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

Abstract

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When 123 I-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 1 H and 13 C 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.

Foam cell formation containing lipid droplets enriched with free cholesterol by hyperlipidemic serum

“…Among a series of monoclonal antibodies against atherosclerotic lesions established in our previous studies, 256C recognizes PC molecules containing polyunsaturated fatty acyl chains as its epitope (8). It should be noted that binding of this antibody to PC is greatly enhanced by the addition of neutral lipids such as FC (8).…”

“…Using this system, we found that the antigenic lipid droplets recognized by monoclonal antibody 256C could be generated in foam cells. This antibody binds to atherosclerotic plaques of WHHL rabbits in vivo and stains immunohistochemically foam cell-enriched lesions (8). We propose that these cell culture conditions provide a useful model system to investigate the behavior of foam cells as well as the cellular basis of atherosclerosis.…”

“…Monoclonal antibodies were raised using homogenates of human atheroma as immunogen, and 256C and FOH1a/DLH3 (DLH3) were prepared from murine ascites as described previously (8)(9)(10). The epitopes of these antibodies were characterized previously; 256C recognizes PC and the binding was enhanced in the presence of cholesterol, and DLH3 recognizes OxPC but not native PC.…”

“…1 To whom correspondence should be addressed. e-mail: t_takano@pharm.teikyo-u.ac.jp characterized (8). This antibody was shown to bind to atheromatous lesions when radiolabeled antibody was injected intravenously into Watanabe heritable hyperlipidemic (WHHL) rabbits.…”

Arteriosclerosis in the Influx and Intravisceral Arteries of the Liver, Kidney and Lung of WHHL Rabbits.

“…There have been many reports on sclerotic lesions of the coronary artery [7,11,13,16] and aorta [4,5,8,9] until now, but there are no reports on the arterial lesions in the influx and intravisceral arteries of the parenchymatous organs, except for the heart and brain [12], in the WHHL rabbit. In the present study, we investigated histopathologically the arteriosclerotic changes in the influx and intravisceral arteries of the liver, kidney and lung, and clarified the degree and progress level of arteriosclerosis in these parenchymatous organs.…”