This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stepan, H. Articles by Walther, T. Search for Related Content PubMed PubMed Citation Articles by Stepan, H. Articles by Walther, T. Related Collections Autoimmunity Female Endocrinology

Relation between Circulating Angiotensin II Type 1 Receptor Agonistic Autoantibodies and Soluble fms-Like Tyrosine Kinase 1 in the Pathogenesis of Preeclampsia

Department of Obstetrics and Gynecology (H.S., R.F.), University of Leipzig, 04109 Leipzig, Germany; Department of Physics (N.W.), University of Potsdam, 14469 Potsdam, Germany; Max-Delbrück Center for Molecular Medicine (G.W.), 13092 Berlin, Germany; Department of Cardiology and Pneumonology (H.-P.S., T.W.), Charité-Campus Benjamin Franklin, 12200 Berlin, Germany; and Department of Pharmacology (T.W.), Erasmus Medical Center, 3000 DR Rotterdam, The Netherlands

Address all correspondence and requests for reprints to: Thomas Walther, Department of Cardiology and Pneumonology, Charité-Universitätsmedizin, Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany. E-mail: thomas.walther{at}charite.de.

	Abstract

Top Abstract Introduction Subjects and Methods Results and Discussion References

 
Context: Placental and circulatory soluble fms-like tyrosine kinase 1 (sFlt1) has proven to be elevated in pregnant women with preeclampsia, a disease characterized by hypertension, proteinuria, and endothelial dysfunction. Recent studies also demonstrated an autoantibody against the angiotensin II type 1 (AT1) receptor (AT1-AA) in that disease.

Objective: Both factors are discussed as key players in the etiology of preeclampsia. However, it has not yet been clarified whether these two circulating factors correlate and whether synergy determines the severity of pathology.

Design: AT1-AA was retrospectively determined by a bioassay and sFlt1 by an ELISA.

Patients: Serum from second-trimester pregnancies with normal or abnormal uterine perfusion and in women at term with or without pregnancy pathology was analyzed.

Results: Most of the preeclamptic patients were characterized by high sFlt1 levels and the presence of AT1-AA, although the agonistic effects of the antibody did not correlate with the sFlt1 concentrations (P = 0.85). Although AT1-AA was also detected in second-trimester pregnancies evidencing abnormal uterine perfusion without later pathology, sFlt1 was not significantly elevated in these pregnancies, compared with those with normal uterine perfusion. However, whereas women with abnormal perfusion and later pregnancy pathology did not differ in AT1-AA, compared with those with normal outcome, sFlt1 was significantly increased. Again, the two factors did not correlate (P = 0.15).

Conclusions: We conclude that AT1-AA bioactivity and sFlt1 concentrations do not correlate, are not mutually dependent, and are thus probably involved in distinct pathogenetic mechanisms. Both factors in combination may not be causative for the early impaired trophoblast invasion and pathological uterine perfusion.

	Introduction

Top Abstract Introduction Subjects and Methods Results and Discussion References

 
PREECLAMPSIA, A SERIOUS pregnancy-specific disorder characterized by proteinuria and hypertension after the 20th week of gestation, is still a leading cause of maternal and neonatal morbidity and mortality and affects about 3–5% of pregnancies worldwide (1). Although the origin of the disease remains unclear, there is growing evidence that one or more placental factors enter the maternal circulation and cause endothelial dysfunction, resulting in hypertension and proteinuria (2). Recently two particular factors have been intensively discussed as markers and possible inductors of the disease.

The autoimmune antibody against the angiotensin (Ang) II receptor type 1 (AT1) was identified by Wallukat et al. (3) as being detectable in preeclamptic patients but not in healthy pregnancies or those with essential hypertension. A possible causality of autoantibody against the AT1 receptor (AT1-AA) in preeclampsia has been postulated becauseAT1 receptor stimulation by this AT1-agonistic AT1-AA in vitro leads to reduced trophoblast invasiveness, a typical feature of preeclampsia (4). The antibody induces Ca²⁺ release in vascular smooth muscle cells and could therefore mediate the vascular alterations in preeclampsia (5). Furthermore, Dechend et al. (6) demonstrated increased activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. This could contribute to oxidative stress and an inflammatory response, which are pathophysiological factors in preeclampsia.

The second candidate, soluble fms-like tyrosine kinase 1 (sFlt1) is a splice variant of the vascular endothelial growth factor receptor Flt1 and a potent inhibitor of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Placental and circulatory sFlt1 was found to be elevated in pregnant women with preeclampsia (7). A clinical study demonstrated that increased sFlt1 plasma concentrations are detectable about 5 wk before the onset of preeclampsia (8).

Interestingly, we found that elevation of both factors as early as the second trimester was not confined to pregnancies with later preeclampsia but also occurred in women with subsequent intrauterine growth restriction (IUGR) or even in isolated cases of abnormal uterine perfusion with a normal course of pregnancy (9, 10). Notably, an abnormal uterine perfusion characterizes pregnancies at risk for these pregnancy complications and precedes their clinical manifestation. However, only approximately one third of the pregnant women with abnormal uterine perfusion develop a complication, whereas the others have a normal course of pregnancy despite the high uteroplacental resistance (11).

A biological link between sFlt1 and AT1-AA may be derived from the observation that Ang II induces an increased sFlt1 production in human proximal tubule cells (12). This finding provides an interesting link to the renin angiotensin system, and thus to the AT1-AA that mimics Ang II effects. Although decisive evidence that one of the two factors is causative for preeclampsia has not yet been provided, it may be hypothesized that concertive regulation is needed to mediate its initial pathology. Thus, the aim of this study was to investigate the possible correlation between AT1-AA and sFlt1 in maternal circulation at the time of manifestation of the pregnancy-induced pathology but especially in second-trimester pregnancies with pathological uterine perfusion because those pregnancies are at high risk for developing pregnancy pathology later on.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results and Discussion References

 
Experimental subjects

All patients gave written informed consent, and the study was approved by the institutional ethics committee. We collected blood consecutively from 15 pregnant women with normal and 36 with pathological uterine perfusion (18th to 22nd wk gestation). All pregnancies were singleton, and at the time of examination, the women were healthy and normotensive. Whereas 25 women had a normal outcome of pregnancy, 11 developed pregnancy disorders (four preeclampsia and seven IUGR). Independently other pregnant women with manifest pathology such as preeclampsia (18 patients) or isolated IUGR (nine patients) were recruited in the third trimester. The second-trimester Doppler investigations were performed using a LOGIQ 9 ultrasound machine (GE, Solingen, Germany) as described before (11). Preeclampsia was defined according to the guidelines of the International Society for the Study of Hypertension in Pregnancy (13). IUGR was defined as a birth weight below the fifth percentile of our reference group adjusted for gestational age and gender.

Methods

A venous blood sample (10 ml) was drawn from each woman into tubes containing EDTA. Immediately after sampling, plasma was separated by centrifugation at 4000 x g for 10 min and frozen at –80 C. AT1-AA activity was measured by the same reference laboratory as first described (3) and modified as recently published (9), and sFlt1 was determined using a commercial ELISA kit (8). Statistical analysis was performed by the Mann-Whitney U test and Pearson’s correlation coefficients where appropriate.

	Results and Discussion

Top Abstract Introduction Subjects and Methods Results and Discussion References

 
Clinical data are summarized in Table 1. Confirming our previous data (9), none of the women with normal uterine perfusion harbored an AT1-AA at the 20th wk of pregnancy, whereas eight of 11 pregnancies with subsequent development of preeclampsia or IUGR, but no sign of a hypertensive disorder, had the agonistic AT1 antibody. Furthermore, test results were positive in 15 of the 25 women with an uneventful pregnancy and normal delivery despite the pathological uterine perfusion. At term, the AT1-AA was positive in 16 of the 18 patients with manifest preeclampsia and in eight of the nine normotensive patients with IUGR. Table 2 summarizes sFlt1 levels and AT1AA detection rates for the independent groups.

View larger version (10K): [in this window] [in a new window]   FIG. 1. Scatter plots between the AT1-AA activity (number is the increase in contractility in beats per 15 sec) and sFlt1 (plasma concentrations in picograms per milliliter) in pregnancies at the second trimester (A) and at the time of manifest disease (third trimester; B); no significant correlation could be found.

The majority of hypotheses that attempt to explain the origin of preeclampsia share the idea that the placenta (as a conditio sine qua non for preeclampsia) produces a toxin that is released into maternal circulation and causes the preeclamptic phenotype (1). However, none of the numerous candidates described could be characterized as the one decisive substance. Two circulating factors, AT1-AA and sFlt1, have recently been discussed as relevant pathophysiological entities in preeclampsia. Although they are both already detectable (AT1-AA) or elevated (sFlt1) before the onset of the disease, neither of them is an exclusive marker for preeclampsia (9, 10). This is reflected by the following observations: 1) not all pregnancies with later pathology or at term are AT1-AA positive; 2) autoantibodies have been detected in pregnancies with abnormal uterine perfusion but a normal outcome; and 3) the detection of AT1-AA and an elevated sFlt1 are also observed in pregnancies with IUGR. Following the idea that Ang II is potent in stimulating sFlt1 (12), the agonistic antibody occurring in the second trimester of pregnancy (9) (or even earlier) could be directly responsible for the development of reduced trophoblast invasiveness but could also, by inducing sFlt1, promote other characteristics of preeclampsia like renal dysfunction. Because both circulating factors are described as being potent in generating endothelial dysfunction, their parallel expression could have additive potency in damaging the endothelium. Thus, although decisive evidence has not yet been provided that one of the two factors is causative for preeclampsia, it may be hypothesized that concertive regulation is needed to mediate its initial pathology. Our investigations thus aimed at answering the question of whether the AT1-AA and sFlt1 are correlated in preeclampsia and whether only the combination of positive AT1-AA and elevated sFlt1 is specific for preeclampsia or even predictive of the disease.

Our study clearly documents that the maternal AT1-AA bioactivity and the sFlt1 concentration do not correlate either before the pregnancy pathology occurs or in the situation of a manifest complication. Besides being substantiated by our statistical analysis, this is most simply illustrated by the observation that part of the patients harboring AT1-AA have normal sFlt1 levels, and patients with excessive sFlt1 are characterized by the absence of the agonistic antibody.

Noteworthy is the fact that no correlation is found, even in the subgroups with pathological uterine perfusion. This is all the more important because it was hypothesized that a combined occurrence and thus a synergistic promotion of pathology could determine whether a pregnancy with uterine perfusion disturbance will develop pathology or will have a normal outcome if neither or only one of the factors is circulating. Our data clearly contradict our working hypothesis.

However, our data keep another interesting finding. Whereas women with abnormal perfusion who develop later pregnancy pathology did not differ in AT1-AA activity, compared with those with abnormal perfusion but normal outcome, sFlt1 was significantly increased in women with later pregnancy pathology.

Nevertheless, it must at least be mentioned that the complicated detection of the agonistic antibody by a bioassay using neonatal rat cardiomyocytes allows only a limited quantitative determination of circulating AT1-AA concentrations (increase in beats per 15 sec of those cardiomyocytes). Therefore, new detection methods have to be developed to make AT1-AA screening faster and more quantitative.

In summary, three major conclusions may be drawn from our study: 1) AT1-AA and sFlt1 values do not correlate in maternal circulation; 2) despite the fact that sFlt1 has proven to be the most potent marker of impending preeclampsia and IUGR and the AT1-AA has the potency to mediate preeclampsia-related pathology, the two factors alone or in combination may not be exclusively causative for early impaired trophoblast invasion and pathological uterine perfusion; and 3) their combination is not decisive in provoking a pathological outcome of pregnancies with altered uterine perfusion.

	Footnotes

 
There is nothing to disclose.

First Published Online March 28, 2006

Abbreviations: Ang, Angiotensin; AT1, Ang II receptor type 1; AT1-AA, autoantibody against the AT1 receptor; IUGR, intrauterine growth restriction; sFlt1, soluble fms-like tyrosine kinase 1.

Received December 12, 2005.

Accepted March 22, 2006.

	References

Top Abstract Introduction Subjects and Methods Results and Discussion References

 

1. Sibai B, Dekker G, Kupferminc M 2005 Pre-eclampsia. Lancet 365:785–799[Medline]
2. Davison JM, Homuth V, Jeyabalan A, Conrad KP, Karumanchi SA, Quaggin S, Dechend R, Luft FC 2004 New aspects in the pathophysiology of preeclampsia. J Am Soc Nephrol 9:2440–2448
3. Wallukat G, Homuth V, Fischer T, Lindschau C, Horstkamp B, Jupner A, Baur E, Nissen E, Vetter K, Neichel D, Dudenhausen JW, Haller H, Luft FC 1999 Patients with preeclampsia develop agonistic autoantibodies against the angiotensin AT1 receptor. J Clin Invest 103:945–952[Medline]
4. Xia Y, Wen H, Bobst S, Day M-C, Kellems RE 2003 Maternal autoantibodies from preeclamptic patients activate angiotensin receptors on human trophoblast cells. J Soc Gynecol Investig 10:82–93[CrossRef][Medline]
5. Thway TM, Shlykov SG, Day MC, Sanborn BM, Gilstrap 3rd LC, Xia Y, Kellems RE 2004 Antibodies from preeclamptic patients stimulate increased intracellular Ca2+ mobilization through angiotensin receptor activation. Circulation 110:1612–1619[Abstract/Free Full Text]
6. Dechend R, Viedt C, Muller DN, Ugele B, Brandes RP, Wallukat G, Park JK, Janke J, Barta P, Theuer J, Fiebeler A, Homuth V, Dietz R, Haller H, Kreuzer J, Luft FC 2003 AT1 receptor agonistic antibodies from preeclamptic patients stimulate NADPH oxidase. Circulation 107:1632–1639[Abstract/Free Full Text]
7. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, Libermann TA, Morgan JP, Sellke FW, Stillman IE, Epstein FH, Sukhatme VP, Karumanchi SA 2003 Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 111:649–658[CrossRef][Medline]
8. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, Sibai BM, Sukhatme VP, Karumanchi SA 2004 Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 350:672–683[Abstract/Free Full Text]
9. Walther T, Wallukat G, Jank A, Bartel S, Schultheiss H-P, Faber R, Stepan H 2005 The angiotensin II AT1 receptor autoimmune antibody reflects fundamental alterations of the utero-placental vasculature. Hypertension 46:1–5[Abstract/Free Full Text]
10. Stepan H, Geide A, Faber R 2004 Soluble fms-like tyrosine kinase 1. N Engl J Med 351:2241–2242[Free Full Text]
11. Stepan H, Heihoff-Klose A, Faber R 2004 Reduced antioxidant capacity in second-trimester pregnancies with pathological uterine perfusion. Ultrasound Obstet Gynecol 23:579–583[Medline]
12. Kim NH, Oh JH, Seo JA, Lee KW, Kim SG, Choi KM, Baik SH, Choi DS, Kang YS, Han SY, Han KH, Ji YH, Cha DR 2005 Vascular endothelial growth factor (VEGF) and soluble VEGF receptor FLT-1 in diabetic nephropathy. Kidney Int 67:167–177[CrossRef][Medline]
13. 2000 Report of the National High Blood Pressure Education Program Working Group on High Blood Pressure in Pregnancy. Am J Obstet Gynecol 183:1–22

This article has been cited by other articles:

				S. Verlohren, D. N. Muller, F. C. Luft, and R. Dechend Immunology in Hypertension, Preeclampsia, and Target-Organ Damage Hypertension, September 1, 2009; 54(3): 439 - 443. [Full Text] [PDF]

				F. Herse, S. Verlohren, K. Wenzel, J. Pape, D. N. Muller, S. Modrow, G. Wallukat, F. C. Luft, C. W.G. Redman, and R. Dechend 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 Hypertension, February 1, 2009; 53(2): 393 - 398. [Abstract] [Full Text] [PDF]

				B. D. LaMarca, J. Gilbert, and J. P. Granger Recent Progress Toward the Understanding of the Pathophysiology of Hypertension During Preeclampsia Hypertension, April 1, 2008; 51(4): 982 - 988. [Full Text] [PDF]

				C. C. Zhou, S. Ahmad, T. Mi, S. Abbasi, L. Xia, M.-C. Day, S. M. Ramin, A. Ahmed, R. E. Kellems, and Y. Xia Autoantibody From Women With Preeclampsia Induces Soluble Fms-Like Tyrosine Kinase-1 Production via Angiotensin Type 1 Receptor and Calcineurin/Nuclear Factor of Activated T-Cells Signaling Hypertension, April 1, 2008; 51(4): 1010 - 1019. [Abstract] [Full Text] [PDF]

				J. S. Gilbert, M. J. Ryan, B. B. LaMarca, M. Sedeek, S. R. Murphy, and J. P. Granger Pathophysiology of hypertension during preeclampsia: linking placental ischemia with endothelial dysfunction Am J Physiol Heart Circ Physiol, February 1, 2008; 294(2): H541 - H550. [Abstract] [Full Text] [PDF]

				J. S. Gilbert, S. A. Babcock, and J. P. Granger Hypertension Produced by Reduced Uterine Perfusion in Pregnant Rats Is Associated With Increased Soluble Fms-Like Tyrosine Kinase-1 Expression Hypertension, December 1, 2007; 50(6): 1142 - 1147. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Stepan, H. Articles by Walther, T. Search for Related Content PubMed PubMed Citation Articles by Stepan, H. Articles by Walther, T. Related Collections Autoimmunity Female Endocrinology