Takanori Nakajima

Lipophilic HMG-CoA reductase inhibitor has an anti-inflammatory effect: Reduction of MRNA levels for interleukin-1β, interleukin-6, cyclooxygenase-2, and p22phox by regulation of peroxisome proliferator-activated receptor α (PPARα) in primary endothelial cells

We examined the effects of four 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (pravastatin, simvastatin, fluvastatin, and cerivastatin) on the production and expression of inflammatory cytokines and on enzyme expression involving prostaglandin and superoxide production in cultured human umbilical vein endothelial cells (HUVEC). All HMG-CoA reductase inhibitors significantly reduced interleukin-1beta and -6 mRNA expression and their protein levels in the culture medium, and also inhibited cyclooxygenase-2 mRNA expression and their protein levels. And these drugs induced peroxisome proliferator-activated receptor alpha (PPARalpha) and PPARgamma mRNA expression and their protein levels in HUVEC and hepatocyte. Moreover, the mRNA levels of p22phox, a 22-kD subunit and the protein levels of p47phox, a 47-kD subunit of nicotine adenine dinucleotide phosphate (NADPH) oxidase, was decreased by treatment with either simvastatin, fluvastatin or cerivastatin, and this effect was reversed by mevalonate, geranylgeraniol, farnesol, and cholesterol. The changes induced by HMG-CoA reductase inhibitors might be due to regulation of cellular cholesterol content level, cellular cholesterol metabolic pathway, and cellular PPARalpha activity, which was related with inflammation. This unique anti-inflammatory effect in addition to its hypolipidemic action, may be beneficial in preventing the vascular complications that are induced by hyperlipidemia.

Bezafibrate has an antioxidant effect: Peroxisome proliferator-activated receptor α is associated with Cu2+, Zn2+-superoxide dismutase in the liver

Administration of bezafibrate in rats significantly reduced the levels of plasma thiobarbituric acid-reactive substances (TBARS) in comparison with those obtained in rats fed a soy or lard chow. Moreover, an elevation of in vitro conjugated diene production and linoleic acid levels in the high-density lipoproteins and low-density lipoproteins induced by a soy or lard chow, was reduced by bezafibrate administration. In addition, the liver Cu2+, Zn2+-superoxide dismutase (SOD) gene expression showed a significant positive correlation with the liver peroxisome proliferator-activated receptor alpha (PPARalpha) mRNA level (R=0.769, p<0.0001). This unique characteristic of bezafibrate, which possesses both a hypolipidemic effect and antioxidant activity, may be beneficial in preventing vascular complications in hyperlipidemia.

Localization of oxidized HDL in atheromatous plaques and oxidized HDL binding sites on human aortic endothelial cells

We examined the localization of oxidized high-density lipoprotein (HDL) in atheromatous plaques and the oxidized HDL binding sites on endothelial cells. Histochemical analysis using CuSO4-oxidized HDL-specific 9F5-3a antibody indicated the presence of oxidized HDL in the intima of atheromatous plaques in human abdominal aortae. The cell surface binding of 125I-oxidized HDL to cultured human aortic endothelial cells (HAEC) was saturable, with an apparent dissociation constant (K d) of 1.43 μmol/L. Competition for 125I-oxidized HDL binding was strong for oxidized HDL, moderate for native HDL and low for acetylated low-density lipoprotein (LDL) or oxidized LDL. Using oxidized HDL as a ligand for blotting, a major 130-kDa band was detected in HAEC. These results suggest that oxidized HDL and its putative binding protein are present in atheromatous plaques and endothelial cells, respectively.

Serum lipid metabolism abnormalities and change in lipoprotein contents in patients with advanced-stage renal disease.

BACKGROUND Arteriosclerosis is the major cause of death in patients with chronic renal failure. There is much interest in the lipid metabolism of patients treated with hemodialysis. METHODS We analyzed low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in chronic renal failure (CRF) patients according to patients on hemodialysis (HD), patients with diabetic nephropathy before initiation of dialysis (DN), and patients with chronic glomerulonephritis in the conservative stage (CGN); and compared the lipid metabolic abnormalities in patients on hemodialysis and those not yet on hemodialysis. We also analyzed the qualitative abnormalities of LDL and HDL and their relationship with the pathological stages. RESULTS Electrophoretic patterns identified small LDL particles and small HDL particles in the three groups, and the degree of denaturation was more enhanced in CRF patients in the conservative stage than in HD patients. For LDL susceptibility to oxidation LDL (oxLDL) by addition of Cu(2+), the lag time was approximately 57 min in healthy controls and CGN patients, but was prolonged to approximately 75 min in HD and DN patients. For HDL susceptibility to oxidation HDL (oxHDL), HD, DN and CGN patients showed lag times shorter than those found in healthy control subjects. These results showed that LDL and HDL in the serum of CRF patients were in a state of enhanced susceptibility to oxidative modification. In Western blot analysis using anti-human-denatured LDL and anti-human-oxidized HDL monoclonal antibodies, bands of low molecular oxLDL at 150-197 kDa were detected in all CRF patients, with marked tailing in CGN patients. Similarly, bands of small oxHDL particles at 110 and 120 kDa were found in HD, DN and CGN patients. CONCLUSIONS Oxidative modification of both LDL and HDL occurs in patients with advanced CRF resulting in small lipoproteins. Increased production of oxLDL and oxHDL is the main cause of lipid metabolic abnormality in CRF patients.

Apolipoprotein A-I concentration in tears in diabetic retinopathy

Background Apolipoprotein concentrations in reflex tears from healthy and noninsulin-dependent diabetes mellitus (NIDDM) subjects were measured and correlated with the stage of diabetic retinopathy. Methods and results The apolipoprotein A-I (apo A-I) concentrations in the tears from NIDDM patients with retinopathy were significantly higher than those from patients with no or negligible retinopathy (P < 0·05), and apo A-I was not detected in healthy subjects by Western blotting. No reactive band was detected on apo A-I by Western blotting for nitrotyrosine, a marker for peroxynitrite oxidation of the tear proteins. Apo A-I concentration in tears was significantly correlated with the stage of retinopathy (r = 0·598, n = 59, P < 0·001). No apo A-I gene expression was detected in the conjunctiva by reverse transcription polymerase chain reaction. Conclusions We conclude that there is increased secretion of native apo A-I from the main lacrimal gland in patients with advanced diabetic retinopathy.

Changes in intestinal alkaline phosphatase isoforms in healthy subjects bearing the blood group secretor and non-secretor

We found the high molecular mass intestinal alkaline phosphatase (HIAP) and normal molecular mass intestinal alkaline phosphatase (NIAP) in serum at fasting and after fatty meal by use of 6.0% polyacrylamide gel electrophoresis (PAGE) in the presence of 1% Triton X-100. HIAP only appeared in serum of Lewis blood group secretors ¿Le(a-b+)¿, and HIAP levels were dependent on ABO blood groups. Among the secretors, the highest activities of HIAP in fasting serum were observed in subjects with blood groups O and B (8.6+/-1.4 U/1; mean+/-SD) and the lowest activities were associated with blood group A (0.7+/-0.2 U/1; mean+/-SD), and the HIAP activities did not change after fatty meal. In contrast, NIAP was present in the serum of both secretors and non-secretors regardless of ABO blood group. Trace amounts of NIAP remained in fasting serum; however serum NIAP activities rose sharply after fatty meal. The remaining ratios of NIAP activity at fasting and 9 h after fatty meal of secretors were approximately the same as those of non-secretors. The electrophoretic mobility on PAGE or the apparent molecular mass estimated by gel filtration of serum NIAP in secretors was slightly different from that in non-secretors. In addition, HIAP can be normalized to NIAP on PAGE in the absence of Triton X-100, and the electrophoretic mobility of normalized-NIAP was identical to that of original NIAP in secretors. Accordingly, it can be concluded that the structure of serum NIAP in the secretor was different from that in the non-secretor, because HIAP is only formed by serum NIAP in the secretor. These results suggest that differences in serum NIAP in the secretor and the non-secretor may be closely related to the appearance of IAP in the circulation.

Reduced alkaline phosphatase activity in diabetic rat bone: a re-evaluation

We found previously that human bone alkaline phosphatase (AP) was glycated by aseptic incubation with glucose, and partially broken down by reactive oxygen species. In this study, we examined whether selective in vivo glycation of AP molecules occurred in bone tissue, using experimental diabetic rats induced by streptozotocin and spontaneously diabetic rats. Additionally, the effects of hyperlipidemia on bone AP activity were examined. Serum AP activity was significantly elevated after incipient onset of diabetes, and the increased activity originated from the intestinal isozyme. High levels of intestinal AP activity were also observed in rats with hyperlipidemia induced by feeding high-fat or high-fructose chow, but the AP activity in bone tissues was maintained at a constant level. AP activity in bone was reduced after the onset of diabetes. The resulting bone AP molecule bound to an aminophenylboronic acid column, which had affinity for glycated proteins, and contained smaller molecular sizes than the native bone AP. These results suggest that elevated levels of serum AP activity originated from the intestinal isozyme accompanied with hyperlipidemia induced by diabetes. In contrast, the reduced serum levels of AP activity in diabetic rats might be dependent on inactivation of bone AP, which was glycated, followed by partial breakdown of bone AP molecules, possibly due to reactive oxygen species.

Characterization of the epitopes specific for the monoclonal antibody 9F5-3a and quantification of oxidized HDL in human plasma

Background: We previously isolated a monoclonal antibody, 9F5-3a, that is specific for HDL oxidized by CuSO4. Methods: We examined the characteristics of the 9F5-3a epitope by Western blot and measured the concentration of oxidized HDL in human plasma by enzyme-linked immunosorbent assay using this antibody. Results: The monoclonal antibody specifically reacted with oxidized HDL in a mixture of HDL, LDL and modified lipoproteins. Oxidation of the HDL particles accelerated cross-linkage of apolipoproteins caused by lipid peroxidation, and the cross-linked apolipoprotein AI selectively reacted with the 9F5-3a antibody. Mean (standard deviation) plasma concentrations of oxidized HDL were 127 (50) μg/L in 23 healthy controls, 191 (65) μg/L in 30 patients with non-insulin-dependent diabetes mellitus (P < 0.01 versus healthy controls) and 200 (87) μg/L in 25 patients with coronary artery disease (P < 0.01 versus healthy controls). The concentrations of oxidized HDL did not correlate with the concentrations of thiobarbituric acid-reactive substances. Conclusions: The results indicate that determination of oxidized HDL concentration may be useful for identifying patients with atherosclerotic disease.