内皮型一氧化氮合成酶基因在重度妊高征胎盘绒毛组织的表达

目的探讨重度妊高征患者胎盘绒毛组织内皮型一氧化氮合成酶ｍＲＮＡ的表达。了解其与妊高征的关系。方法用地高辛标记的内皮型一氧化氮合成酶探针,对９例正常孕妇（正常对照组）及９例重度妊高征孕妇（妊高征组）的胎盘绒毛组织总ＲＮＡ进行点印迹杂交。利用ＬｅｉｃａＱＷＩＮ图像处理及分析系统测量每个杂交点的平均光密度值。结果妊高征组胎盘绒毛组织内皮型一氧化氮合成酶ｍＲＮＡ的表达明显低于正常对照组,两组间差异有极显著性（Ｐ＜０．００１）。结论重度妊高征组胎盘绒毛组织内皮型一氧化氮合成酶的减少,可能是导致胎盘功能不良和胎儿－胎盘循环血流阻力增加的原因之一,并在妊高征的发病中起重要作用     

Endothelial nitric oxide synthase polymorphism in preeclampsia

OBJECTIVE: We wished to determine whether genetic variability in the gene encoding endothelial nitric oxide synthase (eNOS) modifies individual susceptibility to the development of preeclampsia. METHODS: The study involved 132 preeclamptic and 113 healthy control pregnant women who were genotyped for the Glu298Asp polymorphism in the eNOS gene. Chi(2) analysis was used to assess genotype and allele frequency differences between preeclamptic women and controls. RESULTS: A statistically similar allelic distribution of eNOS Glu298Asp polymorphism was observed in the two groups, with the frequency of the variant G allele being 74.6% in the preeclampsia group and 67.7% in the control group (P = .091; odds ratio 1.40, 95% confidence interval 0.95, 2.01). Accordingly, the genotype distribution of the NOS polymorphism in the preeclamptic and control groups was found to be similar (P = .233). CONCLUSION: These genotype data in subjects from eastern Finland were not suggestive of an important contribution of the Glu298Asp polymorphism in the NOS gene on preeclampsia across populations. However, the observed association between the G allele and disease risk, of borderline significance, may imply that other polymorphism(s) in the gene may modify disease risk.     

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.     

胎盘生长因子与子痫前期发病及一氧化氮关系的研究

目的:探讨胎盘生长因子(PLGF)在子痫前期发病中的作用及其与一氧化氮的关系。方法:选择妊娠期高血压疾病患者45例,其中妊娠期高血压10例,轻度子痫前期12例,重度23例;选择同期正常妊娠妇女20例作为对照组。采用免疫组织化学染色法和逆转录-聚合酶链式反应(RT-PCR)检测两组患者胎盘PLGF蛋白及mRNA的表达。采用硝酸盐还原酶法测定两组胎盘组织NO浓度的变化。结果:(1)免疫组化结果显示,轻度和重度子痫前期的胎盘绒毛合体滋养细胞、绒毛间质PLGF表达均显著低于正常妊娠组(P<0.05),妊娠期高血压组与正常组无差别;PLGF在妊娠期高血压、子痫前期组及正常妊娠组分布范围基本一致,主要分布在绒毛合体滋养细胞和间质细胞胞浆,部分血管合体膜上也有表达;(2)轻、重度子痫前期胎盘组织PLGF mRNA平均灰度分别为3.33±0.39、1.97±0.29,显著低于正常妊娠组的平均灰度4.87±0.60(P<0.01);(3)轻、重度子痫前期胎盘组织中NO浓度分别为8.20±5.56μmol/g、6.46±2.25μmol/g,显著低于对照组18.10±7.12μmol/g(P<0.05);妊娠期高血压组胎盘组织NO浓度与对照组差异无显著性;(4)胎盘组织中胎盘生长因子表达水平与胎盘组织NO浓度呈显著正相关(r=0.54,P<0.05)。结论:子痫前期胎盘组织中胎盘生长因子水平降低,NO浓度下降,可能在子痫前期的发病中起一定作用。     

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     

Expression of endothelial and inducible nitric oxide synthases and nitric oxide production in ovine placental and uterine tissues during late pregnancy

We evaluated the expression of endothelial (eNOS) and inducible (iNOS) nitric oxide (NO) synthases, NO production, and the role of angiotensin II (ANG II) in regulating NO production during late ovine pregnancy (day 110-142). Samples of the following tissues were obtained: fetal [cotyledonary (COT)] and maternal [caruncular (CAR)] portions of the placentoma, intercotyledonary fetal chorioallantoic membrane (ICOT) and intercaruncular maternal endometrium (ICAR). Using immunohistochemistry, eNOS positive staining was detected in all four tissues, primarily in the endothelium, chorioallantoic membrane, and luminal and glandular epithelium. For iNOS, the positive staining was observed primarily in stromal cells in ICOT and ICAR. Expression of eNOS and iNOS proteins was confirmed in COT using Western immunoblot. eNOS protein levels increased (P< 0.05) approximately 3.5-fold from day 110 to 130 and then declined at term, whereas no change in iNOS protein levels was observed throughout the days studied. The tissue explants of COT, CAR, ICOT and ICAR were cultured in media in the absence or presence of ANG II (10(-9)or 10(-7) m) for 24 h. Total NO (nitrate and nitrite) levels in the explant-conditioned media were determined by chemiluminesence. In fetal COT, total NO levels increased (P< 0.05) 3.5-fold from day 110 to 130 and then declined (P< 0.05) at term. In ICOT, total NO levels exhibited a gradually increasing trend (r(2)=0.96, P< 0.01) from day 110 to days 130 and 142. In maternal CAR, total NO levels were higher (P< 0.05) on day 130 than those on days 120 and 142, whereas no change in total NO levels was observed in ICAR. ANG II at 10(-7) m treatment decreased (P< 0.05) total NO levels in COT on day 130. Thus, during late ovine pregnancy: (1) eNOS is expressed in COT, CAR, ICOT and ICAR while iNOS is primarily seen in stromal cells of ICOT and ICAR; (2) NO production by COT exhibits a biphasic pattern and parallels the changes in eNOS, but not iNOS protein levels, suggesting that eNOS is a predominant NOS isoform for the NO production; and (3) ANG II may contribute partially to decreases in NO production by COT at term.     

Immuno-electron microscopic localization of endothelial nitric oxide synthase in human placental terminal villous trophoblasts-normal and pre-eclamptic pregnancy

We demonstrated the subcellular localization of endothelial nitric oxide synthase (eNOS) in human placental terminal villous trophoblasts at near term period, and compared the distribution pattern with that in pre-eclamptic trophoblasts, using immunogold electron microscopy. Immunolabelling for eNOS was visible markedly in the syncytial microvilli and syncytial cytoplasm. Semiquantitative analysis showed that the concentration and the distribution pattern of gold particles for eNOS did not significantly differ between normal and pre-eclamptic placental trophoblasts. These results indicated that syncytiotrophoblastic microvilli and cytoplasm were the subcellular localization sites of syncytium-derived eNOS in terminal villi, and that there were no significant differences in this eNOS subcellular distribution pattern between normal and pre-eclamptic syncytiotrophoblasts in regard to immunohistochemically detectable eNOS.     

Endogenous inhibitors of nitric oxide and preeclampsia: A review

Nitric oxide (NO) is a potent vasodilator. NO is synthesized by NO synthases (NOS) and NOS are inhibited by asymmetrical dimethylarginine (ADMA). ADMA is metabolized by dimethylarginine dimethylaminohydrolase (DDAH) and excreted in the kidneys. Lower ADMA levels in pregnant women compared to non-pregnant controls suggest that ADMA has a role in vascular dilatation and blood pressure changes. Several studies show an increase in ADMA levels in pregnancies complicated with preeclampsia. Elevated ADMA levels in preeclampsia are seen before clinical symptoms have developed; these findings suggest that ADMA has a role in the pathogenesis of preeclampsia.     

Doppler analysis and placental nitric oxide synthase expression during fetal growth restriction

Objective. To assess placental nitric oxide (NO) metabolism related to changes in the uteroplacental circulation during fetal growth restriction (FGR).

Methods. The resistance index (RI) from the uterine arteries and pulsatility index (PI) from the umbilical artery were determined by Doppler analysis in 15 patients with FGR and 12 healthy controls, before elective cesarean section. Inducible (iNOS) and endothelial (eNOS) NO synthase expression were measured in placental samples. Immunohistochemistry was performed for iNOS location in the placenta.

Results. During FGR, we observed a significant elevation of iNOS when compared with controls. Conversely, eNOS did not differ between the two groups. A negative correlation with eNOS (r = ?0.85) and a positive correlation with iNOS (r = 0.91) was found correlating to umbilical PI. The iNOS proteins were reduced in syncytiotrophoblast cells and increased in endothelium in the FGR group compared to the controls.

Conclusions. During FGR, placental iNOS expression is significantly increased; this increase possibly represents an adaptive physiological mechanism for overcoming a fetoplacental circulation deficiency.     

Angiotensinogen and endothelial nitric oxide synthase gene polymorphisms among Hispanic patients with preeclampsia

OBJECTIVE: We sought to establish an association between preeclampsia and the methionine to threonine polymorphism at amino acid residue 235 (Met235Thr) in angiotensinogen in a Hispanic population. We looked for a relationship between this allele and the allele in the endothelial nitric oxide synthase gene (NOS3) that produces the A form (NOS3*A) with respect to preeclampsia. STUDY DESIGN: Clinical data were collected from 87 patients with preeclampsia and 53 control subjects. Patients and controls were genotyped for the angiotensinogen polymorphism allele (AGT*T) and the NOS3*A polymorphism. We then compared patients with preeclampsia and control subjects and investigated disease severity within the preeclampsia group as a function of genotype. RESULTS: The AGT*T allelic frequencies among patients with preeclampsia and control subjects were 0.72 and 0.70, respectively (P =.84). The blood pressure of patients with an AGT*T allele who also carried a NOS3*A allele was higher at earlier gestational ages (r = -0.052; P =.02). Analysis suggested that the systolic blood pressure differences were due to gestational age effects and the presence of a NOS3*A allele (P <.10). CONCLUSION: The AGT*T allele was not associated with the development of preeclampsia. Independently of the presence of an AGT*T allele, the NOS3*A allele was associated with a higher blood pressure at an earlier gestational age.     

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.     

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.     

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.     

Association of endothelial nitric oxide synthase gene G894T polymorphism and serum nitric oxide levels in patients with preeclampsia and gestational hypertension

Objective: Pregnancy-induced hypertension is one of the most important cause of maternal-fetal morbidity and mortality. Pregnancy-related hypertensive disorders are usually associated with diminished nitric oxide (NO) levels. We aimed to evaluate the role of serum NO levels and eNOS gene G894T polymorphism on hypertensive disorders of pregnancy.

Methods: Eighty patients with gestational hypertension or preeclampsia, and 80 healthy pregnants were enrolled to analyze serum NO levels and G894T polymorphism of the eNOS gene. NO level was analyzed by high-performance liquid chromatography (HPLC) method. The G894T polymorphism of the eNOS gene was determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP).

Results: There was no significant difference between groups in terms of G894T/eNOS genotype and allele frequencies (p?>?0.05). Serum NO levels were significantly lower in the patients group. In the control group, subjects with thymine-thymine (TT) genotype had significantly lower NO levels when compared to subjects with guanine-guanine (GG) or guanine-thymine (GT) genotype (p?Conclusions: We failed to demonstrate an association between eNOS gene G894T polymorphism and serum NO levels in patients with pregnancy-induced hypertensive disorders. We established a relation between pregnancy-induced hypertension and low NO levels.     

Endothelial nitric oxide synthase polymorphism is not associated with placental abruption in Finnish women

OBJECTIVE: To study whether genetic variability in the gene encoding endothelial nitric oxide synthase (eNOS) affects individual susceptibility to the development of placental abruption during pregnancy. METHODS: One hundred and sixteen pregnant women with placental abruption and 113 healthy controls were genotyped for Glu298Asp polymorphism in the eNOS gene. Chi-square analysis was used to assess the differences in genotype and allele frequencies between the two groups. RESULTS: A statistically similar allelic distribution of eNOS Glu298Asp polymorphism was observed in the two groups, with the frequency of the variant G allele being 66.8% in the abruption group and 68.1% in the control group (OR 0.94, 95% CI: 0.64-1.39; p = 0.76). The genotype distribution of the eNOS polymorphism was also found to be statistically similar (p = 0.72). CONCLUSIONS: The observed genotype data in subjects from eastern Finland suggest that the Glu298Asp polymorphism of the eNOS gene does not contribute to placental abruption in this population.     

Group B Streptococci and inducible nitric oxide synthase: modulation by nuclear factor kappa B and ibuprofen

Group B Streptococci (GBS) neonatal infections cause a complex inflammatory process involving numerous biochemical mediators. Nitric Oxide (NO) is generated by many cell types in response to different inflammatory signals. Nuclear factor kappa B (NFkappaB) plays an important role in the inflammatory process and may induce a number of biochemical mediator genes, including the inducible nitric oxide synthase (iNOS). We tested the hypothesis that GBS induces iNOS gene expression through activation of NFkappaB. We also tested whether ibuprofen (IBU) will suppress iNOS expression by blocking NFkappaB activation. Cerebral microvascular endothelial cells isolated from newborn piglets were harvested for the determination of iNOS gene expression and activation of NFkappaB. GBS significantly induced iNOS mRNA expression (5- to 6-fold, P < .005) and iNOS protein (3- to 4-fold, P < .01) at 24 hours. DNA-NFkappaB binding activity was detected within 15 minutes of GBS treatment and reached a maximal effect at 3 hours. Treatment with IBU significantly suppressed GBS-induced iNOS mRNA expression at 24 hours, and NFkappaB activity at 3 hours, suggesting that suppression of GBS-induced iNOS mRNA expression by IBU occurs by blocking of NFkappaB activation. These data show that NFkappaB activation is an early step in the induction of iNOS gene expression by GBS and that this interaction may play a vital role in the pathogenesis of GBS neonatal infections.