TGF-β Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells

To characterize transforming growth factor-beta (TGF-beta) isoform (TGF-beta 1 similar to 3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including collagen 1 (COL1), COL4, and COL6; alpha-smooth muscle actin (alpha SMA); hypoxia-inducible factor 1 alpha (HIF1 alpha); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1 alpha), a master regulator for mitochondrial respiration;, tight junction-related molecules, Zonula occludens-1 (ZO1) and E-cadherin; and vascular endothelial growth factor (VEGF); (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-beta isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-beta 1 and TGF-beta 3 caused a marked increase in TEER values, and TGF-beta 2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-beta isoforms, among which TGF-beta 1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-beta 2 and TGF-beta 3 or by TGF-beta 1 and TGF-beta 3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-beta isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-beta-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-beta isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.