Oxidant Stress Mechanisms in Preeclampsia

超出提供的空间。先兆子痫导致5-7%的妊娠并发症，是胎儿生长迟缓、早产和产妇死亡的主要原因。先兆子痫的原因尚不清楚。我们认为，过氧化脂质(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，导致内皮细胞和血管平滑肌细胞产生中性粒细胞趋化因子白介素8(IL-8)。血管内膜中IL-8浓度的增加刺激中性粒细胞通过内皮向内膜迁移，在那里它们释放有毒化合物，如作为炎症和细胞功能障碍介质的TNFc、超氧化物(‘O2)和血栓烷(Tx)。以下具体目标将检验这种拟议的病理相互作用的三个方面。特定目的1将验证这样的假设：氧化应激通过核因子-kB(NF-EB)、环氧合酶-2(COX-2)和血栓烷等途径激活中性粒细胞产生有毒化合物，如肿瘤坏死因子(_)、超氧化物和血栓烷。特定目的2将验证氧化应激通过涉及花生四烯酸代谢产物的信号通路刺激人血管平滑肌细胞激活核因子-B和分泌IL-8的假说。具体目标3将验证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细胞迁移的假设。这一目标也将确定先兆子痫妇女的全身组织中是否有中性粒细胞的渗透。抗氧化剂将用于验证氧化应激的作用，IL-8中和抗体用于评估IL-8的重要性，膳食脂肪酸和花生四烯酸途径抑制剂将用于评估它们在改变对氧化应激的反应中的作用。这些研究将使用从未怀孕妇女、正常孕妇和先兆子痫妇女获得的血浆、中性粒细胞和脂肪，以及人类内皮细胞和血管平滑肌细胞的原代细胞培养。方法学将包括细胞转染法和凝胶移位分析，以确定核因子：B的激活；Western印迹检测COX-2；EIA检测细胞因子和二十烷类化合物的水平；丙二醛的分光光度分析，以评估氧化应激；以及独特的实时检测，以确定超氧化物的产生。一种新的跨内皮细胞迁移试验将被用来测量培养中SLCR标记的中性粒细胞跨内皮细胞的迁移。表演网站========================================Section End===========================================

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