Oxidant Stress Mechanisms in Preeclampsia

超出所提供的空间。先兆子痫导致5-7%的妊娠并发症，是胎儿发育迟缓、早产和孕产妇死亡的主要原因。子痫前期的病因尚不清楚。我们认为，脂质过氧化物（LOOH）水平的增加激活了中性粒细胞，以及内皮细胞和血管平滑肌细胞，导致内皮细胞和平滑肌细胞分泌中性粒细胞趋化因子白细胞介素-8 （IL-8）。内膜空间中IL-8浓度的增加刺激中性粒细胞跨内皮迁移到内膜空间，在那里它们释放有毒化合物，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这一提出的病理相互作用的三个方面。特异性目标1将验证氧化应激激活中性粒细胞通过涉及核因子- kb (NF-EB)，环氧化酶-2 （COX-2）和血栓素的途径来制造有毒化合物，如TNF（_，超氧化物和血栓素）的假设。特异性目的2将通过花生四烯酸代谢物的信号通路，验证氧化应激刺激人血管平滑肌细胞NF- _:B的激活和IL-8的合成的假设。特异性目的3将验证氧化应激和子痫前期血浆通过IL-8刺激中性粒细胞跨内皮迁移的假设。这个目的也将确定是否有中性粒细胞浸润到全身组织的妇女先兆子痫。我们将利用抗氧化剂来验证氧化应激的作用，利用IL-8中和抗体来评估IL-8的重要性，利用膳食脂肪酸和花生四烯酸途径抑制剂来评估它们在调节氧化应激反应中的作用。这些研究将使用从未怀孕妇女、正常孕妇和子痫前期妇女获得的血浆、中性粒细胞和脂肪，以及人内皮细胞和血管平滑肌细胞的原代细胞培养物。方法将包括NF-_:B荧光素酶报告载体的细胞转染和凝胶转移试验，以确定NF-_:B的激活；COX-2的Western blot；细胞因子和类二十烷酸水平的EIA；分光光度法测定丙二醛估计氧化应激，和一个独特的实时测定超氧化物的产生。一种新的跨内皮迁移试验将用于测量slcr标记的中性粒细胞在培养中的跨内皮细胞迁移。网站性能 ======================================== 节结束 ===========================================

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===========================================