Search

Purpose To evaluate the role of protein tyrosine phosphatase (PTP) in controlling the integrity of cell–cell junction and intercellular permeability in postconfluent bovine corneal epithelial cells. Methods Confluent cultures of bovine corneal epithelial cells were treated with different concentrations of general phosphate inhibitors andsodium orthovanadate for varying periods. An MTS assay was used to confirm no cellular death under the treatment profile. Immunocytochemical (ICC) analysis was performed to demonstrate protein tyrosine phosphorylation after treatment with sodium orthovanadate, and the effect of sodium orthovanadate on junctional proteins such as p120, α-catenin, occludin, ZO-1, and ZO-2. Western blot analysis was used to analyze the changes in p120, α-catenin, occludin, ZO-1, and ZO-2 after treatment. Paracellular permeability was evaluated by transepithelial electrical resistance (TER). Results During the whole course of treatment, no significant cellular death was noticed. Dose- and time-dependent effects of sodium orthovanadate on protein tyrosine phosphorylation were confirmed by ICC. ICC also demonstrated the dose- and time-dependent effect of sodium orthovanadate on the disruption of p120, α-catenin, occludin, ZO-1, and ZO-2. However, results of Western blot analysis showed no change in the expression levels of p120, α-catenin, occludin, ZO-1, and ZO-2. Dose- and time-dependent increase of paracellular permeability was evaluated by TER. Conclusion Inhibition of protein tyrosine phosphatase activity can increase protein tyrosine phosphorylation. A dose- and time-dependent release of cell–cell contacts and increased transepithelial permeability were found in postconfluent culture of bovine corneal epithelial cells.

PURPOSE: To compare the bacteriostatic effects, corneal cytotoxicity, and ability to seal corneal incisions among fibrin glue and 2 commercially available cyanoacrylate derivatives: N-butyl cyanoacrylate and methoxypropyl cyanoacrylate. METHODS: The bacteriostatic activities of these tissue glues were verified by measuring the zones of bacterial growth inhibition surrounding the adhesive droplets on agar plates inoculated with Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, Escherichia coli, or Mycobacterium chelonae. Corneal cytotoxicity was tested by a direct contact method by using cultured bovine corneal epithelial cells, keratocytes, and corneal endothelial cells challenged with droplets of adhesives. Each of the cells was treated with droplets of adhesives. The ability to seal corneal incisions was verified by calculating the maximum intraocular pressure resistant to leakage of rabbit corneal stab wounds sealed with tissue adhesives. RESULTS: Methoxypropyl cyanoacrylate and N-butyl cyanoacrylate showed bacteriostatic effects against S. aureus, S. pneumoniae, and M. chelonae but not P. aeruginosa and E. coli. In contrast, fibrin glue had no such effects against either Gram-positive or -negative bacteria (P < 0.01). Methoxypropyl cyanoacrylate showed the highest levels of corneal cytotoxicity, followed by N-butyl cyanoacrylate. Fibrin glue, however, showed minimal cytotoxicity (P < 0.01). Methoxypropyl cyanoacrylate and N-butyl cyanoacrylate also displayed a greater ability to seal corneal incisions than that of fibrin glue (P < 0.01). CONCLUSIONS: The bacteriostatic effects, corneal cytotoxicity, and ability to seal corneal incisions differed among the 3 compounds tested. These different properties should be considered when choosing tissue adhesives during corneal surgery.

Baicalein, a pure compound derived from Scutellaria baicalensis Georgi, protected cells from lethal damage in an ischemia-reperfusion model. This study was aimed to investigate the role of opioid receptors in mediating cardioprotection by baicalein against hypoxia-reoxygenation injury. By using chick cardiomyocyte as in vitro model, baicalein was added to the perfusate during 1 h-hypoxia followed by 1 h-reoxygenation. Cell viability was assessed by propidium iodide uptake, while apoptosis was assessed by TUNEL and Hoechst 33342 staining. The expression of opioid receptors mRNA in chicken embryonic myocardium was determined by RT-PCR. Opioid receptor antagonists, protein kinase C inhibitors, and KATP channel blockers were used to determine the presumed signal transduction pathways. The results showed that baicalein (0.1 approximately 5 microM) concentration dependently reduced hypoxia-reoxygenation-induced myocardial death and apoptosis. The cardioprotective effect of baicalein (1 microM) was blocked by pretreatment of nonspecific opioid receptor antagonist (naloxone), opioid mu-receptor (beta-funaltrexamine) and delta-receptor (7-Benzylidenenaltrexone) antagonists, protein kinase C inhibitors (H7 and chelerythrine), and KATP channel blockers (glibenclamide and 5-hydroxydecanoate). Finally, RT-PCR analysis successfully demonstrated the presence of opioid receptors mRNA in chicken embryonic cardiomyocytes. We conclude that the cardioprotective effect of baicalein is mediated via mu-, delta- but not kappa-opioid receptor and their related signal transduction pathways, such as protein kinase C and the KATP channel.

Cell–cell interaction, junctional permeability and contact inhibition are important mechanisms that allow corneal endothelial cells to maintain stable corneal hydration status and also keep these cells in non-proliferative status. Protein tyrosine phosphatases (PTPs) are well known to play an important role in regulating cell–cell contacts, growth and differentiation. Inhibition of PTPs activity by a general PTP inhibitor has been proved to trigger post-confluent rat corneal endothelial cells to reenter cell cycles. In this study, we aimed to evaluate whether protein tyrosine phosphorylation is involved in cell–cell interactions, junctional permeability and cell cycle control in post-confluent, contact inhibited bovine corneal endothelial cells. Confluent cultures of bovine corneal endothelial cells were treated with different concentrations of general phosphatase inhibitor, sodium orthovanadate (vanadate) for several different durations. Protein tyrosine phosphorylation was confirmed by Western blot analysis. Immunocytochemistry was used to evaluate the effect of vanadate on adherens-type junctional proteins by staining of p120, N-cadherin and α-catenin. Paracelluar permeability was evaluated by transepithelial electric resistance. The effect of vanadate on cell cycle progression was confirmed by immunostaining of Ki67. Western blot analysis was used to evaluate the expression level of cell-cycle-associated proteins, including PCNA, cyclin D1, cyclin E and cyclin A. Time-dependent effects of vanadate on protein tyrosine phosphorylation were confirmed by Western blot analysis. ICC demonstrated the effect of vanadate on the disruption of p120, N-cadherin and α-catenin. Time- and dose-effects of vanadate on the severity of p120 disruption were also found. TER demonstrated the time- and dose-effect of vanadte on paracellular permeability. Although cell–cell junctions can be broken through by vanadate, no significant increase of Ki67 positive cells was found among the control group and all groups with different concentrations/durations of vanadate treatment. Western blot also showed no change of PCNA, cyclin D1, cyclin E and cyclin A after treatment with vanadate. In conclusion, protein tyrosine phosphatase inhibition can induce time-dependent release of cell–cell contacts and increase transepithelial permeability in post-confluent cultures of bovine corneal endothelial cells. However, such phenomenon is not enough to promoted cell cycle progression as seen in rat corneal endothelial cells.

PURPOSE. Bevacizumab is a potent recombinant humanized monoclonal antibody directed against vascular endothelial growth factor (VEGF). The purpose of this study was to evaluate the therapeutic effect of subconjunctival injection of bevacizumab on corneal neovascularization (NV) in different rabbit models. METHODS. Several rabbit models of comeal NV were used, including (1) a corneal micropocket assay with VEGF pellet, (2) a comeal micropocket assay with basic fibroblast growth factor (b-FGF) pellets, (3) mechanical limbal injury-induced corneal NV, and (4) an alkali-induced model of corneal NV. Subconjunctival injections of bevacizumab (0.25-2.5 mg) were applied twice per week for 2 to 8 weeks. Digital photographs of the cornea were analyzed to determine the length of corneal NV and the area of cornea covered by NV as a percentage of the total corneal area. Immunohistochemical staining with antihuman IgG antibody labeled with Cy3 was used to determine the detection of intracorneal distribution of bevacizumab after injection. RESULTS. Subconjunctival injection of bevacizumab caused significant inhibition of corneal NV formation as measured by length or surface area in all animal models (P < 0.05). No significant ocular complications were found. Staining of bevacizumab was found in the corneal stroma for 3 to at least 14 days in the different rabbit models. CONCLUSIONS. Subconjunctival injection of bevacizumab is effective in inhibiting corneal NV in several rabbit models. Bevacizumab may diffuse into the corneal stroma and persist for a few days after injection. It may be useful in preventing corneal NV in the acute phase of various kinds of corneal inflammation.