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Plasma cholesteryl ester transfer protein (CETP) induces very high density lipoproteins (VHDL) with a potent anti-atherogenic function
Wednesday 12 October 1994: Poster Abstracts HDL and reverse cholesterol transport
the cells, by a mechanism similar to that described for free apoproteins. These data support the hypothesis that synthetic amphipathic helical peptides can be used to mimic apoproteins not only at the structural but also at the functional level. 11871 bbeur
Comparative turnover studies in rabbits of phosphoIipWpeptide complexes prepared with synthetic amphipatbic helical peptides and of human HDL Ct.*, Defeys R*, Rosseneu Mt, ‘AZ St.-Jan, Lab. Bio-
them., B-8000 Brugge, Belgium; 21rmogenetics N.V. Industriepark Zwiinaarde 7, B 9052 Gent, Belgium
Turnover rates of human HDL and of complexes consisting of a synthetic peptide M4 (Brasseur R et al Biochim Biophyi Acta 1993: 1170: 1-7) and DPPC ohosoholioid were studied in rabbits by monitoring human apo ‘A-I ‘leveis (measured by ELISA), plasma phospholipid and cholesterol levels. Approximately 30 mg phospholipid were injected and samples were taken at 1,5,30,60, 120, 240, 360, 420, 480 min and 24 h. Clearance rates (Clp ml/min) of human apo A-I were 0.091 ml/mm (expressed as apo A-I concentration) with a half-life of 1440 min. Similar clearance rates based upon cholesterol and phospholipid levels were obtained after HDL injection. The clearance rate for me M4-DPPC complexes was 0.086 ml/mitt with a half-life of 750 min. After injection of human HDL into the rabbits, sera were submitted to ultracenttifugation in a sucrose-NaC1 density gradient, enabling the quantification of the increase of apo A-I, phospholipid and cholesterol within the HDL fraction. After injection of the M4-DPPC complex the phospholipid increase was confined to the HDL density fractions. In order to monitor the fate of the peptide moiety of the M4 complex, a fluorescent dansyl group was covalently coupled to the peptide during synthesis. The Iipoproteins were fractionated by gel-chromatography on a Superose 6 HR column (FPLC, Waters) equipped with a fluorescence detector to monitor the dansyl group emission at 518 nm. One and 6 h after injection, respectively, 55 and 26% of the fluorescence intensity was still detected in the HDL fraction. Plasma cholesteryl ester transfer protein (CETP) induces very high density lipoproteins (VHDL) with a potent anti-atherogenic function nS. Ishigami M, Arai T, Sakai N, Hirano K, KamedaTakemura K, Tokunaga K, Matsuzawa Y, 2nd Dept. of Int. Med., Osaka Univ. Med. Sch., 2-2 Yamadaohz, Suita, Osaka 565,
11881
Japan
Plasma CETP transfers cholesteryl ester (CE) from HDL to triglyceride-rich lipoproteins. However, the physiological roles of CETP in the progression and regression of atherosclerosis have not been clarified. The lipoprotein abnormalities in homozygous CETP deficiency are characterized by an increase in HDb and the presence of polydisperse low density lipoproteins (LDL), and large HDL-C like particles enriched with CE and apolipoprotein (apo) E. The objective of the current study was to elucidate the role of CETP in atherogenesis by testing the effect of exogenous CETP on the properties of lipoproteins in CETP-deficient patients. CETP was highly purified from human plasma and added to patient plasma. After adding VLDL or Intrahpid to increase the mass of CE acceptors, the mixture was then incubated at 37°C for I.548 h with inhibition of LCAT. The added CETP induced a time-dependent reduction of large HDL particles along with the appearance of small very high density lipoprotein (VHDL) particles. The VHDL particles were isolated by gel permeation chromatography. The VHDL particles migrated at a pre$ and a position on agarose gel electrophoresis and were composed mainly of phospholipid and protein. Their protein moiety was predominantly apo A-I. The lipoproteins were further tested for their abil-
229
ity to reduce the CE content of mouse peritoneal macrophages during incubation with acetylated LDL. Acetyl LDL caused a dramatic increase of cellular CE, whereas normal HDb added to the medium induced a reduction of cellular CE. The VHDL inhibited CE accumulation much more strongly than normal HDb. Furthermore, the VHDL promoted the efflux of CE more markedly than normal HDLj from macrophages which had been preloaded with acetylated LDL. In conclusion, the data suggest that CETP plays a crucial role in the transformation of HDL particles into VHDL particles, which are functionally very active both in inhibiting cholesterol accumulation and in removing cholesterol from peripheraltissues. Marked hyperalphalipoproteinemia is not a longevity syndrome. Insights from a cohort study in a city where CETP deficiency accumulates Hirano K, Yamashita S, Arai T, Yoshida Y, Takemura K, Matsuzawa Y, 2nd Dept. Int. Med., Osaka Univ. Med. Sch., 2-2 Ya-
11891
madaoka, Suita 565, Osaka, Japan Low levels of HDL have been clearly demonstrated to be sssociated with an increased incidence of coronary heart disease (CHD), strongly suggesting that HDL particles have an anti-atherogenic function. However, there is little information concerning the atherogenicity of marked hyperalphahpoproteinemia (HALP). There is no agreement about whether cholesteryl ester transfer protein (CETP) deficiency is anti-atherogenic or not, even though this disorder is one of the major causes of marked HALPs. Recently we have found a unique area (Omagari City, Akita Prefecture, Japan) where CETP deficiency caused by the intron 14 splicing defect in the CETP gene markedly accumulates. This discovery has made it possible to perform a large populationbased study concerning the atherogenicity of a marked elevation of HDL-C in a genetically more homogeneous population. In Omagati City, the mutation was detected more than 20 times as frequently and the prevalence of a marked HALP with plasma HDL-C 5 100 mg/dl was 5-10 times as high as in other Japanese areas. In subjects aged over 80 years, the prevalence of both marked HALP and the intron 14 splicing defect was significantly lower than in the younger generation. There was a Ushaped relationship between HDL-C levels and the incidence of ischemic ECG changes. In the ranne of HDL-C > 70 me/dl, the incidence increased m proportion to-the levels of HDL-C.This current study suggests that a marked HALP caused by the CETP gene mutation is not a longevity syndrome.
Severe familial primary hypoalphalipoproteinemia in French Canadian kindred: no association with the apo A-I/C-III/A-IV and A-II gene loci, normal LCAT activity, normal triglyceride levels and lack of clinical manifestations of Tangier Disease Marcil M, Boucher B, Solymos BC, Davignon J, Frolich J,m u, Clin. Res. Inst. of Montrial, 110 Pine Ave. West, Mont&al,
11901
Qu f Canada H2 W I R7
A low level of HDL-C is the most common lipoprotein abnormality seen in premature CAD subjects. In most cases, HDL-C reduction in CAD subjects is associated with increased apo Bcontaining lipoproteins. Primary hypoalphaiipoproteinemia is seen in approximately 4% of CAD cases. We report subjects with severe familial HDL deticiencv. (HDL-C <<5th oercentile for age and gender) in a French Canadian kindred, with autosomal codominant inheritance. By inclusion criteria, all subjects were normotriglyceridemic and none were diabetic. Apolipoprotein A-I analysis by PAGE and IEF in affected subjects revealed a normal molecular weight (28 300 kDa) and normal isoelectrofocusing point but a relative increase in pro-apo A-I. which suggests normal secretion of apo
Atherosclerosis X, Montreal, October 1994
the cells, by a mechanism similar to that described for free apoproteins. These data support the hypothesis that synthetic amphipathic helical peptides can be used to mimic apoproteins not only at the structural but also at the functional level. 11871 bbeur
Comparative turnover studies in rabbits of phosphoIipWpeptide complexes prepared with synthetic amphipatbic helical peptides and of human HDL Ct.*, Defeys R*, Rosseneu Mt, ‘AZ St.-Jan, Lab. Bio-
them., B-8000 Brugge, Belgium; 21rmogenetics N.V. Industriepark Zwiinaarde 7, B 9052 Gent, Belgium
Turnover rates of human HDL and of complexes consisting of a synthetic peptide M4 (Brasseur R et al Biochim Biophyi Acta 1993: 1170: 1-7) and DPPC ohosoholioid were studied in rabbits by monitoring human apo ‘A-I ‘leveis (measured by ELISA), plasma phospholipid and cholesterol levels. Approximately 30 mg phospholipid were injected and samples were taken at 1,5,30,60, 120, 240, 360, 420, 480 min and 24 h. Clearance rates (Clp ml/min) of human apo A-I were 0.091 ml/mm (expressed as apo A-I concentration) with a half-life of 1440 min. Similar clearance rates based upon cholesterol and phospholipid levels were obtained after HDL injection. The clearance rate for me M4-DPPC complexes was 0.086 ml/mitt with a half-life of 750 min. After injection of human HDL into the rabbits, sera were submitted to ultracenttifugation in a sucrose-NaC1 density gradient, enabling the quantification of the increase of apo A-I, phospholipid and cholesterol within the HDL fraction. After injection of the M4-DPPC complex the phospholipid increase was confined to the HDL density fractions. In order to monitor the fate of the peptide moiety of the M4 complex, a fluorescent dansyl group was covalently coupled to the peptide during synthesis. The Iipoproteins were fractionated by gel-chromatography on a Superose 6 HR column (FPLC, Waters) equipped with a fluorescence detector to monitor the dansyl group emission at 518 nm. One and 6 h after injection, respectively, 55 and 26% of the fluorescence intensity was still detected in the HDL fraction. Plasma cholesteryl ester transfer protein (CETP) induces very high density lipoproteins (VHDL) with a potent anti-atherogenic function nS. Ishigami M, Arai T, Sakai N, Hirano K, KamedaTakemura K, Tokunaga K, Matsuzawa Y, 2nd Dept. of Int. Med., Osaka Univ. Med. Sch., 2-2 Yamadaohz, Suita, Osaka 565,
11881
Japan
Plasma CETP transfers cholesteryl ester (CE) from HDL to triglyceride-rich lipoproteins. However, the physiological roles of CETP in the progression and regression of atherosclerosis have not been clarified. The lipoprotein abnormalities in homozygous CETP deficiency are characterized by an increase in HDb and the presence of polydisperse low density lipoproteins (LDL), and large HDL-C like particles enriched with CE and apolipoprotein (apo) E. The objective of the current study was to elucidate the role of CETP in atherogenesis by testing the effect of exogenous CETP on the properties of lipoproteins in CETP-deficient patients. CETP was highly purified from human plasma and added to patient plasma. After adding VLDL or Intrahpid to increase the mass of CE acceptors, the mixture was then incubated at 37°C for I.548 h with inhibition of LCAT. The added CETP induced a time-dependent reduction of large HDL particles along with the appearance of small very high density lipoprotein (VHDL) particles. The VHDL particles were isolated by gel permeation chromatography. The VHDL particles migrated at a pre$ and a position on agarose gel electrophoresis and were composed mainly of phospholipid and protein. Their protein moiety was predominantly apo A-I. The lipoproteins were further tested for their abil-
229
ity to reduce the CE content of mouse peritoneal macrophages during incubation with acetylated LDL. Acetyl LDL caused a dramatic increase of cellular CE, whereas normal HDb added to the medium induced a reduction of cellular CE. The VHDL inhibited CE accumulation much more strongly than normal HDb. Furthermore, the VHDL promoted the efflux of CE more markedly than normal HDLj from macrophages which had been preloaded with acetylated LDL. In conclusion, the data suggest that CETP plays a crucial role in the transformation of HDL particles into VHDL particles, which are functionally very active both in inhibiting cholesterol accumulation and in removing cholesterol from peripheraltissues. Marked hyperalphalipoproteinemia is not a longevity syndrome. Insights from a cohort study in a city where CETP deficiency accumulates Hirano K, Yamashita S, Arai T, Yoshida Y, Takemura K, Matsuzawa Y, 2nd Dept. Int. Med., Osaka Univ. Med. Sch., 2-2 Ya-
11891
madaoka, Suita 565, Osaka, Japan Low levels of HDL have been clearly demonstrated to be sssociated with an increased incidence of coronary heart disease (CHD), strongly suggesting that HDL particles have an anti-atherogenic function. However, there is little information concerning the atherogenicity of marked hyperalphahpoproteinemia (HALP). There is no agreement about whether cholesteryl ester transfer protein (CETP) deficiency is anti-atherogenic or not, even though this disorder is one of the major causes of marked HALPs. Recently we have found a unique area (Omagari City, Akita Prefecture, Japan) where CETP deficiency caused by the intron 14 splicing defect in the CETP gene markedly accumulates. This discovery has made it possible to perform a large populationbased study concerning the atherogenicity of a marked elevation of HDL-C in a genetically more homogeneous population. In Omagati City, the mutation was detected more than 20 times as frequently and the prevalence of a marked HALP with plasma HDL-C 5 100 mg/dl was 5-10 times as high as in other Japanese areas. In subjects aged over 80 years, the prevalence of both marked HALP and the intron 14 splicing defect was significantly lower than in the younger generation. There was a Ushaped relationship between HDL-C levels and the incidence of ischemic ECG changes. In the ranne of HDL-C > 70 me/dl, the incidence increased m proportion to-the levels of HDL-C.This current study suggests that a marked HALP caused by the CETP gene mutation is not a longevity syndrome.
Severe familial primary hypoalphalipoproteinemia in French Canadian kindred: no association with the apo A-I/C-III/A-IV and A-II gene loci, normal LCAT activity, normal triglyceride levels and lack of clinical manifestations of Tangier Disease Marcil M, Boucher B, Solymos BC, Davignon J, Frolich J,m u, Clin. Res. Inst. of Montrial, 110 Pine Ave. West, Mont&al,
11901
Qu f Canada H2 W I R7
A low level of HDL-C is the most common lipoprotein abnormality seen in premature CAD subjects. In most cases, HDL-C reduction in CAD subjects is associated with increased apo Bcontaining lipoproteins. Primary hypoalphaiipoproteinemia is seen in approximately 4% of CAD cases. We report subjects with severe familial HDL deticiencv. (HDL-C <<5th oercentile for age and gender) in a French Canadian kindred, with autosomal codominant inheritance. By inclusion criteria, all subjects were normotriglyceridemic and none were diabetic. Apolipoprotein A-I analysis by PAGE and IEF in affected subjects revealed a normal molecular weight (28 300 kDa) and normal isoelectrofocusing point but a relative increase in pro-apo A-I. which suggests normal secretion of apo
Atherosclerosis X, Montreal, October 1994