Abstract
Purpose:
The epithelial-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is associated with the formation of contractile fibrous membranes in proliferative vitreoretionopathy (PVR) and is a significant pathological factor in this disease. In this in vitro study, we investigated the effect of AMP-activated protein kinase (AMPK) on EMT induced in cultured human RPE (ARPE-19) cells.
Methods:
ARPE-19 cells in culture were stimulated with 10 ng/ml tumor necrosis factor (TNF)-α and 5 ng/ml transforming growth factor (TGF)-β2. The effect of 5-aminoimidazole-4-carboxamide (AICAR), an activator of AMPK, and dipridamole (DPY) and 5’-amino-5’-deoxyadenosine (AMDA), inhibitors of AMPK, on EMT of ARPE-19 cells was investigated. The expression of E-cadherin, fibronectin, matrix metalloproteinase (MMP)-2 and MMP-9 by ARPE-19 cells was determined by western blotting and enzyme-linked immunosorbent assay to investigate the mechanism involved in EMT and cell migration.
Results:
After 48 h co-stimulation with TNF-α and TGF-β2, ARPE-19 cells gathered together and formed cell aggregates. This cell aggregation was significantly suppressed by the activation of AMPK. The mean numbers of aggregates per field in control, co-stimulated and co-stimulated, AICAR-treated cultures were 0.0 ± 0.0, 21.0 ± 3.8 and 1.8 ± 2.8, respectively (p < 0.05). This suppressive effect of AICAR was significantly inhibited by pretreatment with DPY or AMDA. As ARPE-19 cells aggregated, E-cadherin expression was suppressed while fibronectin, MMP-2 and MMP-9 expression increased. AMPK activation by AICAR significantly inhibited these changes in the expression of these proteins.
Conclusions:
AMPK activation suppressed EMT of ARPE-19 cells, suggesting novel lines of research for new therapies for PVR.