Siloam Eye Hospital, Seoul, Korea.
Invest Ophthalmol Vis Sci. 2013 Nov 8;54(12):7329-37. doi: 10.1167/iovs.13-12149.
Cigarette smoking (CS) is the most consistent risk factor for advanced age-related macular degeneration (AMD). To verify the molecular basis for CS-induced RPE alterations, RPE cell survival levels after being exposed to CS in relation with VEGF expression and autophagic flux were evaluated.
Cigarette smoking extract (CSE) was added to ARPE-19 cells and hydrogen peroxide (HP) was used as a pure oxidant control. Cell survival was measured by flow cytometry with annexin V-fluorescein isothiocyanate. Cell survival analysis was performed after pretreatment with anti-VEGF or recombinant VEGF. The expression of VEGF-A, VEGF-R1/R2, and soluble VEGF-R1 was determined by semiquantitative RT-PCR. LC3B-I (microtubule-associated protein-1 inhibitors), LC3B-II, and phosphorylation of Akt or Erk were measured with Western blot. Autophagic flux was determined by increasing LC3B-II levels with inhibitors of lysosomal proteases.
Incubation with 5% CSE for 16 hours induced approximately 30% cell death, which was similar to cell death levels when exposed to concentrations of 200 μM HP. Pretreatment with anti-VEGF did not decrease cell survival under CSE, unlike the decrease in cell survival shown with HP. However, supplementation with VEGF rescued CSE-induced RPE cell death. Interestingly, CSE caused an increase in autophagic flux, which was augmented with VEGF pretreatment. Cigarette smoking extract also degraded the total amounts of Akt levels, and VEGF blunted CSE-induced phosphorylation of Erk.
Cigarette smoking extract, similar to HP, affects cell viability and induces expression of VEGF and its receptors. Increased autophagic flux accelerated by treatment of exogenous VEGF may have a role in rescuing CSE-induced RPE cell death.
吸烟是导致年龄相关性黄斑变性（AMD）的最常见的危险因素。为了验证吸烟引起 RPE 改变的分子基础，评估了暴露于香烟烟雾提取物（CSE）后 RPE 细胞的存活水平与 VEGF 表达和自噬流的关系。
将 CSE 添加到 ARPE-19 细胞中，并将过氧化氢（HP）用作纯氧化剂对照。用 Annexin V-异硫氰酸荧光素通过流式细胞术测量细胞存活率。在用抗 VEGF 或重组 VEGF 预处理后进行细胞存活率分析。通过半定量 RT-PCR 测定 VEGF-A、VEGF-R1/R2 和可溶性 VEGF-R1 的表达。用 Western blot 测定 LC3B-I（微管相关蛋白 1 抑制剂）、LC3B-II 和 Akt 或 Erk 的磷酸化。通过用溶酶体蛋白酶抑制剂增加 LC3B-II 水平来确定自噬流。
孵育 5% CSE 16 小时可诱导约 30%的细胞死亡，与暴露于 200 μM HP 时的细胞死亡水平相似。与 HP 一样，用抗 VEGF 预处理不能降低 CSE 下的细胞存活率。然而，VEGF 的补充挽救了 CSE 诱导的 RPE 细胞死亡。有趣的是，CSE 引起自噬流增加，用 VEGF 预处理可进一步增强。香烟烟雾提取物还降解了 Akt 的总量，VEGF 减弱了 CSE 诱导的 Erk 磷酸化。
与 HP 相似，香烟烟雾提取物会影响细胞活力并诱导 VEGF 及其受体的表达。用外源性 VEGF 处理可加速增加的自噬流，可能在挽救 CSE 诱导的 RPE 细胞死亡中起作用。
