Rho-Kinase Inhibition Reduces Myofibroblast Differentiation and Proliferation of Scleral Fibroblasts Induced by Transforming Growth Factor β and Experimental Glaucoma.

PURPOSE

We evaluated prevention of transforming growth factor β (TGFβ)-induced transdifferentiation of cultured scleral fibroblasts to myofibroblasts by rho-associated protein kinase (ROCK) inhibitors. Additionally, we tested whether local delivery of ROCK inhibitors reduced scleral fibroblast proliferation in response to chronic intraocular pressure (IOP) elevation.

METHODS

Primary human peripapillary sclera (PPS) fibroblasts were cultured and treated with TGFβ to induce myofibroblast transdifferentiation, as determined by immunoblot assessment of α smooth muscle actin (SMA) levels and collagen gel contraction. Cells were treated with the ROCK inhibitors Y27632, fasudil, and H1152 before TGFβ treatment. ROCK activity in TGFβ-treated fibroblasts and sclera from ocular hypertensive mice was assessed by measuring phosphorylation of the ROCK substrate MYPT1 at Thr696. Fibroblast proliferation following IOP elevation and ROCK inhibitor treatment was assessed by an enzyme-linked immunosorbent (ELISA) assay.

RESULTS

ROCK inhibitors H1152 (10μM), Y27632 (10 μM), and fasudil (5μM) reduced SMA expression 72%, 85%, and 68%, respectively. Collagen gel contraction was reduced by 36% (< 0.001), 27% (= 0.0003), and 33% (= 0.0019) following treatment with fasudil (25 μM), Y27632 (10 μM), and H1152 (10μM). ROCK activity induced by TGFβ rose 4.74 ± 1.9 times over control at 4 hours (= 0.0004) and 2.4 ± 0.47-fold (= 0.0016) in sclera after IOP elevation. Proliferation of scleral fibroblasts after chronic IOP elevation was reduced 77% by Y27632 (= 0.001) and 84% by fasudil (= 0.0049).

CONCLUSIONS

ROCK inhibitors reduce TGFβ-induced myofibroblast transdifferentiation and glaucoma-induced scleral cell proliferation.

TRANSLATIONAL RELEVANCE

These findings suggest altered fibroblast activity promoted by ROCK inhibitors could modify scleral biomechanics and be relevant to glaucoma treatment.