thapsigargin and Cataract

Epithelial‑to‑mesenchymal transition (EMT) of human lens epithelial cells (HLECs) serve an important role in cataract formation. The endoplasmic reticulum stress response (ER stress) has been demonstrated to regulate EMT in a number of tissues. The aim of the present study was to demonstrate the role of ER stress on EMT in HLECs. HLECs were treated with tunicamycin (TM) or thapsigargin (TG) to disturb ER homeostasis, and 4‑phenylbutyric acid (PBA) or sodium tauroursodeoxycholate (TUDCA) to restore ER homeostasis. Cell morphology was evaluated after 24 h. The long axis and aspect ratio of the cells were analyzed using ImageJ software. The results demonstrated that HLECs adopted an elongated morphology following treatment with TG, and the cellular aspect ratio increased. However, this morphological change was not observed following combination treatment with TG and PBA. Western blot analysis and immunofluorescence staining were used to measure the protein expression levels. A wound‑healing assay was performed to evaluate cell migration. Treatment with TM or TG increased the expression of the ER stress markers glucose‑regulated protein 78, phosphorylated eukaryotic initiation factor 2α, activating transcription factor (ATF)6, ATF4 and inositol‑requiring protein 1α and the EMT markers fibronectin, vimentin, α‑smooth muscle actin and neural cadherin. Furthermore, treatment with TM or TG decreased the expression of the epithelial cell marker epithelial cadherin and enhanced cell migration, which effects were inhibited following treatment with PBA or TUDCA. These results indicates that enhanced ER stress induced EMT and subsequently increased cell migration in HLECs in vitro.

Topics: Cataract; Cell Line; Endoplasmic Reticulum Stress; Epithelial Cells; Epithelial-Mesenchymal Transition; Eye Proteins; Humans; Lens, Crystalline; Phenylbutyrates; Taurochenodeoxycholic Acid; Thapsigargin; Tunicamycin

To investigate, by transmission electron microscopy (TEM), the effect on the posterior capsule of a young rabbit eye of 5-fluorouracil (5-FU) or thapsigargin in a sealed-capsule irrigation device.. St Erik's Eye Hospital, Stockholm, Sweden.. Clear lens extraction was performed unilaterally in eight 4-week-old rabbits. A sealed-capsule irrigation device was irrigated for 2 min with 20 ml of one of the following: balanced salt solution (BSS; n=2), thapsigargin 300 muM (n=2), 5-FU 50 mg/ml (n=2), or 5-FU 25 mg/ml (n=2). The substances were washed out for 10 s with BSS. The eyes were left aphakic. Six weeks postoperatively, the animals were killed, and the posterior capsule was extracted and fixed for TEM. As a control, we also evaluated the capsules from the two fellow eyes in the BSS group that did not undergo surgery.. The ultrastructure of the posterior capsule in eyes irrigated with 5-FU or thapsigargin did not differ from that in the eyes irrigated with BSS or in the eyes that did not have surgery. The membranes had the same ultrastructure with thin collagen fibres on the anterior and posterior face of the posterior capsule and an amorphic matrix.. Thapsigargin or 5-FU used in a sealed-capsule irrigation device does not seem to harm the posterior capsule, which appeared similar to when the capsule is irrigated with BSS.

Topics: Animals; Antimetabolites; Cataract; Enzyme Inhibitors; Fluorouracil; Lens Capsule, Crystalline; Lens, Crystalline; Microscopy, Electron, Transmission; Rabbits; Thapsigargin; Therapeutic Irrigation

To investigate the effect on after-cataract and synechiae formation after clear lens extraction and irrigation with different substances in a sealed-capsule irrigation device.. St Erik's Eye Hospital, Stockholm, Sweden.. Clear lens extraction was performed in one eye of 42 4-week-old rabbits. The sealed-capsule irrigation device was applied and the sealed system was irrigated for 2 min with 20 ml of one of four substances: balanced salt solution (BSS), thapsigargin, 5-fluorouracil (5-FU) 50 mg/ml, or 5-FU 25 mg/ml. The substance then was washed out for 10 s with BSS. The eyes were left aphakic. Formation of after-cataract and synechiae was evaluated during two clinical examinations 3.5 and 5.5 weeks postoperatively, by photographs 5 weeks postoperatively, and by histologic evaluation of haematoxylin-eosin-stained slides after the 6-week end point postoperatively. After-cataract and synechiae were graded on a scale from 0 to 4. Kruskal-Wallis analysis of variance with multiple comparisons was used for statistical analyses.. 5-FU 50 mg/ml prevented after-cataract and synechiae formation best when compared with all other substances at all evaluations. 5-FU 25 mg/ml was not as effective, and thapsigargin was ineffective in this animal model.. 5-FU 50 mg/ml used in the sealed-capsule irrigation device satisfactorily prevents after-cataract and synechiae. Thapsigargin was ineffective in this animal model.

Topics: Animals; Antimetabolites; Cataract; Enzyme Inhibitors; Fluorouracil; Lens, Crystalline; Postoperative Complications; Rabbits; Thapsigargin; Therapeutic Irrigation

Despite recent improvements in IOL design posterior capsule opacification (PCO) remains a significant clinical problem after cataract surgery. The Perfect Capsule device (Milvella, Ltd., Epping, Australia) permits the introduction and subsequent removal of potentially toxic agents into the closed capsular bag. The present purpose was to compare the relative effectiveness of exposing cells within the human capsular bag with a range of stresses with clinical potential and to compare the response to the same agents when applied to rabbit capsular bags and to cultured human lens cells.. Human capsular bags were prepared from donor eyes and sealed with the Perfect Capsule device. Distilled water, 3 M NaCl, 250 microg/mL, 25 mg/mL 5-fluorouracil and 100 microM thapsigargin (Tg) were introduced for 2 minutes. The bags were then perfused with Eagle's minimum essential medium (EMEM) and an IOL inserted before the bags were dissected and pinned to the base of plastic culture dishes. The bags were maintained in EMEM for 28 days and phase images were acquired throughout. Rabbit eyes were prepared and cultured in a similar manner, although tests were limited to 5-FU (25 mg/mL) and Tg (30-300 microM). FHL124 cells were cultured on plastic (EMEM supplemented with 5% FCS), and serum was removed for 24 hours before exposure to the same agents as human bags. Cell survival was assessed by quantifying Coomassie blue staining after 4 days.. Initially, NaCl induced by far the most obvious signs of cell death, especially of anterior cells, followed by 5-FU>water>Tg. However, by 2 weeks, cell death became more apparent in the Tg-exposed bags, and, at the end of 4 weeks, there were no cells surviving. Cells on the posterior capsule were confluent in water-exposed bags (similar to unexposed controls), whereas after NaCl exposure, coverage was incomplete but greater than after 5-FU. In the rabbit bags, exposure to 25 mg/mL 5-FU totally eliminated cells, but 100 microM Tg was ineffective. At the end of a 4-day culture of FHL124 cells exposed for 2 minutes to NaCl, 5-FU or Tg, there were no cells surviving, whereas there was 50% cell survival compared with control cells after water treatment.. Tissue-cultured human lens cells are much more sensitive to short-term hyperosmotic than hyposmotic stress, with a rapid onset of cell death of cultured cells exposed to 3 M NaCl. This finding was confirmed in human capsular bags although adherent fibers appear to offer additional protection to 5-FU which can be overcome by the very hydrophobic Tg. The application of the Perfect Capsule system in concert with thapsigargin provides a promising means of preventing PCO.

Topics: Actins; Animals; Cataract; Cell Culture Techniques; Cell Survival; Fluorescent Antibody Technique, Indirect; Fluorouracil; Humans; Lens Capsule, Crystalline; Lens Implantation, Intraocular; Lens, Crystalline; Lenses, Intraocular; Microscopy, Confocal; Osmotic Pressure; Rabbits; Sodium Chloride; Thapsigargin; Vimentin

The effects of steroids on the steady-state intracellular [Ca(2+)] ([Ca(2+)](i)) and resting Ca(2+) influx in Fura-2-loaded bovine lens epithelial cells were examined to identify potential rapid, non-genomic actions. When administered in the presence of 1-2 mM extracellular Ca(2+) ([Ca(2+)](o)), 100 micro M progesterone produced large (up to 12-fold) and transient (5 min) increases in [Ca(2+)](i). These effects were abolished in EGTA-containing solutions, and were associated with large increases in the rate at which extracellularly administered Mn(2+) quenched the intracellular Fura signal. Lower concentrations of progesterone (10-100 micro M) produced smaller increases in [Ca(2+)](i) that were concentration dependent, and 17beta-estradiol induced large, rapid and brief increases in [Ca(2+)](i) at 100 nM and smaller oscillations in [Ca(2+)](i) at 10 nM. In cells pretreated with thapsigargin, 100 micro M progesterone produced slower increases in [Ca(2+)](i) that were maintained for several minutes. These results demonstrate rapid non-genomic actions of progesterone and estradiol on resting Ca(2+) influx and [Ca(2+)](i) that may involve specific interactions with a recently discovered steroid-binding protein in the plasma membrane of lens epithelial cells.

Topics: Animals; Apoptosis; Biological Transport; Calcium; Cataract; Cattle; Cell Membrane; Cells, Cultured; Electric Capacitance; Enzyme Inhibitors; Epithelial Cells; Estradiol; Lens, Crystalline; Manganese; Progesterone; Thapsigargin

Topics: Cataract; Cataract Extraction; Cell Division; Cell Movement; Drug Delivery Systems; Epithelium; Humans; Lens Capsule, Crystalline; Postoperative Complications; Thapsigargin

Cataract is responsible for rendering several million people blind throughout the world and is also by far the most common cause of low visual acuity. Although cataract surgery is common, routine and effective, posterior capsule opacification (PCO) occurs in 30-50% of patients following modern cataract surgery. This condition arises from stimulated cell growth within the capsular bag after surgery. The resulting decline in visual acuity requires expensive laser treatment, and PCO therefore prevents modern cataract surgery from being carried out routinely in underdeveloped countries. The present study, using a human lens capsular bag culture system, has confirmed that cells from a wide age range of donors proliferate in the absence of added serum protein and explains why PCO is such a common problem even in aged patients. This study also provides one possible solution for PCO by using polymethylmethacrylate (PMMA) implanted intraocular lenses as a drug delivery system. PMMA lenses coated with thapsigargin, a hydrophobic inhibitor of endoplasmic reticulum (ER) (Ca2+)-ATPase, greatly reduced cell growth in the capsular bag at relatively low coating concentrations (200 nM) but, more significantly, induced total cell death of the residual anterior epithelial cells at higher concentrations (>2 microM).

Topics: Calcium-Transporting ATPases; Cataract; Cell Division; Drug Delivery Systems; Endoplasmic Reticulum; Enzyme Inhibitors; Epithelial Cells; Epithelium; Humans; In Vitro Techniques; Lens Capsule, Crystalline; Lenses, Intraocular; Methylmethacrylates; Models, Biological; Thapsigargin