Stefan Verlohren

Predictive Value of the sFlt-1:PlGF Ratio in Women with Suspected Preeclampsia

BACKGROUND The ratio of soluble fms-like tyrosine kinase 1 (sFlt-1) to placental growth factor (PlGF) is elevated in pregnant women before the clinical onset of preeclampsia, but its predictive value in women with suspected preeclampsia is unclear. METHODS We performed a prospective, multicenter, observational study to derive and validate a ratio of serum sFlt-1 to PlGF that would be predictive of the absence or presence of preeclampsia in the short term in women with singleton pregnancies in whom preeclampsia was suspected (24 weeks 0 days to 36 weeks 6 days of gestation). Primary objectives were to assess whether low sFlt-1:PlGF ratios (at or below a derived cutoff) predict the absence of preeclampsia within 1 week after the first visit and whether high ratios (above the cutoff) predict the presence of preeclampsia within 4 weeks. RESULTS In the development cohort (500 women), we identified an sFlt-1:PlGF ratio cutoff of 38 as having important predictive value. In a subsequent validation study among an additional 550 women, an sFlt-1:PlGF ratio of 38 or lower had a negative predictive value (i.e., no preeclampsia in the subsequent week) of 99.3% (95% confidence interval [CI], 97.9 to 99.9), with 80.0% sensitivity (95% CI, 51.9 to 95.7) and 78.3% specificity (95% CI, 74.6 to 81.7). The positive predictive value of an sFlt-1:PlGF ratio above 38 for a diagnosis of preeclampsia within 4 weeks was 36.7% (95% CI, 28.4 to 45.7), with 66.2% sensitivity (95% CI, 54.0 to 77.0) and 83.1% specificity (95% CI, 79.4 to 86.3). CONCLUSIONS An sFlt-1:PlGF ratio of 38 or lower can be used to predict the short-term absence of preeclampsia in women in whom the syndrome is suspected clinically. (Funded by Roche Diagnostics.).

Angiogenic Factors and the Risk of Adverse Outcomes in Women with Suspected Preeclampsia

Background— An imbalance in circulating angiogenic factors plays a central role in the pathogenesis of preeclampsia. Methods and Results— We prospectively studied 616 women who were evaluated for suspected preeclampsia. We measured plasma levels of antiangiogenic soluble fms-like tyrosine kinase 1 (sFlt1) and proangiogenic placental growth factor (PlGF) at presentation and examined for an association between the sFlt1/PlGF ratio and subsequent adverse maternal and perinatal outcomes within 2 weeks. The median sFlt1/PlGF ratio at presentation was elevated in participants who experienced any adverse outcome compared with those who did not (47.0 [25th–75th percentile, 15.5–112.2] versus 10.8 [25th–75th percentile, 4.1–28.6]; P<0.0001). Among those presenting at <34 weeks (n=167), the results were more striking (226.6 [25th–75th percentile, 50.4–547.3] versus 4.5 [25th–75th percentile, 2.0–13.5]; P<0.0001), and the risk was markedly elevated when the highest sFlt1/PlGF ratio tertile was compared with the lowest (odds ratio, 47.8; 95% confidence interval, 14.6–156.6). Among participants presenting at <34 weeks, the addition of sFlt1/PlGF ratio to hypertension and proteinuria significantly improved the prediction for subsequent adverse outcomes (area under the curve, 0.93 for hypertension, proteinuria, and sFlt1/PlGF versus 0.84 for hypertension and proteinuria alone; P=0.001). Delivery occurred within 2 weeks of presentation in 86.0% of women with an sFlt1/PlGF ratio ≥85 compared with 15.8% of women with an sFlt1/PlGF ratio <85 (hazard ratio, 15.2; 95% confidence interval, 8.0–28.7). Conclusions— In women with suspected preeclampsia presenting at <34 weeks, circulating sFlt1/PlGF ratio predicts adverse outcomes occurring within 2 weeks. The accuracy of this test is substantially better than that of current approaches and may be useful in risk stratification and management. Additional studies are warranted to validate these findings.

An automated method for the determination of the sFlt-1/PIGF ratio in the assessment of preeclampsia

OBJECTIVE The angiogenic and antiangiogenic factors soluble fms-like tyrosine kinase (sFlt)-1 and placental growth factor (PIGF) have been implicated in the mechanisms of disease responsible for preeclampsia (PE). Moreover, it has been proposed that the concentrations of these markers in maternal serum/plasma may have predictive value. This study evaluates a newly developed Elecsys (Roche, Penzberg, Germany) assay for sFlt-1 and PIGF and tests the value of the sFlt-1/PIGF ratio in the assessment of PE. STUDY DESIGN This multicenter case-control study included 351 patients: 71 patients with PE and 280 gestational age-matched control subjects from 5 European study centers. A total of 595 serum samples were measured for sFlt-1 and PIGF using an automated platform. RESULTS Maternal serum concentrations of sFlt-1 and PIGF significantly separated healthy women and women with PE. The sFlt-1/PIGF ratio had an area under the receiver operating characteristic curve of 0.95. The best performance was obtained in the identification of early-onset PE (area under the receiver operating characteristic curve of 0.97). CONCLUSION Measurement of sFlt-1 and PIGF and calculation of sFlt-1/PIGF ratio can be performed quickly and in a platform available in clinical laboratories. This is a substantial step forward in bringing the determination of these analytes to clinical practice in obstetrics. We propose that sFlt-1, PIGF, and sFlt-1/PIGF ratio may be of value in the prediction of PE and in the differential diagnosis of patients with atypical presentations of PE, and perhaps in the differential diagnosis of women with chronic hypertension suspected to develop superimposed PE.

Visceral Periadventitial Adipose Tissue Regulates Arterial Tone of Mesenteric Arteries

Periadventitial adipose tissue produces vasoactive substances that influence vascular contraction. Earlier studies addressed this issue in aorta, a vessel that does not contribute to peripheral vascular resistance. We tested the hypothesis that periadventitial adipose tissue modulates contraction of smaller arteries more relevant to blood pressure regulation. We studied mesenteric artery rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared with vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+-containing solutions (60 mmol/L) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+-containing solutions (60 mmol/L), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anticontractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ (Kv) channels with 4-aminopyridine (2 mmol/L) or 3,4-diaminopyridine (1 mmol/L). Blocking other K+ channels with glibenclamide (3 &mgr;mol/L), apamin (1 &mgr;mol/L), iberiotoxin (100 nmol/L), tetraethylammonium ions (1 mmol/L), tetrapentylammonium ions (10 &mgr;mol/L), or Ba2+ (3 &mgr;mol/L) had no effect. Longitudinal removal of half the perivascular tissue reduced the anticontractile effect of fat by almost 50%, whereas removal of the endothelium had no effect. We suggest that visceral periadventitial adipose tissue controls mesenteric arterial tone by inducing vasorelaxation via Kv channel activation in vascular smooth muscle cells.

The sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders and its prognostic potential in preeclamptic patients

OBJECTIVE The soluble fms-like tyrosine kinase (sFlt-1)/placental growth factor (PlGF) ratio is a reliable tool in the assessment of preeclampsia. We tested the hypothesis that the sFlt-1/PlGF ratio is able to identify women at risk for imminent delivery. We characterized the sFlt-1/PlGF ratio in different types of hypertensive pregnancy disorders. STUDY DESIGN We investigated 388 singleton pregnancies with normal pregnancy outcome, 164 with PE, 36 with gestational hypertension, and 42 with chronic hypertension. sFlt-1 and PlGF were measured in serum samples. RESULTS Patients with preeclampsia had a significantly increased sFlt-1/PlGF ratio as compared with controls and with patients with chronic and gestational hypertension in <34 weeks and ≥34 weeks (P < .001). Time to delivery was significantly reduced in women with preeclampsia in the highest quartile of the sFlt-1/PlGF ratio (P < .001). CONCLUSION The sFlt-1/PlGF ratio allows the identification of women at risk for imminent delivery and is a reliable tool to discriminate between different types of pregnancy-related hypertensive disorders.

New Gestational Phase–Specific Cutoff Values for the Use of the Soluble fms-Like Tyrosine Kinase-1/Placental Growth Factor Ratio as a Diagnostic Test for Preeclampsia

To establish gestational phase adapted cutoffs for the use of the soluble fms-like tyrosine kinase-1 (sFlt-1)/placental growth factor (PlGF) ratio as a diagnostic tool for preeclampsia in the clinical setting, a multicenter case–control study including a total of 1149 patients was performed. We report normal values of sFlt-1, PlGF, and the sFlt-1/PlGF ratio based on the analysis of a total of 877 patients with uneventful pregnancy outcome. A total of 234 patients with preeclampsia and a matched cohort consisting of 468 patients with normal pregnancy outcome were compared, and sFlt-1 and PlGF were measured on an automated platform. Separate cutoffs for the sFlt-1/PlGF ratio were determined for the early (20+0–33+6 weeks) and the late gestational phase (34+0 weeks–delivery). For each of the 2 gestational phases, 2 independent cutoffs framing an equivocal zone were determined: the first cutoff with focus on high sensitivity, and the second focusing on high specificity. Between 20+0 and 33+6 weeks, the cutoffs at ⩽33 and ≥85 resulted in a sensitivity/specificity of 95%/94% and 88%/99.5%, respectively. An sFlt-1/PlGF ratio of ⩽33 had the lowest likelihood of a negative test (0.05; 95% confidence interval, 0.02–0.13), whereas values ≥85 had the highest likelihood of a positive test (176; 95% confidence interval, 24.88–1245). After 34+0 weeks, the cutoffs at ⩽33 and ≥110 yielded a sensitivity/specificity of 89.6%/73.1% and 58.2%/95.5%, respectively. The approach to use multiple cutoffs for the early and late gestational phase enhances the diagnostic accuracy of the sFlt-1/PlGF ratio as a diagnostic tool for preeclampsia.

Hypoxia induces dilated cardiomyopathy in the chick embryo: mechanism; intervention; and long-term consequences.

Background Intrauterine growth restriction is associated with an increased future risk for developing cardiovascular diseases. Hypoxia in utero is a common clinical cause of fetal growth restriction. We have previously shown that chronic hypoxia alters cardiovascular development in chick embryos. The aim of this study was to further characterize cardiac disease in hypoxic chick embryos. Methods Chick embryos were exposed to hypoxia and cardiac structure was examined by histological methods one day prior to hatching (E20) and at adulthood. Cardiac function was assessed in vivo by echocardiography and ex vivo by contractility measurements in isolated heart muscle bundles and isolated cardiomyocytes. Chick embryos were exposed to vascular endothelial growth factor (VEGF) and its scavenger soluble VEGF receptor-1 (sFlt-1) to investigate the potential role of this hypoxia-regulated cytokine. Principal Findings Growth restricted hypoxic chick embryos showed cardiomyopathy as evidenced by left ventricular (LV) dilatation, reduced ventricular wall mass and increased apoptosis. Hypoxic hearts displayed pump dysfunction with decreased LV ejection fractions, accompanied by signs of diastolic dysfunction. Cardiomyopathy caused by hypoxia persisted into adulthood. Hypoxic embryonic hearts showed increases in VEGF expression. Systemic administration of rhVEGF165 to normoxic chick embryos resulted in LV dilatation and a dose-dependent loss of LV wall mass. Lowering VEGF levels in hypoxic embryonic chick hearts by systemic administration of sFlt-1 yielded an almost complete normalization of the phenotype. Conclusions/Significance Our data show that hypoxia causes a decreased cardiac performance and cardiomyopathy in chick embryos, involving a significant VEGF-mediated component. This cardiomyopathy persists into adulthood.

Angiogenic growth factors in the diagnosis and prediction of pre-eclampsia

The pathogenesis of pre-eclampsia is still not completely known; however, in the recent decade, there have been tremendous research efforts leading to impressive results highlighting the role of a disturbed angiogenic balance as one of the key features of the disease. Numerous studies have shown the key role of the placenta in the pathogenesis of pre-eclampsia. A shift in the sFlt-1 (soluble Fms-like tyrosine kinase-1)/PlGF (placental growth factor) ratio is associated with the disease. Although pre-eclampsia seems to be a clearly defined disease, clinical presentation, and particularly the dynamics of the clinical course, can vary enormously. The only available tools to diagnose pre-eclampsia are blood pressure measurement and urine protein sampling. However, these tools have a low sensitivity and specificity regarding the prediction of the course of the disease or maternal and perinatal outcomes. The only cure for the disease is delivery, although a timely diagnosis helps in decreasing maternal and fetal morbidity and mortality. The sFlt1/PlGF ratio is able to give additional valuable information on the status and progression of the disease and is apt to be implemented in the diagnostic algorithm of pre-eclampsia. In the present review, we aim to provide an overview of the vast literature on angiogenesis and anti-angiogenesis factors in pre-eclampsia that have been published over the last decade. We introduce work from basic research groups who have focused on the pathophysiological basis of the disease. Furthermore, we review studies with a clinical focus in which the sFlt-1/PlGF ratio has been analysed along with other candidates for routine clinical assessment of pre-eclampsia.

Prevalence of Agonistic Autoantibodies Against the Angiotensin II Type 1 Receptor and Soluble fms-Like Tyrosine Kinase 1 in a Gestational Age–Matched Case Study

We showed earlier that activating autoantibodies against the angiotensin II type 1 (AT1) receptor (AT1-AA) circulate in preeclamptic women. They may be involved in the pathogenesis of preeclampsia. Protein alignment suggests that the binding site for AT1-AAs is highly homologous to the capsid protein VP2 of parvovirus B19. We performed a prospective, nested, case-control study of 30 gestational age–matched women with preeclampsia and 30 normotensive pregnant women. We measured AT1-AA, soluble fms-like tyrosine kinase 1 (sFlt-1), and serum immunoglobulin G against parvovirus B19 proteins. AT1-AAs were present in 70% of preeclamptic patients and absent in 80% of controls. Prediction by AT1-AA was improved in late-onset preeclampsia. The discrimination for sFlt-1 was 96%. We did not find an interaction between sFlt-1 and AT1-AA. A human monoclonal immunoglobulin G antibody against parvovirus B19 VP2-protein showed a positive reaction in the AT1-AA bioassay, which could be blocked by an AT1 receptor blocker, as well as by the epitope amino acid sequence. Immunoglobulin G against parvovirus B19 proteins was similarly distributed between preeclamptic patients and controls and had no significant importance. We detected significantly more AT1-AA in women with an immune response corresponding with parvovirus B19 infection corresponding with a distant viral infection associated with virus elimination. We concluded that AT1-AAs were common in patients with preeclampsia in a prospective case-control study, although sFlt-1 was a superior biomarker. AT1-AA may represent a better marker for late disease, whereas sFlt1 is a better marker for early onset disease.

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background Agonistic autoantibodies directed at the α1-adrenergic receptor (α1-AAB) have been described in patients with hypertension. We implied earlier that α1-AAB might have a mechanistic role and could represent a therapeutic target. Methodology/Principal Findings To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had α1-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α1-adrenergic receptor antibodies (α1-AB). Patient α1-AAB and rabbit α1-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human α1A-adrenergic receptor were incubated with patient α1-AAB and rabbit α1-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient α1-AAB and rabbit α1-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca2+ in cardiomyocytes and induce mesentery artery segment contraction. Conclusions/Significance Patient α1-AAB and rabbit α1-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α1-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.