Department of Ophthalmology, Eye Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226-4812, USA.
Invest Ophthalmol Vis Sci. 2013 Mar 28;54(3):2276-87. doi: 10.1167/iovs.12-11154.
To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis.
ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and β5) and β-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvβ5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy.
Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and β5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type.
After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.
确定 ARPE-19 细胞先前表现出的光动力（PD）诱导的特定光感受器外节（POS）吞噬作用抑制是否与介导 POS 吞噬作用的受体蛋白减少有关，以及 PD 治疗是否用美拉明 540（MC-540）产生导致其抑制非特异性吞噬作用的其他作用。
用 MC-540 或孟加拉玫瑰红（RB）预加载的 ARPE-19 细胞用绿光亚致死照射。通过流式细胞术测量 POS 的吞噬作用，并在照射后 0.5 和 24 小时通过 Western 印迹定量测量 POS 受体蛋白（Mer 酪氨酸激酶受体[MerTK]和整合素亚基αv和β5）和β-肌动蛋白，并与未敏化对照培养物的样品进行比较。通过免疫沉淀 followed by blotting 定量测定完整的整合素异二聚体αvβ5。通过荧光显微镜观察 N-钙粘蛋白、ZO-1 和 F-肌动蛋白的分布。
两种光敏剂介导的轻度 PD 应激产生没有明显形态变化的短暂和可恢复的 MerTK 减少。单独的αv 和β5 整合素亚基也减少，但仅部分恢复。然而，有足够的恢复来支持功能性异二聚体的完全恢复。MC-540 介导的光应激也降低了肌动蛋白的水平，肌动蛋白已知参与无论类型的颗粒内化。
PD 治疗后，POS 受体蛋白丰度和吞噬作用同时减少然后恢复，表明受体蛋白减少有助于吞噬缺陷。MC-540 介导的应激对非特异性吞噬作用的额外抑制可能是由于对胞质蛋白的更广泛影响。数据表明，RPE 细胞中的吞噬作用受体对氧化修饰敏感，这增加了慢性原位氧化应激可能降低 RPE 在光感受器更替中的作用效率的可能性，从而导致视网膜变性。
