Hitoshi Okamura

Association of the missense Glu298Asp variant of the endothelial nitric oxide synthase gene with severe preeclampsia.

Objective: A large number of studies suggest that abnormalities in nitric oxide (NO) synthesis may contribute to the development of preelampsia. We recently identified a variant within exon 7 of the endothelial NO synthase (eNOS) gene: G to T conversion at nucleotide position 894 resulting in replacement of glutamic acid with aspartic acid at codon 298 (Glu298Asp). We analyzed the association between the Glu298Asp eNOS gene variant and preeclampsia. Study design: The study included 152 preeclampsia patients (35 mild, 80 severe, and 37 superimposed) and 170 control subjects. Screeing for the Glu298Asp eNOS gene variant was carried out by analysis of polymerase chain reaction-restriction fragment length polymorphism. Results: The frequency of the Glu298Asp variant was significantly higher in the severe preclampsia group (28.8%) than in the control (14.1%; p < .01), superimposed preeclampsia (8.1%; p < .01), and mild preeclampsia (11.4%; p < .01) groups. Conclusions: We conclude that the presence of the Glu298Asp eNOS gene could be a marker of increased risk of developing severe preeclampsia.

Cytokines in the peritoneal fluid of patients with endometriosis

OBJECTIVE: To determine which of the various cytokines interleukin‐5 (IL‐5), interleukin‐6 (IL‐6) and interleukin‐1 (IL‐1) are important in endometriosis. METHODS: Peritoneal fluid (PF) samples were taken from 55 women at the time of either laparotomy or laparoscopy, and were examined for the levels of IL‐5, IL‐6 and IL‐1 using each cytokine specific sandwich enzyme‐linked immunosorbent assay. Thirty‐two patients had pelvic endometriosis, eight post‐pelvic inflammatory disease, four advanced cancer, three adenomyosis, three benign ovarian tumor, and other diseases. Statistical analysis was performed with the Mann Whitney test, chi‐square test or Fishers test. RESULTS: Both IL‐5 and IL‐6 levels in PF specimens with endometriosis tended to be higher than normal, while the same specimens were mostly interleukin‐1β (IL‐1β) negative. Of great interest was the negative correlation between log (IL‐5) and IL‐6 (Fishers test, P < 0.04). CONCLUSION: These findings support the concept that IL‐5 and IL‐6 but not IL‐1 are associated with endometriosis.

Coelomic metaplasia theory of endometriosis evidence from in vivo studies and an in vitro experimental model

Ultrastructure studies of pelvic peritoneal tissue from women undergoing laparotomy suggest that before endometriosis has become established in the peritoneum, there might be a metaplastic change by peritoneal mesothelial cells into endometrial glandular cells. A new in vitro experimental model of endometriosis using human ovarian surface epithelium cells has shown evidence that endometriotic lesions can arise by a process of metaplasia from the ovarian surface epithelium. In this model, when both ovarian surface epithelium and ovarian stromal cells were cocultured with 17β estradiol in a three-dimensional collagen gel lattice, the ovarian surface epithelium cells formed a lumen structure, surrounded by endometrial stromal cells with an epithelial mesenchymal structure. Immunoreactivity for epithelial membrane antigen and cytokeratin was shown in the glandular cells and cilia, as well as in the microvilli. Electron microscopy showed evidence of tight junctions on cell surfaces. These findings suggest that endometriosis may manifest as a serial change from the adjacent mesothelial cells.

Hypoxia-inducible factor 1 alpha (HIF-1α) gene expression in human ovarian carcinoma

Abstract Hypoxia-inducible factor 1α (HIF-1α) that regulates genes involved in response to hypoxia and promotes neo-angiogenesis, is a transcriptional factor for vascular endothelial cell growth factor (VEGF). The aim of this study was to examine the expression of HIF-1α and VEGF gene expressions and their relation to angiogenesis, clinicopathologic variables and survival in the patient with human ovarian carcinoma. We retrospectively analyzed HIF-1α and VEGF gene expression levels using reverse transcriptase polymerase chain reaction (RT-PCR) in 60 ovarian carcinomas. Intratumoral microvessel density (IMD) was assessed by immunostaining endothelial cells, using anti-CD 31 antibody in frozen sections. The relationships between the expression level of these genes, IMD and clinicopathologic variables were evaluated by Students t-test and chi-square tests. Survival analysis was performed by Kaplan–Meier curves. HIF-1α or VEGF gene expression level was independent of age, clinical stage and histological subtype besides grade of tumor. There was no relationship between HIF-1α or VEGF gene expression level and IMD in all carcinomas (R=0.118 and 0.224, respectively). In addition, a weak association between HIF-1α and VEGF gene expression level was observed (R=0.300, P=0.020). The association between VEGF gene expression and IMD was observed (R=0.501, P=0.016). However, no association between IMD and HIF-1α gene expression was observed. Further, both HIF-1α and VEGF gene expression levels had no effect on survival in the patient with ovarian carcinoma. These results suggest that VEGF upregulated by HIF-1α gene may be involved in angiogenesis of some type of ovarian carcinoma, but the expression levels of both genes have no effect on survival in the patients with ovarian carcinoma.

Pathophysiological dynamics of human ovarian surface epithelial cells in epithelial ovarian carcinogenesis

Epithelial ovarian cancer is responsible for almost half of all the deaths from female genital tract tumors. Major impediments to the clinical treatment of this disease are the relatively asymptomatic progression and a lack of knowledge regarding defined precursor or malignant lesions. Most epithelial ovarian cancers are thought to arise from the transformation of ovarian surface epithelial cells, a single continuous layer of flat-to-cuboidal mesothelial cells surrounding the ovary. To improve our understanding of the pathogenesis of epithelial ovarian cancer, it is necessary to study the biological characteristics of normal ovarian surface epithelial cells. However, this approach has been hampered by the inability to purify and culture such human cells. During the past decade, procedures to isolate and culture human ovarian surface epithelial cells have been developed, and, subsequently, using viral oncogenes, several immortalized cells have been established. This new experimental system is being employed to improve our understanding of the genetic changes leading to the initiation of epithelial ovarian cancer and to identify events in the cancers development. This review mainly describes the biological dynamics of ovarian surface epithelial cells in the pathogenesis of epithelial ovarian cancer, focusing on humans and excluding small animal models.

Characterization of macrophages in the decidual atherotic spiral artery with special reference to the cytology of foam cells

“Acute atherosis” is characteristic in the spiral arteries of the placental bed of preeclampsia and a wide range of pregnancy disorders. The arterial lesion is histologically characterized by fibrinoid necrosis of the vessel walls with infiltration of foam cells, which under a light microscope appears similar to that seen in atherosclerosis. Although acute atherosis is currently considered as atheromatous-like lesions, the precise cellular mechanisms inducing these changes remain unelucidated. By histochemistry, immunohistochemistry, and electron microscopy, we investigated the decidual spiral arteries obtained by placental bed biopsy from 11 preeclamptic and 15 nonpreeclamptic women. In the decidual spiral arteries of preeclamptic patients, acute atherosis was observed in 23.5% (20/85 arteries). Fibrin deposition and accumulation of foam cells were observed more frequently in preeclamptic patients than in nonpreeclamptic patients. Endothelial cells remained in the atheromatous lesion, while the smooth muscle layer surrounding fibrin and foam cells became thin and was finally destroyed. The foam cells were immunohistochemically shown to be macrophages and neutral fat and phospholipids were histochemically demonstrated in them. Ultrastructurally, their cytoplasm was occupied by variously sized lipid droplets and membrane-bound myelin-like granules (myelinosomes). Plasma concentration of monocyte chemoattractant protein 1 (MCP-1), a potent monocyte chemoattractant factor, was significantly elevated in preeclamptic patients compared with normal healthy controls (P ≪ 0.01). In conclusion, injuries to the smooth muscle layer and intramural fibrinoid necrosis may result in infiltration of monocytes into the arterial walls, their maturation into macrophages, and the transformation into foam cells. Considering that atherosclerosis is developed by accumulation of lipid-laden macrophages and migration and proliferation of smooth muscle cells, the roles of macrophages in acute atherosis differ from those in atherosclerosis.

Human Villous Macrophage-Conditioned Media Enhance Human Trophoblast Growth and Differentiation In Vitro

Abstract In human chorionic villi, numerous macrophages, so-called Hofbauer cells, are located adjacent to trophoblasts. To determine the role of the macrophages in the proliferation and differentiation of trophoblasts, cytotrophoblast cells were cultured in serum-free culture-conditioned media of villous macrophages (VMCM), peritoneal macrophages (PMCM), and villous fibroblasts (VFCM). In VMCM, proliferation of cytotrophoblast cells was detected at 24 h by immunocytochemistry with Ki-67-antibody. A large number (P < 0.001) of multinucleated syncytia was formed in VMCM. In VMCM, cytotrophoblast cell fusion was completed by 96 h, which coincided with the peak of hCG secretion and initiation of human placental lactogen (hPL) release. Levels of hCG (P < 0.001) and hPL (P < 0.001) secretion from syncytial cells were significantly higher in VMCM than in PMCM or in VFCM. Concentrations of macrophage colony-stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF) analyzed by ELISA were greater in VMCM than in PMCM or in VFCM, whereas monocyte chemoattractant protein-1 (MCP-1) concentration was high in PMCM. The expression patterns of M-CSF, VEGF, and MCP-1 in villous macrophages and peritoneal macrophages by reverse transcriptase-polymerase chain reaction were similar to their secretion patterns. Thus, villous macrophages have a greater ability to stimulate hCG and hPL secretion than do peritoneal macrophages. This study suggests that macrophages within the villous stroma may stimulate the growth and differentiation of trophoblasts through their secreted substances.

Effect of Medroxyprogesterone Acetate Plus Estradiol on Endothelium-Dependent Vasodilation in Postmenopausal Women

The addition of medroxyprogesterone acetate (MPA) is widely accepted to remove the endometrial-cancerogenic effect of estrogen replacement therapy in postmenopausal women. To evaluate the effect of MPA on endothelial function, we measured flow-mediated vasodilation of brachial arteries after transient occlusion in a randomized, double-blind, placebo-controlled study; we concluded that the addition of MPA attenuates the favorable effects of estradiol on endothelium-dependent vasodilation.

Expression of Regulator of G-Protein Signaling Protein-2 Gene in the Rat Ovary at the Time of Ovulation

Abstract The ovulatory process in mammals begins when an endogenous surge in LH circulates to the ovary and couples with receptors in the plasma membranes of granulosa cells in mature ovarian follicles. This study provides evidence that the ovulatory stimulus includes induction of the gene for regulator of G-protein signaling protein-2 (RGS2). Immature Wistar rats were primed with 10 IU eCG s.c., and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG (a homolog of LH) s.c. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for reverse transcription-polymerase chain reaction differential display to detect gene expression in the stimulated ovarian tissue. Two of the amplified cDNAs that were upregulated within 2 h after the ovaries had been stimulated by hCG were homologous to segments of the mouse gene for RGS2. In situ hybridization indicated that the RGS2 mRNA was expressed in the granulosa layer of mature follicles. In conclusion, the gene for RGS2, which is known to regulate membrane signaling pathways, is transcribed in ovarian follicles in response to an ovulatory dose of gonadotropin.

The Role of c-kit Proto-oncogene during Melanocyte Development in Mouse. In vivo Approach by the In utero Microinjection of Anti-c-kit Antibody

In order to investigate the role of the c‐kit oncogene in the melanoblast development, a rat monoclonal antibody (ACK2) against the mouse c‐kit protein was used to localize cells expressing c‐kit during fetal development. ACK2 was also injected directly into the amniotic cavity of mouse fetuses at successive developmental stages. After birth, the offspring were examined to determine the resulting coat color patterns. c‐kit positive melanoblasts first appeared in dermis of fetuses at 11.5 days postcoitum (dpc). Subsequently, these cells increased in number and migrated dorsolaterally to the ventral region, and by 12.5 dpc some of them began to invade the epidermis. Treatment of fetuses by ACK2 microinjection appeared to affect the pigmentation in the coat, inducing a variety of spotting patterns in offspring, and the location of the spots was closely correlated with gestational stage. ACK2 injection of early fetuses produced major changes in coat color even though few c‐kit positive cells were detectable in the dermal mesenchyme at the time of injection. Large spots were also induced when mid‐stage fetuses with a only few c‐kit positive cells in the dorsal region were injected. By contrast, except for spot formation in the center of ventral region, ACK2 injection did not appear to affect melanogenesis in late stage fetuses that had many c‐kit positive cells.