Abstract

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis.

Highlights

  • Over a decade ago our laboratory used the National Cancer Institute’s Antineoplastic Drug Screen of 60 human cancer cell lines to identify novel inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway

  • We have shown that lethal toxin (LeTx) can perturb developmental vascular growth in a murine model of retinal angiogenesis

  • LeTx treatment results in a transient delay in the formation of the inner plexus layers of retinal vasculature. This is accompanied by an elevation in the concentration of vitreal VEGF that subsequently drives extensive neovascular growth through the inner retina to the outer retinal region. To explain these observations we propose that LeTx acts directly on endothelial cells to prevent growth and remodeling of angiogenic sprouts as they extend into distal region of the retina

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Summary

Introduction

Over a decade ago our laboratory used the National Cancer Institute’s Antineoplastic Drug Screen of 60 human cancer cell lines to identify novel inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway. Our search showed that anthrax lethal toxin (LeTx) had a similar activity profile on cancer cell lines as the mitogen-activated protein kinase kinase (MAPKK or MEK) inhibitor PD98059 [1]. LF was subsequently shown to be a zinc metalloprotease that cleaves the amino termini of MEK 1 and 2 [1,3] as well as MKK 3, 4, 6, and 7 [4,5] and causes their inactivation This leads to inactivation of three MAPK pathways: the extracellular signal-related kinase pathway (ERK), the p38 MAPK pathway (p38), and the c-Jun N-terminal kinase pathway (JNK) [6,7]. The ERK pathway is preferentially activated by growth factors, while the p38 and JNK pathways respond to cellular stresses such as osmotic shock and inflammatory cytokines (reviewed in [8])

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