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Th-P15:101 Functional analysis of chimaeras of the low density- and very low density lipoprotein receptors (LDLR and VLDLR)
P15
Thurs&ty, June 22, 2006: Poster Session Pathophysiology of lipids arm lipoproteins
Th-P15:100 ] M A C R O P H A G E F O A M C E L L F O R M A T I O N M E D I A T E D BY R E C E P T O R - I N D E P E N D E N T MACROPINOCYTOSIS OF NATIVE LDL H. Kruth 1 , B. Zhao 1 , C. Buono 1 , Y. Li 1 , S. Waldo 1, N. Joner 2. 1Nhlbi, National Institutes of Health, Bethesda, USA." 2 Wake Forest Universi~ School of Medicine, Winston-Salent, USA Objective: Rerearcherr believe that incubation of macrophager (mac) only with modified LDL can rerult in cholerterol accumulation rufficient to form foam cellr. We have invertigated whether mac can become foam cellr with unmodified native LDL. M e t h o d s : Human monocyter were differentiated into mac with FBS plur M-CSF and incubated with native 125I-LDL or the fluid tracer, 125I-BSA, to determine the rater of LDL and fluid uptake and cholerterol accumulation. Results: Differentiation of mac from human monocyter in FBS with MCSF produced a mac phenotype demonrtrating rpontaneour fluid-phare uptake of LDL leading to mac cholerterol accumulation. M-CSF differentiated mac rhowed non-competable and non-raturable uptake of LDL that did not downregulate over 48 hourr. Mac endocytorir of fluid could completely account for LDL uptake. Microrcopy rhowed that fluid-phase uptake of LDL war mediated by continuour actin-dependent macropinocytorir of LDL that led to high levelr of cholerterol accumulation (100 increaring to >300 nmoler/mg cell protein) tranrforming the mac into foam cellr. Treatment of mac with LXR agonirtr inhibited macropinocytorir of LDL. Conclusions: Macropinocytorir of LDL by mac differentiated from monocyter under the influence of M-CSF i r a plaurible mechanirm to account for mac foam cell formation in atherorclerotic lerionr. Thir mechanirm of mac foam cell formation doer not depend on LDL modification or mac receptorr. LXR agonirtr may ameliorate atherorclerorir not only by promoting mac cholerterol efflux, but alto by limiting mac uptake of LDL.
Th P 15:101 1
I
I I
FUNCTIONAL ANALYSIS OF CHIMAERAS OF THE LOW DENSITY- AND VERY LOW DENSITY LIPOPROTEIN RECEPTORS (LDLR AND VLDLR)
D.D. Patel, D. Norman, B.L. Knight, A.K. Soutar. MRC Clinical Sciences
Centre, Hamnwrsmith Hospital, LotMon, United Kingdom Objectives: Familial Hypercholerterolemia (FH), one of the commonert inherited direarer of lipoprotein metabolirm, ir caured by defectr in L D L R gene. V L D L R rtrongly referable the L D L R rtructurally but do not bind LDL. The ability of cellr exprerring V L D L R / L D L R chimaerar to bind and degrade LDL war determined. M e t h o d s : Five chimaeric receptor cDNAr were conrtructed by PCR and their requence confirmed; they were tranrfected into Chinere hamrter ovary cellr that lack endogenour L D L R activity (CHO-ldlA7 cellr). Exprerrion of chimaeric receptor proteinr in extractr of Geneticin-rerirtant rtable tranrfectantr war confirmed by immunoblotting with antibodier rpecific to regionr of L D L R and VLDLR. The binding, uptake and degradation of 125I-labelled LDL or bVLDL by cellr war determined. Results: In 1-4L and 1-5L, the firrt five cyrteine-rich ligand binding repeatr of the V L D L R were replaced with repeatr 1-4 or 1-5 of the LDLR. 1-AL comprired repeatr 1-7 plur the epidermal growth factor precurror-like repeat A of LDLR, 1-OL comprired repeatr 1-7 and up to and including the O-linked rugarr domain of L D L R and 1-TM contained binding repeatr 1-7 and up to and including the tranr-membrane domain of the LDLR. All the chimaeric receptor conrtructr were exprerred as mature proteinr on the cell rurface. Cellr exprerring 1-4L and 1-5L did not bind LDL; cellr exprerring 1-AL bound and degraded LDL, but with reduced affinity, while thore exprerring 1-OL, 1-TM or L D L R bound & degraded LDL normally. All cellr bound bVLDL rimilarly. Conclusions: Specific binding of LDL requirer more than cyrteine-rich binding repeatr 1-5 of the LDLR. Funding: MRC I
Th-P15:102 I I N A C T I V A T I O N O F T H E N P X Y X X L D O M A I N O F I
THE LOW-DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN 1 SHOWS INCREASED ATHEROSCLEROSIS
E Gordtr, A. Lauwerr, N. Feyaertr, S. Reekmanr, A. Roebroek. Departnwnt for Human Genetics, Molecular Genetics Section, Universi~ of Leuven, Leuven, Belgium Objectives: The low-denrity lipoprotein receptor (LDLR) related protein 1
515
(LRP1) i r a multifunctional receptor belonging to the L D L R family. The cytoplarmatic domain of LRP1 containr a number of potential rignalling motiver, which t e e m to be relevant for atherorclerorir. By analyrir of knock-in mutationr into the intracellular domain of endogenour murine LRP1 (ICD-LRP1) we want to identify motiver and rignalling pathwayr regulated by LRP1, which are important for the development of atherorclerorir. M e t h o d s : An LRP1 knock-in moure carrying an inactivating mutation in the NPXYXXL (NPVYATL - > AAVAATL) motif into ICD-LRP1 was generated by recombinase mediated casrette exchange (RMCE) (Roebroek et al., MCB, 2006). The NPXYXXL domain containr a motif for endocytorir and a rite of tyrorine phorphorylation. Inactivation of thir domain could porribly influence the PDGF induced rignalling and foam cell formation of varcular rmooth murcle cellr. In the L D L R knockout (LDLR-KO)/LRP1 double mutant we want to evaluate the impact of the LRP1 mutation on the development of atherorclerorir. Results: The LRP1 knock-in moure rhowed a normal level of LRP1 gene exprerrion and a normal phenotype. In a LDLR-KO background thir mutant rhowed compared to the LDLR-KO rignificant increared lerion formation and enlargement of the aorta when put on a high cholerterol diet. C o n d u s i o n : The inactivation of the dirtal NPXYXXL motif rerulted in increared atherorclerorir. Whether thir ir due to the modulating effect of LRP1 on PDGF rignalling or on foam cell formation needr to be determined. Funding: IWT: VIB; FWO
I Th-P 15:103
THE DI-LEUCINE MOTIF CONTRIBUTES TO CLASS A SCAVENGER RECEPTOR-MEDIATED UPTAKE OF ACETYLATED LIPOPROTEINS
Q. Chen, Y.Y. Chen, X.H. Wang, J.J. Beng. Atherosclerosis Researeh Centre, K ~ Laboratoty of Huntan Functional Genomics, Nanjing Medical Universi~, Nanjbtg, China Objective: The di-leucine motif exirtr in the intracellulax domainr of certain cell rurface receptorr, which contributer to the receptor-mediated endocytorir. The aim of the prerent rtudy was to determine the role of the di-leucine motif in clasr A rcavenger receptor (SR-A)-mediated ligand endocytorir. M e t h o d s : cDNA coding for different SR-A mutantr with deletion of the di-leucine rtructure (N3132LM) were tranrfected into Chinere hamrter ovary (CHO) cellr. The exprerrion of the mutantr was detected by obrervation under fluorercence microrcope as well ar wertern blot analyrir. The uptake and binding of DiI-AcLDL by tranrfected CHO cellr war quantitative analyzed by flow cytometry. To explore the role of di-leucine motif in localization of SR-A in clathrin-coated pit, the lyrater of tranrfected cellr were immunoprecipitated by an antibody againrt clathrin. Results: The cellr exprerring the SR-A mutant N3132LM rhowed decreated uptake of the SR-A ligand than wild type SR-A-exprerring cellr. Wertern blot analyrir on immunoprecipitater of cell lyrater indicated the arrociation of the ligand-SR-A mutant N3132LM with clathrin-coated pit. Further truncation of the firrt 27 amino acidr in the N-terminur of the di-leucine motif-mutated SR-A led to further decline in the cellular uptake of acetyl low denrity lipoprotein (AcLDL) when compared to thore without the di-leucine motif mutation. Conclusions: In conjunction with other known motifr, the di-leucine motif playr an important role in SR-A mediated internalization of A c L D L into cell. Funding: The work war rupported by the project of National Baric Rerearch ProgramNational Baric Rerearch Program(973)(TG200056910)and the National Natural Science Foundation of China (No.30370576) to Qi Chen.
I Th-P 1 5 : 1 0 4 I T R I G L Y C E R I D E - R I C H L I P O P R O T E I N S D E R I V E D FROM POMACE OLIVE OIL HAVE DIFFERENTIAL EFFECTS ON THE EXPRESSION OF MRNA FOR MACROPHAGE RECEPTORS I
R. Cabello-Moruno 1, J.S. Perona 1 , E. Montero 2, M. Avella 3, K.M. Botham 3, V. Ruiz-Gutierrez 1 . l b~stituto de la Grasa ( CSIC), Seville, Spain: 2HH, UU,
Virgen del Rocio, Seville, Spain." 3Royal Veterinaty College, London, United Kingdom Objectives: Triglyceride rich lipoproteinr (TRL) comporition changer portprandially according to the type of ingerted fat, affecting metabolirm of the particle. The aim of the prerent rtudy war to evaluate the influence of the unraponifiable fraction prerent in portprandial TRL derived from pomace olive oil (POO-TRL) on the exprerrion of mRNA gener involved in human macrophager foam cell formation, compared with TRL derived from refined olive oil (ROO-TRL).
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
Thurs&ty, June 22, 2006: Poster Session Pathophysiology of lipids arm lipoproteins
Th-P15:100 ] M A C R O P H A G E F O A M C E L L F O R M A T I O N M E D I A T E D BY R E C E P T O R - I N D E P E N D E N T MACROPINOCYTOSIS OF NATIVE LDL H. Kruth 1 , B. Zhao 1 , C. Buono 1 , Y. Li 1 , S. Waldo 1, N. Joner 2. 1Nhlbi, National Institutes of Health, Bethesda, USA." 2 Wake Forest Universi~ School of Medicine, Winston-Salent, USA Objective: Rerearcherr believe that incubation of macrophager (mac) only with modified LDL can rerult in cholerterol accumulation rufficient to form foam cellr. We have invertigated whether mac can become foam cellr with unmodified native LDL. M e t h o d s : Human monocyter were differentiated into mac with FBS plur M-CSF and incubated with native 125I-LDL or the fluid tracer, 125I-BSA, to determine the rater of LDL and fluid uptake and cholerterol accumulation. Results: Differentiation of mac from human monocyter in FBS with MCSF produced a mac phenotype demonrtrating rpontaneour fluid-phare uptake of LDL leading to mac cholerterol accumulation. M-CSF differentiated mac rhowed non-competable and non-raturable uptake of LDL that did not downregulate over 48 hourr. Mac endocytorir of fluid could completely account for LDL uptake. Microrcopy rhowed that fluid-phase uptake of LDL war mediated by continuour actin-dependent macropinocytorir of LDL that led to high levelr of cholerterol accumulation (100 increaring to >300 nmoler/mg cell protein) tranrforming the mac into foam cellr. Treatment of mac with LXR agonirtr inhibited macropinocytorir of LDL. Conclusions: Macropinocytorir of LDL by mac differentiated from monocyter under the influence of M-CSF i r a plaurible mechanirm to account for mac foam cell formation in atherorclerotic lerionr. Thir mechanirm of mac foam cell formation doer not depend on LDL modification or mac receptorr. LXR agonirtr may ameliorate atherorclerorir not only by promoting mac cholerterol efflux, but alto by limiting mac uptake of LDL.
Th P 15:101 1
I
I I
FUNCTIONAL ANALYSIS OF CHIMAERAS OF THE LOW DENSITY- AND VERY LOW DENSITY LIPOPROTEIN RECEPTORS (LDLR AND VLDLR)
D.D. Patel, D. Norman, B.L. Knight, A.K. Soutar. MRC Clinical Sciences
Centre, Hamnwrsmith Hospital, LotMon, United Kingdom Objectives: Familial Hypercholerterolemia (FH), one of the commonert inherited direarer of lipoprotein metabolirm, ir caured by defectr in L D L R gene. V L D L R rtrongly referable the L D L R rtructurally but do not bind LDL. The ability of cellr exprerring V L D L R / L D L R chimaerar to bind and degrade LDL war determined. M e t h o d s : Five chimaeric receptor cDNAr were conrtructed by PCR and their requence confirmed; they were tranrfected into Chinere hamrter ovary cellr that lack endogenour L D L R activity (CHO-ldlA7 cellr). Exprerrion of chimaeric receptor proteinr in extractr of Geneticin-rerirtant rtable tranrfectantr war confirmed by immunoblotting with antibodier rpecific to regionr of L D L R and VLDLR. The binding, uptake and degradation of 125I-labelled LDL or bVLDL by cellr war determined. Results: In 1-4L and 1-5L, the firrt five cyrteine-rich ligand binding repeatr of the V L D L R were replaced with repeatr 1-4 or 1-5 of the LDLR. 1-AL comprired repeatr 1-7 plur the epidermal growth factor precurror-like repeat A of LDLR, 1-OL comprired repeatr 1-7 and up to and including the O-linked rugarr domain of L D L R and 1-TM contained binding repeatr 1-7 and up to and including the tranr-membrane domain of the LDLR. All the chimaeric receptor conrtructr were exprerred as mature proteinr on the cell rurface. Cellr exprerring 1-4L and 1-5L did not bind LDL; cellr exprerring 1-AL bound and degraded LDL, but with reduced affinity, while thore exprerring 1-OL, 1-TM or L D L R bound & degraded LDL normally. All cellr bound bVLDL rimilarly. Conclusions: Specific binding of LDL requirer more than cyrteine-rich binding repeatr 1-5 of the LDLR. Funding: MRC I
Th-P15:102 I I N A C T I V A T I O N O F T H E N P X Y X X L D O M A I N O F I
THE LOW-DENSITY LIPOPROTEIN RECEPTOR RELATED PROTEIN 1 SHOWS INCREASED ATHEROSCLEROSIS
E Gordtr, A. Lauwerr, N. Feyaertr, S. Reekmanr, A. Roebroek. Departnwnt for Human Genetics, Molecular Genetics Section, Universi~ of Leuven, Leuven, Belgium Objectives: The low-denrity lipoprotein receptor (LDLR) related protein 1
515
(LRP1) i r a multifunctional receptor belonging to the L D L R family. The cytoplarmatic domain of LRP1 containr a number of potential rignalling motiver, which t e e m to be relevant for atherorclerorir. By analyrir of knock-in mutationr into the intracellular domain of endogenour murine LRP1 (ICD-LRP1) we want to identify motiver and rignalling pathwayr regulated by LRP1, which are important for the development of atherorclerorir. M e t h o d s : An LRP1 knock-in moure carrying an inactivating mutation in the NPXYXXL (NPVYATL - > AAVAATL) motif into ICD-LRP1 was generated by recombinase mediated casrette exchange (RMCE) (Roebroek et al., MCB, 2006). The NPXYXXL domain containr a motif for endocytorir and a rite of tyrorine phorphorylation. Inactivation of thir domain could porribly influence the PDGF induced rignalling and foam cell formation of varcular rmooth murcle cellr. In the L D L R knockout (LDLR-KO)/LRP1 double mutant we want to evaluate the impact of the LRP1 mutation on the development of atherorclerorir. Results: The LRP1 knock-in moure rhowed a normal level of LRP1 gene exprerrion and a normal phenotype. In a LDLR-KO background thir mutant rhowed compared to the LDLR-KO rignificant increared lerion formation and enlargement of the aorta when put on a high cholerterol diet. C o n d u s i o n : The inactivation of the dirtal NPXYXXL motif rerulted in increared atherorclerorir. Whether thir ir due to the modulating effect of LRP1 on PDGF rignalling or on foam cell formation needr to be determined. Funding: IWT: VIB; FWO
I Th-P 15:103
THE DI-LEUCINE MOTIF CONTRIBUTES TO CLASS A SCAVENGER RECEPTOR-MEDIATED UPTAKE OF ACETYLATED LIPOPROTEINS
Q. Chen, Y.Y. Chen, X.H. Wang, J.J. Beng. Atherosclerosis Researeh Centre, K ~ Laboratoty of Huntan Functional Genomics, Nanjing Medical Universi~, Nanjbtg, China Objective: The di-leucine motif exirtr in the intracellulax domainr of certain cell rurface receptorr, which contributer to the receptor-mediated endocytorir. The aim of the prerent rtudy was to determine the role of the di-leucine motif in clasr A rcavenger receptor (SR-A)-mediated ligand endocytorir. M e t h o d s : cDNA coding for different SR-A mutantr with deletion of the di-leucine rtructure (N3132LM) were tranrfected into Chinere hamrter ovary (CHO) cellr. The exprerrion of the mutantr was detected by obrervation under fluorercence microrcope as well ar wertern blot analyrir. The uptake and binding of DiI-AcLDL by tranrfected CHO cellr war quantitative analyzed by flow cytometry. To explore the role of di-leucine motif in localization of SR-A in clathrin-coated pit, the lyrater of tranrfected cellr were immunoprecipitated by an antibody againrt clathrin. Results: The cellr exprerring the SR-A mutant N3132LM rhowed decreated uptake of the SR-A ligand than wild type SR-A-exprerring cellr. Wertern blot analyrir on immunoprecipitater of cell lyrater indicated the arrociation of the ligand-SR-A mutant N3132LM with clathrin-coated pit. Further truncation of the firrt 27 amino acidr in the N-terminur of the di-leucine motif-mutated SR-A led to further decline in the cellular uptake of acetyl low denrity lipoprotein (AcLDL) when compared to thore without the di-leucine motif mutation. Conclusions: In conjunction with other known motifr, the di-leucine motif playr an important role in SR-A mediated internalization of A c L D L into cell. Funding: The work war rupported by the project of National Baric Rerearch ProgramNational Baric Rerearch Program(973)(TG200056910)and the National Natural Science Foundation of China (No.30370576) to Qi Chen.
I Th-P 1 5 : 1 0 4 I T R I G L Y C E R I D E - R I C H L I P O P R O T E I N S D E R I V E D FROM POMACE OLIVE OIL HAVE DIFFERENTIAL EFFECTS ON THE EXPRESSION OF MRNA FOR MACROPHAGE RECEPTORS I
R. Cabello-Moruno 1, J.S. Perona 1 , E. Montero 2, M. Avella 3, K.M. Botham 3, V. Ruiz-Gutierrez 1 . l b~stituto de la Grasa ( CSIC), Seville, Spain: 2HH, UU,
Virgen del Rocio, Seville, Spain." 3Royal Veterinaty College, London, United Kingdom Objectives: Triglyceride rich lipoproteinr (TRL) comporition changer portprandially according to the type of ingerted fat, affecting metabolirm of the particle. The aim of the prerent rtudy war to evaluate the influence of the unraponifiable fraction prerent in portprandial TRL derived from pomace olive oil (POO-TRL) on the exprerrion of mRNA gener involved in human macrophager foam cell formation, compared with TRL derived from refined olive oil (ROO-TRL).
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006