A novel monoclonal antibody recognizing extracellular space of the atherosclerotic lesions in WHHL rabbits.

为了研究胆固醇酯在动脉粥样硬化主动脉细胞外空间的积聚机制，我们使用Watanabe遗传性高脂血症（WHHL）兔的动脉粥样硬化主动脉的脱脂粗匀浆作为免疫原的复杂混合物制备了抗动脉粥样硬化病变的新型单克隆抗体。通过组织化学筛选抗体，我们能够首次获得特异性识别动脉粥样硬化病变中含有脂质沉积物的细胞外基质（EMRla/212 D）的单克隆抗体。纯化的抗原物质是一种分子量为66 kDa的糖蛋白，其抗原决定簇以唾液酸为主要成分，我们从兔肝cDNA文库中克隆了66 kDa糖蛋白（GP 66）。该克隆跨越编码整个GP 66（456个氨基酸）和信号肽的19个氨基酸的序列。GP 66的核苷酸序列推测为兔玻连蛋白，含有Arg-Gly-Asp细胞附着序列，与人玻连蛋白的氨基酸序列具有76%的同源性。此外，EMRla/212 D识别通过肝素亲和层析纯化的兔玻连蛋白。RNA印迹杂交检测到一个大小相同的转录本在正常和WHHL兔肝。血浆GP 66和肝脏GP 66 mRNA水平没有改变，而在WHHL兔胸主动脉中观察到9倍的GP 66蓄积。这些结果表明，GP 66是兔玻连蛋白，其选择性地在动脉粥样硬化的发展中的动脉粥样硬化的主动脉中积累。

In order to study the mechanisms of cholesteryl ester accumulation in the extracellular space of the atherosclerotic aorta, we prepared novel monoclonal antibodies against atherosclerotic lesions using a delipidated crude homogenate of atherosclerotic aorta from Watanabe-heritable hyperlipidemic (WHHL) rabbits as a complex mixture of immunogens. By screening the antibodies histochemically, we were able to obtain for the first time monoclonal antibodies which specifically recognized the extracellular matrix with lipid-laden deposits (EMRla/212D) in atherosclerotic lesions. The purified antigenic material is a glycoprotein with a molecular mass of 66 kDa, and the epitope of the antigenic material contains sialic acid as a major element.We have isolated from rabbit liver cDNA library clones coding for the 66-kDa glycoprotein (GP66). The clone spans the sequence coding for the entire GP66 (456 amino acids) and 19 amino acids of a signal peptide. GP66 was deduced to be rabbit vitronectin from its nucleotide sequence, containing an Arg-Gly-Asp cell attachment sequence and showing 76% amino acid sequence homology with human vitronectin. Furthermore, EMRla/212D recognized rabbit vitronectin purified by heparin affinity chromatography. RNA blot hybridization detected one transcript of the same size in normal and WHHL rabbit liver. The levels of plasma GP66 and liver GP66 mRNA were not altered, whereas 9-fold greater accumulation of GP66 was observed in the thoracic aorta of WHHL rabbit. These results suggest that GP66 is rabbit vitronectin, which selectively accumulates in the atherosclerotic aorta with the development of atherosclerosis.