Modified Anthrax Toxin Selectively Targets Activated Endothelial Cells: A Novel Therapeutic Approach for the Treatment of Retinal Neovascular Disease

Abstract

Purpose: : The recent introduction of monoclonal antibodies against VEGF for the treatment of retinal and choroidal neovascular disease demonstrates the importance of VEGF in pathological angiogenesis in the eye. Nonetheless, patients with "mature" retinal neovascular lesions are often remarkably insensitive to VEGF inhibition, prompting the exploration of other genes that may serve as appropriate therapeutic targets for these patients. In this regard, the extensive interplay between endothelial cells, soluble factors, and the extracellular matrix is an emerging target for anti-angiogenic therapies. In particular, extracellular proteolysis has been implicated as one of the first and most sustained activities involved in pathological angiogenesis. Our goal is to identify and target key extracellular proteases upregulated by activated endothelial cells to develop new therapies for patients with ischemic retinopathies.

Methods: : Vascular endothelial cells (VECs) were exposed to hypoxia or recombinant VEGF protein and the expression of extracellular proteases was assessed. These results were corroborated in vivo using the oxygen-induced retinopathy (OIR) model for ischemic retinopathy. Modified bacterial (anthrax) toxins - engineered to be active only in the presence of specific proteases - were then used to target activated VECs and their toxicity examined.

Results: : Matrix metalloprotease-2 (MMP-2) RNA and protein are increased several fold in VECs exposed to hypoxia or recombinant VEGF protein in vitro and increase within 24 hours of ocular ischemia in vivo (p<0.05). Moreover, while VEGF RNA levels drop precipitously after 24 hours in the OIR model after return to normoxia, MMP-2 RNA levels remain elevated for at least 72 hours (p<0.05). Using a modified anthrax toxin activated by MMPs, we further observed rapid cell death of activated VECs treated with the toxin.

Conclusions: : We demonstrate that MMP-2 is upregulated in activated VECs by VEGF and can be used therapeutically to specifically target and kill these cells in retinal neovascular lesions. This study provides the foundation for the assessment of modified bacterial toxins as a novel therapeutic approach for patients with ischemic retinopathies.

Keywords: diabetic retinopathy • neovascularization • extracellular matrix