Lu Jonathan, Yang Jun-Hai, Burns Alan R, Chen Hsin-Hung, Tang Daming, Walterscheid Jeffrey P, Suzuki Shinichi, Yang Chao-Yuh, Sawamura Tatsuya, Chen Chu-Huang
Department of Medicine, Baylor College of Medicine, Houston, TX 77030, USA.
Circ Res. 2009 Mar 13;104(5):619-27. doi: 10.1161/CIRCRESAHA.108.190116. Epub 2009 Jan 15.
The lectin-like oxidized LDL receptor LOX-1 mediates endothelial cell (EC) uptake of experimentally prepared copper-oxidized LDL (oxLDL). To confirm the atherogenic role of this receptor cloned against copper-oxLDL, we examined whether it mediates EC uptake of L5, an electronegative LDL abundant in dyslipidemic but not normolipidemic human plasma. Hypercholesterolemic (LDL-cholesterol, >160 mg/dL) human LDL was fractionated into L1-L5, increasingly electronegative, by ion-exchange chromatography. In cultured bovine aortic ECs (BAECs), L5 upregulated LOX-1 and induced apoptosis. Transfection of BAECs with LOX-1-specific small interfering RNAs (siLOX-1) minimized baseline LOX-1 production and restrained L5-induced LOX-1 upregulation. Internalization of labeled L1-L5 was monitored in BAECs and human umbilical venous ECs by fluorescence microscopy. LOX-1 knockdown with siLOX-1 impeded the endocytosis of L5 but not L1-L4. In contrast, blocking LDL receptor with RAP (LDL receptor-associated protein) stopped the internalization of L1-L4 but not L5. Although chemically different, L5 and oxLDL competed for EC entry through LOX-1. Via LOX-1, L5 signaling hampered Akt phosphorylation and suppressed EC expression of fibroblast growth factor-2 and Bcl-2. L5 also selectively inhibited Bcl-xL expression and endothelial nitric oxide synthase phosphorylation but increased synthesis of Bax, Bad, and tumor necrosis factor-alpha. Blocking Akt phosphorylation with wortmannin increased LOX-1 expression, suggesting a modulatory role of Akt in LOX-1 synthesis; L5 upregulated LOX-1 by dephosphorylating Akt. Because endothelial nitric oxide synthase and Bcl-2 activities are Akt-dependent, L5 impairs Akt-mediated growth and survival signals in vascular ECs by way of LOX-1. Thus, the L5/LOX-1 complex may play a critical role in atherogenesis and illuminate important targets for disease intervention.
凝集素样氧化型低密度脂蛋白受体1（LOX-1）介导内皮细胞（EC）摄取实验制备的铜氧化低密度脂蛋白（oxLDL）。为了证实这种针对铜氧化低密度脂蛋白克隆的受体的致动脉粥样硬化作用，我们研究了它是否介导EC摄取L5，L5是一种在血脂异常而非血脂正常的人类血浆中大量存在的带负电荷的低密度脂蛋白。将高胆固醇血症（低密度脂蛋白胆固醇，>160mg/dL）患者的低密度脂蛋白通过离子交换色谱法分离为L1-L5，其负电性逐渐增强。在培养的牛主动脉内皮细胞（BAEC）中，L5上调LOX-1并诱导细胞凋亡。用LOX-1特异性小干扰RNA（siLOX-1）转染BAEC可使基线LOX-1产生最小化，并抑制L5诱导的LOX-1上调。通过荧光显微镜在BAEC和人脐静脉内皮细胞中监测标记的L1-L5的内化情况。用siLOX-1敲低LOX-1可阻碍L5的内吞作用，但不影响L1-L4的内吞。相反，用RAP（低密度脂蛋白受体相关蛋白）阻断低密度脂蛋白受体可阻止L1-L4的内化，但不影响L5的内化。尽管L5和oxLDL在化学性质上不同，但它们通过LOX-1竞争进入EC。通过LOX-1，L5信号传导阻碍Akt磷酸化，并抑制成纤维细胞生长因子-2和Bcl-2在EC中的表达。L5还选择性抑制Bcl-xL表达和内皮型一氧化氮合酶磷酸化，但增加Bax、Bad和肿瘤坏死因子-α的合成。用渥曼青霉素阻断Akt磷酸化可增加LOX-1表达，提示Akt在LOX-1合成中起调节作用；L5通过使Akt去磷酸化上调LOX-1。由于内皮型一氧化氮合酶和Bcl-2的活性依赖于Akt，L5通过LOX-1损害血管内皮细胞中Akt介导的生长和存活信号。因此，L5/LOX-1复合物可能在动脉粥样硬化发生中起关键作用，并为疾病干预提供重要靶点。
