Serum deprivation can suppress receptor-mediated calcium signaling in pterygial-derived fibroblasts.

PURPOSE: Pterygium is characterized as invasive, proliferative fibrovascular altered conjunctival tissue. The extensive vascular network is likely to significantly contribute to the progression of the disease. In the present study, we investigated the effects of reduced serum (to mimic a suppressed blood supply) on cell signaling events and the functional role of the calcium store in cultured, pterygial-derived fibroblasts. METHODS: Pure fibroblast cultures were established from cell outgrowths of pterygial tissue. Growth and migration of pterygial-derived fibroblast was evaluated using a patch growth assay, MTS assay, and a scratch wound assay. Intracellular calcium levels were determined using Fura-2 detection in response to ligand stimulation using a 96-well plate format. RESULTS: A progressive increase in serum concentration resulted in promotion of pterygial cell growth detected using the MTS assay. A significant increase in intracellular calcium level was observed in response to histamine (10 and 100 µM), ATP (10 and 100 µM), acetylcholine (10 and 100 µM) and epidermal growth factor (10 ng/mL) in serum-maintained cells. However, no significant changes were observed when cells were maintained in serum-free medium. Thapsigargin (1 µM), a Ca-ATPase inhibitor, induced a significantly greater increase in intracellular calcium level in the serum-maintained group relative to serum-starved cells. In both cases, elevation of intracellular calcium was reduced when calcium-free bathing medium was used. Preincubation of cells with 1 µM thapsigargin ablated ligand-induced calcium responses. Disruption of calcium signaling through thapsigargin treatment significantly perturbed cell growth and migration. CONCLUSIONS: Receptor-induced calcium signaling activity is suppressed in pterygial-derived fibroblasts in response to serum deprivation. This correlates with reduced growth rates and a depleted endoplasmic reticulum calcium store. The store plays a key role in cell growth and migration of pterygial-derived fibroblasts. Therefore, the strategic reduction of the vascular network in pterygium will affect the calcium store level and in turn affect functional responses associated with pterygia.

A fully human in vitro capsular bag model to permit intraocular lens evaluation.

PURPOSE: To establish a fully human in vitro culture model with which to test the putative effects of intraocular lens (IOL) designs in preventing posterior capsule opacification (PCO) after cataract surgery. METHODS: A sham cataract operation was performed to prepare human capsular bags from donor lenses. In one capsular bag of a donor pair, an intraocular lens (PMMA round-edge IOL or acrylic IOL) was implanted while the other capsular bag remained aphakic. Bags were transferred to a Petri dish and secured anterior-face down using entomological pins. Capsular bags were maintained in Eagle's minimum essential medium supplemented with 2% human serum and 10 ng/mL TGF-ß to drive growth and matrix contraction. RESULTS: In the absence of an IOL, cells appeared within the central posterior capsule at 4.38 ± 0.26 days, whereas in the presence of a PMMA round-edge IOL or an acrylic IOL they appeared at 8 ± 0.41 days and 11 ± 0.7 days, respectively. Immunocytochemical analysis showed an accumulation of cells at the edge of the acrylic IOL and a less evident accumulation with the PMMA round-edge IOL. Moreover, matrix contraction was more prominent in the absence of an IOL but was still apparent, to a lesser degree, in the presence of a PMMA round-edge IOL. The acrylic IOL greatly suppressed matrix contraction. CONCLUSIONS: The authors have developed a fully human in vitro capsular bag system that relates well to clinical observations and permits the testing of novel intraocular lenses.

Regional differences in store-operated Ca2+ entry in the epithelium of the intact human lens.

PURPOSE: An elevated level of Ca(2+) is an important factor in cataract, yet precisely how Ca(2+) enters the lens is unknown. Lens epithelial cells contain a range of G-protein-coupled receptors and receptor tyrosine kinases that induce increases in intracellular Ca(2+). Receptor-associated Ca(2+) influx is, therefore, likely to be an important route for Ca(2+) influx to the lens. The authors investigated stimulated and passive Ca(2+) influx in in situ human lens epithelium. METHODS: Ca(2+) changes in equatorial (E) and central anterior (CA) epithelial cells were monitored with the use of a Ca(2+) indicator (Fluo4) and confocal microscopy. Gene expression was monitored by RT-PCR and immunoblotting. RESULTS: Adenosine triphosphate (ATP) induced Ca(2+) responses that were smaller in CA than E. Ca(2+) store depletion, using ATP (100 microM) or thapsigargin (1 microM), revealed greater relative store capacity and Ca(2+) influx in E. Ca(2+) influx was blocked by La(3+) (0.5 microM) in both regions. Unstimulated Ca(2+) influx was greater in E than CA. Greater expression of Orai1 and STIM1 was detected in E than in CA. CONCLUSIONS: Greater Ca(2+) store capacity and Ca(2+) influx in E compared with CA reflects underlying differences in proliferation and differentiation between the regions. The relatively small resting Ca(2+) influx in CA epithelium suggests that store-operated Ca(2+) entry (SOCE) is the main route of Ca(2+) influx in these cells. Greater resting influx and SOCE in E cells suggests that these are a major route for Ca(2+) influx into the lens. Increased expression of Orai1 and STIM1 in E could account for the differences in Ca(2+) entry. Receptor activation will modulate Ca(2+) influx, and inappropriate activity may contribute to cortical cataract.

Cataract surgery with toric intraocular lenses can optimize uncorrected postoperative visual acuity in patients with marked corneal astigmatism.

PURPOSE: To study the change in visual acuity and refraction after cataract surgery using a toric posterior chamber intraocular lens in patients with astigmatism after penetrating keratoplasty. METHODS: A retrospective case note analysis of cataract surgery involving toric lens implants performed at the Norfolk and Norwich University Hospital was conducted. The pre- and postoperative visual acuities and refractions were recorded. RESULTS: Seven consecutive patients are described (5 men and 2 women) with a mean age of 62 years. They all underwent penetrating keratoplasty, and in every case, all sutures were removed (mean, 11.2 months before cataract surgery). A marked improvement in both unaided visual acuity and astigmatism was shown after the procedure. The average preoperative unaided acuity was 6/120 (range, 6/24 to counting fingers) compared with a postoperative unaided visual acuity average of 6/15 (6/9-6/24). The average preoperative cylinder was 10.12 D (range, 3.40-17.89 D); postoperatively, this fell to 2.75 D (range, 0.75-4.25). CONCLUSIONS: Cataract surgery with toric intraocular lenses allows the correction of high degrees of regular corneal astigmatism. We discussed the potential advantages and complications of performing toric lens cataract surgery as a secondary procedure.

Acanthamoeba keratitis associated with misuse of daily disposable contact lenses.

PURPOSE: To report a case of Acanthamoeba keratitis that occurred in a daily disposable contact lens wearer. METHOD: Case report. RESULTS: A 70-year-old gentleman presented to eye casualty with a red left eye with reduced vision and corneal epithelial changes. Acanthamoeba keratitis was not suspected initially as he was a daily disposable contact lens wearer. This led to a delay of 17 days in initiating treatment. He responded well to topical polyhexamethylene biguanide and propamidine and made a full recovery. CONCLUSION: Acanthamoeba keratitis can occur in patients who reuse daily disposable contact lenses. A diagnosis of Acanthamoeba keratitis can be difficult to make and should be considered in all patients who wear contact lenses, including daily disposable lenses. All patients who wear daily disposable contact lenses should be reminded that the benefits of this modality of contact lens are only possible if the lenses are worn once and thrown away.