withanolides and Corneal-Neovascularization

To develop an animal model for simultaneously eliciting corneal angiogenesis and retinal gliosis that will enable the assessment of inhibitor efficacy on these two pathological processes in separate anatomic sites of the ocular globe.. Four to six week-old mice in a C57BL/6J background were anesthetized and 0.15 N NaOH was applied to the cornea, followed by mechanical scraping of the epithelium from limbus and central cornea. After this injury, mice were treated with vehicle or with an inhibitor (withaferin A [WFA]), which were delivered by intraperitoneal injection, to assess the pharmacological effects on angiogenesis and/or gliosis. Mice were sacrificed after 14 days and tissues (corneas and retinas) were prepared for analysis of corneal neovascularization and retinal gliosis by immunohistochemistry and western blotting, respectively. This protocol was also suited for studying earlier disease end points, for assessment of drug dose efficacy or genetic influences and the entire procedure and this analysis was completed in 16-17 days.. Both corneal angiogenesis and retinal gliosis were maximally sustained at fourteen days following chemical and mechanical injury of the cornea. 1) Injured corneas showed abundant CD31+ staining, with new blood vessels branching out from the limbus to the central cornea. WFA treatment potently inhibited corneal neovascularization. 2) Retinal gliosis in injured mice was associated with upregulated expression of glial fibrillary acidic protein (GFAP) that appeared as polymeric filaments and soluble forms expressed in reactive Müller glial cells. WFA treatment potently downregulated the expression of soluble and filamentous GFAP; the latter protein was fragmented.. We have developed a mouse model for investigating retinal gliosis and corneal neovascularization. We used this model to demonstrate the simultaneous inhibitory effects of WFA on both of these disease processes. Retinal gliosis occurs in several major degenerative conditions of the eye, including age-related macular degeneration, where angiogenesis is also a prevailing pathological feature. Thus, inhibitors of both gliosis and angiogensis used as combination therapy are currently being explored for treatment of such complex diseases. The model presented here affords a very simple preclinical assay for screening combination of drugs or polypharmacological agents and reduces the numbers of animals because of the different anatomic sites of these pathologies. Finally, given that endogenous mediators elicit angiogenesis and gliosis in this model, the combination of genetics and pharmacology can be exploited to study drug mechanisms and for target validation in vivo.

Topics: Animals; Blotting, Western; Cornea; Corneal Injuries; Corneal Neovascularization; Disease Models, Animal; Eye Injuries; Glial Fibrillary Acidic Protein; Gliosis; Humans; Immunochemistry; Injections, Intraperitoneal; Macular Degeneration; Mice; Mice, Inbred C57BL; Neuroglia; Platelet Endothelial Cell Adhesion Molecule-1; Retina; Sodium Hydroxide; Withanolides

The natural product withaferin A (WFA) exhibits antitumor and antiangiogenesis activity in vivo, which results from this drug's potent growth inhibitory activities. Here, we show that WFA binds to the intermediate filament (IF) protein, vimentin, by covalently modifying its cysteine residue, which is present in the highly conserved alpha-helical coiled coil 2B domain. WFA induces vimentin filaments to aggregate in vitro, an activity manifested in vivo as punctate cytoplasmic aggregates that colocalize vimentin and F-actin. WFA's potent dominant-negative effect on F-actin requires vimentin expression and induces apoptosis. Finally, we show that WFA-induced inhibition of capillary growth in a mouse model of corneal neovascularization is compromised in vimentin-deficient mice. These findings identify WFA as a chemical genetic probe of IF functions, and illuminate a potential molecular target for withanolide-based therapeutics for treating angioproliferative and malignant diseases.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Agents; Apoptosis; Binding Sites; Blotting, Western; Cell Line; Corneal Neovascularization; Electrophoresis, Gel, Two-Dimensional; Ergosterol; Fibroblasts; Flow Cytometry; Humans; Mice; Mice, Knockout; Models, Molecular; Molecular Structure; Protein Binding; Vimentin; Withanolides