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

ABSTRACT

To characterize transforming growth factor-ß (TGF-ß) isoform (TGF-ß1~3)-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; α-smooth muscle actin (αSMA); hypoxia-inducible factor 1α (HIF1α); and peroxisome proliferator-activated receptor-gamma coactivator (PGC1α), 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-ß isoforms were observed, and those effects were also different between normoxia and hypoxia conditions (1) TGF-ß1 and TGF-ß3 caused a marked increase in TEER values, and TGF-ß2 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-ß isoforms, among which TGF-ß1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-ß2 and TGF-ß3 or by TGF-ß1 and TGF-ß3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-ß isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-ß-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-ß isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.