Hideki Doi

Remnant lipoprotein levels in fasting serum predict coronary events in patients with coronary artery disease

BACKGROUND Remnant lipoproteins are atherogenic, but assays of remnants have not been available in routine clinical laboratories because of the lack of practical and validated methods. A simple and reliable method for such an assay, using an immunochemical approach, has recently been developed. This study prospectively examined whether remnant lipoprotein levels in fasting serum, measured by our method, may have prognostic value in patients with coronary artery disease (CAD). METHODS AND RESULTS Remnant lipoprotein levels in fasting serum were measured in 135 patients with CAD by an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti-apoB-100 monoclonal antibodies. Patients were followed up for </=36 months until occurrence of 1 of the following clinical coronary events: recurrent or refractory angina pectoris requiring coronary revascularization, nonfatal myocardial infarction, or cardiac death. Kaplan-Meier analysis demonstrated a significantly higher probability of developing coronary events in patients with the highest tertile of remnant levels (>5.1 mg cholesterol/dL; 75th percentile of distribution of remnant levels) than in those with the lowest tertile of remnant levels (</=3.3 mg cholesterol/dL; 50th percentile of the distribution). Higher levels of remnants were a significant and independent predictor of developing coronary events in multivariate Cox hazard analysis including the following covariates: extent of coronary artery stenosis, age, sex, smoking, hypertension, diabetes mellitus, hypercholesterolemia, low HDL cholesterol, and hypertriglyceridemia. CONCLUSIONS Higher levels of remnant lipoproteins in fasting serum predict future coronary events in patients with CAD independently of other risk factors. Thus, measurement of fasting remnant levels, assessed by the current immunoseparation method, may be helpful in assessment of CAD risk.

Circulating Levels of Secretory Type II Phospholipase A2 Predict Coronary Events in Patients with Coronary Artery Disease

Background-The circulating levels of secretory nonpancreatic type II phospholipase A(2) (sPLA(2)) are increased in various chronic inflammatory diseases and the increase in the levels correlates with the disease severity. sPLA(2) may possibly play a role in atherogenesis and is highly expressed in atherosclerotic arterial walls that are known to have inflammatory features. Thus, this study prospectively examined whether circulating levels of sPLA(2) may have a significant risk and prognostic values in patients with coronary artery disease (CAD). Methods and Results-Plasma levels of sPLA(2) were measured in 142 patients with CAD and in 93 control subjects by a radioimmunoassay. The sPLA(2) levels had a significant and positive relations with serum levels of C-reactive protein, a marker of systemic inflammation, and with the number of the traditional coronary risk factors associated with individuals. Multivariate logistic regression analysis showed that higher levels of sPLA(2) (>246 ng/dL; 75th percentile of sPLA(2) distribution in controls) were a significant and independent risk factor for the presence of CAD. In multivariate Cox hazard analysis, the higher levels of sPLA(2) were a significant predictor of developing coronary events (ie, coronary revascularization, myocardial infarction, coronary death) during a 2-year follow-up period in patients with CAD independent of other risk factors, including CRP levels, an established inflammatory predictor. Conclusions-The increase in circulating levels of sPLA(2) is a significant risk factor for the presence of CAD and predicts clinical coronary events independent of other risk factors in patients with CAD; these results may reflect possible relation of sPLA(2) levels with inflammatory activity in atherosclerotic arteries.

Association of Remnant Lipoprotein Levels With Impairment of Endothelium-Dependent Vasomotor Function in Human Coronary Arteries

BACKGROUND It remains undetermined whether triglyceride-rich lipoproteins are an independent risk factor for atherosclerosis. METHODS AND RESULTS The correlation of responses of coronary arterial diameter (quantitative coronary angiography) and coronary blood flow (intracoronary flow wire technique) to intracoronary infusion of acetylcholine (10 and 50 microg/min) with coronary risk factors including remnant lipoprotein levels was statistically analyzed in 106 consecutive subjects with normal coronary angiograms. Remnant lipoproteins were isolated from fasting blood with an immunoaffinity mixed gel containing anti-apolipoprotein (apo) A-1 and anti-apoB-100 monoclonal antibodies. In multivariate stepwise regression analysis, remnant lipoprotein levels had the most significant correlation with abnormal epicardial coronary vasomotor responses to acetylcholine infusion, reflected by impaired dilation or constriction of the epicardial coronary arteries, and the levels also had an inverse and independent correlation with the coronary blood flow increase in response to acetylcholine. In a subgroup of 53 consecutive subjects, constrictor responses of epicardial coronary diameters to intracoronary infusion of NG-monomethyl-L-arginine (50 micromol/min for 4 minutes) at baseline, reflecting the presence of coronary nitric oxide bioactivity, had an inverse and independent correlation with remnant lipoprotein levels by use of multivariate analysis. CONCLUSIONS Remnant lipoprotein levels were independently associated with abnormal endothelium-dependent vasomotor function in large and resistance coronary arteries in humans, indicating that remnant lipoproteins may impair endothelial vasomotor function in human coronary arteries. The decrease in coronary nitric oxide bioactivity may be responsible in part for the inhibitory effects of remnant lipoproteins.

Remnant lipoproteins induce proatherothrombogenic molecules in endothelial cells through a redox-sensitive mechanism.

BACKGROUND Triglyceride-rich lipoproteins (TGLs) are atherogenic. However, their cellular mechanisms remain largely unexplained. This study examined the effects of isolated remnant-like lipoprotein particles (RLPs) on the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and tissue factor (TF), proatherothrombogenic molecules, in cultured human endothelial cells. METHODS AND RESULTS RLPs were isolated from plasma of hypertriglyceridemic patients by use of the immunoaffinity gel mixture of anti-apoA-1 and anti-apoB-100 monoclonal antibodies. The incubation of cells with RLPs significantly upregulated mRNA and protein expression of these molecules. Total TGLs (d<1.006) and LDL had fewer or minimal effects on expression of these molecules compared with RLPs. RLPs increased intracellular oxidant levels, as assessed with an oxidant-sensitive probe. Combined incubation with alpha-tocopherol or N-acetylcysteine, both antioxidants, suppressed RLP-induced increase in expression of these molecules. In patients with higher plasma levels of RLPs, plasma levels of soluble forms of ICAM-1 and VCAM-1 were significantly higher than in patients with lower RLP levels. Treatment with alpha-tocopherol for 1 month decreased levels of the soluble adhesion molecules concomitantly with an increase in resistance of RLPs to oxidative modification in patients with high RLP levels. CONCLUSIONS RLPs upregulated endothelial expression of ICAM-1, VCAM-1, and TF, proatherothrombogenic molecules, partly through a redox-sensitive mechanism. RLPs may have an important role in atherothrombotic complications in hypertriglyceridemic patients.

Impairment of endothelium-dependent relaxation of rabbit aortas by cigarette smoke extract—role of free radicals and attenuation by captopril

The aim of this study was to examine the effects of the water soluble component of cigarette smoke extract (CSE) on endothelium-dependent relaxation (EDR) of isolated rabbit aortas. The incubation with CSE was found to inhibit EDR in a dose-dependent manner. Co-incubation of the aortic strips with superoxide dismutase (SOD), N-acetylcysteine, glutathione or dimethyl sulfoxide (DMSO), free radical scavengers, attenuated the CSE-induced inhibition of the arterial relaxation. Co-incubation of the strips with captopril (3 mM), an angiotensin converting enzyme inhibitor, also attenuated CSE-induced impairment of vasorelaxation. In parallel experiments using cultured human endothelial cells, CSE suppressed endothelial release of NOx, stable metabolites of nitric oxide (NO). SOD, DMSO and captopril attenuated the suppression of NO production by CSE in association with reduction of free radicals, superoxide anions and hydroxyl radicals, in CSE solution. Neither lactate dehydrogenase release from the cultured endothelial cells nor cell death estimated by trypan blue exclusion test was found after the incubation of the cultured endothelial cells with CSE. The results indicate that free radicals in CSE induce the impairment of EDR, which may be partly due to suppression of NO production and is not due to non-specific cytotoxicity by CSE. Captopril attenuates CSE-induced endothelial dysfunction partly through scavenging free radicals.

Remnants of chylomicron and very low density lipoprotein impair endothelium-dependent vasorelaxation

Remnants of chylomicron and very low density lipoprotein (VLDL) have been implicated as potentially atherogenic. Since endothelial dysfunction is an early event in atherosclerosis, we examined effects of the remnants on endothelium-dependent vasorelaxation. The remnant lipoproteins were isolated from postprandial plasma in hyperlipidemic subjects using the immunoaffinity gel mixture of anti apo A-1 and anti apo B-100 monoclonal antibodies and ultracentrifugation. Rabbit aortic strips suspended in the organ chambers were incubated for 2 h with the preparations of lipoproteins and lipids. After incubation, the strips were tested with vasodilators after precontraction with phenylephrine (1 microM). The remnant lipoproteins (750-1500 microg triglyceride/ml) but not VLDL fraction (up to 1500 microg triglyceride/ml) impaired vasorelaxation in responses to acetylcholine, substance P and A23187. Carbamylated or methylated remnant lipoproteins, chemically modified remnant lipoproteins, had comparable impairment of the vasorelaxation as unmodified remnant lipoproteins. Incubation with lipid extracts from the remnant lipoproteins also exerted an inhibitory effect on the vasorelaxation. Relaxation to sodium nitroprusside was fully preserved in all aortas exposed to the lipoprotein preparations. Thus, the remnant lipoproteins impair endothelium-dependent arterial relaxation at the concentrations observed in the plasma in patients with coronary artery disease (500-2000 microg triglyceride of remnant lipoprotein/ml). The impairment may be in apoprotein receptor-independent manner, and the lipids in the remnants seem to contribute to the inhibitory effect. The endothelial dysfunction caused by the remnant lipoproteins may play a role in the high prevalence of atherosclerotic coronary artery disease in postprandial hyperlipidemic patients.

Prognostic value of plasma levels of secretory type II phospholipase A2 in patients with unstable angina pectoris

Plasma levels of secretory nonpancreatic type II phospholipase A2 (sPLA2) are increased in various chronic inflammatory diseases; this increase is correlated with disease severity. sPLA2 plays a possible role in atherogenesis and is highly expressed in atheromatous plaques. Thus, this study prospectively examined whether plasma levels of sPLA2 may have a prognostic value in patients with unstable angina, which is known to have inflammatory features. Plasma levels of sPLA2 were measured in 52 patients with unstable angina, in 107 patients with stable angina, and in 96 control subjects by radioimmunoassay. sPLA2 levels were significantly higher in patients with unstable angina than in those with stable angina and in control subjects. sPLA2 remained elevated after stabilization of disease. The levels were not increased in the blood in the coronary sinus. Kaplan-Meier analysis demonstrated that patients with unstable angina and with the higher sPLA2 levels had a significantly higher probability of developing clinical coronary events during a follow-up period of 2 years compared with those with the lower levels. In multivariate Cox hazard analysis, the higher levels of sPLA2 were a significant predictor of developing coronary events in patients with unstable angina, independent of other risk factors, including C-reactive protein levels, an established inflammatory predictor. In conclusion, the increase in circulating levels of sPLA2 predicts clinical coronary events independently of other risk factors in patients with unstable angina. sPLA2 levels were persistently elevated but the elevated levels may not be derived from coronary circulation.

Biphasic Regulation of Transcription Factor Nuclear Factor-κB Activity in Human Endothelial Cells by Lysophosphatidylcholine Through Protein Kinase C–Mediated Pathway

Lysophosphatidylcholine (lysoPC), which is generated in oxidized LDL (Ox-LDL) and abundantly exists in atherosclerotic arterial walls, has been shown to alter various endothelial functions and induces several endothelial genes expressed in atherosclerotic arterial walls. Nuclear factor-kappa B (NF-kappaB), a pleiotropic transcription factor, plays an important role in regulation of expression of various genes implicated in atherosclerosis. We have previously reported that lysoPC transferred from Ox-LDL to endothelial surface membrane activates endothelial protein kinase C (PKC), leading to modulated endothelial functions. This study was aimed at determining whether lysoPC could modulate activity of transcription factors in cultured human umbilical vein endothelial cells (HUVECs) by using electrophoretic mobility shift assay. LysoPC was found to increase DNA-binding activity of NF-kappaB in HUVECs within 15 minutes, which peaked at 1 to 2 hours and subsequently declined to the baseline level at 6 hours. Lower concentrations (5 to 15 micromol/L) of lysoPC markedly increased NF-kappaB activity, but higher concentration (50 micromol/L) of lysoPC inhibited the activity. Phorbol 12-myristate 13-acetate, a potent activator of PKC, also augmented NF-kappaB activity in HUVECs, mimicking the effects of lysoPC; furthermore, calphostin C and chelerythrine chloride, specific PKC inhibitors, and alpha-tocopherol, a clinically potent PKC inhibitor, suppressed the lysoPC-induced NF-kappaB activation. These results indicate that lysoPC regulates NF-kappaB activity in a biphasic manner dependent on its concentrations and incubation time in human endothelial cells and the endothelial PKC activation may in part be involved in the lysoPC-induced NF-kappaB activation. Thus, the time course and the positive and negative biphasic regulatory actions of lysoPC on NF-kappaB activity in endothelial cells might exhibit a unique effect of lysoPC in arterial walls on the different stages of atherosclerosis.

Improvement of endothelial vasomotor dysfunction by treatment with alpha-tocopherol in patients with high remnant lipoproteins levels.

OBJECTIVES This study sought to examine whether oral intake of alpha-tocopherol, an antioxidant, could improve endothelium-dependent vasorelaxation in patients with high remnant lipoproteins levels. BACKGROUND Remnant lipoproteins are known to be atherogenic and impair endothelium-dependent arterial relaxation, but the underlying mechanisms remain unclear. Oxidative stress is a common feature of various risk factors for atherosclerosis. METHODS Flow-mediated vasodilation of the brachial artery during reactive hyperemia was examined by high resolution ultrasound technique before and at the end of 4 weeks treatment with oral administration of alpha-tocopherol acetate (300 IU/day) or placebo, which was randomly assigned, in 40 patients with high serum levels of remnants and in 30 patients with low remnants levels in the fasting state (>75th percentile and <25th percentile, respectively, of the distribution of remnants levels in 150 consecutive hospitalized patients). RESULTS Before treatment, flow-mediated vasodilation was lower in patients with high remnants levels than in those with low levels (4.1 +/- 0.3% vs. 6.0 +/- 0.5%, p < 0.01). Treatment with alpha-tocopherol but not with placebo significantly increased flow-mediated dilation in patients with high remnants levels (7.5 +/- 0.4% after alpha-tocopherol vs. 4.2 +/- 0.4% after placebo, p < 0.01). In patients with low remnants levels, alpha-tocopherol was not effective. The beneficial effect with alpha-tocopherol in high remnants patients was associated with decrease in plasma levels of thiobarbituric acid reactive substances, an indicator of lipid peroxidation (6.6 +/- 0.3 nmol/ml before alpha-tocopherol vs. 4.6 +/- 0.3 after alpha-tocopherol, p < 0.05). CONCLUSIONS Alpha-tocopherol improved impairment of endothelium-dependent vasodilation in patients with high remnants levels. The increase in oxidative stress may at least partly contribute to endothelial vasomotor dysfunction, in patients with high remnants levels.

Membrane Active Lipids in Remnant Lipoproteins Cause Impairment of Endothelium-Dependent Vasorelaxation

We have recently found that remnant lipoproteins (RLPs) and their lipid fractions impair endothelium-dependent vasorelaxation (EDR). This study was aimed at clarifying mechanisms responsible for RLP-induced endothelial dysfunction in isolated rabbit aortas. RLPs were isolated from plasma in hyperlipidemic subjects by use of the immunoaffinity gel mixture of anti-ApoA1 and anti-ApoB100 monoclonal antibodies and ultracentrifugation. Organ chamber experiments showed that EDR impairment was restored by addition of reduced glutathione (GSH) or N-acetylcysteine, antioxidants, into the incubation buffer containing isolated rabbit aortas and RLPs (0.75 mg of triglyceride/mL). Furthermore, the incubation of isolated human red blood cells (RBCs) with RLP and its lipids converted the normal shape of RBCs to echinocytes, but coincubation with antioxidants suppressed the RLP-induced RBC transformation, suggesting that they exerted oxidative damage on RBC surface membranes. Studies with HPLC and the postcolumn chemiluminescence method showed that RLPs contain a substantial amount of phosphatidylcholine hydroperoxides. Peroxidized phosphatidylcholine also impaired EDR and had echinocytogenic action, both of which were suppressed by N-acetylcysteine. RLPs isolated from the plasma of patients under treatment with alpha-tocopherol, an antioxidant, had a lower level of phosphatidylcholine hydroperoxides (15% of the amount in nontreated patients), which was associated with a lack of the inhibitory action on EDR and with lesser effect on RBC transformation. Oxidative damage caused by lipid components in RLPs, especially peroxidized phospholipids, deteriorates cell surface membrane and may be at least partly responsible for RLP-induced impairment of EDR.