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Ajay E Kuriyan, Hua He, Esdras Arrieta, Varona Sargent, Ying-Ting Zhu, Chen-Wei Su, Nidhi Batra, Harry W Flynn, Jean-Marie A Parel, Scheffer C G Tseng; HC-HA/PTX3, an active matrix component of amniotic membrane, inhibits proliferation and epithelial mesenchymal transition of RPE cells: a potential novel therapy for PVR. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2287. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR), characterized by retinal surface membranes, is the main cause of failure of rhegmatogenous retinal detachments (RRDs). We and others have reported that PVR is mediated by proliferation and epithelial mesenchymal transition (EMT) of RPE cells under the influence of vitreous growth factors. We have recently purified and characterized heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) complex as a unique matrix component from amniotic membrane that exerts anti-inflammatory and anti-EMT effects. HC-HA/PTX3 can be solubilized and injected intravitreally. Therefore, we investigated whether HC-HA/PTX3 may inhibit RPE cell proliferation and EMT in vitro and established rabbit PVR model for future studies.
We first tested if 0.1 to 25 µg/ml HC-HA/PTX3 was toxic to ARPE-19 cells by the Cell Death ELISA in comparison to HA and PBS. We optimized proliferation of ARPE-19 cells measured by BrdU labeling (ELISA and immunofluorescence) regarding the cell density, culturing medium, serum starvation, and addition of different growth factors. EMT was induced by addition of TGFβ1 and measured by nuclear pSMAD2/3 staining. We assessed the inhibitory effect of HC-HA/PTX3 on proliferation and EMT in vitro. Additionally, two New Zealand white (NZW) rabbits underwent cryotherapy, gas displacement of vitreous with intravitreal perfluoron, gas-fluid exchange, and intravitreal injection of NZW RPE cells to induce PVR.
There was no difference in ARPE-19 cell death by addition of HA-HA/PTX3 or HA, or PBS. EGF+FGF induced a 7-fold increase (p<0.05) in proliferation and EGF+FGF+TGFβ induced a 3-fold increase in nuclear pSMAD 2/3 staining. Addition of 25 µg/ml HC-HA/PTX3 inhibited EGF+FGF-induced proliferation 10-fold (p<0.05) and EGF+FGF+TGFβ-induced pSMAD 2/3 staining 2-fold (p<0.05). Addition of 25 µg/ml HA did not inhibit proliferation or nuclear pSMAD 2/3. Both rabbits developed open funnel RDs with PVR membranes four weeks after intravitreal injection of NZW rabbit RPE cells.
HC-HA/PTX3 is a non-toxic, potent inhibitor of RPE cell proliferation and EMT in vitro. We will further test whether intravitreal injection of HC-HA/PTX3 can inhibit PVR formation using the established rabbit model of PVR.
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