The Effects of Anthrax Toxins in an Aortic Ring Model

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Abstract

Bacillus anthracis (B. anthracis) is a major bioterrorism threat in the US today. In the 2001 US outbreak and the recent UK outbreak was that dying patients developed shock resistant to both hemodynamic support with fluids and vasopressors. 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 includes 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 has been be confirmed. In the second (Part 2) and third parts (Part 3), we have investigated the influence of exogenous ET (Part 2) and LT (Part 3) on contraction and relaxation of the aortic ring. Some experiments have been completed and data is being analyzed while other experiments are underway. Findings from this study were presented at the 2012 ATS International Conference.