Inhibitory Effects of Angiotensin II Receptor Blockade on Human Tenon Fibroblast Migration and Reactive Oxygen Species Production in Cell Culture.

PURPOSE: We investigate the effect of angiotensin receptor blockade on the migration of human Tenon fibroblasts (HTF), using irbesartan, an angiotensin II receptor type 1 (AT1R) blocker (ARB) as a potential antifibrotic agent in glaucoma filtration surgery. METHODS: Confluent HTF cultures were scratched with a 1 mL pipette tip and treated with either irbesartan (10, 50, and 100 µg/mL) or angiotensin II (2 µg/mL). The extent of HTF migration up to 30 hours, and cell number and morphology at 72 hours was evaluated. To assess the effect on reactive oxygen species (ROS) level, HTF were treated with either irbesartan (10 µg/mL) or angiotensin II (2 µg/mL) for 24 hours after scratching, and then stained with dihydroethidium (DHE) before evaluation by confocal microscopy. RESULTS: Irbesartan inhibited HTF migration by 50% to 70% compared to controls (P < 0.05). Levels of ROS were almost completely attenuated by irbesartan (DHE fluorescence intensity of 5.68E-09) (P < 0.05). Irbesartan reduced cell numbers by 50% and induced morphologic changes with loss of pseudopods (P < 0.05). Conversely, angiotensin II increased cell numbers up to 4-fold while retaining cell viability. CONCLUSIONS: Irbesartan inhibited HTF migration and ROS production. It also reduced cell numbers and altered HTF morphology. Angiotensin II increased cell number without altering morphology. This initial study warrants future investigations for further potential antifibrotic effects of this drug. TRANSLATIONAL RELEVANCE: This in vitro study focused on investigations of irbesartan's effects on HTF migration, ROS production, as well as HTF cell numbers and morphology. It suggests a potential therapeutic strategy worth further exploration with a view towards postoperative wound healing modulation in glaucoma filtration surgery.

Dendritic cells in the cornea during Herpes simplex viral infection and inflammation.

Herpes simplex keratitis is commonly caused by Herpes simplex virus type 1, which primarily infects eyelids, corneas, or conjunctiva. Herpes simplex virus type 1-through sophisticated interactions with dendritic cells (DCs), a type of antigen-presenting cell)-initiates proinflammatory responses in the cornea. Corneas were once thought to be an immune-privileged region; however, with the recent discovery of DCs that reside in the cornea, this long-held conjecture has been overturned. Therefore, evaluating the clinical, preclinical, and cell-based studies that investigate the roles of DCs in corneas infected with Herpes simplex virus is critical. With in vivo confocal microscopy, animal models, and cell culture experiments, we may further the understanding of the sophisticated interactions of Herpes simplex virus with DCs in the cornea and the molecular mechanism associated with it. It has been shown that specific differentiation of DCs using immunohistochemistry, flow cytometry, and polymerase chain reaction analysis in both human and mice tissues and viral tissue infections are integral to increasing understanding. As for in vivo confocal microscopy, it holds promise as it is the least invasive and a real-time investigation. These tools will facilitate the discovery of various targets to develop new treatments.

Modulation of Fibroblasts in Conjunctival Wound Healing.

Modulating conjunctival wound healing has the potential to improve outcomes after glaucoma filtration surgery and for several ocular disorders, including ocular cicatrial pemphigoid, vernal keratoconjunctivitis, and pterygium. Although anti-inflammatories and antimetabolites have been used with success, these nonspecific agents are not without their complications. The search for novel and more targeted means to control conjunctival fibrosis without such limitations has brought much attention to the regulation of fibroblast proliferation, differentiation, extracellular matrix production, and apoptosis. This review provides an update on where we stand with current antifibrotic agents and outlines the strategies that novel agents use, as they evolve from the bench to the bedside.

Oxidative stress and reactive oxygen species: a review of their role in ocular disease.

For many years, oxidative stress arising from the ubiquitous production of reactive oxygen species (ROS) has been implicated in the pathogenesis of various eye diseases. While emerging research has provided some evidence of the important physiological role of ROS in normal cell function, disease may arise where the concentration of ROS exceeds and overwhelms the body's natural defence against them. Additionally, ROS may induce genomic aberrations which affect cellular homoeostasis and may result in disease. This literature review examines the current evidence for the role of oxidative stress in important ocular diseases with a view to identifying potential therapeutic targets for future study. The need is particularly pressing in developing treatments for conditions which remain notoriously difficult to treat, including glaucoma, diabetic retinopathy and age-related macular degeneration.

Intereye comparison of femtosecond laser-assisted cataract surgery capsulotomy and manual capsulorhexis edge strength.

PURPOSE: To compare the breaking force required to tear the explanted capsule after femtosecond laser-assisted cataract surgery in the worse eye and manual cataract surgery in the contralateral eye. SETTING: Personaleyes, Sydney, Australia. DESIGN: Prospective nonrandomized case study. METHOD: Patients with bilateral cataract had femtosecond laser-assisted cataract surgery with the Lensx laser in the eye with worse vision and manual cataract surgery in the contralateral eye. Each explanted capsule was stretched mechanically, and the breaking force and strain in grams (g) were compared. When a large contralateral difference in capsule strength was found, scanning electron microscopy (SEM) was applied to determine whether morphologic imperfections were present in a case with a weak capsule. RESULTS: Paired samples of 78 eyes of 39 patients were tested. The mean breaking force was not significantly different between manual capsulorhexes (2.3 g ± 2.0 [SD]) and femtosecond laser capsulotomies (2.0 ± 2.2 g, P = .52). The breaking strain for the manual samples (33.8% ± 18.3%) and for the femtosecond laser samples (34.6% ± 18.6%) were also not significantly different (P = .81). In 5 patients, in the femtosecond group, the capsules required considerably less force to break than the capsules in the manual group. However, the SEM images of these samples did not show specific laser imperfections. CONCLUSION: In paired human eyes, the capsulotomies created by a femtosecond laser with a contact lens interface were as strong as manual capsulorhexes.

Transection and explantation of intraocular lenses using femtosecond lasers.

We present 2 cases in which a femtosecond laser was used in vitro to transect hydrophilic acrylic intraocular lenses (IOLs). At 8 µJ with 3 µm spot separation and 6 µm line separation, no charring occurred and there was no increase in total organic carbon. In vivo, the IOLs were successfully transected in the capsular bag (Case 1, opaque IOL) and the sulcus (Case 2, subluxated IOL post-pneumatic displacement of submacular hemorrhage) and explanted through a clear corneal incision (∼3.0 mm). At 3 months, in Case 1, the corrected distance visual acuity (CDVA) improved from 6/24 to 6/5, astigmatism improved by 0.23 diopters, and endothelial cell density (ECD) remained unchanged (1935 to 2210 cells/mm2); in Case 2, the CDVA was hand motion, astigmatism remained unchanged, and ECD decreased (1960 to 1600 cells/mm2), possibly as a result of complex surgery. Femtosecond IOL transection and explantation may be a clinically safe and feasible option for surgeons.

A mouse retinal explant model for use in studying neuroprotection in glaucoma.

The study aims to validate mouse retinal explants for use as an efficient tool to investigate neuroprotective therapies for treatment in diseases such as glaucoma. Eyes from C57BL/6 mice were enucleated immediately post-mortem to make two retinal explants per eye. Explants were cultured at an air/medium interface on membrane inserts for 7 days ex-vivo. Explants were treated either with Z-VAD-FMK (a pan-caspase inhibitor; 100 µM) or vehicle. Retinal Ganglion cell (RGC) density was analysed by ßIII tubulin and RNA-binding protein with multiple splicing (RBPMS) immunohistochemistry. Caspase activity was measured using Caspase 3/7 glo assay and western blot. Caspase-3 expression was quantified using RT-PCR and western blotting. Retinal explants treated with Z-VAD-FMK demonstrated a 1.5-fold (p = 0.027) increase in number of surviving RGCs on day 4 compared to the control treatment using ßIII tubulin staining. RGC viability was 2-fold (p = 0.002) higher in RGC stained with RBPMS on day 1 compared to control. There was no RBPMS staining of RGCs beyond day 1 in either treatment. The caspase activity was 4.75 and 5.5-fold (p = 0.002 and 0.004 respectively) higher in control as compared to treatment with Z-VAD-FMK on day 1, 2 respectively. Increase in caspase activity in control group was also confirmed by western blot for day 1 protein lysates. Caspase-3 mRNA expression was 4.75-fold higher in Z-VAD-FMK treated explants compared to control on day 1 (p < 0.001). Culture conditions appropriate to retinal explant culture for investigation of RGC apoptosis was identified. Retinal cultures at day 4 were ideal for detecting neuroprotection using ßIII tubulin staining. RBPMS acts as a viability marker as well as to define best time point for investigation of apoptosis related signalling pathways which is day 1. These findings suggest that mouse retinal explants are good model for studying ganglion cell specific apoptosis and are applicable to diseases such as glaucoma.

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

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-1ß. 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-1ß. CONCLUSION: BLF accelerated healing of corneal alkali injury in BALB/c mice which was associated with suppression of IL-1 and reduced infiltrating cells.

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

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.