for LDL-receptors (LDLr), LDLr-related protein (LRP), lectin-like oxLDL receptor (LOX) and scavenger receptors SR-A2, SR-B1 and CD36 were determined by real time RT-PCR. Results: All time points at which TRL were obtained and both experimental oils influenced mRNA expression for the studied genes. LDLr gene expression was decreased by all types of TRL, whereas that for LRP was unaffected in all cases, mRNA levels for LOX were raised only by POO-TRL obtained at 4 hours. The levels of mRNA for CD36 were increased by all TRL types, whereas SR-A2 and SR-B1 mRNA levels increased or decreased depending on the oil and the postpradial time point. Conclusions: TRL from two MUFA-rich oils with different unsaponifiable composition affected differentially the gene expression of receptors involved in lipoprotein uptake and foam cell formation. Funding: CICYT AGL2002-00195, AGL2005-00572, FISG03-140PRE DIMED ]
T h - P 1 5 : 1 0 5 ] H U M A N P D Z K 1 I N C R E A S E H U M A N SRBI (CLA-1) A E X P R E S S I O N IN T H E M O U S E L I V E R H. Komori I , Y. LTeda - , T. Kita I . 1Departments of Cardiovascular Medicine, Kyoto Universi~ GrcMuate School of Medicine, Kyoto, Japan: 2Horizontal Medical Research Organization, Kyoto Universi~ GrcMuate School of Medicine, Kyoto, Japan Previously, we reported that sufficient CLA-1 protein is not expressed compared with the RNA expression in the liver of human SRBI (CLA-1) BAC transgenic mouse. Investigating the regulatory mechanism of CLA-1 expression, we focused on a molecule named PDZK1. PDZK1, a PDZ domain containing protein was reported to interact with SRBI and serve an essential role in post-transcriptional regulation of hepatic SRBI. In this study we hypothesized that human PDZK1 is necessary for human SRBI (CLA-1) expression. We generated human PDZK1 BAC transgenic mouse injecting human BAC clone containing 34Kb upstream as well as the full length of the gene. In the transgenic mouse human PDZK1 was expressed in the liver, the kidney, the ovary, and the adrenal glands while endogenous PDZK1 was expressed in the liver, the kidney, and the intestine. The human PDZK1 BAC transgenic mouse shows no significant change in the expression level of hepatic SRBI. The plasma lipid levels were not influenced either. To evaluate the effects of human PDZK1 on CLA-1 protein expression, we cross-bred human PDZK1 mouse with SRBI-/- CLA-1 mouse. Western blot analysis showed CLA-1 expression increased in the liver with the human PDZK1 transgene compared with that in the CLA-1 transgenic mouse. In conclusion human PDZK1 is necessary for CLA-1 expression on mouse liver. 1 T h - P 1 5 : 1 0 6 ] A D V A N C E D G L Y C A T I O N E N D P R O D U C T S (AGE), IN CONTRAST TO OXLDL, DO NOT STIMULATE MACROPHAGE GENE EXPRESSION VIA CD36 Collot-Teixeira 1
1,
J. Martin 1 , S. , C. McDermott-Roe S. Clogenson 1 , J.L. McGregor 1'2'3. 1 Genomics arm Atherothrombosis, Thrombosis Research
Institute, LotMon, United Kingdom." 2 Cardiovascular Division, Kcl, Universi~ of London, LotMon, United Kingdom." 31nserm U689, HOpital Lariboisiere, Paris, France Proteins indiabetics react with glucose to form Advanced glycation End products (AGE). Indeed, atherothrombotic lesions ans its clinical complications axe thought to be accelerated in diabetics by AGE. Monocytes/macrophages, playing a critical role in atherothrombosis, axe reported to bind AGE via CD36. Investigate the binding of AGE to CD36 and assay the resulting expression of genes implicated in inflammation, apoptosis and plaque rupture on: a) PMA-treated human monocytic cell line (THP1), b) HEK293 expressing or not CD36 and c) in 7 days cultured monocyte derived macrophages (MDM) isolated from blood of healthy donors. THP1, HEK293 or M D M were treated with AGE (Sigma, UK) for 24 hours. Total RNA, extracted with RNeasy Minikit (Qiagen, UK), was retro-transcribed. Gene expressions were monitored using TaqMan quantitative PCR (Q-PCR). THP1 and transfected HEK293 (5.105 cells/mL) were grown on coverslips. Binding was perfolmed by incubating cells for 90rain at 4C with AGE (501tg/mL) labeled with rhodamine (lh, RT). THP1 cells bound rhodamine-labeled AGE but no binding was observed on HEK293 expressing CD36. In contrast, rhodamine-labeled
OxLDL (501~g/mL) and anti-CD36 antibody bound to all cells. Surprisingly, AGE (lO01~g/mL, 24h) did not induce any changes in expression of 14 investigated genes in THP-1. AGE-treated M D M cells only showed 50% increase of IL-8 expression. Under our experimental conditions, AGE does not bind to CD36 nor affect the expression of most genes in macrophages induced by OxLDL. Other receptors and different gene candidate's pathways may induced in macrophages by AGE. Funding: G. Weston foundation I
Th-P 1 5 : 1 0 7 II H I G H G L U C O S E E N H A N C E S T H E E X P R E S S I O N O F C D 3 6 A N D T H E LIPID A C C U M U L A T I O N IN THP-1 MACROPHAGES Y.-L. Tan, G.-H. Yi, Y. Zeng, L. Sun, J.-T. Feng, D.-X. Zeng, X.-B. Yin, J. Wang, J.-H. Xia, A.-E Chen. b~stitute of Cardiovascular Disease, Nanhua
Universi~, Hengyang, Chbta Objective: CD36 is one of the major scavenger receptors for oxLDL which induces macrophage foam cell formation. This study was aimed to investigate the effect of high glucose on the expression of CD36 and the lipid accumulation in THP- 1 macrophages. M e t h o d s : THP-1 cells were induced to differentiate into macrophages by PMA(160nmol/L) co-incubation for 24h.THP-1 macrophages were treated with 50mg/L oxLDL and 20 m M D-glucose for different times (6, 12, 24, 36, 48h). The mRNA and protein levels of CD36 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The lipid accumulation was detected by oil red O stain. The cholesterol and cholesteryl ester contents of cells were determined by high performance liquid chromatography. Results: The expression of CD36 was up-regulated after high glucose treatment. The relative density increased 48.56% under the condition of 20 m M D-glucose treatment, P<0.05, n=3), and it was in a dose and time dependence. The lipid accumulation was enhanced, and the cholesterol and cholesteryl ester levels increased by 51.97% under the condition of 20 m M D-glucose treatment, P<0.05, n=3 after high glucose stimulation. The endocytotic process was significantly suppressed by an anti-CD36 neutralizing antibody. C o n d u s i o n s : High glucose up-regulates the express of CD36 and the lipid accumulation in THP-1 macrophage. This may contribute to the acceleration of atherosclerosis in patients with diabetes. Funding: This work was supported by the National Natural Science Foundation of China(NSFC) (NO.30570938). I
Th 1 p 1 5 : 1 0 8 1 I R E G U L A T I O N O F T H E C D 3 6 E X P R E S S I O N A N D C H O L E S T E R O L I N F L U X IN T H P - 1 M A C R O P H A G E BY P P A R G.H. Yi 1 , Z.C. Mo -, X. Chen 1 , Z. Wang 1 , Z. Ren 1 , C.K. Tang 1 , L.S. Liu 1, Y.Z. Yang 1. l b~stitut e of Cardiovascular Research, Nanhua
Universi~,Hengyang, Hurrah, Chitta; 2Department of Histology arm Embryology, Nanhua Universi~,Hengyang, Hunan, Chbta B a c k g r o u n d : CD36 is thought to play a significant role in atherosclerotic foam cell development because of their ability to bind and internalize oxLDL. Peroxisome proliferator-activated receptor g a m m a (PPARy has been implicated that play a critical role in lipid metabolism. The interest has focused on the role of PPARy in modulating macrophage cholesterol and lipid homeostasis. This study, we investigated the effect of PPARy on CD36 expression and cholesterol influx in THP-1 macrophage. M e t h o d s : After exposure of the cultured THP-1 macrophage to ciglitazone and antisense PPARy oligonucleotide for 24 hours, [3H]-labeled cholesterol influx was determined by FJ-2107P type liquid scintillator. CD36 mRNA and protein level were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively. Results: Ciglitazone elevated CD36 in both protein and mRNA levels, and increased cholesterol influx in THP-1 macrophage. The levels of cholesterol influx were 20.3%,28.6%,37.2%,44.3%,48.7% respectively. Antisense PPARy oligonucleotide inhibited cholesterol influx in THP-1 macrophage and decreased CD36 expression. C o n d u s i o n s : Our date demonstrate that PPARy may play an important role in cholesterol influx and modulating CD36 expression in THP-1 macrophage.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
516
Thurs&ty, June 22, 2006: Poster Session Pathophysiology of lipids arm lipoproteins
M e t h o d s : 9 normolipidemic men received two different test meals enriched with POO or ROO. Postprandial blood samples were collected at 2, 4 and 6 hours and TRL isolated by ultracentrifugation. THP-1 monocytes were differentiated into macrophages and incubated for 24h with TRL. mRNA levels for LDL-receptors (LDLr), LDLr-related protein (LRP), lectin-like oxLDL receptor (LOX) and scavenger receptors SR-A2, SR-B1 and CD36 were determined by real time RT-PCR. Results: All time points at which TRL were obtained and both experimental oils influenced mRNA expression for the studied genes. LDLr gene expression was decreased by all types of TRL, whereas that for LRP was unaffected in all cases, mRNA levels for LOX were raised only by POO-TRL obtained at 4 hours. The levels of mRNA for CD36 were increased by all TRL types, whereas SR-A2 and SR-B1 mRNA levels increased or decreased depending on the oil and the postpradial time point. Conclusions: TRL from two MUFA-rich oils with different unsaponifiable composition affected differentially the gene expression of receptors involved in lipoprotein uptake and foam cell formation. Funding: CICYT AGL2002-00195, AGL2005-00572, FISG03-140PRE DIMED ]
T h - P 1 5 : 1 0 5 ] H U M A N P D Z K 1 I N C R E A S E H U M A N SRBI (CLA-1) A E X P R E S S I O N IN T H E M O U S E L I V E R H. Komori I , Y. LTeda - , T. Kita I . 1Departments of Cardiovascular Medicine, Kyoto Universi~ GrcMuate School of Medicine, Kyoto, Japan: 2Horizontal Medical Research Organization, Kyoto Universi~ GrcMuate School of Medicine, Kyoto, Japan Previously, we reported that sufficient CLA-1 protein is not expressed compared with the RNA expression in the liver of human SRBI (CLA-1) BAC transgenic mouse. Investigating the regulatory mechanism of CLA-1 expression, we focused on a molecule named PDZK1. PDZK1, a PDZ domain containing protein was reported to interact with SRBI and serve an essential role in post-transcriptional regulation of hepatic SRBI. In this study we hypothesized that human PDZK1 is necessary for human SRBI (CLA-1) expression. We generated human PDZK1 BAC transgenic mouse injecting human BAC clone containing 34Kb upstream as well as the full length of the gene. In the transgenic mouse human PDZK1 was expressed in the liver, the kidney, the ovary, and the adrenal glands while endogenous PDZK1 was expressed in the liver, the kidney, and the intestine. The human PDZK1 BAC transgenic mouse shows no significant change in the expression level of hepatic SRBI. The plasma lipid levels were not influenced either. To evaluate the effects of human PDZK1 on CLA-1 protein expression, we cross-bred human PDZK1 mouse with SRBI-/- CLA-1 mouse. Western blot analysis showed CLA-1 expression increased in the liver with the human PDZK1 transgene compared with that in the CLA-1 transgenic mouse. In conclusion human PDZK1 is necessary for CLA-1 expression on mouse liver. 1 T h - P 1 5 : 1 0 6 ] A D V A N C E D G L Y C A T I O N E N D P R O D U C T S (AGE), IN CONTRAST TO OXLDL, DO NOT STIMULATE MACROPHAGE GENE EXPRESSION VIA CD36 Collot-Teixeira 1
1,
J. Martin 1 , S. , C. McDermott-Roe S. Clogenson 1 , J.L. McGregor 1'2'3. 1 Genomics arm Atherothrombosis, Thrombosis Research
Institute, LotMon, United Kingdom." 2 Cardiovascular Division, Kcl, Universi~ of London, LotMon, United Kingdom." 31nserm U689, HOpital Lariboisiere, Paris, France Proteins indiabetics react with glucose to form Advanced glycation End products (AGE). Indeed, atherothrombotic lesions ans its clinical complications axe thought to be accelerated in diabetics by AGE. Monocytes/macrophages, playing a critical role in atherothrombosis, axe reported to bind AGE via CD36. Investigate the binding of AGE to CD36 and assay the resulting expression of genes implicated in inflammation, apoptosis and plaque rupture on: a) PMA-treated human monocytic cell line (THP1), b) HEK293 expressing or not CD36 and c) in 7 days cultured monocyte derived macrophages (MDM) isolated from blood of healthy donors. THP1, HEK293 or M D M were treated with AGE (Sigma, UK) for 24 hours. Total RNA, extracted with RNeasy Minikit (Qiagen, UK), was retro-transcribed. Gene expressions were monitored using TaqMan quantitative PCR (Q-PCR). THP1 and transfected HEK293 (5.105 cells/mL) were grown on coverslips. Binding was perfolmed by incubating cells for 90rain at 4C with AGE (501tg/mL) labeled with rhodamine (lh, RT). THP1 cells bound rhodamine-labeled AGE but no binding was observed on HEK293 expressing CD36. In contrast, rhodamine-labeled
OxLDL (501~g/mL) and anti-CD36 antibody bound to all cells. Surprisingly, AGE (lO01~g/mL, 24h) did not induce any changes in expression of 14 investigated genes in THP-1. AGE-treated M D M cells only showed 50% increase of IL-8 expression. Under our experimental conditions, AGE does not bind to CD36 nor affect the expression of most genes in macrophages induced by OxLDL. Other receptors and different gene candidate's pathways may induced in macrophages by AGE. Funding: G. Weston foundation I
Th-P 1 5 : 1 0 7 II H I G H G L U C O S E E N H A N C E S T H E E X P R E S S I O N O F C D 3 6 A N D T H E LIPID A C C U M U L A T I O N IN THP-1 MACROPHAGES Y.-L. Tan, G.-H. Yi, Y. Zeng, L. Sun, J.-T. Feng, D.-X. Zeng, X.-B. Yin, J. Wang, J.-H. Xia, A.-E Chen. b~stitute of Cardiovascular Disease, Nanhua
Universi~, Hengyang, Chbta Objective: CD36 is one of the major scavenger receptors for oxLDL which induces macrophage foam cell formation. This study was aimed to investigate the effect of high glucose on the expression of CD36 and the lipid accumulation in THP- 1 macrophages. M e t h o d s : THP-1 cells were induced to differentiate into macrophages by PMA(160nmol/L) co-incubation for 24h.THP-1 macrophages were treated with 50mg/L oxLDL and 20 m M D-glucose for different times (6, 12, 24, 36, 48h). The mRNA and protein levels of CD36 were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The lipid accumulation was detected by oil red O stain. The cholesterol and cholesteryl ester contents of cells were determined by high performance liquid chromatography. Results: The expression of CD36 was up-regulated after high glucose treatment. The relative density increased 48.56% under the condition of 20 m M D-glucose treatment, P<0.05, n=3), and it was in a dose and time dependence. The lipid accumulation was enhanced, and the cholesterol and cholesteryl ester levels increased by 51.97% under the condition of 20 m M D-glucose treatment, P<0.05, n=3 after high glucose stimulation. The endocytotic process was significantly suppressed by an anti-CD36 neutralizing antibody. C o n d u s i o n s : High glucose up-regulates the express of CD36 and the lipid accumulation in THP-1 macrophage. This may contribute to the acceleration of atherosclerosis in patients with diabetes. Funding: This work was supported by the National Natural Science Foundation of China(NSFC) (NO.30570938). I
Th 1 p 1 5 : 1 0 8 1 I R E G U L A T I O N O F T H E C D 3 6 E X P R E S S I O N A N D C H O L E S T E R O L I N F L U X IN T H P - 1 M A C R O P H A G E BY P P A R G.H. Yi 1 , Z.C. Mo -, X. Chen 1 , Z. Wang 1 , Z. Ren 1 , C.K. Tang 1 , L.S. Liu 1, Y.Z. Yang 1. l b~stitut e of Cardiovascular Research, Nanhua
Universi~,Hengyang, Hurrah, Chitta; 2Department of Histology arm Embryology, Nanhua Universi~,Hengyang, Hunan, Chbta B a c k g r o u n d : CD36 is thought to play a significant role in atherosclerotic foam cell development because of their ability to bind and internalize oxLDL. Peroxisome proliferator-activated receptor g a m m a (PPARy has been implicated that play a critical role in lipid metabolism. The interest has focused on the role of PPARy in modulating macrophage cholesterol and lipid homeostasis. This study, we investigated the effect of PPARy on CD36 expression and cholesterol influx in THP-1 macrophage. M e t h o d s : After exposure of the cultured THP-1 macrophage to ciglitazone and antisense PPARy oligonucleotide for 24 hours, [3H]-labeled cholesterol influx was determined by FJ-2107P type liquid scintillator. CD36 mRNA and protein level were determined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting respectively. Results: Ciglitazone elevated CD36 in both protein and mRNA levels, and increased cholesterol influx in THP-1 macrophage. The levels of cholesterol influx were 20.3%,28.6%,37.2%,44.3%,48.7% respectively. Antisense PPARy oligonucleotide inhibited cholesterol influx in THP-1 macrophage and decreased CD36 expression. C o n d u s i o n s : Our date demonstrate that PPARy may play an important role in cholesterol influx and modulating CD36 expression in THP-1 macrophage.
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006