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144 Overexpression of human apolipoprotein A-I in transgenic rabbits
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144 Overe8pmeIon of b~manl pdipoprotein A-I in transgenk rabbita Florence Emmawel, Jean biichel CnilIaud. Nathalte Hennuyer*. Catherine Fieve~. Graciela Capho*. Jean Charler Fruchati. Louis Marie HoudebineW. Patrice Den@ and Nicolas Duverger RPR-Gcnocll, Cardiovascular departcment, Vitry sur Seine. #lNRA, Jouy co Josas. ‘Pasteur Institute, Lie, France. Cholesterol in High density lipoproteins (HDL) and apolipopmtein (ape) A-I levels arc inversely correlated with coronary heart diseases. Gvcrexprcssion of humao apoA-I in several strains of mice has been shown to protect against atherosclerosis dcvclopmcnt. However, lipoprotein metabolism in mice are far from that in human. Concerning this aspect, the rabbit model presents scvcral advantage with the presence of cholesteryl ester transfer protein or with the apoB-containing lipoproteins as major cholcaterol carriers in plasma. Therefore, in order to study the role of apoA-I in lipoprotein metabolism and assessits efficacy to retard atbcrosclcrosis progression in another animal model, we produced human apoA-I transgcnic rabbits (New Zealand White background, NZW) with the use of an 11-kh gcnomic apoA-I fragment containing a liver-specific promoter. Traosgenic animals cxprcsscd human tracsgcnc in the liver whereas, in rabbits, apoA-I is normally produced by the intestine. In one of five tmnsgenic lies established, human apoA-I concentration wss superior to 175 mg/dl whereas rabbit apoA-I levels wcrc signiticantly rcduccd. In this lie, HDL-cholesterol levels were 2-fold increased in comparison with that of non haosgcnic littermate. Protective effect of apoA-I overexpression was evaluated in rabbits fed a cholesterol rich diet for 14 weeks. Plasma levels of athcrogcnic apoB-containing lipoproteins (>lOOil n&U) were matched between transgcnic and control snimals. HDL-cholestcrol levels in traosgcnic rabbits were still approximately twice that of the contml rabbits. At the end of the experiment, the amount of aortic surface area covered by the lesions as well as the amount of lipid accumulation in the aorta wcrc significantly less in tmnsgenic rabbits compared with control rabbits, confirming tbe bcncficial properties of apoA-I-containing lipoproteins. Furthermore, human apoA-1 transgenic rabbits were crossed with Watanabe rabbits, a relevant model of familial hypercholestcrolemia, which develop spontaneously severe athcrosclcrosis. Human apoA-I lcvcls in human apoA-I Watanabe rabbits, 101 mg/dl, were lower than that in traosgcnic NZW rabbits. Rabbit apoA-I levels were similar between human apoA-I Watanabe rabbits and control rabbits. However, HDL-cholestcrol concentration were still 2-fold higher in transgenie versus non traosgcnic Watanabe rabbits. These data established the fundamental role of apoA-I in HDL metabolism and in the atherosclerosis process.
145 Use of adenovirsl vectors to buxease reverse cholesterol transport. Bart De Geest, D&irk Collen, Paul Holvoet. Center for Molecular and Vascular Biology, University of Leuven Adcnoviros-mediated gene tmasfer by systemic administration of recombinant replication-defective adenoviruses results in a highly efficient transduction of hcpatocytes in viva. Major advantages of gene transfer by adenovimscs arc tbe capability to transduce both replicating and non-replicating cells and the easy production of adenovims stocks of high titer. The most important limitation of current technology is the transient nature of tramgene expression which is principally due to an immune response against the traosgene and viral gene products. Potent transient effects on lipoprotein metabolism have been demonstrated using recombit adcnoviroses expressing the LDL receptor, the VLDL receptor, 7 a-hydroxylaac, apo E, APOBEC-1 and a tramdominant negative mutant of APOBECl A recombinant adcnoviral vector containing the rabbit LDL receptor cDNA not only lowcrcd LDL cholesterol levels, but also inorcascd HDL cholesterol levels and ape A-I lcvcls by 30@-400% in Watanabe Heritable Hyperlipidemic rabbits. Administration of an adenovirus containing the human ape E gcnc to ape E deficient mice led to a nomnlisation of total cholesterol levels aud a more than threefold increase of HDLcholcsterol. Using an adcnoviral v&or expressing human ape A-I under control of the CMV-promoter, we found a 2.8-fold incrcasc of HDL-cholcstcrol levels in C57BU6J mice and a 2.7-fold incrcasc of HDL cholesterol in C57BU6J ape E deficient mice &r administration of 4 x 109 p.f.u. The HDL cholcstcrol / non-HDL cholesterol ratio inoreascd from 0.063 i 0.003 in control ape E deficient mice to 0.16 * 0.015 tbrcc days after injection. Human ape A-I levels were nearly undetectable 3 weeks atIer injection. Previously, intmdoction of a human ape A-I tramgene in ape E deficient mice has been shown to bc a potent suppressor of atherosclerosis. Similarly, the transient increase in HDL cholesterol levels induced by adcoovirus mediated gem transfer of human ape A-I in ape E deficient mice led to a profound reduction in m&time formation after endothelial denudation of the common carotid artety. Because of the potent anti-athemgenic effects of human apolipoprotcin A-I as shown in transgenic mice, the clear inverse relationship bctwccn HDL cholcstcrol and cardiovsscular disease lo humans and the absence of a rclationsldp bchvcen HDL cholesterol and nm~ovascular disease, adenovbus mediated gene transfer with apolipoprotcio A-I is particularly attractive. However, the hansient nature of gene expression remains the major
146 Adenoviml-mcdiatcd gene transfer of eodothelld nitric oxide ayntbasc to the rabbit carotid artery rItcrs voscolar mactMty. I. Kullo, G. Mazes, R. Schwartz, I! Gloviak-t, 7: Crony. Z.S. Katwic. I: O’Bnkn Mayo Chic, Rochester, MN, USA. We tested the effect of overexpression of cndothclial nitric oxide synthasc gene in the rabbit carotid artery by adcnoviral mediated gene ban&r. Rabbit carotid arteries were surgically isolated and exposed to an adenoviral vector encoding cNOS (AdeNOS) and ao adcnovird vector emding Bgalactosidase on the other side (RCial). Vector solutions at a wncenttatkm of 1010pfu/l were instilled intral~ally for 20 minutes or delivered to the pmiadventitial sheath of the vessel. Arteries were harvested 4 days later for inummosmining, vasomotor stodies and measurement of cGMP levels and NOS activity. Intiuminal admiition resulted in cndotheliom specific gcnc transfer, while advcntitial delivery resulted in advcntitial specific gene transfer. In both delivery models cGMP levels were si@icantly higher in the AdcNOS transduced vessels. Maximal contmctions to phcnylephtine were diminished io both models (Intraluminal: 6.0 f 0.3 grams vs 5.0 + 0.2 gnuns, p < 0.02; Adventitisl: 5.6 f 0.2 grams vs. 4.6 f 0.2 grams, p < 0.05.). This effect WBSabolished in the presence of the NOS inhibitor L-NMMA. After in!mluminal administration, relaxations to acetylcholinc obtained during submaximal con&actions to phcnylephrinc were significantly enhanced in the AdeNOS artcries (-IogECrc 7.4 + 0.08~s 7.09 + 0.09, P < 0.02). After advcotitial admiistration, relaxations to calcium ionophore obtained during submaximal contractions to phenylcphrioe were significantly enhanced in the AdcNOS vessels (-log Ejc 7.77 f 0.08 vs 7.45 f 0.07, P < 0.02. We conclude that overexpression of eNOS in the cndotheliom or advcntitia results in altered vasorcactivity. These results suggest that gene transfer of eNOS may be beneficial in the treatment of vasospasm and cndothclial dysfunction.
147 Metabolic role of apoUpopmtein A-II studies in traosgenic mice M. Cloes, C. de Geitere. A .Boullie~ B. De@y, G. Tremp, N. Duverger, J.C. Fruchort and G.R. Castro SERLIA - Institut Pastcur -Lille - France Gcncell Division - Rhone Poulcnc Rarer - Vitry sur Seine - France High density lipoprotein (HDL) levels are inversely corrclatcd with tbe incidence of atherosclerotic cardiovascular diseases. HDL may protect against atbcrosclcmsis by promoting tmcqxt of excess cholesterol from peripheral tissues to liver, a process termed rcvcrx cholesterol transport (RCT). In addition, HDL protects low density lipoproteins from oxidative damage known to contribute to the onset and progression of atberosclcrosis The major HDL proteins arc spolipoprotcin (apo)A-I and apoA-II. Some evidence suggests that apoA-I is directly protective against atherosclerosis, but relatively little is known about the role of apoA-II. Contrasting results have been obtained in mice overexpressing human (h) or murinc (m) ape A-II. hapoA-II, a homodimeric protein, seems to confer protection with regard to athcrogencsis, in contrast, overproduction of mapoA-Il (that is monomeric) has been proposed to be .&erogenic. To forther investigate the role of apoA-II we measured the initials steps of RCT and the panloxonase activity in both types of mice. To determine if the observed differences were related to the mono or dimeric forms we studied tmnsgeoic mice for hapo A-II rendered monomeric by (Cyd+Ser) mutation @apoA-IImon). mapoA-II mice had cHDL values 84% higher than control mice, their sor HDL had a capacity to promote cellular cholesterol cfflux that was twice the one of controls. LCAT activity measured with exogenous substrate was not different to the activity of control scra (31.8 f 1.8 vs 33.8 * 2 %) but the endogcnous estcrification capacity was higher (16 * 2.5 vs 7.2 f 0.2 %), hapoA-II mice with normal cHDL and hapoA-IImon with reduced cHDL had lower capacity to efflux cholesterol and to ester@ it than control mice. Paroxonase activities for mapoA-II, hapoA-II and hapoA-IImon were respectively 98,83 and 73% of control mice serum activity Our results are a clear indication that the metabolic diffbrcnces observed between m and h apoA-11 arc not related to the mono or dimcric forms, and above all that RCT is a more complex pathway and that increased activity of some steps is not a proof that peripheral cholcstcml will bc efficiently eliminated.
144 Overe8pmeIon of b~manl pdipoprotein A-I in transgenk rabbita Florence Emmawel, Jean biichel CnilIaud. Nathalte Hennuyer*. Catherine Fieve~. Graciela Capho*. Jean Charler Fruchati. Louis Marie HoudebineW. Patrice Den@ and Nicolas Duverger RPR-Gcnocll, Cardiovascular departcment, Vitry sur Seine. #lNRA, Jouy co Josas. ‘Pasteur Institute, Lie, France. Cholesterol in High density lipoproteins (HDL) and apolipopmtein (ape) A-I levels arc inversely correlated with coronary heart diseases. Gvcrexprcssion of humao apoA-I in several strains of mice has been shown to protect against atherosclerosis dcvclopmcnt. However, lipoprotein metabolism in mice are far from that in human. Concerning this aspect, the rabbit model presents scvcral advantage with the presence of cholesteryl ester transfer protein or with the apoB-containing lipoproteins as major cholcaterol carriers in plasma. Therefore, in order to study the role of apoA-I in lipoprotein metabolism and assessits efficacy to retard atbcrosclcrosis progression in another animal model, we produced human apoA-I transgcnic rabbits (New Zealand White background, NZW) with the use of an 11-kh gcnomic apoA-I fragment containing a liver-specific promoter. Traosgenic animals cxprcsscd human tracsgcnc in the liver whereas, in rabbits, apoA-I is normally produced by the intestine. In one of five tmnsgenic lies established, human apoA-I concentration wss superior to 175 mg/dl whereas rabbit apoA-I levels wcrc signiticantly rcduccd. In this lie, HDL-cholesterol levels were 2-fold increased in comparison with that of non haosgcnic littermate. Protective effect of apoA-I overexpression was evaluated in rabbits fed a cholesterol rich diet for 14 weeks. Plasma levels of athcrogcnic apoB-containing lipoproteins (>lOOil n&U) were matched between transgcnic and control snimals. HDL-cholestcrol levels in traosgcnic rabbits were still approximately twice that of the contml rabbits. At the end of the experiment, the amount of aortic surface area covered by the lesions as well as the amount of lipid accumulation in the aorta wcrc significantly less in tmnsgenic rabbits compared with control rabbits, confirming tbe bcncficial properties of apoA-I-containing lipoproteins. Furthermore, human apoA-1 transgenic rabbits were crossed with Watanabe rabbits, a relevant model of familial hypercholestcrolemia, which develop spontaneously severe athcrosclcrosis. Human apoA-I lcvcls in human apoA-I Watanabe rabbits, 101 mg/dl, were lower than that in traosgcnic NZW rabbits. Rabbit apoA-I levels were similar between human apoA-I Watanabe rabbits and control rabbits. However, HDL-cholestcrol concentration were still 2-fold higher in transgenie versus non traosgcnic Watanabe rabbits. These data established the fundamental role of apoA-I in HDL metabolism and in the atherosclerosis process.
145 Use of adenovirsl vectors to buxease reverse cholesterol transport. Bart De Geest, D&irk Collen, Paul Holvoet. Center for Molecular and Vascular Biology, University of Leuven Adcnoviros-mediated gene tmasfer by systemic administration of recombinant replication-defective adenoviruses results in a highly efficient transduction of hcpatocytes in viva. Major advantages of gene transfer by adenovimscs arc tbe capability to transduce both replicating and non-replicating cells and the easy production of adenovims stocks of high titer. The most important limitation of current technology is the transient nature of tramgene expression which is principally due to an immune response against the traosgene and viral gene products. Potent transient effects on lipoprotein metabolism have been demonstrated using recombit adcnoviroses expressing the LDL receptor, the VLDL receptor, 7 a-hydroxylaac, apo E, APOBEC-1 and a tramdominant negative mutant of APOBECl A recombinant adcnoviral vector containing the rabbit LDL receptor cDNA not only lowcrcd LDL cholesterol levels, but also inorcascd HDL cholesterol levels and ape A-I lcvcls by 30@-400% in Watanabe Heritable Hyperlipidemic rabbits. Administration of an adenovirus containing the human ape E gcnc to ape E deficient mice led to a nomnlisation of total cholesterol levels aud a more than threefold increase of HDLcholcsterol. Using an adcnoviral v&or expressing human ape A-I under control of the CMV-promoter, we found a 2.8-fold incrcasc of HDL-cholcstcrol levels in C57BU6J mice and a 2.7-fold incrcasc of HDL cholesterol in C57BU6J ape E deficient mice &r administration of 4 x 109 p.f.u. The HDL cholcstcrol / non-HDL cholesterol ratio inoreascd from 0.063 i 0.003 in control ape E deficient mice to 0.16 * 0.015 tbrcc days after injection. Human ape A-I levels were nearly undetectable 3 weeks atIer injection. Previously, intmdoction of a human ape A-I tramgene in ape E deficient mice has been shown to bc a potent suppressor of atherosclerosis. Similarly, the transient increase in HDL cholesterol levels induced by adcoovirus mediated gem transfer of human ape A-I in ape E deficient mice led to a profound reduction in m&time formation after endothelial denudation of the common carotid artety. Because of the potent anti-athemgenic effects of human apolipoprotcin A-I as shown in transgenic mice, the clear inverse relationship bctwccn HDL cholcstcrol and cardiovsscular disease lo humans and the absence of a rclationsldp bchvcen HDL cholesterol and nm~ovascular disease, adenovbus mediated gene transfer with apolipoprotcio A-I is particularly attractive. However, the hansient nature of gene expression remains the major
146 Adenoviml-mcdiatcd gene transfer of eodothelld nitric oxide ayntbasc to the rabbit carotid artery rItcrs voscolar mactMty. I. Kullo, G. Mazes, R. Schwartz, I! Gloviak-t, 7: Crony. Z.S. Katwic. I: O’Bnkn Mayo Chic, Rochester, MN, USA. We tested the effect of overexpression of cndothclial nitric oxide synthasc gene in the rabbit carotid artery by adcnoviral mediated gene ban&r. Rabbit carotid arteries were surgically isolated and exposed to an adenoviral vector encoding cNOS (AdeNOS) and ao adcnovird vector emding Bgalactosidase on the other side (RCial). Vector solutions at a wncenttatkm of 1010pfu/l were instilled intral~ally for 20 minutes or delivered to the pmiadventitial sheath of the vessel. Arteries were harvested 4 days later for inummosmining, vasomotor stodies and measurement of cGMP levels and NOS activity. Intiuminal admiition resulted in cndotheliom specific gcnc transfer, while advcntitial delivery resulted in advcntitial specific gene transfer. In both delivery models cGMP levels were si@icantly higher in the AdcNOS transduced vessels. Maximal contmctions to phcnylephtine were diminished io both models (Intraluminal: 6.0 f 0.3 grams vs 5.0 + 0.2 gnuns, p < 0.02; Adventitisl: 5.6 f 0.2 grams vs. 4.6 f 0.2 grams, p < 0.05.). This effect WBSabolished in the presence of the NOS inhibitor L-NMMA. After in!mluminal administration, relaxations to acetylcholinc obtained during submaximal con&actions to phcnylephrinc were significantly enhanced in the AdeNOS artcries (-IogECrc 7.4 + 0.08~s 7.09 + 0.09, P < 0.02). After advcotitial admiistration, relaxations to calcium ionophore obtained during submaximal contractions to phenylcphrioe were significantly enhanced in the AdcNOS vessels (-log Ejc 7.77 f 0.08 vs 7.45 f 0.07, P < 0.02. We conclude that overexpression of eNOS in the cndotheliom or advcntitia results in altered vasorcactivity. These results suggest that gene transfer of eNOS may be beneficial in the treatment of vasospasm and cndothclial dysfunction.
147 Metabolic role of apoUpopmtein A-II studies in traosgenic mice M. Cloes, C. de Geitere. A .Boullie~ B. De@y, G. Tremp, N. Duverger, J.C. Fruchort and G.R. Castro SERLIA - Institut Pastcur -Lille - France Gcncell Division - Rhone Poulcnc Rarer - Vitry sur Seine - France High density lipoprotein (HDL) levels are inversely corrclatcd with tbe incidence of atherosclerotic cardiovascular diseases. HDL may protect against atbcrosclcmsis by promoting tmcqxt of excess cholesterol from peripheral tissues to liver, a process termed rcvcrx cholesterol transport (RCT). In addition, HDL protects low density lipoproteins from oxidative damage known to contribute to the onset and progression of atberosclcrosis The major HDL proteins arc spolipoprotcin (apo)A-I and apoA-II. Some evidence suggests that apoA-I is directly protective against atherosclerosis, but relatively little is known about the role of apoA-II. Contrasting results have been obtained in mice overexpressing human (h) or murinc (m) ape A-II. hapoA-II, a homodimeric protein, seems to confer protection with regard to athcrogencsis, in contrast, overproduction of mapoA-Il (that is monomeric) has been proposed to be .&erogenic. To forther investigate the role of apoA-II we measured the initials steps of RCT and the panloxonase activity in both types of mice. To determine if the observed differences were related to the mono or dimeric forms we studied tmnsgeoic mice for hapo A-II rendered monomeric by (Cyd+Ser) mutation @apoA-IImon). mapoA-II mice had cHDL values 84% higher than control mice, their sor HDL had a capacity to promote cellular cholesterol cfflux that was twice the one of controls. LCAT activity measured with exogenous substrate was not different to the activity of control scra (31.8 f 1.8 vs 33.8 * 2 %) but the endogcnous estcrification capacity was higher (16 * 2.5 vs 7.2 f 0.2 %), hapoA-II mice with normal cHDL and hapoA-IImon with reduced cHDL had lower capacity to efflux cholesterol and to ester@ it than control mice. Paroxonase activities for mapoA-II, hapoA-II and hapoA-IImon were respectively 98,83 and 73% of control mice serum activity Our results are a clear indication that the metabolic diffbrcnces observed between m and h apoA-11 arc not related to the mono or dimcric forms, and above all that RCT is a more complex pathway and that increased activity of some steps is not a proof that peripheral cholcstcml will bc efficiently eliminated.