Oxidized lipids and microvesicle particles as effectors for chemical threats to skin

摘要 与化学战有关的制剂，包括砷和氮的威胁正在增加 而且目前还没有直接的解毒剂来减轻有害的皮肤和全身毒性。 预防死亡的对策。虽然大多数这些药物的相关细胞毒性作用是介导的， 由于它们作为烷基化剂的能力，在理解详细的烷基化反应中存在显著的知识差距。 这些水疱剂如何引起皮肤和全身毒性作用的分子机制，因此， 解毒剂的研制目前的建议是建立在科学的前提下，暴露于各种亲- 氧化应激源，包括烷化剂化疗剂和热烧伤（TBI）， 与乙醇（ETOH），产生有效的生物活性脂质介质，血小板活化因子（PAF）， 酶促和非酶促通过活性氧（ROS）。包括我们在内的研究表明 这些PAF激动剂诱导局部炎症以及多系统器官功能障碍（MOD）。 重要的是，最近的研究表明，小膜结合囊泡称为微泡颗粒 (MVP)通过酸性鞘磷脂酶（aSMase）酶产生的，响应于各种刺激而从细胞释放。 压力源这些MVP可以作为有效的信号传导剂，因为它们能够携带细胞核和细胞质 件.更重要的是，目前的建议是建立在我们的发现，化疗剂， 和TBI+ETOH通过其损伤角质形成细胞的能力，产生PAF激动剂，其通过MVP旅行以诱导 局部（皮肤）和全身反应。使用抗氧化剂和PAFR表达/无效细胞系， 药理学/遗传学抑制aSMase酶，我们的研究表明PAFR参与 aSMase活化中的信号传导导致载有PAF的MVP释放。基于这些令人信服的证据， 我们假设化学战相关因子通过其产生活性氧的能力，产生PAF激动剂， 和MVP以PAFR-aSM酶依赖性方式从人和鼠角质形成细胞中分离，其介导了 皮肤细胞毒性以及全身MOD效应。提出了两个目标来检验我们的假设。目标1将 使用经验证的体外和离体模型和药理学试剂来确定PAFR的作用， 化学战相关剂中的下游信号通路和aSMase酶诱导PAF 激动剂和MVP生成。待测试的试剂是氮/硫化合物和砷化合物。目标2将使用 PAFR/aSM酶表达和缺陷小鼠模型，以及PAFR和aSM酶的药理学抑制剂， aSMase，以确定PAF负载MVP生成在局部和全身反应中的作用， 氮芥暴露。该项目的成功完成将（一）填补重要的机械空白， 验证新的工具，以允许调节载有PAF的MVP生成，从而减轻化学战- ii）解决PAFR-aSM酶在氮芥诱导的局部细胞凋亡中的功能作用。 和全身MOD效应，以提供新的治疗方法。

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.