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

## TL;DR

This study shows how different TGF-β forms and oxygen levels affect human retinal cells in 2D and 3D models, influencing their function and structure.

## Contribution

The study reveals distinct biological effects of TGF-β isoforms on RPE cells under normoxia and hypoxia in 2D and 3D models.

## Key findings

- TGF-β1 and TGF-β3 significantly increased trans-epithelial electrical resistance (TEER) values in RPE cells.
- TGF-β isoforms reduced 3D spheroid size, with TGF-β1 being the most effective under both normoxia and hypoxia.
- TGF-β isoforms altered mitochondrial and glycolytic functions differently depending on oxygen levels and culture conditions.

## Abstract

To characterize transforming growth factor-β (TGF-β) isoform (TGF-β1~3)-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-β isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-β1 and TGF-β3 caused a marked increase in TEER values, and TGF-β2 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-β isoforms, among which TGF-β1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-β2 and TGF-β3 or by TGF-β1 and TGF-β3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-β isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-β-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-β isoforms and oxygen levels, suggesting that pathological conditions including epithelial–mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.