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.
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