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

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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 (10μM), Y27632 (10 μM), and fasudil (5μM) 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 μM), Y27632 (10 μM), and H1152 (10μM). 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.

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Most cited references 40

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Specificity and mechanism of action of some commonly used protein kinase inhibitors.

S P Davies, H Reddy, M Caivano … (2000)

The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.

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Fibrosis--a common pathway to organ injury and failure.

Dan L Longo, Don C. Rockey, P Darwin Bell … (2015)

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Fibroblasts and myofibroblasts: what are we talking about?

Martina Baum, Frank Duffy (2011)

Cardiac myocytes, although large enough to make up most of the heart volume, are only a minority of cells within the heart with fibroblasts and blood vessel components (endothelial and smooth muscle cells) making up the remainder of the heart. In recent years, there has been increasing interest in the nonmyocyte population within the heart. This is attributable, in part, to our increasing understanding of the biology of the nonmyocyte cell types and additionally it is the result of our awakening realization that these cells are not static but rather that they are dynamic in nature indicating that they play a more active role in cardiac function than previously imagined. Studies now show that fibroblasts are involved in formation of the extracellular matrix and they control the size of the extracellular matrix. Additionally, they participate in the repair process by differentiating into myofibroblasts, which are cells involved in the inflammatory response to injury. Myofibroblasts migrate to the sites of injury where they produce cytokines, thus enhancing the inflammatory response. This review discusses both structural and functional differences between the two cell types and examines the different roles of these two different cell types in the heart.

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Author and article information

Journal

Journal ID (nlm-ta): Transl Vis Sci Technol

Journal ID (iso-abbrev): Transl Vis Sci Technol

Journal ID (hwp): tvst

Journal ID (pmc): Transl Vis Sci Technol

Journal ID (publisher-id): TVST

Title: Translational Vision Science & Technology

Publisher: The Association for Research in Vision and Ophthalmology

ISSN (Electronic): 2164-2591

Publication date Collection: November 2018

Publication date (Electronic): 14 November 2018

Volume: 7

Issue: 6

Electronic Location Identifier: 6

Affiliations

[1 ]Department of Ophthalmology, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

[2 ]Center for Nanomedicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

[3 ]Glaucoma Center of Excellence, The Johns Hopkins University School of Medicine, Baltimore, MD, USA

Author notes

Correspondence: Ian Pitha, John Hopkins University, Wilmer Eye Institute, 600 N Wolfe St, Baltimore, MD 21287, USA. e-mail: ipitha1@ 123456jhmi.edu

Article

Serial Item and Contribution ID: tvst-07-06-04 Publisher ID: TVST-18-1000

DOI: 10.1167/tvst.7.6.6

PMC ID: 6238981

SO-VID: 7fff70dc-6795-4a32-8d55-8b9c8308720a

License:

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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

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Abstract

Purpose

Methods

Results

Conclusions

Translational Relevance

Related collections

Karger: Ophthalmology

Most cited references 40

Specificity and mechanism of action of some commonly used protein kinase inhibitors.

Fibrosis--a common pathway to organ injury and failure.

Fibroblasts and myofibroblasts: what are we talking about?

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