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Department of Obstetrics and Gynecology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Shikata, Okayama 700-8558, Japan
Address all correspondence and requests for reprints to: Hisashi Masuyama, M.D., Ph.D., 2-5-1, Shikata, Okayama 700-8558, Japan. E-mail: masuyama{at}cc.okayama-u.ac.jp.
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Abstract |
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Objectives: We examined possible links between sEng, VEGF, sVEGFR, and adipocytokines in the pathophysiology of PE.
Study Design: We performed a cross-sectional study in 30 PE patients and controls matched for gestational age and body mass index. Blood samples were collected soon after disease onset. We measured serum concentrations of leptin, adiponectin, sEng, VEGF, placental growth factor (PlGF), and sVEGFR [soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble fetal liver kinase 1 (sFlk-1)], and examined the placental protein content of sEng and sFlt-1.
Results: sEng concentrations in PE patients (60.9 ± 28.8 ng/ml) were significantly higher than those in controls (11.2 ± 4.4 ng/ml). There was a significant correlation between sEng and sFlt-1 or PlGF. Moreover, there were significant differences in mean blood pressure between the high and low sEng groups, and in proteinuria between the high and low sFlt-1 groups, and significant differences in placental sEng and sFlt-1 contents between patients with and without severe hypertension or proteinuria. sEng was also correlated positively with adiponectin levels and negatively with the leptin to adiponectin ratio.
Conclusions: Along with sFlt-1 and PlGF, sEng might play a role in the pathophysiology of PE, especially in elevating blood pressure, while the association with hypoadiponectinemia and the high leptin to adiponectin ratio in pregnancy seem to be risk factors for PE.
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Introduction |
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Endoglin (Eng) is a component of the receptor complex for TGF-ß and interacts efficiently with TGF-ß (12). It modulates the TGF-ß signaling pathway by interacting with TGF-ß receptors I and II, and a proliferation-associated, hypoxia-inducible protein abundantly expressed in angiogenic endothelial cells and the cytotrophoblast. Several studies have suggested that Eng is a pro-angiogenic component that protects endothelial cells under hypoxia-induced apoptosis and regulates nitric oxide-dependent vasodilatation (13, 14, 15). On the other hand, soluble endoglin (sEng) is an antiangiogenic protein thought to impair TGF-ß1 binding to cell surface receptors and to decrease endothelial nitric oxide signaling (16, 17). Recent reports have demonstrated that sEng, which is placental in origin, is elevated in the sera of preeclamptic individuals, increasing with disease severity and falling after delivery, and that administration of both sFlt1 and sEng produces a severe PE-like animal model with hypertension, proteinuria, glomerular endotheliosis, and features of HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome (16, 18). These data suggest that sEng plays an important role in the pathophysiology of PE.
In addition to alterations in angiogenesis, there is an association between markers of insulin resistance and PE, and insulin resistance syndrome has been observed before, during, and after this pregnancy complication (19). Moreover, recent reports have suggested that insulin signaling and angiogenesis are intimately related (20, 21), and that insulin regulates the expression of genes involved in angiogenesis, including the expression of VEGF mRNA, and VEGF signaling also activates Akt/protein kinase B phosphorylation in the insulin signaling pathway (22, 23). These data suggest that alternations in angiogenesis and insulin resistance may have an additive effect that leads to alterations in critical cellular functions, endothelial cell injury, and, subsequently, increased risk of developing PE. Adipose tissue functions as a highly specialized endocrine and paracrine tissue, producing an array of adipocytokines such as leptin, TNF-
, and adiponectin. Such factors have local and systemic biological effects, and play important roles in insulin signaling and the development of metabolic diseases (24, 25).
In this study, to determine possible links among sEng, angiogenic factors, and adipocytokines in the pathophysiology of PE, we first examined serum concentrations of sEng in pregnant women with PE and healthy pregnant women. Second, to determine any relationships between sEng and angiogenic factors, we measured levels of the circulating angiogenic factors VEGF and PlGF, and the soluble VEGF receptors sFlt-1 and sFlk-1 in women with PE and in healthy pregnant women. Third, we examined the association of sEng and sFlt-1 with severity of PE, and finally, whether there are significant correlations between sEng and adipocytokines in PE and healthy pregnant women.
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Patients and Methods |
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A total of 60 pregnant Japanese women who visited the Department of Obstetrics and Gynecology, Okayama University Hospital, Japan, were included in the present study. Of these 60 women, 30 had severe PE, and 30 were controls matched for age, gestational age, parity, and body mass index (BMI), with normotensive pregnancies. There were 10 age-matched nonpregnant women, 20 healthy pregnant women in the first trimester, and those in the second trimester also included as volunteers. According to the definition of the Japan Society of Obstetrics and Gynecology, PE was defined as a persistent elevation of systolic blood pressure to 140 mm Hg and diastolic blood pressure to 90 mmHg on two occasions several hours apart, with proteinuria of more than 300 mg in a 24-h urine collection. Severe PE was defined as either severe hypertension (systolic blood pressure > 160 mm Hg or diastolic blood pressure > 110 mm Hg) or severe proteinuria (>2.0 g protein in a 24-h urine collection). None of the patients with PE had any prior history of renal disorder or essential hypertension. The healthy pregnant women had no history of illness and no form of hypertension or renal disorder. PE patients were also subdivided using the median values of sEng, sFlt-1, or sFlt-1/PlGF as cutoff points: those with a high-circulating sEng level [high sEng group, 
55 ng/ml (n = 15)]; low-circulating sEng level [low sEng group, <55 ng/ml (n = 15)]; high-circulating sFlt-1 level [high sFlt-1 group, 5300 pg/ml (n = 15)]; low-circulating sFlt-1 level [low sFlt-1 group, <5300 pg/ml (n = 15)]; high-circulating sFlt-1/PlGF ratio [high sFlt-1/PlGF group, 33 (n = 15)]; and low-circulating sFlt-1/PlGF ratio [low sFlt-1/PlGF group, <33 (n = 15)]. Clinical records were carefully reviewed, and some patients were interviewed further at the time of sample collection, with those not meeting the aforementioned criteria being eliminated from the study. The study was approved by the Institutional Ethical Review Board of Okayama University Hospital, and all subjects gave informed consent. Blood samples were collected from PE patients soon after disease onset. All PE patients underwent termination within 1 wk from the time of sample collection, due to severe PE. None of the subjects received any medication before blood sampling. Samples from healthy pregnant women in their second or third trimester were matched with PE patients by age, gestational week, parity, and BMI to avoid possible bias. All patients, including healthy controls, visited Okayama University, and all blood samples (5 ml from each patient) were collected between 2000 and 2006. Immediately after sample collection, the serum was separated by centrifugation and stored at –80 C until use. The average time of freezer storage in PE patients (2.4 ± 1.6 yr) was not significantly different from that of the controls (2.1 ± 1.3 yr).
ELISA for angiogenic factors and adipocytokines
Serum levels of VEGF, PlGF, sFlt-1, sFlk-1, adiponectin, leptin, and sEng were determined by ELISA (R&D Systems, Minneapolis, MN), following the manufacturer’s instructions. All samples were examined in duplicate, and the mean values of individual sera were used for statistical analysis. The minimum detectable concentrations in the assays for VEGF, PlGF, sFlt-1, sFlk-1, adiponectin, leptin, and sEng were 5.0, 7.0, 5.0, 5.0, 3.0, 0.5, and 0.1 ng/ml, respectively. The intraassay and interassay coefficients of variation for VEGF were less than 4.5% and 7.0%, respectively; PlGF, 3.6% and 11.0%; sFlt-1, 3.8% and 7.0%; sFlk-1, 3.6% and 6.9%; adiponectin, less than 3.5% and 7.0%; leptin, less than 3.0% and 7.5%; and sEng, 3.0% and 6.5%.
Western blot analysis
Protein extracts were obtained from placental tissues of patients with PE and healthy controls using tissue protein extraction reagent (Pierce, Rockford, IL) according to the manufacturer’s protocol, and stored at –80 C until analysis. Equivalent amounts of protein (50 µg/sample) from each extract were determined by bicinchoninic acid protein assay (Pierce), then solubilized in sodium dodecyl sulfate buffer [0.05 M Tris/HCl, 2% sodium dodecyl sulfate, 6% mercaptoethanol, and 10% glycerol (pH 6.8)]. Tissue extracts were separated on 10% SDS-PAGE, along with molecular weight markers, and subsequently transferred to polyvinylidene fluoride membranes (Amersham Biosciences, Piscataway, NJ). Nonspecific protein binding was blocked with 3% nonfat dry milk in Tris-buffered saline for 1 h at room temperature. The blots were incubated with either rabbit polyclonal antibody raised against sFlt-1 (1:200 dilution; Zymed Laboratories, San Francisco, CA) or mouse monoclonal antibody raised against sEng (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) at 4 C overnight. Rabbit polyclonal antibody raised against ß-actin (1:1000 dilution; Santa Cruz Biotechnology) was used to confirm equal loading. The blots were then washed before incubation with horseradish peroxidase-conjugated antirabbit or mouse secondary antibody (1:2000 dilution; Santa Cruz Biotechnology) for 1 h at room temperature. The blots were then washed, and specific signals were detected using the ECL chemiluminescence system (Amersham Biosciences) according to the manufacturer’s instructions. The amount of each protein was quantified densitometrically using an Image Scanner (CanoScan D 2400U; Canon, Tokyo, Japan) and Bio Image BQ 2.0 software (Bio Image, Ann Arbor, MI).
Statistical analysis
All values are expressed as the mean ± SD. The Kruskal-Wallis and Scheffé’s tests were used for intergroup comparisons of clinical parameters and serum levels of PlGF, sFlt-1, sFlk-1, adiponectin, leptin, and sEng. The correlations between sEng and several parameters were analyzed using Spearman’s rank correlation. Statistical analysis was performed with StatView software (Abacus Concepts, Berkeley, CA). P < 0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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When we compared variables (e.g. gestational age, maternal age, smoking habit, BMI before pregnancy) between PE and the controls, we found no significant differences. However, mean blood pressure [diastolic blood pressure + (systolic blood pressure – diastolic blood pressure)/3 mm Hg] in PE was significantly higher than that in healthy pregnant women (Table 1
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To evaluate the gestational pattern of sEng level and sFlt-1/PlGF ratio, we first examined the serum levels of sEng and the sFlt-1/PlGF ratio in healthy pregnant and nonpregnant women. sEng levels were significantly elevated in women in their third trimester compared with levels in nonpregnant women and those in their first trimester (Fig. 1A). sFlt-1/PlGF ratios were significantly increased in women in the first trimester compared with nonpregnant women; the ratios decreased in the second trimester and then increased again in the third trimester. There was a significant decrease of the ratios in the second trimester compared with those in the first and third trimester and nonpregnant women (Fig. 1A). We also examined serum levels of sEng and sFlt-1/PlGF ratios in PE patients and normal controls. The serum concentration of sEng and sFlt-1/PlGF ratios in PE patients was significantly increased compared with those in healthy pregnant women (Fig. 1B). There were also significant differences in sFlt-1 and PlGF levels between PE patients and healthy controls, which is consistent with previous reports by our own and other groups (6, 7, 8, 9, 10, 11) (Table 2).
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We examined the correlation between sEng and other angiogenic factors (PlGF, sFlt-1, sFlk-1, and sFlt-1/PlGF ratios) in PE patients and normal controls; VEGF was not detected in any samples. We used ELISA for VEGF, which detects only free VEGF, therefore, free VEGF might be captured by excess circulating sFlt-1. There was a significant positive correlation between sEng and sFlt-1 in PE patients, but not controls (Fig. 2A). sEng levels were also negatively correlated with PlGF in PE patients, but not controls (Fig. 2B), and there were no significant correlations between sEng and sFlk-1 in either group (Fig. 2C). In addition, there was a significant positive correlation between sEng and sFlt-1/PlGF ratios in PE patients, but not controls (Fig. 2D).
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There was a significant difference in the mean blood pressure between the high sEng and low sEng groups among the PE patients. Moreover, the mean blood pressure in the high sFlt-1 and sFlt-1/PlGF ratio groups was elevated compared with that in the low sFlt-1 and sFlt-1/PlGF ratio groups, but the difference was not significant (Fig. 3A). In contrast, there was a significant difference in proteinuria between the high and low sFlt-1 groups, and between the high and low sFlt-1/PlGF ratio groups (Fig. 3B). In the high sEng group, proteinuria was elevated compared with that in the low sEng group, but not significantly.
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As shown in Fig. 4, placental sFlt-1 and sEng protein content in the placenta of women with PE was significantly increased compared with that in healthy controls. In addition, we also observed significant differences in sEng between PE patients with and without severe hypertension, and significant differences in sFlt-1 between PE patients with and without severe proteinuria. Although both Eng and sEng were up-regulated in placental tissues, the sEng/Eng ratio showed no significant difference among healthy controls and subgroups of PE patients (data not shown).
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We examined the correlation between sEng and adipocytokines by analyzing the correlation with adiponectin in PE and control patients. There was a significant positive correlation between sEng and adiponectin in PE patients, but not controls (Fig. 5A). There was no significant correlation between sEng and leptin in either group (Fig. 5B). In addition, sEng levels were negatively correlated with the leptin to adiponectin ratio, which has been reported to be a more efficacious parameter of insulin resistance than adiponectin or leptin alone (26), in PE patients, but not controls (Fig. 5C). There was no significant correlation between sEng and BMI (data not shown).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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As previously documented (16, 18), serum sEng concentrations are elevated in women in their third trimester compared with the first trimester and nonpregnant women. These data suggest that the placenta is the primary source of circulating sEng in healthy pregnant women. Here, we examined sEng levels in healthy pregnancy and in PE patients. In our study, sEng levels in PE were significantly higher than those in normal pregnancy, consistent with previous reports (16, 18). Next, we examined the correlation between sEng and VEGFs, other angiogenic factors that have been shown to play an important role in the pathophysiology of PE. Serum sEng concentrations were shown to be correlated positively with sFlt-1 and negatively with PlGF, suggesting that the production of sEng, as well as sFlt-1, in the placenta might be enhanced under hypoxic conditions. We also examined the relationship between severity of PE and sEng plus sFlt-1 because previous reports have indicated that there are significant differences in both sFlt-1 and sEng levels between mild and severe PE (11, 16). Accordingly, we revealed that levels of circulating angiogenic factors sFlt-1 and sEng were correlated with PE severity. In particular, in our study sEng levels were significantly correlated with blood pressure, while sFlt-1 was correlated with proteinuria. We also observed a significant difference in placental expression of sEng and sFlt-1 proteins between PE patients with severe hypertension and proteinuria. It has also been reported that there is a positive correlation between the plasma concentration of sFlt-1 and the degree of proteinuria among patients with PE (27). These data suggest that these angiogenic factors might have different roles in the pathophysiology of PE and that sEng might play a relatively important role in elevating blood pressure in PE.
We also examined the correlations between sEng and adipocytokines, leptin, adiponectin, and the leptin to adiponectin ratio. There was a positive correlation between sEng and adiponectin in PE patients, but not healthy controls. We have previously reported that there is a significant correlation between adiponectin and PlGF or sFlt-1 in a group of PE patients and controls, which was different from the group examined in this study (28). Although the adiponectin levels in PE pregnancies are significantly higher than those in normal pregnancies (28, 29, 30, 31), in the present study, PE patients with relatively low adiponectinemia showed lower sEng levels, as well as low sFlt-1 levels. Adiponectin has been demonstrated to have pro-angiogenic, antiatherogenic, and antiinflammatory functions in the endothelium (32, 33, 34). Moreover, hypoadiponectinemia is associated with impaired endothelium-dependent vasodilatation and reduced blood flow (35, 36), and is an independent risk factor for hypertension (37). These data suggest that adiponectin might maintain endothelial function, and its deficiency might lead to endothelial dysfunction/hypertension. Thus, the elevation of circulating adiponectin might be a physiological response to the endothelial dysfunction caused by angiogenic factors derived from the placenta in PE patients. On the contrary, hypoadiponectinemia might exaggerate rather mild endothelial dysfunction caused by a relatively lower sEng level, and lead to PE. We also demonstrated that sEng level is negatively correlated with the leptin to adiponectin ratio in PE patients, but not healthy controls. Insulin resistance and the resultant hyperinsulinemia are characteristic of normal pregnancy, and there appears to be an exaggeration of insulin resistance and associated metabolic changes in women whose pregnancy is complicated by hypertension (19); therefore, pregnant women with high insulin resistance might suffer from PE in correlation with a relatively low sEng concentration. Thadhani et al. (38) have demonstrated that women with low levels of PlGF in the first trimester are at increased risk for developing subsequent PE, and this risk is exaggerated in women who also have low levels of SHBG, which is a surrogate insulin resistance marker. These data indicate that women with preexisting alterations in insulin sensitivity have an exaggerated response to alterations in circulating angiogenic factors, including sEng, and both alterations may interact to magnify the risk for PE.
Together, our results suggest that, along with other angiogenic factors such as sFlt-1 and PlGF, sEng might play a role in the pathophysiology of PE, especially in elevating blood pressure, and that the association with hypoadiponectinemia and a high leptin to adiponectin ratio in pregnancy might be risk factors for PE. This was only a small-scale cross-sectional study of Japanese patients with PE; therefore, we will examine a larger sample size with a gradation in level of PE severity, which will improve the precision of our findings.
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Footnotes |
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Disclosure Statement: The authors have nothing to disclose.
First Published Online April 10, 2007
Abbreviations: BMI, Body mass index; Eng, endoglin; PE, preeclampsia; PlGF, placental growth factor; sEng, soluble endoglin; sFlk-1, soluble fetal liver kinase 1; sFlt-1, soluble fms-like tyrosine kinase 1; sVEGFR, soluble VEGFR; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor.
Received October 26, 2006.
Accepted April 3, 2007.
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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