Interferon-α and angiogenic dysregulation in pregnant lupus patients who develop preeclampsia

Abstract

Objective: To investigate whether an elevated interferon-α (IFNα) level early in pregnancy is associated with poor pregnancy outcomes and to examine the relationship of an elevated IFNα level to angiogenic imbalance.

Methods: Women were enrolled in a longitudinal case-control study of pregnant patients with lupus. Serum samples obtained monthly throughout pregnancy were assayed for IFNα and for the antiangiogenic factor soluble Flt-1 and the proangiogenic factor placenta growth factor (PlGF). Each of 28 patients with systemic lupus erythematosus (SLE) with a poor pregnancy outcome was matched to an SLE patient with an uncomplicated pregnancy and to a pregnant healthy control. The effects of IFNα and/or soluble Flt-1 on human endothelial cells and endothelial cell-trophoblast interactions were assessed.

Results: Compared to SLE patients with uncomplicated pregnancies, patients with preeclampsia had increased IFNα levels before clinical symptoms. Patients without autoimmune disease who developed preeclampsia did not have increased IFNα levels. In SLE patients with low IFNα levels, marked angiogenic imbalance (higher soluble Flt-1, lower PlGF, and higher soluble Flt-1:PlGF ratios) preceded maternal manifestations of preeclampsia, whereas in SLE patients with high IFNα levels, preeclampsia occurred without evidence of systemic angiogenic imbalance. Treatment of human endothelial cells with soluble Flt-1 induced expression of sFLT1 messenger RNA, and IFNα dramatically amplified responses to soluble Flt-1. In a model of spiral artery transformation, only the combination of IFNα and soluble Flt-1 disrupted the ability of trophoblast cells to remodel endothelial tube structures.

Conclusion: Our findings identify a new mechanism by which IFNα induces an antiangiogenic milieu and increases the sensitivity of endothelial cells to soluble Flt-1, and suggest that elevated IFNα levels may contribute to the pathogenesis of preeclampsia in some pregnant patients with SLE.

Trial registration: ClinicalTrials.gov NCT00198068.

Copyright © 2015 by the American College of Rheumatology.

Figures

Figure 1. IFN-α Activity in PROMISSE Study Patients

Samples from 6 - 27 weeks’ gestation (average 3.6/patient), a period that antedated clinically-apparent pregnancy complications, were assayed for IFN-α activity using the WISH reporter cell assay. Data are reported as median values of RE of MX1 (an IFN-α-responsive gene) from all samples collected for each patient. Lines represent 25-75th percentile. (A) Median values of RE MX1 are shown for healthy controls (n=28 patients), SLE patients without poor outcomes (No Outcome) (n=27) and SLE patients with poor outcomes (Outcome) (n=28). *Healthy controls vs. SLE/Outcome: p<0.004; Healthy controls vs. SLE/No Outcome: p=0.07; SLE/Outcome vs. SLE/No Outcome: p=0.23. (B) Median values of RE MX1 are shown for SLE patients with poor outcomes other than PE (n=14) and SLE patients with PE (n=14). (**p=0.006). (C) Samples from healthy, non-autoimmune patients who developed PE (Non-AI PE, n=9), SLE patients without PE (no outcomes and outcomes but not PE) (SLE, no PE, n=41) and SLE patients with PE (SLE, PE, n=14) were assayed and data presented group by weeks of gestation. *p=0.02; **p=0.04; ***p=0.008; ****p<0.005. Non-AI, non-autoimmune; PE, preeclampsia

Figure 2. Angiogenic Factor Levels in SLE Patients Who Developed Preeclampsia

SLE patients were grouped as low IFN-α (n=22) or high IFN-α (n=17). Plasma from 18-21 weeks was assayed for sFlt1 and PlGF by ELISA. Lines represent 25-75th percentile. (A) Median levels of sFlt1 are shown in low IFN-α group with PE (n=5) compared to those without PE (no outcome and non PE outcomes) (n=17) (*p=0.08, Wilcoxon rank sum) and in high IFN-α patients with PE (n=7) compared to those without PE (n=10). (B) Median levels of PlGF are similarly compared in these groups (**p=0.03). (C) Ratios of sFlt1/PlGF are shown (***p=0.07). For reference, in healthy controls enrolled in the PROMISSE study the median levels of angiogenic factors at 18-21 weeks were: sFlt1 1026 pg/ml, PlGF 249 pg/ml, and sFlt1/PlGF ratio 6.21.

Figure 3. Regulation of gene expression in HUVECs by IFN-α and sFLT1

The transcriptional regulation of sFLT1 (A) and MX-1 (B) in HUVECs following treatment with recombinant IFN-α and sFlt1 was analyzed by q-RT PCR. HUVECs were incubated for 6 h with no treatment (None), IFN-α (100 U/ml), sFlt1 (5 ng/ml) or both proteins. The fold of gene expression was calculated using ΔΔCt normalized to the housekeeping gene GAPDH and to the baseline condition without treatment. Results represent the mean ± standard error of fold-gene expression of at least 5 different experiments; *:p<0.05 by two-tailed Wilcoxon matched-pairs signed rank test.

Figure 4. HTR8 cells stabilize HEEC tubes over time

HEEC cells were seeded in matrigel overnight to allow tube formation. Media was then removed from the wells and replaced with or without the first trimester extravillous trophoblast cell line, HTR8. After 24hrs, in absence of HTR8 cells, HEEC tubes are established. However, by 48hrs, the HEEC tubes begin to destabilize and by 72hrs they are mostly disintegrated. In contrast, in the presence of HTR8 cells, the tube structures are maintained over 72hrs, and increase in size as the trophoblast cells remodel the endothelium.

Figure 5. Effect of IFN-α and sFlt1 on endothelial-trophoblast interactions

HTR8 cells were added to HEEC tube cultures in the presence of no treatment (NT), IFN-α (100U/ml), sFlt1 (5ng/ml) or both (IFN-α + sFtl-1) and cultured for 24hrs. (A) HEEC (red)-HRT8 (green) interactions were visualized by fluorescent microscopy. Images are from one representative experiment. Co-localization of the cells is seen as yellow. (B) Bar chart shows the % inhibition of the number of tubes per field expressed as the mean ± SEM relative to the NT control from 4 independent experiments. For NT controls, the number of tubes was 18.1 ± 1.0. *p<0.05 by ANOVA with Bonferroni correction.