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Spontaneous remodeling of HDL particles at acidic pH enhances their capacity to induce cholesterol efflux from human macrophage foam cells

2012; Elsevier BV; Volume: 53; Issue: 10 Linguagem: Inglês

10.1194/jlr.m028118

1539-7262

Su Duy Nguyen, Katariina Öörni, Miriam Lee‐Rueckert, Tero Pihlajamaa, Jari Metso, Matti Jauhiainen, Petri T. Kovanen,

Atherosclerosis and Cardiovascular Diseases

HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL2 and HDL3 subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL3 than in HDL2. Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects. HDL particles may enter atherosclerotic lesions having an acidic intimal fluid. Therefore, we investigated whether acidic pH would affect their structural and functional properties. For this purpose, HDL2 and HDL3 subfractions were incubated for various periods of time at different pH values ranging from 5.5 to 7.5, after which their protein and lipid compositions, size, structure, and cholesterol efflux capacity were analyzed. Incubation of either subfraction at acidic pH induced unfolding of apolipoproteins, which was followed by release of lipid-poor apoA-I and ensuing fusion of the HDL particles. The acidic pH-modified HDL particles exhibited an enhanced ability to promote cholesterol efflux from cholesterol-laden primary human macrophages. Importantly, treatment of the acidic pH-modified HDL with the mast cell-derived protease chymase completely depleted the newly generated lipid-poor apoA-I, and prevented the acidic pH-dependent increase in cholesterol efflux. The above-found pH-dependent structural and functional changes were stronger in HDL3 than in HDL2. Spontaneous acidic pH-induced remodeling of mature spherical HDL particles increases HDL-induced cholesterol efflux from macrophage foam cells, and therefore may have atheroprotective effects. HDL are composed of varying subpopulations of particles that are highly heterogeneous in size, physicochemical properties, metabolism, and in their anti-atherogenic functions (1Navab M. Reddy S.T. Van Lenten B.J. Fogelman A.M. HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms.Nat. Rev. Cardiol. 2011; 8: 222-232Crossref PubMed Scopus (456) Google Scholar, 2Meurs I. Van Eck M. Van Berkel T.J. High-density lipoprotein: key molecule in cholesterol efflux and the prevention of atherosclerosis.Curr. Pharm. Des. 2010; 16: 1445-1467Crossref PubMed Scopus (37) Google Scholar). The particles undergo dynamic remodeling in circulation by several plasma factors, such as LCAT (3Liang H.Q. Rye K.A. Barter P.J. Remodelling of reconstituted high density lipoproteins by lecithin:cholesterol acyltransferase.J. Lipid Res. 1996; 37: 1962-1970Abstract Full Text PDF PubMed Google Scholar), cholesteryl ester transfer protein (CETP) (4Rye K.A. Hime N.J. Barter P.J. Evidence that cholesteryl ester transfer protein-mediated reductions in reconstituted high density lipoprotein size involve particle fusion.J. Biol. Chem. 1997; 272: 3953-3960Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), phospholipid transfer protein (PLTP) (5Lusa S. Jauhiainen M. Metso J. Somerharju P. Ehnholm C. The mechanism of human plasma phospholipid transfer protein-induced enlargement of high-density lipoprotein particles: evidence for particle fusion.Biochem. J. 1996; 313: 275-282Crossref PubMed Scopus (145) Google Scholar), hepatic lipase, and endothelial lipase (6Lund-Katz S. Phillips M.C. High density lipoprotein structure-function and role in reverse cholesterol transport.Subcell. Biochem. 2010; 51: 183-227Crossref PubMed Scopus (178) Google Scholar). Such physiological HDL remodeling can lead to destabilization of HDL structure, and in some cases also to the release of lipid-free/lipid-poor apoA-I from the particles and to particle fusion (7Lie J. de Crom R. Jauhiainen M. van Gent T. van Haperen R. Scheek L. Jansen H. Ehnholm C. van Tol A. Evaluation of phospholipid transfer protein and cholesteryl ester transfer protein as contributors to the generation of pre beta-high-density lipoproteins.Biochem. J. 2001; 360: 379-385Crossref PubMed Scopus (42) Google Scholar). Similarly, serum opacity factor (8Gillard B.K. Courtney H.S. Massey J.B. Pownall H.J. Serum opacity factor unmasks human plasma high-density lipoprotein instability via selective delipidation and apolipoprotein A-I desorption.Biochemistry. 2007; 46: 12968-12978Crossref PubMed Scopus (39) Google Scholar) has been found to destabilize HDL particles with ensuing fusogenic formation of larger particles and concomitant release of lipid-free/lipid-poor apoA-I. Thus, generation of larger HDL particles coupled with the release of apoA-I appears to be a seminal characteristic of circulating HDL particles when exposed to various factors involved in their physiological remodeling. The ability of HDL to remove excess cholesterol from macrophages in the arterial intima, i.e., their ability to initiate the macrophage-specific reverse cholesterol transport pathway, is a key anti-atherogenic action of HDL. The various subclasses of HDL particles have distinct abilities to stimulate cellular cholesterol efflux, the mature spherical α-migrating HDL particles preferring ABCG1-mediated and the lipid-poor preβ-migrating apoA-I preferring ABCA1-mediated cholesterol efflux (9Asztalos B.F. de la Llera-Moya M. Dallal G.E. Horvath K.V. Schaefer E.J. Rothblat G.H. Differential effects of HDL subpopulations on cellular ABCA1- and SR-BI-mediated cholesterol efflux.J. Lipid Res. 2005; 46: 2246-2253Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Importantly, the ability of HDL to remove cholesterol from macrophage foam cells may be compromised when HDL particles are modified by enzymatic or nonenzymatic processes such as oxidation, glycosylation, nitrosylation/chlorination, or by proteolysis (10Lee-Rueckert M. Kovanen P.T. Extracellular modifications of HDL in vivo and the emerging concept of proteolytic inactivation of prebeta-HDL.Curr. Opin. Lipidol. 2011; 22: 394-402Crossref PubMed Scopus (21) Google Scholar). Acidic pH of the extracellular fluid is a common characteristic of inflammatory tissue sites (11Lardner A. The effects of extracellular pH on immune function.J. Leukoc. Biol. 2001; 69: 522-530Crossref PubMed Google Scholar, 12Bellocq A. Suberville S. Philippe C. Bertrand F. Perez J. Fouqueray B. Cherqui G. Baud L. Low environmental pH is responsible for the induction of nitric-oxide synthase in macrophages. Evidence for involvement of nuclear factor-kappaB activation.J. Biol. Chem. 1998; 273: 5086-5092Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) and, importantly, it has also been observed in human atherosclerotic lesions (13Naghavi M. John R. Naguib S. Siadaty M.S. Grasu R. Kurian K.C. van Winkle W.B. Soller B. Litovsky S. Madjid M. et al.pH Heterogeneity of human and rabbit atherosclerotic plaques; a new insight into detection of vulnerable plaque.Atherosclerosis. 2002; 164: 27-35Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Accordingly, circulating HDL particles entering such lesions may be exposed to acidic pH. On the basis of the fact that HDL particles are sensitive to various physiological perturbations, we hypothesized that the structural and functional properties of the particles are also sensitive to changes in pH of the medium in which they are suspended. Our data show that at acidic pH, HDL particles undergo spontaneous remodeling, with formation of lipid-poor apoA-I displaying preβ mobility and fusion of the α-migrating HDL particles, and that the generated mixture of preβ-HDL and fused α-HDL possesses an enhanced ability to promote cholesterol efflux from cultured human macrophage foam cells. The enhanced efflux-inducing capacity was shown to be primarily due to the de novo generation of the preβ-HDL particles. LDL (d = 1.019–1.050 g/ml), HDL2 (d = 1.063–1.125 g/ml), and HDL3 (d = 1.125–1.210 g/ml) were prepared from freshly isolated plasma of healthy volunteers obtained from the Finnish Red Cross by sequential flotation ultracentrifugation using KBr for density adjustment (14McPherson P.A. Young I.S. McKibben B. McEneny J. High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation.J. Lipid Res. 2007; 48: 86-95Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Ultracentrifugation was carried out in a Beckman OptimaTM TLX system table top ultracentrifuge using a Beckman fixed-angle rotor (TLA-100.3) at 541,000 g. This method subfractionates HDL from plasma in only 6 h, and it yielded HDL preparations that were almost totally devoid (<0.7% of total HDL) of the minor preβ-migrating component composed of lipid-poor apoA-I found to be present in the HDL3 subfraction isolated by the traditional lengthy ultracentrifugation protocol (typically 5% to 8% of total HDL) (15Lee M. Metso J. Jauhiainen M. Kovanen P.T. Degradation of phospholipid transfer protein (PLTP) and PLTP-generated pre-beta-high density lipoprotein by mast cell chymase impairs high affinity efflux of cholesterol from macrophage foam cells.J. Biol. Chem. 2003; 278: 13539-13545Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The HDL2 and HDL3 subfractions were washed by reflotation for 2 h at densities of 1.125 and 1.21 g/ml, respectively. Lipoprotein purity was assessed by size-exclusion chromatography (SEC), agarose gel electrophoresis, and nondenaturing gradient polyacrylamide gel electrophoresis (NDGGE). No detectable activities of LCAT, PLTP, or CETP were present in the HDL2 or HDL3 preparations, when determined using assays described previously in detail (16Jauhiainen M. Dolphin P.J. Human plasma lecithin-cholesterol acyltransferase. An elucidation of the catalytic mechanism.J. Biol. Chem. 1986; 261: 7032-7043Abstract Full Text PDF PubMed Google Scholar, 17Jauhiainen M. Metso J. Pahlman R. Blomqvist S. van Tol A. Ehnholm C. Human plasma phospholipid transfer protein causes high density lipoprotein conversion.J. Biol. Chem. 1993; 268: 4032-4036Abstract Full Text PDF PubMed Google Scholar). Lipoprotein stock solutions were dialyzed against PBS, pH 7.4, containing 1 mM EDTA, filtered, purged with nitrogen, and then stored at 4°C, and used within 2 weeks, during which no changes in protein or lipid composition or particle size were observed. The amounts of lipoproteins are expressed in terms of their total protein concentrations, which were determined by bicinchoninic acid protein assay kit (Pierce; Rockford, IL) using BSA as the standard. LDL was acetylated by repeated additions of acetic anhydride (18Basu S.K. Goldstein J.L. Anderson G.W. Brown M.S. Degradation of cationized low density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts.Proc. Natl. Acad. Sci. USA. 1976; 73: 3178-3182Crossref PubMed Scopus (823) Google Scholar), and acetyl-LDL was radiolabeled by treatment with [3H]cholesteryl linoleate ([1,2-3H]cholesteryl linoleate, Amersham Pharmacia) (19Brown M.S. Dana S.E. Goldstein J.L. Receptor-dependent hydrolysis of cholesteryl esters contained in plasma low density lipoprotein.Proc. Natl. Acad. Sci. USA. 1975; 72: 2925-2929Crossref PubMed Scopus (167) Google Scholar). The specific activities of the [3H]CE acetyl-LDL preparations ranged from 50 to 100 dpm/ng protein. Lipid-free apoA-I was purified according to a previously published procedure (20Pussinen P.J. Jauhiainen M. Metso J. Pyle L.E. Marcel Y.L. Fidge N.H. Ehnholm C. Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I.J. Lipid Res. 1998; 39: 152-161Abstract Full Text Full Text PDF PubMed Google Scholar). The results are presented for lipoprotein preparations derived from a single donor, and similar data were also obtained for lipoprotein preparations derived from three other donors. HDL2 or HDL3 (0.1–2 mg protein/ml) was incubated in either 20 mM MES (pH 5.5, 5.75, 6.0, 6.25, or 6.5), 20 mM PIPES (pH 6.5 or 7.0), or 20 mM Tris (pH 7.0 or 7.5) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2 for the indicated times. All incubations were carried out at 37°C. After incubation, the samples were centrifuged at 10,000 g for 10 min, and the particle sizes of the HDL-containing supernatants were analyzed using a high-resolution SEC Superose HR6 column connected to the Amersham-Pharmacia (GE Healthcare) AKTA chromatography system. Typically, a 50 µl aliquot was injected into the column and eluted with PBS buffer at a flow rate of 0.5 ml/min, and fractions (0.5 ml) were collected for protein and lipid analyses. Kinetics of the elution profile changes in SEC were analyzed using the Unicorn 5.2 software. The particle size was assessed by a calibration curve (R2= 0.98) using a gel filtration calibration kit (GE Healthcare). Proteins were resolved in 12.5% SDS-PAGE under reducing conditions, transferred to nitrocellulose, and immunoblotted either with anti-human apoA-I polyclonal antibody (21Siggins S. Jauhiainen M. Olkkonen V.M. Tenhunen J. Ehnholm C. PLTP secreted by HepG2 cells resembles the high-activity PLTP form in human plasma.J. Lipid Res. 2003; 44: 1698-1704Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) or with anti-human apoA-II monoclonal antibody (22Pussinen P.J. Jauhiainen M. Ehnholm C. ApoA-II/apoA-I molar ratio in the HDL particle influences phospholipid transfer protein-mediated HDL interconversion.J. Lipid Res. 1997; 38: 12-21Abstract Full Text PDF PubMed Google Scholar). The amounts of total cholesterol were measured using the Amplex Red cholesterol assay kit (Invitrogen) according to the manufacturer's instructions. Phospholipids were measured by a fluorometric assay (23Nanjee M.N. Gebre A.K. Miller N.E. Enzymatic fluorometric procedure for phospholipid quantification with an automated microtiter plate fluorometer.Clin. Chem. 1991; 37: 868-874Crossref PubMed Scopus (26) Google Scholar) using Amplex Red reagent to enhance its sensitivity. Essentially, in this method, choline-containing phospholipids (PCs) are hydrolyzed by phospholipase D (Sigma P0065) into free choline, which is further oxidized with choline oxidase to yield H2O2. The formed H2O2 then reacts stoichiometrically with the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) in the presence of HRP, and ultimately forms the fluorescent compound resorufin. Finally, the fluorescence was measured with a VICTOR3 multilabel plate reader (Perkin Elmer; Finland) using an excitation wavelength of 544 nm and an emission wavelength of 595 nm. HDL samples (8 µg as total protein) were loaded onto self-prepared 4–30% polyacrylamide gradient gels (8.0 cm × 8.0 cm) and run at 125 V under nondenaturing conditions overnight at 4°C to reach equilibrium and then stained with Coomassie blue. HDL particle size was determined based on the use of high-molecular-weight electrophoresis calibration standards as molecular size markers. Particle size was also assessed by negative staining electron microscopy (24Oorni K. Hakala J.K. Annila A. Ala-Korpela M. Kovanen P.T. Sphingomyelinase induces aggregation and fusion, but phospholipase A2 only aggregation, of low density lipoprotein (LDL) particles. Two distinct mechanisms leading to increased binding strength of LDL to human aortic proteoglycans.J. Biol. Chem. 1998; 273: 29127-29134Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). For this purpose, HDL samples (3 μl from HDL stock, 1 mg/ml) were dried on carbon-coated grids, after which 3 μl of 1% potassium phosphotungstate, pH 7.4, was added and also dried on the grids. The samples were viewed and photographed in a JEOL 1200EX electron microscope at the Institute for Biotechnology, Department of Electron Microscopy, Helsinki, Finland. For the determination of the size distribution of the lipoprotein particles, the diameters of 100 randomly selected particles were measured from the electron micrographs. Agarose gel electrophoresis (0.6% gel) was carried out using the Paragon electrophoresis system according to the instructions of the manufacturer (Beckman Coulter, Inc.). Proteins were transferred from the agarose gel to the polyvinylidene difluoride (PVDF) membrane by pressure blotting. ApoA-I was identified using a monoclonal anti-human apoA-I antibody (Abcam; UK), followed by an HRP-conjugated anti-rabbit IgG as the secondary antibody (Dako; Denmark). To further examine the effect of pH on the formation of preβ-HDL, HDL2 or HDL3 (0.1–1 mg/ml) was incubated in either 20 mM MES (pH 5.5–6.5), 20 mM PIPES (pH 6.5 or 7.0), or 20 mM Tris (pH 7.5) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2 at 37°C for different periods of time (0–48 h). The preβ-HDL and α-HDL contents were quantified by two-dimensional crossed immunoelectrophoresis (25van Haperen R. van Tol A. Vermeulen P. Jauhiainen M. van Gent T. van den Berg P. Ehnholm S. Grosveld F. van der Kamp A. de Crom R. Human plasma phospholipid transfer protein increases the antiatherogenic potential of high density lipoproteins in transgenic mice.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1082-1088Crossref PubMed Scopus (187) Google Scholar), and the amount of preβ-HDL was expressed as a percentage of the sum of the preβ- and α-mobile areas. Samples of HDL3 or LDL (50 μg/ml) were analyzed by circular dichroism (CD) before and after their incubation at pH 7.5 or 5.5. The treated samples were placed in 0.1 cm quartz cuvettes, and CD spectra were recorded on a JASCO J-715 spectropolarimeter (Japan Spectroscopic Co.; Tokyo, Japan) in the region of 190–250 nm with a step size of 0.5 nm, scan speed of 50 nm per min, band width of 1 nm, and 1 s response. The cell-holder compartment was thermostatically maintained at 37 ± 0.1°C. For each sample, five spectra were averaged, and blank measurements were subtracted. The kinetics of apolipoprotein unfolding were monitored by recording CD signals at 222 nm. Molar ellipticity ([Θ]) was calculated from the equation: [Θ] = (MRW)* Θ/10lc, where Θ is a measured ellipticity in degrees, l is the cuvette path length (0.1 cm), c is the protein concentration (g/ml), and the mean residue weight (MRW) is obtained from the molecular weight and the number of amino acids. The α-helix contents were calculated from the equation using [Θ] at 222 nm: percent α-helix = [(-[Θ] 222+ 3,000)/(36,000 + 3,000)] * 100 (26Tanaka M. Dhanasekaran P. Nguyen D. Ohta S. Lund-Katz S. Phillips M.C. Saito H. Contributions of the N- and C-terminal helical segments to the lipid-free structure and lipid interaction of apolipoprotein A-I.Biochemistry. 2006; 45: 10351-10358Crossref PubMed Scopus (68) Google Scholar, 27Lee-Rueckert M. Lappalainen J. Leinonen H. Pihlajamaa T. Jauhiainen M. Kovanen P.T. Acidic extracellular environments strongly impair ABCA1-mediated cholesterol efflux from human macrophage foam cells.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 1766-1772Crossref PubMed Scopus (23) Google Scholar). HDL2 or HDL3 (1 mg/ml) was incubated in either 20 mM MES (pH 5.5) or 20 mM Tris (pH 7.5) buffer containing 150 mM NaCl, 2 mM MgCl2, and 2 mM CaCl2 at 37°C. After 24 h incubation, samples were dialyzed extensively against 5 mM Tris (pH 7.4) containing 150 mM NaCl, 1 mM EDTA. Aliquots of the dialyzed HDL fractions (1 mg/ml) were then incubated in the absence or presence of 200 BTEE units/ml of recombinant human chymase (kindly provided by Teijin Ltd. Hino, Tokyo, Japan). The enzymatic activity of chymase was measured and 1 U of chymase activity was defined, as described by Woodbury et al., (28Woodbury R.G. Everitt M.T. Neurath H. Mast cell proteases.Methods Enzymol. 1981; 80: 588-609Crossref PubMed Scopus (75) Google Scholar). After incubation, the samples were kept on ice, and 50 µl aliquots were analyzed by SEC. Aliquots (200 µl) were withdrawn, and soybean trypsin inhibitor (SBTI; final concentration: 100 µg/ml) was added to the samples to fully inhibit chymase activity (15Lee M. Metso J. Jauhiainen M. Kovanen P.T. Degradation of phospholipid transfer protein (PLTP) and PLTP-generated pre-beta-high density lipoprotein by mast cell chymase impairs high affinity efflux of cholesterol from macrophage foam cells.J. Biol. Chem. 2003; 278: 13539-13545Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Aliquots of the various chymase-treated preparations and their corresponding nontreated HDL preparations were then added to macrophage foam cell cultures, and their abilities to promote cholesterol efflux were examined. Human monocytes were isolated from buffy coats (Finnish Red Cross Blood Transfusion Center; Helsinki, Finland) by centrifugation in Ficoll-Paque gradient as described (29Saren P. Welgus H.G. Kovanen P.T. TNF-alpha and IL-1beta selectively induce expression of 92-kDa gelatinase by human macrophages.J. Immunol. 1996; 157: 4159-4165PubMed Google Scholar). Washed cells were suspended in DMEM supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, counted, and seeded on 24 well-plates (1.5 million cells per well). After 1 h, nonadherent cells were removed and the medium was replaced with macrophage-serum-free medium (Gibco) supplemented with 1% penicillin-streptomycin and 10 ng/ml of granulocyte macrophage colony-stimulating factor (Biosite; San Diego, CA). The medium was then changed every 2 to 3 days throughout the culture period. The monocyte-derived macrophages were incubated in DMEM (pH 7.4) containing 25 μg/ml of [3H]CE-acetyl-LDL for 24 h to induce the formation of foam cells. To measure cholesterol efflux, macrophages were washed with PBS, and fresh media containing the various cholesterol acceptors (25 µg protein/ml) were added. After incubation for 16 h, the media were collected centrifuged at 300 g for 10 min to remove cellular debris, and the radioactivity in the supernatants was determined by liquid scintillation counting. Cells were solubilized with 0.2 M NaOH, and analyzed for radioactivity. Cholesterol efflux was expressed as the percentage radioactivity in the medium relative to the sum of total radioactivity present in the medium and the cells. Cholesterol efflux to the incubation medium in the absence of any added cholesterol acceptors was considered as basal efflux, and was subtracted from the efflux values obtained in the presence of acceptors. Results are reported as mean ± SD. Statistical significance (P < 0.05) was determined by two-tailed Student's t-test. HDL3 and HDL2 were incubated at different pH values (5.5-7.5) for 24 h, after which aliquots of the incubation media were analyzed by SEC. Figure 1A shows that by using this methodology, non-incubated HDL3 (nHDL3) eluted as a single peak, and that the same applied to HDL3 preparations incubated for 24 h at pH 7.5, 7.0, or 6.5. In contrast, incubation of HDL3 at pH 6.0 and below led to substantial changes in the elution profile of HDL3, the major peak (I) having a shorter elution time and a minor peak (II) appearing only slightly earlier than did the isolated and purified lipid-free apoA-I, which served as a standard. Thus, the SEC analysis revealed that when compared with the original Peak I (nHDL3), the particles in the major peak had an increased size, whereas the particles in the newly appeared Peak II were much smaller. Analysis of pH-treated HDL3 using the highly sensitive two-dimensional crossed-immunoelectrophoresis revealed that incubation at pH 6.5 and below resulted in the formation of preβ-migrating particles and that the lower the pH, the higher was the amount of preβ-HDL formed (Fig. 1B). To estimate the sizes of the HDL3 particles during size conversion, the fractions of the major peak were pooled and analyzed by 4–30% NDGGE. This analysis confirmed the presence of enlarged HDL particles after incubation at pH 5.5 (Fig. 1C). Compared with HDL3, similar but less-remarkable effects were observed when HDL2 was incubated at pH 5.5 (see supplementary Fig. I). In contrast, LDL, which lacks apoA-I but contains a nonexchangeable and nonreleasable apolipoprotein, apoB-100, was fully resistant to the above-shown acidic pH-induced effects on HDL (see supplementary Fig. IIA). We next used negative staining electron microscopy to visualize the particle morphology both before and after incubation of HDL3 at acidic pH. Before the incubation, HDL3 appeared as spherical particles with a mean diameter of 8.3 ± 1.2 nm (range 6–11 nm) (Fig. 1D). Incubation of HDL3 at pH 5.5 caused a significant change in the particle size distribution (mean diameter 10.8 ± 2.3 nm; range 7–16 nm), and revealed a remarkable increase in the population of larger particles. Indeed, we found that 60% of the acidic pH-treated particles were within the size range of 9–13 nm. We also observed that large particles with diameters of 12–16 nm, not present in the nHDL3, appeared. Taken together, these results show that upon exposure to pH 5.5, of the ultracentrifugally isolated HDL3 and HDL2, the HDL3 particles in particular undergo remodeling, which consists of two stages: release of smaller apoA-I-containing particles with preβ-mobility and formation of enlarged α-migrating HDL particles. Next, we investigated the rate of the formation of enlarged HDL particles and the detachment of apoA-I. For this purpose, HDL3 was incubated at pH 5.5 and aliquots of supernatants at different time points up to 48 h were analyzed by SEC. The formation of enlarged HDL particles (Peak I) and the release of apoA-I with preβ-mobility (Peak II), were both time-dependent (Fig. 2A, B). To obtain more-precise information about the time-dependent effect of acidic pH on HDL size, the average sizes of particles in Peaks I and II were calculated using gel filtration calibration of proteins with known Stokes diameters. Native HDL3 particles were 8.8 ± 0.1 nm in diameter, and incubation at pH 5.5 progressively increased their size (Table 1). The size of the particles in Peak II remained unchanged (7.5 ± 0.2 nm), a finding consistent with previous reports on the size of preβ1-HDL (30Duong P.T. Weibel G.L. Lund-Katz S. Rothblat G.H. Phillips M.C. Characterization and properties of pre beta-HDL particles formed by ABCA1-mediated cellular lipid efflux to apoA-I.J. Lipid Res. 2008; 49: 1006-1014Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 31Clay M.A. Barter P.J. Formation of new HDL particles from lipid-free apolipoprotein A-I.J. Lipid Res. 1996; 37: 1722-1732Abstract Full Text PDF PubMed Google Scholar). To quantitatively determine the generation of preβ-HDL, the HDL particles were analyzed by two-dimensional crossed-immunoelectrophoresis. Incubation of HDL3 at pH 5.5 resulted in the formation of preβ-HDL in a time-dependent manner (Fig. 2C, left). After 6 h incubation at pH 5.5, the fraction of the newly generated preβ-HDL fraction amounted to 11% of the total HDL, and it increased to 27% after 48 h incubation. In contrast, at neutral pH 7.5, even after 48 h incubation, the majority of HDL3 migrated in the α-position and only a negligible amount (always <1%) of preβ-HDL was detected (Fig. 2C; right). Similar, but less-profound, data were also found for HDL2 (see supplementary Fig. IIIA, B), again revealing that HDL3 is more susceptible to acidic pH remodeling than is HDL2.TABLE 1.Particle size of HDL3 at pH 5.5 as a function of timeIncubation TimePeak I Diameterhnm69.1 ± 0.1aP < 0.05 versus 0 h (nHDL3).129.3 ± 0.2aP < 0.05 versus 0 h (nHDL3).249.8 ± 0.3aP < 0.05 versus 0 h (nHDL3).4810.1 ± 0.2aP < 0.05 versus 0 h (nHDL3).HDL3 (1 mg/ml) was incubated in 20 mM MES (pH 5.5) for the indicated times. The samples were analyzed by SEC, and particle sizes of Peaks I and II were calculated using a gel filtration calibration kit.a P < 0.05 versus 0 h (nHDL3). Open table in a new tab HDL3 (1 mg/ml) was incubated in 20 mM MES (pH 5.5) for the indicated times. The samples were analyzed by SEC, and particle sizes of Peaks I and II were calculated using a gel filtration calibration kit. We next examined whether incubation of HDL at pH 5.5 would exert differential effects on HDL protein and lipid distribution. Before incubation, nHDL3 elution profiles for protein, total cholesterol, and phospholipids fully coincided, and incubation at acidic pH induced similar shifts in all the elution profiles of the main peak (data not shown). However, the small HDL particles (Peak II) appeared to contain only minor amounts of phospholipids and cholesterol. Detailed analysis indicated that of the total protein, 88 ± 4% and 12 ± 0.6% were in Peaks I and II, respectively, and that the corresponding values for total phospholipids were 94 ± 3% and 6.4 ± 0.3%, and for total cholesterol, 95 ± 3% and 5.4 ± 0.3%. Compared with nHDL3, the enlarged HDL3 particles (Peak I) contained less protein and slightly more lipids, whereas the preβ-HDL particles (Peak II) were rich in protein and had lower amounts of lipids (Table 2). Immunoblot analysis (data not shown) revealed the presence of both apoA-I and apoA-II in the enlarged HDL particles (Peak I), whereas only apoA-I was detected in the preβ-HDL particles (Peak II).