Estrogen increases inducible nitric oxide synthase gene expression

OBJECTIVE: The goal of this study was to determine whether estrogen increases the expression of the inducible nitric oxide synthase gene. STUDY DESIGN: An inducible nitric oxide synthase fusion gene was created with its promoter and the reporter gene, luciferase. COS cells were transfected transiently with the fusion gene and cotransfected with an estrogen receptor-alpha expression plasmid to ensure the presence of an estrogen receptor. Cells were then exposed to estradiol (1 nmol/L and 10 nmol/L) or a cytokine mix that consisting of tumor necrosis factor-alpha, interleukin-1beta, and interferon gamma. Gene expression was measured in relative light units. RESULTS: Estradiol increased the expression of inducible nitric oxide synthase by an average of 31.2% in the COS cells that were cotransfected with estrogen receptor compared with -10.4% in cells without estrogen receptor (P =.006). CONCLUSION: Inducible nitric oxide synthase expression was increased with the addition of estrogen. These data support previous studies that demonstrated the inflammatory effects of estrogen and provides further insight into the mechanism by which estrogen might have an impact on the cardiovascular system.     

The effect of inducible nitric oxide synthase on postoperative adhesion formation in rats

OBJECTIVE: To evaluate the effect of iNOS on adhesion formation and to assess whether inhibition of iNOS expression affected adhesion formation according to adhesion maturation days. STUDY DESIGN: Forty Wistar Albino rats were subjected to standardized lesion by cecal abrasion and parietal peritoneal defect and were randomly divided into four groups. Group I (control) received no treatment; groups II-IV received N-acetyl-cystein (NAC) 15 mg/100 g per day intramuscularly on days 4-14, 0-14 and 0-3, respectively, after surgery. On the postoperative 14th day adhesion score, tissue iNOS expression, inflammatory cell reaction (ICR) and tissue fibrosis score were determined. RESULTS: Inflammation score of groups I and II was lower than that of groups III and IV (P < 0.05). Adhesion scores and tissue fibrosis of group II were significantly lower than that of the other groups (P < 0.001). CONCLUSION: iNOS inhibition during the first 3 days postoperatively caused a delay in the resolution of inflammatory cell reaction. On the other hand, when inhibited after the first 3 days, adhesion formation and fibrosis were reduced both clinically and histopathologically.     

Polymorphisms of the endothelial nitric oxide synthase gene in women with vulvar cancer

OBJECTIVE: Nitric oxide (NO) is involved in angiogenesis and tumor growth. We attempted to establish an association between two polymorphisms of the endothelial nitric oxide synthase (NOS3) gene and vulvar cancer. METHODS: We used peripheral venous blood sampling, DNA extraction, and polymerase chain reaction (PCR) and pyrosequencing to genotype 68 women with vulvar cancer and 227 healthy Caucasian women for the presence of the intron 4 27-bp-repeat [NOS3*A] and exon 7 Glu298Asp polymorphisms. RESULTS: The presence of a polymorphic NOS3*A allele (26.2% vs. 24.6%; OR: 1.01; 95% CI: 0.6-2.0; P = 0.9) or a polymorphic NOS3 exon 7 Glu298Asp allele (41.2% vs. 53.7%; OR: 0.6; 95% CI: 0.3-1.0; P = 0.09) was not associated with vulvar cancer. Within the vulvar cancer group, the presence of a polymorphic NOS3*A or a polymorphic NOS3 exon 7 Glu298Asp allele was not associated with clinico-pathological parameters such as advanced tumor stage, groin lymph node involvement, tumor grading, and age at diagnosis. Survival analysis demonstrated that the presence of a polymorphic NOS3*A allele was associated with a significantly reduced disease-free survival time (P = 0.03), whereas the presence of the polymorphic NOS3 exon 7 Glu298Asp allele was not associated with disease-free survival (P = 0.5). CONCLUSIONS: We are the first to report on NOS3 polymorphisms in vulvar cancer. We found that allelic variation within intron 4, but not ithin exon 7 of NOS3, influences the length of disease-free survival, but not the biological phenotype of vulvar cancer.     

Polymorphisms of the endothelial nitric oxide synthase gene in women with vulvar cancer

Objective. Nitric oxide (NO) is involved in angiogenesis and tumor growth. We attempted to establish an association between two polymorphisms of the endothelial nitric oxide synthase (NOS3) gene and vulvar cancer.Methods. We used peripheral vanous blood sampling, DNA extraction, and polymerase chain reaction (PCR) and pyrosequencing to genotype 68 women with vulvar cancer and 227 healthy Caucasian women for the presence of the intron 4 27-bp-repeat [NOS3*A] and exon 7 Glu298Asp polymorphisms.Results. The presence of a polymorphic NOS3*A allele (26.2% vs. 24.6%; OR: 1.01; 95% CI: 0.6–2.0; P = 0.9) or a polymorphic NOS3 exon 7 Glu298Asp allele (41.2% vs. 53.7%; OR: 0.6; 95% CI: 0.3–1.0; P = 0.09) was not associated with vulvar cancer. Within the vulvar cancer group, the presence of a polymorphic NOS3*A or a polymorphic NOS3 exon 7 Glu298Asp allele was not associated with clinico-pathological parameters such as advanced tumor stage, groin lymph node involvement, tumor grading, and age at diagnosis. Survival analysis demonstrated that the presence of a polymorphic NOS3*A allele was associated with a significantly reduced disease-free survival time (P = 0.03), whereas the presence of the polymorphic NOS3 exon 7 Glu298Asp allele was not associated with disease-free survival (P = 0.5).Conclusions. We are the first to report on NOS3 polymorphisms in vulvar cancer. We found that allelic variation within intron 4, but not within exon 7 of NOS3, influences the length of disease-free survival, but not the biological phenotype of vulvar cancer.     

Expression of inducible nitric oxide synthase and cyclooxygenase-2 in ovarian cancer: correlation with clinical outcome

OBJECTIVES: Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) play a critical role in cancer development. We investigated iNOS and COX-2 expression in relation to clinical outcome in 78 International Federation of Gynecology and Obstetrics (FIGO) stage III ovarian serous carcinoma with a low grade of differentiation (G3). METHODS: Disease-free interval and cause-specific survival rates (Kaplan-Meier method) were compared using the log rank test. A multivariate analysis (Cox-proportional hazards models) was used to determine the independent effect of each variable on prognosis. Fisher's exact test was used to analyze the distribution of iNOS and COX-2 expression according to clinical complete response to chemotherapy and to the presence of a brief disease-free interval (< or =12 months). RESULTS: Overall 60 and 125 months cause-specific survival rates were 32% and 11%, respectively. In univariate analysis, iNOS (P=0.005 and P=0.003, respectively), COX-2 (P=0.002 and P=0.007, respectively), residual disease after surgery (P=0.017 and P=0.032, respectively), and FIGO stage (P=0.008 and P=0.025, respectively) were associated with survival and a disease-free interval. In multivariate analysis (Cox proportional hazards models), the factors that were found to be significantly independent predictors of disease relapse as well as survival were iNOS (P=0.014 and P=0.001, respectively), COX-2 expression (P=0.007 and P=0.029, respectively), and FIGO stage (P=0.026 and P=0.05, respectively). iNOS and COX-2 expressions were correlated with a brief disease-free interval (P=0.001) and clinical complete response to first-line chemotherapy (P=0.038 and P=0.033, respectively). CONCLUSIONS: The evaluation of iNOS and COX-2 expression may give additional prognostic information concerning the clinical outcome of patients with ovarian carcinoma and may encourage them to select more tailored therapies.     

Endothelial nitric oxide synthase expression in leiomyoma and parental myometrium

The aim of this study was to determine the expression of endothelial nitric oxide synthase (eNOS) in leiomyomatous tissue as a possible mediator of the growth process. Nine patients had myoma enucleation during the follicular phase. The myomata and adjacent myometrial tissues were fixed in formol until study. Immunohistochemical localization of leiomyomatous and myometrial tissue eNOS expression was performed using specific monoclonal antibodies to eNOS. Statistical comparisons were made using the Mann-Whitney Wilcoxon rank-sum test for the expression of eNOS. The test was considered significant when p values were <0.05. Immunostaining for eNOS was seen in the cytoplasm of myometrial endothelial and smooth muscle cells in both tissue sections. eNOS expression was significantly higher in the smooth muscle cells of leiomyomata, compared to parental myometrium (p < 0.0005). The expression of eNOS in vascular endothelial cells was not different in the leiomyoma and myometrium (p > 0.05). To our knowledge, this is the first study to document marked expression of eNOS in leiomyomatous tissue, compared to parental myometrium. We also conclude that the mechanism(s) of the growth-promoting effect of estrogen on leiomyomata is mediated by more synthesis of NO.     

Inducible nitric oxide synthase expression by peritoneal macrophages in endometriosis-associated infertility.

OBJECTIVE: Determine whether peritoneal macrophages from women with endometriosis-associated infertility express more inducible nitric oxide synthase (NOS2) and produce more NO than fertile controls. DESIGN: Unblinded clinical study. PATIENT(S): Nine infertile women with endometriosis and nine normal fertile women undergoing laparoscopy.Intervention(s): Peritoneal fluid and macrophages were collected. Cells were also cultured with the NOS2 inducers interferon-alpha (IFN-alpha) or IFN-gamma plus lipopolysaccharide (LPS). MAIN OUTCOME MEASURE(S): Peritoneal fluid NO levels, peritoneal macrophage NOS activity, and peritoneal macrophage NOS2 protein expression. RESULT(S): NOS enzyme activity was higher in peritoneal macrophages from endometriosis patients. Immunoblots demonstrated NOS2 protein only in peritoneal macrophages from women with endometriosis. Peritoneal fluid NO concentration was similar in the two groups, but total peritoneal fluid NO content was higher in endometriosis patients. After 3 days' culture, peritoneal macrophages from women with endometriosis produced more NO in response to IFN-alpha or IFN-gamma plus LPS than controls. CONCLUSION(S): Peritoneal macrophages from women with endometriosis-associated infertility express higher levels of NOS2, have higher NOS enzyme activity, and produce more NO in response to immune stimulation in vitro. As high levels of NO adversely affect sperm, embryos, implantation, and oviductal function, reducing peritoneal fluid NO production or blocking NO effects may improve fertility in women with endometriosis.     

Estradiol 17-beta and progesterone modulate inducible nitric oxide synthase and high mobility group box 1 expression in human endometrium

The aim of the present study is to investigate the effects of ovarian sex steroid hormones on the expression and the release of several locally active substances by human endometrium. Specific objectives are (1) to ascertain if estradiol 17-beta (E2) and progesterone modulate inducible nitric oxide synthase (iNOS) expression and nitric oxide release; (2) to determine whether human endometrium can express High Mobility Group Box 1 (HMGB1), a multifunctional cytokine, and whether sexual steroid hormones can modulate this expression; and (3) to evaluate whether nitric oxide can influence HMGB1 expression in this tissue. Endometrial tissue was obtained from 40 healthy premenopausal women who underwent hysteroscopy for suspected benign gynecological conditions. Endometrium was incubated with E2, progesterone, or sodium nitroprusside, a nitric oxide donor. Nitrite assay was used to quantify stable nitric oxide metabolites in culture medium, and Western blot analysis was used to detect iNOS and HMGB1. Incubation of endometrium with E2 results in an increase in iNOS expression and nitric oxide metabolite production. The opposite effect is obtained by incubating tissues with progesterone. HMGB1 is expressed by human endometrium, and its expression is increased by E2 and decreased by progesterone. Incubation with sodium nitroprusside results in a reduction in HMGB1 expression. Both E2 and progesterone modulate iNOS expression and nitric oxide production in human endometrium. HMGB1 is expressed in the human endometrium, and its expression is modulated by E2, progesterone, and nitric oxide.     

Psychosexual aspects of the evaluation and management of vulvar vestibulitis.

OBJECTIVE: This article describes the structure and outcome of a collaboration between a gynecologist and a psychologist in evaluating and treating 45 consecutive women with vulvar vestibulitis. STUDY DESIGN: Women were interviewed by the psychologist in a structured format and also filled out questionnaires. Vulvar lesions were defined by clinical examination and colposcopy, and a conservative local excision was performed, without mobilization of the vagina. Postoperatively, women were offered sexual counseling including Kegel exercises, vaginal dilation, and couple therapy. Follow-up data were gathered a mean of 8 months after treatment. RESULTS: Of the 32 women who had both surgical excision of vulvar lesions and contact with the psychologist, 50% were much improved in perceived pain, 41% were somewhate improved, and 9% were unimproved. Factors predictive of an improved outcome included willingness to have psychologic treatment, higher socioeconomic status, and self-report of specific, localized areas of vulvar pain rather than vague, diffuse pain. Parous women were more likely to improve. Those who reported increased pain intensity premenstrually had poorer outcomes. CONCLUSIONS: Vulvar vestibulitis may be a syndrome that results from interacting pathophysiologic and psychologic factors, so that a comprehensive treatment approach is beneficial. Women who have diffuse genital pain or who refuse psychologic intervention may be poor candidates for surgery.     

Localized increase in nitric oxide production and the expression of nitric oxide synthase isoforms in rat uterus with experimental intrauterine infection.

OBJECTIVE: We recently reported that nitric oxide was associated with increased mortality among pregnant rats with intrauterine infection. In our current study we investigated the expression of different isoforms of nitric oxide synthases and nitric oxide in the nonpregnant rat uterus with experimental intrauterine infection. STUDY DESIGN: Pathogenic Escherichia coli was inoculated into the uterine lumen of ovariectomized rats. Animals were killed after inoculation, and uterine horns were collected for assessing nitric oxide production with high-performance liquid chromatography and nitric oxide synthase (type II and type III) protein expression with Western immunoblotting and immunofluorescence methods. RESULTS: (1) Nitric oxide production increased in the infected uterine horn in a time-dependent manner after intrauterine infection but did not increase in the uninfected horn. (2) Nitric oxide synthase type III protein contents did not show a difference between infected and uninfected horns, and type III nitric oxide synthase was expressed by the epithelial cells and smooth muscle cells. (3) Type II nitric oxide synthase was abundantly expressed in infected horns but was not expressed in uninfected horns. Immunofluorescence data indicated that macrophages and natural killer cells, located in the endometrial layer clustering around epithelial cells, expressed type II protein. CONCLUSION: We suggest that localized increase in type II nitric oxide synthase expression and nitric oxide production occurs in response to intrauterine infection and that the nitric oxide system may play a role in host response to restrict the infection.     

胎盘组织中诱导型一氧化氮合酶和巨噬细胞的表达与妊娠高血压综合征发病的关系

目的　通过测定妊娠高血压综合征 (妊高征 )孕妇胎盘组织中诱导型一氧化氮合酶和巨噬细胞的表达 ,探讨其在妊高征发病中的作用。方法　应用免疫组织化学方法检测 30例妊高征孕妇 (妊高征组 ,其中轻度 2例 ,中度 7例 ,重度 2 1例 )和 2 2例正常妊娠妇女 (对照组 )胎盘组织中诱导型一氧化氮合酶和巨噬细胞的表达。结果　 (1)妊高征组孕妇胎盘组织中诱导型一氧化氮合酶和巨噬细胞表达的阳性率分别为 (2 9± 0 7) %和 (3 0± 0 8) % ,对照组孕妇分别为 (2 1± 0 8) %和 (2 1±0 7) % ,两组比较 ,差异有极显著性 (P <0 0 1)。 (2 )轻、中度妊高征孕妇胎盘组织中诱导型一氧化氮合酶和巨噬细胞表达的阳性率分别为 (2 8± 0 7) %和 (2 8± 1 0 ) % ,重度妊高征孕妇分别为 (3 0±0 7) %和 (3 1± 0 8) % ,两者之间比较 ,差异无显著性 (P >0 0 5 )。结论　妊高征患者胎盘组织中诱导型一氧化氮合酶和巨噬细胞均有高表达 ,这可能是引起胎盘功能不良和胎儿 胎盘循环阻力改变的原因之一 ,并可能在妊高征的发病中起重要作用     

The effects of cervical application of inhibitors of inducible nitric oxide synthase, cyclooxygenase-1, and cyclooxygenase-2 on delivery in rats

OBJECTIVE: To investigate the effect of cervical application of nonselective and selective inhibitors of nitric oxide synthases--N-nitro-L-arginine methyl ester, L-N-iminoethyl-lysine, and aminoguanidine--as well as inhibitors of cyclooxygenases--indomethacin, and nimesulide--on timing of delivery and fetal death and disease in pregnant rats. STUDY DESIGN: In a series of experimental protocols, timed-pregnant Sprague-Dawley rats (length of pregnancy, 22 days) were randomly allocated to daily cervical applications of (1) 0.04 mg (n = 6), 0.4 mg (n = 6), 4 mg (n = 6), or 40 mg (n = 6) L-N-iminoethyl-lysine or vehicle (n = 12) on days 19 to 22 of pregnancy; (2) 50 mg aminoguanidine (n = 6), 150 mg aminoguanidine (n = 6), or vehicle (n = 10) on days 19 to 22 of pregnancy; (3) 3 mg indomethacin (n = 6) or vehicle (n = 6) on days 19 to 22 of pregnancy; (4) 12.5 mg/kg nimesulide (n = 8), 25 mg/kg nimesulide (n = 8), 50 mg/kg nimesulide (n = 12), or vehicle (n = 12) on days 19 to 22 of pregnancy and 50 mg/kg nimesulide (n = 23) or vehicle (n = 23) on days 14 to 22 of pregnancy; (5) 50 mg/kg nimesulide (n = 10), 50 mg aminoguanidine plus 50 mg/kg nimesulide (n = 10), 50 mg aminoguanidine (n = 10), or vehicle (n = 10) on days 14 to 22 of pregnancy. The following variables were evaluated: proportion of animals that were delivered on day 23, time to delivery of the first pup (midnight on day 22 was considered to be 0 hour), number of stillborn pups, and average pup weight of each litter. RESULTS: Unlike L-N-iminoethyl-lysine, aminoguanidine, and indomethacin, 50 mg/kg nimesulide applied on the cervix daily for 8 days significantly increased the proportion of animals that were delivered on day 23 (18 of 23 versus 7 of 23; P =.003) and the time to delivery of the first pup by a mean of 10.8 hours (P <.001). Shorter treatment with nimesulide for 4 days increased only the time to delivery of the first pup at the 25-mg/kg dosage (P =.008). Simultaneous application of aminoguanidine and nimesulide significantly (P =.008) prolonged pregnancy to a degree similar to nimesulide alone. The experiment with N-nitro-L-arginine methyl ester was aborted because of severe maternal side effects. Unlike pups in the L-N-iminoethyl-lysine, aminoguanidine, and nimesulide groups, significantly more pups in the indomethacin group died in utero compared with the control group (36.1% versus 3.1%; P <.001), and the surviving pups had lower birth weights (P <.001). CONCLUSIONS: In an animal model, nimesulide was effective in delaying the onset of labor, was well tolerated during pregnancy, and affected cervical ripening directly independent of progesterone withdrawal. Conversely, cervical application of nitric oxide synthase and nonselective cyclooxygenase inhibitors do not extend the duration of pregnancy in the dosages studied, and some are associated with significant adverse effects in the mothers and fetuses.     

A hypothesis for preeclampsia: adenosine and inducible nitric oxide synthase in human placental microvascular endothelium

Preeclampsia is a human syndrome characterized by elevated maternal blood pressure and proteinuria. It is the main cause of maternal morbidity and mortality, and fetal metabolic disturbances in developed and developing countries. Fetal complications in preeclampsia have been related with lower placental blood flow. The placenta lacks of innervation, thus vascular tone regulation depends on endothelial release of vasoactive molecules such as adenosine and nitric oxide (NO). Information about NO synthesis and its action in the feto-placental vasculature in preeclamptic pregnancies is controversial mainly due to the use of different methodological approaches, severity of the disease and cell type that had been analyzed. A high plasma level of adenosine has been reported in umbilical vein from preeclampsia compared with normal pregnancies. Since this nucleoside is mainly involved in the regulation of vascular tone and angiogenesis, perhaps through the modulation of potassium channels, it is suggested that it would be involved in the maintenance of normal feto-placental function. In this review we hypothesize a potential adenosine-mediated, NO-dependent mechanism accounting for the feto-placental reduced blood flow exhibited in preeclampsia. We summarize the potential mechanisms involved in the modulation of inducible NO synthase expression and activity in preeclampsia, emphasizing metabolic alterations in the placenta microvascular endothelial function. The involvement of adenosine, nucleoside membrane transporters and adenosine receptors, nuclear factor kappa B (NF-kappaB), potassium channels and angiogenesis, are also discussed regarding abnormal endothelial function in preeclampsia.