Higashide Megumi, Watanabe Megumi, Sato Tatsuya, Ogawa Toshifumi, Umetsu Araya, Suzuki Soma, Furuhashi Masato, Ohguro Hiroshi, Nishikiori Nami
Departments of Ophthalmology, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
Departments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Chuo-ku, Sapporo 060-8556, Japan.
Biomedicines. 2024 Sep 30;12(10):2228. doi: 10.3390/biomedicines12102228.
To study the effects of all-trans retinoic acid (ATRA) on TGF-β2-induced effects of human retinal pigment epithelium cells under normoxia and hypoxia conditions. Two-dimensionally (2D) and three-dimensionally (3D) cultured ARPE19 cells were subjected to cellular functional analyses by transepithelial electrical resistance (TEER) and an extracellular flux assay (2D), measurement of levels of reactive oxygen species (ROS), gene expression analyses of , , , , and (2D), and physical property analyses (3D). Under a normoxia condition, treatment with 100 nM ATRA substantially decreased barrier function regardless of the presence of 5 ng/mL TGF-β2 in 2D ARPE19 monolayer cells. Under a hypoxia condition, treatment with ATRA conversely increased barrier function, but the effect was masked by a marked increase in effects induced by TGF-β2. Although ATRA alone did not affect cellular metabolism and ROS levels in 2D ARPE cells, treatment with ATRA under a hypoxia condition did not affect ROS levels but shifted cellular metabolism from mitochondrial respiration to glycolysis. The changes of cellular metabolism and ROS levels were more pronounced with treatment of both ATRA and TGF-β2 independently of oxygen conditions. Changes in mRNA expressions of some of the above genes suggested the involvement of synergistical regulation of cellular functions by TGF-β2 and hypoxia. In 3D ARPE spheroids, the size was decreased and the stiffness was increased by either treatment with TGF-β2 or ATRA, but these changes were unexpectedly modulated by both ATRA and TGF-β2 treatment regardless of oxygen conditions. The findings reported herein indicate that TGF-β2 and hypoxia synergistically and differentially induce effects in 2D and 3D cultured ARPE19 cells and that their cellular properties are significantly altered by the presence of ATRA.
研究全反式维甲酸（ATRA）在常氧和缺氧条件下对转化生长因子-β2（TGF-β2）诱导的人视网膜色素上皮细胞效应的影响。对二维（2D）和三维（3D）培养的ARPE19细胞进行跨上皮电阻（TEER）和细胞外通量分析（2D）、活性氧（ROS）水平测定、 、 、 、 和 的基因表达分析（2D）以及物理性质分析（3D），以进行细胞功能分析。在常氧条件下，在2D ARPE19单层细胞中，无论是否存在5 ng/mL TGF-β2，用100 nM ATRA处理都会显著降低屏障功能。在缺氧条件下，ATRA处理反而增加了屏障功能，但该效应被TGF-β2诱导的显著增加所掩盖。尽管单独使用ATRA不影响2D ARPE细胞的细胞代谢和ROS水平，但在缺氧条件下用ATRA处理不影响ROS水平，但将细胞代谢从线粒体呼吸转变为糖酵解。无论氧气条件如何，同时用ATRA和TGF-β2处理时，细胞代谢和ROS水平的变化更为明显。上述一些基因的mRNA表达变化表明TGF-β2和缺氧对细胞功能有协同调节作用。在3D ARPE球状体中，用TGF-β2或ATRA处理都会使球状体大小减小、硬度增加，但无论氧气条件如何，ATRA和TGF-β2联合处理意外地调节了这些变化。本文报道的研究结果表明，TGF-β2和缺氧在2D和3D培养的ARPE19细胞中协同且差异地诱导效应，并且ATRA的存在会显著改变它们的细胞特性。
