Composition-function analysis of HDL subpopulations: influence of lipid composition on particle functionality

Abstract

The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low preβ-1 functionality (low-F) (ABCA1-dependent cholesterol-efflux normalized for preβ-1 concentration) and controls who had either low-F or high preβ-1 functionality (high-F). Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC-MS. The average number of lipid molecules decreased from 422 in the large spherical α-1 particles to 57 in the small discoid preβ-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in α-mobility but not in preβ-1 particles. Preβ-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and triglyceride), indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with preβ-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I. The composition-function relationship of HDL particles and its effects on the mechanisms driving coronary heart disease (CHD) is poorly understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated five different-sized HDL subpopulations from CHD patients who had low preβ-1 functionality (low-F) (ABCA1-dependent cholesterol-efflux normalized for preβ-1 concentration) and controls who had either low-F or high preβ-1 functionality (high-F). Molecular numbers of apoA-I, apoA-II, and eight major lipid classes were determined in each subpopulation by LC-MS. The average number of lipid molecules decreased from 422 in the large spherical α-1 particles to 57 in the small discoid preβ-1 particles. With decreasing particle size, the relative concentration of free cholesterol (FC) decreased in α-mobility but not in preβ-1 particles. Preβ-1 particles contained more lipids than predicted; 30% of which were neutral lipids (cholesteryl ester and triglyceride), indicating that these particles were mainly remodeled from larger particles not newly synthesized. There were significant correlations between HDL-particle functionality and the concentrations of several lipids. Unexpectedly, the phospholipid:FC ratio was significantly correlated with large-HDL-particle functionality but not with preβ-1 functionality. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the cholesterol efflux capacity of HDL particles. Functionality and lipid composition of HDL particles are significantly correlated and probably both are influenced by the lipid-binding capacity of apoA-I. Although epidemiological studies indicate that HDL plays a significant role in coronary heart disease (CHD) (1Gordon D.J. Probstfield J.L. Garrison R.J. Neaton J.D. Castelli W.P. Knoke J.D. Jacobs D.R. Bangdiwala S. Tyroler H.A. High-density lipoprotein cholesterol and cardiovascular disease. Four prospective American studies.Circulation. 1989; 79: 8-15Crossref PubMed Scopus (2479) Google Scholar, 2Ballantyne C.M. Herd J.A. Ferlic L.L. Dunn J.K. Farmer J.A. Jones P.H. Schein J.R. Gotto A.M. Influence of low HDL on progression of coronary artery disease and response to fluvastatin therapy.Circulation. 1999; 99: 736-743Crossref PubMed Scopus (172) Google Scholar, 3Nicholls S.J. Tuzcu E.M. Sipahi I. Grasso A.W. Schoenhagen P. Hu T. Wolski K. Crowe T. Desai M.Y. Hazen S.L. Statins, high-density lipoprotein cholesterol, and regression of coronary atherosclerosis.JAMA. 2007; 297: 499-508Crossref PubMed Scopus (596) Google Scholar), clinical trials of HDL-targeted therapies have recently failed (4Keech A. Simes R.J. Barter P. Best J. Scott R. Taskinen M.R. Forder P. Pillai A. Davis T. Glasziou P. FIELD Study Investigators et al.Effects of long-term fenofibrate therapy on cardiovascular events in 9795 people with type 2 diabetes mellitus (the FIELD study): randomised controlled trial.Lancet. 2005; 366: 1849-1861Abstract Full Text Full Text PDF PubMed Scopus (2488) Google Scholar, 5AIM-HIGH Investigators Boden W.E. Probstfield J.L. Anderson T. Chaitman B.R. Desvignes-Nickens P. Koprowicz K. McBride R. Teo K. Weintraub W. Niacin in patients with low HDL-cholesterol levels receiving intensive statin therapy.N. Engl. J. Med. 2011; 365: 2255-2267Crossref PubMed Scopus (2033) Google Scholar, 6Schwartz G.G. Olsson A.G. Abt M. Ballantyne C.M. Barter P.J. Brumm J. Chaitman B.R. Holme I.M. Kallend D. Leiter L.A. dal-OUTCOMES Investigators et al.Effects of dalcetrapib in patients with a recent acute coronary syndrome.N. Engl. J. Med. 2012; 367: 2089-2099Crossref PubMed Scopus (1405) Google Scholar). Therefore, there is a need to better understand the mechanisms by which HDL influences CHD risk and how the anti-atherogenic properties of HDL can be manipulated for preventive and therapeutic purposes. HDL is the most complex lipoprotein class comprised of various particles that differ in size, composition, and function, including cholesterol efflux and anti-oxidative and anti-inflammatory activities. HDL's functions are determined, or at least significantly influenced, by the size and chemical composition of its particles. We have shown that the concentrations of apoA-I-containing HDL particles (e.g., large α-1 and small preβ-1) are significantly better predictors of CHD risk than HDL-C levels in both primary and secondary prevention and that an increase in α-1 level is significantly associated with decreased progression of coronary artery stenosis as assessed by angiography (7Asztalos B.F. Cupples L.A. Demissie S. Horvath K.V. Cox C.E. Batista M.C. Schaefer E.J. High-density lipoprotein subpopulation profile and coronary heart disease prevalence in male participants of the Framingham Offspring Study.Arterioscler. Thromb. Vasc. Biol. 2004; 24: 2181-2187Crossref PubMed Scopus (250) Google Scholar, 8Asztalos B.F. Collins D. Cupples L.A. Demissie S. Horvath K.V. Bloomfield H.E. Robins S.J. Schaefer E.J. Value of high-density lipoprotein (HDL) subpopulations in predicting recurrent cardiovascular events in the Veterans Affairs HDL Intervention Trial.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 2185-2191Crossref PubMed Scopus (232) Google Scholar, 9Asztalos B.F. Batista M. Horvath K.V. Cox C.E. Dallal G.E. Morse J.S. Brown G.B. Schaefer E.J. Change in alpha1 HDL concentration predicts progression in coronary artery stenosis.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 847-852Crossref PubMed Scopus (114) Google Scholar). Recently, it has been documented that the cell-cholesterol efflux capacity (CEC) of HDL predicts incident and prevalent CVD risk more accurately than HDL-C (10Khera A.V. Cuchel M. de la Llera-Moya M. Rodrigues A. Burke M.F. Jafri K. French B.C. Phillips J.A. Mucksavage M.L. Wilensky R.L. Cholesterol efflux capacity, high-density lipoprotein function, and atherosclerosis.N. Engl. J. Med. 2011; 364: 127-135Crossref PubMed Scopus (1382) Google Scholar, 11Rohatgi A. Khera A. Berry J.D. Givens E.G. Ayers C.R. Wedin K.E. Neeland I.J. Yuhanna I.S. Rader D.R. de Lemos J.A. HDL cholesterol efflux capacity and incident cardiovascular events.N. Engl. J. Med. 2014; 371: 2383-2393Crossref PubMed Scopus (816) Google Scholar, 12Saleheen D. Scott R. Javad S. Zhao W. Rodrigues A. Picataggi A. Lukmanova D. Mucksavage M.L. Luben R. Billheimer J. Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study.Lancet Diabetes Endocrinol. 2015; 3: 507-513Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). CEC, via ABCA1 and scavenger receptor BI (SR-BI), is HDL-particle specific; preβ-1 particles are responsible for about 60% of cell-cholesterol efflux via the ABCA1 pathway, and large-HDL particles (α-1 and α-2) are responsible for about 80% of cell-cholesterol efflux via the SR-BI pathway (13Asztalos B.F. Horvath K.V. Schaefer E.J. High-density lipoprotein cell-cholesterol and coronary heart disease Thromb. Vasc. Biol. PubMed Scopus Google Scholar, B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. effects of HDL subpopulations on and cholesterol 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). not on the concentration but on the functionality of HDL particles. CHD patients have than preβ-1 concentrations but than preβ-1 functionality (ABCA1-dependent normalized for preβ-1 concentration) (13Asztalos B.F. Horvath K.V. Schaefer E.J. High-density lipoprotein cell-cholesterol and coronary heart disease Thromb. Vasc. Biol. PubMed Scopus Google Scholar). The relationship between HDL composition and is not understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. We isolated and five different-sized apoA-I-containing HDL subpopulations and determined the numbers of apoA-I, apoA-II, and eight major lipid classes particle in each we how the lipid composition correlated with the functionality of the small and the large HDL the of cell-cholesterol efflux via the ABCA1 and SR-BI the hypothesis that HDL-particle functionality is significantly influenced by the lipid composition of the we in had CHD and low preβ-1 functionality (low-F) had CHD and low-F and had CHD and high preβ-1 functionality Preβ-1 functionality was as by apoA-I concentration in preβ-1 particles. controls were with preβ-1 functionality not than 2 of the preβ-1 functionality of the CHD and high-F controls were with at least 2 than the preβ-1 functionality of the CHD CHD patients and were from the of a clinical Schaefer E.J. Desai T. M. Using in Study et of with the clinical on progression of coronary in and patients PubMed Scopus Google Scholar). in had coronary artery disease coronary artery or or of CHD on an or by by the clinical not to and at least with on were and in and in a of statin and an of at least controls and were from a large of or or diabetes and in and in of an level and a level were an cholesterol, and HDL-C concentrations were and apoA-I concentration was an apoA-I concentrations in five different-sized HDL subpopulations and preβ-1 were determined by by for apoA-I and as B.F. R.L. M. J.R. Horvath K.V. Schaefer E.J. of apoA-I-containing HDL subpopulations in patients with coronary heart Thromb. Vasc. Biol. PubMed Scopus Google Scholar). HDL and anti-oxidative capacity were at B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. effects of HDL subpopulations on and cholesterol 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, M. S.J. A.M. for HDL that is in the of or Full Text Full Text PDF PubMed Google Scholar). to the was to by to for and the was by was in with 2 of ABCA1 was by with of was for to and was to the cholesterol in the efflux was as the efflux from efflux was from efflux was as the between and was as the of cholesterol from to the were as cholesterol efflux HDL anti-oxidative capacity was by the of to or the of in the of a M. S.J. A.M. for HDL that is in the of or Full Text Full Text PDF PubMed Google Scholar). was in at and at in the for with isolated as concentration) in the or of as for HDL was determined with a at an of and an of The anti-oxidative capacity of was in anti-oxidative capacity that the was for a of was tested in with the in efflux and anti-oxidative capacity were normalized to a and HDL particles were isolated from a the HDL particles were from the of by and α-mobility and a of were in a of were a on a and at in the from the the and was on each The and α-mobility were on a the HDL particles were by The and were on the of and in at for the the size α-mobility HDL subpopulations and were on the in each the the of the preβ-1 subpopulation was by a shown in The five HDL particle were from the and the HDL particles were from the the the apoA-I-containing HDL particles in each were by HDL particles were from the by each to an about via a to the of HDL particles on the of the The HDL particles were from the which was with the size and of the isolated HDL each was to of which were with on a by of apoA-I each was at the of a was for apoA-I and apoA-II, the was by the was with from lipid The of in the were as apoA-I ester free cholesterol (FC) and The concentrations were in the preβ-1 and with of the concentrations the of for of the The were in Jones J.L. J.R. of free cholesterol, and ester by and PubMed Scopus Google Scholar, A. J. K. Jones J. et and of a high for of in and lipoprotein in and PubMed Scopus Google Scholar, Taskinen M.R. M. K. M. of lipid in lipoprotein PubMed Scopus Google Scholar). a of each HDL was with and Jones M.L. J.L. et with for of PubMed Scopus Google Scholar). The was an with a a and an particle size and particle size, for apoA-I and and for were relative to by the most and for each and lipid the were with and of lipids and Jones J.L. J.R. of free cholesterol, and ester by and PubMed Scopus Google Scholar), a of each was with of with to a and with of each was an with a was with and and were for and of classes and a of each was with of with to a and with a of A. J. K. Jones J. et and of a high for of in and lipoprotein in and PubMed Scopus Google Scholar). each were an with a particle an was in and of were to be most in by and number of for for for for and for class concentrations were on the of the normalized to the of the The number of apoA-I in the particles of each different-sized subpopulation was by The were on in HDL subpopulation was on for apoA-I and and and apoA-I were in each as size were to by with primary to either apoA-I or and secondary The were in a Molecular we not the of HDL the and lipid concentrations were normalized for apoA-I concentration by in each isolated HDL HDL particle number in each of the five HDL subpopulations was by apoA-I concentrations with the number of apoA-I molecules and the various lipids were as number Preβ-1 particle functionality was as cholesterol efflux by apoA-I concentration in preβ-1 particles and large-HDL-particle functionality was as cholesterol efflux by apoA-I concentration in α-1 particles. that were not were for the were as in between were The correlations between functionality and lipid composition of small and large (α-1 α-2) HDL particles were as correlation and their between were via are but the was to the type for correlation to of the low-F and are in with CHD patients had significantly and significantly HDL-C Although apoA-I concentrations were there were significant in the of apoA-I in HDL particles the with high-F and low-F controls, CHD patients had significantly concentrations of the small preβ-1 particles and and significantly concentration of the large α-1 particles and was significantly in the CHD with the low-F but with the high-F Preβ-1 functionality was in the low-F and CHD and significantly in both with the high-F was in functionality was significantly and the anti-oxidative capacity of was significantly in the CHD with the of the from the from the (low-F) from the from the (low-F) for from the from the (low-F) from the from the (low-F) HDL subpopulations Preβ-1 for from the from the from the (low-F) α-1 from the from the (low-F) from the from the (low-F) functions from the from the (low-F) functionality was by with preβ-1 concentration from the from the functionality was by with the concentration of α-1 and from the from the (low-F) from the (low-F) are capacity is in total from the from the (low-F) for functionality was by with preβ-1 concentration functionality was by with the concentration of α-1 and in a are capacity is in total to particle numbers in each of the five different-sized HDL the number of apoA-I molecules particle was assessed by and on the size profile of the apoA-I and the The average number of molecules apoA-I was determined on the We that α-1 particles contained apoA-I and molecules particles contained apoA-I and molecules and particles contained apoA-I and molecules The and preβ-1 particles contained apoA-I molecules and an of by and small of apoA-I not was in each isolated HDL of the five apoA-I-containing HDL subpopulations from are in The average number of total lipid molecules particle decreased with particle size from 422 in the large α-1 particles to 57 in the small preβ-1 particles. and were the most lipids in each and were the least With decreasing particle size, the relative concentration of decreased in α-mobility particles but not in preβ-1 particles. were in The relative concentration of was significantly in preβ-1 particles with the average of the lipid in preβ-1 particles and in large preβ-1 the and were significantly and with the average of the α-mobility particles There were in the and numbers of lipid molecules particle in each HDL the particle numbers and the lipid of HDL subpopulations are in the α-1 the CHD had significantly and molecules and more and molecules and a ratio with both The and were significantly in the CHD with the high-F but not with the low-F the CHD had significantly concentration and ratio with both preβ-1 there were significant in the concentrations of lipids the The high-F had a significantly ratio with the low-F and CHD and significantly ratio with the CHD composition of HDL subpopulations in high-F and low-F controls and CHD is as from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the from the are in number of molecules HDL is as from the from the in a are in number of molecules HDL the correlations between the functionality and the lipid of preβ-1 particles are in the There were significant positive correlations between preβ-1 functionality and the of total lipids and in the high-F the CHD and low-F the of preβ-1 functionality was to low correlation the CHD there were significant positive correlations between the functionality and the as as the ratio of preβ-1 particles. for the correlation between the functionality and the concentration of preβ-1 was significantly between the high-F and the CHD There were significant correlations between preβ-1 functionality and the concentrations of and or the and in either The between the functionality and the lipid of large HDL particles are shown in the high-F there were positive correlations between large-HDL-particle functionality and the concentrations of and total lipids as as the ratio the CHD large-HDL-particle functionality correlated with the concentrations of and and with the ratio for the correlations between large-HDL-particle functionality and the concentrations of and as as the ratio were significantly between the CHD and the high-F There were significant correlations between large-HDL-particle functionality and the concentrations of and or the and in either The relationship between HDL composition and is not understood. We tested the hypothesis that the functionality of HDL particles is significantly influenced by their lipid composition. Using a novel 3D-separation method, we isolated and five different-sized apoA-I-containing HDL subpopulations and determined the numbers of apoA-I, apoA-II, and eight major lipid classes of the particles in each We associated the lipid and the functionality of the small and the large HDL the of cell-cholesterol efflux via the ABCA1 and SR-BI the average number of lipid molecules particle decreased with decreasing particle size Unexpectedly, the small preβ-1 particles contained an average of 57 lipid of which were neutral lipids and has been and are as it has been shown that a of and is on the of HDL and with decreasing particle size, their on the Taskinen M.R. M. K. M. of lipid in lipoprotein PubMed Scopus Google Scholar, R. M. S. lipids are to in a in PubMed Scopus Google Scholar). high neutral lipid on the the particle more and the with The responsible for the of the lipid composition of the discoid HDL particles is not it is that it is by the lipid-binding capacity of apoA-I at the S. M. D. Phillips M.C. responsible for the of high Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). we that the lipid composition of the HDL subpopulations is influenced by apoA-I HDL particles and lipids from the to the particles. and were the most lipids in each HDL subpopulation with A. M. the of the HDL Full Text Full Text PDF PubMed Scopus Google Scholar), we have that was the most by and in each we not the of et M. W. A. R. M. A. high-density particles are in to cholesterol and Thromb. Vasc. Biol. PubMed Scopus Google who a of in large HDL particles with small particles. Preβ-1 particles contained a of and and a of with α-mobility particles. the major was and correlated with both and in preβ-1 the that the of are for neutral lipids on the of lipoprotein particles S. M. D. Phillips M.C. responsible for the of high Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that the of a particle is a of its and a the to ratio with a in particle Therefore, we that the ratio increase with decreasing particle the ratio was in α-mobility particles with a size The that the ratio with size that the subpopulations are apoA-I on the of particles be and more the size to the size of the that is not for in the ratio be the lipids on particles R. M. S. lipids are to in a in PubMed Scopus Google Scholar). The average and were the in the small preβ-1 particles the that these particles are the major of from and are not the for B.F. R.L. M. J.R. Horvath K.V. Schaefer E.J. of apoA-I-containing HDL subpopulations in patients with coronary heart Thromb. Vasc. Biol. PubMed Scopus Google Scholar, D. K. A. I. P. of the role of in cholesterol efflux isolated Thromb. Vasc. Biol. PubMed Scopus Google Scholar). We that the of preβ-1 particles from the large α-1 as these particles are to to the of and and apoA-I and its lipids are as preβ-1 particles. apoA-I which are HDL particles can lipids from larger size particles to preβ-1 particles. we have shown that functionality (ABCA1-dependent normalized for was significantly and in CHD patients as with controls, that more preβ-1 particles in CHD (13Asztalos B.F. Horvath K.V. Schaefer E.J. High-density lipoprotein cell-cholesterol and coronary heart disease Thromb. Vasc. Biol. PubMed Scopus Google Scholar). We that the lipid composition of preβ-1 particles was in CHD patients and significantly to there was large in preβ-1 functionality controls with a of as low-F as CHD Therefore, to better understand the relationship between the lipid composition and the functionality of preβ-1 we CHD patients who had low-F and to who had either high-F or low-F and low-F to there were significant in the of preβ-1 particles the we the correlations between the functionality and the lipid of preβ-1 we significant positive correlations between preβ-1 functionality and the of total and in the high-F the correlations between preβ-1 functionality and concentration were significantly between the high-F and the CHD for the that preβ-1 particles need to efflux cholesterol we that the ratio in preβ-1 particles be correlated with preβ-1 functionality. the that the ratio was the in the high-F and was not significantly correlated with preβ-1 functionality in either not not the that a high ratio preβ-1 particles better of these and recently that a positive correlation between the lipid-binding capacity and the of apoA-I A. T. K. M. K. K. T. K. M. K. et of HDL functionality for coronary risk Med. PubMed Google Scholar), we that both the lipid composition of preβ-1 and the lipid-binding capacity of apoA-I the functionality of preβ-1 particles. We have in to large-HDL-particle functionality normalized for the concentrations of α-1 and was significantly in CHD patients as with controls (13Asztalos B.F. Horvath K.V. Schaefer E.J. High-density lipoprotein cell-cholesterol and coronary heart disease Thromb. Vasc. Biol. PubMed Scopus Google Scholar). We significant correlations between the functionality and the and concentrations of large HDL particles in the CHD which were significantly from the positive correlations in the high-F the hypothesis that the lipid and the cholesterol efflux of apoA-I were in CHD patients with high-F the concentration correlated the ratio correlated with the functionality of large HDL particles in CHD these as assessed by an were not in cholesterol in by the low cholesterol in the is an is that from an efflux not how the particles efflux cholesterol in The of apoA-I lipids to HDL anti-oxidative capacity is we between the anti-oxidative capacity of of and the concentrations of either HDL-C or apoA-I (13Asztalos B.F. Horvath K.V. Schaefer E.J. High-density lipoprotein cell-cholesterol and coronary heart disease Thromb. Vasc. Biol. PubMed Scopus Google Scholar). the there was a positive correlation between the anti-oxidative capacity of and the of the large α-1 particles is that and apoA-I, several lipid and molecules with capacity and that a for HDL in molecules with The of is that we have novel on the of the major apoA-I-containing HDL subpopulations a and method, which to the better of the composition-function relationship of has several We have a small number of and high that the to significant CHD patients were on statin which have influenced the lipid composition the efflux capacity of HDL particles with who not statin several lipids were not We have documented a of five different-sized apoA-I-containing HDL We have shown that the lipid composition of the different-sized α-mobility particles was and that the small discoid preβ-1 particles were more in and and had and with α-mobility particles. We have shown that preβ-1 particles contained more lipids than of which 30% were neutral lipids and indicating that these particles were mainly remodeled from larger particles not newly synthesized. CHD were with controls, we more significant in the of the large HDL particles than that of the small preβ-1 particles. We have significant correlations between HDL particle functionality and the concentrations of several lipids. There was significant positive correlation between particle functionality and total lipids in high-F controls, indicating that the lipid-binding capacity of apoA-I plays a major role in the of HDL particles. indicate a complex between the lipid-binding capacity of apoA-I and the lipid composition and the functionality of HDL particles.

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