The Effects of Anthrax Toxins in an Aortic Ring Model

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Anthrax produces two binary type toxins, lethal toxin (LT) and edema toxin (ET), which are closely associated with the bacterias lethal effects. Both LT and ET reduce blood pressure in animal models. Using the isolated perfused rat heart, we showed previously that while ET has positive chronotropic and inotropic effects (increased heart rate and ventricular contraction), it also increased coronary flow suggesting a vasodilator effect. LT in that model had some negative inotropic effects but only at very high levels. Each toxin includes a common component called protective antigen (PA) necessary for cellular uptake of the toxins toxic moieties; lethal factor (LF) for LT and edema factor (EF) for ET. LT is zinc metalloprotease that inactivayes mitogen activated protein kinase kinase pathways and has the potential to alter cellular Ca2+ metabolism. ET has calmodulin dependent adenylate cyclase activity that increases intracellular cyclic adenosine monophosphate (cAMP) level and decreases intracellular Ca2+. However, to what degree hypotension with these toxins reflects their direct effect on arterial dilation (relaxation) as opposed to indirect effects (e.g. increased endothelial permeability with extravasation of fluid, myocardial depression, inappropriate urinary losses) is unclear. However, we hypothesize that both toxins can cause vasodilation and the present study will employ an isolated rat aortic ring assay to investigate this. In the aortic ring assay, standardized lengths of isolated rat aorta (4 mm) are connected to pressure transducers while immersed in a bath of physiologic solution. These aortic ring sections are then challenged with agents with known constrictor or relaxant properties or with experimental agents, and the level of resulting contractile or relaxant forces are measured. Normal arterial constriction and relaxation entails several pathways, but by using agents that are known to selectively influence these pathways, it is possible to decipher how an unknown agent might be altering arterial tone. The aortic ring assay was developed in the late 1970s and has been used extensively since then to investigate the pathophysiology and treatment of abnormal states of arterial tone (i.e. relaxation-dilation or constriction). The present protocol originally included three parts. In the initial part (Part 1), the stability of the aortic ring preparation in our lab and its ability to contract with increasing concentrations of phenylephrine (PE) or relax with increasing concentrations of acetylcholine was confirmed. In the second (Part 2) and third parts (Part 3), we investigated the influence of exogenous ET (Part 2) and LT (Part 3) on contraction and relaxation of the aortic ring. These studies were subsequently published (Li Y, et al. B. anthracis edema toxin increases cAMP levels and inhibits phenylephrine-stimulated contraction in a rat aortic ring model. Am J Physiol. 2013. 305: H238-H250). Additional studies have examined the mechanisms underlying the vaso-relaxant effects of anthrax edema toxin as well as the potential for agents to interfere with the toxins vascular effects ex vivo and in vivo. Work from this protocol was presented at the 2015 International Conference of the American Thoracic Society. One studying examining the role of nitric oxide in edema toxins effects was published in 2016. Li Y, Cui X, Xu W, Ohanjanian L, Sampath-Kumar H, Suffredini D, Moayeri M, Leppla S, Fitz Y, Eichacker PQ. Nitric oxide production contributes to Bacillus anthracis edema toxin-associated arterial hypotension and lethality: ex vivo and in vivo studies in the rat. Am J Physiol Heart Circ Physiol. 2016 Sep 1;311(3):H781-93. doi: 10.1152/ajpheart.00163.2016. Epub 2016 Jul 22.PMID: 27448553 Additional studies have now shown that ETs adenyl cyclase activity and hypotensive effects are related to activation of both protein kinase A (PKA), and exchange protein activated by cAMP (EPAC) pathways. A manuscript is in preparation. At this time work continues to further differentiate the downstream events contributing to anthrax edema toxins potent vasodilatory effects. A manuscript is in preparation. At this time work continues to further differentiate the downstream events contributing to anthrax edema toxins potent vasodilatory effects.