PATHOGENESIS OF COCAETHYLENE-INDUCED VASCULITIS

描述(由申请人提供)：脉管炎是可卡因成瘾者的常见病理结果，其中80%的人共同滥用可卡因和乙醇。我们相信，在可卡因滥用的研究中使用可卡因(CE，可卡因和乙醇的结合物，在体内产生)将准确地代表致病后遗症。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，并建议证明阳离子在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)可诱导促炎活性和通透性。暴露在CE或生理盐水中的HUVEC单层将被分析CE(气相色谱-质谱仪)、电解液(电解液分析仪)和氧气/二氧化碳水平(气体分析仪)。固定单层用于细胞间隙形成计数(银染)和表面活化标记(免疫组织化学)。目的#2：人脐静脉内皮细胞暴露CE可激活促炎信号/基因表达通路。CE或生理盐水处理的单层将使用凝胶迁移率改变分析和超转移、核糖核酸酶保护实验和Western印迹来检测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和血管细胞黏附分子-1。目的#3：CE暴露后HUVEC的激活与高持续的细胞质阳离子通量有关。单层HUVEC将用CE和荧光分光光度记录的细胞内阳离子流量(钙、镁和离子)处理。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。拟议的目标将进一步深入了解人类产生CE所造成的致病后遗症。

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.