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Expression of endothelial nitric oxide synthase traffic inducer in the placentas of women with pre-eclampsia
International Journal of Gynecology and Obstetrics (2005) 89, 103 — 107
www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Expression of endothelial nitric oxide synthase traffic inducer in the placentas of women with pre-eclampsia W. XiangT, H. Chen, X. Xu, M. Zhang, R. Jiang Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan-430030, China Received 20 July 2004; received in revised form 16 December 2004; accepted 16 December 2004
KEYWORDS NOSTRIN; Nitric oxide synthase; Placenta; Pre-eclampsia
Abstract Objective: To investigate the expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) in the placenta of women with pre-eclampsia (PE) and discuss its role in the pathogenesis of PE. Methods: Western blot analysis was used to detect the expression of NOSTRIN and endothelial nitric oxide synthase (eNOS). The activity of eNOS in placental tissue was assayed by spectrophotometry. Serum and placental NO2/NO3, the stable metabolic end product of NO, was measured using nitrate reductase. Results: Western blot analysis showed that the protein level of NOSTRIN was significantly higher in women with PE than in the control group ( Pb0.01). However, no significant difference between groups was observed in the expression of placental eNOS ( PN0.05). The activity of eNOS was significantly decreased in women with PE (13.727F3.58 U/mg) compared with the control group (21.69F3.84 U/mg) ( Pb0.01). The placental levels of NO2/NO3 were significantly lower in women with PE (27.53F8.51 Amol/mg) than in the control group (44.38F9.59 Amol/mg) ( Pb0.01). The levels of serum NO2/NO3 were significantly lower in women with PE (48.56F8.49 Amol/L) than in the control group (65.37F9.61 Amol/L) ( Pb0.01). A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in the placental tissues of women with PE (r= 0.57, Pb0.01). Conclusion: The level of NOSTRIN expression in the placental tissues of women with PE is increased and is negatively correlated with the activity of eNOS— which may play an important role in the pathogenesis of PE. D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
T Corresponding author. Tel.: +86 27 83662698; fax: +86 27 83692550. E-mail address: [email protected] (W. Xiang). 0020-7292/$ - see front matter D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.12.041
104
W. Xiang et al.
1. Introduction
2. Materials and methods
Pre-eclampsia (PE), the most common serious complication of pregnancy, is characterized by vasoconstriction, dysfunction of the vascular endothelium, and hypertension. Despite decades of intensive research, the pathogenesis of PE remains incompletely understood. Since 1992 [1], nitric oxide (NO)-mediated vasoconstriction has been thought to contribute to the development of the syndrome and attention has been focused on the relationship between NO levels and PE. There are many studies suggesting maternal levels of NO may be increased [2], decreased [3] or unaltered [4] in women with PE. Nitric oxide, an important mediator of biological processes such as neurotransmission, inflammatory response, and vascular homeostasis [5], is synthesized from l-arginine by a family of nitric oxide synthases (NOSs). Nitric oxide derived from endothelial cells acts as a potent vasodilator via several mechanisms such as second messenger cyclic guanine monophosphate (cGMP) [6]. It leads to smooth muscle relaxation by acceleration of the intracellular binding of free calcium and is known for its ability to inhibit platelet aggregation and adhesion to vascular endothelial cells. Three NOS isoenzymes have been identified, of which endothelial NOS (eNOS) is known to be expressed by human placental syncytiotrophoblasts, extravillous trophoblasts [7], and vascular endothelial cells. The endothelial isoform eNOS is a key enzyme of the cardiovascular system that contributes to vascular homeostasis through tightly regulated NO production [8].The level of eNOS expression can be altered by various stimuli. Shear stress and estrogen can increase eNOS expression both in vitro and in vivo [9], and eNOS interacting proteins (NOSIPs) and endothelial nitric oxide synthase traffic inducer (NOSTRIN) have been reported to regulate eNOS activity [10,11]. A large number of studies [12,13] have shown that abnormalities in eNOS activity may contribute to the development of preeclampsia. In a study of the yeast 2-hybrid system using the eNOS oxygenase domain as bait, Zimmermann et al. [11] identified a previously uncharacterized eNOSinteracting protein that they dubbed NOSTRIN. NOSTRIN was observed to induce a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern, which was followed by a significant inhibition of NO release [11]. In the present study, we detected the expression of NOSTRIN in placental tissue form healthy pregnant women and women with PE, and discussed the role of NOSTRIN in the pathogenesis of PE.
2.1. Patients Placental samples were obtained from 11 healthy pregnant women and 11 women with PE at the time of their cesarean section. The study was approved by the local ethics committee and informed consent was obtained from all patients. Pre-eclampsia was defined by hypertension and proteinuria after the 20th week of pregnancy; a blood pressure of 160/ 110 mm Hg or greater; urine albumin of 5 g/24 h or greater; and/or presence of various symptoms such as persistent headache and/or visual impairment and seizures, as described in text books. The placentas of women with twin pregnancies were not included, nor were any of the placentas from mothers with primary hypertension and cardiovascular disease, renal disease, hepatic disease, diabetes, or other infectious or autoimmune diseases.
2.2. Treatment of samples Placentas were obtained from all women at cesarean section. A specimen of placental tissue of at least 2.0 cm2.0 cm2.0 cm was taken at the central site of the placenta, avoiding calcified and hemorrhagic areas, and rinsed with isotonic saline solution to remove blood. Peripheral venous blood samples were obtained from all women before the cesarean section was performed. Serums were taken after centrifugation at 3000 g/min for 15 minutes. The samples were stored at 80 8C until analysis was performed.
2.3. Western blot analysis Samples were harvested with lysis buffer containing 150 mM NaCl, 50 mM Tris—HCl (pH 7.4), 0.1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 50 mM NaF, and phenylmethylsulfonyl fluoride, as previously described [14]. Then electrophoresis of 100 Ag of total protein was performed on 10% SDS-polyacrylamide gels and the protein was transferred to a nitrocellulose membrane (Zhongshan Biotech, Beijing, China). The membrane was blocked with 3% bovine serum antibody (BSA, Hyclone, USA) for 2 h, and incubated with polyclonal antibodies against NOSTRIN (a gift from Prof. Zimmermann, Institute for Biochemistry and Institute for Pathology, University of Frankfurt, Germany); eNOS (NOS3 [C-20], Santa Cruz Biotechnology, USA) (NOS3 [C-20] is an affinity-purified rabbit polyclonal antibody raised against a peptide mapping at the carboxy terminus
Expression of endothelial nitric oxide synthase traffic inducer Table 1
105
¯ Fs) Level of NOSTRIN and eNOS protein (x
NOSTRIN eNOS
Group
Patient no.
Area (picture element)
OPTDM
OPTDI
Control PE Control PE
11 11 11 11
458 1859T 1298 1256TT
0.390378 0.537124T 0.488106 0.483182TT
178.79 998.51T 633.56 606.88TT
Abbreviations: OPTDM, optical density; OPTDI, optical density index. T Pb0.01 vs. control group (NOSTRIN). TT PN0.05 vs. control group (eNOS).
of NOS3 of human origin); and h-actin (Zhongshan, China). The membrane was then incubated with a secondary antibody (goat antirabbit Ig, Santa Cruz Biotechnology) conjugated to horseradish peroxidase (Amersham, Japan) and developed with a chemiluminescent assay (Pierce, USA). An image band analysis system (FR-980, China) was used to determine the concentrations of NOSTRIN and eNOS.
group. The optical density index of bands were markedly higher in the PE group compared with the control group ( Pb0.01) but there was no significant difference in the protein expression of placental eNOS between the 2 groups ( PN0.05) (Table 1). Four specimens of healthy placental tissue and 5 specimens of placentas from women with PE are shown in Figs. 1 and 2.
2.4. eNOS activity and serum and placental NO2/NO3
3.2. eNOS activity assay
eNOS activity in placental tissues was assayed by spectrophotometry and NO2/NO3 was measured with nitrate reductase following the kit manufacturer’s instructions (NanJing Biology Institute, China).
Spectrophotometry was used to assay the activity of eNOS following the kit manufacturers’ instructions. We found that eNOS activity was decreased in the PE group compared with the control group (13.73F3.58 U/mg vs. 21.69F3.84 U/ mg; Pb0.01).
2.5. Statistical analysis
3.3. NO2/NO3 assay
The data were analyzed using SPSS 10.0 software (SPSS, Chicago, USA). The correlation between the level of NOSTRIN and eNOS activity was determined by correlation analysis. Pb0.05 indicated statistical significance.
The NO2/NO3 assay was performed with nitrate reductase according to the kit manufacturers’ instructions. Placental NO2/NO3 was significantly lower in PE patients than in the control group (27.53F8.51 Amol/mg vs. 44.38F9.59 Amol/mg; Pb0.01). The concentrations of serum NO2/NO3 were significantly lower in PE patients than in the control group (48.56F8.49 Amol/L vs. 65.37F9.61 Amol/L; Pb0.01).
3. Results 3.1. Western blot analysis of NOSTRIN and eNOS NOSTRIN monomer (58 kDa) and eNOS monomer (135 kDa) were found in all tissues examined. Light bands of NOSTRIN were found in the control group, whereas stronger bands were found in the PE
3.4. Correlation between the expression of NOSTRIN and the activity of eNOS A correlation analysis was used to determine the correlation between NOSTRIN levels and eNOS activity in the PE group. The results indicated that
Figure 1 Level of NOSTRIN protein in placental tissue specimens from women with PE and healthy controls. Lanes 1, 2, 5, 6: healthy placenta specimens; lanes 3, 4, 7, 8, 9: placental tissue specimens from women with PE.
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Figure 2 Level of eNOS protein in placental tissue specimens from healthy controls and women with PE. Lanes 1, 2, 5, 6: healthy placenta specimens; lanes 3, 4, 7, 8, 9: placental tissue specimens from women with PE.
a significant negative correlation existed between them (r= 0.57, Pb0.01).
4. Discussion A large number of studies suggest that abnormalities in NO synthesis may contribute to the development of pre-eclampsia. Smarason et al. [2] suggested that serum nitrate levels are increased; Diejomaoh et al. [4] suggested that serum nitrate levels are unchanged; and Seligman et al. [15] suggested that circulating levels of nitrite are decreased in women with pre-eclampsia. Seligman demonstrated a significant decrease of serum nitrite level in preeclampsia and a negative correlation of serum nitrite levels with systolic and diastolic blood pressure. The Var et al. [16] study has shown that serum NO is also decreased in pre-eclampsia and the low level of NO plays an important role in pathophysiology of PE. Our data show that serum NO2/NO3 in women with was significantly lower than that in the control group (48.56F8.49 Amol/L vs. 65.37F9.61 Amol/L), which supports the concept that diminished NO synthesis contributes to the pathophysiologic changes seen in pre-eclampsia. Nitric oxide has a very short half-life time, as it quickly undergoes oxidation into its stable metabolites. Therefore, more stable results could be expected by studies on NOS activity in biopsy specimens of the placental bed. We used spectrophotometry to assay the activity of eNOS and found that eNOS activity was significantly lower in the PE group than that in the control group, which is consistent with the results of previous studies [17,18] but conflicts with the study by Shaamash and Rajagopal [19,20]. We also found placental NO was significantly lower in women with PE than that in the control group (27.53F8.51 Amol/mg vs. 44.38F9.59 Amol/mg), which suggests that low eNOS activity results in less production of placental NO. Low levels of placental NO can effect a decrease in serum NO, and low levels of serum NO can lead to hypertension and increase venous thrombosis, vasospasm, and poor perfusion of the placenta [6]. All these pathological changes can aggravate endothelial injury and the dysfunction of associated organs, and eventually lead to PE. Our results suggest that a decreased eNOS activity may
be involved in the pathogenesis of PE via its impact on NO production. We determined the expression of a particular eNOS interaction protein, NOSTRIN, which was identified by Zimmermann et al. [11]. We found that NOSTRIN levels were significantly higher in the placentas of women with PE than in the placentas of women having a normal pregnancy. We also found that a significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in the placental tissues of women with PE. Our results suggest that an increased expression of placental NOSTRIN results in reduced eNOS activity and less production of placental and serum NO, and that lower levels of serum NO may destroy the balance between vasoconstriction and vasodilatation and lead to endothelial injury and dysfunction, which ultimately results in PE and influences its severity. We also found that there was no significant difference in levels of eNOS expression between placental tissues of women having normal pregnancies and placental tissues of women with PE. This finding indicates that NOSTRIN overexpression results in changes in eNOS activity rather than eNOS concentration, and that placental hypoxia, which is associated with PE, did not induce upregulation of eNOS—which is in agreement with a previous study [21]. Whether NOSTRIN is the original etiological factor or a key factor of PE is poorly understood. In summary, the present study confirms that the level of NOSTRIN expression increases in women with PE, and that it is negatively correlated with the activity of eNOS. This finding indicates that these may be involved in the pathogenesis of PE, and that overexpression of NOSTRIN, which leads to endothelial cell injury, should be considered an important risk factor. But large studies are required to determine the causes of NOSTRIN overexpression and the biological effects of NOSTRIN on endothelial cells and the diagnostic value of its expression in women having PE.
References [1] Zeeman GG, Dekker GA. Endothelial function in normal and preeclampsia pregnancy: a hypothesis. Eur J Obstet, Gynecol, Reprod Biol 1992;43:113 – 22. [2] Smarason AK, Allman KG, Young D, Redman CW. Elevated levels of serum nitrate, a stable end product of nitric oxide,
Expression of endothelial nitric oxide synthase traffic inducer
[3]
[4]
[5] [6] [7]
[8]
[9]
[10]
[11]
[12]
in women with pre-eclampsia. Br J Obstet Gynaecol 1997; 104:538 – 43. Davidge ST, Stranko CP, Roberts JM. Urine but not plasma nitric oxide metabolites are decreased in women with preeclampsia. Am J Obstet Gynecol 1996;174:1008 – 13. Diejomaoh FM, Omu AE, Al-Busiri N, Taher S, Al-Othman S, Fatinikun T. Nitric oxide prodcution is not altered in preeclampsia. Arch Gynecol Obstet 2004 May;269(4):237 – 43. Bredt DS, Snyder SH. Nitric oxide: a physiologic messenger molecule. Annu Rev Biochem 1994;63:175 – 95. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol 1997;272:R441 – 63. Li H, Wallerath T, Forstermann U. Physiological mechanisms regulating the expression of endothelial-type NO synthase. Nitric Oxide 2002;7:132 – 47. Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ. Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 2001; 276:16587 – 91. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M. Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions. Circ Res 1995;76:536 – 43. Dedio J, Konig P, Wohlfart P, Schroeder C, Kummer W, Muller-Esterl W. NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 2001;15:19 – 89. Zimmermann K, Opitz N, Dedio J. NOSTRIN: a protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A 2002;99:16510 – 2. Napolitano M, Miceli F, Calce A. Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/endothelial nitric oxide synthase mRNA iso-
[13]
[14] [15]
[16]
[17]
[18]
[19]
[20]
[21]
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forms from normal and preeclamptic placentas. J Clin Endocrinol Metab 2000;85:2318 – 23. Myatt L, Eis AL, Brockman DE, Greer IA, Lyall F. Endothelial nitric oxide synthase in placental villous tissue from normal, pre-eclamptic and intrauterine growth restricted pregnancies. Hum Reprod 1997;12:167 – 72. Harlow E, Lane D. Antibodies: a laboratory manual. Cold Spring Harbor (NY)7 Cold Spring Harbor Laboratory; 1988. Seligman SP, Buyon JP, Clancy RM. The role of nitric oxide in the pathogenesis of preeclampsia. Am J Obstet Gynecol 1994;171:944 – 8. Var A, Yildirim Y, Onur E, Kuscu NK, Uyanik BS, Goktalay K. Endothelial dysfunction in preeclampsia: increased homocysteine and decreased nitric oxide levels. Gynecol Obstet Investig 2003;56:221 – 4. Morris NH, Sooranna SR, Learmont JG. Nitric oxide synthase activity in placental tissue from normatensive, preeclampsic and growth retarded pregnancies. Br J Obstet Gynaecol 1995;102:711 – 4. Tong X, Li S, Zhao Y. Expression of endothelial nitric oxide synthase gene in placental villus of preeclampsia. Zhonghua Fuchanke Zazhi 1998;33:527 – 8. Shaamash AH, Elsnosy ED, Makhlouf AM. Maternal and fetal serum nitric oxide (NO) concentrations in normal pregnancy, pre-eclampsia and eclampsia. Int J Gynaecol Obstet 2000;68:207 – 14. Rajagopal M, Moodley J, Chetty R. Nitric oxide: does it have an etiological role in pre-eclampsia? Acta Obstet Gynecol Scand 2003;82:216 – 9. Orange SJ, Painter D, Horvath J, Yu B, Trent R, Hennessy A. Placental endothelial nitric oxide synthase localization and expression in normal human pregnancy and pre-eclampsia. Clin Exp Pharmacol Physiol 2003;30:376 – 81.
www.elsevier.com/locate/ijgo
CLINICAL ARTICLE
Expression of endothelial nitric oxide synthase traffic inducer in the placentas of women with pre-eclampsia W. XiangT, H. Chen, X. Xu, M. Zhang, R. Jiang Department of Obstetrics and Gynecology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan-430030, China Received 20 July 2004; received in revised form 16 December 2004; accepted 16 December 2004
KEYWORDS NOSTRIN; Nitric oxide synthase; Placenta; Pre-eclampsia
Abstract Objective: To investigate the expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) in the placenta of women with pre-eclampsia (PE) and discuss its role in the pathogenesis of PE. Methods: Western blot analysis was used to detect the expression of NOSTRIN and endothelial nitric oxide synthase (eNOS). The activity of eNOS in placental tissue was assayed by spectrophotometry. Serum and placental NO2/NO3, the stable metabolic end product of NO, was measured using nitrate reductase. Results: Western blot analysis showed that the protein level of NOSTRIN was significantly higher in women with PE than in the control group ( Pb0.01). However, no significant difference between groups was observed in the expression of placental eNOS ( PN0.05). The activity of eNOS was significantly decreased in women with PE (13.727F3.58 U/mg) compared with the control group (21.69F3.84 U/mg) ( Pb0.01). The placental levels of NO2/NO3 were significantly lower in women with PE (27.53F8.51 Amol/mg) than in the control group (44.38F9.59 Amol/mg) ( Pb0.01). The levels of serum NO2/NO3 were significantly lower in women with PE (48.56F8.49 Amol/L) than in the control group (65.37F9.61 Amol/L) ( Pb0.01). A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in the placental tissues of women with PE (r= 0.57, Pb0.01). Conclusion: The level of NOSTRIN expression in the placental tissues of women with PE is increased and is negatively correlated with the activity of eNOS— which may play an important role in the pathogenesis of PE. D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.
T Corresponding author. Tel.: +86 27 83662698; fax: +86 27 83692550. E-mail address: [email protected] (W. Xiang). 0020-7292/$ - see front matter D 2005 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijgo.2004.12.041
104
W. Xiang et al.
1. Introduction
2. Materials and methods
Pre-eclampsia (PE), the most common serious complication of pregnancy, is characterized by vasoconstriction, dysfunction of the vascular endothelium, and hypertension. Despite decades of intensive research, the pathogenesis of PE remains incompletely understood. Since 1992 [1], nitric oxide (NO)-mediated vasoconstriction has been thought to contribute to the development of the syndrome and attention has been focused on the relationship between NO levels and PE. There are many studies suggesting maternal levels of NO may be increased [2], decreased [3] or unaltered [4] in women with PE. Nitric oxide, an important mediator of biological processes such as neurotransmission, inflammatory response, and vascular homeostasis [5], is synthesized from l-arginine by a family of nitric oxide synthases (NOSs). Nitric oxide derived from endothelial cells acts as a potent vasodilator via several mechanisms such as second messenger cyclic guanine monophosphate (cGMP) [6]. It leads to smooth muscle relaxation by acceleration of the intracellular binding of free calcium and is known for its ability to inhibit platelet aggregation and adhesion to vascular endothelial cells. Three NOS isoenzymes have been identified, of which endothelial NOS (eNOS) is known to be expressed by human placental syncytiotrophoblasts, extravillous trophoblasts [7], and vascular endothelial cells. The endothelial isoform eNOS is a key enzyme of the cardiovascular system that contributes to vascular homeostasis through tightly regulated NO production [8].The level of eNOS expression can be altered by various stimuli. Shear stress and estrogen can increase eNOS expression both in vitro and in vivo [9], and eNOS interacting proteins (NOSIPs) and endothelial nitric oxide synthase traffic inducer (NOSTRIN) have been reported to regulate eNOS activity [10,11]. A large number of studies [12,13] have shown that abnormalities in eNOS activity may contribute to the development of preeclampsia. In a study of the yeast 2-hybrid system using the eNOS oxygenase domain as bait, Zimmermann et al. [11] identified a previously uncharacterized eNOSinteracting protein that they dubbed NOSTRIN. NOSTRIN was observed to induce a profound redistribution of eNOS from the plasma membrane to vesicle-like structures matching the NOSTRIN pattern, which was followed by a significant inhibition of NO release [11]. In the present study, we detected the expression of NOSTRIN in placental tissue form healthy pregnant women and women with PE, and discussed the role of NOSTRIN in the pathogenesis of PE.
2.1. Patients Placental samples were obtained from 11 healthy pregnant women and 11 women with PE at the time of their cesarean section. The study was approved by the local ethics committee and informed consent was obtained from all patients. Pre-eclampsia was defined by hypertension and proteinuria after the 20th week of pregnancy; a blood pressure of 160/ 110 mm Hg or greater; urine albumin of 5 g/24 h or greater; and/or presence of various symptoms such as persistent headache and/or visual impairment and seizures, as described in text books. The placentas of women with twin pregnancies were not included, nor were any of the placentas from mothers with primary hypertension and cardiovascular disease, renal disease, hepatic disease, diabetes, or other infectious or autoimmune diseases.
2.2. Treatment of samples Placentas were obtained from all women at cesarean section. A specimen of placental tissue of at least 2.0 cm2.0 cm2.0 cm was taken at the central site of the placenta, avoiding calcified and hemorrhagic areas, and rinsed with isotonic saline solution to remove blood. Peripheral venous blood samples were obtained from all women before the cesarean section was performed. Serums were taken after centrifugation at 3000 g/min for 15 minutes. The samples were stored at 80 8C until analysis was performed.
2.3. Western blot analysis Samples were harvested with lysis buffer containing 150 mM NaCl, 50 mM Tris—HCl (pH 7.4), 0.1 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 50 mM NaF, and phenylmethylsulfonyl fluoride, as previously described [14]. Then electrophoresis of 100 Ag of total protein was performed on 10% SDS-polyacrylamide gels and the protein was transferred to a nitrocellulose membrane (Zhongshan Biotech, Beijing, China). The membrane was blocked with 3% bovine serum antibody (BSA, Hyclone, USA) for 2 h, and incubated with polyclonal antibodies against NOSTRIN (a gift from Prof. Zimmermann, Institute for Biochemistry and Institute for Pathology, University of Frankfurt, Germany); eNOS (NOS3 [C-20], Santa Cruz Biotechnology, USA) (NOS3 [C-20] is an affinity-purified rabbit polyclonal antibody raised against a peptide mapping at the carboxy terminus
Expression of endothelial nitric oxide synthase traffic inducer Table 1
105
¯ Fs) Level of NOSTRIN and eNOS protein (x
NOSTRIN eNOS
Group
Patient no.
Area (picture element)
OPTDM
OPTDI
Control PE Control PE
11 11 11 11
458 1859T 1298 1256TT
0.390378 0.537124T 0.488106 0.483182TT
178.79 998.51T 633.56 606.88TT
Abbreviations: OPTDM, optical density; OPTDI, optical density index. T Pb0.01 vs. control group (NOSTRIN). TT PN0.05 vs. control group (eNOS).
of NOS3 of human origin); and h-actin (Zhongshan, China). The membrane was then incubated with a secondary antibody (goat antirabbit Ig, Santa Cruz Biotechnology) conjugated to horseradish peroxidase (Amersham, Japan) and developed with a chemiluminescent assay (Pierce, USA). An image band analysis system (FR-980, China) was used to determine the concentrations of NOSTRIN and eNOS.
group. The optical density index of bands were markedly higher in the PE group compared with the control group ( Pb0.01) but there was no significant difference in the protein expression of placental eNOS between the 2 groups ( PN0.05) (Table 1). Four specimens of healthy placental tissue and 5 specimens of placentas from women with PE are shown in Figs. 1 and 2.
2.4. eNOS activity and serum and placental NO2/NO3
3.2. eNOS activity assay
eNOS activity in placental tissues was assayed by spectrophotometry and NO2/NO3 was measured with nitrate reductase following the kit manufacturer’s instructions (NanJing Biology Institute, China).
Spectrophotometry was used to assay the activity of eNOS following the kit manufacturers’ instructions. We found that eNOS activity was decreased in the PE group compared with the control group (13.73F3.58 U/mg vs. 21.69F3.84 U/ mg; Pb0.01).
2.5. Statistical analysis
3.3. NO2/NO3 assay
The data were analyzed using SPSS 10.0 software (SPSS, Chicago, USA). The correlation between the level of NOSTRIN and eNOS activity was determined by correlation analysis. Pb0.05 indicated statistical significance.
The NO2/NO3 assay was performed with nitrate reductase according to the kit manufacturers’ instructions. Placental NO2/NO3 was significantly lower in PE patients than in the control group (27.53F8.51 Amol/mg vs. 44.38F9.59 Amol/mg; Pb0.01). The concentrations of serum NO2/NO3 were significantly lower in PE patients than in the control group (48.56F8.49 Amol/L vs. 65.37F9.61 Amol/L; Pb0.01).
3. Results 3.1. Western blot analysis of NOSTRIN and eNOS NOSTRIN monomer (58 kDa) and eNOS monomer (135 kDa) were found in all tissues examined. Light bands of NOSTRIN were found in the control group, whereas stronger bands were found in the PE
3.4. Correlation between the expression of NOSTRIN and the activity of eNOS A correlation analysis was used to determine the correlation between NOSTRIN levels and eNOS activity in the PE group. The results indicated that
Figure 1 Level of NOSTRIN protein in placental tissue specimens from women with PE and healthy controls. Lanes 1, 2, 5, 6: healthy placenta specimens; lanes 3, 4, 7, 8, 9: placental tissue specimens from women with PE.
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W. Xiang et al.
Figure 2 Level of eNOS protein in placental tissue specimens from healthy controls and women with PE. Lanes 1, 2, 5, 6: healthy placenta specimens; lanes 3, 4, 7, 8, 9: placental tissue specimens from women with PE.
a significant negative correlation existed between them (r= 0.57, Pb0.01).
4. Discussion A large number of studies suggest that abnormalities in NO synthesis may contribute to the development of pre-eclampsia. Smarason et al. [2] suggested that serum nitrate levels are increased; Diejomaoh et al. [4] suggested that serum nitrate levels are unchanged; and Seligman et al. [15] suggested that circulating levels of nitrite are decreased in women with pre-eclampsia. Seligman demonstrated a significant decrease of serum nitrite level in preeclampsia and a negative correlation of serum nitrite levels with systolic and diastolic blood pressure. The Var et al. [16] study has shown that serum NO is also decreased in pre-eclampsia and the low level of NO plays an important role in pathophysiology of PE. Our data show that serum NO2/NO3 in women with was significantly lower than that in the control group (48.56F8.49 Amol/L vs. 65.37F9.61 Amol/L), which supports the concept that diminished NO synthesis contributes to the pathophysiologic changes seen in pre-eclampsia. Nitric oxide has a very short half-life time, as it quickly undergoes oxidation into its stable metabolites. Therefore, more stable results could be expected by studies on NOS activity in biopsy specimens of the placental bed. We used spectrophotometry to assay the activity of eNOS and found that eNOS activity was significantly lower in the PE group than that in the control group, which is consistent with the results of previous studies [17,18] but conflicts with the study by Shaamash and Rajagopal [19,20]. We also found placental NO was significantly lower in women with PE than that in the control group (27.53F8.51 Amol/mg vs. 44.38F9.59 Amol/mg), which suggests that low eNOS activity results in less production of placental NO. Low levels of placental NO can effect a decrease in serum NO, and low levels of serum NO can lead to hypertension and increase venous thrombosis, vasospasm, and poor perfusion of the placenta [6]. All these pathological changes can aggravate endothelial injury and the dysfunction of associated organs, and eventually lead to PE. Our results suggest that a decreased eNOS activity may
be involved in the pathogenesis of PE via its impact on NO production. We determined the expression of a particular eNOS interaction protein, NOSTRIN, which was identified by Zimmermann et al. [11]. We found that NOSTRIN levels were significantly higher in the placentas of women with PE than in the placentas of women having a normal pregnancy. We also found that a significant negative correlation existed between the expression of NOSTRIN and the activity of eNOS in the placental tissues of women with PE. Our results suggest that an increased expression of placental NOSTRIN results in reduced eNOS activity and less production of placental and serum NO, and that lower levels of serum NO may destroy the balance between vasoconstriction and vasodilatation and lead to endothelial injury and dysfunction, which ultimately results in PE and influences its severity. We also found that there was no significant difference in levels of eNOS expression between placental tissues of women having normal pregnancies and placental tissues of women with PE. This finding indicates that NOSTRIN overexpression results in changes in eNOS activity rather than eNOS concentration, and that placental hypoxia, which is associated with PE, did not induce upregulation of eNOS—which is in agreement with a previous study [21]. Whether NOSTRIN is the original etiological factor or a key factor of PE is poorly understood. In summary, the present study confirms that the level of NOSTRIN expression increases in women with PE, and that it is negatively correlated with the activity of eNOS. This finding indicates that these may be involved in the pathogenesis of PE, and that overexpression of NOSTRIN, which leads to endothelial cell injury, should be considered an important risk factor. But large studies are required to determine the causes of NOSTRIN overexpression and the biological effects of NOSTRIN on endothelial cells and the diagnostic value of its expression in women having PE.
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