- Email: [email protected]
Tu-P8:318 Modulation of plasma HDL by intestine specific activation of the liver X receptor
Tues&ty, June 20, 2006: Poster Session P8 Basic science (2tM part)
ITu-P8:318
I
I M O D U L A T I O N O F P L A S M A H D L BY I N T E S T I N E i
SPECIFIC ACTIVATION OF THE LIVER X RECEPTOR
J.K. Kruit, R. Havinga, R. Bovorhof, F. Kuipora. Universi~ Medical Center
Gronbzgen, Department of Pediatrics, Gronbzgen, The NetherlatMs Agoniata for tho Livor X Rocoptor (LXR) havo potontially anti-athoroaclorotic actiona, howovor, LXR activation in rodonta alao loada to incroaaod lipogonoaia, hyportriglycoridomia and hopatic atoatoaia. Tho conaoquoncoa of LXR activation in tho livor axo woll atudiod, yot, thoao of L X R activation apocifically in tho intoatino axo loaa known. In thia atudy wo oxaminod tho phyaiological offocta of tho LXR agoniat GW3965 on plasma lipid profilo and choloatorol abaorption and aocrotion by tho intoatino in rata. For thia purpoao, Wiatar rata woro troatod with tho aynthotic L X R agoniat GW3965 (10 mg/kg/day for 6 daya), which lod to an intoatino-apocific L X R roaponao: No changoa in hopatic lipid loyola or in gono oxproaaion of L X R targot gonoa in tho livor woro obaorvod upon GW3965 troatmont. Biliaxy choloatorol and phoapholipid output romainod unaYfoctod upon GW3965 troatmont. LXR activation, howovor, did incroaao focal noutral atorol output by 63% and roducod fractional choloatorol abaorption by 45%. Intoatinal oxproaaion of tho Abe tranaportora A b c a l and Abcg5, both involvod in choloatorol tranaport was incroaaod 11- and 7-fold upon LXR activation. Plasma total choloatorol loyola woro unaffoctod, howtrot, FPLC analyaia on poolod aamploa ahowod an incroaao in HDL choloatorol by 20%, which ia a thorapoutically rolovant riao. Thoao roaulta indicato that intoatino-apocific activation of L X R loada to a potontially anti-athoroaclorotic roaponao without advorao offocta on triglycorido motaboliam. Funding: Thia atudy was fundod by tho Nothorlanda Heart Foundation grant 2001B043 I
T u - P 8 : 3 1 9 ]i A N O V E L L X R R E S P O N S I V E E L E M E N T E X I S T S IN A T P - B I N D I N G C A S S E T T E T R A N S P O R T E R G1 (ABCG1) GENE PROMOTER S. Abo 1, Y. Uohara 1'2 , S. Miura 1 , S. Furuyama 1 , C. Kitaguchi 2 , H. Urata 1 , T. Yamada 2 , K. Saku 1.1Departmentof Cardiology, Fukuokzt Universi~,
Fukuoka, Japan: 2Department of lntemal Medicine, Fukuokzt Universi~, Fukuoka, Japan B a c k g r o u n d : ATP-binding caaaotto tranaportor G1 (ABCG1) ia a mombrano choloatorol tranaportor bolong to ABC tranaportor family as woll as ABCA1. Rocontly, it has appoarod that tho ABCG1 gono ia involvod in HDL-modiatod collular choloatorol offlux and playa an antiathorogonic action. M e t h o d : HEK293 colla woro tranafoctod with luciforaso gono conatructa containing tho human wild-typo (WT) full longth and ita dolotod or pointmutatod ABCG1 promotor. In oloctrophorotic mobility a t i l t aaaaya (EMSA) oxporimont, HoLa nudoar oxtracta woro incubatod with tho ABCG1 promotor in tho proaonco or abaonco of LXR alpha/bota antibody. Roaulta and Concluaiona: Liganda for LXR and RXR oxtromoly atimulatod ABCG1 gono tranacription on a promotor locatod upatroam of oxonl. Sinco tho loaion, -303 to -163, waa found aa an important for roaponao to LXR on tho ABCG1 promotor by uaing tho dolotion conatracta, wo confirmod tho oxact loaion for roaponao to LXR/RXR by making a point-mutatod promotor. Aa tho roaulta, loaion -243/-228 on upatroam of oxonl was dotorminod for LXR/RXR roaponaivo (DR4) olomont as two analogoua of A G G T C A with 4 nuclootidoa apacoa. Tho olomont waa alao appoarod to bind to a LXR protoina in tho E M S A asaay. Thoao roaulta provido tho firat ovidonco for oxiatonco LXR roaponaivo alto in tho ABCG1 promotor locatod on upatroam of oxon 1. Our obaorvationa may h a r t an important rolo for a thorapoutic target to athoroacloroaia.
I Tu-P8:320 I L Y S O P H O S P H A T I D I C
ACID INDUCES NEOINTIMA AND VSMC DEDIFFERENTIATION VIA PPARG G. Tigyi I , N. Makaxova I , L. Balazs ~ , C. Zhan~o3 , D. Bakor 3 . 1Dept. of
Physiology, Universi~ of Tetmessee, Memphis, USA." 2Dept. of Pathology, Memphis, USA."3Dept. of Medicine, Universi~ of Tetmessee, Memphis, USA Objective: Idontify noointima-inducing lipida in oxidizod LDL M e t h o d s : Mass apoctromotry, noointima modol, ligand binding, roportor gono asaaya, immunohiatology Results: Wo provido ovidonco that a brief oxpoauro to oithor alkyl ether analoga of tho growth factor-liko phoapholipid lyaophoaphatidic acid (LPA), producta gonoratod during tho oxidativo modification of low donaity lipoprotoin, or to unaaturatod acyl forms of LPA induct progroaaivo formation of noointima in vivo in a rat carotid artery modol. Thia offoct ia complotoly inhibitod by protroatmont with PPARg antagoniat GW9662 and mimickod by
255
PPARg agoniata Roaiglitazono. In contraat to these lipida, atoaxoyl-oxovaloryl phoaphatidylcholino, EGF, PDGF or VEGF faMod to olicit noointima. Tho atructuro-activity rolationahip for noointima induction by LPA analoga in vivo is idontical to that of PPARg activation in vitro and diaparato from that of LPA G protein-coupled rocoptor activation. LPA olicita noointima in L P A I & L P A 2 doublo knockout mico. Noointima-inducing LPA analoga uprogulatod tho CD36 acavongor rocoptor in vitro and in vivo. Molocular forms of LPA that olicit noointima formation in vivo olicit phonotypic modulation of vascular amooth muaclo cella in vitro through PPARg. C o n d u s i o n : Soloct form of LPA art important novel ondogonoua PPARg liganda capablo of modiating vascular romodoling and that activation of tho nucloax tranacription factor PPARg ia nocoaaary for noointima formation by compononta of oxidized LDL. Funding: NIH NHLBI 79004
Tu-P8:321
RXR-GAMMA AND FXR VARIANTS AFFECT SERUM CHOLESTEROL LEVELS MAINLY THROUGH LIPOPROTEIN LIPASE LEVELS
A. Nohara I , M. Tsuchida 2 , M. Takata 2, S. Katsuda 2, K. Miwa 2, M. Kawashiri 2, A. Inazu 3, J. Kobayashi 4, J. Koizumi 5, H. Mabuchi I .
1Department of Lipidology, Kanazawa Universi~. Kanazawa, Japan: 2Molecular Genetics of Cardiovascular Disorders, Kanazawa University, Kanazawa, Japan." 3School of Health Sciences, Kanazawa Universi~, Kanazawa, Japan." 4Department of Lifes~le-Related Disease, Kanazawa Universi~, Kanazawa, Japan." 5Department of General Medicine, Katta2awa Universi~ Hospital, Kana2awa, Japan B a c k g r o u n d s a n d Objective: Rocontly, some nudoax rocoptora art shown to play important roles in onorgy homooataaia. Wo idontifiod gonotic variants of these nucloar rocoptor genes in pationta with dyalipidomia, and invoatigatod tho influonco of these variants. M e t h o d s : Wo acroonod all tho coding rogiona of PPARalpha, PPARgamma2, PPARdolta, FXR, LXRalpha and R X R g a m m a genes in 160 hyporlipidomic pationta by PCR-DGGE analyaia. Clinical impacta of tho idontifiod varianta were ovaluatod in 176 coronary angiography porformod pationta using PCR-RFLE Results: Wo idontifiod PPARalpha G395E, PPARgamma2 P12A, R X R g a m m a G14S, and FXR - l g - > t varianta. R X R g a m m a S14 carriora had aignificantly lower HDL-C (52 4- 19 va. 38 4- 9 mg/dl, p=0.01). FXR - l t carriora without diabotoa had lower HDL-C (57 4- 22 va. 49 4- 14 mg/dl, p=0.03) and lower LDL-C (159 4- 59 va. 136 4- 57 mg/dl, p=0.03). R X R g a m m a S 14 and FXR - l t caxriora had aignificantly lower poat-hopaxin lipoprotoin lipaao (LPL) loyola, and multiplo rogroaaion analyaia ahowod that LPL loyola were tho main contributor to aorum choloatorol loyola in these variant caxriora. C o n d u s i o n : R X R g a m m a and FXR varianta ahowod asaociation with aorum choloatorol loyola proaumably through diroct or indiroct LPL rogulation.
Tu-P8:322 ] T H E D I E T A R Y A N T I O X I D A N T R E S V E R A T R O L AFFECTS REDOX CHANGES OF PPAR-ALFA ACTIVITY • .1 ~ F.E Mancini -, E Iannolli.~- , A. Mooli.1 , V. Zaxrilli 1 , D. Tramontano 3. 1Dept.
of Biol. Ela'iron. Sci., Univ. of Satmio, Benevento, Italy: 2Dept. of Biochem. Med. Biotech., Univ. of Napoli Federico II, Napoli, Italy: 3Dept. of BPCM L. Califano, Univ. of Napoli Federico II, Napoli, Italy Objective: Gono-onvironmont interaction lies beneath tho pathogonoaia of tho most widespread diaoaaoa, and nutrition ia among tho environmental factors with tho highoat impact on human health. Tho mochaniama involved in this interaction aro still undoax. In thia study wo investigated whether roavoratrol, an antioxidant polyphonol of rod wine, can influence tho expression and function of several genes, including PPARa, a transcriptional factor regulating gono expression when activated by endogenous or exogenous long-chadn fatty adds. Ita activation results in a significant protection from cardiovascular diseases in humans. Metbods: Eloctromobility shift assay, Western Blot analysis and GSH dotormination were employed to analyzo tho effect of rodox balanco (DEM versus rosvoratrol) in cultured cells. Results: PPARa is rodox-sonsitivo, as it displays reduced DNA-binding activity following in vivo-troatmont of tho cells with 1 mmol/L diothylmaloato (DEM), a glutathiono-doploting agent. 100 I~mol/L rosvoratrol has a dual effect on PPARa activity: it prevents to a large extent tho DEM-inducod reduction of DNA-binding activity at earlier time points, when tho effect of
XIV bztemational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
ITu-P8:318
I
I M O D U L A T I O N O F P L A S M A H D L BY I N T E S T I N E i
SPECIFIC ACTIVATION OF THE LIVER X RECEPTOR
J.K. Kruit, R. Havinga, R. Bovorhof, F. Kuipora. Universi~ Medical Center
Gronbzgen, Department of Pediatrics, Gronbzgen, The NetherlatMs Agoniata for tho Livor X Rocoptor (LXR) havo potontially anti-athoroaclorotic actiona, howovor, LXR activation in rodonta alao loada to incroaaod lipogonoaia, hyportriglycoridomia and hopatic atoatoaia. Tho conaoquoncoa of LXR activation in tho livor axo woll atudiod, yot, thoao of L X R activation apocifically in tho intoatino axo loaa known. In thia atudy wo oxaminod tho phyaiological offocta of tho LXR agoniat GW3965 on plasma lipid profilo and choloatorol abaorption and aocrotion by tho intoatino in rata. For thia purpoao, Wiatar rata woro troatod with tho aynthotic L X R agoniat GW3965 (10 mg/kg/day for 6 daya), which lod to an intoatino-apocific L X R roaponao: No changoa in hopatic lipid loyola or in gono oxproaaion of L X R targot gonoa in tho livor woro obaorvod upon GW3965 troatmont. Biliaxy choloatorol and phoapholipid output romainod unaYfoctod upon GW3965 troatmont. LXR activation, howovor, did incroaao focal noutral atorol output by 63% and roducod fractional choloatorol abaorption by 45%. Intoatinal oxproaaion of tho Abe tranaportora A b c a l and Abcg5, both involvod in choloatorol tranaport was incroaaod 11- and 7-fold upon LXR activation. Plasma total choloatorol loyola woro unaffoctod, howtrot, FPLC analyaia on poolod aamploa ahowod an incroaao in HDL choloatorol by 20%, which ia a thorapoutically rolovant riao. Thoao roaulta indicato that intoatino-apocific activation of L X R loada to a potontially anti-athoroaclorotic roaponao without advorao offocta on triglycorido motaboliam. Funding: Thia atudy was fundod by tho Nothorlanda Heart Foundation grant 2001B043 I
T u - P 8 : 3 1 9 ]i A N O V E L L X R R E S P O N S I V E E L E M E N T E X I S T S IN A T P - B I N D I N G C A S S E T T E T R A N S P O R T E R G1 (ABCG1) GENE PROMOTER S. Abo 1, Y. Uohara 1'2 , S. Miura 1 , S. Furuyama 1 , C. Kitaguchi 2 , H. Urata 1 , T. Yamada 2 , K. Saku 1.1Departmentof Cardiology, Fukuokzt Universi~,
Fukuoka, Japan: 2Department of lntemal Medicine, Fukuokzt Universi~, Fukuoka, Japan B a c k g r o u n d : ATP-binding caaaotto tranaportor G1 (ABCG1) ia a mombrano choloatorol tranaportor bolong to ABC tranaportor family as woll as ABCA1. Rocontly, it has appoarod that tho ABCG1 gono ia involvod in HDL-modiatod collular choloatorol offlux and playa an antiathorogonic action. M e t h o d : HEK293 colla woro tranafoctod with luciforaso gono conatructa containing tho human wild-typo (WT) full longth and ita dolotod or pointmutatod ABCG1 promotor. In oloctrophorotic mobility a t i l t aaaaya (EMSA) oxporimont, HoLa nudoar oxtracta woro incubatod with tho ABCG1 promotor in tho proaonco or abaonco of LXR alpha/bota antibody. Roaulta and Concluaiona: Liganda for LXR and RXR oxtromoly atimulatod ABCG1 gono tranacription on a promotor locatod upatroam of oxonl. Sinco tho loaion, -303 to -163, waa found aa an important for roaponao to LXR on tho ABCG1 promotor by uaing tho dolotion conatracta, wo confirmod tho oxact loaion for roaponao to LXR/RXR by making a point-mutatod promotor. Aa tho roaulta, loaion -243/-228 on upatroam of oxonl was dotorminod for LXR/RXR roaponaivo (DR4) olomont as two analogoua of A G G T C A with 4 nuclootidoa apacoa. Tho olomont waa alao appoarod to bind to a LXR protoina in tho E M S A asaay. Thoao roaulta provido tho firat ovidonco for oxiatonco LXR roaponaivo alto in tho ABCG1 promotor locatod on upatroam of oxon 1. Our obaorvationa may h a r t an important rolo for a thorapoutic target to athoroacloroaia.
I Tu-P8:320 I L Y S O P H O S P H A T I D I C
ACID INDUCES NEOINTIMA AND VSMC DEDIFFERENTIATION VIA PPARG G. Tigyi I , N. Makaxova I , L. Balazs ~ , C. Zhan~o3 , D. Bakor 3 . 1Dept. of
Physiology, Universi~ of Tetmessee, Memphis, USA." 2Dept. of Pathology, Memphis, USA."3Dept. of Medicine, Universi~ of Tetmessee, Memphis, USA Objective: Idontify noointima-inducing lipida in oxidizod LDL M e t h o d s : Mass apoctromotry, noointima modol, ligand binding, roportor gono asaaya, immunohiatology Results: Wo provido ovidonco that a brief oxpoauro to oithor alkyl ether analoga of tho growth factor-liko phoapholipid lyaophoaphatidic acid (LPA), producta gonoratod during tho oxidativo modification of low donaity lipoprotoin, or to unaaturatod acyl forms of LPA induct progroaaivo formation of noointima in vivo in a rat carotid artery modol. Thia offoct ia complotoly inhibitod by protroatmont with PPARg antagoniat GW9662 and mimickod by
255
PPARg agoniata Roaiglitazono. In contraat to these lipida, atoaxoyl-oxovaloryl phoaphatidylcholino, EGF, PDGF or VEGF faMod to olicit noointima. Tho atructuro-activity rolationahip for noointima induction by LPA analoga in vivo is idontical to that of PPARg activation in vitro and diaparato from that of LPA G protein-coupled rocoptor activation. LPA olicita noointima in L P A I & L P A 2 doublo knockout mico. Noointima-inducing LPA analoga uprogulatod tho CD36 acavongor rocoptor in vitro and in vivo. Molocular forms of LPA that olicit noointima formation in vivo olicit phonotypic modulation of vascular amooth muaclo cella in vitro through PPARg. C o n d u s i o n : Soloct form of LPA art important novel ondogonoua PPARg liganda capablo of modiating vascular romodoling and that activation of tho nucloax tranacription factor PPARg ia nocoaaary for noointima formation by compononta of oxidized LDL. Funding: NIH NHLBI 79004
Tu-P8:321
RXR-GAMMA AND FXR VARIANTS AFFECT SERUM CHOLESTEROL LEVELS MAINLY THROUGH LIPOPROTEIN LIPASE LEVELS
A. Nohara I , M. Tsuchida 2 , M. Takata 2, S. Katsuda 2, K. Miwa 2, M. Kawashiri 2, A. Inazu 3, J. Kobayashi 4, J. Koizumi 5, H. Mabuchi I .
1Department of Lipidology, Kanazawa Universi~. Kanazawa, Japan: 2Molecular Genetics of Cardiovascular Disorders, Kanazawa University, Kanazawa, Japan." 3School of Health Sciences, Kanazawa Universi~, Kanazawa, Japan." 4Department of Lifes~le-Related Disease, Kanazawa Universi~, Kanazawa, Japan." 5Department of General Medicine, Katta2awa Universi~ Hospital, Kana2awa, Japan B a c k g r o u n d s a n d Objective: Rocontly, some nudoax rocoptora art shown to play important roles in onorgy homooataaia. Wo idontifiod gonotic variants of these nucloar rocoptor genes in pationta with dyalipidomia, and invoatigatod tho influonco of these variants. M e t h o d s : Wo acroonod all tho coding rogiona of PPARalpha, PPARgamma2, PPARdolta, FXR, LXRalpha and R X R g a m m a genes in 160 hyporlipidomic pationta by PCR-DGGE analyaia. Clinical impacta of tho idontifiod varianta were ovaluatod in 176 coronary angiography porformod pationta using PCR-RFLE Results: Wo idontifiod PPARalpha G395E, PPARgamma2 P12A, R X R g a m m a G14S, and FXR - l g - > t varianta. R X R g a m m a S14 carriora had aignificantly lower HDL-C (52 4- 19 va. 38 4- 9 mg/dl, p=0.01). FXR - l t carriora without diabotoa had lower HDL-C (57 4- 22 va. 49 4- 14 mg/dl, p=0.03) and lower LDL-C (159 4- 59 va. 136 4- 57 mg/dl, p=0.03). R X R g a m m a S 14 and FXR - l t caxriora had aignificantly lower poat-hopaxin lipoprotoin lipaao (LPL) loyola, and multiplo rogroaaion analyaia ahowod that LPL loyola were tho main contributor to aorum choloatorol loyola in these variant caxriora. C o n d u s i o n : R X R g a m m a and FXR varianta ahowod asaociation with aorum choloatorol loyola proaumably through diroct or indiroct LPL rogulation.
Tu-P8:322 ] T H E D I E T A R Y A N T I O X I D A N T R E S V E R A T R O L AFFECTS REDOX CHANGES OF PPAR-ALFA ACTIVITY • .1 ~ F.E Mancini -, E Iannolli.~- , A. Mooli.1 , V. Zaxrilli 1 , D. Tramontano 3. 1Dept.
of Biol. Ela'iron. Sci., Univ. of Satmio, Benevento, Italy: 2Dept. of Biochem. Med. Biotech., Univ. of Napoli Federico II, Napoli, Italy: 3Dept. of BPCM L. Califano, Univ. of Napoli Federico II, Napoli, Italy Objective: Gono-onvironmont interaction lies beneath tho pathogonoaia of tho most widespread diaoaaoa, and nutrition ia among tho environmental factors with tho highoat impact on human health. Tho mochaniama involved in this interaction aro still undoax. In thia study wo investigated whether roavoratrol, an antioxidant polyphonol of rod wine, can influence tho expression and function of several genes, including PPARa, a transcriptional factor regulating gono expression when activated by endogenous or exogenous long-chadn fatty adds. Ita activation results in a significant protection from cardiovascular diseases in humans. Metbods: Eloctromobility shift assay, Western Blot analysis and GSH dotormination were employed to analyzo tho effect of rodox balanco (DEM versus rosvoratrol) in cultured cells. Results: PPARa is rodox-sonsitivo, as it displays reduced DNA-binding activity following in vivo-troatmont of tho cells with 1 mmol/L diothylmaloato (DEM), a glutathiono-doploting agent. 100 I~mol/L rosvoratrol has a dual effect on PPARa activity: it prevents to a large extent tho DEM-inducod reduction of DNA-binding activity at earlier time points, when tho effect of
XIV bztemational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006