Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Division of Vision Research, National Institute of Sensory Organs, National Hospital Organization Tokyo Medical Center, Tokyo, Japan.
Mol Vis. 2021 Jan 20;27:61-77. eCollection 2021.
The effects of aqueous mediators possibly increasing the outflow resistance, transforming growth factor-β1 (TGF-β1), TGF-β2, autotaxin (ATX), and lysophosphatidic acid (LPA) on human trabecular meshwork (hTM) cells and monkey Schlemm's canal endothelial (SCE) cells were characterized and compared, and the effects of intracameral application of these mediators on intraocular (IOP) elevation were also examined.
Cells were treated with TGF-β1, TGF-β2, ATX, LPA, or vehicle, and mRNA and protein expression levels of α-SMA, COL1A1, fibronectin, β-catenin, and ZO-1 were examined with real-time quantitative PCR (RT-qPCR) or immunofluorescence analyses or both. The permeability of cell monolayers was measured by determining the transendothelial electrical resistance (TEER) or with the fluorescein isothiocyanate (FITC)-dextran permeability assay. IOP was evaluated in rabbit eyes after intracameral administration of the mediators.
All mediators induced upregulation of α-SMA, COL1A1, and fibronectin in hTM cells. The effect of TGF-β2 on mRNA expression of fibrotic markers was statistically significantly greater than that of TGF-β1. The effects of ATX and LPA indicated the time-dependent difference in the upregulation of α-SMA, COL1A1, and fibronectin. The TEER and FITC-dextran permeability of the SCE cells was evaluated after treatment with TGF-β1 and TGF-β2, but no statistically significant change was observed within 24 h. ATX and LPA also reduced permeability statistically significantly after 3 h and 0.5 h, respectively, and the effect of LPA was more rapid compared to that of ATX. Statistically significant IOP elevation was observed in rabbit eyes as early as 0.5-2.0 h after ATX and LPA treatment and at 24 h after treatment with TGF-β2.
TGF-β2 and ATX and LPA regulate aqueous outflow by modulation of hTM cells and SCE cells, and differences in timing between the effects of each mediator were observed. ATX and LPA showed more rapid effects on IOP elevation than TGF-β2. It was suggested that TGF-β2 and ATX/LPA are involved in increases of IOP, but the timing and sustainability differ between mediators, and they may play specific roles in different glaucoma subtypes.
研究可能增加房水流出阻力的水性介质转化生长因子-β1（TGF-β1）、TGF-β2、自分泌酶（ATX）和溶血磷脂酸（LPA）对人眼小梁细胞（hTM）和猴氏管内皮细胞（SCE）的作用，并进行比较，同时观察这些介质在房内应用对眼内压（IOP）升高的影响。
用 TGF-β1、TGF-β2、ATX、LPA 或对照处理细胞，用实时定量 PCR（RT-qPCR）或免疫荧光分析或两者结合的方法检测α-SMA、COL1A1、纤维连接蛋白、β-连环蛋白和 ZO-1 的 mRNA 和蛋白表达水平。通过测定跨内皮电阻（TEER）或用荧光素异硫氰酸酯（FITC）-葡聚糖通透性测定法来测量细胞单层的通透性。在兔眼内给予这些介质后，评估眼内压。
所有介质均诱导 hTM 细胞中α-SMA、COL1A1 和纤维连接蛋白的上调。TGF-β2 对纤维化标志物的 mRNA 表达的影响明显大于 TGF-β1。ATX 和 LPA 的作用表明，α-SMA、COL1A1 和纤维连接蛋白的上调存在时间依赖性差异。用 TGF-β1 和 TGF-β2 处理 SCE 细胞后，评估 TEER 和 FITC-葡聚糖通透性，但 24 h 内无统计学显著变化。ATX 和 LPA 分别在 3 h 和 0.5 h 后统计学显著降低通透性，且 LPA 的作用比 ATX 更快。ATX 和 LPA 处理后，兔眼的眼内压早在 0.5-2.0 h 就明显升高，TGF-β2 处理后 24 h 升高。
TGF-β2 和 ATX/LPA 通过调节 hTM 细胞和 SCE 细胞来调节房水流出，并且观察到每种介质作用之间的时间差异。ATX 和 LPA 对眼压升高的作用比 TGF-β2 更快。提示 TGF-β2 和 ATX/LPA 参与了眼压升高，但不同介质之间的时间和可持续性不同，它们可能在不同的青光眼亚型中发挥特定作用。
