Barry Quill

The Effect of Graded Cyclic Stretching on Extracellular Matrix–Related Gene Expression Profiles in Cultured Primary Human Lamina Cribrosa Cells

PURPOSE Cyclic stretching of the glial fibrillary acidic protein (GFAP)-negative lamina cribrosa (LC) cell in vitro is associated with transcriptomic changes in genes involved in extracellular matrix (ECM) dynamics in vivo, thereby implicating this cell type in the pathophysiologic changes of the optic nerve head (ONH) in glaucoma. The purpose of the study was to determine whether exposure to different grades of mechanical stretch progressively alters the expression of ECM genes in cultured LC cells. METHODS Primary cultures of human LC cells from three separate donors were maintained in static culture or exposed to low-level strain (3% ± 0.5% elongation, 1 Hz) for 24 hours. A baseline comparison of the expression of 62 genes involved in ECM dynamics was performed with low-density gene arrays (LDAs). The 3% protocol was used in a 24-hour period of baseline dynamic low-level stretch, and gene expression was compared with that occurring in a further 24-hour exposure to a 12.5% or a 20% stretch. Gene expression levels were determined by qRT-PCR. RESULTS LC cells displayed a nonlinear, transcriptional response to the mechanical stretch. Ten ECM-related and growth factor genes were altered by 3% strain versus static culture (nine downregulated and one upregulated). Increasing strain from 3% to 20% resulted in a significant increase in expression of 15 ECM-elated genes. Only one gene (epidermal growth factor) was increased between the 3% and 12.5% strains. CONCLUSIONS Low-level, pulsatile, cyclic strain resets a lower baseline expression of several glaucoma-associated ECM genes. The LC ECM gene response occurs above a fourfold increase in baseline strain (12.5% strain) in vitro. The study supports the use of a nonstatic baseline when studying the effect of stretch (or strain) on the activation of ONH-derived, ECM-producing cells.

Activation of stretch-activated channels and maxi-K+ channels by membrane stress of human lamina cribrosa cells.

PURPOSE The lamina cribrosa (LC) region of the optic nerve head is considered the primary site of damage in glaucomatous optic neuropathy. Resident LC cells have a profibrotic potential when exposed to cyclical stretch. However, the mechanosensitive mechanisms of these cells remain unknown. Here the authors investigated the effects of membrane stretch on cell volume change and ion channel activity and examined the associated changes in intracellular calcium ([Ca(2+)](i)). METHODS The authors used primary LC cells obtained from normal human donor eyes. Confocal microscopy was used to investigate the effect of hypotonic cell membrane stretch on cell volume changes. Whole-cell patch-clamp and calcium imaging techniques were used to investigate the effect of hypotonicity on ion channel(s) activity and [Ca(2+)](i) changes, respectively. RT-PCR was used to examine for the maxi-K(+) signature in LC cells. RESULTS In this study, LC cells showed significant volume changes in response to hypotonic cell swelling. The authors characterized a large conductance K(+) channel (maxi-K(+)) in LC cells and demonstrated its increased activity during cell membrane hypotonic stretch. RT-PCR revealed the presence of maxi-K(+) signature in LC cells. The authors showed the [Ca(2+)](i) and maxi-K(+) channels to be dependent on extracellular Ca(2+) and inhibited by gadolinium, which blocks stretch-activated channels (SACs). Pretreatment with thapsigargin, which blocks the release of Ca(2+) from endoplasmic reticulum stores, showed no significant difference in [Ca(2+)](i) concentration on hypotonic swelling. CONCLUSIONS The results show that hypotonic stress of human LC cells activates SAC and Ca(2+)-dependent maxi-K(+) channels and that the increase in [Ca(2+)](i) during cell swelling was predominantly from extracellular sources (or intracellular stores other than the endoplasmic reticulum). These findings improve the understanding of how LC cells respond to cell membrane stretch. Further experiments in this area may reveal future targets for novel therapeutic intervention in the management of glaucoma.

Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells

BackgroundDisease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell’s constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes.MethodsThe number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed.ResultsA significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors.ConclusionsIncreased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.

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

Purpose This study examines the effect of the L-type calcium channel blocker verapamil on mechanical strain-induced 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.

Evaluation of the Effect of Hypercapnia on Vascular Function in Normal Tension Glaucoma.

INTRODUCTION Altered ocular perfusion and vascular dysregulation have been reported in glaucoma. The aim of this paper was to evaluate the vascular response to a hypercapnic stimulus. METHODS Twenty normal tension glaucoma (NTG) patients and eighteen age- and gender-matched controls had pulsatile ocular blood flow (POBF) measurements, systemic cardiovascular assessment, and laser Doppler digital blood flow (DBF) assessed. Measurements were taken at baseline, after 10-minutes rest, in the stable sitting and supine positions and following induction and stabilization of hypercapnia, which induced a 15% increase in end-tidal pCO2. The POBF response to hypercapnia was divided into high (>20%) and low responders (<20%). RESULTS 65% of NTG patients had a greater than 41% increase in POBF following CO2 rebreathing (high responders). These high responders had a lower baseline POBF, lower baseline DBF, and a greater DBF response to thermal stimulus. CONCLUSION NTG patients that have a greater than 20% increase in POBF after a hypercapnic stimulus have lower baseline POBF and DBF values. This suggests that there is impaired regulation of blood flow in a significant subgroup of NTG patients. This observation may reflect a generalised dysfunction of the vascular endothelium.