Oxidant Stress Mechanisms in Preeclampsia

先兆子痫的氧化应激机制

• 批准号: 6829114
• 负责人: SCOTT W WALSH
• 金额: $ 25.87万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-12-05 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6829114
• 关键词: antioxidants; clinical research; fatty acids; female; gel mobility shift assay; human pregnant subject; human tissue; immunocytochemistry; interleukin 8; lipid peroxides; muscle cells; neutrophil; nuclear factor kappa beta; oxidative stress; preeclampsia; prostaglandin endoperoxide synthase; spectrometry; superoxides; thromboxanes; tissue /cell culture; transfection /expression vector; tumor necrosis factor alpha; vascular endothelium; western blottings; women' s health

EXCEED THE SPACE PROVIDED. Preeclampsia complicates 5-7% of pregnancies, and is a leading cause of fetal growth retardation, premature delivery and maternal death. The cause of preeclampsia is not known. We propose that increased levels of lipid peroxides (LOOH) activate neutrophils, as well as endothelial and vascular smooth muscle cells, resulting in the elaboration of the neutrophil chemokine interleukin-8 (IL-8) from the endothelial and smooth muscle cells. Increased concentrations of IL-8 in the intimal space stimulate transendothelial migration of the neutrophils to the intimal space where they release toxic compounds, such as TNFc(, superoxide ('O2) and thromboxane (TX) that are mediators of inflammation and cell dysfunction. The following Specific Aims will test three arms of this proposed pathologic interaction. Specific Aim 1 will test the hypothesis that oxidative stress activates neutrophils to elaborate toxic compounds, such as TNF(_, superoxide and thromboxane, by a pathway involving nuclear factor-kB (NF-EB), cyclooxygenase-2 (COX-2) and thromboxane. Specific Aim 2 will test the hypothesis that oxidative stress stimulates the activation of NF- _:B and the elaboration of IL-8 by human vascular smooth muscle cells by a signaling pathway involving arachidonic acid metabolites. Specific Aim 3 will test the hypothesis that oxidative stress and preeclamptic plasma stimulate transendothelial migration of neutrophils via IL-8. This aim will also determine if there is infiltration of neutrophils into systemic tissue of women with preeclampsia. Antioxidants will be used to verify the role of oxidative stress, IL-8 neutralizing antibody to assess the importance of IL-8, and dietary fatty acids and arachidonic acid pathway inhibitors to assess their role in modifying responses to oxidative stress. These studies will use plasma, neutrophils and fat obtained from nonpregnant women, normal pregnant women and women with preeclampsia, and primary cell cultures of human endothelial cells and vascular smooth muscle cells. Methodologies will include cell transfection of an NF-_:B luciferase reporter vector and gel shift assay to determine NF-_:B activation; Western blot for COX-2; EIA for cytokine and eicosanoid levels; spectrophotometric assay of MDA to estimate oxidative stress, and an unique real time assay to determine superoxide generation. A novel transendothelial migration assay will be used to measure the migration of SlCr-labeled neutrophils across endothelial cells in culture. PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间。先兆子痫使 5-7% 的妊娠变得复杂，并且是胎儿生长迟缓、早产和孕产妇死亡的主要原因。先兆子痫的原因尚不清楚。我们认为，脂质过氧化物 (LOOH) 水平的增加会激活中性粒细胞以及内皮细胞和血管平滑肌细胞，从而导致内皮细胞和平滑肌细胞产生中性粒细胞趋化因子白细胞介素 8 (IL-8)。内膜间隙中 IL-8 浓度的增加刺激中性粒细胞跨内皮迁移至内膜间隙，并在此释放有毒化合物，例如 TNFc(、超氧化物 ('O2) 和血栓素 (TX)，它们是炎症和细胞功能障碍的介质。以下具体目标将测试所提出的病理相互作用的三个方面。具体目标 1 将测试以下假设： 氧化应激通过涉及核因子-kB (NF-EB)、环氧合酶-2 (COX-2) 和血栓素的途径，激活中性粒细胞合成有毒化合物，如 TNF(_)、超氧化物和血栓素。具体目标 2 将检验氧化应激刺激 NF-_:B 的激活和人类对 IL-8 的合成这一假设。 血管平滑肌细胞通过涉及花生四烯酸代谢物的信号通路。具体目标 3 将检验氧化应激和先兆子痫血浆通过 IL-8 刺激中性粒细胞跨内皮迁移的假设。这一目标还将确定中性粒细胞是否浸润到先兆子痫女性的全身组织中。将使用抗氧化剂来验证其作用 氧化应激、IL-8 中和抗体以评估 IL-8 的重要性，以及膳食脂肪酸和花生四烯酸途径抑制剂以评估其在改变氧化应激反应中的作用。这些研究将使用从非孕妇、正常孕妇和先兆子痫女性获得的血浆、中性粒细胞和脂肪，以及人内皮细胞和血管平滑肌的原代细胞培养物 细胞。方法将包括 NF-_:B 荧光素酶报告载体的细胞转染和凝胶位移测定以确定 NF-_:B 激活； COX-2 的蛋白质印迹；细胞因子和类二十烷酸水平的 EIA； MDA 分光光度测定法可评估氧化应激，并采用独特的实时测定法测定超氧化物的产生。一种新型的跨内皮迁移测定将用于测量 SlCr 标记的中性粒细胞在培养物中穿过内皮细胞的迁移。表演网站==========================================章节结束===============================================