Objective
Soluble fms-like tyrosine kinase (sFLT-1) is a potent antiangiogenic growth factor that has been found to be markedly elevated in preeclampsia. In healthy pregnancy, serum sFLT-1 concentrations are 50-fold higher than in the nonpregnant state. The functional significance of this physiologic elevation in serum sFLT-1 in normal pregnancy is unknown. We hypothesized that sFLT-1 regulates placental cytotrophoblast invasion and that lower levels of sFLT-1 would be observed locally in invasive placentas (accreta/increta/percreta).
Study Design
We performed a retrospective case-control study comparing placental sFLT-1 expression in hysterectomy specimens from 3 groups: group 1, focally invasive placenta; group 2, normal invasion from the same specimen; and group 3, normal invasion associated with placenta previa. Immunohistochemistry for sFLT-1 was performed, and staining intensity was graded on a scale from 1+ (weak) to 5+ (strong).
Results
We identified 10 hysterectomy specimens from women with invasive placentation and 3 with placenta previa. The median sFLT-1 staining score for group 1 was 1.75 compared to 4.0 for group 2 ( P = .01). A significant difference was also found between group 1 and group 3 ( P = .01). When comparing depth of invasion, there was a trend toward lower staining score as depth of invasion increased ( P = .11). Interobserver agreement for immunohistochemistry scoring was 87%.
Conclusion
Lower levels of sFLT-1 protein expression were associated with invasive placentation suggesting a critical functional role for sFLT-1 in regulation of placental invasion.
Soluble fms-like tyrosine kinase (sFLT-1) is a secreted splice variant of fms-like tyrosine kinase (FLT-1) that binds circulating vascular endothelial growth factor (VEGF) and placental growth factor and is believed to contribute to the pathogenesis of preeclampsia. VEGF is a potent angiogenic growth factor that increases angiogenesis, nitric oxide, and vasodilatory prostaglandins. VEGF supports endothelial health and blood pressure regulation. VEGF is produced by some cancers and serves to promote angiogenesis supporting tumor growth. Treatment with bevacizumab (Avastin; Genentech, Inc., San Francisco, CA), which blocks VEGF, has been used effectively to suppress tumor growth. A syndrome associated with treatment with VEGF inhibitors characterized by hypertension, proteinuria, and cerebral edema, similar in some degree to preeclampsia, has been reported.
In normal pregnancy, VEGF supports endothelial health and vasodilatation. Angiogenesis, necessary for placental development, is also supported by VEGF. sFLT-1 irreversibly binds to circulating VEGF, preventing its binding to endothelial cell receptors and expression of its physiologic effects.
sFLT-1 overexpression in rodents has produced a model of preeclampsia characterized by hypertension, proteinuria, and characteristic renal glomerular changes of endotheliosis. Affected animals can be rescued by treatment with VEGF. Levine et al have reported that in normal pregnancy circulating sFLT-1 is elevated 20- to 50-fold higher with mean peak serum concentrations >2000 pg/mL at term, compared to nonpregnant state (∼100 pg/mL). In preeclamptic pregnancies, sFLT-1 is overexpressed in the placenta and the mean peak serum concentration is increased 2- to 5-fold compared to normotensive pregnancies. While human and animal studies support a pathogenic role for sFLT-1 in preeclampsia, the physiologic role of elevated sFLT-1 in normal pregnancy is unknown.
In normal pregnancy, extravillous cytotrophoblasts invade the decidua and the spiral arterioles replacing maternal vascular endothelium with endothelialized trophoblast of fetal origin resulting in the remodeling of spiral arteries into low-resistance vessels that are generally unresponsive to vascular control. Excessive placental invasion occurs when trophoblast invades through the decidua and into the myometrium and adjacent pelvic organs, in some cases resulting in catastrophic hemorrhage at delivery. Excessive invasion is particularly common when the placenta implants over uterine scars where vascular potential is presumably reduced. While the molecular mechanisms of trophoblast invasion are complex, prior cell culture and immunohistochemistry studies suggest that VEGF may promote trophoblast invasion.
We therefore hypothesized that sFLT-1 may operate at the trophoblast-decidual interface to regulate placental invasion and facilitate detachment of the placenta after delivery of the fetus. To address this question, we sought to evaluate whether sFLT-1 protein expression was decreased at sites of excessive placental invasion compared with normal placentation.
Materials and Methods
Study population
We performed a retrospective case-control study comparing sFLT-1 protein expression in hysterectomy specimens from pregnancies complicated by excessive placental invasion (placenta percreta, increta, or accreta) and by placenta previa without excessive invasion collected from 1996 through 2006. Group 1 was composed of histologic sections of focally invasive placenta obtained from hysterectomy specimens with invasive placentation. Group 2 was composed of histologic sections with normal invasion from the same specimens (internal control). Group 3 was composed of sections from with normal invasion from hysterectomies due to hemorrhage associated with placenta previa (external control). Inclusion criteria consisted of a pathologic diagnosis of invasive placenta or a clinical diagnosis of placenta previa without evidence of excessive invasion with gestational ages between 20-40 weeks. Subjects with known risk factors for preeclampsia, fetal growth restriction, abruption, or chorioamnionitis were excluded. We identified 10 cases and 3 external controls with archived hysterectomy tissue samples. The study received institutional review board approval at the University of Washington. Clinical data were obtained from each subject’s medical record: subject age, estimated gestational age at delivery, medical history, history of hypertensive disease, fetal growth restriction, placental abruption, or chorioamnionitis.
Immunohistochemistry
From stored tissue blocks from hysterectomy specimen samples were obtained demonstrating areas of focal invasion and areas of normal invasion. Tissue was formalin fixed, paraffin embedded, and stained with hematoxylin and eosin using routine methods prior to sFLT-1 staining. FLT-1 was identified using a goat antihuman VEGFR-1/FLT-1 antibody (catalog no. AF321; R and D Systems, Minneapolis, MN) as described previously and an antigoat cell and tissue staining kit (catalog no. CTS 008; R and D Systems). Antigen retrieval was performed using citrate buffer and microwave heating. Staining for sFLT-1 was based on the formation of the avidin-biotin complex with primary antibodies that reacted with sFLT-1. Visualization was based on enzymatic conversion of a chromogenic substrate 3,3′-diaminobenzidine into a colored brown precipitate by horseradish peroxidase at the sites of antigen localization.
Preparation was made for all samples with a primary antibody dilution of 1:100 and was performed without knowledge of the identity of the samples. Digital photomicrographs were taken for use in grading at identified areas of normal and excessive invasion. Evaluation of the sections stained for sFLT-1 was performed by a 2 investigators blinded to the histologic diagnosis. Grading of villous trophoblast sFLT-1 staining was done using a semiquantitative ordinal scale as follows: 5 (strong focal trophoblast staining >75%), 4 (50-74% of the trophoblast showing staining), 3 (25-49% staining), and 2 (1-24% staining). A guide for grading was provided with each photomicrograph ( Figure ).
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