Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

Abstract

Preeclampsia, a syndrome affecting 5% of pregnancies, causes substantial maternal and fetal morbidity and mortality. The pathophysiology of preeclampsia remains largely unknown. It has been hypothesized that placental ischemia is an early event, leading to placental production of a soluble factor or factors that cause maternal endothelial dysfunction, resulting in the clinical findings of hypertension, proteinuria, and edema. Here, we confirm that placental soluble fms-like tyrosine kinase 1 (sFlt1), an antagonist of VEGF and placental growth factor (PlGF), is upregulated in preeclampsia, leading to increased systemic levels of sFlt1 that fall after delivery. We demonstrate that increased circulating sFlt1 in patients with preeclampsia is associated with decreased circulating levels of free VEGF and PlGF, resulting in endothelial dysfunction in vitro that can be rescued by exogenous VEGF and PlGF. Additionally, VEGF and PlGF cause microvascular relaxation of rat renal arterioles in vitro that is blocked by sFlt1. Finally, administration of sFlt1 to pregnant rats induces hypertension, proteinuria, and glomerular endotheliosis, the classic lesion of preeclampsia. These observations suggest that excess circulating sFlt1 contributes to the pathogenesis of preeclampsia.

Figure 1

mRNA and protein expression of sFlt1 in preeclampsia. (a) mRNA expression of placental sFlt1 from three patients with preeclampsia (P1, P2, and P3) and three normotensive term pregnancies (N1, N2, and N3) were determined by Northern blot analysis. The higher band (7.5 kb) is the full-length Flt1 mRNA, and the lower, more abundant band (3.4 kb) is the alternatively spliced sFlt1 mRNA. GAPDH is included as a loading control, and the location of 28S is indicated by an arrowhead. Patients P1 and P2 had severe preeclampsia, whereas patient P3 had mild preeclampsia. (b) ELISA was performed for sFlt1 on serum from patients with mild preeclampsia (PE), severe preeclampsia and from normotensive pregnant women at term (normal) as described in Table 1. Patients with preterm deliveries were included as additional controls to rule out changes due to gestational age. The numbers of patients tested are shown in parentheses on the x-axis. Serum samples were collected before delivery (t = 0) and 48 hours after delivery (t = 48). *P < 0.05 and **P < 0.01 as compared with normotensive controls.

Figure 2

Free VEGF and free PlGF levels are decreased in the serum of patients with preeclampsia. (a) Standard curve for recombinant human VEGF protein was generated in the absence (control) or in the presence of two different doses of recombinant human sFlt1-Fc using the ELISA kit for measurement of human VEGF protein, as described in Methods. (b) Standard curve for recombinant human PlGF protein was generated in the absence (control) or in the presence of two different doses of recombinant human sFlt1-Fc using the ELISA kit for measurement of human PlGF protein, as described in Methods. (c) Free VEGF levels (pg/ml) at the time of delivery (t = 0) were determined by ELISA for the four patient groups described in Figure 1b and Table 1. (d) Free PlGF levels (pg/ml) at the time of delivery were determined by ELISA for the four patient groups described in Figure 1b and Table 1. The numbers of patients tested are shown in parentheses on the x-axis. *P < 0.05 and **P < 0.01 as compared with normotensive controls.

Figure 3

Preeclampsia is an antiangiogenic state due to excess sFlt1. Endothelial tube assay was performed using serum from four normal pregnant controls and four patients with preeclampsia before and after delivery. A representative experiment from one normal control and one patient with preeclampsia is shown. (a) t = 0 (5% serum from a normal pregnant woman at term). (b) t = 48 (5% serum from a normal pregnant woman 48 hours after delivery). (c) t = 0 plus exogenous sFlt1 (10 ng/ml). (d) t = 0 (5% serum from a preeclamptic woman before delivery). (e) t = 48 (5% serum from a preeclamptic woman 48 hours after delivery). (f) t = 0 plus exogenous VEGF (10 ng/ml) and PlGF (10 ng/ml). The tube assay was quantified, and the mean tube length ± SEM in pixels is given at the bottom of each panel for all the patients analyzed. Recombinant human VEGF, human PlGF, and human sFlt1-Fc were used for the assays.

Figure 4

sFlt1 inhibits VEGF- and PlGF-induced vasodilation of renal microvessel. (a) The relaxation responses of rat renal arterioles to sFlt1, VEGF, and PlGF at three different doses were measured. S, sFlt1; V, VEGF; P, PlGF. V+ and P+ represent responses to VEGF and PlGF in the presence of sFlt1 at 100 ng/ml. A positive change reflects an increase in vessel diameter. (b) Microvascular relaxation responses were measured at physiological doses of VEGF (100 pg/ml), PlGF (500 pg/ml), sFlt1 (10 ng/ml), VEGF (100 pg/ml) plus PlGF (500 pg/ml), or VEGF (100 pg/ml) plus PlGF (500 pg/ml) plus sFlt1 (10 ng/ml). Each experiment was repeated in six different rat renal microvessels, and data are reported as means ± SEM. *P < 0.05 as compared with VEGF plus PlGF. Reagents used for the assays were recombinant rat VEGF, mouse PlGF, and mouse sFlt1-Fc.

Figure 5

Western blot analysis for PlGF expression in pregnant rats versus nonpregnant rats. Western blot analysis for PlGF levels in the systemic circulation of rats was performed using blood specimens from two nonpregnant and two pregnant rats (early third trimester) as described in Methods. Lanes 1 and 2 represent 1 ng and 10 ng of recombinant mouse PlGF protein used as a positive control. Twenty microliters of concentrated (10-fold) serum specimens from two nonpregnant rats (lanes 3 and 4) and two pregnant rats in the early third trimester (lanes 5 and 6) were used, and shown is a representative Western blot. The blot shows almost absent PlGF in the nonpregnant rats and expression of PlGF protein in pregnant rats.

Figure 6

sFlt1 induces glomerular endotheliosis. (a) Histopathological analysis of renal tissue from one representative Fc-treated pregnant rat (upper panel), one sFlt1–treated pregnant rat (middle panel), and one sFlk1-Fc–treated pregnant rat (lower panel) is shown here. H&E stain shows capillary occlusion in the sFlt1 treated animal with enlarged glomeruli and swollen endothelial cells compared to Fc control animal and sFlk1-Fc control animals. PAS stain of the sFlt1–treated rat demonstrates PAS-negative swollen cytoplasm of endocapillary cells (endotheliosis). Numerous protein resorption droplets are also seen in the PAS section. These pathologic changes are absent in the Fc-treated rat as well as the sFlk1-Fc–treated pregnant rats. All light photomicrographs were taken at ×60 (original magnification). (b) Electron microscopy (EM) and immunofluorescence (IF) for fibrin was performed for the same rats shown in Figure 6a. Electron micrographs of glomeruli from an sFlt1–treated rat (lower panel) confirmed cytoplasmic swelling of the endocapillary cells. There is relative preservation of the podocyte foot processes and the basement membranes. Immunofluorescence for fibrin shows foci of fibrin deposition within the glomeruli of sFlt1–treated rats but not Fc-treated rats. The immunofluorescence pictures were taken at ×40 and the electron micrographs were taken at ×2400 (original magnification). All figures are reproduced at identical magnifications. (c) Histopathological analysis of one representative nonpregnant rat treated with low-dose sFlt1 is shown here. Low-power (×30, original magnification) Masson trichrome staining of renal tissue from the low-dose sFlt1–treated rat shows varying glomerular size representing focal endotheliosis. This degree of variation in glomerular involvement was only noted in the low-dose group. Higher-power H&E staining and PAS staining showed segmental endotheliosis and protein resorption droplets with preservation of basement membranes.