3) 血管内皮から観た妊娠高血圧症候群の病態とその発症予知(シンポジウム2:周産期「妊娠高血圧症候群の基礎と臨床-予防・治療の新戦略に向けて」,第65回日本産科婦人科学会・学術講演会)

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Pregnancy induced hypertension(PIH) is characterized by the failure of cytotrophoblast infiltration into decidua. The failure harms the vasodilatory function of vascular endothelium and the placental vasculogenesis to keep the blood circulation increased during pregnancy. In this study, we investigated the involvement of inflammation in the vascular endothelial dysfunction and disturbed vasculogenesis in PIH and evaluate the possibility of the prediction of the occurrence of PIH using inflammatory serum factors. Serum factors derived from PIH patients stimulated endothelial cells. We studied the change of endothelial intracellular Ca^<++> concentration and the oscillation by serum derived from PIH patients using calcium flowed meter or confocal laser microscopy. As a result, serum factors derived from PIH patients activated the endothelial function through the increase of Ca^<++> concentration and oscillation of Ca〜<++>. Inflammation could be involved in the endothelial activation in PIH. Adhesion (rolling-adhesion) between leukocyte and endothelium is an important process for the beginning of inflammation and the expression of adhesion molecules is important on the cells. In this study, we demonstrated that leukocyte function-associated antigen-1 (LFA-1) was strongly expressed on neutrophils and lymphocytes derived from peripheral blood of PIH patients. On the other hand, intercellular adhesion molecule-1 (ICAM-1) was strongly expressed on endothelial cells by serum derived from PIH. It is thought that LFA-1 and ICAM-1 plays an important role in the inflammatory process in the pathogenesis of PIH. Adhesion molecules are known to be produced and released into peripheral blood by alternative splicing. Therefore, inflammation increases the serum concentration of adhesion molecules. The serum level of soluble ICAM-1 (sICAM-1), sE-selectin, and TNFα was already increased in the 1st trimester of PIH patients. We found that inflammation was already developed in early pregnancy of PIH patients. Reactive oxygen species (ROS) is also involved in inflammation and the pathophysiology of PIH. We studied the relationship between ROS and the pathogenesis of PIH. The serum level of creatol, oxidative metabolite of creatinine, and nitric oxide metabolites (NOx) was measured by HPLC-fluorescence antibody method. Serum creatol concentration was significantly increased since the 1st trimester through to the 3rd trimester in PIH. Additionally, serum concentration of NOx was significantly decreased in the1st trimester in PIH; however, that was not different in the 3rd trimester. Serum derived from normal pregnant women or PIH patients was added on cultured endothelial cells and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (gp91^<phox>・p22^<phox>) mRNA, induced nitric oxide synthase (iNOS) mRNA, and endothelial NOS (eNOS) mRNA was measured using semi-quantitative RT-PCR. The expression of NADPH oxidase gp91^<phox> and iNOS mRNA was increased by serum derived from PIH patients. The increase of NADPH oxidase mRNA was inhibited by anti TNFα antibody or angiotensin II (AngII) receptor subtype 1 blocker (AT1 blocker). On the other hand, the increase of iNOS mRNA was inhibited by AT2 blocker and stimulated more by AT1 blocker. Since immunohistochemistry demonstrated the strong expression of nitrotyrosin in PIH placenta it is thought that increased ROS and NO could induce the placental dysfunction through the formation of peroxynitrite in the 3rd trimester. Furthermore, we demonstrated that ROS could already disturb the placentation in early pregnancy. It is possible that the development of PIH is facilitated by inflammation at the implantation. In this study, CD4OL DNA was transfected into zygotes and those were transferred into uteri of pseudopregnant mice. After the conception blood pressure and proteinuria was measured every day. Maternal spleen cells were retrieved from the mice and the intracellular cytokines of helper T cells were measured by flow cytometry. Placenta was also studied by hematoxylin-eosin staining and kidney was studied by PAS staining. Inflammation induced by CD4OL caused hypertension and proteinuria. The profile of helper T cell was changed to Th1 dominancy in the mice. Placental labyrinth was narrowed and fibrotic in some parts. Renal vascular lumen was narrowed by increased mesangial matrix in the glomeruli. Vasculogenesis is known to be disturbed in PIH placenta. We demonstrated that the number of circulating EPC was measured by flow cytometry and the proliferation was measured by LDL/lectin assay. The number was not different between normal pregnancy and PIH; however, the proliferation was significantly increased in PIH. We also demonstrated that the chemotaxis of EPC was induced by VEGF and the proliferation was induced by P1GF. The chemotaxis and proliferation was inhibited by anti fms-related tyrosine kinase 1 (Flt-1) antibody. This means that soluble Flt-1 (sFlt-1), whish is known to be increased in PIH, could inhibit the chemotaxis of EPC from bone marrow into peripheral blood. Chloride conductance regulatory protein (pICln) DNA or siRNA was transfected into trophoblast cell line (TBT) or EPC cell line (BME) by lipofectamine method. As a result, pICln reduced the production of sFlt-1 in TBT; on the contrary, pICln stimulated the production of sFlt-1 in BME. It is thought that pICln is an important factor to influence the production of sFlt-1 in placenta. Serum-induced disturbance of vasculogenesis and dysfunction of vascular endothelium is important for the pathogenesis of PIH. We demonstrated that the serum factors were related to inflammation from the 1st trimester through to the 3rd trimester. Especially, sFlt-1 is critical for vasculogenesis in the pathogenesis of PIH through the dysfunction of EPC. It is thought that pICln could regulate the production of sFlt-1 in the placenta. In this study, we also showed the inflammatory serum factors were significantly increased since the 1st trimester. The combination of the analysis of the serum factors might lead to the realization of the prediction of PIH in the 1st trimester.
2013-11-01

3) 血管内皮から観た妊娠高血圧症候群の病態とその発症予知(シンポジウム2:周産期「妊娠高血圧症候群の基礎と臨床-予防・治療の新戦略に向けて」,第65回日本産科婦人科学会・学術講演会)

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