Nano-Graphene Oxide-Promoted Epithelial-Mesenchymal Transition of Human Retinal Pigment Epithelial Cells through Regulation of Phospholipase D Signaling.

Abstract

Nano-graphene oxide (Nano-GO) is an extensively studied multifunctional carbon nanomaterial with attractive applications in biomedicine and biotechnology. However, few studies have been conducted to assess the epithelial-to-mesenchymal transition (EMT) in the retinal pigment epithelium (RPE). We aimed to determine whether Nano-GO induces EMT by regulating phospholipase D (PLD) signaling in human RPE (ARPE-19) cells. The physicochemical characterization of Nano-GO was performed using a Zetasizer, X-ray diffraction, Fourier-transform infrared spectroscopy, and transmission electron microscopy. RPE cell viability assays were performed, and the migratory effects of RPE cells were evaluated. RPE cell collagen gel contraction was also determined. Intracellular reactive oxygen species (ROS) levels were determined by fluorescence microscopy and flow cytometry. Immunofluorescence staining and western blot analysis were used to detect EMT-related protein expression. Phospholipase D (PLD) enzymatic activities were also measured. Nano-GO significantly enhanced the scratch-healing ability of RPE cells, indicating that the RPE cell migration ability was increased. Following Nano-GO treatment, the RPE cell penetration of the chamber was significantly promoted, suggesting that the migratory ability was strengthened. We also observed collagen gel contraction and the generation of intracellular ROS in RPE cells. The results showed that Nano-GO induced collagen gel contraction and intracellular ROS production in RPE cells. Moreover, immunofluorescence staining and western blot analysis revealed that Nano-GO significantly regulated key molecules of EMT, including epithelial-cadherin, neural-cadherin, α-smooth muscle actin, vimentin, and matrix metalloproteinases (MMP-2 and MMP-9). Interestingly, Nano-GO-induced RPE cell migration and intracellular ROS production were abrogated in PLD-knockdown RPE cells, indicating that PLD activation played a crucial role in the Nano-GO-induced RPE EMT process. We demonstrate for the first time that Nano-GO promotes RPE cell migration through PLD-mediated ROS production. We provide preliminary evidence to support the hypothesis that Nano-GO has adverse health effects related to RPE damage.