Electronegative LDLs from familial hypercholesterolemic patients are physicochemically heterogeneous but uniformly proapoptotic

Abstract

A highly electronegative fraction of human plasma LDLs, designated L5, has distinctive biological activity that includes induction of apoptosis in bovine aortic endothelial cells (BAECs). This study was performed to identify a relationship between LDL density, electronegativity, and biological activity, namely, the induction of apoptosis in BAECs. Plasma LDLs from normolipidemic subjects and homozygotic familial hypercholesterolemia subjects were separated into five subfractions, with increasing electronegativity from L1 to L5, and into seven subfractions according to increasing density, D1 to D7. L1 to L5 were also separated according to density, and D1 to D7 were separated according to charge. The density profiles of L1 to L5 were similar (maximum density = 1.030 ± 0.002 g/ml). Induction of apoptosis by all seven density subfractions was confined to the highly electronegative fraction, L5, and within each density subfraction the magnitude of apoptosis correlated with the L5 content. Electronegative LDL is heterogeneous with respect to density and composition, and induction of apoptosis is more strongly associated with LDL electronegativity than with LDL size or density. A highly electronegative fraction of human plasma LDLs, designated L5, has distinctive biological activity that includes induction of apoptosis in bovine aortic endothelial cells (BAECs). This study was performed to identify a relationship between LDL density, electronegativity, and biological activity, namely, the induction of apoptosis in BAECs. Plasma LDLs from normolipidemic subjects and homozygotic familial hypercholesterolemia subjects were separated into five subfractions, with increasing electronegativity from L1 to L5, and into seven subfractions according to increasing density, D1 to D7. L1 to L5 were also separated according to density, and D1 to D7 were separated according to charge. The density profiles of L1 to L5 were similar (maximum density = 1.030 ± 0.002 g/ml). Induction of apoptosis by all seven density subfractions was confined to the highly electronegative fraction, L5, and within each density subfraction the magnitude of apoptosis correlated with the L5 content. Electronegative LDL is heterogeneous with respect to density and composition, and induction of apoptosis is more strongly associated with LDL electronegativity than with LDL size or density. Plasma levels of LDL-cholesterol are positively associated with the risk for coronary heart disease (CHD). Although some models suggest that oxidized LDLs are an important initiating factor for atherogenesis, their identity and structure in vivo are not known. Small, dense low density lipoprotein (sdLDL), another CHD risk factor, may be mechanistically linked to atherosclerosis (1Mackey R.H. Kuller L.H. Sutton-Tyrrell K. Evans R.W. Holubkov R. Matthews K.A. Lipoprotein subclasses and coronary artery calcium in postmenopausal women from the Healthy Women Study..Am. J. 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Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC..Free Radic. Biol. Med. 1991; 11: 247-253Crossref PubMed Scopus (101) Google Scholar) isolated by ion-exchange methods is mechanistically linked to atherosclerosis through its cytotoxicity to endothelial cells (ECs). LDL− isolated from the plasma of patients with familial hypercholesterolemia and diabetes induces in vitro EC apoptosis and inflammatory responses through the induction of monocyte chemotactic protein-1 and interleukin-8 production (15Chen C.H. Jiang T. Yang J.H. Jiang W. Lu J. Marathe G.K. Pownall H.J. Ballantyne C.M. McIntyre T.M. Henry P.D. et al.Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription..Circulation. 2003; 107: 2102-2108Crossref PubMed Scopus (135) Google Scholar, 16Sanchez-Quesada J.L. Camacho M. Anton R. Benitez S. Vila L. Ordonez-Llanos J. Electronegative LDL of FH subjects: chemical characterization and induction of chemokine release from human endothelial cells..Atherosclerosis. 2003; 166: 261-270Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Thus, the increased plasma LDL− concentrations in FH and diabetic patients (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1083-1090Crossref PubMed Scopus (91) Google Scholar, 18Moro E. Zambon C. Pianetti S. Cazzolato G. Pais M. Bittolo Bon G. Electronegative low density lipoprotein subform (LDL−) is increased in type 2 (non-insulin-dependent) microalbuminuric diabetic patients and is closely associated with LDL susceptibility to oxidation..Acta Diabetol. 1998; 35: 161-164Crossref PubMed Scopus (35) Google Scholar) may increase CHD risk. The molecular interactions associated with the biological effects of LDL− are unknown. The altered conformation of apolipoprotein B-100 (apoB-100) in LDL− is a factor in its reduced binding to the LDL receptor (19Lund-Katz S. Laplaud P.M. Phillips M.C. Chapman M.J. Apolipoprotein B-100 conformation and particle surface charge in human LDL subspecies: implication for LDL receptor interaction..Biochemistry. 1998; 37: 12867-12874Crossref PubMed Scopus (117) Google Scholar). Whereas one study showed that the densest LDL subfractions were richest in LDL− (18%) (20Sevanian A. Hwang J. Hodis H. Cazzolato G. Avogaro P. Bittolo-Bon G. Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations..Arterioscler. Thromb. Vasc. Biol. 1996; 16: 784-793Crossref PubMed Scopus (143) Google Scholar), another reported more LDL− in both sdLDL and buoyant LDL from hyperlipemic subjects (21Sanchez-Quesada J.L. Benitez S. Otal C. Franco M. Blanco-Vaca F. Ordonez-Llanos J. Density distribution of electronegative LDL in normolipemic and hyperlipemic subjects..J. Lipid Res. 2002; 43: 699-705Abstract Full Text Full Text PDF PubMed Google Scholar). Reports on the distribution of LDL− over the entire LDL density range have not appeared; this information could reveal the homogeneity of LDL− and provide clues about its formation if it were associated with a narrow density range within the broader range of whole LDL. Here, we report the density distribution of LDL− isolated from hypercholesterolemic (FH) and normolipidemic (NL) subjects as assessed by equilibrium density gradient ultracentrifugation (EDGUC) (22Hosken B.D. Cockrill S.L. Macfarlane R.D. Metal ion complexes of EDTA: a solute system for density gradient ultracentrifugation analysis of lipoproteins..Anal. Chem. 2005; 77: 200-207Crossref PubMed Scopus (19) Google Scholar) and an assay of biological effects, EC apoptosis. All reagents were purchased from Sigma (St. Louis, MO). This study was conducted using LDL isolated from homozygotic FH and NL subjects. FH subjects (two females aged 33 and 41 years and one male aged 6 years when the project started) had total plasma cholesterol > 500 mg/dl. The diagnoses of homozygotic FH were made on the bases of DNA analyses of leukocytes and clinical features characteristic of the disease. The FH subjects were treated with cholesterol as and in to LDL LDL was performed using a plasma was from by the plasma and were and by ultracentrifugation to LDL. The NL aged had total cholesterol and LDL cholesterol mg/dl. were by using an and were to plasma by the for of and the project and were from plasma subjects LDLs were isolated from plasma by between = and = C.Y. Yang Yang M. Smith C.V. of by of low density lipoproteins to in Radic. Biol. Med. 1997; 23: PubMed Scopus Google Scholar), by = and and with in LDL was separated according to charge using a on a an with A and (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1083-1090Crossref PubMed Scopus (91) Google Scholar). in A was the and with a gradient of in and LDL fractions were according to concentration into five subfractions, L1 through L5 and The isolated fractions were and within 2 LDL isolated by in with = was to A gradient of to = and 1.030 by the of L. P. F. S. M. Chapman M.J. is a of the oxidative susceptibility of dense Thromb. Vasc. Biol. 1999; 19: PubMed Scopus Google Scholar) was over the was for the gradient was in with a The was and seven LDL subfractions with increasing density from D1 to D7 were the was also not as LDL LDL of was to and with of a to a of = g/ml). a density solute are to of in was and with the LDL for the were for 6 and in a The density on the of various in the was to an were in a a a and a with a (22Hosken B.D. Cockrill S.L. Macfarlane R.D. Metal ion complexes of EDTA: a solute system for density gradient ultracentrifugation analysis of lipoproteins..Anal. Chem. 2005; 77: 200-207Crossref PubMed Scopus (19) Google Scholar). The and were to each other on an in a The and were with for and The were to an using The of to the of the were A was from to the and of the to the is The LDL profile was a of LDL subfractions were to a with in and with a of The profiles are as were conducted as (15Chen C.H. Jiang T. Yang J.H. Jiang W. Lu J. Marathe G.K. Pownall H.J. Ballantyne C.M. McIntyre T.M. Henry P.D. et al.Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription..Circulation. 2003; 107: 2102-2108Crossref PubMed Scopus (135) Google Scholar, M. S. H. K. H. C. Pownall H.J. Chen H. M. Yang C. of in endothelial cells by low density lipoproteins subfractions from patients with familial Full Text Full Text PDF PubMed Scopus Google Scholar). bovine aortic endothelial cells were in with and bovine of the cells were cell were to and for with LDL subfractions, or as a were for with to nuclear and with and to was according to the by were with in of lipoprotein of was to of and in and (15Chen C.H. Jiang T. Yang J.H. Jiang W. Lu J. Marathe G.K. Pownall H.J. Ballantyne C.M. McIntyre T.M. Henry P.D. et al.Low-density lipoprotein in hypercholesterolemic human plasma induces vascular endothelial cell apoptosis by inhibiting fibroblast growth factor 2 transcription..Circulation. 2003; 107: 2102-2108Crossref PubMed Scopus (135) Google Scholar). was to each of a of in by for The was in for and in are as ± were by for or by or with or modified with In FH patients and NL respectively, the electronegative L5, for and of total plasma LDL. the and for and not The density distribution of and was by using The and distributed and the of LDL was in ± for and ± for and ± for and ± for Although the subfraction had similar the of both and were ± and ± the the of the and were LDL NL and FH and five charged LDL subfractions (two NL and FH were with the and in a The distribution of a as an with an 1.030 The of the suggests and the small between and is of a LDL The density of five charged LDL subfractions of were similar and also a the in the of density by equilibrium density gradient ultracentrifugation and of by chromatography for LDL charged density distribution of a was determined using by of a gradient ultracentrifugation with The of density the was by the of from within the Density of LDL charged LDL subfractions, for normolipidemic and FH subjects determined the The LDL in each of from to on was by and analysis 6 of ultracentrifugation determined by the for FH density and charged subfractions using chromatography with 6 The for FH charged subfractions are and for density subfractions are of density by equilibrium density gradient ultracentrifugation and of by chromatography for LDL charged density distribution of a was determined using by of a gradient ultracentrifugation with The of density the was by the of from within the Density of LDL charged LDL subfractions, for normolipidemic and FH subjects determined the The LDL in each of from to on was by and analysis 6 of ultracentrifugation determined by the for FH density and charged subfractions using chromatography with 6 The for FH charged subfractions are and for density subfractions are In the density profiles of all charged LDL subfractions from FH patients in were similar to each other from of distributed a range of than LDL. of the LDL was from FH or NL the LDL− was distributed the entire range of Thus, charge is not associated with a density range of LDL from NL or FH charged LDL are distributed the entire range of for NL and in the of various subfractions isolated according to density and charge were determined by to the increase with size, the buoyant subfractions were associated with the LDL the densest subfractions to the LDLs The fraction of total LDL was in and with of and In of by charge fractions with similar and the and of the charged subfractions, respectively, had of and All charged LDL subfractions were by between of and Thus, the of charged LDL subfractions are similar and of charge. of the size of LDL from its (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1083-1090Crossref PubMed Scopus (91) Google Scholar) and of each W. Pownall H.J. of human low-density lipoproteins according to 2003; PubMed Scopus (28) Google Scholar) from to and for charged LDL subfractions from NL and FH familial in a familial were with various concentrations of and apoptosis was assessed according to the by showed that the effects of were and to were with various LDL subfractions, to and according to the and respectively, were highly = and = induction of apoptosis by to was of the induction of apoptosis by the various and of each LDL density showed that the electronegative fraction in each density the fraction not The L5 of each density fraction, D1 through as assessed from the as L5 by ion-exchange chromatography, were and Induction of apoptosis by the D1 through was with respect to their L5 = The effects associated with L5 that electronegativity is more closely linked to apoptosis than LDL size or effects of charged effects of charged subfractions endothelial In the a of the effects to the of to a of from the of and is of from one of effects of density subfractions from and a ± showed the activity The electronegativity of FH density subfractions and were by Although density subclasses were of charged their as a The that and are the and electronegative LDL density showed the by and that the of density are also Although (21Sanchez-Quesada J.L. Benitez S. Otal C. Franco M. Blanco-Vaca F. Ordonez-Llanos J. Density distribution of electronegative LDL in normolipemic and hyperlipemic subjects..J. Lipid Res. 2002; 43: 699-705Abstract Full Text Full Text PDF PubMed Google Scholar) that the buoyant and dense subfractions of were with this has to gradient and of the L5, an is not confined to a narrow density or size In both FH and NL all charged subfractions and LDL a similar density in their with that of total LDL. Thus, the fractions with the LDL also have the L5 the of L5 to the total from is the density L5 in both FH and NL subjects were also broader than of the more of its broader density L5 for a of total LDL the buoyant and dense of LDL density. A density distribution by for with a for L5 for a similar density A for L5 a broader distribution than that for with a The broader distribution the of L5 buoyant and dense subfractions of the of L5 is in the Although some have reported a between sdLDL and this is by and low HDL-cholesterol concentration of low density lipoprotein particle with 15: PubMed Scopus Google Scholar, G. Y. G. J. M.J. of small and dense LDL concentration in and men and 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), both of are CHD risk Therefore, LDL particle size is not an CHD risk In vitro have modified LDL in the of human Whereas in vitro of the biological activity of sdLDL are L5 induces apoptosis of (15Chen C.H. Jiang T. Yang J.H. Jiang W. Lu J. Marathe G.K. Pownall H.J. Ballantyne C.M. McIntyre T.M. 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Thus, LDL− could be one of modified lipoprotein that are atherogenic in vivo and that are to that activity by all L5 density subfractions that by LDL density sdLDL more L5 and its biological activity, it could be that of LDL according to charge is more closely linked to than to size or density. other is that the magnitude of apoptosis by various LDL density subfractions is to the of L5 in that fraction, of density. this relationship for density subfractions that had similar of L5 of total LDL. This is important because it that a small of L5 can a biological within a of and that LDL is not a of apoptosis. This for the that L5 is in various of LDL and that of L5 by other LDL fractions not the magnitude of apoptosis. that L5 is a of human plasma with the to by the of the vascular The molecular of the association between LDL electronegativity and its cytotoxicity to endothelial may be of one is not and may according to the identity of the In the biological effects of L5 may be to a with distinctive or and of composition, and Lipoprotein charge is altered by and and by the conformation of on the lipoprotein of could some charged their to the total charge on the in the charge on with and have to in conformation and S. Phillips M.C. The molecular for the in charge between and density Biol. Chem. Full Text PDF PubMed Google Scholar). in the of charged could their charge and that of the total In type diabetic electronegative and LDLs are by in the of and factor that are of oxidation and J.L. Benitez S. A. M. G. Vila L. Camacho M. R. Ordonez-Llanos J. The inflammatory of electronegative low-density lipoprotein from type diabetic patients are related to increased factor 2005; PubMed Scopus Google Scholar). The of LDL− from FH patients is altered (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1083-1090Crossref PubMed Scopus (91) Google Scholar). The of and for and respectively, could to electronegativity, because the for is Although some that the of the surface of LDL− are from of LDL (17Yang C.Y. Raya J.L. Chen H.H. Chen C.H. Abe Y. Pownall H.J. Taylor A.A. Smith C.V. Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins..Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1083-1090Crossref PubMed Scopus (91) Google Scholar, T. Bittolo-Bon G. R. 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The by ion-exchange chromatography and can be by the of the charged in LDL and by in the the In the through the that exchange In when whole LDL is an ion-exchange in each particle to a on the is the is with the electronegativity are and the and the electronegative fractions are and through the as the with other with to or charge to an exchange whole is of L1 to L5, be to as some of the of its This was by from the of to and to the American to and the American Heart to apolipoprotein B-100 bovine aortic endothelial cell coronary heart disease endothelial cell equilibrium density gradient ultracentrifugation familial hypercholesterolemic electronegative low density lipoprotein of a normolipidemic size-exclusion chromatography dense low density lipoprotein