Kaplan M, Williams K J, Mandel H, Aviram M
Lipid Research Laboratory, The Bruce Rappaport Faculty of Medicine, Technion, the Rappaport Family Institute for Research in the Medical Sciences, and Rambam Medical Center, Haifa, Israel.
Arterioscler Thromb Vasc Biol. 1998 Apr;18(4):542-53. doi: 10.1161/01.atv.18.4.542.
Macrophage binding sites for oxidized LDL (Ox-LDL) include class A scavenger receptors (SR-As), the CD-36 molecule, and an additional but hitherto unidentified binding site. Because cell-surface glycosaminoglycans (GAGs) were previously shown to be involved in the cellular uptake of native LDL and lipoprotein(a), several strategies to assess the participation of heparan sulfate (HS) and chondroitin sulfate (CS) in macrophage catabolism of Ox-LDL were used. First, incubation of J-774 A.1 macrophage-like cells with either heparinase or chondroitinase, or with both enzymes together, reduced the binding, uptake, and degradation of 125I-Ox-LDL by 20% to 45%, in comparison with control nontreated cells, while catabolism of 125I-labeled acetylated LDL (Ac-LDL) and native LDL were unaffected. Second, the proteoglycan (PG) cellular content was increased by cell enrichment with exogenous GAGs or by using human monocyte-derived macrophages from two patients with Sanfilippo mucopolysaccharidosis, which are characterized by cellular HS accumulation. In these macrophages, cellular uptake of 125I-Ox-LDL increased, while catabolism of 125I-Ac-LDL and native LDL were unaffected. Experiments using conditioned media from control, heparinase-digested, or chondroitinase-digested macrophages indicated that neither secreted GAGs nor released digestion products played any role in Ox-LDL catabolism. To evaluate potential interactions between cell-surface GAGs and known receptors for Ox-LDL, we used excess unlabeled Ac-LDL to block SR-As or anti-CD-36 antibodies to block CD-36, and then examined the catabolism of 125I-Ox-LDL by GAG-enriched or -depleted macrophages. Both excess unlabeled Ac-LDL and anti-CD-36 antibodies reduced 125I-Ox-LDL catabolism, but only excess unlabeled Ac-LDL completely abolished the increase in 125I-Ox-LDL catabolism on GAG enrichment of the cells, indicating a cooperation between exogenous GAGs and cell-surface SR-As in the catabolism of OX-LDL. Moreover, the addition of GAGases to macrophages that were preincubated with anti-CD-36 antibodies and excess Ac-LDL further reduced macrophage degradation of Ox-LDL in comparison with cells that were pretreated only with anti-CD-36 antibodies and Ac-LDL, indicating a more complex role for endogenous GAGs. Overall, these studies demonstrate a substantial contribution of macrophage-associated GAGs in the catabolism of Ox-LDL, which is mediated in part by a cooperation between GAGs and cell-surface SR-As.
氧化型低密度脂蛋白（Ox-LDL）的巨噬细胞结合位点包括A类清道夫受体（SR-As）、CD-36分子以及另一个迄今尚未明确的结合位点。由于此前已表明细胞表面糖胺聚糖（GAGs）参与天然低密度脂蛋白和脂蛋白(a)的细胞摄取，因此采用了多种策略来评估硫酸乙酰肝素（HS）和硫酸软骨素（CS）在巨噬细胞对Ox-LDL的分解代谢中的作用。首先，与对照未处理细胞相比，用肝素酶或软骨素酶单独处理J-774 A.1巨噬细胞样细胞，或两种酶一起处理，可使125I-Ox-LDL的结合、摄取和降解减少20%至45%，而125I标记的乙酰化低密度脂蛋白（Ac-LDL）和天然低密度脂蛋白的分解代谢不受影响。其次，通过用外源性GAGs使细胞富集或使用来自两名患有Sanfilippo黏多糖贮积症患者的人单核细胞衍生巨噬细胞（其特征为细胞内HS积累），可增加蛋白聚糖（PG）的细胞含量。在这些巨噬细胞中，125I-Ox-LDL的细胞摄取增加，而125I-Ac-LDL和天然低密度脂蛋白的分解代谢不受影响。使用来自对照、肝素酶消化或软骨素酶消化巨噬细胞的条件培养基进行的实验表明，分泌的GAGs和释放的消化产物在Ox-LDL分解代谢中均不起任何作用。为了评估细胞表面GAGs与已知的Ox-LDL受体之间的潜在相互作用，我们使用过量未标记的Ac-LDL来阻断SR-As或抗CD-36抗体来阻断CD-36，然后检测富含或缺乏GAGs的巨噬细胞对125I-Ox-LDL的分解代谢。过量未标记的Ac-LDL和抗CD-36抗体均降低了125I-Ox-LDL的分解代谢，但只有过量未标记的Ac-LDL完全消除了细胞富含GAGs时125I-Ox-LDL分解代谢的增加，这表明外源性GAGs与细胞表面SR-As在Ox-LDL分解代谢中存在协同作用。此外, 与仅用抗CD-36抗体和Ac-LDL预处理的细胞相比，向预先用抗CD-36抗体和过量Ac-LDL孵育的巨噬细胞中添加GAGases进一步降低了巨噬细胞对Ox-LDL的降解，这表明内源性GAGs的作用更为复杂。总体而言，这些研究表明巨噬细胞相关GAGs在Ox-LDL的分解代谢中起重要作用，这部分是由GAGs与细胞表面SR-As之间的协同作用介导的。
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