Perturbation of mouse retinal vascular morphogenesis by anthrax lethal toxin.
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.
Bromberg-White
Jennifer L
JL
Laboratory of Cancer and Developmental Cell Biology, Van Andel Research Institute, Grand Rapids, Michigan, United States of America.
Boguslawski
Elissa
E
Duesbery
Nicholas S
NS
D000818:Animals; D000881:Anthrax / Q000378:metabolism / Q000503:physiopathology; D000942:Antigens, Bacterial / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D015151:Immunoblotting; D020935:MAP Kinase Signaling System; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009024:Morphogenesis / Q000187:drug effects; D018919:Neovascularization, Physiologic; D012160:Retina / Q000187:drug effects*; D012171:Retinal Vessels / Q000187:drug effects*; D015398:Signal Transduction; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D042461:Vascular Endothelial Growth Factor A; C030325:anthrax toxin