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ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice

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Science  18 Aug 2017:
Vol. 357, Issue 6352, pp. 707-713
DOI: 10.1126/science.aam6607
  • Fig. 1 Zygotic deletion of Ela causes midgestation lethality due to cardiovascular defects and phenocopies loss of Apj.

    (A) Exon 3 of murine Ela was flanked with loxp sites and excised with cre recombinase to generate the ElaΔ allele lacking the ELA mature peptide (MP) coding region. (B) Schematic of cDNA from WT and ElaΔ alleles. SP, signal peptide. (C) Semi-qPCR of Ela locus from genomic DNA (gDNA) and cDNA. Primer locations are indicated in (B). (D) Distribution of genotypes at E10.5 and at weaning from intercrosses and ElaΔ/Δ (mother) x Ela+/Δ (father) crosses. %P, penetrance; L, number of litters. Data were tested using a chi-square test with 1 degree of freedom for significant deviation from the expected distribution. (E to G) At E10.5, Ela+/Δ embryos are indistinguishable from WT, whereas 43% (n = 17 of 39) of ElaΔ/Δ embryos and 14% (n = 3 of 22) of ApjΔ/Δ embryos display cardiovascular defects along with IUGR. Scale bars, 1 mm. (H to J) At E10.5, Ela+/Δ yolk sacs have normal vitelline vessels, whereas affected ElaΔ/Δ and ApjΔ/Δ embryos have avascular yolk sacs with a ruffled appearance. Scale bars, 1 mm. (K to M) CD31 staining of Ela+/Δ, ElaΔ/Δ, and ApjΔ/Δ yolk sacs reveals poorly matured vasculature in mutant embryos. Scale bars, 50 μm. (N to P) CD31 staining of Ela+/Δ, ElaΔ/Δ, and ApjΔ/Δ head vasculature at E10.5. Scale bars, 300 μm. (Q to S) CD31 (green) and SMA (red) staining of Ela+/Δ, ElaΔ/Δ, and ApjΔ/Δ hearts. Scale bars, 300 μm. (T) In situ hybridization of Ela at E8, showing mRNA localization in the region overlying the developing heart tube (ht) and chordal neural hinge (cnh). Scale bar, 200 μm. (U and V) RNAScope of Ela and Apj in E8 embryo within its decidua showing expression in the primitive foregut (fg) and hindgut (hg) endoderm. Arrowheads indicate the start of Ela expression in the chorionic trophoblast. Scale bars, 100 μm. (W and X) RNAScope of Ela and Apj in E8 yolk sac layers adhering to underlying decidua. en, endoderm; me, mesoderm. Scale bars, 40 μm.

  • Fig. 2 ELA is a pregnancy hormone required for placental angiogenesis.

    (A and B) At E9, Ela is expressed in the chorionic plate (cp) of the chorioallantoic placenta, and its receptor Apj is expressed in fetal allantoic endothelial cells. d, decidua. Scale bars, 1 mm. (C and D) At E10.5, Ela expression in the placenta labyrinth (lb) is restricted to STs, whereas Apj expression is restricted to endothelial cells adjacent to STs. Scale bars, 100 μm. (C′ and D′) Higher magnification showing Ela expression in STs surrounding maternal blood spaces (mbs) and Apj expression in endothelial cells (EC) lining fetal blood spaces (fbs). Scale bars, 200 μm. (E and F) ELA can be detected by immunohistochemistry using an ELA-specific antibody (α C) in WT E10.5 labyrinth in cells lining blood spaces (arrowheads) but not in ElaΔ/Δ placentas. (G) ELISA detects circulating ELA in maternal serum harvested at indicated gestational days (GD). n = number of mice assayed at each gestational time point. NP, nonpregnant. Error bars indicate SEM of three independent experiments. Data were tested with one-way analysis of variance (ANOVA) (red asterisk) and with two-sample Student’s t test (black asterisks). (H) ELISA of GD 10.5 maternal serum harvested from WT or ElaΔ/Δ mothers mated with the WT or ElaΔ/Δ fathers, indicating a maternal and zygotic origin of circulating ELA during pregnancy. n = number of mice assayed at each gestational time point. Error bars, SEM of three independent experiments. Data were tested using one-way ANOVA. In (G) and (H), ELA detected in maternal zygotic knockout is attributed to assay background. (I and J) Hematoxylin and eosin staining of Ela+/Δ and ElaΔ/Δ E10.5 placentas showing poor invasion and angiogenesis of ElaΔ/Δ placentas. Scale bars, 250 μm. al, allantois. (K and L) CD31/Pecam-1 staining of E10.5 Ela+/Δ and ElaΔ/Δ showing a paucity of fetal endothelial cells in the labyrinth. Scale bars, 100 μm. (M and N) Alpp (placenta alkaline phosphatase) staining of E10.5 Ela+/Δ and ElaΔ/Δ placentas showing lack of trophoblasts in the labyrinth. Scale bars, 50 μm. *P < 0.05, **P < 0.01 from indicated tests of significance.

  • Fig. 3 Loss of Ela causes hypoxic response and up-regulation of a pro-angiogenic program.

    (A) Schematic of RNA-seq experiment of E9.5 WT versus ElaΔ/Δ labyrinths. (B and C) GSEA analysis of ElaΔ/Δ (classes 1 and 3) showing an up-regulation of hypoxic response and pro-angiogenic genes in ElaΔ/Δ labyrinths, even in morphologically normal class 1 placentas. (D) Gene ontology analysis of genes up-regulated in ElaΔ/Δ labyrinths. In red are pathways enriched in tip cells. P values are derived from a binomial distribution with Bonferroni correction. (E) GSEA detects an up-regulation of endothelial tip cell genes in class 1 ElaΔ/Δ labyrinths. (F) qPCR validation of tip cell–enriched and angiogenic genes in ElaΔ/Δ labyrinths (n = 6 WT; n = 6 ElaΔ/Δ). Error bars indicate SEM of two independent experiments. (G) Esm1 immunofluorescence on E9.5 placenta vibratome sections taken from medial planes containing the maternal central canal. Dotted line marks the position of the transitional zone. al, allantois; lb, labyrinth; d, decidua. Scale bars, 40 μm. (H) Number of Esm1+ cells per section (n = 6 WT; n = 7 ElaΔ/Δ; each section represents a distinct placenta). (I) Seventy-fifth percentile integrated density of Esm1+ cells in each placental sample quantified in (H). Data presented as arbitrary units (A.U.). (J) β-galactosidase (LacZ transgene in AplnΔ allele) staining of Ela+/+;Apln+/Δ and ElaΔ/Δ;Apln+/Δ placentas indicate increased Apln expression in ElaΔ/Δ labyrinths. Scale bars, 300 μm. Data are depicted as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 of two-sample Student’s t test.

  • Fig. 4 Endogenous ELA prevents preeclampsia (PE), and exogenous ELA administration rescues PE symptoms in Ela-deficient mice.

    (A) Urine protein/creatinine ratios from GD 15 pregnant mothers (♀) of indicated genotype mated with fathers (♂) of indicated genotypes. Each dot represents an individual mouse. Error bars indicate SEM. (B) Repeated tail-cuff systolic blood pressure measurements of WT mothers (n = 7, mated to WT fathers) and ElaΔ/Δ mothers (n = 5, mated to ElaΔ/Δ fathers) at the indicated gestational age. Dotted line indicates day of parturition. Error bars indicate SEM. Two-way ANOVA analysis detected a significant interaction between time and genotype, F(7,49) = 2.074; P = 0.0413; i.e., ElaΔ/Δ females developed significantly higher systolic BP compared to the controls as pregnancy progressed. Asterisks indicate significance of two-sample unpaired t test between WT and ElaΔ/Δ on the indicated GD. (C) BP readings from (B) calculated in the form of delta BP (BP of indicated GD minus baseline nonpregnant BP of the same mother). Each dot represents BP of one mouse averaged over 20 readings; error bars indicate SEM. Two-way ANOVA test detected a statistically significant difference in mean delta BP between WT and ElaΔ/Δ mice; F(1,16) = 11.28, P = 0.0100. Asterisks indicate significance of two-sample unpaired t test. (D) Weight of pups at E18.5 collected by caesarean section. Each dot represents one pup. Error bars indicate SEM. (E) Urine protein/creatinine ratios of WT mothers (mated to WT fathers) (black squares) and ElaΔ/Δ mothers (mated to ElaΔ/Δ fathers) (red circles) implanted at GD 7 with infusion pumps containing either phosphate-buffered saline (PBS) (closed symbols) or synthetic ELA peptide (open symbols) measured at GD 15. Each dot represents one mouse; error bars indicate SEM. (F) Systolic delta BP measurements of subjects in (E) measured at GD 14, 16, and 18. Each dot represents one mouse; error bars indicate SEM. Two-way ANOVA test detected a statistically significant difference in mean delta BP between ElaΔ/Δ + PBS and ElaΔ/Δ + ELA mice; F(1,16) = 6.938, P = 0.0300. Asterisks indicate significance of two-sample unpaired t test. (G) Immunohistochemistry of ELA with α C biotinylated ELA-specific antibody on human first trimester (8 + 3 weeks) placental formalin-fixed paraffin-embedded sections. Scale bars, 200 μm. (H) Transwell invasion assay using Jar choriocarcinoma cells cultured in the presence of increasing concentrations of synthetic ELA peptide. Each dot represents the mean of three wells; error bars indicate SEM of three independent experiments. (I) Working model: mouse ELA, produced by ST cells, signals to APJ expressed on fetal endothelial cells (ECs) to facilitate normal placental angiogenesis. ELA also enters the maternal circulation, where it acts systemically to prevent symptoms of preeclampsia during pregnancy. Tb, trophoblast. Data are depicted as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 of two-sample Student’s t test, unless otherwise stated.

Supplementary Materials

  • ELABELA deficiency promotes preeclampsia and cardiovascular malformations in mice

    Lena Ho, Marie Van Dijk, Sam Tan Jian Chye, Daniel M. Messerschmidt, Serene C. Chng, Sheena Ong, Ling Ka Yi, Souad Boussata, Grace Hui-Yi Goh, Gijs B. Afink, Chin Yan Lim, N. Ray Dunn, Davor Solter, Barbara B. Knowles, Bruno Reversade

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S6
    • Table S1
    • References

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