- Email: [email protected]
Mo-P3:227 Easy-to-use system for testing efficiency of targets for RNA interference
Mon&ty, June 19, 2006: Poster Session P3 Gene therapy attcl stem cells
96
Conclusion: Although electrotransfer enhances ApoE expression from skeletal muscle, a more efficient delivery vector, such as recombinant AAV, will be needed to achieve therapeutic levels. Funding: British Heart Foundation (FS/03/082/15926).
I Mo-P3:227
I EASY-TO-USE SYSTEM FOR TESTING EFFICIENCY OF TARGETS FOR RNA INTERFERENCE
N.M. Kashirina, P.N. Rutkevich, E.V. Yanushevskaya, M.M. Peklo, M.A. Slinkin, T.N. Vlasik, A.Y. Shevelev. bzst. of Eaperimental Cardiology,
Russian Cardiology Researeh and Industrial Complex, Moscow, Russia
Mo-P3:2251 P Y R R O L E - I M I D A Z O L E
POLYAMIDE TARGETING HUMAN LECTIN-LIKE OXIDIZED LDL RECEPTOR 1 GENE REDUCED ENDOTHELIAL CELL A P O P T O S I S I N D U C E D BY A N G I O T E N S I N II A N D OXIDIZED LDL
T. Ueno I , N. Fukuda I , K. Tahira I , R. S u z u k i 1 , T. Matsumoto I , H. Matsuda I , A. Tsunemi I , K. Matsumoto I , H. Sugiyama 2, T. Sawamura 3 . 1Department
of Medicine, Nihon Universi~ School of Medicbte, Itabashi, Tokyo, Japan: 2Department of Chemistt T, Kyoto Graduate School of Science, Sakyo-Ku,Kyoto, Japan," 3Department of Bioscience, National Cardiovascular Center Researeh Institute, Suita, Osa~t, Japan Objective: Polyamide containing N-methylimidazole and N-methylpyrrole amino acids (pyrrole-imidazole polyamide:Py-Im) can be combined in antiparallel side-by-side dimeric complex with the minor groove of DNA in a sequence specific manner. Py-Im are effective inhibitors of tissue specific and general transcription factors as well as viral repressors and transactivators. Recently, importance of Lectin-like oxidized low density lipoprotein receptor-1 (Lox-1) has been reported as a major factor contributing to the pathogenesis of coronary atherosclerosis. In this work, we have designed the Py-Im against Lox-1 gene, and evaluated the effect on the gene transcription. Methods and Results: Py-Im was designed to target the AP-1 binding site of Lox-1 gene, and synthesized by solid phase methods. Py-Im significantly inhibited Lox-1 promoter activity in 293cells determined by the luciferase assay. Lox-1 mRNA expression were also inhibited by Py-Im at the conscentration of 10-9M Human umbilical vein endotherial cell (HUBEC) determined by the real-time PCR method. HUBEC were treated by Py-Im targeting LOX1 gene, and apoptosis was assessed by Hoechst stain, TUNEL or Annexin V method. Treatment for 72 hours in LOX1 Py-Im decreased apoptosis induced by angiotensin II and oxidative L D loading by all assays. Conclusion: This novel therapeutic agent, Py-Im, could specifically inhibit Lox-1 gene expression and by reducing the promoter activity of this gene. Py-Im seems to be a powerful tool for the new strategy for transcription inhibition therapy. Molecular recognition of DNA by small molecules could impact and evolve human medicine.
Mo-P3:226 I S T A B L E T R A N S D U K T I O N O F M U R I N E B O N E MARROW LIN-C-KIT+ SUBPOPULATION WITH HIV-BASED PSEUDOVIRAL PARTICLES N.V. Radukhina, O.P. Ilyinskaya, P.N. Rutkevich, T.N. Vlasik, T.I. Aref'Eva, I.N. Rybalkirl, M.M. Peklo, E.V. Janushevskaya, E.M. Taraxak. Russian
Cardiology Researeh Industrial Complex, Moskow, Russia Objectives: In a number of recent publications, bone marrow (BM) progenitor cells were shown to take part in cardiovascular damage reparation. If this is really the case, of great importance would be the possibility to modify the behaviour of those cells. Cell transduction with the use of lentiviral vectors allows for the stable gene transfer into non-dividing cells including stem cells. This research was performed in order to develop an optimal system for studying lentiviral transduction of hematopoietic precursor cells. M e t h o d s : Murine BM subpopulation enriched in hematopoietic progenitor Lin-c-Kit+-cells, was stained with MAB to CD34, Sca-1 and c-Kit and then analyzed using FACS. Cells were transduced with HIV-based pseudoviral particles encoding GFP and analyzed for GFP expression by FACS analysis. Lethally irradiated murine females of CBAxC57B1/6 line were injected with 2000 transduced Lin-c-Kit+-cells from murine males of the same line to obtain spleen colonies. Large colonies were isolated from the spleens of the chimeras, and PCR was performed in some colonies to detect WPRE-fragment from viral genome. Results: One week after transduction, 27% of cultured hematopoietic progenitor Lin-c-Kit+-cells expressed GFP. DNA of a number of spleen colonies contained WPRE-fragment. Conclusions: HIV-based genetic construction can stably and effectively transduce hematopoietic progenitor Lin-c-Kit+-cells. Transduced Lin-c-Kit+cells bearing transgene can give rise to normal spleen colonies in vivo and restore hematopoiesis of lethally irradiated animals. Funding: This research was supported by CRDF grant RUB1-576-MO-04.
Objective: RNA interference (RNAi) is an efficient way to silence gene expression and can be used for preventing intimal hyperplasia and restenosis by silencing cell-cycle regulators such as cdc-2, cdk-2, etc. RNAi computational design is limited in the prediction of siRNA efficiency. Our goal was to develop an easy-to-use system for direct measuring of RNAi target sequence efficiency. M e t h o d s : 3 vectors were generated, one producing double-stranded siRNA (effector), and two others expressing Luciferase (Luc) gene fused to siRNA target sequence (reporters). Cells HEK-293 were transiently cotransfected with the mixture of effector and respective reporter vectors. Relative Luc activity was measured and compared for different target sequences. Results: Two sets of target sequences (19 nt long) against Green Fluorescent Protein (CopGFP) and Human Transferrin receptor (CD71) were designed. 2 of 10 targets for CopGFP and 4 of 10 targets for CD71 demonstrated significant silencing (less than 20% of Luc activity). In most cases, silencing efficiency was not greatly affected by a target position (up- or downstream) relevant to the Luc coding region. These data were confirmed by stable silencing of CopGFP and CD71 in cells transduced with lentiviral constructs expressing selected siRNAs. C o n d u s i o n s : We developed a reliable siRNA test system enabling direct evaluation of the siRNA target potential and avoiding the need of real-time RT-PCR. It can be especially appropriate when the gene of interest is not available as a full-size cDNA. Funding: The work is supported by the Russian Ministry of Health and Mona Ltd., Russia THE FIBROCYTE IMo-P3:2291 1~ 1~
IN I N T I M A L H Y P E R P L A S I A
R.L. Vaxcoe '-, H. Medbury '-, M. Vicaretti 1'2 , A. Guiffre 1'2, J.P. Fletcher 1'2 .
1Universi~ of Sydney, Sydney, Australia." 2 Westmead Hospital, Westmead, Australia Objectives: Experimental animal studies have shown that the intimal hyperplasia (IH) responsible for occlusion after successful revasculaxization procedures may be partially caused by a bone marrow derived cell that migrates to the site of vascular injury. Concurrent studies have demonstrated an extensive role in wound healing for the circulating fibrocyte. We aimed to trace the path of the circulating cell that contributes to IH and determine if it is the fibrocyte. M e t b o d s mid Results: We established an in vitro model whereby purified monocytes from six healthy human volunteers were cultured into fibrocytes. These cells were morphometrically similar to the vascular smooth muscle cell (VSMC) found in IH and expressed alpha-smooth muscle actin (alpha-SMA) as well as CD34, CD45 and Collagen I (Col I), markers indicative of the fibrocyte. In an in vivo ovine carotid artery synthetic patch graft model CFSE labeled circulating leukocytes were observed throughout the graft as well as in the neointima in eighteen sheep. These cells were shown to produce collagen and alpha-SMA at 1, 2 and 4 weeks. These cells then underwent immunohistochemical analysis and were found to express a set of markers unique to the fibrocyte (CD34, CD45, Vimentin and alpha-SMA) and also to double stain for CD34 and alpha-SMA. Conclusions: IH in an ovine carotid artery patch graft model is partially derived from a haematopoietic circulating progenitor cell that acquires mesenchymal features as it matures at the site of injury. Funding: None.
IMo-P3:2301 AV ENGEFWR - ~MCEDT1H3 3O/DC DO3 4F PEUNRDI FOYTIHNEGL I A L P R O G E N I T O R C E L L S F R O M RAT B O N E M A R R O W ~ Z.Y. Tong 1 , B.L. Hu 1, Z.S. Jiang 1 , G.H. Li 1 , F. Yao 1 , Z. Wan.,e l '-, Y.Z. Yang 1 G.X. Wang 2. 1 The bzstitute of Cardiovascular Disease, Nanhua
Universi~, Heng Yang, Hunan, China: 2Bioengineering College of Chongqing Universi~ and Key Lab for Biomechanics/Tissue Engineering of Ministty of Education, Chongqing, China B a c k g r o u n d : Methods of isolating endothelial progenitor cells OEPCs) have been established, such as immunomagnetic beads and adherence culture, But there are still lacking of an easy and applied method.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
96
Conclusion: Although electrotransfer enhances ApoE expression from skeletal muscle, a more efficient delivery vector, such as recombinant AAV, will be needed to achieve therapeutic levels. Funding: British Heart Foundation (FS/03/082/15926).
I Mo-P3:227
I EASY-TO-USE SYSTEM FOR TESTING EFFICIENCY OF TARGETS FOR RNA INTERFERENCE
N.M. Kashirina, P.N. Rutkevich, E.V. Yanushevskaya, M.M. Peklo, M.A. Slinkin, T.N. Vlasik, A.Y. Shevelev. bzst. of Eaperimental Cardiology,
Russian Cardiology Researeh and Industrial Complex, Moscow, Russia
Mo-P3:2251 P Y R R O L E - I M I D A Z O L E
POLYAMIDE TARGETING HUMAN LECTIN-LIKE OXIDIZED LDL RECEPTOR 1 GENE REDUCED ENDOTHELIAL CELL A P O P T O S I S I N D U C E D BY A N G I O T E N S I N II A N D OXIDIZED LDL
T. Ueno I , N. Fukuda I , K. Tahira I , R. S u z u k i 1 , T. Matsumoto I , H. Matsuda I , A. Tsunemi I , K. Matsumoto I , H. Sugiyama 2, T. Sawamura 3 . 1Department
of Medicine, Nihon Universi~ School of Medicbte, Itabashi, Tokyo, Japan: 2Department of Chemistt T, Kyoto Graduate School of Science, Sakyo-Ku,Kyoto, Japan," 3Department of Bioscience, National Cardiovascular Center Researeh Institute, Suita, Osa~t, Japan Objective: Polyamide containing N-methylimidazole and N-methylpyrrole amino acids (pyrrole-imidazole polyamide:Py-Im) can be combined in antiparallel side-by-side dimeric complex with the minor groove of DNA in a sequence specific manner. Py-Im are effective inhibitors of tissue specific and general transcription factors as well as viral repressors and transactivators. Recently, importance of Lectin-like oxidized low density lipoprotein receptor-1 (Lox-1) has been reported as a major factor contributing to the pathogenesis of coronary atherosclerosis. In this work, we have designed the Py-Im against Lox-1 gene, and evaluated the effect on the gene transcription. Methods and Results: Py-Im was designed to target the AP-1 binding site of Lox-1 gene, and synthesized by solid phase methods. Py-Im significantly inhibited Lox-1 promoter activity in 293cells determined by the luciferase assay. Lox-1 mRNA expression were also inhibited by Py-Im at the conscentration of 10-9M Human umbilical vein endotherial cell (HUBEC) determined by the real-time PCR method. HUBEC were treated by Py-Im targeting LOX1 gene, and apoptosis was assessed by Hoechst stain, TUNEL or Annexin V method. Treatment for 72 hours in LOX1 Py-Im decreased apoptosis induced by angiotensin II and oxidative L D loading by all assays. Conclusion: This novel therapeutic agent, Py-Im, could specifically inhibit Lox-1 gene expression and by reducing the promoter activity of this gene. Py-Im seems to be a powerful tool for the new strategy for transcription inhibition therapy. Molecular recognition of DNA by small molecules could impact and evolve human medicine.
Mo-P3:226 I S T A B L E T R A N S D U K T I O N O F M U R I N E B O N E MARROW LIN-C-KIT+ SUBPOPULATION WITH HIV-BASED PSEUDOVIRAL PARTICLES N.V. Radukhina, O.P. Ilyinskaya, P.N. Rutkevich, T.N. Vlasik, T.I. Aref'Eva, I.N. Rybalkirl, M.M. Peklo, E.V. Janushevskaya, E.M. Taraxak. Russian
Cardiology Researeh Industrial Complex, Moskow, Russia Objectives: In a number of recent publications, bone marrow (BM) progenitor cells were shown to take part in cardiovascular damage reparation. If this is really the case, of great importance would be the possibility to modify the behaviour of those cells. Cell transduction with the use of lentiviral vectors allows for the stable gene transfer into non-dividing cells including stem cells. This research was performed in order to develop an optimal system for studying lentiviral transduction of hematopoietic precursor cells. M e t h o d s : Murine BM subpopulation enriched in hematopoietic progenitor Lin-c-Kit+-cells, was stained with MAB to CD34, Sca-1 and c-Kit and then analyzed using FACS. Cells were transduced with HIV-based pseudoviral particles encoding GFP and analyzed for GFP expression by FACS analysis. Lethally irradiated murine females of CBAxC57B1/6 line were injected with 2000 transduced Lin-c-Kit+-cells from murine males of the same line to obtain spleen colonies. Large colonies were isolated from the spleens of the chimeras, and PCR was performed in some colonies to detect WPRE-fragment from viral genome. Results: One week after transduction, 27% of cultured hematopoietic progenitor Lin-c-Kit+-cells expressed GFP. DNA of a number of spleen colonies contained WPRE-fragment. Conclusions: HIV-based genetic construction can stably and effectively transduce hematopoietic progenitor Lin-c-Kit+-cells. Transduced Lin-c-Kit+cells bearing transgene can give rise to normal spleen colonies in vivo and restore hematopoiesis of lethally irradiated animals. Funding: This research was supported by CRDF grant RUB1-576-MO-04.
Objective: RNA interference (RNAi) is an efficient way to silence gene expression and can be used for preventing intimal hyperplasia and restenosis by silencing cell-cycle regulators such as cdc-2, cdk-2, etc. RNAi computational design is limited in the prediction of siRNA efficiency. Our goal was to develop an easy-to-use system for direct measuring of RNAi target sequence efficiency. M e t h o d s : 3 vectors were generated, one producing double-stranded siRNA (effector), and two others expressing Luciferase (Luc) gene fused to siRNA target sequence (reporters). Cells HEK-293 were transiently cotransfected with the mixture of effector and respective reporter vectors. Relative Luc activity was measured and compared for different target sequences. Results: Two sets of target sequences (19 nt long) against Green Fluorescent Protein (CopGFP) and Human Transferrin receptor (CD71) were designed. 2 of 10 targets for CopGFP and 4 of 10 targets for CD71 demonstrated significant silencing (less than 20% of Luc activity). In most cases, silencing efficiency was not greatly affected by a target position (up- or downstream) relevant to the Luc coding region. These data were confirmed by stable silencing of CopGFP and CD71 in cells transduced with lentiviral constructs expressing selected siRNAs. C o n d u s i o n s : We developed a reliable siRNA test system enabling direct evaluation of the siRNA target potential and avoiding the need of real-time RT-PCR. It can be especially appropriate when the gene of interest is not available as a full-size cDNA. Funding: The work is supported by the Russian Ministry of Health and Mona Ltd., Russia THE FIBROCYTE IMo-P3:2291 1~ 1~
IN I N T I M A L H Y P E R P L A S I A
R.L. Vaxcoe '-, H. Medbury '-, M. Vicaretti 1'2 , A. Guiffre 1'2, J.P. Fletcher 1'2 .
1Universi~ of Sydney, Sydney, Australia." 2 Westmead Hospital, Westmead, Australia Objectives: Experimental animal studies have shown that the intimal hyperplasia (IH) responsible for occlusion after successful revasculaxization procedures may be partially caused by a bone marrow derived cell that migrates to the site of vascular injury. Concurrent studies have demonstrated an extensive role in wound healing for the circulating fibrocyte. We aimed to trace the path of the circulating cell that contributes to IH and determine if it is the fibrocyte. M e t b o d s mid Results: We established an in vitro model whereby purified monocytes from six healthy human volunteers were cultured into fibrocytes. These cells were morphometrically similar to the vascular smooth muscle cell (VSMC) found in IH and expressed alpha-smooth muscle actin (alpha-SMA) as well as CD34, CD45 and Collagen I (Col I), markers indicative of the fibrocyte. In an in vivo ovine carotid artery synthetic patch graft model CFSE labeled circulating leukocytes were observed throughout the graft as well as in the neointima in eighteen sheep. These cells were shown to produce collagen and alpha-SMA at 1, 2 and 4 weeks. These cells then underwent immunohistochemical analysis and were found to express a set of markers unique to the fibrocyte (CD34, CD45, Vimentin and alpha-SMA) and also to double stain for CD34 and alpha-SMA. Conclusions: IH in an ovine carotid artery patch graft model is partially derived from a haematopoietic circulating progenitor cell that acquires mesenchymal features as it matures at the site of injury. Funding: None.
IMo-P3:2301 AV ENGEFWR - ~MCEDT1H3 3O/DC DO3 4F PEUNRDI FOYTIHNEGL I A L P R O G E N I T O R C E L L S F R O M RAT B O N E M A R R O W ~ Z.Y. Tong 1 , B.L. Hu 1, Z.S. Jiang 1 , G.H. Li 1 , F. Yao 1 , Z. Wan.,e l '-, Y.Z. Yang 1 G.X. Wang 2. 1 The bzstitute of Cardiovascular Disease, Nanhua
Universi~, Heng Yang, Hunan, China: 2Bioengineering College of Chongqing Universi~ and Key Lab for Biomechanics/Tissue Engineering of Ministty of Education, Chongqing, China B a c k g r o u n d : Methods of isolating endothelial progenitor cells OEPCs) have been established, such as immunomagnetic beads and adherence culture, But there are still lacking of an easy and applied method.
XIV bztentational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006