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Hua He, Ajay E. Kuriyan, Chen-Wei Su, Yuan Zhang, Megha Mahabole, Ying-Ting Zhu, Esdras A. Quintero, Nidhi Rehlan, Harry W Flynn, Jean-Marie A Parel, Scheffer C G Tseng; Inhibition of Proliferation and Epithelial Mesenchymal Transition via Wnt and TGF-β Signaling Pathway in an in vitro Cell Culture Based-PVR Model by HC-HA/PTX3 Purified from Amniotic Membrane. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5384. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) is the main cause of failure of rhegmatogenous retinal detachments and characterized by proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells under the influence of vitreous growth factors. Recently, we have purified a heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) complex from human amniotic membrane (AM) which retains AM’s anti-inflammatory, anti-scarring, and anti-angiogenic activity. Herein, we investigate whether HC-HA/PTX3 can prevent PVR by inhibiting proliferation and EMT of RPE cells in vitro.
EGF(10 ng/ml) and FGF-2 (10 ng/ml) or together with TGF-β were used to induce proliferation and EMT in human ARPE-19 cells with or without HC-HA/PTX3. The cell viability was determined by MTT and WST-1, the proliferation was measured by BrdU ELISA, WST-1 assay as well as BrdU and β-catenin immunostaining, while EMT was examined by immunostaining of phosphorylated Smad2/3 and a-smooth muscle actin. The migration and collagen gel contraction in ARPE-19 and human primary RPE cells were also assayed.
We have established an in vitro cell culture-based PVR model by defining the cell density, growth factors, and measurement methods. HC-HA/PTX3 dose-dependently inhibited proliferation and EMT of RPE cells stimulated by EGF/FGF-2 and TGF-βs, respectively, without any cytotoxic effect on the normal RPE cells. In addition, HC-HA/PTX3 and HA inhibited migration of EGF+FGF-2+TGF-β1 stimulated ARPE-19 cells but HC-HA/PTX3 but not HA inhibited collagen gel contraction in both primary human RPE and cell line ARPE-19 cells. The inhibition of proliferation of EMT by HC-HA/PTX3 is mediated by down-regulation of canonical signaling pathways of Wnt (β-catenin) and TGF-β (Smad2/3), respectively.
HC-HA/PTX3 is a non-toxic, potent inhibitor of RPE cell proliferation and EMT in vitro. Hence, its efficacy in preventing PVR can be tested in our established rabbit PVR model.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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