Abstract
Purpose: :
Chemical and alkali burns cause extensive damage to the anterior aspects of the eye resulting in corneal neovascularization and fibrosis. The pathological induction of alkali-induced retinal gliosis is a largely unexplored facet of clinical medicine and basic scientific research. Here we have exploited withaferin A (WFA) to probe these disease pathways by focusing on its newly discovered vimentin-targeting activity (Bargagna-Mohan et al., Chemistry & Biology 2007).
Methods: :
Under anesthesia, mouse eyes were treated by brief application of 0.15 N NaOH and corneal epithelium was removed to induce angiogenesis. Wild-type 129 Svev or vimentin-deficient (vim-/-) mice were systemically treated with injection of withaferin A for seven days and killed. Their eyes were processed either for cryo-embedded immunohistochemical analysis with epifluorescence detection, western blot analysis or corneal flatmount CD31 staining. IFs in Muller cells were characterized from digital images by computer-assisted quantification and data validated by statistical analysis.
Results: :
In the injured cornea, WFA exerts potent anti-angiofibrotic activity at 2 mg/kg as demonstrated by decreased capillary density, inhibition of polyubiquitinated protein expression, inhibition of NF-ΚB activation and downregulation of vimentin and -smooth muscle actin expression in injured tissues. Both suppression of corneal angiogenesis and protein ubiquitination by WFA were abrogated in vim-/- mice. Interestingly, even mild alkali injury caused upregulation of vimentin and GFAP expression by day 7 post-injury in retinal Muller glia, a hallmark of gliosis. WFA treatment in wild-type mice caused potent downregulation of vimentin expression in retinal ganglion and Muller cells, and GFAP-stained IFs were reduced in density and filament length. This filament density and length reduction phenocopies the functional perturbation of GFAP in the injured state produced by genetic deficiency of vimentin. WFA treatment of vim-/- mice did not further reduce GFAP expression.
Conclusions: :
Gliosis undermines several retinal pathological conditions, including age-related macular degeneration, diabetic retinopathy and glaucoma where currently there is no treatment for this disease axis. Our findings identify WFA as an important drug-like agent having broad polypharmacological activities that could be exploited for a wide range of diseases driven by type III IF overexpression.
Keywords: neovascularization • Muller cells • signal transduction: pharmacology/physiology