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Generation of APOE2- and APOE4-secreting cell lines by targeting of wild-type APOE3 cells using in-situ chimeraplasty
166
Miranda
gene polymorphrisms. The aim of our study was to evaluate the Lp(a) levels and apo(a) phenotypes in SH patients compared to healthy controls as well as the influence of L-thyroxine substitution therapy on Lp(a) values in relation with the apo(a) isoform size. Lp(a) levels were measured in 52 SH patients before and after the restoration of a euthyroid state along with 66 age and sex-matched healthy controls. Apo(a) isoform size was determined by SDS agarose gel electrophoresis followed by immunoblotting and development via chemiluminescence. SH patients exhibited increased Lp(a) levels compared to controls (12.25 mg/dl vs. 6.4 mg/dl [p-0.008]) but this was not due to differences in the frequencies of apo(a) phenotypes. In subjects with high molecular weight apo(a) isoforms (HMW), Lp(a) concentrations were higher in patients than in the control group (6.2 mg/dl vs. 3.8 mg/dl [p-0.004]). On the other hand, subjects of both groups with low molecular weight (LMW) isoforms had similar Lp(a) levels (25.8 mg/dl vs. 22.0 mg/dl for patients and controls respectively, [p-0.12]). Finally, L-thyroxine treatment resulted in a reduction of Lp(a) levels by 17 per cent (12.25 mg/dl pre-treatment vs. 10,15 mg/dl post-treatment [p-0.05]). This effect was independent ofapo(a) isoform size. ~6--~ FIBRINOGEN POLYMORPHISMS AND CEREBROVASCULAR DISEASE I.I. Correia, S. Viegas, P. Nogueira, M.T.E De Seixas, A.M.S.M. Miranda.
National Institute of Health, Lisbon, Portugal The cerebrovascular disease is the main cause of death in Portugal and the mortality rate of this disease is the highest in Europe. Plasma fibrinogen is a major risk factor for thrombosis and ischaemic heart disease. There is evidence that genetic variation in the beta-fibrinogen gene is associated with raised plasma fibrinogen levels. The aim of this study was to investigate the association of +1689T/G, -148C/T, Bcl I, Arg448Lys beta-fibrinogen polymorphisms and Thr312Ala alpha-fibrinogen polymorphism with the risk of cerebrovascular disease. Three hundred and one subjects (recruited from 23 Portuguese Hospitals) were genotyped by RFLP: 149 control subjects with plasmatic fibrinogen and lipid values within the reference range and 152 individuals who had an ischaemic stroke before 65 years. There was a significant difference in genotypic frequency in cases versus control subjects for Bcl I (p-0, 0297) and Arg448Lys (p-0,046) beta-fibrinogen polymorphisms. There was no statistical significant difference in the genotype distribution between cases and control subjects for +1689T/G (p-0,265), -148 C/T (p-0,282) and Thr312Ala (p-0,657) fibrinogen polymorphisms. It is concluded that the less frequent allele of fibrinogen polymorphisms +1689T/G, -148C/T, and Thr312Ala are not associated significantly with cerebrovascular disease. The rare alleles of the Bcl I, Arg448Lys betafibrinogen polymorphisms were positively associated with cerebrovascular disease and these last two nucleotide substitutions in the beta-fibrinogen gene are polymorphisms with a significant impact in cerebrovascular disease. ~6--~ INHIBITION OF PLATELET ACTIVATION AND FIBRINOGEN BINDING TO GPIIB/IIIA USING SYNTHETIC PEPTIDE-ANALOGUES DERIVED F R O M T H E GPIIB SUBUNIT J.V. Mitsios 1, A.P. Tambaki 1, M. Abatzis 1, N. Biffs 1, M. Sakarellos-Daitsiotis 1, C. Sakarellos 1, K. Soteriadou 4, L.K. Michalis 2, J.A. Goudevenos2, M. Elisaf3, D. Tsoukatos 1, V. Tsikaris 1, D.A. Sideris 2, A.D. Tselepis 1. JDepartment of Chemistry, 2Department of Cardiology,
3Department of Internal Medicine, University of loannina," 4Hellenic Pasteur Institute, Athens, Greece Platelet glycoprotein GPIIb/IIIa, is an inducible receptor which binds to ligands, containing the Arg-Gly-Asp (RGD) sequence, primarily found on fibrinogen (Fg), thereby mediating platelet aggregation and further activation, through an outside-in signaling pathway. RGD containing peptides are potent inhibitors of GPIIb/IIIa-Fg interactions, however, they maintain the receptor in its high affinity state. We synthesized five 20-peptides, covering the extracellular sequence 289 356 of the GPIIb subunit. Their effect on ADP-induced platelet aggregation, ATP secretion, as well as the binding of FITC-labeled Fg (FITC-Fg), and the binding of PAC-1 (a specific antibody against the activated receptor) were determined by flow cytometry. Among them, the 20-peptide 313 332 (YMESRADRKLAEVGRVYLFL) exhibited the most potent inhibitory effect on platelet aggregation (70%inhibition) and ATP secretion (100% inhibition). Furthermore, this peptide completely inhibited the binding of FITC-Fg on ADP activated platelets, whereas it did not affect the binding of PAC-1. To investigate the amino acid sequence responsible for this effect, we synthesized several 8-peptide analogues of
the above sequence. Only the 8-peptides containing the ESRAD sequence maintained the inhibitory properties associated with the original 20-peptide. The RGDS peptide that is known to bind to the receptor inhibited platelet aggregation and ATP secretion, as well as the binding of FITC-Fg and PAC-1. The 20-peptide analogue 313 332 of the GPIIb subunit inhibits platelet aggregation and secretion interacting with Fg rather than the receptor itself, possibly through the amino acid sequence ESRAD. Such peptides may be used as potent inhibitors of platelet aggregation and further activation. ~ - - ~ REGULATION OF ENDOTHELIAL APOPTOSIS BY SHEAR STRESS X. Jin2, M. Mitsumata 1, T. Yamane 2, Y. Yoshida 2. INihon University,
Tokyo; 2 yamanashi Medical School, Yamanashi, Japan To disclose the anti-atherosclerotic mechanisms of steady laminar shear stress, we analyzed the expression of human inhibitor of apoptosis protein-2 (HIAP-2), whose gene was selected from a cDNA library of sheared endothelial cells (ECs), on ECs. HIAP-2 was dose-independently and timedependently induced in ECs by shear stress, regulated at the transcriptional level. HIAP-2 expression was also identified in vivo. Shear stress-mediated inhibition of EC apoptosis was associated with the inhibition of caspase-3 activity, and this association was inhibited by the transfection of IAP inhibitor, Smac. These results suggest that the shear stress prevents EC apoptosis via negative regulation of caspase-3 by the increment of HIAP-2. [~'-~ GENERATION OF APOE2- AND APOE4-SECRETING CELL LINES BY TARGETING OF WILD-TYPE APOE3 CELLS USING IN-SITU CHIMERAPLASTY Z. Mohri 1, A.D. Tagalakis 1'2, G. Dickson2, J.S. Owen 1. 1Department of Medicine, Royal Free and University College Medical School, London," 2Department of Biochemistry, Royal Holloway University of London, Egham, Surrey, UK Apolipoprotein E (apoE), a 34-kDa circulating protein, has three common isoforms due to single nucleotide polymorphisms. The rarest variant, apoE2, differs from wild-type apoE3 by a R158C substitution, and causes Type III hyperlipidaemia. ApoE4 (Cll2R) is a risk factor for smoking-related heart disease and restenosis. These associations reflect key roles for apoE in cholesterol transport and cell signalling. Potentially, the new technology of targeted gene repair using synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct simple genetic defects. Our aim is to target THP1 monocyte-macrophages and HepG2 hepatoblastoma ceils to produce new cultured cell lines secreting apoE2 or apoE4 particles. These will facilitate comparative studies into differential biological activities of apoE isoforms. Initially, we targeted recombinant CHO cells secreting apoE3 with 68-mer apoE3-to-apoE2/E4 chimeraplasts complexed to polyethylenimine (PEI); the expected conversions were detected. After verifying that THP1 and HepG2 cells were derived from homozygous apoE3/3 donors, we investigated ways to increase conversion efficiency, including linear vs. branched PEI, centrifugation (to enhance membrane-cell contact), and modifying the PEI with melittin (to perturb membranes). Linear PEI alone proved the best reagent for our transfections. Direct sequencing and PCRRFLP analysis confirmed a dose-dependent conversion in HepG2. THP1 ceils were harder to transfect, but a small percentage were converted. Conversion stability was assessed by repeated passaging and freeze-thaw studies. An ELISA was established to quantify secreted apoE protein, while isoelectric focusing & immunoblotting will verify phenotypic changes. Our final goal is to isolate apoE2 and apoE4 clones. This study was supported by the British Heart Foundation. [~6--~ A STUDY TO DEMONSTRATE THE UTILITY OF HELP LDL APHERESIS TREATMENT F O R PATIENTS W I T H NON-ST ELEVATION ACUTE CORONARY SYNDROME P.M. Moriarty 1, C.A. Gibson 1, K. Flechsenhar2, M.S. Mayo 1, J.L. Vacek 1, P. Tadros 1, S.D. Owens 1, P. Delafontaine 1. 1University of Kansas, School
of Medicine, Kansas City, KS, USA," 2B. Braun, Melsungen, Germany The HELPME trial will establish the safety of HELP therapy in patients with non-ST elevation acute coronary syndrome and determine the utility of rapid and aggressive cholesterol lowering with reduction of inflammatory markers and rheological improvement by HELP therapy as an early intervention in acute coronary syndromes.
73rd EAS Congress
Miranda
gene polymorphrisms. The aim of our study was to evaluate the Lp(a) levels and apo(a) phenotypes in SH patients compared to healthy controls as well as the influence of L-thyroxine substitution therapy on Lp(a) values in relation with the apo(a) isoform size. Lp(a) levels were measured in 52 SH patients before and after the restoration of a euthyroid state along with 66 age and sex-matched healthy controls. Apo(a) isoform size was determined by SDS agarose gel electrophoresis followed by immunoblotting and development via chemiluminescence. SH patients exhibited increased Lp(a) levels compared to controls (12.25 mg/dl vs. 6.4 mg/dl [p-0.008]) but this was not due to differences in the frequencies of apo(a) phenotypes. In subjects with high molecular weight apo(a) isoforms (HMW), Lp(a) concentrations were higher in patients than in the control group (6.2 mg/dl vs. 3.8 mg/dl [p-0.004]). On the other hand, subjects of both groups with low molecular weight (LMW) isoforms had similar Lp(a) levels (25.8 mg/dl vs. 22.0 mg/dl for patients and controls respectively, [p-0.12]). Finally, L-thyroxine treatment resulted in a reduction of Lp(a) levels by 17 per cent (12.25 mg/dl pre-treatment vs. 10,15 mg/dl post-treatment [p-0.05]). This effect was independent ofapo(a) isoform size. ~6--~ FIBRINOGEN POLYMORPHISMS AND CEREBROVASCULAR DISEASE I.I. Correia, S. Viegas, P. Nogueira, M.T.E De Seixas, A.M.S.M. Miranda.
National Institute of Health, Lisbon, Portugal The cerebrovascular disease is the main cause of death in Portugal and the mortality rate of this disease is the highest in Europe. Plasma fibrinogen is a major risk factor for thrombosis and ischaemic heart disease. There is evidence that genetic variation in the beta-fibrinogen gene is associated with raised plasma fibrinogen levels. The aim of this study was to investigate the association of +1689T/G, -148C/T, Bcl I, Arg448Lys beta-fibrinogen polymorphisms and Thr312Ala alpha-fibrinogen polymorphism with the risk of cerebrovascular disease. Three hundred and one subjects (recruited from 23 Portuguese Hospitals) were genotyped by RFLP: 149 control subjects with plasmatic fibrinogen and lipid values within the reference range and 152 individuals who had an ischaemic stroke before 65 years. There was a significant difference in genotypic frequency in cases versus control subjects for Bcl I (p-0, 0297) and Arg448Lys (p-0,046) beta-fibrinogen polymorphisms. There was no statistical significant difference in the genotype distribution between cases and control subjects for +1689T/G (p-0,265), -148 C/T (p-0,282) and Thr312Ala (p-0,657) fibrinogen polymorphisms. It is concluded that the less frequent allele of fibrinogen polymorphisms +1689T/G, -148C/T, and Thr312Ala are not associated significantly with cerebrovascular disease. The rare alleles of the Bcl I, Arg448Lys betafibrinogen polymorphisms were positively associated with cerebrovascular disease and these last two nucleotide substitutions in the beta-fibrinogen gene are polymorphisms with a significant impact in cerebrovascular disease. ~6--~ INHIBITION OF PLATELET ACTIVATION AND FIBRINOGEN BINDING TO GPIIB/IIIA USING SYNTHETIC PEPTIDE-ANALOGUES DERIVED F R O M T H E GPIIB SUBUNIT J.V. Mitsios 1, A.P. Tambaki 1, M. Abatzis 1, N. Biffs 1, M. Sakarellos-Daitsiotis 1, C. Sakarellos 1, K. Soteriadou 4, L.K. Michalis 2, J.A. Goudevenos2, M. Elisaf3, D. Tsoukatos 1, V. Tsikaris 1, D.A. Sideris 2, A.D. Tselepis 1. JDepartment of Chemistry, 2Department of Cardiology,
3Department of Internal Medicine, University of loannina," 4Hellenic Pasteur Institute, Athens, Greece Platelet glycoprotein GPIIb/IIIa, is an inducible receptor which binds to ligands, containing the Arg-Gly-Asp (RGD) sequence, primarily found on fibrinogen (Fg), thereby mediating platelet aggregation and further activation, through an outside-in signaling pathway. RGD containing peptides are potent inhibitors of GPIIb/IIIa-Fg interactions, however, they maintain the receptor in its high affinity state. We synthesized five 20-peptides, covering the extracellular sequence 289 356 of the GPIIb subunit. Their effect on ADP-induced platelet aggregation, ATP secretion, as well as the binding of FITC-labeled Fg (FITC-Fg), and the binding of PAC-1 (a specific antibody against the activated receptor) were determined by flow cytometry. Among them, the 20-peptide 313 332 (YMESRADRKLAEVGRVYLFL) exhibited the most potent inhibitory effect on platelet aggregation (70%inhibition) and ATP secretion (100% inhibition). Furthermore, this peptide completely inhibited the binding of FITC-Fg on ADP activated platelets, whereas it did not affect the binding of PAC-1. To investigate the amino acid sequence responsible for this effect, we synthesized several 8-peptide analogues of
the above sequence. Only the 8-peptides containing the ESRAD sequence maintained the inhibitory properties associated with the original 20-peptide. The RGDS peptide that is known to bind to the receptor inhibited platelet aggregation and ATP secretion, as well as the binding of FITC-Fg and PAC-1. The 20-peptide analogue 313 332 of the GPIIb subunit inhibits platelet aggregation and secretion interacting with Fg rather than the receptor itself, possibly through the amino acid sequence ESRAD. Such peptides may be used as potent inhibitors of platelet aggregation and further activation. ~ - - ~ REGULATION OF ENDOTHELIAL APOPTOSIS BY SHEAR STRESS X. Jin2, M. Mitsumata 1, T. Yamane 2, Y. Yoshida 2. INihon University,
Tokyo; 2 yamanashi Medical School, Yamanashi, Japan To disclose the anti-atherosclerotic mechanisms of steady laminar shear stress, we analyzed the expression of human inhibitor of apoptosis protein-2 (HIAP-2), whose gene was selected from a cDNA library of sheared endothelial cells (ECs), on ECs. HIAP-2 was dose-independently and timedependently induced in ECs by shear stress, regulated at the transcriptional level. HIAP-2 expression was also identified in vivo. Shear stress-mediated inhibition of EC apoptosis was associated with the inhibition of caspase-3 activity, and this association was inhibited by the transfection of IAP inhibitor, Smac. These results suggest that the shear stress prevents EC apoptosis via negative regulation of caspase-3 by the increment of HIAP-2. [~'-~ GENERATION OF APOE2- AND APOE4-SECRETING CELL LINES BY TARGETING OF WILD-TYPE APOE3 CELLS USING IN-SITU CHIMERAPLASTY Z. Mohri 1, A.D. Tagalakis 1'2, G. Dickson2, J.S. Owen 1. 1Department of Medicine, Royal Free and University College Medical School, London," 2Department of Biochemistry, Royal Holloway University of London, Egham, Surrey, UK Apolipoprotein E (apoE), a 34-kDa circulating protein, has three common isoforms due to single nucleotide polymorphisms. The rarest variant, apoE2, differs from wild-type apoE3 by a R158C substitution, and causes Type III hyperlipidaemia. ApoE4 (Cll2R) is a risk factor for smoking-related heart disease and restenosis. These associations reflect key roles for apoE in cholesterol transport and cell signalling. Potentially, the new technology of targeted gene repair using synthetic RNA/DNA oligonucleotides (chimeraplasts) can permanently correct simple genetic defects. Our aim is to target THP1 monocyte-macrophages and HepG2 hepatoblastoma ceils to produce new cultured cell lines secreting apoE2 or apoE4 particles. These will facilitate comparative studies into differential biological activities of apoE isoforms. Initially, we targeted recombinant CHO cells secreting apoE3 with 68-mer apoE3-to-apoE2/E4 chimeraplasts complexed to polyethylenimine (PEI); the expected conversions were detected. After verifying that THP1 and HepG2 cells were derived from homozygous apoE3/3 donors, we investigated ways to increase conversion efficiency, including linear vs. branched PEI, centrifugation (to enhance membrane-cell contact), and modifying the PEI with melittin (to perturb membranes). Linear PEI alone proved the best reagent for our transfections. Direct sequencing and PCRRFLP analysis confirmed a dose-dependent conversion in HepG2. THP1 ceils were harder to transfect, but a small percentage were converted. Conversion stability was assessed by repeated passaging and freeze-thaw studies. An ELISA was established to quantify secreted apoE protein, while isoelectric focusing & immunoblotting will verify phenotypic changes. Our final goal is to isolate apoE2 and apoE4 clones. This study was supported by the British Heart Foundation. [~6--~ A STUDY TO DEMONSTRATE THE UTILITY OF HELP LDL APHERESIS TREATMENT F O R PATIENTS W I T H NON-ST ELEVATION ACUTE CORONARY SYNDROME P.M. Moriarty 1, C.A. Gibson 1, K. Flechsenhar2, M.S. Mayo 1, J.L. Vacek 1, P. Tadros 1, S.D. Owens 1, P. Delafontaine 1. 1University of Kansas, School
of Medicine, Kansas City, KS, USA," 2B. Braun, Melsungen, Germany The HELPME trial will establish the safety of HELP therapy in patients with non-ST elevation acute coronary syndrome and determine the utility of rapid and aggressive cholesterol lowering with reduction of inflammatory markers and rheological improvement by HELP therapy as an early intervention in acute coronary syndromes.
73rd EAS Congress