Oxidized lipids and microvesicle particles as effectors for chemical threats to skin - Abstract
The threats to chemical warfare-associated agents, including arsenicals and nitrogen mustards are increasing
exceedingly, and no direct antidote is currently available to mitigate the deleterious cutaneous and systemic
responses to prevent mortality. Though the associated cytotoxic effects of most of these agents are mediated
due to their ability to act as alkylating agents, a significant knowledge gap exits in the understanding of detailed
molecular mechanisms of how these vesicants cause cutaneous and systemic toxic effects, and thus, the
development of antidotes. The current proposal is built upon the scientific premise that exposure to various pro-
oxidative stressors, including alkylating chemotherapeutic agents, and thermal burn injuries (TBI) when coupled
with ethanol (ETOH) produce the potent bioactive lipid mediator, Platelet-activating factor (PAF) by both
enzymatically and non-enzymatically via reactive oxygen species (ROS). Studies, including ours, have shown
that these PAF agonists induce local inflammation, as well as multi-system organ dysfunction (MOD).
Importantly, recent studies have indicated that small membrane-bound vesicles known as microvesicle particles
(MVP), generated via acid sphingomyelinase (aSMase) enzyme, are released from cells in response to various
stressors. These MVP can act as potent signaling agents due to their ability to carry nuclear and cytoplasmic
components. More importantly, the current proposal is built upon our discovery that chemotherapeutic agents,
and TBI+ETOH via their ability to damage keratinocytes, generate PAF agonists which travel via MVP to induce
the local (cutaneous) and systemic responses. Using antioxidants and PAFR-expressing/null cell lines and
pharmacologic/genetic inhibition of aSMase enzyme, our studies have implicated the involvement of the PAFR
signaling in aSMase activation resulting in PAF-laden MVP release. Based upon these compelling evidences,
we hypothesize that chemical warfare-associated agents via their ability to generate ROS, produce PAF agonists
and MVP from human and murine keratinocytes in a PAFR-aSMase-dependent manner, which mediate the
cutaneous cytotoxic as well as systemic MOD effects. Two aims are proposed to test our hypothesis. Aim 1 will
use validated in vitro and ex vivo models and pharmacologic agents to determine the roles of the PAFR,
downstream signaling pathways, and aSMase enzyme in chemical warfare-associated agents-induce PAF
agonists and MVP generation. Agents to be tested are nitrogen/sulfur mustards and an arsenical. Aim 2 will use
PAFR/aSMase-expressing and deficient mouse models, as well as pharmacological inhibitors of PAFR and
aSMase to determine the role of PAF-laden MVP generation in the local and systemic responses by topical
nitrogen mustargen exposure. Successful completion of this project will (i) fill important mechanistic gaps in and
validate novel tools to allow the modulation of PAF-laden MVP generation to mitigate chemical warfare-
associated agents’ effects; and ii) address functional roles of PAFR-aSMase in nitrogen mustard-induced local
and systemic MOD effects, to provide novel treatment approaches.
