Purchase this article with an account.
J. T. Durham, I. M. Herman; Inhibition of Retinal Angiogenesis: A Central Role for β-Actin Dependent Cytoskeletal Remodeling. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2051.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Retinal capillary endothelial cell (RCEC) migration and morphogenesis, both central steps in angiogenesis, require cytoskeletal-dependent remodeling. The β-actin network is enriched in highly motile cytoplasm and modulated by the isoactin-specific barbed-end capping protein, βcap73. We hypothesize that over-expression of βcap73 in the retinal microvasculature could disrupt angiogenesis by capping β-actin-filament assembly thus impairing cellular migration and microvascular morphogenesis.
βcap73, previously cloned from a bovine retinal endothelial cell library, was engineered into an Ad 5, E1/E3-deleted adenovirus (Ad-βcap73). For migration studies, RCEC monolayers, infected with Ad-βcap73 or control Ad-GFP, were mechanically injured, and time-lapse imaging was performed. For in vitro angiogenesis models, RCEC were plated on Matrigel, and infected either coincidently with seeding, or after a significant period of capillary formation. For apoptosis analyses, infected RCEC were either fixed and labeled with fluorescent markers of nuclei, cytoskeleton, and activated caspase-3, or analyzed via Western blotting for activated caspase-3 and cleaved PARP.
Ad-βcap73 elicits a robust RCEC rounding response that occurs concomitantly with cytoskeletal disruption. Also, Ad-βcap73 inhibits RCEC migration by 50%. Ad-βcap73 inhibits in vitro angiogenesis by 62% in tube number and in total tube length as compared to controls. Ad-βcap73 causes angiogenic catastrophe of nascent remodeling capillaries in vitro as noted by a 55% decrease in tube number and a 61% decrease in total tube length. We reveal that βcap73 infection activates a caspase-3 mediated cell death response which, in part, inhibits angiogenesis.
Altogether, these findings suggest that RCEC-specific targeting and βcap73 over-expression may represent an innovative therapeutic approach capable of abrogating pathologic angiogenesis. Ongoing experiments are aimed at validating this hypothesis in vivo, therein inhibiting pathologic angiogenesis that accompanies diabetic retinopathy or age-related macular degeneration.
This PDF is available to Subscribers Only