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Hua He, Yuan Zhang, Megha Mahabole, Chen-Wei Su, Scheffer C G Tseng; Deactivation of CD44-mediated Endocytosis by Heavy Chain-Hyaluronic Acid/Pentrixin3 in Human Retinal Pigment Epithelium. Invest. Ophthalmol. Vis. Sci. 2017;58(8):252.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferation and epithelial mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells are two hallmarks of proliferative vitreoretinopathy (PVR). Recently, we have found a heavy chain-hyaluronic acid/pentraxin 3 (HC-HA/PTX3) complex purified from human amniotic membrane inhibits both proliferation and EMT of RPE cells in vitro. Herein, we investigate whether the suppression of PVR by HC-HA/PTX3 is through CD44-mediated endocytosis.
Proliferation of ARPE-19 cells stimulated by growth factors and cytokines was measured by BrdU labeling. α-smooth muscle actin expression in TGF-βs-stimulated RPE cells was detected by immunostaining. CD44-mediated endocytosis was detected by incubating FITC-conjugated HA (20 µg/ml) for 24 hours after RPE cells were pre-treated with or without 5 units/ml hyaluronidase for 20 minutes at 37 °C. Expression of CD44 and phosphorylation of Merlin, TAK1, and Smad2/3 were detected by quantitative real-time PCR and Western blot.
Among PVR-related growth factors and cytokines, EGF, FGF-2, and TGF-βs are most potent in induction of proliferation and EMT in RPE cells, respectively. CD44-mediated endocytosis of HA by unstimulated RPE cells is weak, but is significantly increased by hyaluronidase digestion of extracellular matrix. Pretreatment of HC-HA/PTX3 (25 µg/ml) inhibits CD44-mediated endocytosis of HA, much stronger than high molecular mass of HA (> 4000 kDa). Accordingly, HC-HA/PTX3 also down-regulates p-Merlin (Ser518), p-TAK1 (Thr184/187), and p-Smad2 (Ser465/467), which all are related to CD44-mediated endocytosis.
Suppression of CD44-mediated endocytosis by HC-HA/PTX3 could lead to prevention of proliferation and EMT in PVR.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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