Journal of Human Hypertension (2015), 1–7 © 2015 Macmillan Publishers Limited All rights reserved 0950-9240/15 www.nature.com/jhh
ORIGINAL ARTICLE
Expression levels of urotensin II are associated with endoplasmic reticulum stress in patients with severe preeclampsia W-Y He1,3, G-J Chen1,3, X Lai1, F Wu1, C-S Tang2 and A-H Zhang1 Hypertensive disorders in pregnancy remain a leading cause of maternal and perinatal mortality and morbidity. We aim to study urotensin II (UII) and its association with the markers of endoplasmic reticulum stress (ERS) in placentas of patients with severe preeclampsia (SPE). Thirty-three patients with hypertensive disorders in pregnancy and twenty-two healthy pregnant women designated as healthy controls were recruited. Expression levels of UII, UII receptor (GPR14) and the markers of ERS in placenta specimens of patients were performed. Plasma and urinary UII levels were measured by radioimmunoassay method. Our study showed that the plasma levels of UII in patients with hypertensive disorders during pregnancy were significantly higher than that of the healthy control group. However, the urinary levels of UII had no difference in two groups. The expression level of mRNA and protein of UII, CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose regulation protein 78 in placentas of SPE was significantly increased. Immunohistochemical analyses show that the expression levels of UII and ERS markers were mainly located in the cytoplasm of placental trophoblastic cells. Moreover, expression level of UII mRNA and protein was positively correlated with that of the markers of ERS. The positive correlation between UII and ERS markers expression level also corresponded with the level of patient’s systolic blood pressure and proteinuria. In conclusion, we first verify that expression of UII is associated with ERS in patients with SPE. Our results indicate that UII may trigger ERS in placental trophoblastic cells in patients with preeclampsia. Journal of Human Hypertension advance online publication, 16 April 2015; doi:10.1038/jhh.2015.28
INTRODUCTION Hypertensive disorders in pregnancy remain a leading cause of maternal and perinatal mortality and morbidity. The pathogenesis is associated with intense vasospasm, which presents as hypertension, poor organ perfusion affecting the kidney, the central nervous system and the liver.1 It is generally defined as newly developed hypertension (systolic blood pressure ⩾ 140 mm Hg or diastolic blood pressure ⩾ 90 mm Hg) with or without substantial proteinuria (⩾300 mg in 24 h) at or after 20 weeks gestation. Pathogenesis in hypertensive disorders in pregnancy remains largely unknown; the leading hypotheses strongly rely on disturbed placental function in early pregnancy.1 Among them, endothelial dysfunction is frequently emphasized in the pathogenesis of hypertensive disorders in pregnancy.2 At present, the only known definitive treatment for these diseases is the delivery of fetus and placenta. Therefore, in order to develop a less invasive or alternative approach to treating these diseases, it is urgent to recognize new pathogenic factors and discover other pathogenic models that lead to hypertensive disorders in pregnancy. Endoplasmic reticulum stress (ERS), which is essentially accumulation of unfolded or misfolded proteins in endoplasmic reticulum, has recently been identified as an important process involved in the pathology of preeclampsia.3,4 The most important marker of ERS is glucose regulation protein 78 (GRP78), and severe
ERS can activate several signal molecules, such as C/EBP homologous protein (CHOP).2,3 Urotensin II (UII) is a vasoactive, somatostatin-like cyclic peptide of 11 amino acids initially isolated from the urophysis of the goby fish. It is widely expressed in the cardiovascular system as well as in the central nervous system, kidney, spleen, small intestine, thymus, prostate, pituitary, adrenal gland, placenta and colonic mucosa.5–7 UII is generally agreed to be the most potent endogenous vasoconstrictor discovered to date.6 It has been demonstrated that UII interacts with a G-protein-coupled receptor known as GPR14 or urotensin receptor (UT), and the binding is functionally coupled to calcium mobilization, which may participate in the pathophysiology of hypertensive disorders in pregnancy.1,8 Moreover, UII binds with its receptor and triggers phospholipidase C activation, and inositol 1,4,5 triphosphate receptors dependent pathway which induces calcium releasing from endoplasmic reticulum,9 and Mekahli et al.10 also found that the depletion of endoplasmic reticulum calcium was associated with ERS. Since both UII and ERS are associated with lowered calcium concentration in the endoplasmic reticulum, we speculate that UII may also be correlated with ERS in its role in the development of hypertensive disorder in pregnancy. The aim of our study is to assess the role of UII, a vasoactive peptide, in the pathophysiology of hypertensive disorders in pregnant patients.
1 Department of Nephrology, Peking University Third Hospital, Beijing, China and 2Department of Pathology and Physiology, Peking University Health Science Center, Beijing, China. Correspondence: Professor A-H Zhang, Department of Nephrology, Peking University Third Hospital, No. 49 North Garden Road, Beijing 100191, China. E-mail: [email protected] 3 These authors contributed equally to this work. Received 19 November 2014; revised 20 February 2015; accepted 26 February 2015
UII associated with ERS in severe preeclampsia W-Y He et al
2
MATERIALS AND METHODS Study subjects Thirty-three patients with hypertensive disorders during pregnancy and twenty-two healthy pregnant women designated as control group were recruited from the inpatient obstetric ward at the Peking University Third Hospital from October 2012 to March 2013. Histological studies, real-time PCR and western blot analysis were performed on placental tissues of 25 patients with hypertensive disorders during pregnancy (5 patients with gestational hypertension (GH), 8 patients with mild preeclampsia (MPE) and 12 patients with severe preeclampsia (SPE)). Same set of studies were also performed on the placental tissues of 25 patients with normal pregnancies from June 2012 to March 2013. All 25 placentas from patients with hypertensive disorder during pregnancy were belong to the same patients. Although we originally recruited 33 patients with hypertensive disorders during pregnancy, we failed to collect six placentas with GH and two placentas with MPE for missing the suitable collecting time. Human placental tissues were obtained just after spontaneous labor or caesarean operation. All participants signed their written informed consent. Then, these patients with hypertensive disorders in pregnancy were classified into three subgroups according to their blood pressures and proteinuria levels. These subgroups included GH, MPE and SPE. For the GH group, 11 patients were included and GH was defined as persistent hypertension, systolic blood pressure ⩾ 140 mm Hg or diastolic blood pressure equal to 90 mm Hg, without proteinuria, developed after 20 weeks of gestation. For the MPE group, 10 patients were included and MPE was defined as persistent hypertension, systolic blood pressure ⩾ 140 mm Hg or diastolic blood pressure equal to 90 mm Hg, with proteinuria (⩾0.3 g in a 24 h urine collection or ⩾ 1 g according to a dipstick test), developed after 20 weeks of gestation.3 For the SPE group, 12 patients were included and SPE was defined as systolic blood pressure greater than 160 mm Hg or diastolic blood pressure greater than 110 mm Hg on 2 occasions each 6 h apart with proteinuria exceeding 2 g in a 24-h period or 2–4+ on dipstick testing.2 The inclusion criteria of patients were that patients must have hypertensive disorders, singleton pregnancies with a living fetus and gestational ages of 32 weeks. Pregnancies with infection, cancer, liver disease, kidney disease or heart disease were excluded from both study groups. Healthy pregnant women with no prior pregnancy complications were included as controls. Detailed histories were obtained from all patients. Demographic data including age, body weight, height and so on were recorded. Body mass indices of all patients were calculated. Blood pressures of all patients were measured with sphygmomanometer at 0800 hours by qualified physicians. Complete blood counts, routine urinary and biochemical analysis were all performed as well. Estimated glomerular filtration rate was assessed by simplified modification of diet in renal disease (MDRD) equation = 186 × (Scr)−1.154 × (Age)−0.203 × (0.742 if female).
Immunohistochemical analyses Histological studies were performed on placental tissues of patients with hypertensive disorders during pregnancies and healthy pregnancies. All participants signed their written informed consents. The placental tissues were embedded in optimal cutting temperature compound. The tissues were sectioned at a thickness of 10 μm. For immunohistochemical analysis, 5% hydrogen peroxide was used to deplete endogenous peroxidase activity. Following pre-incubation with 5% bovine serum albumin for 30 min to prevent nonspecific staining, the sections were incubated with rabbit anti-human UII (1:50), GPR14 (1:100), CHOP (1:100) and GRP78 antibody (1:200) at 4 °C overnight (all antibodies were bought from Abcam Inc., Cambridge, UK, and all other chemicals were of analytical grade from commercial suppliers (Beijing Guangquyuan chemical reagent company, Beijing, China)). The sections were then incubated with horseradish peroxidase-coupled goat anti-rabbit IgG antibody for 20 min, followed by incubation with strep-avidin-biotinperoxidase complex for 20 min at 37 °C. The peroxidase was visualized by incubation with 3, 3′-diaminobenzidine in the dark for 3 min for UII, GPR14 antibody, and 1.5 min for GRP78, CHOP antibody. The sections were counterstained with hematoxylin, dehydrated and observed under a light microscope. Negative controls were established using PBS as a substitute for the primary antibody. Positive staining was indicated by brown deposits. The integral optical density (IOD) was measured with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA), and the result was determined as the sum of five different fields (one in the center and four in the periphery) of each section. The IOD of the target protein was defined as Journal of Human Hypertension (2015), 1 – 7
the sum of the optical densities of all the positive pixels in the image, which represents the quantity of the targeted protein. The pathological image analysis system was used to calculate the IOD of positive staining for UII, GPR14, CHOP and GRP78.
UII measurement using radioimmunoassay Plasma UII was measured using a radioimmunoassay method according to our previous publication and other literatures.6,11 Plasma samples of 3ml were deproteinated with 0.75 ml of 2 mol l− 1 hydrochloric acid. After centrifugation for 20 min at 6000 g, the supernatant was loaded onto cartridges that had been activated with 3 ml of 100% methanol and 3ml of double-distilled deionized water. The cartridges were then washed twice with 3 ml of 0.1% trifluoroacetic acid and eluted with 3 ml of 60% acetonitrile in 0.1% of trifluoroacetic acid. The eluants were freeze-dried overnight and resuspended in 250 μl of radioimmunoassay buffer. One hundred microliters of standard UII or assay sample were incubated overnight at 48 °C with 100 μl rabbit antiserum. One hundred microliters of labeled 125I-urotensin II (Phoenix Pharmaceuticals, Inc. Belmont, CA, USA) were added to each tube and incubated for an additional 24 h. Antibodybound UII was precipitated using a goat anti-rabbit antiserum and normal horse serum. Using a gamma counter, the amount of bound 125I-urotensin II was measured as picograms per milliliter.
RNA isolation and quantitative real-time PCR analysis for placenta Total RNA was extracted from the placentas of both patients with hypertensive disorders during pregnancy and healthy pregnancies by using TRIzol Reagent (Invitrogen. Ltd, Carlsbad, CA, USA). RNA was reverse transcribed into first-strand complementary DNA using GoScript Reverse Transcription System (A5001, Promega Co., Madison, WI, USA). A real-time quantitative PCR method was used to detect the changes in UII, CHOP and GRP78 mRNA levels. Primers for human UII, CHOP and GRP78 were designed using Primer Express software (Applied Biosystems, Sangon Biotech (Shanghai) Co., Ltd, Shanghai, China). Quantitative real-time PCR was carried out in a total volume of 20 μl using a Bio-Rad iQ5 detector (Applied Biosystems) to determine the threshold cycle (Ct) value. Each sample was run and analyzed in triplicate. For the UII gene, the forward primers was 5′-GCACTGTTTGCTTTGGACTCC-3′ and the reverse primer was 5′-TGGTCGTCCATGCACAGATT-3′, the forward primer of UII receptor (GPR14) gene was 5′-CACGGGCACCATTGGGACTC-3′ and the reverse primer was 5′-CGCCAGGTTGACCACGTAGAC-3′; the forward primer of CHOP was 5′-GGAAACAGAGTGGTCATTCCC-3′ and the reverse primer was 5′-CTGCTTGAGCCGTTCATTCTC-3′; the forward primer of GRP78 was 5′-CAT CACGCCGTCCTATGTCG-3′ and the reverse primer was 5′-CGTCAAAGAC CGTGTTCTCG-3′. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control, the forward primer was 5′-AACGGATTTGGTCGTATTGGGC-3′ and the reverse primer was 5′-TCGCTCCTGGAAGATGGTGATG-3′. Relative quantitation values were calculated using the 2–ΔΔCt method as fold changes in the target gene related to the expression level of GAPDH. Three-step real-time PCR of denaturing, annealing and extension reactions proceeded for 40 cycles at 15 s at 95 °C, 1 min at 59 °C (UII ), and 30 s at 72 °C. For UII, UII receptor, CHOP, GRP78 and GAPDH, the annealing temperature is 58 °C.
Western blot analysis for placentas Proteins were extracted from the placentas of both patients with hypertensive disorders during pregnancy and healthy pregnancies. Immunoprecipitation and western blot analysis of UII, GPR14, GRP78 and CHOP were performed. Briefly, the protein samples were denatured at 95 °C for 5 min separated on a 10% SDS-PAGE gel before being transferred to nitrocellulose filter (NC) membranes (Applygen Technologies Inc., Beijing, China). Membranes were subsequently incubated with primary rabbit polyclonal anti-UII, anti-GPR14, anti-GRP78 and anti-CHOP antibodies (1:1000; ab21685, Abcam) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibodies (1:500, Zhongshan Gold Bridge Biotechnology Co., Ltd, Beijing, China). Semiquantitative grayscale intensity was generated with Odssey Software v1.2 (Samsung Corporation, Gyeonggi-Do, Korea).
Statistical analyses All analyses were performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). Results were reported as mean ± standard deviation (x ± s). Nonparametric data were tested by Mann–Whitney test. One-way analysis of variance was © 2015 Macmillan Publishers Limited
UII associated with ERS in severe preeclampsia W-Y He et al
3 applied in statistical analysis. Spearman’s rank correlation was used for correlation analyses. P values below 0.05 were accepted to be significant.
RESULTS Clinical characteristics of the study subjects Table 1 summarized the clinical characteristics of the study subjects. A total of 55 subjects were included. As expected, significantly elevated systolic blood pressure, elevated diastolic blood pressure, elevated plasma creatinine and lower estimated by glomerular filtration rate (eGFR), lowered plasma total protein and lowered albumin level were observed in patients with hypertensive disorder in pregnancy. Elevated hepatic enzymes, such as alanine aminotransferase, were also observed in pregnancies with hypertensive disorders in comparison to those of normal control (NC) group. The mean plasma UII levels were significantly higher in patients with hypertensive disorders during pregnancy than those of NC (P = 0.003). In contrast, analysis of two groups revealed no significant difference between the urinary UII Table 1. Comparison of clinical characteristics of the study subjects included (n = 55) Normal controls (n = 22) Age (years) BMI (kg m −2) GA (weeks) SBP (mmHg) DBP (mmHg) Urinary protein level (g per 24 h) WBC (*109 l − 1) Hb (g l − 1) PLT (*109 l − 1) ALT (U l − 1) AST (U l − 1) TP (g l − 1) Alb (g l − 1) Urea (mmol l − 1) Cr (umol l − 1) eGFRb (ml min − 1) Glu (mmol l − 1) Plasma UII (pg ml − 1) Urinary UII (pg ml − 1)
Hypertensive P value disorders in pregnancy (n = 33)
30.59 ± 3.35 27.36 ± 3.00 37.09 ± 2.02 119.73 ± 11.00 74.86 ± 7.29
31.79 ± 5.39 30.65 ± 4.64 35.91 ± 2.31 139.12 ± 15.09 89.45 ± 10.08 0.49(0.16, 2.85)
0.315 0.005 0.056 0.000 0.000
8.75 ± 2.21 112.55 ± 16.47 174.18 ± 69.28 14.36 ± 10.46 15.59 ± 2.65 62.95 ± 4.47 35.73 ± 2.46 2.99 ± 1.27 51.09 ± 11.91 121.23 ± 11.45 4.29 ± 0.38 29.93 ± 9. 95
10.04 ± 2.53 114.91 ± 19.30 173.45 ± 60.48 19.03 ± 12.57 23.27 ± 8.53 57.18 ± 8.08 32.09 ± 5.90 4.17 ± 1.33 62.42 ± 15.08 108.19 ± 13.31 4.62 ± 0.746 42.01 ± 16.30
0.056 0.629 0.968 0.142 0.000 0.004 0.008 0.002 0.005 0.019 0.059 0.003
11.19 (8.22, 25.67) 16.61 (13.06, 22.72) 0.45
Abbreviations: Alb, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; Cr, blood creatinine; GA, gestational age; DBP, diastolic blood pressure; Glu, glucose; Hb, hemoglobin, PLT, platelet; SBP, systolic blood pressure; TP, total protein; UII, urotensin II; WBC, white blood cell. P values below 0.05 have been accepted to be significant. Normal distribution data are presented as means ± s.d. Abnormal distribution data are presented as median, 25 percentile and 75 percentile.
Table 2.
levels of subjects with hypertensive disorders in pregnancy and those of NCs. UII level analysis according to the severity of hypertensive disorder during pregnancy Elevated plasma UII level was observed in SPE group when compared with GH group and MPE group (Po0.01). Also, no statistically significant difference in terms of urinary UII level was detected among groups (P40.05; see Table 2). Moreover, neither plasma UII nor urinary UII level was not correlated with the level of serum albumin and estimated glomerular filtration rate. There were four patients with elevated plasma UII in SPE group (plasma UII concentrations were 74, 68, 72 and 69 pg ml − 1). Their clinical and laboratory findings were different from others for they all had partial HELLP syndrome, such as mild anemia, mild thrombocytopenia, increased aspartate aminotransferase level of ⩾ 40 IU l − 1, increased alanine aminotransferase level of ⩾ 40 IU l − 1 or severe proteinuria. Pathological analyses of placentas We discovered vessel congestion and edema of placental villi. The mesenchymal cells were reduced comparatively, and significant deposition of cellulose could be found. In addition, little necrosis and calcification could be observed, especially in patients with SPE (Figure 1a and b). Immunohistochemical analyses of expression level of UII, GPR14, GRP78 and CHOP in placental tissue Immunochemistry analysis of placental tissue showed that expression level of UII and its receptor GPR14 in SPE group were highly increased and were mainly located in the cytoplasm of placental trophoblastic cells; however, UII and GPR14 expression level were only moderately increased in MPE and GH groups and weakly expressed in NC group (Figures 2a–h). Our results demonstrated that the expression level of UII and GPR14 (IOD) increased significantly in patients with SPE in pregnancy in comparison with that of the healthy controls, GH and MPE group (Figures 2i and k). The expression levels of GRP78 and CHOP were mainly located in the cytoplasm of placental trophoblastic cells and few were located in vascular endothelial cells as shown by immunohistochemical analyses. The expression level of GRP78 and CHOP was increased significantly in patients with hypertensive disorders in pregnancy than that of the NC group (Figure 3a–i). By immunochemical semi-quantitive analysis, IODs of GRP78 and CHOP were significantly higher in SPE group than that of NC, GH or MPE groups (Figures 3j and k). mRNA and protein expression levels of UII, GPR14, CHOP and GRP78 in placentas with hypertensive disorders during pregnancy and in placentas of normal pregnancy Our results showed that there was no significant difference in mRNA expression levels of UII, UII receptor, CHOP and GRP78 among MPE, GH and normal pregnancy groups (data not shown), but significantly elevated mRNA expression levels of UII (mean = 9.5-fold), CHOP (mean = 9.6-fold) and GRP78 (mean = 5.1-fold) in
Comparison of plasma and urinary UII levels of patients according to the severity of disease
Plasma UII level (pg ml − 1) Urinary UII level (pg ml − 1)
GH group (n = 11)
MPE group (n = 10)
SPE group (n = 12)
34.13 ± 8.36 13.63 (8.36, 16.61)
33.83 ± 9.32 18.70 (14.67, 22.68)
56.05 ± 17.34* 21.88 (13.07, 28.14)
Abbreviations: GH, gestational hypertension; MPE, mild preeclampsia; SPE, severe preeclampsia. P values below 0.05 have been accepted to be significant. Normal distribution data are presented as means ± s.d. Abnormal distribution data are presented as median, 25 percentile and 75 percentile. *Po0.01 compared with GH group and MPE group.
© 2015 Macmillan Publishers Limited
Journal of Human Hypertension (2015), 1 – 7
UII associated with ERS in severe preeclampsia W-Y He et al
4
Figure 1. HE slides of placenta tissue of SPE patients under light microscope ((a): X10; (b): X200). Vessel congestion and edema of placental villi could be found in the slides. Reduced mesenchymal cell, significant deposition of cellulose and little necrosis and calcification could be observed.
Figure 2. Immunochemistry analysis in placental tissue showed that expression level of UII and its receptor GPR14 in SPE group were highly increased and were mainly located in the cytoplasm of placental trophoblastic cells. Expression levels of UII and GPR14 are moderately increased in MPE and GH groups but very weak expression level in NC group (see arrow). (a–h) UII in SPE (a), MPE (b), GH (c) NC (d); GPR14 in SPE group (e) GPR14 in MPE (f) GPR14 in GH (g) GPR14 in NC group (h). IOD of UII (i) in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.006) and MPE group (P = 0.000). No significant differences were observed between NC, GH and MPE group. IOD of GPR14 (k) in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.001) and MPE group (P = 0.001). No significant differences were observed between NC and GH, and MPE and SPE group.
placentas with SPE were observed in comparison with that of the placentas with normal pregnancy (Figure 4). Moreover, there were no significant differences in protein expression levels of UII, UII receptor, CHOP, GRP78 among the placentas of MPE, GH and normal pregnancy groups (data not shown), but protein expression level of UII (mean = 1.7-fold), CHOP (mean = 2.2-fold) and GRP78 (mean = 1.5-fold) in placentas with SPE was significantly Journal of Human Hypertension (2015), 1 – 7
higher as demonstrated by western blot in comparison with that of normal pregnancy group (Figure 5). Correlations between expression level of UII and markers of ERS The plasma UII level had positive correlation with the level of systolic blood pressure (r = 0.459, P = 0.001) and amount of © 2015 Macmillan Publishers Limited
UII associated with ERS in severe preeclampsia W-Y He et al
5
Figure 3. The expression levels of GRP78 (a–d) and CHOP were mainly located in the cytoplasm of placental trophoblastic cells (f–i) and few were located in vascular endothelial cell (e) by immunochemistry analysis. Both of them in the SPE group (a, f) were significantly increased in expression levels of GRP78 and CHOP, while the expression levels of GRP78 and CHOP were moderately increased in MPE (b, f), GH group (c, g) and were nearly deficient in NC group (d, h). IOD of GRP78 in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.045) and MPE group (P = 0.01; j). Expression level of CHOP (i) in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.000) and MPE group (P = 0.000; k).
proteinuria (r = 0.607, P = 0.001); however, no correlation was found with the level of creatinine estimated by the filtration rate calculated by MDRD equation. Expression levels of UII mRNA in placentas with SPE also were positively correlated with the amount of proteinuria (r = 0.73, P = 0.014) and the level of systolic blood pressure (r = 0.64, P = 0.017). The expression level of UII mRNA was positively correlated with the expression level of CHOP mRNA (r = 0.789, P = 0.001) and GRP78 mRNA (r = 0.689, P = 0.011) in placentas with SPE. The protein expression level of UII was also found to be positively correlated with that of CHOP (r = 0.71, P = 0.003) and GRP78 (r = 0.69, P = 0.015). © 2015 Macmillan Publishers Limited
DISCUSSION The pathogenesis of hypertensive disorders in pregnancy is the subject of much debate but it is generally believed to be a type of placental dysfunction that resulted from arteriolar vasoconstriction with abnormal invasion of the spiral arteries by cytotrophoblast cells.1–3 In contrast to normal pregnancy, the synthesis and release of vasoactive factors are enhanced, which induce widespread injury of maternal vascular endothelium. This results in increased formation of endothelin, thromboxane, superoxide, increased vascular sensitivity to angiotensin II and decreased formation of vasodilators, such as nitric oxide and Journal of Human Hypertension (2015), 1 – 7
UII associated with ERS in severe preeclampsia W-Y He et al
6 prostacyclin. These endothelial abnormalities, in turn, cause hypertension by impairing renal function and increasing total peripheral resistance.2,7 UII, one of the potent endogenous vasoconstrictors, was found to be significantly higher in patients with essential hypertension, severe coronary artery disease, ischemic cardiomyopathy, congestive heart failure, diabetes mellitus, renal failure and portal
Figure 4. mRNA expression levels of UII, CHOP and GRP78 in placentas with hypertensive disorders in pregnancy and normal pregnancy. There were significantly higher mRNA expression levels of UII (mean = 9.5-fold), CHOP (mean = 9.6-fold) and GRP78 (mean = 5.1-fold) in placentas with SPE compared with normal pregnancy by real-time PCR method (**P o0.01 compared with normal pregnancy).
hypertension caused by liver cirrhosis.5,12 Balat et al. reported a significant increase in circulating levels of UII in preeclampsia,6 whereas others reported no difference of UII level between normal pregnancy and pregnancy with preeclampsia.8 In our study, we found that the level of UII was significantly higher in the plasma of patients with hypertensive disorders during pregnancy than those of patients with healthy pregnancies. However, the urinary levels of UII had no differences between patients with hypertensive disorders during pregnancy and patients from healthy pregnant group. According to Matsushita’ study, urinary UII is primarily derived from a renal source.7 Although both groups of patients excrete UII in the urine, we speculate that the renal UII expression level does not upregulate in either patients with hypertensive disorder during pregnancy or NCs. From our study, we were able to show that the expression levels of UII and UII receptor were increased in the placentas of patients with hypertensive disorders during pregnancy. However, real-time PCR and western blot methods only showed increased mRNA and protein expression levels for UII but not UII receptor. Although immunochemistry method demonstrated an increase in the expression level of both UII and its receptor for the hypertensive placenta, this may result from unspecific staining. The cause for the increased concentrations of UII in maternal plasma in hypertensive disorder in pregnancy is unclear. According to the previous study, plasma levels of UII had been measured in healthy individuals, and the circulated UII levels were relatively low.7 This finding suggests that UII is not predominantly a circulating hormone. To be more specific, the increased circulating UII in patients with hypertension disorders during pregnancy may come from the secretion of UII in placenta. It may result from deficient spiral artery conversion, causing decreased uteroplacental blood flow that can result in either hypoxia or ischemia-reperfusion. Gould et al. also reported that under hypoxic conditions, endothelial cells or trophoblastic cells produce more UII and, thus, may contribute to the increased UII circulating levels.1 We found that the level of plasma UII in SPE group was significantly higher than that of the MPE group and GH group. In addition, we also found that plasma UII level had positive correlation with blood pressure and 24 h urinary proteinuria level,
Figure 5. Protein expression levels of UII, CHOP and GRP78 in placentas with hypertensive disorders in pregnancy and normal pregnancy. There were significantly higher protein expression levels of UII ( mean = 1.7-fold ), CHOP (mean = 2.2-fold) and GRP78 (mean = 1.5-fold) in placentas with SPE by western blot compared with normal pregnancy (**P o0.01 compared with normal pregnancy, *Po0.05 compared with normal pregnancy). Journal of Human Hypertension (2015), 1 – 7
© 2015 Macmillan Publishers Limited
UII associated with ERS in severe preeclampsia W-Y He et al
7 which were associated with the outcome of the pregnancy. This suggests that plasma UII levels are also associated with the development of the disease. In our study, we also found that UII protein is mainly located in the cytoplasm of placental trophoblastic cells as shown by immunohistochemistry studies. This indicates that the UII not only affects the vascular endothelial cells, but also affects the trophoblastic cells. This would be a reason for placental ischemia/ hypoxia in hypertensive disorders during pregnancy. Also, the expression level of UII in placenta had positive correlation with blood pressure and proteinuria. This, in turn, is associated with the increase in plasma UII level and, thus, the progression of the disease. ERS, a major regulator of cell homeostasis through its involvement in post-translational protein modifications and folding, has recently been identified as an important process involved in the pathology of preeclampsia.2–4 Activation of pro-inflammatory pathway may also occur under ERS. The proinflammtory environment is thought to lead to maternal endothelial cell dysfunction, resulting in the syndrome of hypertension and proteinuria.13 In our present study, we found the expression levels of CHOP and GRP78 mRNA and protein were significantly increased in placental tissue in patients with SPE compared with that of the patients with normal pregnancy. Moreover, their expression levels were mainly located in placental trophoblastic cells as shown by immunochemistry method. Also, the expression level of UII mRNA and protein was positively correlated with the expression level of CHOP and GRP78 mRNA and protein. ERS markers in placentas all had positive correlation with blood pressure and proteinuria. Our results indicate that ERS in placental trophoblastic cells may play some important roles in the development of hypertensive disorder in pregnancy in addition to endothelial cell. Recent study showed that endothelin-1 could induce ERS in trophoblasts through the PLC-IP (3) pathway.13 The physiological mechanisms of UII are similar in some ways to endothelin-1, because hypertensive disorders in pregnancy are associated with increased level of plasma UII, which is a vasoactive peptide that has the potential to disrupt ER Ca2+ homeostasis. It is reported that UII can induce calcium releasing from endoplasmic reticulum,9 and Mekahli et al. also showed that loss of luminal calcium in endoplasmic reticulum causes ERS and activates an unfolded protein response.10 With the findings of these studies, we speculate that UII may induce ERS, and our results verified that they had positive correlation. Our results indicate that UII may induce placental trophoblastic cell ERS. Future studies are needed to test this hypothesis. For instance, we can treat trophoblastic cells with UII in vitro and also use animal model to test whether UII can induce the expression of ERS markers that can subsequently induce apoptosis of the trophoblastic cells. According to the previous studies, UII can constrict blood vessel, induce cell proliferation, atherosclerosis and regulate soluble vascular endothelial growth factor (VEGF) receptor 1 secretion by placental tissues,1,2,6 which may be associated with the pathogenesis of hypertensive disorder during pregnancy. From our study, we demonstrated for the first time that the expression level of UII had a positive correlation with ERS markers in trophoblastic cell of placental tissue in patients with SPE. The findings in this study can be the basis of a new pathogenic model for preeclampsia and potential foundation for the development of a less invasive treatment for this disease. CONCLUSION In summary, our present study shows that plasma level and expression level of UII in placental tissue are associated with
© 2015 Macmillan Publishers Limited
development of hypertensive disorder during pregnancy. We are the first to verify that the expression level of UII mRNA and protein are associated with expression level of CHOP and GRP78 mRNA and protein in SPE, and their expression levels are mainly located in trophoblastic cells in placentas. Our results indicate that UII may trigger ERS in placental trophoblastic cells, which can be a new pathogenic model for hypertensive disorder during pregnancy. What is known about topic ● Expression level of UII is associated with endoplasmic reticulum stress in patient with severe preeclampsia What this study adds ● We are the first to verify that the expression level of UII mRNA and protein are associated with expression level of CHOP and GRP78 mRNA and protein in severe preeclampsia, and their expression levels are mainly located in trophoblastic cells in placentas ● Our results indicate that UII may trigger ERS in placental trophoblastic cells, which may be a new pathogenic model in the development of hypertensive disorder during pregnancy
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation (Grant No. 81170706 and Grant No 81341022) to A-H Zhang.
REFERENCES 1 Gould PS, Gu M, Liao JQ, Ahmad S, Cudmore MJ, Ahmed A et al. Up regulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia. Hypertension 2010; 56: 172–178. 2 Liane LA, Loset LM, Mundal SB, Fenstad MH, Johnson MP, Eide IP. Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and without pre-eclampsia. Placenta 2011; 32: 823–829. 3 Eric AP, Dadelszen PV, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet 2010; 376: 631–644. 4 Veerbeek JH, Tissot Van Patot MC, Burton GJ, Yung HW. Endoplasmic reticulum stress is induced in the human placenta during labour. Placenta 2015; 36: 88–92. 5 Ames RS, Sarau HM, Chambers JK. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 40: 282–286. 6 Balat O, Aksoy F, Kutlar I, Ugur MG, Iyikosker H, Balat A et al. Increased plasma levels of Urotensin-II in preeclampsia–eclampsia: a new mediator in pathogenesis? Eur J Obstet Gynecol Reprod Biol 2005; 120: 33–38. 7 Matsushita M, Shichiri M, Imai T, Iwashina M, Tanaka H, Takasu N et al. Coexpression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 2001; 19: 2185–2190. 8 Cowley E, Thompson JP, Sharpe P, Waugh J, Ali N, Lambert DG. Effects of preeclampsia on maternal plasma, cerebrospinal fluid, and umbilical cord urotensin II concentrations: a pilot study. Br J Anaesth 2005; 95: 495–499. 9 Jarry M, Diallo M, Lecointre C. The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte cell activity through common and distinct mechanisms: involvement in cell proliferation. Biochem J 2010; 428: 113–124. 10 Mekahli D, Bultynck G, Parys JB, Smedt HD, Missiaen L. Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb Perspect Biol 2011; 3: 1–30. 11 Bai Q, Zhang J, Zhang AH, Cheng LT, He L, Fan MH et al. Roles of human urotensin II in volume resistance hypertension in peritoneal dialysis patients. Ren Fail 2012; 340: 713–717. 12 Totsune K, Takahashi K, Arihara Z, Sone M, Ito S, Murakami O. Increased plasma urotensin II level s in patients with diabetes mellitus. Clin Sci 2003; 104(1): 1–5. 13 Jain A, Olovsson M, Burton GJ, Yung HW. Endothelin-1 induces endoplasmic reticulum stress by activating the PLC-IP (3) pathway: implications for placental pathophysiology in preeclampsia. Am J Pathol 2012; 180: 2309–2320.
Journal of Human Hypertension (2015), 1 – 7
ORIGINAL ARTICLE
Expression levels of urotensin II are associated with endoplasmic reticulum stress in patients with severe preeclampsia W-Y He1,3, G-J Chen1,3, X Lai1, F Wu1, C-S Tang2 and A-H Zhang1 Hypertensive disorders in pregnancy remain a leading cause of maternal and perinatal mortality and morbidity. We aim to study urotensin II (UII) and its association with the markers of endoplasmic reticulum stress (ERS) in placentas of patients with severe preeclampsia (SPE). Thirty-three patients with hypertensive disorders in pregnancy and twenty-two healthy pregnant women designated as healthy controls were recruited. Expression levels of UII, UII receptor (GPR14) and the markers of ERS in placenta specimens of patients were performed. Plasma and urinary UII levels were measured by radioimmunoassay method. Our study showed that the plasma levels of UII in patients with hypertensive disorders during pregnancy were significantly higher than that of the healthy control group. However, the urinary levels of UII had no difference in two groups. The expression level of mRNA and protein of UII, CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose regulation protein 78 in placentas of SPE was significantly increased. Immunohistochemical analyses show that the expression levels of UII and ERS markers were mainly located in the cytoplasm of placental trophoblastic cells. Moreover, expression level of UII mRNA and protein was positively correlated with that of the markers of ERS. The positive correlation between UII and ERS markers expression level also corresponded with the level of patient’s systolic blood pressure and proteinuria. In conclusion, we first verify that expression of UII is associated with ERS in patients with SPE. Our results indicate that UII may trigger ERS in placental trophoblastic cells in patients with preeclampsia. Journal of Human Hypertension advance online publication, 16 April 2015; doi:10.1038/jhh.2015.28
INTRODUCTION Hypertensive disorders in pregnancy remain a leading cause of maternal and perinatal mortality and morbidity. The pathogenesis is associated with intense vasospasm, which presents as hypertension, poor organ perfusion affecting the kidney, the central nervous system and the liver.1 It is generally defined as newly developed hypertension (systolic blood pressure ⩾ 140 mm Hg or diastolic blood pressure ⩾ 90 mm Hg) with or without substantial proteinuria (⩾300 mg in 24 h) at or after 20 weeks gestation. Pathogenesis in hypertensive disorders in pregnancy remains largely unknown; the leading hypotheses strongly rely on disturbed placental function in early pregnancy.1 Among them, endothelial dysfunction is frequently emphasized in the pathogenesis of hypertensive disorders in pregnancy.2 At present, the only known definitive treatment for these diseases is the delivery of fetus and placenta. Therefore, in order to develop a less invasive or alternative approach to treating these diseases, it is urgent to recognize new pathogenic factors and discover other pathogenic models that lead to hypertensive disorders in pregnancy. Endoplasmic reticulum stress (ERS), which is essentially accumulation of unfolded or misfolded proteins in endoplasmic reticulum, has recently been identified as an important process involved in the pathology of preeclampsia.3,4 The most important marker of ERS is glucose regulation protein 78 (GRP78), and severe
ERS can activate several signal molecules, such as C/EBP homologous protein (CHOP).2,3 Urotensin II (UII) is a vasoactive, somatostatin-like cyclic peptide of 11 amino acids initially isolated from the urophysis of the goby fish. It is widely expressed in the cardiovascular system as well as in the central nervous system, kidney, spleen, small intestine, thymus, prostate, pituitary, adrenal gland, placenta and colonic mucosa.5–7 UII is generally agreed to be the most potent endogenous vasoconstrictor discovered to date.6 It has been demonstrated that UII interacts with a G-protein-coupled receptor known as GPR14 or urotensin receptor (UT), and the binding is functionally coupled to calcium mobilization, which may participate in the pathophysiology of hypertensive disorders in pregnancy.1,8 Moreover, UII binds with its receptor and triggers phospholipidase C activation, and inositol 1,4,5 triphosphate receptors dependent pathway which induces calcium releasing from endoplasmic reticulum,9 and Mekahli et al.10 also found that the depletion of endoplasmic reticulum calcium was associated with ERS. Since both UII and ERS are associated with lowered calcium concentration in the endoplasmic reticulum, we speculate that UII may also be correlated with ERS in its role in the development of hypertensive disorder in pregnancy. The aim of our study is to assess the role of UII, a vasoactive peptide, in the pathophysiology of hypertensive disorders in pregnant patients.
1 Department of Nephrology, Peking University Third Hospital, Beijing, China and 2Department of Pathology and Physiology, Peking University Health Science Center, Beijing, China. Correspondence: Professor A-H Zhang, Department of Nephrology, Peking University Third Hospital, No. 49 North Garden Road, Beijing 100191, China. E-mail: [email protected] 3 These authors contributed equally to this work. Received 19 November 2014; revised 20 February 2015; accepted 26 February 2015
UII associated with ERS in severe preeclampsia W-Y He et al
2
MATERIALS AND METHODS Study subjects Thirty-three patients with hypertensive disorders during pregnancy and twenty-two healthy pregnant women designated as control group were recruited from the inpatient obstetric ward at the Peking University Third Hospital from October 2012 to March 2013. Histological studies, real-time PCR and western blot analysis were performed on placental tissues of 25 patients with hypertensive disorders during pregnancy (5 patients with gestational hypertension (GH), 8 patients with mild preeclampsia (MPE) and 12 patients with severe preeclampsia (SPE)). Same set of studies were also performed on the placental tissues of 25 patients with normal pregnancies from June 2012 to March 2013. All 25 placentas from patients with hypertensive disorder during pregnancy were belong to the same patients. Although we originally recruited 33 patients with hypertensive disorders during pregnancy, we failed to collect six placentas with GH and two placentas with MPE for missing the suitable collecting time. Human placental tissues were obtained just after spontaneous labor or caesarean operation. All participants signed their written informed consent. Then, these patients with hypertensive disorders in pregnancy were classified into three subgroups according to their blood pressures and proteinuria levels. These subgroups included GH, MPE and SPE. For the GH group, 11 patients were included and GH was defined as persistent hypertension, systolic blood pressure ⩾ 140 mm Hg or diastolic blood pressure equal to 90 mm Hg, without proteinuria, developed after 20 weeks of gestation. For the MPE group, 10 patients were included and MPE was defined as persistent hypertension, systolic blood pressure ⩾ 140 mm Hg or diastolic blood pressure equal to 90 mm Hg, with proteinuria (⩾0.3 g in a 24 h urine collection or ⩾ 1 g according to a dipstick test), developed after 20 weeks of gestation.3 For the SPE group, 12 patients were included and SPE was defined as systolic blood pressure greater than 160 mm Hg or diastolic blood pressure greater than 110 mm Hg on 2 occasions each 6 h apart with proteinuria exceeding 2 g in a 24-h period or 2–4+ on dipstick testing.2 The inclusion criteria of patients were that patients must have hypertensive disorders, singleton pregnancies with a living fetus and gestational ages of 32 weeks. Pregnancies with infection, cancer, liver disease, kidney disease or heart disease were excluded from both study groups. Healthy pregnant women with no prior pregnancy complications were included as controls. Detailed histories were obtained from all patients. Demographic data including age, body weight, height and so on were recorded. Body mass indices of all patients were calculated. Blood pressures of all patients were measured with sphygmomanometer at 0800 hours by qualified physicians. Complete blood counts, routine urinary and biochemical analysis were all performed as well. Estimated glomerular filtration rate was assessed by simplified modification of diet in renal disease (MDRD) equation = 186 × (Scr)−1.154 × (Age)−0.203 × (0.742 if female).
Immunohistochemical analyses Histological studies were performed on placental tissues of patients with hypertensive disorders during pregnancies and healthy pregnancies. All participants signed their written informed consents. The placental tissues were embedded in optimal cutting temperature compound. The tissues were sectioned at a thickness of 10 μm. For immunohistochemical analysis, 5% hydrogen peroxide was used to deplete endogenous peroxidase activity. Following pre-incubation with 5% bovine serum albumin for 30 min to prevent nonspecific staining, the sections were incubated with rabbit anti-human UII (1:50), GPR14 (1:100), CHOP (1:100) and GRP78 antibody (1:200) at 4 °C overnight (all antibodies were bought from Abcam Inc., Cambridge, UK, and all other chemicals were of analytical grade from commercial suppliers (Beijing Guangquyuan chemical reagent company, Beijing, China)). The sections were then incubated with horseradish peroxidase-coupled goat anti-rabbit IgG antibody for 20 min, followed by incubation with strep-avidin-biotinperoxidase complex for 20 min at 37 °C. The peroxidase was visualized by incubation with 3, 3′-diaminobenzidine in the dark for 3 min for UII, GPR14 antibody, and 1.5 min for GRP78, CHOP antibody. The sections were counterstained with hematoxylin, dehydrated and observed under a light microscope. Negative controls were established using PBS as a substitute for the primary antibody. Positive staining was indicated by brown deposits. The integral optical density (IOD) was measured with Image-Pro Plus 6.0 (Media Cybernetics, Silver Spring, MD, USA), and the result was determined as the sum of five different fields (one in the center and four in the periphery) of each section. The IOD of the target protein was defined as Journal of Human Hypertension (2015), 1 – 7
the sum of the optical densities of all the positive pixels in the image, which represents the quantity of the targeted protein. The pathological image analysis system was used to calculate the IOD of positive staining for UII, GPR14, CHOP and GRP78.
UII measurement using radioimmunoassay Plasma UII was measured using a radioimmunoassay method according to our previous publication and other literatures.6,11 Plasma samples of 3ml were deproteinated with 0.75 ml of 2 mol l− 1 hydrochloric acid. After centrifugation for 20 min at 6000 g, the supernatant was loaded onto cartridges that had been activated with 3 ml of 100% methanol and 3ml of double-distilled deionized water. The cartridges were then washed twice with 3 ml of 0.1% trifluoroacetic acid and eluted with 3 ml of 60% acetonitrile in 0.1% of trifluoroacetic acid. The eluants were freeze-dried overnight and resuspended in 250 μl of radioimmunoassay buffer. One hundred microliters of standard UII or assay sample were incubated overnight at 48 °C with 100 μl rabbit antiserum. One hundred microliters of labeled 125I-urotensin II (Phoenix Pharmaceuticals, Inc. Belmont, CA, USA) were added to each tube and incubated for an additional 24 h. Antibodybound UII was precipitated using a goat anti-rabbit antiserum and normal horse serum. Using a gamma counter, the amount of bound 125I-urotensin II was measured as picograms per milliliter.
RNA isolation and quantitative real-time PCR analysis for placenta Total RNA was extracted from the placentas of both patients with hypertensive disorders during pregnancy and healthy pregnancies by using TRIzol Reagent (Invitrogen. Ltd, Carlsbad, CA, USA). RNA was reverse transcribed into first-strand complementary DNA using GoScript Reverse Transcription System (A5001, Promega Co., Madison, WI, USA). A real-time quantitative PCR method was used to detect the changes in UII, CHOP and GRP78 mRNA levels. Primers for human UII, CHOP and GRP78 were designed using Primer Express software (Applied Biosystems, Sangon Biotech (Shanghai) Co., Ltd, Shanghai, China). Quantitative real-time PCR was carried out in a total volume of 20 μl using a Bio-Rad iQ5 detector (Applied Biosystems) to determine the threshold cycle (Ct) value. Each sample was run and analyzed in triplicate. For the UII gene, the forward primers was 5′-GCACTGTTTGCTTTGGACTCC-3′ and the reverse primer was 5′-TGGTCGTCCATGCACAGATT-3′, the forward primer of UII receptor (GPR14) gene was 5′-CACGGGCACCATTGGGACTC-3′ and the reverse primer was 5′-CGCCAGGTTGACCACGTAGAC-3′; the forward primer of CHOP was 5′-GGAAACAGAGTGGTCATTCCC-3′ and the reverse primer was 5′-CTGCTTGAGCCGTTCATTCTC-3′; the forward primer of GRP78 was 5′-CAT CACGCCGTCCTATGTCG-3′ and the reverse primer was 5′-CGTCAAAGAC CGTGTTCTCG-3′. For glyceraldehyde 3-phosphate dehydrogenase (GAPDH) control, the forward primer was 5′-AACGGATTTGGTCGTATTGGGC-3′ and the reverse primer was 5′-TCGCTCCTGGAAGATGGTGATG-3′. Relative quantitation values were calculated using the 2–ΔΔCt method as fold changes in the target gene related to the expression level of GAPDH. Three-step real-time PCR of denaturing, annealing and extension reactions proceeded for 40 cycles at 15 s at 95 °C, 1 min at 59 °C (UII ), and 30 s at 72 °C. For UII, UII receptor, CHOP, GRP78 and GAPDH, the annealing temperature is 58 °C.
Western blot analysis for placentas Proteins were extracted from the placentas of both patients with hypertensive disorders during pregnancy and healthy pregnancies. Immunoprecipitation and western blot analysis of UII, GPR14, GRP78 and CHOP were performed. Briefly, the protein samples were denatured at 95 °C for 5 min separated on a 10% SDS-PAGE gel before being transferred to nitrocellulose filter (NC) membranes (Applygen Technologies Inc., Beijing, China). Membranes were subsequently incubated with primary rabbit polyclonal anti-UII, anti-GPR14, anti-GRP78 and anti-CHOP antibodies (1:1000; ab21685, Abcam) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit antibodies (1:500, Zhongshan Gold Bridge Biotechnology Co., Ltd, Beijing, China). Semiquantitative grayscale intensity was generated with Odssey Software v1.2 (Samsung Corporation, Gyeonggi-Do, Korea).
Statistical analyses All analyses were performed using SPSS 19.0 (SPSS, Inc., Chicago, IL, USA). Results were reported as mean ± standard deviation (x ± s). Nonparametric data were tested by Mann–Whitney test. One-way analysis of variance was © 2015 Macmillan Publishers Limited
UII associated with ERS in severe preeclampsia W-Y He et al
3 applied in statistical analysis. Spearman’s rank correlation was used for correlation analyses. P values below 0.05 were accepted to be significant.
RESULTS Clinical characteristics of the study subjects Table 1 summarized the clinical characteristics of the study subjects. A total of 55 subjects were included. As expected, significantly elevated systolic blood pressure, elevated diastolic blood pressure, elevated plasma creatinine and lower estimated by glomerular filtration rate (eGFR), lowered plasma total protein and lowered albumin level were observed in patients with hypertensive disorder in pregnancy. Elevated hepatic enzymes, such as alanine aminotransferase, were also observed in pregnancies with hypertensive disorders in comparison to those of normal control (NC) group. The mean plasma UII levels were significantly higher in patients with hypertensive disorders during pregnancy than those of NC (P = 0.003). In contrast, analysis of two groups revealed no significant difference between the urinary UII Table 1. Comparison of clinical characteristics of the study subjects included (n = 55) Normal controls (n = 22) Age (years) BMI (kg m −2) GA (weeks) SBP (mmHg) DBP (mmHg) Urinary protein level (g per 24 h) WBC (*109 l − 1) Hb (g l − 1) PLT (*109 l − 1) ALT (U l − 1) AST (U l − 1) TP (g l − 1) Alb (g l − 1) Urea (mmol l − 1) Cr (umol l − 1) eGFRb (ml min − 1) Glu (mmol l − 1) Plasma UII (pg ml − 1) Urinary UII (pg ml − 1)
Hypertensive P value disorders in pregnancy (n = 33)
30.59 ± 3.35 27.36 ± 3.00 37.09 ± 2.02 119.73 ± 11.00 74.86 ± 7.29
31.79 ± 5.39 30.65 ± 4.64 35.91 ± 2.31 139.12 ± 15.09 89.45 ± 10.08 0.49(0.16, 2.85)
0.315 0.005 0.056 0.000 0.000
8.75 ± 2.21 112.55 ± 16.47 174.18 ± 69.28 14.36 ± 10.46 15.59 ± 2.65 62.95 ± 4.47 35.73 ± 2.46 2.99 ± 1.27 51.09 ± 11.91 121.23 ± 11.45 4.29 ± 0.38 29.93 ± 9. 95
10.04 ± 2.53 114.91 ± 19.30 173.45 ± 60.48 19.03 ± 12.57 23.27 ± 8.53 57.18 ± 8.08 32.09 ± 5.90 4.17 ± 1.33 62.42 ± 15.08 108.19 ± 13.31 4.62 ± 0.746 42.01 ± 16.30
0.056 0.629 0.968 0.142 0.000 0.004 0.008 0.002 0.005 0.019 0.059 0.003
11.19 (8.22, 25.67) 16.61 (13.06, 22.72) 0.45
Abbreviations: Alb, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMI, body mass index; Cr, blood creatinine; GA, gestational age; DBP, diastolic blood pressure; Glu, glucose; Hb, hemoglobin, PLT, platelet; SBP, systolic blood pressure; TP, total protein; UII, urotensin II; WBC, white blood cell. P values below 0.05 have been accepted to be significant. Normal distribution data are presented as means ± s.d. Abnormal distribution data are presented as median, 25 percentile and 75 percentile.
Table 2.
levels of subjects with hypertensive disorders in pregnancy and those of NCs. UII level analysis according to the severity of hypertensive disorder during pregnancy Elevated plasma UII level was observed in SPE group when compared with GH group and MPE group (Po0.01). Also, no statistically significant difference in terms of urinary UII level was detected among groups (P40.05; see Table 2). Moreover, neither plasma UII nor urinary UII level was not correlated with the level of serum albumin and estimated glomerular filtration rate. There were four patients with elevated plasma UII in SPE group (plasma UII concentrations were 74, 68, 72 and 69 pg ml − 1). Their clinical and laboratory findings were different from others for they all had partial HELLP syndrome, such as mild anemia, mild thrombocytopenia, increased aspartate aminotransferase level of ⩾ 40 IU l − 1, increased alanine aminotransferase level of ⩾ 40 IU l − 1 or severe proteinuria. Pathological analyses of placentas We discovered vessel congestion and edema of placental villi. The mesenchymal cells were reduced comparatively, and significant deposition of cellulose could be found. In addition, little necrosis and calcification could be observed, especially in patients with SPE (Figure 1a and b). Immunohistochemical analyses of expression level of UII, GPR14, GRP78 and CHOP in placental tissue Immunochemistry analysis of placental tissue showed that expression level of UII and its receptor GPR14 in SPE group were highly increased and were mainly located in the cytoplasm of placental trophoblastic cells; however, UII and GPR14 expression level were only moderately increased in MPE and GH groups and weakly expressed in NC group (Figures 2a–h). Our results demonstrated that the expression level of UII and GPR14 (IOD) increased significantly in patients with SPE in pregnancy in comparison with that of the healthy controls, GH and MPE group (Figures 2i and k). The expression levels of GRP78 and CHOP were mainly located in the cytoplasm of placental trophoblastic cells and few were located in vascular endothelial cells as shown by immunohistochemical analyses. The expression level of GRP78 and CHOP was increased significantly in patients with hypertensive disorders in pregnancy than that of the NC group (Figure 3a–i). By immunochemical semi-quantitive analysis, IODs of GRP78 and CHOP were significantly higher in SPE group than that of NC, GH or MPE groups (Figures 3j and k). mRNA and protein expression levels of UII, GPR14, CHOP and GRP78 in placentas with hypertensive disorders during pregnancy and in placentas of normal pregnancy Our results showed that there was no significant difference in mRNA expression levels of UII, UII receptor, CHOP and GRP78 among MPE, GH and normal pregnancy groups (data not shown), but significantly elevated mRNA expression levels of UII (mean = 9.5-fold), CHOP (mean = 9.6-fold) and GRP78 (mean = 5.1-fold) in
Comparison of plasma and urinary UII levels of patients according to the severity of disease
Plasma UII level (pg ml − 1) Urinary UII level (pg ml − 1)
GH group (n = 11)
MPE group (n = 10)
SPE group (n = 12)
34.13 ± 8.36 13.63 (8.36, 16.61)
33.83 ± 9.32 18.70 (14.67, 22.68)
56.05 ± 17.34* 21.88 (13.07, 28.14)
Abbreviations: GH, gestational hypertension; MPE, mild preeclampsia; SPE, severe preeclampsia. P values below 0.05 have been accepted to be significant. Normal distribution data are presented as means ± s.d. Abnormal distribution data are presented as median, 25 percentile and 75 percentile. *Po0.01 compared with GH group and MPE group.
© 2015 Macmillan Publishers Limited
Journal of Human Hypertension (2015), 1 – 7
UII associated with ERS in severe preeclampsia W-Y He et al
4
Figure 1. HE slides of placenta tissue of SPE patients under light microscope ((a): X10; (b): X200). Vessel congestion and edema of placental villi could be found in the slides. Reduced mesenchymal cell, significant deposition of cellulose and little necrosis and calcification could be observed.
Figure 2. Immunochemistry analysis in placental tissue showed that expression level of UII and its receptor GPR14 in SPE group were highly increased and were mainly located in the cytoplasm of placental trophoblastic cells. Expression levels of UII and GPR14 are moderately increased in MPE and GH groups but very weak expression level in NC group (see arrow). (a–h) UII in SPE (a), MPE (b), GH (c) NC (d); GPR14 in SPE group (e) GPR14 in MPE (f) GPR14 in GH (g) GPR14 in NC group (h). IOD of UII (i) in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.006) and MPE group (P = 0.000). No significant differences were observed between NC, GH and MPE group. IOD of GPR14 (k) in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.001) and MPE group (P = 0.001). No significant differences were observed between NC and GH, and MPE and SPE group.
placentas with SPE were observed in comparison with that of the placentas with normal pregnancy (Figure 4). Moreover, there were no significant differences in protein expression levels of UII, UII receptor, CHOP, GRP78 among the placentas of MPE, GH and normal pregnancy groups (data not shown), but protein expression level of UII (mean = 1.7-fold), CHOP (mean = 2.2-fold) and GRP78 (mean = 1.5-fold) in placentas with SPE was significantly Journal of Human Hypertension (2015), 1 – 7
higher as demonstrated by western blot in comparison with that of normal pregnancy group (Figure 5). Correlations between expression level of UII and markers of ERS The plasma UII level had positive correlation with the level of systolic blood pressure (r = 0.459, P = 0.001) and amount of © 2015 Macmillan Publishers Limited
UII associated with ERS in severe preeclampsia W-Y He et al
5
Figure 3. The expression levels of GRP78 (a–d) and CHOP were mainly located in the cytoplasm of placental trophoblastic cells (f–i) and few were located in vascular endothelial cell (e) by immunochemistry analysis. Both of them in the SPE group (a, f) were significantly increased in expression levels of GRP78 and CHOP, while the expression levels of GRP78 and CHOP were moderately increased in MPE (b, f), GH group (c, g) and were nearly deficient in NC group (d, h). IOD of GRP78 in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.045) and MPE group (P = 0.01; j). Expression level of CHOP (i) in SPE group was significantly higher than NC group (P = 0.000), GH group (P = 0.000) and MPE group (P = 0.000; k).
proteinuria (r = 0.607, P = 0.001); however, no correlation was found with the level of creatinine estimated by the filtration rate calculated by MDRD equation. Expression levels of UII mRNA in placentas with SPE also were positively correlated with the amount of proteinuria (r = 0.73, P = 0.014) and the level of systolic blood pressure (r = 0.64, P = 0.017). The expression level of UII mRNA was positively correlated with the expression level of CHOP mRNA (r = 0.789, P = 0.001) and GRP78 mRNA (r = 0.689, P = 0.011) in placentas with SPE. The protein expression level of UII was also found to be positively correlated with that of CHOP (r = 0.71, P = 0.003) and GRP78 (r = 0.69, P = 0.015). © 2015 Macmillan Publishers Limited
DISCUSSION The pathogenesis of hypertensive disorders in pregnancy is the subject of much debate but it is generally believed to be a type of placental dysfunction that resulted from arteriolar vasoconstriction with abnormal invasion of the spiral arteries by cytotrophoblast cells.1–3 In contrast to normal pregnancy, the synthesis and release of vasoactive factors are enhanced, which induce widespread injury of maternal vascular endothelium. This results in increased formation of endothelin, thromboxane, superoxide, increased vascular sensitivity to angiotensin II and decreased formation of vasodilators, such as nitric oxide and Journal of Human Hypertension (2015), 1 – 7
UII associated with ERS in severe preeclampsia W-Y He et al
6 prostacyclin. These endothelial abnormalities, in turn, cause hypertension by impairing renal function and increasing total peripheral resistance.2,7 UII, one of the potent endogenous vasoconstrictors, was found to be significantly higher in patients with essential hypertension, severe coronary artery disease, ischemic cardiomyopathy, congestive heart failure, diabetes mellitus, renal failure and portal
Figure 4. mRNA expression levels of UII, CHOP and GRP78 in placentas with hypertensive disorders in pregnancy and normal pregnancy. There were significantly higher mRNA expression levels of UII (mean = 9.5-fold), CHOP (mean = 9.6-fold) and GRP78 (mean = 5.1-fold) in placentas with SPE compared with normal pregnancy by real-time PCR method (**P o0.01 compared with normal pregnancy).
hypertension caused by liver cirrhosis.5,12 Balat et al. reported a significant increase in circulating levels of UII in preeclampsia,6 whereas others reported no difference of UII level between normal pregnancy and pregnancy with preeclampsia.8 In our study, we found that the level of UII was significantly higher in the plasma of patients with hypertensive disorders during pregnancy than those of patients with healthy pregnancies. However, the urinary levels of UII had no differences between patients with hypertensive disorders during pregnancy and patients from healthy pregnant group. According to Matsushita’ study, urinary UII is primarily derived from a renal source.7 Although both groups of patients excrete UII in the urine, we speculate that the renal UII expression level does not upregulate in either patients with hypertensive disorder during pregnancy or NCs. From our study, we were able to show that the expression levels of UII and UII receptor were increased in the placentas of patients with hypertensive disorders during pregnancy. However, real-time PCR and western blot methods only showed increased mRNA and protein expression levels for UII but not UII receptor. Although immunochemistry method demonstrated an increase in the expression level of both UII and its receptor for the hypertensive placenta, this may result from unspecific staining. The cause for the increased concentrations of UII in maternal plasma in hypertensive disorder in pregnancy is unclear. According to the previous study, plasma levels of UII had been measured in healthy individuals, and the circulated UII levels were relatively low.7 This finding suggests that UII is not predominantly a circulating hormone. To be more specific, the increased circulating UII in patients with hypertension disorders during pregnancy may come from the secretion of UII in placenta. It may result from deficient spiral artery conversion, causing decreased uteroplacental blood flow that can result in either hypoxia or ischemia-reperfusion. Gould et al. also reported that under hypoxic conditions, endothelial cells or trophoblastic cells produce more UII and, thus, may contribute to the increased UII circulating levels.1 We found that the level of plasma UII in SPE group was significantly higher than that of the MPE group and GH group. In addition, we also found that plasma UII level had positive correlation with blood pressure and 24 h urinary proteinuria level,
Figure 5. Protein expression levels of UII, CHOP and GRP78 in placentas with hypertensive disorders in pregnancy and normal pregnancy. There were significantly higher protein expression levels of UII ( mean = 1.7-fold ), CHOP (mean = 2.2-fold) and GRP78 (mean = 1.5-fold) in placentas with SPE by western blot compared with normal pregnancy (**P o0.01 compared with normal pregnancy, *Po0.05 compared with normal pregnancy). Journal of Human Hypertension (2015), 1 – 7
© 2015 Macmillan Publishers Limited
UII associated with ERS in severe preeclampsia W-Y He et al
7 which were associated with the outcome of the pregnancy. This suggests that plasma UII levels are also associated with the development of the disease. In our study, we also found that UII protein is mainly located in the cytoplasm of placental trophoblastic cells as shown by immunohistochemistry studies. This indicates that the UII not only affects the vascular endothelial cells, but also affects the trophoblastic cells. This would be a reason for placental ischemia/ hypoxia in hypertensive disorders during pregnancy. Also, the expression level of UII in placenta had positive correlation with blood pressure and proteinuria. This, in turn, is associated with the increase in plasma UII level and, thus, the progression of the disease. ERS, a major regulator of cell homeostasis through its involvement in post-translational protein modifications and folding, has recently been identified as an important process involved in the pathology of preeclampsia.2–4 Activation of pro-inflammatory pathway may also occur under ERS. The proinflammtory environment is thought to lead to maternal endothelial cell dysfunction, resulting in the syndrome of hypertension and proteinuria.13 In our present study, we found the expression levels of CHOP and GRP78 mRNA and protein were significantly increased in placental tissue in patients with SPE compared with that of the patients with normal pregnancy. Moreover, their expression levels were mainly located in placental trophoblastic cells as shown by immunochemistry method. Also, the expression level of UII mRNA and protein was positively correlated with the expression level of CHOP and GRP78 mRNA and protein. ERS markers in placentas all had positive correlation with blood pressure and proteinuria. Our results indicate that ERS in placental trophoblastic cells may play some important roles in the development of hypertensive disorder in pregnancy in addition to endothelial cell. Recent study showed that endothelin-1 could induce ERS in trophoblasts through the PLC-IP (3) pathway.13 The physiological mechanisms of UII are similar in some ways to endothelin-1, because hypertensive disorders in pregnancy are associated with increased level of plasma UII, which is a vasoactive peptide that has the potential to disrupt ER Ca2+ homeostasis. It is reported that UII can induce calcium releasing from endoplasmic reticulum,9 and Mekahli et al. also showed that loss of luminal calcium in endoplasmic reticulum causes ERS and activates an unfolded protein response.10 With the findings of these studies, we speculate that UII may induce ERS, and our results verified that they had positive correlation. Our results indicate that UII may induce placental trophoblastic cell ERS. Future studies are needed to test this hypothesis. For instance, we can treat trophoblastic cells with UII in vitro and also use animal model to test whether UII can induce the expression of ERS markers that can subsequently induce apoptosis of the trophoblastic cells. According to the previous studies, UII can constrict blood vessel, induce cell proliferation, atherosclerosis and regulate soluble vascular endothelial growth factor (VEGF) receptor 1 secretion by placental tissues,1,2,6 which may be associated with the pathogenesis of hypertensive disorder during pregnancy. From our study, we demonstrated for the first time that the expression level of UII had a positive correlation with ERS markers in trophoblastic cell of placental tissue in patients with SPE. The findings in this study can be the basis of a new pathogenic model for preeclampsia and potential foundation for the development of a less invasive treatment for this disease. CONCLUSION In summary, our present study shows that plasma level and expression level of UII in placental tissue are associated with
© 2015 Macmillan Publishers Limited
development of hypertensive disorder during pregnancy. We are the first to verify that the expression level of UII mRNA and protein are associated with expression level of CHOP and GRP78 mRNA and protein in SPE, and their expression levels are mainly located in trophoblastic cells in placentas. Our results indicate that UII may trigger ERS in placental trophoblastic cells, which can be a new pathogenic model for hypertensive disorder during pregnancy. What is known about topic ● Expression level of UII is associated with endoplasmic reticulum stress in patient with severe preeclampsia What this study adds ● We are the first to verify that the expression level of UII mRNA and protein are associated with expression level of CHOP and GRP78 mRNA and protein in severe preeclampsia, and their expression levels are mainly located in trophoblastic cells in placentas ● Our results indicate that UII may trigger ERS in placental trophoblastic cells, which may be a new pathogenic model in the development of hypertensive disorder during pregnancy
CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation (Grant No. 81170706 and Grant No 81341022) to A-H Zhang.
REFERENCES 1 Gould PS, Gu M, Liao JQ, Ahmad S, Cudmore MJ, Ahmed A et al. Up regulation of urotensin II receptor in preeclampsia causes in vitro placental release of soluble vascular endothelial growth factor receptor 1 in hypoxia. Hypertension 2010; 56: 172–178. 2 Liane LA, Loset LM, Mundal SB, Fenstad MH, Johnson MP, Eide IP. Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and without pre-eclampsia. Placenta 2011; 32: 823–829. 3 Eric AP, Dadelszen PV, Duvekot JJ, Pijnenborg R. Pre-eclampsia. Lancet 2010; 376: 631–644. 4 Veerbeek JH, Tissot Van Patot MC, Burton GJ, Yung HW. Endoplasmic reticulum stress is induced in the human placenta during labour. Placenta 2015; 36: 88–92. 5 Ames RS, Sarau HM, Chambers JK. Human urotensin-II is a potent vasoconstrictor and agonist for the orphan receptor GPR14. Nature 1999; 40: 282–286. 6 Balat O, Aksoy F, Kutlar I, Ugur MG, Iyikosker H, Balat A et al. Increased plasma levels of Urotensin-II in preeclampsia–eclampsia: a new mediator in pathogenesis? Eur J Obstet Gynecol Reprod Biol 2005; 120: 33–38. 7 Matsushita M, Shichiri M, Imai T, Iwashina M, Tanaka H, Takasu N et al. Coexpression of urotensin II and its receptor (GPR14) in human cardiovascular and renal tissues. J Hypertens 2001; 19: 2185–2190. 8 Cowley E, Thompson JP, Sharpe P, Waugh J, Ali N, Lambert DG. Effects of preeclampsia on maternal plasma, cerebrospinal fluid, and umbilical cord urotensin II concentrations: a pilot study. Br J Anaesth 2005; 95: 495–499. 9 Jarry M, Diallo M, Lecointre C. The vasoactive peptides urotensin II and urotensin II-related peptide regulate astrocyte cell activity through common and distinct mechanisms: involvement in cell proliferation. Biochem J 2010; 428: 113–124. 10 Mekahli D, Bultynck G, Parys JB, Smedt HD, Missiaen L. Endoplasmic-reticulum calcium depletion and disease. Cold Spring Harb Perspect Biol 2011; 3: 1–30. 11 Bai Q, Zhang J, Zhang AH, Cheng LT, He L, Fan MH et al. Roles of human urotensin II in volume resistance hypertension in peritoneal dialysis patients. Ren Fail 2012; 340: 713–717. 12 Totsune K, Takahashi K, Arihara Z, Sone M, Ito S, Murakami O. Increased plasma urotensin II level s in patients with diabetes mellitus. Clin Sci 2003; 104(1): 1–5. 13 Jain A, Olovsson M, Burton GJ, Yung HW. Endothelin-1 induces endoplasmic reticulum stress by activating the PLC-IP (3) pathway: implications for placental pathophysiology in preeclampsia. Am J Pathol 2012; 180: 2309–2320.
Journal of Human Hypertension (2015), 1 – 7
