TGF-β-induced IOP elevations are mediated by RhoA in the early but not the late fibrotic phase of open angle glaucoma

Lisa J Hill; Ben Mead; Chloe N Thomas; Simon Foale; Elena Feinstein; Martin Berry; Richard J Blanch; Zubair Ahmed; Ann Logan

TGF-β-induced IOP elevations are mediated by RhoA in the early but not the late fibrotic phase of open angle glaucoma

Lisa J Hill, Ben Mead, Chloe N Thomas, Simon Foale, Elena Feinstein, Martin Berry, Richard J Blanch, Zubair Ahmed, Ann Logan

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Abstract

Purpose: Elevations in intraocular pressure (IOP) are associated with the development of glaucoma and loss of sight. High transforming growth factor-β (TGF-β) 1 levels in the eye's anterior chamber can lead to dysfunctional contractions through RhoA signaling in trabecular meshwork (TM) cells and IOP spikes. Sustained high TGF-β levels leads to TM fibrosis and sustained increases in IOP. We investigated whether inhibiting RhoA, using a siRNA-mediated RhoA (siRhoA), controls IOP by altering TM expression of fibrosis and contractility-related proteins in a rodent model of glaucoma.

Methods: TGF-β was injected intracamerally twice a week into adult Sprague Dawley rats, and IOP was recorded with tonometry. Animals were euthanized on day 7 and 35 with TM expression of fibrosis and contractility-related proteins, as well as survival of retinal ganglion cells (RGCs) assessed with immunohistochemistry. siRNA against RhoA or enhanced green fluorescent protein (EGFP) was also injected intracamerally into select animals. Successful RhoA knockdown was determined with quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, and the effects of the knockdown on the parameters above analyzed.

Results: TGF-β caused increased TM contractile proteins and IOP spikes by day 7, sustained increases in IOP from day 15, and TM fibrosis at day 35. siRhoA abolished the transient 7 day IOP rise but not the later sustained IOP increase (due to fibrosis). At 35 days, TGF-β-related RGC loss was not prevented with siRhoA treatment.

Conclusions: We conclude that RhoA signaling mediates the early IOP rise induced by TM cellular changes associated with contractility but not the sustained IOP elevation caused by TM fibrosis. Thus, RhoA therapies offer a clinically relevant opportunity for IOP management, likely through the modulation of TM contractility, but appear to be ineffective in the amelioration of fibrosis.

Original language	English
Pages (from-to)	712-726
Number of pages	15
Journal	Molecular Vision
Volume	24
Publication status	Published - 29 Oct 2018

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@article{9b5494d8f736427aad2c176c03f6a231,

title = "TGF-β-induced IOP elevations are mediated by RhoA in the early but not the late fibrotic phase of open angle glaucoma",

abstract = "Purpose: Elevations in intraocular pressure (IOP) are associated with the development of glaucoma and loss of sight. High transforming growth factor-β (TGF-β) 1 levels in the eye's anterior chamber can lead to dysfunctional contractions through RhoA signaling in trabecular meshwork (TM) cells and IOP spikes. Sustained high TGF-β levels leads to TM fibrosis and sustained increases in IOP. We investigated whether inhibiting RhoA, using a siRNA-mediated RhoA (siRhoA), controls IOP by altering TM expression of fibrosis and contractility-related proteins in a rodent model of glaucoma.Methods: TGF-β was injected intracamerally twice a week into adult Sprague Dawley rats, and IOP was recorded with tonometry. Animals were euthanized on day 7 and 35 with TM expression of fibrosis and contractility-related proteins, as well as survival of retinal ganglion cells (RGCs) assessed with immunohistochemistry. siRNA against RhoA or enhanced green fluorescent protein (EGFP) was also injected intracamerally into select animals. Successful RhoA knockdown was determined with quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, and the effects of the knockdown on the parameters above analyzed.Results: TGF-β caused increased TM contractile proteins and IOP spikes by day 7, sustained increases in IOP from day 15, and TM fibrosis at day 35. siRhoA abolished the transient 7 day IOP rise but not the later sustained IOP increase (due to fibrosis). At 35 days, TGF-β-related RGC loss was not prevented with siRhoA treatment.Conclusions: We conclude that RhoA signaling mediates the early IOP rise induced by TM cellular changes associated with contractility but not the sustained IOP elevation caused by TM fibrosis. Thus, RhoA therapies offer a clinically relevant opportunity for IOP management, likely through the modulation of TM contractility, but appear to be ineffective in the amelioration of fibrosis.",

author = "Hill, {Lisa J} and Ben Mead and Thomas, {Chloe N} and Simon Foale and Elena Feinstein and Martin Berry and Blanch, {Richard J} and Zubair Ahmed and Ann Logan",

year = "2018",

month = oct,

day = "29",

language = "English",

volume = "24",

pages = "712--726",

journal = "Molecular Vision",

issn = "1090-0535",

}

TY - JOUR

T1 - TGF-β-induced IOP elevations are mediated by RhoA in the early but not the late fibrotic phase of open angle glaucoma

AU - Hill, Lisa J

AU - Mead, Ben

AU - Thomas, Chloe N

AU - Foale, Simon

AU - Feinstein, Elena

AU - Berry, Martin

AU - Blanch, Richard J

AU - Ahmed, Zubair

AU - Logan, Ann

PY - 2018/10/29

Y1 - 2018/10/29

N2 - Purpose: Elevations in intraocular pressure (IOP) are associated with the development of glaucoma and loss of sight. High transforming growth factor-β (TGF-β) 1 levels in the eye's anterior chamber can lead to dysfunctional contractions through RhoA signaling in trabecular meshwork (TM) cells and IOP spikes. Sustained high TGF-β levels leads to TM fibrosis and sustained increases in IOP. We investigated whether inhibiting RhoA, using a siRNA-mediated RhoA (siRhoA), controls IOP by altering TM expression of fibrosis and contractility-related proteins in a rodent model of glaucoma.Methods: TGF-β was injected intracamerally twice a week into adult Sprague Dawley rats, and IOP was recorded with tonometry. Animals were euthanized on day 7 and 35 with TM expression of fibrosis and contractility-related proteins, as well as survival of retinal ganglion cells (RGCs) assessed with immunohistochemistry. siRNA against RhoA or enhanced green fluorescent protein (EGFP) was also injected intracamerally into select animals. Successful RhoA knockdown was determined with quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, and the effects of the knockdown on the parameters above analyzed.Results: TGF-β caused increased TM contractile proteins and IOP spikes by day 7, sustained increases in IOP from day 15, and TM fibrosis at day 35. siRhoA abolished the transient 7 day IOP rise but not the later sustained IOP increase (due to fibrosis). At 35 days, TGF-β-related RGC loss was not prevented with siRhoA treatment.Conclusions: We conclude that RhoA signaling mediates the early IOP rise induced by TM cellular changes associated with contractility but not the sustained IOP elevation caused by TM fibrosis. Thus, RhoA therapies offer a clinically relevant opportunity for IOP management, likely through the modulation of TM contractility, but appear to be ineffective in the amelioration of fibrosis.

AB - Purpose: Elevations in intraocular pressure (IOP) are associated with the development of glaucoma and loss of sight. High transforming growth factor-β (TGF-β) 1 levels in the eye's anterior chamber can lead to dysfunctional contractions through RhoA signaling in trabecular meshwork (TM) cells and IOP spikes. Sustained high TGF-β levels leads to TM fibrosis and sustained increases in IOP. We investigated whether inhibiting RhoA, using a siRNA-mediated RhoA (siRhoA), controls IOP by altering TM expression of fibrosis and contractility-related proteins in a rodent model of glaucoma.Methods: TGF-β was injected intracamerally twice a week into adult Sprague Dawley rats, and IOP was recorded with tonometry. Animals were euthanized on day 7 and 35 with TM expression of fibrosis and contractility-related proteins, as well as survival of retinal ganglion cells (RGCs) assessed with immunohistochemistry. siRNA against RhoA or enhanced green fluorescent protein (EGFP) was also injected intracamerally into select animals. Successful RhoA knockdown was determined with quantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry, and the effects of the knockdown on the parameters above analyzed.Results: TGF-β caused increased TM contractile proteins and IOP spikes by day 7, sustained increases in IOP from day 15, and TM fibrosis at day 35. siRhoA abolished the transient 7 day IOP rise but not the later sustained IOP increase (due to fibrosis). At 35 days, TGF-β-related RGC loss was not prevented with siRhoA treatment.Conclusions: We conclude that RhoA signaling mediates the early IOP rise induced by TM cellular changes associated with contractility but not the sustained IOP elevation caused by TM fibrosis. Thus, RhoA therapies offer a clinically relevant opportunity for IOP management, likely through the modulation of TM contractility, but appear to be ineffective in the amelioration of fibrosis.

M3 - Article

C2 - 30429640

SN - 1090-0535

VL - 24

SP - 712

EP - 726

JO - Molecular Vision

JF - Molecular Vision

ER -

TGF-β-induced IOP elevations are mediated by RhoA in the early but not the late fibrotic phase of open angle glaucoma

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