Piia Vuorela

Release and Complex Formation of Soluble VEGFR-1 from Endothelial Cells and Biological Fluids

One of the key molecules promoting angiogenesis is the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF or VEGF-A), which acts through two high-affinity receptor tyrosine kinases (VEGFR), VEGFR-1 (or Flt-1) and VEGFR-2 (or KDR/Flk-1). It was shown before that a soluble variant of VEGFR-1 (sVEGFR-1) can be generated by differential splicing of the flt-1 mRNA. This soluble receptor is an antagonist to VEGF action, reducing the level of free, active VEGF-A, and therefore, plays a pivotal role in the generation of vascular diseases like pre-eclampsia or intra-uterine growth retardation. Here we show that sVEGFR-1 is produced by cultured human microvascular and macrovascular endothelial cells and a human melanoma cell line. The soluble receptor is mainly complexed with ligands; only 5–10% remains detectable as free, uncomplexed receptor protein. Furthermore, we show the time course of total and free sVEGFR-1 release together with its putative ligands, VEGF-A and placenta growth factor (PlGF), from macrovascular endothelial cells. The release of sVEGFR-1 was quantitatively measured in two different ELISA types. The release of sVEGFR-1 was strongly enhanced by phorbol-ester (PMA); the cells produced up to 22 ng/ml of sVEGFR-1 after 48 hours. The expression of VEGF-A and PlGF was moderately influenced by PMA. We also show a hypoxia-induced increase of sVEGFR-1 expression in cells cultured from placenta, a tissue that has a high flt-1 gene expression. Moreover, we demonstrate that sVEGFR-1 in amniotic fluids acts as a sink for exogenous VEGF165 and PlGF-2. Here, for the first time, to what extent recombinant ligands have to be added to compensate for the sink function of amniotic fluids was analyzed. In conclusion, human endothelial cells produce high levels of sVEGFR-1, which influences the availability of VEGF-A or related ligands. Therefore, sVEGFR-1 may reduce the ligand binding to transmembrane receptors and interfere with their signal transduction.

Amniotic fluid--soluble vascular endothelial growth factor receptor-1 in preeclampsia.

Objective To measure the levels of the soluble receptor for the potent angiogenic agent vascular endothelial growth factor (VEGF) in amniotic fluid (AF) in healthy and complicated pregnancies, and compare them with levels of erythropoietin, another factor upregulated by hypoxia. Methods We assessed amniotic fluid from the second (n = 35, gestational weeks 14–19) and third (n = 29) trimesters of healthy women, and from the third trimesters of preeclamptic (n = 22) and diabetic women with (n = 11) or without preeclampsia (n = 34) and from women with fetal growth restriction (FGR) (n = 14) for soluble VEGF receptor-1 (VEGFR-1) by enzyme-linked immunosorbent assay. Results In early normal pregnancy, AF-soluble VEGFR-1 levels were higher (median 22 ng/mL, range 2.3–29.5 ng/mL) than in the third trimester (median 13 ng/mL, range 0.5–32 ng/mL; P < .05). In preeclamptic women during the third trimester, levels were higher (median 20 ng/mL, range 10.5–37 ng/mL; P < .05) than healthy controls. The lowest third-trimester levels were in diabetic women (median 11 ng/mL, range 0.5–27 ng/mL). In women with preeclampsia and diabetes, AF-soluble VEGFR-1 levels remained lower (median 13, range 6–32 ng/mL; P < .05) than in women with preeclampsia alone. Amniotic fluid levels of soluble VEGFR-1 in women with FGR (median 19.5 ng/mL, range 5–40 ng/mL) did not statistically differ from those of controls. The AF levels of soluble VEGFR-1 did not correlate with those of erythropoietin. Soluble VEGFR-1 was clearly detectable (median 14 ng/mL, range 9–22 ng/mL) in culture media from placental biopsies (n = 20). Conclusion Preeclampsia is associated with increased levels of soluble VEGFR-1, which are independent of erythropoietin, another hypoxia-inducible factor.

First trimester hyperglycosylated human chorionic gonadotrophin in serum - a marker of early-onset preeclampsia.

INTRODUCTION Recent studies indicate that treatment with low-dose aspirin may reduce the risk of preeclampsia. Thus, early prediction of preeclampsia is needed. Low serum concentrations of hyperglycosylated human chorionic gonadotrophin (hCG-h) are associated with early pregnancy loss. We therefore studied whether it may serve as an early marker of preeclampsia. METHODS A nested case-control study included 158 women with subsequent preeclampsia, 41 with gestational hypertension, 81 normotensive women giving birth to small-for-gestational-age (SGA) infants and 427 controls participating in first trimester screening for Downs syndrome between 8 and 13 weeks of gestation. Gestational-age-adjusted multiples of medians (MoMs) were calculated for serum concentrations of hCG-h, the free beta subunit of hCG (hCGβ) and pregnancy-associated plasma placental protein A (PAPP-A) and the proportion of hCG-h to hCG (%hCG-h). Clinical risk factors including mean arterial pressure (MAP) and parity were also included in the risk calculation. RESULTS In women with subsequent preeclampsia %hCG-h was lower than in controls (median MoM 0.92, P < 0.001), especially in 29 cases with early-onset preeclampsia (0.86, P < 0.001), in which PAPP-A also was reduced (0.95, P = 0.001). At 90% specificity for prediction of early-onset preeclampsia, sensitivity was 56% (95% confidence interval, 52-61%) for %hCG-h, 33% (28-37%) for PAPP-A, and 69% (51-83%) for the combination of these with first trimester MAP and parity. The area under the receiver-operating characteristic (ROC) curve for the combination of all these was 0.863 (0.791-0.935). CONCLUSIONS hCG-h is a promising first trimester marker for early-onset preeclampsia. Addition of PAPP-A and maternal risk factors may improve the results.

Maternal Serum Angiopoietin-1 and -2 and Tie-2 in Early Pregnancy Ending in Preeclampsia or Intrauterine Growth Retardation

CONTEXT The antiangiogenic growth factor angiopoietin-2 (Ang-2) antagonizes, whereas angiopoietin-1 (Ang-1) activates the endothelial cell-specific tyrosine kinase receptor-2 (Tie-2). In preeclampsia, circulating concentrations of Ang-1 are increased and those of Ang-2 and Tie-2 are decreased. OBJECTIVE We wanted to study whether maternal serum concentrations of Ang-1, Ang-2, and Tie-2 are altered at gestational wk 12-15 or 16-20 in women with subsequent preeclampsia or intrauterine growth retardation (IUGR). DESIGN This was a case-control study. SETTING The study was conducted in Helsinki University Central Hospital, a tertiary referral center. PATIENTS This study comprised 124 pregnant women, of whom 49 developed preeclampsia and 16 gave birth to infants with IUGR, and 59 healthy women served as controls. MAIN OUTCOME MEASURES Serum concentrations of Ang-1, Ang-2, and Tie-2 were assessed by ELISA. Data were combined with our earlier data on soluble VEGF receptor (sVEGFR)-1. RESULTS At gestational wk 12-15, the median concentrations of Ang-1, Ang-2, or Tie-2 were all similar between the study groups. At 16-20 wk, Ang-2 concentrations were higher in women with subsequent preeclampsia [25.0 ng/ml, 19.3-39.5 ng/ml; median, interquartile range (IQR)] than in the controls (17.7 ng/ml, 10.8-27.4 ng/ml, P = 0.006). The odds ratio of high Ang-2 concentrations for subsequent preeclampsia was 4.2 (95% confidence interval 1.4-12.6; P = 0.011) and high Ang-2 combined with high sVEGFR-1, 6.4 (95% confidence interval 2.2-18.7; P = 0.001). CONCLUSION Maternal serum Ang-2 concentrations are increased prior to preeclampsia. High concentrations of both Ang-2 and sVEGFR-1 indicate subsequent disease.

Endothelial Tie receptor antigen in maternal and cord blood of healthy and preeclamptic subjects

Objective To measure the vascular endothelial receptor tyrosine kinase Tie, essential in the process of angiogenesis, in blood from healthy and preeclamptic pregnant women, in umbilical cord blood from both, and in blood from nonpregnant women. Methods A total of 143 women participated in four arms of the study. Blood samples were collected from 54 healthy nonlaboring pregnant women (gestational weeks 14–41). Samples were collected immediately prepartum and postpartum from another 40 healthy women (15 delivered vaginally and 25 by cesarean) and 15 preeclamptic women (all delivered by cesarean). Arterial and venous cord samples were collected, when possible, from infants born by cesarean. Single blood samples were drawn from 34 nonpregnant controls. Of these, weekly samples from 11 were drawn during one menstrual cycle. A time-resolved fluoroimmunoassay was developed for the detection of the soluble extra-cellular domain of Tie. Results Maternal serum Tie levels decreased with advancing gestational age after 26 weeks (r = .6, P < .001). They were significantly higher in healthy women at term (median 233 ng/mL, range 152–414 ng/mL) compared with nonpregnant controls (median 173 ng/mL, range 107–333 ng/mL, P < .001) or with preeclamptic women at term (median 152 ng/mL, range 90–372 ng/mL, P < .05). This difference between healthy and preeclamptic women persisted on the first postpartum day (median 221 ng/mL, range 128–343 and median 152 ng/mL, range 90–372 ng/mL, respectively, P < .05). The highest levels of serum Tie receptor were observed in umbilical arterial and venous blood (median 240 ng/mL, range 174–474 ng/mL and median 340 ng/mL, range 245–690 ng/mL, respectively). In nonpregnant women, serum Tie levels did not vary with menstrual cycle. Conclusion The high levels of the extracellular domain of Tie in healthy term maternal and cord blood may indicate a role for Tie in the vascular development of human fetuses and placentas.

Maternal serum endostatin at gestational weeks 16-20 is elevated in subsequent pre-eclampsia but not in intrauterine growth retardation.

Objective. Endostatin, an important anti‐angiogenic factor produced by endothelial cells, is elevated in established pre‐eclampsia. We measured maternal serum endostatin concentrations in early pregnancy associated with later pre‐eclampsia and intrauterine growth retardation (IUGR). Design. Retrospective case–control study. Setting. University Central Hospital. Sample. Serum samples were collected at 12–15 and 16–20 gestational weeks from a total of 124 pregnant women of whom 49 developed pre‐eclampsia, 16 gave birth to infants with IUGR without pre‐eclampsia, and 59 remained normotensive giving birth to healthy, normal‐weight infants. Methods. Enzyme‐linked immunosorbent assay. Main outcome measures. Endostatin concentrations in serum. Results. At 12–15 gestational weeks, there was no difference in median endostatin concentrations between the groups. At 16–20 gestational weeks, the median endostatin concentration was higher in the women with subsequent pre‐eclampsia (p = 0.026), especially preceding a later severe form of the disease (p = 0.041), than in the controls. The results were further confirmed by receiver operating characteristic (ROC) analysis showing an area under the curve (AUC) of 0.64 (95% confidence interval: 0.50–0.81) for endostatin to identify subsequent pre‐eclampsia, and 0.71 (0.53–0.89) in cases of severe pre‐eclampsia. Optimal cut‐off values were determined and used for calculations of sensitivity and specificity, which were 80 and 52% (cut‐off value = 58.0 μg/L) in pre‐eclampsia, and 80 and 65% (cut‐off value = 65.5 μg/L) in the severe form of the disease. Conclusions. The concentrations of endostatin in maternal serum at 16–20 weeks’ of gestation are associated with an increased risk of pre‐eclampsia but not IUGR.

Cost‐effectiveness of hysterectomy for benign gynecological conditions: a systematic review

The objective of this study was to assess the cost‐effectiveness of hysterectomy performed for benign indications. Hysterectomy remains the most common major gynecological operation in the Western world. Rates of hysterectomy have not declined as expected with the introduction of new treatment options. Furthermore, use of laparoscopic techniques varies widely within the Nordic countries. We designed a systematic review in a University Central Hospital. The sample included all published studies regarding the cost‐effectiveness of hysterectomy performed for benign indications (n = 1666). Medline, Cochrane Library, PsycINFO, CINAHL, and Nursing databases were searched. Inclusion criteria were the availability of pre‐ and post‐intervention health‐related quality of life measures (HRQoL) and data on costs. HRQoL, costs, and cost‐effectiveness of treatment were the main outcome measures. Studies (n = 24) focused on treatment of symptomatic fibroids (n = 8), treatment of heavy menstrual bleeding (n = 10), various surgical techniques (n = 5) and the effect of various indications for hysterectomy (n = 2). Follow‐up periods varied from 4 months to over 10 years. SF/RAND‐36 or EQ‐5D measures and societal cost perspective were most commonly used. Only 11 studies used individual patient data. HRQoL following hysterectomy was generally good but costs were high. The cost‐effectiveness depended on indication, age, and duration of follow‐up. The cost‐effectiveness of hysterectomy has been surprisingly poorly studied. Conclusions are difficult to draw due to different study designs, indications, follow‐up times, and HRQoL instruments used. Rates of hysterectomy have declined less than expected with the introduction of new treatment modalities. Costs of surgery are high. Laparoscopic hysterectomy seems to be the least cost‐effective, although further data from original patient cohorts with long‐term follow‐up are needed.

Serum hyperglycosylated human chorionic gonadotrophin at 14-17 weeks of gestation does not predict preeclampsia.

Low first‐trimester serum concentrations of hyperglycosylated human chorionic gonadotrophin (hCG‐h) predict later preeclampsia. We studied whether serum hCG‐h at 14–17 weeks of pregnancy also predicts preeclampsia alone or combined with placental growth factor (PlGF) and soluble vascular endothelial growth factor 1 (sVEGFR‐1).

Expression of Vascular Endothelial Growth Factor in Peripheral Blood Cells of Preeclamptic Women

Objective: Preeclampsia is associated with platelet and endothelial dysfunction. Vascular endothelial growth factor (VEGF) is found in peripheral blood leukocytes and released from platelets on activation. We analyzed the content of (VEGF) in peripheral blood cells of preeclamptic women. Methods: The VEGF content of platelets, mononuclear white blood cells, and granulocytes of peripheral blood were analyzed from 12 women with preeclampsia, 19 healthy pregnant women, and 20 nonpregnant women. Protein released from lysed cells was analyzed by enzyme‐linked immunoassay (ELISA). Results: Platelet VEGF content of preeclamptic women (0.081 ng/109 cells, 0.016 to 2.7 ng/109 cells; median, range) was similar to that of healthy pregnant women (0.31 ng/109 cells, 0.013 to 0.92 ng/109 cells) and that of nonpregnant (0.073 ng/109 cells, 0.012 to 0.76 ng/109 cells) women. Likewise, the VEGF content of granulocytes was similar in preeclamptic (18.5 ng/109 cells, 1.2 to 193 ng/109 cells), healthy pregnant (25.3 ng/109 cells, 0.8 to 441 ng/109 cells), and nonpregnant (29 ng/109 cells, 0.25 to 200 ng/109 cells) women. In mononuclear cells, the VEGF content of healthy pregnant women was higher (4.4 ng/109 cells, 0.13 to 13.7 ng/109 cells) than in nonpregnant women (1.7 ng/109 cells, 0.15 to 11.4 ng/109 cells, P < 0.05). Also, the mononuclear cell VEGF content of preeclamptic women (8.2 ng/109 cells, 0.04 to 23 ng/109 cells) tended to be higher than in nonpregnant women (P ≈ 0.07). Conclusion: Uncomplicated pregnancy is associated with an elevated VEGF content of mononuclear cells. Preeclampsia does not seem to affect the VEGF content of maternal peripheral blood mononuclear cells, granulocytes, or platelets.

Effect of an in vitro fertilization program on serum CA 125, tumor-associated trypsin inhibitor, free β-subunit of human chorionic gonadotropin, and common α-subunit of glycoprotein hormones ☆

OBJECTIVE To investigate the impact of an IVF program on serum levels of tumor markers CA 125, tumor-associated trypsin inhibitor, free hCG beta-subunit, and free glycoprotein hormone alpha-subunit. DESIGN A prospective controlled clinical study. SETTING Outpatient university infertility clinic. PATIENT(S) Seventy-one infertile patients (with tubal occlusion, pelvic endometriosis, or unexplained infertility) undergoing IVF and nine control women with regular menstrual cycles. INTERVENTION(S) Serial blood sampling before, during, and after IVF, or during one ovulatory menstrual cycle in the controls. MAIN OUTCOME MEASURE(S) Serum levels of CA 125, tumor-associated trypsin inhibitor, hCG-beta, and glycoprotein hormone-alpha. RESULT(S) Before IVF, all tumor markers were within the normal range except for CA 125, which was elevated in patients with endometriosis. IVF led to significant increases in CA 125 and glycoprotein hormone-alpha that differed from the changes seen during normal menstrual cycles. The luteal phase increase in CA 125 correlated with levels of E(2) and P and the number of follicles. Two months after IVF, levels of CA 125 were 12% higher than levels before treatment. Tumor-associated trypsin inhibitor and hCG-beta revealed no cyclicity. CONCLUSION(S) An IVF regimen increased the release of CA 125 and glycoprotein hormone-alpha. The CA 125 elevation after IVF implies a persistent effect of ovarian hyperstimulation on CA 125 release.