Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells

B. Quill, M. Irnaten, N. G. Docherty, E. M. McElnea, D. M. Wallace, A. F. Clark, C. J. O'Brien

Research output: Contribution to journalArticle

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Abstract

Purpose: This study examines the effect of the L-type calcium channel blocker verapamil on mechanical straininduced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Methods: Changes in LC cell intracellular calcium [Ca2+]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Results: Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i ( p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). Conclusions: This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma.

Original languageEnglish
Pages (from-to)1009-1014
Number of pages6
JournalBritish Journal of Ophthalmology
Volume99
Issue number7
DOIs
StatePublished - 1 Jul 2015

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Calcium Channels
Extracellular Matrix
Calcium Channel Blockers
Verapamil
Chondroitin Sulfate Proteoglycans
L-Type Calcium Channels
Genes
Optic Disk
Transforming Growth Factors
Calcium
Collagen
Cell Membrane
Fura-2
Fluorescent Dyes
Confocal Microscopy
Glaucoma
Disease Progression
Cations
Cell Culture Techniques
Fluorescence

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Quill, B. ; Irnaten, M. ; Docherty, N. G. ; McElnea, E. M. ; Wallace, D. M. ; Clark, A. F. ; O'Brien, C. J. / Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells. In: British Journal of Ophthalmology. 2015 ; Vol. 99, No. 7. pp. 1009-1014.
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abstract = "Purpose: This study examines the effect of the L-type calcium channel blocker verapamil on mechanical straininduced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Methods: Changes in LC cell intracellular calcium [Ca2+]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15{\%}) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Results: Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i ( p<0.05). Exposure to cyclical mechanical strain (15{\%} strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). Conclusions: This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma.",
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Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells. / Quill, B.; Irnaten, M.; Docherty, N. G.; McElnea, E. M.; Wallace, D. M.; Clark, A. F.; O'Brien, C. J.

In: British Journal of Ophthalmology, Vol. 99, No. 7, 01.07.2015, p. 1009-1014.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Calcium channel blockade reduces mechanical strain-induced extracellular matrix gene response in lamina cribrosa cells

AU - Quill, B.

AU - Irnaten, M.

AU - Docherty, N. G.

AU - McElnea, E. M.

AU - Wallace, D. M.

AU - Clark, A. F.

AU - O'Brien, C. J.

PY - 2015/7/1

Y1 - 2015/7/1

N2 - Purpose: This study examines the effect of the L-type calcium channel blocker verapamil on mechanical straininduced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Methods: Changes in LC cell intracellular calcium [Ca2+]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Results: Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i ( p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). Conclusions: This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma.

AB - Purpose: This study examines the effect of the L-type calcium channel blocker verapamil on mechanical straininduced extracellular matrix genes in optic nerve head lamina cribrosa (LC) cells. Methods: Changes in LC cell intracellular calcium [Ca2+]i following hypotonic cell membrane stretch were measured with the fluorescent probe fura-2/AM. Fluorescence intensity was measured, after labelling, by calcium (Ca2+) imaging confocal microscopy. Confluent human LC cell cultures were serum starved for 24 h prior to exposure to cyclical mechanical strain (1 Hz, 15%) for 24 h in the presence or absence of verapamil (10 mm). Transforming growth factor-β 1 (TGF-β1), collagen 6A3 (COL6A3) and chondroitin sulfate proteoglycan 2 (CSPG2) mRNA expression levels were assessed by quantitative RT-PCR. Results: Hypotonic cell membrane stretch of LC cells from normal donors significantly increased [Ca2+]i ( p<0.05). Exposure to cyclical mechanical strain (15% strain) produced a statistically significant increase in the three matrix genes that were examined (TGF-β1, COL6A3 and CSPG2). This response in both cyclical and mechanical stretch was significantly reduced by pretreating LC cells with the L-type calcium channel blocker verapamil (p<0.05). Conclusions: This study provides evidence of a novel mechanotransduction pathway linking mechanical strain, cation channel function and the induction of LC cell matrix gene transcription. This highlights the potential involvement of calcium influx in the activation of matrix remodelling responses in the optic nerve head and supports the rationale that calcium channel blockers may attenuate disease progression in glaucoma.

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U2 - 10.1136/bjophthalmol-2014-306093

DO - 10.1136/bjophthalmol-2014-306093

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AN - SCOPUS:84937724143

VL - 99

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EP - 1014

JO - British Journal of Ophthalmology

JF - British Journal of Ophthalmology

SN - 0007-1161

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