ABSTRACT
It has been suggested that unoprostone isopropyl (UNO) has potent neuroprotective activity in the retina. The effect of sustained transscleral UNO delivery to the posterior segment of the eye on photoreceptor degeneration was evaluated. UNO was loaded into a device made of poly(ethyleneglycol) dimethacrylate by polydimethylsiloxane mold-based UV-curing. The amount of UNO diffusing from these devices was measured using high-performance liquid chromatography. The polymeric devices that released UNO at 1.8 µg/day were implanted on the sclerae of S334ter rats at postnatal 21 days, and electroretinograms (ERGs) were compared with those of topical application and placebo devices. Retinal thickness was evaluated by histological examination. Western blots of specimens 4 weeks after implantation were performed. ERGs showed that the UNO-loaded device prevented the reduction of ERG amplitudes 2 and 4 weeks after implantation, compared with results using a placebo device or topical application. Histological examination showed that the UNO-loaded device prevented the reduction of retinal thickness, and Western blots of specimens indicated that the UNO-loaded device decreased expression of ERK1/2, phosphorylated ERK1/2, and caspase-3. A device that provided sustained UNO administration protected against retinal degeneration in rhodopsin mutant rats, and thus, may have translational potential as a sustainable method to administer drugs to treat retinitis pigmentosa. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1730-1737, 2016.
Subject(s)
Dinoprost/analogs & derivatives , Mutation , Retina/metabolism , Retinitis Pigmentosa/metabolism , Retinitis Pigmentosa/therapy , Sensory Rhodopsins/genetics , Animals , Dinoprost/pharmacology , Gene Expression Regulation , Rats , Rats, Mutant Strains , Retinitis Pigmentosa/genetics , Sensory Rhodopsins/metabolismABSTRACT
Controlled transscleral co-delivery of two drugs, edaravone (EDV) and unoprostone (UNO), using a platform that comprises a microfabricated reservoir, controlled-release cover, and drug formulations, which are made of photopolymerized poly(ethyleneglycol) dimethacrylates, shows synergistic retinal neuroprotection against light injury in rats when compared with single-drug-loaded devices. The device would offer a safer therapeutic method than intravitreal injections for retinal disease treatments.
Subject(s)
Drug Delivery Systems/instrumentation , Drug Delivery Systems/methods , Retina/metabolism , Retinal Diseases/drug therapy , Administration, Ophthalmic , Animals , Antipyrine/administration & dosage , Antipyrine/analogs & derivatives , Antipyrine/pharmacokinetics , Dinoprost/administration & dosage , Dinoprost/analogs & derivatives , Dinoprost/pharmacokinetics , Drug Combinations , Edaravone , Equipment Design , Methacrylates/chemistry , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/pharmacokinetics , Polyethylene Glycols/chemistry , Polymethacrylic Acids/chemistry , Prostheses and Implants , Rats , Retina/radiation effects , Retinal Diseases/etiology , Retinal Diseases/metabolism , Retinal Diseases/prevention & control , Sclera/surgeryABSTRACT
LIM-kinases (LIMKs) play crucial roles in various cell activities, including migration, division, and morphogenesis, by phosphorylating and inactivating cofilin. Using a bimolecular fluorescence complementation assay to detect the actin-cofilin interaction, we screened LIMK1 inhibitors and identified two effective inhibitors, damnacanthal (Dam) and MO-26 (a pyrazolopyrimidine derivative). These compounds have already been shown to inhibit Lck, a Src family tyrosine kinase. However, in vitro kinase assays revealed that Dam inhibited LIMK1 more effectively than Lck. Dam suppressed LIMK1-induced cofilin phosphorylation and deceleration of actin retrograde flow in lamellipodia in N1E-115 cells. Dam impaired CXCL12-induced chemotactic migration of Jurkat T lymphocytes and Jurkat-derived, Lck-deficient JCaM1.6 cells and also inhibited serum-induced migration and invasion of MDA-MB-231 breast carcinoma cells. These results suggest that Dam has the potential to suppress cell migration and invasion primarily through the inhibition of LIMK kinase activity. Topical application of Dam also suppressed hapten-induced migration of epidermal Langerhans cells in mouse ears. Dam provides a useful tool for investigating cellular and physiological functions of LIMKs and holds promise for the development of agents against LIMK-related diseases. The bimolecular fluorescence complementation assay system used in this study will provide a useful method to screen for inhibitors of various protein kinases.
Subject(s)
Anthraquinones/pharmacology , Cell Movement/drug effects , Lim Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pseudopodia/drug effects , Actins/antagonists & inhibitors , Actins/genetics , Actins/metabolism , Animals , COS Cells , Cell Line, Tumor , Chemokine CXCL12/pharmacology , Chlorocebus aethiops , Cofilin 1/antagonists & inhibitors , Cofilin 1/genetics , Cofilin 1/metabolism , Gene Expression Regulation , High-Throughput Screening Assays , Humans , Jurkat Cells , Langerhans Cells/cytology , Langerhans Cells/drug effects , Langerhans Cells/metabolism , Lim Kinases/genetics , Lim Kinases/metabolism , Mice , Microscopy, Fluorescence , Pseudopodia/metabolism , Pyrimidines/pharmacology , Signal TransductionABSTRACT
Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin.
Subject(s)
Actin Cytoskeleton/drug effects , Actin Depolymerizing Factors/antagonists & inhibitors , Actins/antagonists & inhibitors , Cytochalasin D/pharmacology , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Animals , COS Cells , Chlorocebus aethiopsABSTRACT
The bimolecular fluorescence complementation (BiFC) assay is a method for visualizing protein-protein interactions in living cells. To visualize the cofilin-actin interaction in living cells, a series of combinations of the N- and C-terminal fragments of Venus fused upstream or downstream of cofilin and actin were screened systematically. A new pair of split Venus fragments, Venus (1-210) fused upstream of cofilin and Venus (210-238) fused downstream of actin, was the most effective combination for visualizing the specific interaction between cofilin and actin in living cells. This pair of Venus fragments was also effective for detecting the active Ras-dependent interaction between H-Ras and Raf1 and the Ca(2+)-dependent interaction between calmodulin and its target M13 peptide. In vitro BiFC assays using the pair of purified BiFC probes provided the means to detect the specific interactions between cofilin and actin and between H-Ras and Raf1. In vivo and in vitro BiFC assays using the newly identified pair of Venus fragments will serve as a useful tool for measuring protein-protein interactions with high specificity and low background fluorescence and could be applied to the screening of inhibitors that block protein-protein interactions.
