Anthrax poses a considerable public health burden because of its potential use as a biological weapon. Although vaccination against the bacteria might offer the best protection, there remains a pressing need to successfully treat anthrax complications. Antibiotics can stop the progression of the infection, but are of limited use once a damaging or lethal amount of anthrax toxin has been produced by the bacteria. Anthrax toxin is believed to damage tissues through the action of its protein components particularly the Lethal Factor (LF), which causes severe vascular leak and tissue edema. Our laboratory has been studying the regulation of the pulmonary vascular endothelial permeability barrier and has identified signaling pathways that weaken the barrier and those that augment it. We have shown that p38 MAP kinase activation leading to HSP27 phosphorylation by the kinase MK2 augments the endothelial permeability barrier through strengthening adhesive forces mediated by actin stress fiber and vimentin intermediate filament network formation. Since a major molecular activity of the LF component of anthrax toxin is blocking p38 activation, downstream HSP27 phosphorylation and its barrier augmenting effect are postulated to be blocked in response to anthrax toxin. The overall hypothesis to be tested in this proposal is that anthrax toxin produces some of its effects through disrupting the endothelial permeability barrier via blocking p38-MK2 activation and HSP27 phosphorylation. As a result phospho-HSP27 is unable to form and mediate permeability barrier augmentation through its action on actin and vimentin, resulting in increased endothelial permeability and edema. Endothelial barrier permeability and vascular leak caused by anthrax lethal toxin are also postulated to be blocked or reversed by activating HSP27 phosphorylation. Since LF causes most of the symptoms of anthrax we will characterize the action of Lethal Toxin (LT) which is a combination of LF and Protective Antigen (PA). PA is another component of the anthrax toxin that mediates the entry of LF into cells. We will carry out experiments in rat pulmonary microvascular endothelial cells because they form a tight permeability barrier in culture and LF is believed to act directly on endothelial cells. In addition we will carry out in vivo experiments in Fisher 244 rats which have been shown to be sensitive to anthrax LF.
In Aim 1 we will determine the kinetics of anthrax LT action on p38-MK2-HSP27 signaling and cytoskeletal remodeling as related to endothelial monolayer permeability.
In Aim 2 we will evaluate induction of HSP27 phosphorylation as a mechanism to protect against anthrax LT-induced permeability and edema in cell culture and in vivo. We expect our experiments to demonstrate that blocking HSP27 phosphorylation by LT causes permeability and leak through inhibiting actin- and vimentin-mediated barrier augmentation. Furthermore, we expect our experiments to show that targeting HSP27 phosphorylation is a mechanistically sound approach to treat anthrax and to demonstrate the effectiveness of that approach in animal models of anthrax.
Because of the lethal threat of anthrax exposure there is a pressing need to successfully treat its complications, and while antibiotics can stop the progression of the infection, they are of limited use once a damaging or lethal amount of anthrax toxin has been produced by the bacteria. The studies proposed in this application focus on a potential mechanism by which the anthrax toxin can cause permeability and vascular leak, which has been associated with its lethality. By evaluating approaches that block the molecular action of anthrax toxin on endothelial permeability barrier-regulating pathways these studies might lead to successful treatment of anthrax and similar agents that cause permeability and vascular leak.
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