Shin-ichiroh Kawai

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.

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.

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.