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Tu-P8:292 How aspirin modulates the TGF-B pathway in human vascular smooth muscle cells
Tues&ty, June 20, 2006: Poster Session P8 Basic science (2nd part) Morphologically, AGEt and RAGEt show both an axtracallular (matrix) and an intracallulax localization (macrophagas, lymphocitas, smooth muscla calls). Thasa rasults confirm a ralation batwaan AGEt and RAGEt also suggasting tha possibla involvamant of AGE-RAGE complax in plaqua complication. Funding: PAR 2004 Univarsity of Siana
P8
B A S I C S C I E N C E (2nd P A R T )
I
I Tu-P8:291 I H U M A N M A T U R I N G M A S T C E L L S E N H A N C E T i C E L L P R O L I F E R A T I O N IN VITRO J.A. Trosian, K.A. Lindstadt, ET. Kovanan. Wihuri Research btstitute,
Helsb~ki, Finland Objective: Our laboratory has shown praviously that mast calls (MC) ara abundant in tha shouldar ragions of atharosclarotic plaquas, whara also T calls ara prasant. MC pracursors infiltrata sitas of inflammation whara thay diffarantiata to matura MC. Wa invastigatad tha affact of tha maturation procass on tha prolifarativa rasponsa of T calls in vitro. Methods and Results: Praconditionad madium was obtainad from unstimulatad human cord blood-darivad MC culturas at diffarant tima points of call maturation. Culturing of T calls in praconditionad madium showad an anhancad prolifarativa rasponsa to CD3CD28 stimulus. Tha dagraa of tha prolifarativa rasponsa dapandad on tha dagraa of MC maturation. Haat traatmant of tha madium abolishad tha rasponsa. It has baan shown praviously that mousa MC-darivad TNF-alpha inducas T call prolifaration, but wa datactad only vary low concantrations of TNF-alpha (< 1.2 pg/ml) in tha praconditionad madium, and nautralizing it with an antibody had no affact on tha anhancad T call prolifaration. Conclusions: Our rasults show that unstimulatad human maturing mast calls anhanca T call prolifaration in vitro by sacrating a solubla protain madiator(s) othar than TNF-alpha. Funding: This rasearch projact has baan supportad by a Marie Curia Early Staga Rasaarch Training Fallowship of tha Europaan Community's Sixth Framawork Programma undar contract numbar 504926. Wihuri Rasaarch Instituta is maintainad by tha Janny and Antti Wihuri Foundation.
I
I Tu-P8:292 Ii H O W ASPIRIN MODULATES THE TGF-B PATHWAY IN H U M A N V A S C U L A R S M O O T H M U S C L E C E L L S
S. Radondo, E. Ruiz, A. Gordillo-Moscoso, T. Talarina. Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
Objectives: Antiprolifarativa affact of aspirin in rat vascular smooth muscla calls (VSMC) has been linkad to TGF-B. Wa aimad to axplora whathar aspirin was able to dacraasa human VSMC prolifaration through an autocrina loop of TGF-B 1 sacration. Methods: Human VSMC were obtained from patiants undargoing coronary artery by-pass grafting, culturad using tha axplant tachniqua and maintainad in RPMI-10% FCS. Cell prolifaration was assassad by BrdU incorporation (ELISA). Cytotoxicity and apoptosis were assassad by LDH assay and DNA fragmantation (ELISA), raspactivaly. TGF-B1 sacration was quantifiad by ELISA from cell conditionad madia and TGF-B-R-II axprassion was maasurad by RT-PCR. Results: Aspirin dacraasad human VSMC prolifaration in a dose dapandant mannar from 0.5 to 2mM. At 2mM, aspirin dacraasad DNA synthasis from 101.0-4-3.205% to 71.84-4-2.733%. Co-incubation with anti-TGF-B1 antibody was able to incraasa prolifaration from 71.84-t-2.733% to 94.53-t-5.965%. At this concantration, aspirin inducad naithar cytotocity nor apoptosis up to 48h. Aspirin 2mM inducad TGF-B1 sacration from 0 to 6h. Howavar, mRNA synthasis of TGF-B-R-II remained unchangad. Conclusions: At antiinflammatory concantrations (2mM) aspirin was able to dacraasa human vascular smooth muscla cell DNA synthasis by inducing sacration of TGF-B 1. Funding: This work was fundad by Fondo da Invastigacionas Sanitarias FISS 01/0815 (Haalth Research Fund from tha Spanish Ministry of Haalth), and by RECAVA. Wa thank tha Cardiac Surgary Sarvica (Hospital Clfnico San Carlos, Madrid) for providing us tha intarnal mammary artarias.
249
Tu-P8:293 ] T R A N I L A S T I N H I B I T S T R A N S D I F F E R E N T I A T I O N OF MACROPHAGES INTO SMOOTH M U S C L E - L I K E CELLS K. Ninomiya I , Y. Fujioka I , A. Takahashi I , A. Fukuda I , Y. Ishikawa 2, M. Yokoyama I . 1Division of Cardiovascular and Respirator 7 Medicitw,
Kobe Universi~ Graduate School of Medicine, Kobe, Japan." 2Facul~ of Health Sciences, Kobe Universi~ School of Medicine, Kobe, Japan Objective: Wa have praviously raportad that monocytas/macrophagas linaaga transdiffarantiatas into smooth muscle-lika calls via transforming growth factor-beta (TGF-bata) signaling in vitro (2004 AHA, Naw Orlaans and 2005 EAS, Praqua) and it may contributa to vascular ramodaling. Wa axaminad whathar an anti-allergic agant, Tranilast, supprassas tha transdiffarantiation in vitro. Methods and Results: Using rat paritonaal axcudata macrophagas, wa invastigatad tha axprassions of smooth muscla spacific diffarantiation markar protains such as alpha-SM actin (SMA), ambryonic myosin haavy chain isoform (SMamb) and calponin by Wastarn Bloting. At day 1, more than 95% of culturad monocytas were positiva for macrophaga-spacific antigan ( C D l l b and ED1) but nagativa for SMA, calponin and SMamb. Altar 7 days in 10% FBS cultura condition, robust induction of SMC spacific markars were obsarvad. Traatmant of macrophagas with Tranilast (300 mcmol/1) supprassad smooth muscla spacific markar appaaranca (about 0.2- 0.5 fold). Flowcytomatric analysis ravaalad at day 7,Tranilast dacraasad SMA-positiva cell populations by 0.25-fold less than that of control (10%FBS alona). Condusions: Thasa rasults indicatad that Tranilast supprassas transdiffarantiation of macrophagas into smooth muscle-lika calls and partly bacausa of tha supprassiva affact, Tranilast pravants naointimal thickaning and post angioplasty rastanosis. 1
Tu-P8:294 // E S S E N T I A L R O L E OF INCREASED RELEASE OF MCP-1 IN THE M E C H A N I S M OF PLATELETD E R I V E D GROWTH FACTOR (PDGF)-INDUCED P R O L I F E R A T I O N A N D M I G R A T I O N O F VASCULAR SMOOTH MUSCLE CELLS K. Nakano, K. Egashira, K. Sunagawa. Depcwtmet~t of Cardiovascular Medicbw, Graduate School of Medical Science, Kyushu Universi~, Fukuokzt, Japan Objective: Wa and othars have raportad a cantral role of monocyta chamoattractant protain-1 (MCP-1)-madiatad inflammation in tha pathoganasis of atharosclarosis. Wa also raportad calcium channal blockars (CCBs) raducad atharosclarosis possibly through raducad MCP-1 axprassion. Racant raports suggast tha prasanca of MCP-1 racaptor on vascular smooth muscla calls (VSMCs). Tharafora, incraasad axprassion of MCP-1 might ba involvad in tha machanism of prolifaration/migration of VSMCs, which might contributa to atharoganasis. Mathods and Rasults: PDGF-inducad prolifaration was datarminad by cell counting, and migration was avaluatad by tha Boydan's chambar mathod. Traatmant with a dominant-nagativa inhibitor of MCP-1 pravantad prolifaration/migration of VSMCs inducad not only by MCP-1 at 10 ng/mL, but also by PDGF at 10 ng/mL. To axamina contribution of MCP-1 ralaasa, MCP-1 lavals in suparnant was maasurad by ELISA. PDGF incraasad tha MCP-1 lavals from 3.4 -4- 0.6 to 33.5 -4- 12.0 ng/mg. Calcium channal blockar (azalnidipina at 1, 10, and 100 nM) raducad PDGF-inducad prolifaration/migration of VSMCs and MCP-1 ralaasa in a dosa-dapandant mannar. Condusions: Thasa data suggast that incraasad ralaasa of MCP- 1 madiatad via Ca influx is involvad critically in tha machanism of PDGF-inducad mitoganic action of MCP-1. This study supports tha notion that MCP-1 functions not only as tha chamokina for monocytas, but also as a critical mitogan of VSMCs in tha procass of atharosclarotic vascular disaasa. Funding: This study was supportad by Grants-in-Aid for Sciantific Research from tha Ministry of Education, Scianca, and Cultura, Japan.
Tu-P8:295 ] N U C L E A R P H O S P H A T I D Y L I N O S I T O L (3,4,5)TRISPHOSPHATE R E G U L A T I O N IN VASCULAR SMOOTH MUSCLE CELLS PROLIFERATION/DIFFERENTIATION
M. Laffar~ua, S. Gayral, A. Fougarat, P. Dalaris, N. Malat, M. Douillon. Lml Dept, Insernl U563, Toulouse, France Vascular smooth muscla calls (VSMC) prolifaration/diffaranciation is ona of tha kay procassas in tha davaloppamant of vascular pathologias such as
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
P8
B A S I C S C I E N C E (2nd P A R T )
I
I Tu-P8:291 I H U M A N M A T U R I N G M A S T C E L L S E N H A N C E T i C E L L P R O L I F E R A T I O N IN VITRO J.A. Trosian, K.A. Lindstadt, ET. Kovanan. Wihuri Research btstitute,
Helsb~ki, Finland Objective: Our laboratory has shown praviously that mast calls (MC) ara abundant in tha shouldar ragions of atharosclarotic plaquas, whara also T calls ara prasant. MC pracursors infiltrata sitas of inflammation whara thay diffarantiata to matura MC. Wa invastigatad tha affact of tha maturation procass on tha prolifarativa rasponsa of T calls in vitro. Methods and Results: Praconditionad madium was obtainad from unstimulatad human cord blood-darivad MC culturas at diffarant tima points of call maturation. Culturing of T calls in praconditionad madium showad an anhancad prolifarativa rasponsa to CD3CD28 stimulus. Tha dagraa of tha prolifarativa rasponsa dapandad on tha dagraa of MC maturation. Haat traatmant of tha madium abolishad tha rasponsa. It has baan shown praviously that mousa MC-darivad TNF-alpha inducas T call prolifaration, but wa datactad only vary low concantrations of TNF-alpha (< 1.2 pg/ml) in tha praconditionad madium, and nautralizing it with an antibody had no affact on tha anhancad T call prolifaration. Conclusions: Our rasults show that unstimulatad human maturing mast calls anhanca T call prolifaration in vitro by sacrating a solubla protain madiator(s) othar than TNF-alpha. Funding: This rasearch projact has baan supportad by a Marie Curia Early Staga Rasaarch Training Fallowship of tha Europaan Community's Sixth Framawork Programma undar contract numbar 504926. Wihuri Rasaarch Instituta is maintainad by tha Janny and Antti Wihuri Foundation.
I
I Tu-P8:292 Ii H O W ASPIRIN MODULATES THE TGF-B PATHWAY IN H U M A N V A S C U L A R S M O O T H M U S C L E C E L L S
S. Radondo, E. Ruiz, A. Gordillo-Moscoso, T. Talarina. Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain
Objectives: Antiprolifarativa affact of aspirin in rat vascular smooth muscla calls (VSMC) has been linkad to TGF-B. Wa aimad to axplora whathar aspirin was able to dacraasa human VSMC prolifaration through an autocrina loop of TGF-B 1 sacration. Methods: Human VSMC were obtained from patiants undargoing coronary artery by-pass grafting, culturad using tha axplant tachniqua and maintainad in RPMI-10% FCS. Cell prolifaration was assassad by BrdU incorporation (ELISA). Cytotoxicity and apoptosis were assassad by LDH assay and DNA fragmantation (ELISA), raspactivaly. TGF-B1 sacration was quantifiad by ELISA from cell conditionad madia and TGF-B-R-II axprassion was maasurad by RT-PCR. Results: Aspirin dacraasad human VSMC prolifaration in a dose dapandant mannar from 0.5 to 2mM. At 2mM, aspirin dacraasad DNA synthasis from 101.0-4-3.205% to 71.84-4-2.733%. Co-incubation with anti-TGF-B1 antibody was able to incraasa prolifaration from 71.84-t-2.733% to 94.53-t-5.965%. At this concantration, aspirin inducad naithar cytotocity nor apoptosis up to 48h. Aspirin 2mM inducad TGF-B1 sacration from 0 to 6h. Howavar, mRNA synthasis of TGF-B-R-II remained unchangad. Conclusions: At antiinflammatory concantrations (2mM) aspirin was able to dacraasa human vascular smooth muscla cell DNA synthasis by inducing sacration of TGF-B 1. Funding: This work was fundad by Fondo da Invastigacionas Sanitarias FISS 01/0815 (Haalth Research Fund from tha Spanish Ministry of Haalth), and by RECAVA. Wa thank tha Cardiac Surgary Sarvica (Hospital Clfnico San Carlos, Madrid) for providing us tha intarnal mammary artarias.
249
Tu-P8:293 ] T R A N I L A S T I N H I B I T S T R A N S D I F F E R E N T I A T I O N OF MACROPHAGES INTO SMOOTH M U S C L E - L I K E CELLS K. Ninomiya I , Y. Fujioka I , A. Takahashi I , A. Fukuda I , Y. Ishikawa 2, M. Yokoyama I . 1Division of Cardiovascular and Respirator 7 Medicitw,
Kobe Universi~ Graduate School of Medicine, Kobe, Japan." 2Facul~ of Health Sciences, Kobe Universi~ School of Medicine, Kobe, Japan Objective: Wa have praviously raportad that monocytas/macrophagas linaaga transdiffarantiatas into smooth muscle-lika calls via transforming growth factor-beta (TGF-bata) signaling in vitro (2004 AHA, Naw Orlaans and 2005 EAS, Praqua) and it may contributa to vascular ramodaling. Wa axaminad whathar an anti-allergic agant, Tranilast, supprassas tha transdiffarantiation in vitro. Methods and Results: Using rat paritonaal axcudata macrophagas, wa invastigatad tha axprassions of smooth muscla spacific diffarantiation markar protains such as alpha-SM actin (SMA), ambryonic myosin haavy chain isoform (SMamb) and calponin by Wastarn Bloting. At day 1, more than 95% of culturad monocytas were positiva for macrophaga-spacific antigan ( C D l l b and ED1) but nagativa for SMA, calponin and SMamb. Altar 7 days in 10% FBS cultura condition, robust induction of SMC spacific markars were obsarvad. Traatmant of macrophagas with Tranilast (300 mcmol/1) supprassad smooth muscla spacific markar appaaranca (about 0.2- 0.5 fold). Flowcytomatric analysis ravaalad at day 7,Tranilast dacraasad SMA-positiva cell populations by 0.25-fold less than that of control (10%FBS alona). Condusions: Thasa rasults indicatad that Tranilast supprassas transdiffarantiation of macrophagas into smooth muscle-lika calls and partly bacausa of tha supprassiva affact, Tranilast pravants naointimal thickaning and post angioplasty rastanosis. 1
Tu-P8:294 // E S S E N T I A L R O L E OF INCREASED RELEASE OF MCP-1 IN THE M E C H A N I S M OF PLATELETD E R I V E D GROWTH FACTOR (PDGF)-INDUCED P R O L I F E R A T I O N A N D M I G R A T I O N O F VASCULAR SMOOTH MUSCLE CELLS K. Nakano, K. Egashira, K. Sunagawa. Depcwtmet~t of Cardiovascular Medicbw, Graduate School of Medical Science, Kyushu Universi~, Fukuokzt, Japan Objective: Wa and othars have raportad a cantral role of monocyta chamoattractant protain-1 (MCP-1)-madiatad inflammation in tha pathoganasis of atharosclarosis. Wa also raportad calcium channal blockars (CCBs) raducad atharosclarosis possibly through raducad MCP-1 axprassion. Racant raports suggast tha prasanca of MCP-1 racaptor on vascular smooth muscla calls (VSMCs). Tharafora, incraasad axprassion of MCP-1 might ba involvad in tha machanism of prolifaration/migration of VSMCs, which might contributa to atharoganasis. Mathods and Rasults: PDGF-inducad prolifaration was datarminad by cell counting, and migration was avaluatad by tha Boydan's chambar mathod. Traatmant with a dominant-nagativa inhibitor of MCP-1 pravantad prolifaration/migration of VSMCs inducad not only by MCP-1 at 10 ng/mL, but also by PDGF at 10 ng/mL. To axamina contribution of MCP-1 ralaasa, MCP-1 lavals in suparnant was maasurad by ELISA. PDGF incraasad tha MCP-1 lavals from 3.4 -4- 0.6 to 33.5 -4- 12.0 ng/mg. Calcium channal blockar (azalnidipina at 1, 10, and 100 nM) raducad PDGF-inducad prolifaration/migration of VSMCs and MCP-1 ralaasa in a dosa-dapandant mannar. Condusions: Thasa data suggast that incraasad ralaasa of MCP- 1 madiatad via Ca influx is involvad critically in tha machanism of PDGF-inducad mitoganic action of MCP-1. This study supports tha notion that MCP-1 functions not only as tha chamokina for monocytas, but also as a critical mitogan of VSMCs in tha procass of atharosclarotic vascular disaasa. Funding: This study was supportad by Grants-in-Aid for Sciantific Research from tha Ministry of Education, Scianca, and Cultura, Japan.
Tu-P8:295 ] N U C L E A R P H O S P H A T I D Y L I N O S I T O L (3,4,5)TRISPHOSPHATE R E G U L A T I O N IN VASCULAR SMOOTH MUSCLE CELLS PROLIFERATION/DIFFERENTIATION
M. Laffar~ua, S. Gayral, A. Fougarat, P. Dalaris, N. Malat, M. Douillon. Lml Dept, Insernl U563, Toulouse, France Vascular smooth muscla calls (VSMC) prolifaration/diffaranciation is ona of tha kay procassas in tha davaloppamant of vascular pathologias such as
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006