Novel NADPH oxidase inhibitor VAS2870 suppresses TGF‑β‑dependent epithelial‑to‑mesenchymal transition in retinal pigment epithelial cells

Abstract

NADPH oxidases (NOXs) are important in the pathophysiology of fibrotic diseases. The expression and activity of NOXs are regulated by growth factors, including transforming growth factor (TGF-β). The proliferation of retinal pigment epithelial (RPE) cells following epithelial- to-mesenchymal transition (EMT) is a major pathological change involved in proliferative vitreoretinopathy (PVR). The aim of the present study was to determine the effects of the novel NOX inhibitor VAS2870 on the TGF-β-dependent expression of NOX4 and associated cellular events in RPE cells. Cell viability was examined using a Cell Counting Kit-8 assay and cell cycle progression was detected by flow cytometric analysis. Immunofluorescence analysis and western blot analysis were performed to assess EMT. It was found that TGF-β increased the expression of NOX4 and that pre-incubation with VAS2870 eliminated this effect. Additionally, TGF-β promoted RPE migration and increased EMT. Pre-incubation with VAS2870 significantly prevented TGF-β2-induced EMT by decreasing the levels of α-smooth muscle actin and E-cadherin, and also inhibited the migratory ability of the RPE cells, as demonstrated by scratch assays. Finally, VAS2870 suppressed the proliferation of RPE cells, and led to G1-phase cell cycle arrest and a significant downregulation of the expression of cyclin D1. In conclusion, the pharmacological inhibition of NOX may be a promising tool for the treatment of PVR.

On the Clinical Pharmacology of Reactive Oxygen Species

“…Evidence in eye disease has been preclinically published in retinopathy of prematurity (Deliyanti and Wilkinson-Berka, 2015) but could very likely be extended to diabetic retinopathy. VAS2870, a NOX4 inhibitor from Vasopharm (claimed in WO/2005/ 111041), has recently been reported to inhibit the proliferation of retinal pigment epithelial cells after their epithelial-to-mesenchymal transition, a significant pathologic change involved in proliferative vitreoretinopathy (Yang et al, 2018). The Swedish company Glucox Biotech AB is also focused on NOX4 inhibitors and is reported to be looking into diabetes, Marfan syndrome, stroke, and cancer.…”

Blockade of mTORC1‐NOX signaling pathway inhibits TGF‐β1‐mediated senescence‐like structural alterations of the retinal pigment epithelium

“…Meanwhile, the upregulation of NOX4 is an essential mechanism underlying TGF‐β1‐induced oxidative stress, leading to EMT of the RPE 26 . Several clinical trials have tested the effect of using NOX inhibitors to treat chronic inflammatory disease and fibrotic disease in many organs, including the retina and RPE 20,23,41 . In this study, we showed that AICAR and GKT137831 protected against structural alterations in the RPE tissue induced by intravitreal TGF‐β1 injection in vivo and by incubating ARPE‐19 cells with TGF‐β1 in vitro.…”

“…Therefore, we require attention that this model is not a disease model but more suitable as a screening model for AMD therapeutics. Furthermore, there have already been many reports to examine not only the EMT‐inducing effects of TGF‐β in RPE cells, 18‐22 but also amelioration of TGF‐β1‐induced EMT and fibrosis by AICAR and GKT137831 in many organs 23,28,44‐46 . However, to our knowledge, this is the first study to investigate morphometric abnormalities and EMT of the RPE in vivo.…”

“…Intracellular cytoskeletal rearrangement and ECM alteration induced by TGF‐β signaling leads to a decrease in structural integrity and morphometric changes in RPE cells 17 . Thus far, researchers have mainly performed in vitro experiments to examine the EMT‐inducing effects of TGF‐β1 or TGF‐β2 in RPE cells 18‐22 . Among the TGF‐β‐related signal regulators, mammalian target of rapamycin C1 (mTORC1) and NADPH oxidase (NOX) have been reported to be involved in cellular senescence and EMT 23‐25 .…”

VAS2870 Inhibits Histamine-Induced Calcium Signaling and vWF Secretion in Human Umbilical Vein Endothelial Cells

“…VAS2870 in micromolar concentrations is widely used to suppress NOX activity in different types of cells [42,43,44,45]. The IC50 for VAS2870’s effect on NOX activity in a cell-free system with membranes and cytosol from human neutrophils was 10.6 μM, and in intact HL-60 cells the IC50 was 2 μM [43].…”