PATHOGENESIS OF COCAETHYLENE-INDUCED VASCULITIS

描述（由申请人提供）：血管炎是可卡因成瘾者常见的病理结果，其中80%的人同时滥用可卡因和乙醇。我们认为，在可卡因滥用研究中使用可卡因（CE，可卡因和乙醇的一种结合物，在体内产生）将准确地代表致病性后遗症。我们的长期目标是更好地了解ce相关血管毒性的细胞和亚细胞机制，并提出证明阳离子在ce相关血管毒性中的作用。目的1:CE暴露于人脐静脉内皮细胞（HUVEC）诱导促炎激活和通透性。暴露于CE或生理盐水的HUVEC单层将被分析CE（气相色谱-质谱），电解质（电解质分析仪）和氧气/二氧化碳水平（气体分析仪）。将对固定的单层进行染色，以计数细胞间隙形成（银染色）和表面活化标记（免疫组织化学）。目的2：暴露于CE的HUVEC激活促炎信号/基因表达途径。CE或盐水处理的单层将使用电泳迁移量转移试验和超转移、RNAse保护试验和Western blot检测RNA和蛋白质的表达和活性变化。靶标是信号和激活标记物，包括核因子kB、白细胞介素-8和血管细胞粘附分子-1。目的3：暴露于CE后HUVEC激活与高持续的细胞质阳离子通量有关。单层HUVEC将用CE处理，并用荧光光谱法记录细胞内阳离子通量（钙、镁和水合氢离子）。流式细胞术和/或Western blotting将用于监测钙通道密度随时间的变化。拟议的目的将进一步深入了解人类CE产生的致病性后遗症。

DESCRIPTION (provided by applicant): Vasculitis is a common pathological outcome in cocaine addicts, 80% of whom co-abuse cocaine and ethanol. We believe that the use of cocaethylene (CE, a conjugate of cocaine and ethanol, produced in vivo) in studies of cocaine abuse will accurately represent pathogenic sequelae. Our long-term goal is to better understand the cellular and subcellular mechanisms of CE-associated vasculotoxicity, and propose to demonstrate the role of cations in CE-associated vasculotoxicity. Aim #1: CE exposure in human umbilical vein endothelial cells (HUVEC) induces pro-inflammatory activation and permeability. HUVEC monolayers exposed to CE or saline will be analyzed for CE (Gas Chromatography-Mass Spectrometry), and electrolyte (electrolyte analyzers) and oxygen/carbon dioxide levels (gas analyzers). Fixed monolayers will be stained for counting of intercellular gap formations (silver stain), and surface markers of activation (immunohistochemistry). Aim #2: CE exposure in HUVEC activates pro-inflammatory signaling/gene expression pathways. CE or saline treated monolayers will be tested for RNA and protein expression and activity changes utilizing electrophoretic mobility shift assay and supershift, RNAse protection assay, and Western blot. Targets are signaling and activation markers, and include Nuclear Factor kB, interleukin-8, and Vascular Cell Adhesion Molecule-1. Aim #3: HUVEC activation after exposure to CE is associated with high sustained cytoplasmic cation flux. Monolayers of HUVEC will be treated with CE and intracellular cation flux (calcium, magnesium, and hydronium) recorded utilizing fluorescence spectrometry. Flow cytometry and/or Western blotting will be used to monitor changes in calcium channel density over time. The proposed Aims will provide further insight into the pathogenic sequelae resulting from production of CE in humans.