Oxidant Stress Mechanisms in Preeclampsia

描述（由申请人提供）：先兆子痫使5-7%的妊娠复杂化，并且是胎儿生长迟缓、早产和孕产妇死亡的主要原因。先兆子痫的病因尚不清楚。我们提出，脂质过氧化物（LOOH）水平的增加激活中性粒细胞，以及内皮细胞和血管平滑肌细胞，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的阐述。内膜空间中IL-8浓度的增加刺激嗜中性粒细胞跨内皮迁移到内膜空间，在内膜空间中它们释放毒性化合物，例如TNF α、超氧化物（O2-）和血栓烷（TX），它们是炎症和细胞功能障碍的介质。以下特定目的将测试该拟议病理相互作用的三个方面。具体目标1将检验以下假设：氧化应激通过涉及核因子-kB（NF-κ B）、环氧合酶-2（考克斯-2）和血栓烷的途径激活中性粒细胞以产生毒性化合物，如TNF α、超氧化物和血栓烷。具体目标2将检验氧化应激通过涉及花生四烯酸代谢物的信号传导途径刺激NF-κ B活化和人血管平滑肌细胞产生IL-8的假设。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。这一目标也将确定是否有中性粒细胞浸润到先兆子痫妇女的全身组织。抗氧化剂将用于验证氧化应激的作用，IL-8中和抗体将用于评估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其在改变氧化应激反应中的作用。这些研究将使用从非妊娠女性、正常妊娠女性和先兆子痫女性中获得的血浆、中性粒细胞和脂肪，以及人内皮细胞和血管平滑肌细胞的原代细胞培养物。方法学将包括NF-κ B荧光素酶报告载体的细胞转染和确定NF-κ B活化的凝胶迁移试验;考克斯-2的蛋白质印迹;细胞因子和类花生酸水平的EIA; MDA的分光光度测定以估计氧化应激，以及确定超氧化物生成的独特的真实的时间测定。一种新的跨内皮迁移试验将用于测量51铬标记的中性粒细胞跨培养内皮细胞的迁移。

DESCRIPTION (provided by applicant): 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 TNFalpha, 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 TNFalpha, superoxide and thromboxane, by a pathway involving nuclear factor-kB (NF-kappaB), cyclooxygenase-2 (COX-2) and thromboxane. Specific Aim 2 will test the hypothesis that oxidative stress stimulates the activation of NF- kappaB 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-kappaB luciferase reporter vector and gel shift assay to determine NF-kappaB 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 51Cr-labeled neutrophils across endothelial cells in culture.