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J Biol Chem, Vol. 274, Issue 35, 24828-24837, August 27, 1999

Presence of Phospholipid-Neutral Lipid Complex Structures in Atherosclerotic Lesions as Detected by a Novel Monoclonal Antibody

Masahiro Mori, Hiroyuki Itabe, Keizo Takatoku^§, Keiji Shima, Jun Inoue, Masaru Nishiura^§, Hideyo Takahashi^¶, Hiro Ohtake^¶, Ryuichiro Sato, Yusuke Higashi, Tsuneo Imanaka, Shiro Ikegami^¶, and Tatsuya Takano

From the  Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan, ^§ Daiichi Radioisotope Laboratories, Ltd., Chiba 289-1517, Japan, and the ^¶ Laboratory of Organic and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa 199-0195, Japan

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When ¹²³I-labeled ASH1a/256C antibody is injected intravenously into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purified by high performance liquid chromatography, was shown to be phosphatidylcholine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing ¹H and ¹³C nuclear magnetic resonance, Fourier transfer-infrared spectrum, and mass spectrometry. The antibody did not react with other classes of phospholipids or neutral lipids when tested using an enzyme-linked immunosorbent assay. When PC was mixed with either cholesterol, cholesteryl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal and WHHL rabbits were fractionated using sucrose density gradient ultracentrifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions from WHHL rabbits was twice as high as that of normal rabbits, and there was an enormous difference in the antigenic activity in these fractions. The content of cholesterol in the cellular membrane fraction of WHHL rabbits was approximately 50 times higher than that of normal rabbits. Addition of neutral lipids to the cellular membrane fraction of normal rabbit markedly increased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-neutral lipid complex domains are formed in atherosclerotic lesions.

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