介导巨噬细胞摄取氧化修饰极低密试脂蛋白的受体

用未标记氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）、ｎ－ＶＬＤＬ、乙酰ＬＤＬ竞争＾１２５Ｉ－ｏｘ－ＶＬＤＬ与巨噬细胞的结合。在浓度为２００μｇ蛋白／ｍｌ时,分别抑制标记ｏｘ－ＶＬＤＬ结合量的７０￣７８％、６０￣７０％和２５￣３５％。用未标记ｏｘ－ＶＬＤＬ竞争＾１２５Ｉ－ｎ－ＶＬＤＬ与巨噬细胞的结合,能抑制７７％。结果说明ｏｘ－ＶＬＤＬ主要通过ｎ－ＶＬＤＬ受体进入巨噬细胞。以ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤ     

介导巨噬细胞摄取氧化修饰极低密度脂蛋白的受体

用未标记氧化修饰极低密度脂蛋白（ｏｘ－ＶＬＤＬ）、ｎ－ＶＬＤＬ、乙酰ＬＤＬ竞争１２５Ｉ－ｏｘ－ＶＬＤＬ与巨噬细胞的结合。在浓度为２００μｇ蛋白／ｍｌ时,分别抑制标记ｏｘ－ＶＬＤＬ结合量的７０～７８％、６０～７０％和２５～３５％。用未标记ｏｘ－ＶＬＤＬ竟争１２５Ｉ－ｎ－ＶＬＤＬ与巨噬细胞的结合,能抑制７７％。结果说明ｏｘ－ＶＬＤＬ主要通过ｎ－ＶＬＤＬ受体进入巨噬细胞。以ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤＬ进行交叉竞争时,ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤＬ自身可抑制标记ｏｘ－ＶＬＤＬ或ｏｘ－ＬＤＬ的７５～８２％,而ｏｘ－ＶＬＤＬ或ｏｘ－ＬＤＬ的交叉竞争仅３８～４０％。表明ｏｘ－ＶＬＤＬ与ｏｘ－ＬＤＬ有部分共同的构象与巨噬细胞的脂蛋白受体结合,但ｏｘ－ＶＬＤＬ不是经ｏｘ－ＬＤＬ受体被巨噬细胞摄取。     

蛋白激酶C抑制剂对U937细胞清道夫受体功能的影响

为了解细胞内蛋白质磷酸化水平对清道夫受体功能的影响,用蛋白激酶Ｃ抑掉剂形孢菌素（ｓｔａｕｒｏｓｐｏｒｉｎｅ,ＳＴＡ）处理人Ｕ９３７细胞,分别测定对照组和处理组细胞对碘标记的氧化低密度脂蛋白（＾１２５Ｉ）ｏｘ－ＬＤＬ的降解,结合,细胞表面受体复合物的内移以及细胞内脂质蓄积的程度,并利用放射自显影方法观察药物对细胞表面受体表达的影响,结果发现ＳＴＡ可以促进细胞结合（＾１２５Ｉ）ｏｘ－ＬＤＬ增加细胞表面     

巨噬细胞新型氧化低密度脂蛋白结合蛋白的研究

小鼠腹腔巨噬细胞（ＭＰＭ）膜上存在能结合氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的Ａ类清道夫受体（ＳＲ－Ａ）,但用配体印迹技术研究制备ＭＰＭ膜蛋白,发现还存在一种新型ｏｘ－ＬＤＬ膜结合蛋白,其分子量低于ＳＲ－Ａ,为９２ｋＤ它不结合乙酰化低密度脂蛋白ａｃ－ＬＤＬ）,与配体的结合也不受还原剂的影响,但唾液酸酶处理则明显减弱其与ｏｘ－ＬＤＬ的结合,未标记ｏｘ－ＬＤＬ能竞争性抑制（＾１２５Ｉ）ｏｘ－ＬＤＬ与９２ｋ     

蛋白激酶C对大鼠缺血海马突触体谷氨酸摄取的调控作用

采用大鼠海马脑片体外缺血模型,观察海马突触体内蛋白激酶Ｃ（ＰＫＣ）活性的变化,以及这种变化对突触体谷氨酸（ＧＬＵ）摄取的影响。结果显示：海马脑片体外“缺血”１０ｍｉｎ,其突触体内ＰＫＣ活性基本不变,而缺血３０ｍｉｎ,突触体内ＰＫＣ活性显著上升（Ｐ＜０．０１,ｎ＝６）;非Ｎ－甲基－Ｄ－天门冬氨酸（ＮＭＤＡ）受体拮抗剂ＤＮＱＸ有效地抑制ＰＫＣ活性的同时,可降低胞外ＧＬＵ的堆积,而ＮＭＤＡ受体阻断剂ＡＰ＿５无作用。进一步实验证明,ＰＫＣ激动剂ＰＤＢ浓度依赖性地抑制突触体对３Ｈ－ＧＬＵ的摄取（ＩＣ５０＝１３１±１０μｍｏｌ／Ｌ）,此抑制作用可由ＰＫＣ抑制剂Ｈ－７（１００μｍｏｌ／Ｌ）抵消。提示脑缺血诱发ＧＬＵ堆积的作用机理可能是：脑缺血引发钙内流导致ＧＬＵ过量释放,ＧＬＵ又通过突触前非ＮＭＤＡ受体激活ＰＫＣ,抑制其自身摄取,正反馈性加重胞外ＧＬＵ的堆积。     

大鼠卵巢绒毛膜促性腺激素受体在CHO中的表达和扩增

本文报道了利用二氢叶酸还原酶放大系统将大鼠ＬＨ／ｈＣＧ受体（记为Ｆ－ｈＣＧＲ）及其胞外肽段（记为Ｔ－ｈＣＧＲ）在中国苍鼠卵巢细胞（ＣＨＯ）中的表达。ＳＤＳ－ＰＡＧＥ分析表明,Ｆ－ｈＣＧＲ为一条蛋白质带,其表观分子量为９２ｋｄ,而Ｔ－ｈＣＧＲ为３５ｋｄ和３７ｋｄ两条带。表达受体对其配基ｈＣＧ表现出高的亲合力,Ｆ－ｈＣＧＲ的解离常数为７×１０－９ｍｏｌ／Ｌ,Ｔ－ｈＣＧＲ为６．４×１０－９ｍｏｌ／Ｌ。表达Ｆ－ｈＣＧＲ的转染ＣＨＯ细胞可结合１２５Ｉ－ｈＣＧ,而表达Ｔ－ｈＣＧＢ者不结合１２５Ｉ－ｈＣＧ。这提示Ｆ－ｈＣＧＲ主要存在于细胞质膜表面上。表达Ｆ－ｈＣＧＲ的转染ＣＨＯ细胞能刺激。ｃＡＭＰ的形成,而表达Ｔ－ｈＣＧＲ者不能刺激ｃＡＭＰ形成。免疫荧光定位结果表明,Ｔ－ｈＣＧＲ主要分布于质膜的细胞质侧以及胞内其他一些细胞器膜上。用免疫亲和层析可以得到纯化的Ｔ－ｈＣＧＲ。     

N42－A对神经生长因子诱导PC12细胞分化的抑制作用

研究表明,缺乏神经生长因子（ＮＧＦ）的营养支持是Ａｌｚｈｅｉｍｅｒ＇ｓ等神经元退行性疾病发生发展的重要原因,而ＮＧＦ和／或ＮＧＦ受体的过度表达则与一些神经系统肿瘤的发生发展有着十分密切的因果关系。采用（１２５）Ⅰ－ＮＧＦ受体特异结合实验作为ＮＧＦ受体活性物质筛选实验模型从中药牛膝中筛选出了能强烈地抑制（１２５）Ⅰ－ＮＧＦ受体结合的活性成分Ｎ４２－Ａ（ⅠＣ（５０）＝６．１８±３．４３,ｎ＝４）;细胞培养实验表明,Ｎ４２－Ａ对ＮＧＦ诱导大鼠嗜铬神经瘤ＰＣｌ２细胞的分化也具有很强的剂量依赖性抑制作用（对０．１ｎｍｏｌ／Ｌ和０．２ｎｍｏｌ／ＬＮＧＦ诱导的大鼠嗜铬神经病ＰＣ１２细胞轴突生长的半数抑制浓度分别为６μｇ／ｍＬ和２１μｇ／ｍＬ）。这表明,Ｎ４２－Ａ是神经元上介导ＮＧＦ诱导轴突生长的特异受体抑制剂,不仅对ＮＧＦ及其受体过度表达所致的神经系统肿瘤的防治具有潜在的应用价值,而且对Ａｌｚｈｅｉｍｅｒ＇ｓ等神经元退行性疾病防治药物的开发研究具有十分重要的意义。     

I型胶原对巨噬细胞摄取氧化低密度脂蛋白的作用

为探讨胶原的存在对细胞摄取氧化低密度脂蛋白（ｏｘ－ＬＤＬ）的影响,本研究在体外制成Ｉ型胶原凝胶和巨噬细胞实验体系,ＬＤＬ经Ｃｕ＾２＋催化氧化,丙二醛（ＭＤＡ）及乙酰化修饰后,与胶原的结合能力明显增强,但４－羟基壬烯醛（ＨＮＥ）修饰的ＬＤＬ与胶原的结合能力反应不如天然ＬＤＬ。当小鼠腹腔巨噬细胞培养在胶原凝胶上时,其对ｏｘ－ＬＤＬ的摄取明显减少,这时大部分ｏｘ－ＬＤＬ为胶原凝胶所结合,如用细胞松弛素Ｄ     

动脉壁细胞氧化修饰极低密度脂蛋白

将正常极低密度脂蛋白（ｎ－ＶＬＤＬ）分别与动脉壁的三种主要细胞：巨噬细胞（Ｍφ）、内皮细胞（ＥＣ）、平滑肌细胞（ＳＭＣ）温育２４小时后,ＶＬＤＬ的ＴＢＡＴＳ值明显增高,琼脂糖电泳速度加快,溶血卵磷脂与卵磷脂（ＰＣ）的比值明显升高,载脂蛋白的Ｂ１００（ａｐｏＢ１００）发生降解,未见集中的降解区带,ａｐｏ遥相对含量在各组无明显变化,加入抗氧化剂２,６－叔丁基甲苯能抑制上述变化,这些结果表明：动脉壁的三     

天然及氧化修饰脂蛋白对人动脉平滑肌细胞原癌基因表达的影响

动脉平滑肌细胞（ＳＭＣ）的增殖在动脉粥样硬化（ＡＳ）的形成过程中极其重要。我们在建立人主动脉ＳＭＣ体外培养方法的基础上,观察了ＬＤＬ,ＶＬＤＬ及ＨＤＬ和相应的氧化修饰型脂蛋白对培养人ＳＭＣｓｉｓ,ｊｕｎ,Ｈ－ｒａｓ原癌基因及Ｒｂ抗癌基因转录表达的影响。结果表明：（１）ＨＤＬ对ＳＭＣｓｉｓ,ｊｕｎ,ｒａｓ基因表达无影响;（２）ＬＤＬ和ＶＬＤＬ有使这些基因表达增加的趋势;（３）ｏｘ－ＬＤＬ,ｏｘ－ＶＬＤＬ和ｏｘ－ＨＤＬ具有使ＳＭＣｓｉｓ,ｊｕｎ,和ｒａｓ基因表达显著增强的作用（Ｐ＜０．０１）,且其作用较相应的天然脂蛋白大（Ｐ＜０．０１）;（４）天然和氧化修饰型脂蛋白对Ｒｂ基因表达均无影响。据上述结果推测：ＬＤＬ,ＶＬＤＬ,ｏｘ－ＬＤＬ,ｏｘ－ＶＬＤＬ和ｏｘ－ＨＤＬ的致ＡＳ作用可能与刺激ＳＭＣｓｉｓ,ｊｕｎ和ｒａｓ原癌基因表达增加有关。     

Uptake of type IV hypertriglyceridemic VLDL by cultured macrophages is enhanced by interferon-gamma.

Hypertriglyceridemic (HTG) very low density lipoproteins (VLDL) from subjects with type IV hyperlipoproteinemia induce both cholesteryl ester (CE) and triglyceride (TG) accumulation in cultured J774 macrophages. We examined whether the cytokine interferon-gamma (IFN-gamma), which is expressed by lymphocytes in atherosclerotic lesions, would modulate macrophage uptake of HTG -VLDL. Incubation of cells with HTG -VLDL alone significantly increased cellular CE and TG mass 17- and 4.3-fold, respectively, while cellular free cholesterol (FC) was unaffected. Pre-incubation of cells with IFN-gamma (50 U/ml) prior to incubation with HTG -VLDL caused a marked enhancement in cellular CE and TG 27- and 6-fold over no additions (controls), respectively, and a 1.5-fold increase in FC. IFN-gamma increased low density lipoprotein (LDL)-induced cellular CE 2-fold compared to LDL alone. IFN-gamma did not enhance the uptake of type III (apoE2/E2) HTG -VLDL or VLDL from apoE knock-out mice. Incubations in the presence of a lipoprotein lipase (LPL) inhibitor or an acylCoA:cholesterol acyltransferase (ACAT) inhibitor demonstrated that the IFN-gamma-enhanced HTG -VLDL uptake was dependent on LPL and ACAT activities. IFN-gamma significantly increased the binding and degradation of 125I-labeled LDL. Binding studies with 125I-labeled alpha2-macroglobulin, a known LDL receptor-related protein (LRP) ligand, and experiments with copper-oxidized LDL indicated that the IFN-gamma-enhanced uptake was not due to increased expression of the LRP or scavenger receptors. Thus, IFN-gamma may promote foam cell formation by accelerating macrophage uptake of native lipoproteins. IFN-gamma-stimulated CE accumulation in the presence of HTG -VLDL occurs via a process that requires receptor binding-competent apoE and active LPL. IFN-gamma-enhanced uptake of both HTG -VLDL and LDL is mediated by the LDL-receptor and requires ACAT-mediated cholesterol esterification.     

巨噬细胞对正常人极低密度脂蛋白亚组分代谢的研究

本文研究了小鼠腹腔巨噬细胞对正常人极低密度脂蛋白(N-VLDL)两种亚组分VLDL_1和VLDL_3的代谢。两种亚组分都能以受体方式和非特异性方式被巨噬细胞摄取和降解。在受体途径中以VLDL_1的摄入量居多。对胞内甘油三酯(TG)的堆积作用以VLDL_1较强,对胆固醇酶(CE)的堆积则以VLDL_3较强。表明两者在促进巨噬细胞向泡沫细胞转变中的作用有所不同。     

Differential effects of low-density lipoprotein and chylomicron remnants on lipid accumulation in human macrophages

The effects of low-density lipoprotein (LDL) and chylomicron remnants on lipid accumulation in human monocyte-derived macrophages (HMDMs) and in macrophages derived from the human monocyte cell line THP-1 were compared. The HMDMs or THP-1 macrophages were incubated with LDL, oxidized LDL (oxLDL), chylomicron remnant-like particles (CMR-LPs), or oxidized CMR-LPs (oxCMR-LPs), and the amount and type of lipid accumulated were determined. As expected, the lipid content of both cell types was increased markedly by oxLDL but not LDL, and this was due to a rise in cholesterol, cholesteryl ester (CE), and triacylglycerol (TG) levels. In contrast, both CMR-LPs and oxCMR-LPs caused a considerable increase in cellular lipid in HMDMs and THP-1 macrophages, but in this case there was a greater rise in the TG than in the cholesterol or CE content. Lipid accumulation in response to oxLDL, CMR-LPs, and oxCMR-LPs was prevented by the ACAT inhibitor CI976 in HMDMs but not in THP-1 macrophages, where TG levels remained markedly elevated. The rate of incorporation of [(3)H]oleate into CE and TG in THP-1 macrophages was increased by oxLDL, CMR-LPs, and oxCMR-LPs, but incorporation into TG was increased to a greater extent with CMR-LPs and oxCMR-LPs compared with oxLDL. These results demonstrate that both CMR-LPs and oxCMR-LPs cause lipid accumulation in human macrophages comparable to that seen with oxLDL and that oxidation of the remnant particles does not enhance this effect. They also demonstrate that a greater proportion of the lipid accumulated in response to CMR-LPs compared with oxLDL is TG rather than cholesterol or CE and that this is associated with a higher rate of TG synthesis. This study, therefore, provides further evidence to suggest that chylomicron remnants have a role in foam cell formation that is distinct from that of oxLDL.     

Glycated phosphatidylethanolamine promotes macrophage uptake of low density lipoprotein and accumulation of cholesteryl esters and triacylglycerols.

Non-enzymatic glycation of low density lipoprotein (LDL) has been suggested to be responsible for the increase in susceptibility to atherogenesis of diabetic individuals. Although the association of lipid glycation with this process has been investigated, the effect of specific lipid glycation products on LDL metabolism has not been addressed. This study reports that glucosylated phosphatidylethanolamine (Glc-PtdEtn), the major LDL lipid glycation product, promotes LDL uptake and cholesteryl ester (CE) and triacylglycerol (TG) accumulation by THP-1 macrophages. Incubation of THP-1 macrophages at a concentration of 100 micrograms/ml protein LDL specifically enriched (10 nmol/mg LDL protein) with synthetically prepared Glc-PtdEtn resulted in a significant increase in CE and TG accumulation when compared with LDL enriched in non-glucosylated PtdEtn. After a 24-h incubation with LDL containing Glc-PtdEtn, the macrophages contained 2-fold higher CE (10.11 +/- 1.54 micrograms/mg cell protein) and TG (285.32 +/- 4.38 micrograms/mg cell protein) compared with LDL specifically enriched in non-glucosylated PtdEtn (CE, 3.97 +/- 0.95, p < 0.01 and TG, 185.57 +/- 3.58 micrograms/mg cell protein, p < 0.01). The corresponding values obtained with LDL containing glycated protein and lipid were similar to those of LDL containing Glc-PtdEtn (CE, 11.9 +/- 1.35 and TG, 280.78 +/- 3.98 micrograms/mg cell protein). The accumulation of both neutral lipids was further significantly increased by incubating the macrophages with Glc-PtdEtn LDL exposed to copper oxidation. By utilizing the fluorescent probe, 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate (DiI), a 1.6-fold increase was seen in Glc-PtdEtn + LDL uptake when compared with control LDL. Competition studies revealed that acetylated LDL is not a good competitor for DiI Glc-PtdEtn LDL (5-6% inhibition), whereas glycated LDL gave an 80% inhibition, and LDL + Glc-PtdEtn gave 93% inhibition of uptake by macrophages. These results indicate that glucosylation of PtdEtn in LDL accounts for the entire effect of LDL glycation on macrophage uptake and CE and TG accumulation and, therefore, the increased atherogenic potential of LDL in hyperglycemia.     

巨噬细胞极低密度脂蛋白受体的调节作用

本文研究了小鼠腹腔巨噬细胞极低密度脂蛋白(VLDL)受体的调节。用富含甘油三酯(TG)的VLDL与小鼠巨噬细胞预温育后,细胞结合~(125)I-VLDL的最大结合容量(Bmax)比对照细胞只降低5％(1071/1127ng/mg细胞蛋白质),当细胞内TG增加到对照的2.5倍时,细胞摄取及降解~(125)I-VLDL的量分别下降40％和22％;乙酰-低密度脂蛋白(AC-LDL)预温育的细胞结合~(125)I-VLDL的Bmax比对照细胞只降低14％(633/831ng/mg细胞蛋白质),当细胞内胆固醇(Ch)增加到对照的23倍时,细胞摄取及降解~(125)I-VLDL的量只分别下降10％和21％。此后随着细胞内TG或Ch含量的增加,摄取及降解~(125)I-VLDL的量仍保持不变。 结论:细胞内TG或Ch含量对VLDL受体的调节作用微弱,与TG相比,Ch的调节作用更弱。     

Elevated triglyceride content diminishes the capacity of high density lipoprotein to deliver cholesteryl esters via the scavenger receptor class B type I (SR-BI)

The selective uptake of high density lipoprotein (HDL) cholesteryl ester (CE) by the scavenger receptor class B type I (SR-BI) is well documented. However, the effect of altered HDL composition, such as occurs in hyperlipidemia, on this important process is not known. This study investigated the impact of variable CE and triglyceride (TG) content on selective uptake. CE selective uptake by Y1 and HepG2 cells was strongly affected by modification of either the CE or TG content of HDL. Importantly, TG, like CE, was selectively taken up by a dose-dependent, saturable process in these cells. As shown by ACTH up-regulation and receptor overexpression experiments, SR-BI mediated the selective uptake of both CE and TG. With in vitro modified HDLs of varying CE and TG composition, the selective uptake of CE and TG was dependent on the abundance of each lipid within the HDL particle. Furthermore, total selective uptake (CE + TG) remained constant, indicating that these lipids competed for cellular uptake. These data support a novel mechanism whereby SR-BI binds HDL and mediates the incorporation of a nonspecific portion of the HDL lipid core. In this way, TG directly affects the ability of HDL to donate CE to cells. Processes that raise the TG/CE ratio of HDL will impair the delivery of CE to cells via this receptor and may compromise the efficiency of sterol balancing pathways such as reverse cholesterol transport.     

Possible role for intracellular cholesteryl ester transfer protein in adipocyte lipid metabolism and storage

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester (CE) and triglyceride (TG) between lipoproteins in plasma. However, short term suppression of CETP biosynthesis in cells alters cellular cholesterol homeostasis, demonstrating an intracellular role for CETP as well. The consequences of chronic CETP deficiency in lipid-storing cells normally expressing CETP have not been reported. Here, SW872 adipocytes stably expressing antisense CETP cDNA and synthesizing 20% of normal CETP were created. CETP-deficient cells had 4-fold more CE but an approximately 3-fold decrease in cholesterol biosynthesis. This phenotype of cholesterol overload is consistent with the observed 45% reduction in low density lipoprotein receptor and 2.5-fold increase in ABCA1 levels. However, cholesterol mass in CETP-deficient adipocytes was actually reduced. Strikingly, CETP-deficient adipocytes stored <50% of normal TG, principally reflecting reduced synthesis. The hydrolysis of cellular CE and TG in CETP-deficient cells was reduced by >50%, although hydrolase/lipase activity was increased 3-fold. Notably, the incorporation of recently synthesized CE and TG into lipid storage droplets in CETP-deficient cells was just 40% of control, suggesting that these lipids are inefficiently transported to droplets where the hydrolase/lipase resides. The capacity of cellular CETP to transport CE and TG into storage droplets was directly demonstrated in vitro. Overall, chronic CETP deficiency disrupts lipid homeostasis and compromises the TG storage function of adipocytes. Inefficient CETP-mediated translocation of CE and TG from the endoplasmic reticulum to their site of storage may partially explain these defects. These studies in adipocytic cells strongly support a novel role for CETP in intracellular lipid transport and storage.     

Lipid biosynthesis and metabolism of native and acetylated low density lipoproteins in macrophages stimulated by zymosan in vivo and in vitro]

The effects of zymosan on lipid metabolism in mouse peritoneal macrophages (MPM) in vitro and in vivo were studied with special reference to the following parameters: i) 14C-oleate incorporation into cholesteryl esters (CE), triglycerides (TG), and phospholipids (PL) in MPM incubated with low density lipoproteins (LDL) and acetylated LDL; ii) cholesteryl-14C-oleate-acetyl LDL uptake and 125I-acetyl LDL degradation; iii) oxidative modification of LDL. Zymosan administered to mice caused significant stimulation of 14C-oleate incorporation into CE, TG, and PL with no effect on 3H-cholesterol (Ch) incorporation into CE or 3H-glycerol incorporation into TG and PL in MPM. The 14C-oleate incorporation into cellular lipids was unaffected by 18-hour incubation of MPM with zymosan (100-500 micrograms/ml) but increased after incubation of unstimulated MPM with blood serum and peritoneal fluid harvested harvested from zymosan-treated mice. One possible explanation of this phenomenon is oleyl-CoA formation induction in cytokine-stimulated MPM in vivo. Zymosan decreased the Ch-14C-oleate-acetyl LDL uptake, 125I-acetyl LDL degradation, and Ch esterification in the presence of acetyl LDL in MPM both in vitro and in vivo. An increase in Ch esterification after incubation of MPM with zymosan for 6-18 hours in the presence of LDL was accompanied by an increase in lipid peroxidation of LDL and its electrophoretic mobility. The data obtained suggest that the macrophage acetyl LDL receptor pathway may be inhibited by zymosan and that cytokines released from zymosan-stimulated cells may influence the generation of foam cells.     

Regulation of LTP-I secretion from human monocyte-derived macrophages by differentiation and cholesterol accumulation in vitro

Human macrophages in vitro synthesize and secrete the cholesteryl ester (CE) transfer protein, LTP-I. The effect of differentiation of monocyte-to-macrophage on the synthesis and secretion of LTP-I cholesteryl ester transfer activity was investigated. One marker of macrophage differentiation is expression of the 'scavenger' receptor, which mediates macrophage uptake and degradation of acetylated low-density lipoprotein. Monocytes secreted very little detectable CE transfer activity in the first 24 h following cell isolation. Both CE transfer activity and scavenger receptor activity increased with time in culture. Thus, although circulating monocytes probably do not secrete CE transfer activity, tissue macrophages such as hepatic Kupffer cells may contribute to plasma CE transfer activity. Resident macrophages of the arterial wall are derived from circulating monocytes which enter the vessel wall where they differentiate into macrophages. Such macrophages are the principal source of lipid-laden foam cells of the atherosclerotic plaque. Cholesterol accumulation results when uptake of lipoprotein cholesterol overwhelms the capacity of macrophages to excrete cholesterol. Since LTP-I is postulated to function in reverse cholesterol transport, the effect on LTP-I secretion of loading macrophages with cholesterol was determined after exposure of macrophages to acetylated-LDL or free cholesterol (FC). Cholesterol loading by both these maneuvers resulted in dose-dependent increases in macrophage secretion of CE transfer activity, and there was a significant positive correlation between CE transfer activity secreted and accumulation of CE. Thus, LTP-I may function at the cellular level in maintenance of lipid homeostasis: macrophage LTP-I secretion may be a protective mechanism in response to excess cholesterol accumulation in resident macrophages of the arterial wall.     

Negatively cooperative binding of high-density lipoprotein to the HDL receptor SR-BI

Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 μg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.