Osamu Miyazaki

LCAT-Dependent Conversion of Preβ1-HDL into α-Migrating HDL is Severely Delayed in Hemodialysis Patients

ABSTRACT. Preβ1-HDL is a minor HDL subfraction that acts as an efficient initial acceptor of cell-derived free cholesterol. During 37°C incubation, plasma preβ1-HDL decreases over time due to its conversion to α-migrating HDL by lecithin:cholesterol acyltransferase (LCAT). This conversion may be delayed in hemodialysis patients who have decreased LCAT activity. To clarify whether LCAT-dependent conversion of preβ1-HDL to α-migrating HDL is delayed in hemodialysis patients, preβ1-HDL concentrations were determined in 45 hemodialysis patients and 45 gender-matched control subjects before and after 37°C incubation with and without the LCAT inhibitor. It was found that the baseline preβ1-HDL concentration in hemodialysis patients was more than twice that in the controls (44.9 ± 21.4 versus 19.8 ± 6.7 mg/L apoAI; P versus 97 ± 17% of baseline; P preβ1 ) was the same for samples from hemodialysis patients exhibiting normal (≥1.03 mmol/L) and low HDL-cholesterol levels (32 ± 32 versus 28 ± 23% of baseline; NS). DR preβ1 was positively correlated with LCAT activity ( r = 0.617; P

Probucol markedly reduces HDL phospholipids and elevated preβ1-HDL without delayed conversion into α-migrating HDL: Putative role of angiopoietin-like protein 3 in probucol-induced HDL remodeling

Probucol is a unique hypolipidemic agent that increases cholesteryl ester transfer protein (CETP) activity. Enhanced CETP-mediated conversion of high-density lipoprotein (HDL) partly explains the probucol-induced decrease in HDL cholesterol and increase in plasma prebeta1-HDL (native lipid-poor HDL) concentrations. However, HDL cholesterol is reduced in patients that are completely deficient in CETP. Angiopoietin-like protein 3 (ANGPTL3) is an endogenous suppressor of endothelial lipase that promotes the hydrolysis of HDL phospholipids and may generate prebeta1-HDL. To determine whether probucol decreases ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL, we measured these parameters before and after a 4-week probucol treatment in 39 hypercholesterolemic patients and age- and sex-matched controls. The median ANGPTL3 had decreased from 143 to 113 microg/L by week 4 (p<0.05). High-performance liquid chromatography revealed that probucol decreased the phospholipid content of very large (13.5-15 nm) and large (12.1 nm) HDL particles predominantly by 65% (p<0.01) and 53% (p<0.001), respectively. The change in ANGPTL3, but not CETP mass, was positively correlated with that in large HDL phospholipids (r=0.455, p<0.05). The absolute and relative concentrations of prebeta1-HDL increased by 14% (p<0.01) and 60% (p<0.001), respectively. The conversion rate of prebeta1-HDL into alpha-migrating HDL by lecithin-cholesterol acyltransferase did not change significantly. In conclusion, probucol decreases plasma ANGPTL3 and HDL phospholipids while increasing prebeta1-HDL. We speculate that probucol induces HDL remodeling via an endothelial lipase-mediated pathway.

Analytical performance of a sandwich enzyme immunoassay for preβ1-HDL in stabilized plasma

We have established an immunoassay for preβ1-HDL (the initial acceptor of cellular cholesterol) using a monoclonal antibody, MAb55201. Because preβ1-HDL is unstable during storage, fresh plasma must be used for preβ1-HDL measurements. In this study, we describe a method of stabilizing preβ1-HDL, and evaluate the analytical performance of the immunoassay for preβ1-HDL. Fresh plasma was stored under various conditions with or without a pretreatment consisting of a 21-fold dilution into 50% (v/v) sucrose. Preβ1-HDL concentration was measured by immunoassay. In nonpretreated samples, preβ1-HDL decreased significantly from the baseline after 6 h at room temperature. Although preβ1-HDL was more stable at 0°C than at room temperature, it increased from 30.2 ± 8.5 (SE) to 56.5 ± 5.5 mg/l apolipoprotein A-I (apoA-I) (P < 0.001) in hyperlipidemics, and from 18.4 ± 1.2 to 37.9 ± 3.3 mg/l apoA-I (P < 0.001) in normolipidemics after 5-day storage. After 30-day storage at −80°C, preβ1-HDL increased from 29.0 ± 4.0 to 38.0 ± 5.7 mg/l apoA-I (P < 0.001) in hyperlipidemics, whereas it did not change in normolipidemics. In pretreated samples, preβ1-HDL concentration did not change significantly under any of the above conditions. Moreover, preβ1-HDL concentrations determined by immunoassay correlated with those determined by native two-dimensional gel electrophoresis (n = 24, r = 0.833, P < 0.05). An immunoassay using MAb55201 with pretreated plasma is useful for clinical measurement of preβ1-HDL.

Plasma CCN2 (connective tissue growth factor; CTGF) is a potential biomarker in idiopathic pulmonary fibrosis (IPF)

BACKGROUNDnIdiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal pulmonary fibrotic disease and useful biomarkers are required to diagnose and predict disease activity. CCN2 (connective tissue growth factor; CTGF) has been reported as one of the key profibrotic factors associated with transforming growth factor-β (TGF-β), and its assay has potential as a non-invasive measure in various fibrotic diseases. Recently, we developed a new subtraction method for determination of plasma CCN2 levels. We examined the utility of plasma CCN2 levels as a surrogate marker in IPF.nnnMETHODSnPlasma CCN2 levels were calculated in 33 patients with IPF, 14 patients with non-IPF idiopathic interstitial pneumonias (IIPs) and 101 healthy volunteers by sandwich enzyme-linked immunosorbent assay (ELISA) using specific monoclonal antibodies for two distinct epitopes of human CCN2. We evaluated the utility of plasma CCN2 levels by comparison with clinical parameters.nnnRESULTSnPlasma CCN2 levels were significantly higher in patients with IPF than in those with non-IPF IIPs and healthy volunteers. Importantly, plasma CCN2 levels showed significantly negative correlation with 6-month change of forced vital capacity (FVC) in patients with IPF.nnnCONCLUSIONSnPlasma CCN2 is a potential biomarker for IPF.

Delineation of the Role of Pre-β1-HDL in Cholesterol Efflux Using Isolated Pre-β1-HDL

OBJECTIVEnThe role of pre-beta1-high density lipoprotein (pre-beta1-HDL) in cholesterol efflux was investigated by separating human plasma into purified pre-beta1-HDL and pre-beta1-HDL-deficient plasma by using a monoclonal antibody specifically reacting with pre-beta1-HDL.nnnMETHODS AND RESULTSnWhen compared with whole plasma, pre-beta1-HDL-deficient plasma was equally efficient in promoting cholesterol efflux from human skin fibroblasts and THP-1 human macrophage cells. When added at the same apolipoprotein A-I concentration, pre-beta1-HDL was less effective than whole plasma in promoting cholesterol efflux from fibroblasts but equally effective in promoting cholesterol efflux from THP-1 cells. However, pre-beta1-HDL-deficient plasma reconstituted with 16% pre-beta1-HDL was more active than whole plasma, demonstrating that pre-beta1-HDL does promote cholesterol efflux actively. The amount of cellular cholesterol present in reisolated pre-beta1-HDL was 1.5- to 2-fold greater after incubation of the cells with whole plasma than after incubation of the cells with pre-beta1-HDL-deficient plasma or plasma treated with the anti-pre-beta1-HDL antibody. However, the anti-pre-beta1-HDL antibody did not inhibit cholesterol efflux.nnnCONCLUSIONSnWe conclude that whereas pre-beta1-HDL is capable of taking up cellular cholesterol, its presence in plasma is not essential for cholesterol efflux, at least in vitro. Instead, pre-beta1-HDL may be the first product of apolipoprotein A-I lipidation during the formation of HDL but may not play a major role in transferring cellular cholesterol to HDL.

Formation of preβ1-HDL during lipolysis of triglyceride-rich lipoprotein

Prebeta1-HDL, a putative discoid-shaped high-density lipoprotein (HDL) is known to participate in the retrieval of cholesterol from peripheral tissues. In this study, to clarify potential sources of this lipoprotein, we conducted heparin injection on four Japanese volunteer men and found that serum triglyceride (TG) level decreased in parallel with the increase in serum nonesterified fatty acids and plasma lipoprotein lipase (LPL) protein mass after heparin injection. Plasma prebeta1-HDL showed considerable increases at 15 min after the heparin injection in all of the subjects. In contrast, serum HDL-C levels did not change. Gel filtration with fast protein liquid chromatography system (FPLC) study on lipoprotein profile revealed that in post-heparin plasma, low-density lipoprotein and alphaHDL fractions did not change, whereas there was a considerable decrease in very low-density lipoprotein (VLDL) fraction and an increase in prebeta1-HDL fraction when compared with those in pre-heparin plasma. We also conducted in vitro analysis on whether prebeta1-HDL was produced during VLDL lipolysis by LPL. One hundred microliters of VLDL extracted from pooled serum by ultracentrifugation was incubated with purified bovine milk LPL at 37 degrees C for 0-120 min. Prebeta1-HDL concentration increased in a dose dependent manner with increased concentration of added LPL in the reaction mixture and with increased incubation time, indicating that prebeta1-HDL was produced during lipolysis of VLDL by LPL. Taken these in vivo and in vitro analysis together, we suggest that lipolysis of VLDL particle by LPL is an important source for formation of prebeta1-HDL.

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

Subtraction method for determination of N-terminal connective tissue growth factor

Background Connective tissue growth factor (CTGF) may be a potential marker of fibrosis. However, platelet-derived CTGF may be released into the plasma by platelet activation during or after blood collection, thereby interfering with accurate determination of the true plasma CTGF level. Plasma CTGF exists as the N-terminal CTGF fragment (N-fragment), composed of modules 1 and 2, whereas platelet CTGF exists as full-length CTGF (full-length), composed of modules 1–4. We perceived the need to develop a method for distinguishing between the N-fragment and full-length CTGF levels, so that the true plasma and serum CTGF (N-fragment) levels could be accurately determined. Methods Full-length levels were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using two monoclonal antibodies recognizing modules 1 and 4, respectively (M1/4 ELISA). Total CTGF (full-length CTGF plus N-terminal CTGF) levels were determined by a sandwich ELISA using two monoclonal antibodies recognizing modules 1 and 2, respectively (M1/2 ELISA). N-terminal CTGF levels were determined by subtracting the full-length levels from the total CTGF levels. Results Both the M1/2 and M1/4 ELISAs showed good analytical performance. When the CTGF levels of plasma and serum collected simultaneously from the same subject were compared, the N-fragment levels determined by the subtraction method were the same, in spite of the fact that full-length CTGF was present in the sample. Conclusion N-fragment levels in plasma and serum can be accurately determined by this subtraction method, even if full-length CTGF in platelets is released during or after blood collection.

Plasma connective tissue growth factor levels as potential biomarkers of airway obstruction in patients with asthma

BACKGROUNDnBronchial asthma is a chronic inflammatory disorder characterized by airway hyperresponsiveness and airflow limitation. Connective tissue growth factor (CTGF), one of the key profibrotic factors associated with transforming growth factor β, may be related to airway remodeling in asthma. However, no data are available on the association between plasma CTGF levels and clinical and physiologic parameters in patients with asthma. Recently, we developed a novel subtraction method for determination of plasma CTGF levels.nnnOBJECTIVEnTo investigate the utility of plasma CTGF level as a surrogate biomarker in asthma.nnnMETHODSnPlasma CTGF levels were measured in 67 patients with stable asthma and 81 healthy volunteers, using the subtraction method. We evaluated correlations between plasma CTGF levels and clinical and physiologic parameters in patients with asthma.nnnRESULTSnPlasma CTGF levels were higher in patients with asthma than in healthy volunteers. Asthmatic patients with a percentage of predicted forced expiratory volume in 1 second (FEV1) less than 80% had significantly higher levels of plasma CTGF than those with a percentage of predicted FEV1 of 80% or more. In patients with asthma, plasma CTGF levels had significantly negative correlations with forced vital capacity (FVC), FEV1, percentage of predicted FEV1, FEV1/FVC ratio, forced expiratory flow at 50% of the FVC (FEF50%), percentage of predicted FEF50%, forced expiratory flow at 75% of the FVC (FEF75%), and percentage of predicted FEF75%, parameters that reflect the degree of airway obstruction. Plasma CTGF levels were negatively correlated with Asthma Control Test scores, a patient-based index of clinical control of asthma.nnnCONCLUSIONnPlasma CTGF may be a potential biomarker for stable asthma when evaluating the degree of persistent airway obstruction.nnnTRIAL REGISTRATIONnumin.ac.jp/ctr Identifier: UMIN000013081.

Plasma preβ1-HDL level is elevated in unstable angina pectoris

Pre beta1-HDL, a minor HDL subfraction consisting of apolipoprotein A-I (apoA-I), phospholipids and unesterified cholesterol, plays an important role in reverse cholesterol transport. Plasma pre beta1-HDL levels have been reported to be increased in patients with coronary artery disease (CAD) and dyslipidemia. To clarify the clinical significance of measuring plasma pre beta1-HDL levels, we examined those levels in 112 patients with CAD, consisting of 76 patients with stable CAD (sCAD) and 36 patients with unstable angina pectoris (uAP), and in 30 patients without CAD as controls. The pre beta1-HDL levels were determined by immunoassay using a specific monoclonal antibody (Mab55201) that we established earlier. The mean pre beta1-HDL level in the CAD patients was significantly higher than the level in the controls (34.8+/-12.9 mg/L vs. 26.6+/-6.9 mg/L, p<0.001). In addition, the mean pre beta1-HDL level was markedly higher in the uAP subgroup than in the sCAD subgroup (43.1+/-11.5mg/L vs. 30.9+/-11.7 mg/L, p<0.0001). These tendencies remained even after excluding dyslipidemic subjects. These results suggest that elevation of the plasma pre beta1-HDL level is associated with the atherosclerotic phase of CAD and may be useful for identifying patients with uAP.