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Ian F Pitha, Ericka Oglesby, Elizabeth Cone-Kimball, Julie Schaub, Mary Pease, Harry A Quigley; RHO-kinase-mediated regulation of myofibroblast differentiation in scleral fibroblasts during experimental glaucoma. Invest. Ophthalmol. Vis. Sci. 2018;59(9):6038.
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© ARVO (1962-2015); The Authors (2016-present)
In glaucoma, intraocular pressure(IOP)-generated stress is conducted to the optic nerve head through the peripapillary sclera (PPS). Altering the mechanical properties of the sclera can affect the extent of glaucomatous damage and protect against axonal injury. Fibroblasts are the main cellular component of the sclera and could potentially regulate PPS biomechanics. Additionally, experimental glaucoma induces differentiation of PPS fibroblasts to a profibrotic, myofibroblast phenotype. We hypothesize that rho-kinase signaling is necessary for myofibroblast differentiation of PPS fibroblasts in experimental glaucoma.
Primary, human PPS fibroblasts were cultured, treated with rho-kinase inhibitors, and exposed to TGFβ to induce myofibroblast phenotype as determined by αSMA immunoblot and collagen plug contraction assay. Rho-associated protein kinase (ROCK) activity was then evaluated using an ELISA-based method that measures phosphorylation of the ROCK target MYPT1 when incubated with lysates from TGFβ-treated PPS fibroblasts and sclera from CD1 mice with bead—induced glaucoma.
TGFβ treatment of PPS fibroblasts significantly induced ROCK activity as soon as 1 hour (3.6±0.3 fold over control, p=0.008), peaked at 4 hours (4.7±1.9 fold over control, p=0.0004), and was not present 24 hours after treatment. ROCK activity was significantly induced in mouse scleral lysates 7 days (3.2±1.8 fold over control eyes, p=0.03) after induction of IOP elevation. Treatment with rho-kinase inhibitors fausidil, Y-27632, and H1152 inhibited TGFβ-induced αSMA expression and collagen plug contraction compared to controls at micomolar dosages, however, did not affect canonical TGFb signaling as determined by SMAD2/3 phosphorylation.
Bead-induced glaucoma was associated with transient induction of ROCK activity in scleral tissue. In cultured PPS fibroblasts, rho-kinase signaling can be targeted to inhibit myofibroblast differentiation of scleral fibroblasts. These results support the hypothesis that rho-kinase signaling participates in myofibroblast differentiation of PPS fibroblasts and could regulate PPS biomechanics in glaucoma. Future work will determine the role of PPS fibroblast rho-kinase signaling in regulating axonal damage.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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