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Weiming Mao, Michela Montecchi-Palmer, Abbot F Clark, ; TGFβ2 induces cross-linked actin network formation in the trabecular meshwork via Smad and non-Smad pathways. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3296.
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Glaucoma is the leading cause of blindness worldwide. In primary open angle glaucoma (POAG), the most important risk factor is elevated intraocular pressure (IOP). This IOP elevation is due to increased outflow resistance in the trabecular meshwork (TM). Glaucomatous TM changes include loss of TM cells, excessive deposition of extracellular matrix, as well as formation of cross-linked actin networks (CLANs). It is believed that CLANs increase TM cell stiffness, which contribute to elevated outflow resistance and IOP. CLANs are induced by TGFβ2, a POAG-associated growth factor. In this study, we investigated whether TGFβ2 induces CLANs via the Smad pathway or non-Smad pathway or both. We also explored whether Rho kinase (ROCK) inhibitors are able to inhibit CLAN formation
Primary HTM cells were cultured in DMEM-low glucose medium containing 10% fetal bovine serum and antibiotics. Cells were grown to confluency on coverslips before treatment with or without 5ng/ml TGFβ2, 5uM SB431542 (TGFβ receptor inhibitor), 10nM BMP4 (TGFβ pathway inhibitor), 10uM SIS3 (Smad3 inhibitor), 10uM SP600125 (JNK inhibitor), or 10uM Y27362 (ROCK inhibitor) for 1 or 10 days. After treatment, cells were fixed in 2% paraformaldehyde/PBS, washed, blocked, and stained with phalloidin-Alex 488 and DAPI. Also, cells and conditioned medium were harvested for western immunoblotting (WB). CLANs were counted in a masked manner, and 10 regions were counted using fluorescent microscopy with 600x magnification. CLAN ratios were calculated as CLAN positive cells vs. DAPI positive cells. CLAN ratios between groups were compared by using one-way ANOVA.
TGFβ2 significantly induced CLAN formation in HTM cell cultures compared to untreated cells (n=6, p<0.05). This induction was inhibited by BMP4, TGFβ receptor inhibitor, Smad inhibitor, JNK inhibitor, as well as ROCK inhibitor (n=6, p<0.05 compared to TGFβ2 treated cells). TGFβ receptor and ROCK inhibitors completely inhibited CLAN induction (p<0.05 compared to TGFb2 treated cells), while BMP4, Smad and JNK showed only partial inhibition. As a positive control, Western immunoblotting showed that TGFb2 induction of fibronectin was inhibited by all these inhibitors.
TGFβ2 induces CLAN formation in HTM cells via both the Smad and non-Smad pathways. This induction can be inhibited by TGFβ pathway inhibitors as well as the ROCK inhibitor Y27632.
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