Publications by authors named "Qian Garrett"

30 Publications

  • Page 1 of 1

Genetic factors and molecular mechanisms in dry eye disease.

Ocul Surf 2018 04 10;16(2):206-217. Epub 2018 Mar 10.

University of New South Wales, New South Wales, Australia.

Dry eye disease (DED) is a complex condition with a multifactorial etiology that can be difficult to manage successfully. While external factors are modifiable, treatment success is limited if genetic factors contribute to the disease. The purpose of this review is to compile research describing normal and abnormal ocular surface function on a molecular level, appraise genetic studies involving DED or DED-associated diseases, and introduce the basic methods used for conducting genetic epidemiology studies.
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http://dx.doi.org/10.1016/j.jtos.2018.03.003DOI Listing
April 2018

Upregulation of miR-195 accelerates oxidative stress-induced retinal endothelial cell injury by targeting mitofusin 2 in diabetic rats.

Mol Cell Endocrinol 2017 09 6;452:33-43. Epub 2017 May 6.

Central Laboratory, The First Hospital of Hebei Medical University, Shijiazhuang, PR China; Burn Engineering Center of Hebei Province, Shijiazhuang, PR China. Electronic address:

This study was performed to investigate the oxidative stress-induced miRNA changes in relation to pathogenesis of diabetic retinopathy (DR) and to establish a functional link between miRNAs and oxidative stress-induced retinal endothelial cell injury. Our results demonstrated that oxidative stress could induce alterations of miRNA expression profile, including up-regulation of miR-195 in the diabetic retina or cultured HMRECs after exposed to HO or HG (P < 0.05). Oxidative stress also resulted in a significant reduction of MFN2 expression in diabetic retina or HMRECs (P < 0.05). Overexpression of miR-195 reduced MFN2 protein levels, and induced tube formation and increased permeability of diabetic retinal vasculature. The luciferase reporter assay confirmed that miR-195 binds to the 3' -untranslated region (3'-UTR) of MFN2 mRNA. This study suggested that miR-195 played a critical role in oxidative stress-induced retinal endothelial cell injury by targeting MFN2 in diabetic rats.
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http://dx.doi.org/10.1016/j.mce.2017.05.009DOI Listing
September 2017

Treatment Practices and Outcomes of Meibomian Gland Dysfunction at a Tertiary Center in Southern India.

Eye Contact Lens 2018 Sep;44 Suppl 1:S138-S143

Brien Holden Vision Institute (L.L., J.L.F., E.B.P.), Sydney, New South Wales, Australia; Vision Cooperative Research Centre (L.L., J.L.F., E.B.P.), Sydney, New South Wales, Australia; School of Optometry and Vision Science (L.L., Q.G., J.L.F., E.B.P.), The University of New South Wales, Sydney, New South Wales, Australia; The University of Notre Dame Australia (Q.G.), Sydney, New South Wales, Australia; and L V Prasad Eye Institute (P.K.V.), Hyderabad, India.

Objective: To describe the current treatment practices for meibomian gland dysfunction (MGD) at a tertiary eye center, together with the subjective outcomes and compliance behaviors of patients.

Methods: This retrospective cohort study reviewed medical records for MGD severity grading, treatment prescribed, and follow-up schedule. In addition, participants were surveyed to gauge subjective outcomes and treatment adherence.

Results: Eight hundred ten patients were diagnosed with "MGD" or "meibomitis" and had a total of 14 different treatment combinations prescribed. In 3.0% of cases, there was no treatment specified. As MGD severity increased, it became more likely that management would be applied and this was also associated with significantly longer treatment durations (P=0.02) and shorter follow-up periods (P<0.001). Posttreatment subjective outcomes and treatment adherence surveys had a response rate of 36.7% and 24.1% respectively. Overall, 53.5% reported sustained improvement, 40.7% no improvement, and 5.7% experienced temporary relief. Although no treatment regimen seemed to be more efficacious than others, patients showed greater adherence when using topical reagents compared with lid hygiene measures (P≤0.002).

Conclusion: Clinicians, in this large tertiary eye center, use a wide range of treatment regimens to manage MGD. This suggests the need for development of standard management protocols. Whether alone, or in combination, no MGD treatment significantly improved subjective symptoms, a result that may be influenced by compliance behaviors. Use of topical reagents (eye drops or ointment) seemed to be associated with the best compliance. Future focus on more effective MGD treatments is needed to improve practical outcomes.
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http://dx.doi.org/10.1097/ICL.0000000000000356DOI Listing
September 2018

The efficacy and safety of a novel posterior scleral reinforcement device in rabbits.

Mater Sci Eng C Mater Biol Appl 2016 May 14;62:233-41. Epub 2016 Jan 14.

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China. Electronic address:

Purpose: To evaluate the efficacy and safety of posterior scleral reinforcement (PSR) device for myopia suppression in rabbits' eyes.

Methods: PSR surgery was performed on the normal 12 8-week-old New Zealand white rabbits' right eyes. To determine efficacy of the device, ophthalmic examination would be taken at pre-operation and post-operation (1 week, 1 month, 3 months, 6 months, and 1 year), such as A-ultrasound, diopter and B-ultrasound. Evaluation of safety were based on the following indicators: intraocular pressure (IOP), slit lamp, fundus photography, fundus fluorescein angiography and pathological examination after surgery. The efficacy and safety of PSR device were evaluated by comparison (treated eyes and contralateral eyes) of pre and post-operation.

Results: The novel PSR device could significantly shorten axial length (preoperative axial length: 16.36 ± 0.14 mm, postoperative 1 week, 1 month, 3 months, 6 months and 1 year axial lengths: 15.03 ± 0.28 mm, 15.23 ± 0.32 mm, 15.39 ± 0.31 mm, 15.45 ± 0.22 mm and 15.45 ± 0.22 mm; P=0.00037<0.001) in the treated eyes (right eyes) after surgery. At different postoperative time points, the B-ultrasound images showed that the PSR located in appropriate position and supported the posterior sclera very well. At the same time, IOP of treated eyes kept a relatively stable level (preoperative IOP: 12.56 ± 2.01 mmHg, postoperative IOP: ranging from 11.33 ± 1.23 mmHg to 13.44 ± 2.19 mmHg, P>0.05) post-operation 1 year. During observation period, there was no significant inflammatory reaction and complications such as anterior chamber flare, empyema, endophthalmitis, vitreous hemorrhage, retina detachment and retinal choroid neovascularization by slit lamp, fundus photography and fundus fluorescein angiography. In addition, there were no pathologic changes be found by comparison treated eyes group and contralateral group eyes based on pathological examinations.

Conclusions: In vivo study, effectively and safely, the novel PSR device can inhibit rabbits' axial length elongation during postoperative 1 year. This study demonstrates that this novel PSR could be a potential treatment approach for myopia.
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http://dx.doi.org/10.1016/j.msec.2015.12.046DOI Listing
May 2016

Study of retinal vessel oxygen saturation in ischemic and non-ischemic branch retinal vein occlusion.

Int J Ophthalmol 2016 18;9(1):99-107. Epub 2016 Feb 18.

Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Sun Yat-sen University, Guangzhou 510060, Guangdong Province, China.

Aim: To explore how oxygen saturation in retinal blood vessels is altered in ischemic and non-ischemic branch retinal vein occlusion (BRVO).

Methods: Fifty BRVO eyes were divided into ischemic (n=26) and non-ischemic (n=24) groups, based on fundus fluorescein angiography. Healthy individuals (n=52 and n=48, respectively) were also recruited as controls for the two groups. The mean oxygen saturations of the occluded vessels and central vessels were measured by oximetry in the BRVO and control groups.

Results: In the ischemic BRVO group, the occluded arterioles oxygen saturation (SaO2-A, 106.0%±14.3%), instead of the occluded venule oxygen saturation (SaO2-V, 60.8%±9.4%), showed increases when compared with those in the same quadrant vessels (SaO2-A, 86.1%±16.5%) in the contralateral eyes (P<0.05). The oxygen saturations of the central vessels showed similar trends with those of the occluded vessels. In the non-ischemic BRVO group, the occluded and central SaO2-V and SaO2-A showed no significant changes. In both the ischemic and non-ischemic BRVOs, the central SaO2-A was significantly increased when compared to healthy individuals.

Conclusion: Obvious changes in the occluded and central SaO2-A were found in the ischemic BRVO group, indicating that disorders of oxygen metabolism in the arterioles may participate in the pathogenesis of ischemic BRVO.
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http://dx.doi.org/10.18240/ijo.2016.01.17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4768501PMC
March 2016

Measurement of consensual accommodation in vision-impaired eyes.

Optom Vis Sci 2014 Jul;91(7):752-9

*BSc(Optom) †BOptom ‡DNB §PhD ∥BSc **MS ††PhD, FAAO L V Prasad Eye Institute, Hyderabad, India (PV, MT, VSS); Vision Cooperative Research Centre, Sydney, New South Wales, Australia (PV, MT, QG, SD, VSS); Brien Holden Vision Institute, New South Wales, Australia (LD, TJN, QG, SD, AH); School of Optometry and Vision Science, University of New South Wales, New South Wales, Australia (QG, AH); and Bascom Palmer Eye Institute, University of Miami, Miller School of Medicine, Miami, Florida (AH).

Purpose: To measure the accommodative response in unsighted or profoundly vision impaired (PVI) eyes when accommodation is elicited in the fellow, sighted eye.

Methods: Eighty-eight unilaterally PVI subjects (UPS) and 97 bilaterally sighted subjects (BSS) (10 to 45 years) were enrolled. Subjects had clear ocular media for auto-refraction and could steadily fixate targets with the sighted eye. For BSS, a long-pass filter was placed in front of one eye to simulate unilateral blindness. Both eyes were measured with a Shin-Nippon auto-refractor while fixating a 4/40 letter at 4 m and then an N8 letter at 40 cm and at 33 cm. Accommodation was calculated as the difference between distance and near refraction.

Results: Only subjects with repeatable alignment between measurements were included in the analyses (64 UPS, 95 BSS). Results were analyzed using t test and a generalized linear mixed model including age, sightedness, distance spherical equivalent, and accommodation as factors. The t test found no significant difference between eyes for UPS (p = 0.981 at 40 cm and p = 0.663 at 33 cm). For BSS, the sighting eye produced statistically significant but only slightly greater amounts of accommodation than the filtered eye (0.098 diopters [D], p = 0.002 at 40 cm and 0.189 D, p < 0.001 at 33 cm). The generalized linear mixed model found no difference between BSS and UPS in terms of difference in accommodation between eyes (p = 0.128 at 40 cm and p = 0.157 at 33 cm).

Conclusions: The PVI eyes of unilaterally PVI individuals display similar accommodative response to their fellow, sighted eyes when accommodation is elicited by near target of up to 3 D to the fellow eye. However, the difference in accommodative response between PVI and fellow, sighted eye is related to the amount of accommodation elicited.
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http://dx.doi.org/10.1097/OPX.0000000000000294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090593PMC
July 2014

Release of betaine and dexpanthenol from vitamin E modified silicone-hydrogel contact lenses.

Curr Eye Res 2015 Mar 15;40(3):267-73. Epub 2014 May 15.

Department of Chemical Engineering, University of Florida , Gainesville, FL , USA .

Purpose: To develop a contact lens system that will control the release of an osmoprotectant and a moisturizing agent with the aim to reduce symptoms of ocular dryness.

Materials And Methods: Profiles of the release of osmoprotectant betaine and moisturizing agent dexpanthenol from senofilcon A and narafilcon B contact lenses were determined in vitro under sink conditions. Both types of lenses were also infused with vitamin E to increase the duration of drug release due to the formation of the vitamin E diffusion barriers in the lenses. The release profiles from vitamin E-infused lenses were compared with those from the control lenses.

Results: Both dexpanthenol and betaine are released from commercial silicone hydrogel lenses for only about 10 min. Vitamin E loadings into contact lenses at about 20-23% can increase the release times to about 10 h, which is about 60 times larger compared to the control unmodified lenses.

Conclusions: Vitamin E-loaded silicone hydrogel contact lenses released betaine and dexpanthenol in a controlled fashion.
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http://dx.doi.org/10.3109/02713683.2014.917192DOI Listing
March 2015

Fluorescein staining and physiological state of corneal epithelial cells.

Cont Lens Anterior Eye 2014 Jun 12;37(3):213-23. Epub 2013 Dec 12.

Brien Holden Vision Institute, Sydney, Australia; School of Optometry and Vision Science, The University of New South Wales, Sydney, Australia.

Purpose: To evaluate the physiological status of corneal epithelial cells exhibiting fluorescein staining.

Methods: Fluorescein staining properties of corneal epithelial cells under normal and stressed conditions were studied using cell-culture (human corneal limbal epithelial cells - HCLE) and organ-culture (rabbit) models. Stress stimuli comprised exposure to hypotonicity, hypertonicity, preservatives, scratch, and alkaline wounding. In addition to fluorescein, cells were stained with Hoechst-33342 (HO), Propidium-iodide (PI), and Annexin-V (AN-V) to identify live, dead and apoptotic cells. Clinical-slit-lamp and fluorescence confocal-microscopic (FCM) observations were performed. FCM images were quantified for fluorescence intensity using Image-J software.

Results: Healthy HCLE cells uniformly took up fluorescein to a moderate degree with a mean grey value of 62 ± 24 (mean ± SD) on a scale of 0-256 (no unit). Fluorescence levels similar to those observed prior to stress were associated with healthy cells. Apoptotic cells showed the highest fluorescence (138 ± 38). Dead cells showed minimal fluorescence (23 ± 7) that was similar to the background (20 ± 11, p>0.05). Observations in whole rabbit eyes were in general agreement with these cell culture findings.

Conclusions: The clinical observation of corneal staining with fluorescein suggests the presence of epithelial cells that are undergoing apoptosis but does not indicate dead cells. Under in vitro or ex vivo conditions, healthy cells took up fluorescein at levels that were lower than those of apoptotic cells and thus, are not likely to be perceived as exhibiting staining during clinical observation. Sodium fluorescein may be considered as a probe for apoptotic epithelial cells.
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http://dx.doi.org/10.1016/j.clae.2013.11.003DOI Listing
June 2014

Decrease in hyperosmotic stress-induced corneal epithelial cell apoptosis by L-carnitine.

Mol Vis 2013 19;19:1945-56. Epub 2013 Sep 19.

Brien Holden Vision Institute, Sydney, Australia.

Purpose: To characterize the osmoprotective properties of L-carnitine on human corneal epithelial cell volume and apoptosis during hyperosmotic stress.

Methods: Human corneal limbal epithelial (HCLE) cells were exposed to culture medium at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) with or without L-carnitine (10 mM). Induction of apoptosis was detected by quantifying the proteolytic activity of caspase-8, caspase-9, and caspase-3/7 using caspase activity assays, the expression of tumor necrosis factor (TNF)-α with enzyme-linked immunosorbent assay, and annexin V/propidium iodide staining of HCLE cells evaluated with confocal microscopy and flow cytometry. Cell volume changes in response to hyperosmotic stress were analyzed using flow cytometry.

Results: After the HCLE cells were exposed to hyperosmotic medium (500 mOsm), the percentage of shrunken cells and damaged/dead cells (stained positively for annexin V and/or propidium iodide) was six- and three-fold, respectively, higher than that under isotonic conditions (300 mOsm). This was paralleled by an increase in TNF-α concentration in media and caspase-8, -9, and -3/7 activities (six-, four-, ten-, and twelve-fold, respectively; all showing p < 0.001). Addition of L-carnitine during hyperosmotic stress partly restored cell volume and significantly reduced the concentration of TNF-α released (p = 0.005) and caspase-9 activity (p = 0.0125). Addition of L-carnitine reduced the percentage of hyperosmolarity-induced damaged/dead cells to levels observed under isotonic conditions.

Conclusions: L-carnitine can regulate human corneal epithelial cell volume under hyperosmotic stress and ameliorate hyperosmotic stress-induced apoptosis.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782369PMC
March 2014

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PLoS One 2013 19;8(8):e71948. Epub 2013 Aug 19.

Vision CRC, Sydney, Australia ; School of Optometry and Vision Science, University of New South Wales, Sydney, Australia.

Purpose: This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.

Methods: Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.

Results: The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.

Conclusions: Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071948PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747068PMC
April 2014

Efficacy of osmoprotectants on prevention and treatment of murine dry eye.

Invest Ophthalmol Vis Sci 2013 Sep 19;54(9):6287-97. Epub 2013 Sep 19.

School of Ophthalmology and Optometry, Wenzhou Medical College, Wenzhou, China.

Purpose: To evaluate the efficacy of osmoprotectants on prevention and treatment of dry eye in a murine model.

Methods: Dry eye was induced in mice by using an intelligently controlled environmental system (ICES). Osmoprotectants betaine, L-carnitine, erythritol, or vehicle (PBS) were topically administered to eyes four times daily following two schedules: schedule 1 (modeling prevention): dosing started at the beginning of housing in ICES and lasted for 21 or 35 days; schedule 2 (modeling treatment): dosing started after ICES-housed mice developed dry eye (day 21), continuing until day 35. Treatment efficacy was evaluated for corneal fluorescein staining; corneal epithelial apoptosis by TUNEL and caspase-3 assays; goblet cell numbers by PAS staining; and expression of inflammatory mediators, TNF-α, IL-17, IL-6, or IL-1β by using RT-PCR on days 0, 14, 21, and/or 35.

Results: Compared with vehicle, prophylactic administration of betaine, L-carnitine, or erythritol significantly decreased corneal staining and expression of TNF-α and IL-17 on day 21 (schedule 1). Treatment of mouse dry eye with osmoprotectants significantly reduced corneal staining on day 35 compared with day 21 (schedule 2). Relative to vehicle, L-carnitine treatment of mouse dry eye for 14 days (days 21 to 35) resulted in a significant reduction in corneal staining, number of TUNEL-positive cells, and expression of TNF-α, IL-17, IL-6, or IL-1β, as well as significantly increased the number of goblet cells.

Conclusions: Topical application of betaine, L-carnitine, or erythritol systematically limited progression of environmentally induced dry eye. L-carnitine can also reduce the severity of such dry-eye conditions.
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http://dx.doi.org/10.1167/iovs.13-12081DOI Listing
September 2013

Bovine lactoferrin promotes corneal wound healing and suppresses IL-1 expression in alkali wounded mouse cornea.

Curr Eye Res 2013 Nov 30;38(11):1110-7. Epub 2013 Jul 30.

Brien Holden Vision Institute , Sydney, NSW , Australia and.

Purpose: Using an in vitro cell culture model, bovine lactoferrin (BLF) stimulates healing of alkali-induced human corneal epithelial wounds. The present study examined the efficacy of BLF in promoting healing of corneal injury in vivo and explored BLF modulation of interleukin-1 (IL-1) during wound healing.

Methods: Alkali injury was induced to BALB/c mice by exposure of the mouse cornea to a sodium hydroxide (NaOH)-soaked filter disc for 2 min. The corneal surface was irrigated after the injury with saline. Topical BLF in phosphate buffered saline (PBS) (10 µl, 62.5 μM), bovine serum albumin (BSA) (10 µl, 62.5 μM in PBS) or PBS only (10 µl) were applied three times daily to both the alkali-injured and uninjured eyes for 3 d. Wound healing was assessed using 0.1% fluorescein staining under slit lamp microscope. The corneas at 6 h, 24 h or 3 d post-injury and treatment were excised and examined histologically, homogenized corneal tissue was evaluated for expression of IL-1α and IL-1β.

Results: After 6 h post-wounding and treatment no significant reduction of wound area was observed between treatments and infiltrating cells or IL-1 expression were not elevated in any group. By 24 h, BLF-treatment resulted in accelerated wound closure (100%) compared to PBS and BSA treatment (70% and 65%, respectively). BLF treatment reduced infiltrating cells compared to controls and no elevation of IL-1, whereas controls displayed elevated infiltrating cells and increased levels of IL-1. After 3 d, mice treated with BLF exhibited complete wound closure while control corneas still exhibited some minor defects. Resolution of inflammation with minimal remaining infiltrating cells was observed in all corneas by day 3, coincident to normal levels of IL-1α and IL-1β.

Conclusion: BLF accelerated healing of corneal alkali injury in BALB/c mice which was associated with suppression of IL-1 and reduced infiltrating cells.
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http://dx.doi.org/10.3109/02713683.2013.811259DOI Listing
November 2013

Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress.

Exp Eye Res 2013 Mar 12;108:33-41. Epub 2012 Dec 12.

Brien Holden Vision Institute, Sydney, Australia.

Elevated tear osmolarity is one of the key pathological factors in dry eye leading to ocular discomfort associated with damage to the ocular surface and inflammation. The aim of this study was to determine the capacity of the organic osmolyte, betaine, to act as an osmoprotectant against hypertonic stress-induced human corneal epithelial cell shrinkage and apoptosis using in vitro cell culture models. Human corneal limbal epithelial (HCLE) cells exposed to culture medium for 16 h at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) in the presence or absence of betaine (5 or 10 mM) were evaluated for cell volume changes; cell viability; and apoptosis. Betaine (10 mM) ameliorated hyperosmotically induced reduction of cell volume (from 27% reduction to 11%) and resulted in increased mitochondrial activity (by 17%) and an increase in viable cell numbers (by 12%) compared to controls (exposure to hyperosmotic medium without betaine). Hyperosmotically shocked HCLE cells in the presence of betaine (10 mM) halved the number of damaged cells (apoptotic/necrotic) compared to cells in the absence of betaine. The presence of betaine (at 5 or 10 mM) significantly reduced the activity of caspase-8, -9 and -3/7 and release of TNF-α was also reduced by 34% or 55% after exposure of HCLE to 500 mOsm in the presence of 5 or 10 mM betaine, respectively. Using polyclonal antibody against Betaine/GABA transporter 1 (BGT-1), we detected the presence of BGT-1 in HCLE. We demonstrated that the transport of betaine was facilitated by increased osmolarity. In conclusion, betaine stabilized corneal epithelial cell volume under hyperosmotic stress and limited hyperosmotic stress-induced HCLE apoptosis.
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http://dx.doi.org/10.1016/j.exer.2012.12.001DOI Listing
March 2013

High refractive index polysiloxane as injectable, in situ curable accommodating intraocular lens.

Biomaterials 2012 Aug 15;33(23):5659-71. Epub 2012 May 15.

Materials Science and Engineering, CSIRO, Bayview Avenue, Clayton, VIC 3168, Australia.

Functionalised siloxane macromonomers, with properties designed for application as an injectable, in situ curable accommodating intraocular lens (A-IOL), were prepared via re-equilibration of a phenyl group-containing polysiloxane of very high molecular weight with octamethylcyclotetrasiloxane (D₄) and 2,4,6,8-tetra(n-propyl-3-methacrylate)-2,4,6,8-tetramethyl-cyclotetrasiloxane (D₄(AM)) in toluene using trifluoromethanesulfonic acid as a catalyst. Hexaethyldisiloxane was used as an end group to control the molecular weight of the polymer. The generated polymers had a consistency suitable for injection into the empty lens capsule. The polymers contained a low ratio of polymerisable groups so that, in the presence of a photo-initiator, they could be cured on demand in situ within 5 min under irradiation of blue light to form an intraocular lens within the lens capsule. All resulting polysiloxane soft gels had a low elastic modulus and thus should be able to restore accommodation. The pre-cure viscosity and post-cure modulus of the generated polysiloxanes were controlled by the end group and D₄(AM) concentrations respectively in the re-equilibration reactions. The refractive index could be precisely controlled by adjusting the aromatic ratio in the polymer to suit such application as an artificial lens. Lens stretching experiments with both human and non-human primate cadaver lenses of different ages refilled with polysiloxane polymers provided a significant increase in amplitude of accommodation (up to 4 D more than that of the respective natural lens). Both in vitro cytotoxicity study using L929 cell lines and in vivo biocompatibility study in rabbit models demonstrated the non-cytotoxicity and ocular biocompatibility of the polymer.
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http://dx.doi.org/10.1016/j.biomaterials.2012.04.052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3371592PMC
August 2012

A preliminary study of changes in tear film proteins in the feline eye following nictitating membrane removal.

Vet Ophthalmol 2012 May 7;15(3):164-71. Epub 2011 Oct 7.

Vision CRC, Sydney, NSW, Australia.

Objective: To investigate the influence of nictitating membrane (third eyelid) removal on selected proteins in feline tears.

Animal Studied: Domestic short-haired cats (7-17 months; 2.6-5.2 kg) were used.

Procedures: Eye-flush tears were collected periodically for up to 18 weeks from both eyes of animals with nictitating membranes removed, but nictitating gland left intact, (n = 4) or with nictitating membranes intact (n = 4). Tear comparisons were based on total protein content (TPC) using micro bicinchoninic acid assay, immunoglobulin A (IgA), and matrix-metalloproteinase (MMP)-9 measurements using sandwich enzyme-linked immunosorbent assay (ELISA) and tear gelatinase activity using gelatin zymography. Expression of MMP-2 and -9 in nictitating membranes removed at baseline (week 0) and eyes collected at 18 weeks were also investigated in histological sections using immunoperoxidase for visualization.

Results: Nictitating membrane removal did not significantly change TPC and MMP-9 in tears within the first 4 weeks. MMP-9 was not detected by ELISA in tears from eyes without nictitating membranes from week 5 onwards. IgA (%IgA of TPC) data varied between animals. Gelatin zymography showed increased MMP-2 and -9 activity in tears from eyes without nictitating membranes at week 1 and a decrease following week 2 post-surgery. MMP-2 and -9 were immunolocalised to conjunctival goblet cells of removed nictitating membranes and to the conjunctival epithelium, respectively. After 18 weeks, the distribution of MMPs in tissue was comparable between eyes with and without nictitating membranes.

Conclusions: Based on this preliminary study, nictitating membrane removal appeared to cause long-term changes in expression of tear proteins, including reduced MMP-9 expression.
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http://dx.doi.org/10.1111/j.1463-5224.2011.00955.xDOI Listing
May 2012

Mechanisms of superficial micropunctate corneal staining with sodium fluorescein: the contribution of pooling.

Cont Lens Anterior Eye 2012 Apr 13;35(2):81-4. Epub 2011 Sep 13.

The Brien Holden Vision Institute, Sydney, Australia.

Purpose: To establish if sodium fluorescein (SFL) dye accumulation within intercellular spaces on the ocular surface contributes to the appearance of superficial punctate corneal staining.

Methods: Thirteen subjects bilaterally wore PureVision™ lenses that had been pre-soaked in ReNu MultiPlus® multipurpose solution. After 1h of lens wear, corneal staining with SFL was assessed using a standard slit-lamp technique. Participants who presented with bilateral, corneal staining were selected for further evaluation. A randomly selected eye was rinsed with saline three times. Fellow eyes (control) received no rinsing. After each rinse, the appearance of SFL staining was recorded without any further instillation of the dye. To eliminate any confounding effects of staining due to residual fluorescein in the tear menisci, corneal staining was induced in freshly excised, isolated, rabbit eyes by topical administration of 0.001% PHMB and staining, rinsing and grading were performed as above.

Results: Nine out of 13 subjects presented with bilateral diffuse corneal staining (mean grade±SD: 2.4±0.7). The mean staining grades in test and control eyes respectively after each of the three rinses were (1) 2.41±0.41, 2.25±0.69 (p=0.9); (2) 2.34±0.79, 2.1±0.83 (p=0.8); and (3) 1.71±0.65, 1.60±0.79 (p=0.6) there was no significant reduction in staining with rinsing (p>0.05) and no difference was observed between test and control eyes at any sampling-point. Similar observations made in ex vivo rabbit eyes replicated these results.

Conclusions: Pooling or accumulation of SFL solution within intercellular spaces does not appear to contribute to the appearance of superficial micropunctate corneal staining.
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http://dx.doi.org/10.1016/j.clae.2011.08.008DOI Listing
April 2012

Bovine lactoferrin structures promoting corneal epithelial wound healing in vitro.

Invest Ophthalmol Vis Sci 2011 Apr 25;52(5):2719-26. Epub 2011 Apr 25.

Brien Holden Vision Institute, Sydney, NSW, Australia.

Purpose: To use an in vitro alkali-induced wound model to identify structures of bovine lactoferrin (BLF) that contribute to the promotion of human corneal epithelial healing.

Methods: BLF N-lobe and C-lobe were separated using limited proteolysis and purified by preparative chromatography. Isoforms of BLF were separated according to serine protease activity. Catalytic activities of isoforms and lobes were quantified by hydrolysis of a synthetic serine protease substrate. The promotion of healing by cognate moieties, lactoferricin-B, BLF isoforms, and BLF in various forms-iron-free, iron-saturated, deglycosylated, zwitterionic detergent exposed, chaotrope denatured, disulfide reduced-was assessed on alkali wounding of confluent monolayers of human corneal epithelial cells.

Results: The C-lobe of BLF (6.4-128 μM) promoted greater wound healing than native-BLF or N-lobe. BLF (12.8 μM) promoted wound closure in an iron-free, iron-bound, or deglycosylated state or after exposure to zwitterionic detergent. Healing was not stimulated by chaotropically denatured or disulfide reduced BLF (12.8 μM) or by lactoferricin-B (12.8 μM). Proteolytically active BLF (0.6 μM) promoted wound closure at a lower concentration than proteolytically inactive BLF (12 μM). This proteolytic activity was localized to the N-lobe.

Conclusions: The C-lobe is the primary promoter of BLF-stimulated corneal epithelial wound closure in vitro and is effective at concentrations ≥6.4 μM. Increased healing from BLF occurs with the native conformation and is unaffected by glycosylation or iron saturation. To a lesser extent, proteolytic activity of the N-lobe also improves healing rates. The BLF C-lobe may be a novel treatment for corneal lesions with delayed healing.
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http://dx.doi.org/10.1167/iovs.10-6352DOI Listing
April 2011

A comparison of basal and eye-flush tears for the analysis of cat tear proteins.

Acta Ophthalmol 2011 Feb;89(1):e75-81

Vision CRC, Sydney, NSW, Australia.

Purpose: To identify a rapid and effective tear collection method providing sufficient tear volume and total protein content (TPC) for analysis of individual proteins in cats.

Methods: Domestic adult short-haired cats (12-37 months; 2.7-6.6 kg) were used in the study. Basal tears without stimulation and eye-flush tears after instillation of saline (10 μl) were collected using microcapillary tubes from animal eyes either unwounded control or wounded with 9-mm central epithelial debridement giving four groups with n = 3. Tear comparisons were based on total time and rate for tear collection, TPC using micro bicinchoninic acid (BCA), tear immunoglobulin A (IgA), total matrix-metalloproteinase (MMP)-9 concentration using sandwich enzyme-linked immunosorbent assay (ELISA) and MMP-9 activity.

Results: Eye-flush tears were collected significantly faster than basal tears in wounded eyes with higher rates for tear collection in unwounded control and wounded eyes. TPC was significantly lower in eye-flush tears compared to basal tears. The relative proportion of tear IgA normalized to TPC (% IgA of TPC) was not significantly different between basal and eye-flush tears. In unwounded control eyes, MMP-9 was slightly higher in eye-flush than in basal tears; activity of MMP-9 in both tear types was similar. In wounded eyes, eye-flush tears showed highest MMP-9 levels and activity on Day 1, which subsequently decreased to Day 7. MMP-9 activity in basal tears from wounded eyes did not display changes in expression.

Conclusions: Eye-flush tears can be collected rapidly providing sufficient tear volume and TPC. This study also indicates that eye-flush tears may be more suitable than basal tears for the analysis of MMPs following corneal wounding.
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http://dx.doi.org/10.1111/j.1755-3768.2010.02082.xDOI Listing
February 2011

Transport of L-carnitine in human corneal and conjunctival epithelial cells.

Mol Vis 2010 Sep 4;16:1823-31. Epub 2010 Sep 4.

Brien Holden Vision Institute, The University of New South Wales, Sydney, NSW, Australia.

Purpose: Previously we demonstrated expression and localization of carnitine/organic cation transporters, OCTN1 and OCTN2, in human corneal and conjunctival epithelia. The present study aimed to examine the characteristics of L-carnitine transporters in cultured human limbal corneal (HCLE) and conjunctival epithelial (HCjE) cells.

Methods: Time-course, Na(+)-dependence, kinetics, energy- and pH- dependence of L-carnitine transport were investigated by monitoring L-[(3)H]carnitine uptake into HCLE and HCjE cells. To determine the specificity of action, competition and inhibition studies were performed.

Results: The uptake of L-carnitine into HCLE and HCjE cells was saturable and time-dependent. An Eadie-Hofstee plot showed two distinct components: a high- and a low- affinity carnitine transport system in HCLE and/or HCjE cells. L-carnitine transport was significantly inhibited by the metabolic inhibitors (sodium azide, dinitrophenol, iodoacetic acid). The L-carnitine analogs (D-carnitine, acetyl-L-carnitine and γ-butyrobetaine), tetraethylammonium (TEA), 2-amino-2-norbornane carboxylic acid (BCH), strongly inhibited uptake of L-[(3)H]carnitine. Uptake of L-[(3)H]carnitine also required the presence of Na(+) in the external medium and the uptake activity was maximal at pH 5.5. The anti-OCTN2 antibody blocked L-carnitine uptake in both HCLE and HCjE cells whereas the anti-OCTN1 antibody did not significantly block L-carnitine uptake.

Conclusions: L-carnitine is transported into HCLE and HCjE cells by an active carrier mediated transport system that is time-, Na(+)-, energy- and pH- dependent. The carnitine/organic cation transporter OCTN2 appears to play a dominant role in this process.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2956661PMC
September 2010

Role of carnitine in disease.

Nutr Metab (Lond) 2010 Apr 16;7:30. Epub 2010 Apr 16.

Institute for Eye Research, Sydney, New South Wales, Australia.

Carnitine is a conditionally essential nutrient that plays a vital role in energy production and fatty acid metabolism. Vegetarians possess a greater bioavailability than meat eaters. Distinct deficiencies arise either from genetic mutation of carnitine transporters or in association with other disorders such as liver or kidney disease. Carnitine deficiency occurs in aberrations of carnitine regulation in disorders such as diabetes, sepsis, cardiomyopathy, malnutrition, cirrhosis, endocrine disorders and with aging. Nutritional supplementation of L-carnitine, the biologically active form of carnitine, is ameliorative for uremic patients, and can improve nerve conduction, neuropathic pain and immune function in diabetes patients while it is life-saving for patients suffering primary carnitine deficiency. Clinical application of carnitine holds much promise in a range of neural disorders such as Alzheimer's disease, hepatic encephalopathy and other painful neuropathies. Topical application in dry eye offers osmoprotection and modulates immune and inflammatory responses. Carnitine has been recognized as a nutritional supplement in cardiovascular disease and there is increasing evidence that carnitine supplementation may be beneficial in treating obesity, improving glucose intolerance and total energy expenditure.
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http://dx.doi.org/10.1186/1743-7075-7-30DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861661PMC
April 2010

Onset time course of solution induced corneal staining.

Cont Lens Anterior Eye 2010 Aug 15;33(4):199-201. Epub 2010 Mar 15.

The Institute for Eye Research, Sydney NSW, Australia.

Purpose: To evaluate the early phase time course of solution induced corneal staining.

Methods And Materials: A double masked, single centred, prospective clinical trial was conducted. Twenty-five participants, either experienced or new contact lens wearers, participated in the study. Corneal staining response to short term use of ReNu MultiPlus Multipurpose Solution and PureVision silicon hydrogel contact lens with fluorescein was observed using standard techniques after 15, 30, 45, 60 and 120 min of lens wear and graded according to the IER scale. Measurements were carried out on separate days for each time point, in random order.

Results: Mean extent of staining was greater in test than in control eyes at all time points except baseline. In test eyes, the degree of staining increased successively at each time point after insertion, up to, but not beyond, 60 min. For those participants presenting with staining, maximum severity and frequency were both observed at 60 min and were significantly greater (p < 0.05) than at 15, 30, and 45 min.

Conclusion: Solution induced corneal staining gradually increased after lens insertion to a maximum at 1h. This level was maintained until at least 2h post-insertion.
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http://dx.doi.org/10.1016/j.clae.2010.02.004DOI Listing
August 2010

Care regimen and lens material influence on silicone hydrogel contact lens deposition.

Optom Vis Sci 2009 Mar;86(3):251-9

Institute for Eye Research, Sydney, Australia.

Purpose: To quantitatively detect proteins and cholesterol extracted from worn silicone hydrogel contact lenses and determine the effect of various lens care solutions on deposit accumulation.

Methods: Contact lenses, made from different polymers and worn on a daily wear schedule with different lens care solutions, were collected. Lipid and protein deposits were extracted by methanol:chloroform (1:1, v/v) and protein extraction solution (containing urea and surfactant), respectively. Lipid extracts were separated and cholesterol quantified using thin layer chromatography. Protein extracts were quantified using standard techniques.

Results: Among all lenses tested, Balafilcon A lenses exhibited greatest extracted cholesterol (4.1 to 8.2 microg/lens) and total protein (5.4 to 23.2 microg/lens). AQuify was the most effective solution in reducing extracted deposits, especially extracted protein, from Balafilcon A lenses. AQuify and Opti-Free RepleniSH solutions were most effective in reducing extracted cholesterol from Senofilcon A and Galyfilcon A lenses, respectively. Use of Opti-Free Express solution resulted in more extracted protein from Lotrafilcon B lenses than use of other solutions. Generally, Lotrafilcon B, Senofilcon A, and Galyfilcon A lenses accumulated relatively low amount of proteins. Lotrafilcon B lenses accumulated the least amount of cholesterol deposit among all lenses tested regardless of solution used.

Conclusions: Lens polymer (possibly associated with surface characteristics) is a prominent factor affecting lipid and protein accumulation. Within a lens polymer type, lens care solutions exhibit varying effectiveness in reducing protein and lipid accumulation.
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http://dx.doi.org/10.1097/OPX.0b013e318196a74bDOI Listing
March 2009

Bovine lactoferrin stimulates human corneal epithelial alkali wound healing in vitro.

Invest Ophthalmol Vis Sci 2009 Apr 5;50(4):1636-43. Epub 2008 Dec 5.

Institute for Eye Research, Sydney, New South Wales, Australia.

Purpose: The purpose of this study was to investigate the effect of bovine lactoferrin (BLF) on human corneal epithelial wound healing using an in vitro alkali-induced wound model and to understand its role in promoting wound healing.

Methods: Confluent human corneal limbal epithelial (HCLE) cells wounded using 0.5 microL of 0.1 M sodium hydroxide were treated with BLF (0, 0.1, 1, 2.5, and 5 mg/mL) or anti-human interleukin-6 (IL-6) receptor neutralizing antibody (anti-IL-6 antibody; 1, 10, and 50 microg/mL) or tyrphostin AG1295 (an inhibitor of platelet-derived growth factor [PDGF] receptor kinase; 1 and 10 microM), IL-6, or PDGF-BB. The conditioned medium collected for BLF treatment (0 and 5 mg/mL) was analyzed using a protein array for a number of cytokines/growth factors involved in corneal wound healing. A preliminary animal study using mice was carried out to determine the effect of BLF on alkali wounds.

Results: BLF at 2.5 and 5 mg/mL promoted wound healing (P<0.01). During wound closure, BLF upregulated PDGF-BB 180-fold and IL-6 10-fold compared with control. Treatment with tyrphostin AG1295 (10 microM; P<0.01) or anti-IL-6 antibody (50 microg/mL; P<0.01) in the presence of BLF inhibited wound closure, whereas the addition of exogenous IL-6 and PDGF-BB promoted wound closure. Preliminary animal studies have shown that BLF (5 mg/mL) promotes alkali wound healing in vivo.

Conclusions: These results suggest that BLF at >or=2.5 mg/mL stimulates HCLE wound healing, and this stimulation is mediated through the upregulation of PDGF or IL-6.
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http://dx.doi.org/10.1167/iovs.08-1882DOI Listing
April 2009

Proteomic analysis of protein deposits on worn daily wear silicone hydrogel contact lenses.

Mol Vis 2008 7;14:2016-24. Epub 2008 Nov 7.

Institute for Eye Research, Sydney, NSW, Australia.

Purpose: Previous studies have demonstrated deposition of tear proteins onto worn contact lenses. In this study, we used proteomic techniques to analyze the protein deposits extracted from worn daily wear silicone hydrogel contact lenses in combination with different lens care solutions.

Methods: Worn lenses were collected and protein deposits extracted using urea and surfactant. Protein extracts were desalted, concentrated, and then separated using one-dimensional gel electrophoresis. Individual protein components in extracts were identified using liquid chromatography combined with tandem mass spectrometry (LC-MS-MS) after trypsin digestion.

Results: One-dimensional gel electrophoresis revealed that lysozyme and other small proteins (around 20 kDa) were the most abundant proteins in the extracts. LC-MS-MS revealed a wide array of proteins in lens extracts with lysozyme and lipocalin 1 being the most commonly identified in deposit extracts.

Conclusions: Worn contact lenses deposit a wide array of proteins from tear film and other sources. Protein deposit profiles varied and were specific for each contact lens material.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2579937PMC
March 2009

Expression and localization of carnitine/organic cation transporter OCTN1 and OCTN2 in ocular epithelium.

Invest Ophthalmol Vis Sci 2008 Nov 18;49(11):4844-9. Epub 2008 Jul 18.

Institute for Eye Research, The University of New South Wales, NSW, Australia.

Purpose: The existence of an organic cation transport process in rabbit cornea and conjunctiva that mediates absorption of carnitine has previously been suggested. This study was conducted to determine the expression and localization of the carnitine/organic cation transporter (OCTN1 and OCTN2) in corneal or conjunctival epithelium.

Methods: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used for OCTN1 and OCTN2 mRNA expression in cultured human corneal-limbal epithelial (HCLE) or human conjunctival epithelial (HCjE) cells. Immunofluorescence staining with polyclonal antibody against human OCTN1 or OCTN2 was performed to investigate transporter expression in ocular epithelial cells or rabbit corneal and conjunctival epithelium. Polarity of the transporter expression was determined using Western blot analysis of the apical or basal membrane proteins extracted from the cultured cells. Apical or basal uptake of [H(3)]-L-carnitine was determined using the polarized epithelial cells grown onto collagen-coated porous filter support.

Results: OCTN1 and OCTN2 mRNA expression was detected in HCLE and HCjE cells of rabbits and humans. OCTN1 and OCTN2 were predominately localized in the apical membranes of the cells. HCLE and HCjE cells were able to take up L-carnitine; most carnitine uptake occurred through the apical surfaces.

Conclusions: This report is the first to document OCTN1 and OCTN2 expression in human corneal and conjunctival epithelial cells. These findings suggest potential involvement of OCTN1 and OCTN2 in the transport of carnitine in ocular tissues.
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http://dx.doi.org/10.1167/iovs.07-1528DOI Listing
November 2008

Carboxymethyl cellulose stimulates rabbit corneal epithelial wound healing.

Curr Eye Res 2008 Jul;33(7):567-73

Institute for Eye Research, Sydney, New South Wales, Australia.

Purpose: Previously, we reported carboxymethyl cellulose (CMC) binding to human corneal epithelial cells and promoting corneal epithelial wound closure in vitro. Using an animal model, the efficacy of CMC in promoting corneal wound healing was examined.

Materials And Methods: Following corneal epithelial wounding of NZ white rabbits, CMC (0.2% or 1.0%) or control vehicle (PBS) was administered topically (4 times daily for 3 days) to wounded and unwounded eyes with or without contact lens wear. Wound healing in response to the treatments was measured as percentage reduction of fluorescein-stained wound area 0 to 72 hr post-wounding. Corneas were examined histologically and expression of zonula occludens-1 (ZO-1) tight-junction was detected by immunohistochemistry.

Results: Percentage wound reduction in CMC-treated groups was significantly greater than controls (p < 0.05) at 24 and 32 hr. Complete wound closure was observed by 48 hr in 100% of CMC-treated eyes compared to 45% of vehicle-treated eyes. CMC also promoted wound closure dose-dependently. Epithelial cells formed an intact layer following CMC-treatment whereas vehicle-treated cells were less ordered. Strong ZO-1 expression in corneal epithelia of CMC-treated eyes was observed at 72 hr. Contact lens wear appeared to delay wound closure compared to without lens wear during CMC-treatment (p = 0.001).

Conclusions: CMC promoted dose-dependent corneal epithelial wound healing. CMC stimulated ZO-1 expression, indicating accelerated corneal epithelial resistance barrier regeneration.
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http://dx.doi.org/10.1080/02713680802140213DOI Listing
July 2008

Carboxymethylcellulose binds to human corneal epithelial cells and is a modulator of corneal epithelial wound healing.

Invest Ophthalmol Vis Sci 2007 Apr;48(4):1559-67

Institute for Eye Research, University of New South Wales, Sydney, NSW 2052, Australia.

Purpose: In this study, the ability of carboxymethylcellulose (CMC), used in artificial tear formulations, to interact with corneal-epithelial-cells (HCECs) and facilitate corneal epithelial wound healing was investigated.

Methods: HCECs were incubated with fluorescein-labeled CMC (F-CMC). CMC-epithelial binding was measured by spectrophotometry. The effect on F-CMC binding by hyaluronic acid (HA) or glucose was measured after preincubation in HA, mAb to CD44, or glucose, or mAb to GluT-1. F-CMC binding to fibronectin or collagen was measured by incubating proteins with F-CMC. The wound widths were measured 18 hours after confluent HCECs were scratch wounded. The ability of CMC to induce cell chemotaxis, proliferation, or migration was measured by quantitative assay. The efficacy of CMC in promoting epithelial wound healing was also tested in a rabbit epithelial scrape-wound model.

Results: CMC remained bound to the HCECs for 2 hours. Preincubation of HCECs with glucose or mAb to GluT-1, but not with HA or mAb to CD44, reduced the binding of CMC to HCECs from 43.7% to 67.2% or 10.9% to 25.3%, respectively. CMC bound significantly to fibronectin (3.1-fold) or collagen (9.3-fold) compared with the control (BSA), and such binding enhanced cell adhesion. CMC stimulated re-epithelialization of HCECs scratched in vitro and in vivo rabbit cornea epithelial scrape wounds. CMC stimulated cell migration but not proliferation.

Conclusions: CMC probably binds to HCECs through interaction of its glucopyranose subunits with glucose transporters. CMC binding to the matrix proteins stimulated HCEC attachment, migration, and re-epithelialization of corneal wounds.
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http://dx.doi.org/10.1167/iovs.06-0848DOI Listing
April 2007

Involvement of CTGF in TGF-beta1-stimulation of myofibroblast differentiation and collagen matrix contraction in the presence of mechanical stress.

Invest Ophthalmol Vis Sci 2004 Apr;45(4):1109-16

Wound Healing Research Unit, Institute of Ophthalmology, London, United Kingdom.

Purpose: This study was undertaken to investigate the role of connective tissue growth factor (CTGF) in fibroblast-to-myofibroblast differentiation and fibroblast-mediated collagen matrix contraction in the presence of mechanical stress.

Methods: An in vitro three-dimensional contraction model of human corneal-fibroblast-seeded collagen lattices (FSCLs) in the presence of mechanical stress generated by attaching the lattices to the culture well was used to measure FSCL contraction. FSCLs were treated with CTGF; TGF-beta1; serum-free (SF) control medium; or TGF-beta1 plus antisense oligodeoxynucleotides to CTGF; TGF-beta1 plus scrambled-sequence oligodeoxynucleotide to CTGF; or TGF-beta antibody. Expression of alpha-smooth muscle actin (alpha-SMA) by fibroblasts in FSCLs was detected by immunostaining and confocal microscopy, whereas ELISA was used for the fibroblasts cultured on plastic. The conditioned media were analyzed by ELISA for CTGF production.

Results: Exogenous CTGF stimulated significantly less collagen matrix contraction and myofibroblast differentiation than TGF-beta1, but similar to that stimulated by SF. TGF-beta1 stimulated fibroblasts to express CTGF. CTGF antisense oligodeoxynucleotide inhibited TGF-beta1-stimulated myofibroblast differentiation and FSCL contraction. Exogenous CTGF circumvented the inhibitory effects of CTGF antisense on FSCL contraction. TGF-beta antibody significantly inhibited FSCL contraction and myofibroblast differentiation under mechanical stress and SF control conditions.

Conclusions: In the presence of mechanical stress, CTGF is necessary for TGF-beta1-stimulation of myofibroblast differentiation and subsequent collagen matrix contraction, but CTGF alone is not sufficient to induce myofibroblast differentiation and collagen matrix contraction. Thus, TGF-beta1 appears to regulate multiple genes that are essential for fibroblast-mediated contraction of stressed matrix, one of which is CTGF.
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http://dx.doi.org/10.1167/iovs.03-0660DOI Listing
April 2004

Mediation of transforming growth factor-beta(1)-stimulated matrix contraction by fibroblasts: a role for connective tissue growth factor in contractile scarring.

Am J Pathol 2003 Nov;163(5):2043-52

Epithelial Repair and Regeneration Group, Wound Healing Research Unit, Divisions of Pathology and Cell Biology, Institute of Ophthalmology, Bath Street, London EC1V 9EL, United Kingdom.

Excessive cell-mediated tissue contraction after injury can lead to morbid contractile scarring in the body. In the eye this can cause blindness because of posterior capsule opacification, proliferative vitroretinopathy, failure of glaucoma filtration surgery, and corneal haze. During repair, transforming growth factor (TGF)-beta and connective tissue growth factor (CTGF) genes are co-ordinately expressed. Although TGF-beta and CTGF stimulate new matrix deposition, their role and regulation during contractile scarring is unknown. In this study, an in vitro model of collagen matrix contraction culminating from tractional forces generated by fibroblasts showed that both TGF-beta(1) and CTGF-stimulated contraction. Using a specific anti-sense oligodeoxynucleotide to CTGF the procontractile activity of TGF-beta(1) was found to be mediated by CTGF. During contraction fibroblasts produced similar levels of matrix metalloproteinases (MMPs)-2 and -9 with TGF-beta(1) or CTGF and a modest increase in MMP-1 with CTGF only (indicated by zymography and enzyme-linked immunosorbent assay). The requirement of MMPs for contraction was demonstrated using a broad-spectrum synthetic inhibitor. This study demonstrates a new function for CTGF in mediating matrix contraction by fibroblasts involving MMPs and suggests a novel regulatory mechanism for TGF-beta-stimulated contraction. Inhibition of CTGF activity or gene transcription could be a suitable target for anti-scarring therapies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1892432PMC
http://dx.doi.org/10.1016/s0002-9440(10)63562-6DOI Listing
November 2003

Matrix metalloproteinase inhibition modulates fibroblast-mediated matrix contraction and collagen production in vitro.

Invest Ophthalmol Vis Sci 2003 Mar;44(3):1104-10

Wound Healing Research Unit, Department of Pathology and Glaucoma, Institute of Ophthalmology and Moorfields Eye Hospital, London, United Kingdom.

Purpose: To investigate the effect of matrix metalloproteinase (MMP) inhibition on fibroblast-mediated matrix contraction and production.

Methods: Free-floating fibroblast-populated type I collagen lattices were prepared with human Tenon's capsule fibroblasts. Lattice areas were photographed and digitally analyzed to indicate the degree of lattice contraction. Quantitative competitive reverse transcription-polymerase chain reaction (QCRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to quantify mRNA and protein respectively for MMP-1, -2, and -3 by fibroblasts during lattice contraction. Gelatin zymography demonstrated activity of MMPs released into the conditioned medium of contracting lattices. Concentrations of the broad-spectrum MMP inhibitors ilomastat, CellTech (Slough, UK), and BB-94 were added to the contracting fibroblast-populated collagen lattices. Secreted C-terminal propeptide of type I collagen was measured in conditioned medium of contracting lattices by ELISA. Fibroblast proliferation in the presence of concentrations of ilomastat was measured by using the reagent water-soluble tetrazolium-1 (WST-1).

Results: During contraction of type I collagen lattices, Tenon's capsule fibroblasts expressed MMP-1, -2, and -3 mRNA and protein. Zymography demonstrated the release of four gelatinolytic species into the conditioned medium of contracting lattices (57, 72, 91, and 100 kDa). Inclusion of MMP inhibitors in the zymogram-developing buffer reduced the proteolytic activity of the detected bands. MMP inhibition (1-100 microM) significantly reduced fibroblast-mediated collagen lattice contraction (P < 0.05), and this effect was found to be reversible. Ilomastat also significantly inhibited production of collagen in a dose-dependent manner (P < 0.05). No effect on fibroblast proliferation was found in the presence of ilomastat.

Conclusions: MMPs are produced during Tenon's capsule fibroblast-mediated collagen lattice contraction. Broad-spectrum MMP inhibition significantly reduced matrix contraction and production without cell toxicity. Future clinical use of MMP inhibitors may be possible, because MMP inhibition significantly reduces fibroblast functions associated with contractile scarring.
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http://dx.doi.org/10.1167/iovs.02-0412DOI Listing
March 2003
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