Rho-kinase inhibition reduces myofibroblast differentiation and proliferation of scleral fibroblasts induced by transforming growth factor β and experimental glaucoma

Ian Pitha, Ericka Oglesby, Amanda Chow, Elizabeth Kimball, Mary Ellen Pease, Julie Schaub, Harry A Quigley

Research output: Contribution to journalArticle

Abstract

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

Original languageEnglish (US)
Article number6
JournalTranslational Vision Science and Technology
Volume7
Issue number6
DOIs
StatePublished - Nov 1 2018

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rho-Associated Kinases
Myofibroblasts
Transforming Growth Factors
Fibroblasts
Glaucoma
Intraocular Pressure
Sclera
Collagen
Smooth Muscle
Muscle
Actins
Gels
Enzyme-Linked Immunosorbent Assay
Phosphorylation
Biomechanics
Cell proliferation
Therapeutics
Protein Kinase Inhibitors
Intercellular Signaling Peptides and Proteins
Biomechanical Phenomena

Keywords

  • Fasudil
  • H1152
  • Intraocular pressure
  • Peripapillary sclera
  • ROCK
  • Y27632

ASJC Scopus subject areas

  • Biomedical Engineering
  • Ophthalmology

Cite this

@article{63cd4c367fbf49fda1790c5f46f7a678,
title = "Rho-kinase inhibition reduces myofibroblast differentiation and proliferation of scleral fibroblasts induced by transforming growth factor β and experimental glaucoma",
abstract = "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 (10lM), Y27632 (10 lM), and fasudil (5lM) reduced SMA expression 72{\%}, 85{\%}, and 68{\%}, respectively. Collagen gel contraction was reduced by 36{\%} (P < 0.001), 27{\%} (P = 0.0003), and 33{\%} (P = 0.0019) following treatment with fasudil (25 lM), Y27632 (10 lM), and H1152 (10lM). ROCK activity induced by TGFβ rose 4.74 ± 1.9 times over control at 4 hours (P = 0.0004) and 2.4 ± 0.47-fold (P = 0.0016) in sclera after IOP elevation. Proliferation of scleral fibroblasts after chronic IOP elevation was reduced 77{\%} by Y27632 (P = 0.001) and 84{\%} by fasudil (P = 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.",
keywords = "Fasudil, H1152, Intraocular pressure, Peripapillary sclera, ROCK, Y27632",
author = "Ian Pitha and Ericka Oglesby and Amanda Chow and Elizabeth Kimball and Pease, {Mary Ellen} and Julie Schaub and Quigley, {Harry A}",
year = "2018",
month = "11",
day = "1",
doi = "10.1167/tvst.7.6.6",
language = "English (US)",
volume = "7",
journal = "Translational Vision Science and Technology",
issn = "2164-2591",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "6",

}

TY - JOUR

T1 - Rho-kinase inhibition reduces myofibroblast differentiation and proliferation of scleral fibroblasts induced by transforming growth factor β and experimental glaucoma

AU - Pitha, Ian

AU - Oglesby, Ericka

AU - Chow, Amanda

AU - Kimball, Elizabeth

AU - Pease, Mary Ellen

AU - Schaub, Julie

AU - Quigley, Harry A

PY - 2018/11/1

Y1 - 2018/11/1

N2 - 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 (10lM), Y27632 (10 lM), and fasudil (5lM) reduced SMA expression 72%, 85%, and 68%, respectively. Collagen gel contraction was reduced by 36% (P < 0.001), 27% (P = 0.0003), and 33% (P = 0.0019) following treatment with fasudil (25 lM), Y27632 (10 lM), and H1152 (10lM). ROCK activity induced by TGFβ rose 4.74 ± 1.9 times over control at 4 hours (P = 0.0004) and 2.4 ± 0.47-fold (P = 0.0016) in sclera after IOP elevation. Proliferation of scleral fibroblasts after chronic IOP elevation was reduced 77% by Y27632 (P = 0.001) and 84% by fasudil (P = 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.

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

KW - Fasudil

KW - H1152

KW - Intraocular pressure

KW - Peripapillary sclera

KW - ROCK

KW - Y27632

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U2 - 10.1167/tvst.7.6.6

DO - 10.1167/tvst.7.6.6

M3 - Article

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