Oxidant Stress Mechanisms in Preeclampsia

先兆子痫使5-7%的妊娠复杂化，并且是胎儿生长迟缓的主要原因， 早产和产妇死亡。先兆子痫的病因尚不清楚。我们建议增加 脂质过氧化物（LOOH）水平激活中性粒细胞以及内皮和血管平滑肌 细胞，导致中性粒细胞趋化因子白细胞介素-8（IL-8）从内皮细胞和 平滑肌细胞内膜间隙中IL-8浓度升高刺激跨内皮细胞 中性粒细胞迁移到内膜空间，在那里它们释放有毒化合物，如TNFc（， 超氧化物（1 O2）和血栓素（TX），它们是炎症和细胞功能障碍的介质。的 以下具体目标将测试这种拟议的病理相互作用的三个方面。具体目标1将测试 假设氧化应激激活嗜中性粒细胞产生毒性化合物，如TNF α， 超氧化物和血栓素，通过涉及核因子-kB（NF-EB）、环氧合酶-2（考克斯-2）的途径 和血栓素。具体目标2将检验氧化应激刺激NF-κ B活化的假设。 _：B和人血管平滑肌细胞通过一个信号通路产生IL-8， 花生四烯酸代谢产物。具体目标3将检验氧化应激和先兆子痫 血浆通过IL-8刺激嗜中性粒细胞的跨内皮迁移。这一目标也将决定是否有 先兆子痫妇女全身组织中性粒细胞浸润。抗氧化剂将用于验证 氧化应激的作用，IL-8中和抗体，以评估IL-8的重要性，和膳食脂肪 酸和花生四烯酸途径抑制剂，以评估它们在改变对氧化应激的反应中的作用。 这些研究将使用从非妊娠女性、正常妊娠女性和正常妊娠女性中获得的血浆、中性粒细胞和脂肪。 以及人内皮细胞和血管内皮细胞的原代细胞培养物 平滑肌细胞方法学将包括NF-κ B：B荧光素酶报告载体的细胞转染， 凝胶迁移试验测定NF-_：B活化; Western印迹法测定考克斯-2; EIA法测定细胞因子和类花生酸 水平; MDA的分光光度法测定，以估计氧化应激，以及独特的真实的时间测定， 测定超氧化物生成。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物中穿过内皮细胞的迁移。

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.