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J. T. Durham, I. M. Herman; Endothelial-Specific and Drug-Inducible βcap73 Expression Impairs Retinal Microvascular Remodeling in a Mouse Model of Retinopathy of Prematurity. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3654.
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Retinal capillary endothelial cell (RCEC) migration, a critical step in angiogenesis, is driven by cytoskeletal remodeling, and is mainly achieved by the dynamic regulation of the β-actin network. Previous work reveals that the β-actin network is enriched in motile cytoplasm of RCEC during microvascular morphogenesis, and is modulated by the isoactin-specific capping protein, βcap73. We predict that regulated RCEC βcap73 over-expression could disrupt angiogenesis by perturbing β-actin-dynamics. Indeed, recent work has shown that adenoviral-βcap73 RCEC infection disrupts cytoskeletal architecture, inhibits motility and morphogenesis, and, induces anoikis, or detachment-dependent apoptosis.
We are validating our in vitro work with a ‘two-mouse’ transgenic approach in retinopathy of prematurity (ROP) studies. We have subcloned βcap73 into a tetracycline-inducible vector, pTRE2, and confirmed expression using cell culture transfection. Using this vector, we have established several transgenic lines, and crossed these mice with a second strain that express the tetracycline transactivator under the control of an endothelial-specific promoter to achieve endothelial-specific and drug-inducible βcap73 in offspring positive for both transgenes.
Cells co-transfected with the pTet-On and pTRE2-βcap73 vectors express a myc-tagged version of this molecule when exposed to 1µg/ml of doxycycline. Earliest protein expression occurs at 6h post drug addition, and persists up to 48h. Also, cells over-expressing βcap73 undergo a marked morphogenic alteration compared to non-transfected cells. Preliminary data reveal that in our double transgenic mice, neovascular tuft formation is impaired by 41% in the mouse ROP model.
Our preliminary findings suggest that endothelial-specific targeting and βcap73 over-expression may represent an innovative therapeutic approach capable of abrogating pathologic ocular angiogenesis that accompanies wet age-related macular degeneration or diabetic retinopathy.
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