Lymphatic mimicry in maternal endothelial cells promotes placental spiral artery remodeling

Abstract

Molecular heterogeneity of endothelial cells underlies their highly specialized functions during changing physiological conditions within diverse vascular beds. For example, placental spiral arteries (SAs) undergo remarkable remodeling to meet the ever-growing demands of the fetus - a process which is deficient in preeclampsia. The extent to which maternal endothelial cells coordinate with immune cells and pregnancy hormones to promote SA remodeling remains largely unknown. Here we found that remodeled SAs expressed the lymphatic markers PROX1, LYVE1, and VEGFR3, mimicking lymphatic identity. Uterine natural killer (uNK) cells, which are required for SA remodeling and secrete VEGFC, were both sufficient and necessary for VEGFR3 activation in vitro and in mice lacking uNK cells, respectively. Using Flt4Chy/+ mice with kinase inactive VEGFR3 and Vegfcfl/fl Vav1-Cre mice, we demonstrated that SA remodeling required VEGFR3 signaling, and that disrupted maternal VEGFR3 signaling contributed to late-gestation fetal growth restriction. Collectively, we identified a novel instance of lymphatic mimicry by which maternal endothelial cells promote SA remodeling, furthering our understanding of the vascular heterogeneity employed for the mitigation of pregnancy complications such as fetal growth restriction and preeclampsia.

Keywords: Lymph; Mouse models; Reproductive Biology; Reproductive biochemistry; Vascular Biology.

Figure 1. SAs acquire lymphatic characteristics during SAR.

(A) Tissue sections of mouse placenta at time points before and after SAR. PROX1 is sporadically expressed at low levels on the SA endothelium at E11.5 and E13.5 (per embryonic day, n = 7–9 total placentas from 3 litters, with 1–4 placentas from each litter). White arrowheads mark PROX1⁺ nuclei. Scale bars: 20 μm. (B) PROX1-RFP⁺ SA endothelium at E11.5 and E13.5. Scale bars: 50 μm. (C and D) LYVE1 and VEGFR3 expression is low or absent in SAs at E11.5, but are highly expressed at E13.5 (per embryonic day, n = 8–12 total placentas from 3–4 litters, with 2–4 placentas from each litter). Scale bars: 50 μm. (E) Tissue sections of rat placenta at E11.5 show absent VEGFR3 expression while at E13.5 and E18.5 there is robust VEGFR3 expression in SAs. Cytokeratin 7⁺ (CK7) invasive trophoblasts do not express VEGFR3 (per embryonic day, n = 4–6 total placentas from 3 litters, with 1–2 placentas from each litter). Scale bars: 100 μm. (F) A model summarizing features of lymphatic mimicry in SAs during remodeling. SMC, smooth muscle cell.

Figure 2. Endothelial ERK activation during SAR is blunted in uNK-deficient mice.

(A) Il2rγ^tm1Wjl mice do not have DBA lectin⁺ uNK cells in the mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis (DB) (per group, n = 6 total placentas from 3 litters with 2 placentas from each litter). JZ, junctional zone. Scale bars: 300 μm. (B) WT SAs exhibit increased VEGFR3 expression and loss of smooth muscle cells from E11.5 to E13.5. Il2rγ^tm1Wjl SAs have VEGFR3 expression at E13.5, but SMC coverage remains high. Scale bars: 50 μm. (C) p-ERK staining increases in the SA endothelium from E11.5 to E13.5 in WT, but not in Il2rγ^tm1Wjl mice. Scale bars: 50 μm. (D) Quantification of p-ERK and (E) VEGFR3 mean fluorescence intensity (MFI) in the SA endothelium of WT and Il2rγ^tm1Wjl placentas at E11.5 and E13.5 (per group, n = 6–7 total placentas from 3 litters with 1–3 placentas from each litter; 2-way ANOVA with Bonferroni posttest, P < 0.0001 for D and E). In all graphs, the red horizontal line represents the mean. ***P < 0.001 versus control.

Figure 3. Loss of VEGFR3 signaling is sufficient to impair SAR.

(A and B) The density of uNK cells in the placental decidua of Flt4^Chy/+ mice is similar to WT at E13.5 (per group, n = 6 total placentas from 3 litters with 2 placentas from each litter; unpaired t test). (C) VEGFR3 signaling–deficient Flt4^Chy/+ mice retain more SA SMC coverage (blue) compared with WT, even while total expression of VEGFR3 is unchanged. (D) Quantification of endothelial ERK phosphorylation (p-ERK) of WT and Flt4^Chy/+ mice at E13.5 (per group, n = 5 total placentas from 3 litters with 1–2 placentas from each litter; 2-way ANOVA with Bonferroni posttest, P = 0.0002). (E) Quantification of the percentage of SA perimeter covered in αSM-actin⁺ SMCs of WT and Flt4^Chy/+ mice at E13.5 (per group, n = 6–9 total placentas from 3 litters with 2–3 placentas from each litter; 2-way ANOVA with Bonferroni posttest, P = 0.0001). (F) H&E staining of SAs in WT and Flt4^Chy/+ mice at E11.5 and E13.5. (G and H) Quantification of the luminal area and wall thickness (ratio of vessel wall to lumen area) of SAs in WT and Flt4^Chy/+ mice at E11.5 and E13.5 (per group, n = 21–35 total placentas from 3 litters with 4–13 placentas from each litter; 2-way ANOVA with Bonferroni posttest, P < 0.0001 for G and H). (I–K) Flt4^Chy/+ mice exhibit fetal growth restriction and increased placental weights at E18.5 (per genotype, n = 33–44 total embryos and placentas from 3 litters with 9–16 embryos from each litter; unpaired t test). All scale bars: 50 μm. In all graphs the red horizontal line represents the mean. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 versus control.

Figure 4. Conditioned media from uNK cells activates VEGFR3.

(A) Representative images of p-VEGFR3 staining in treated LECs. Scale bar: 20 μm. (B) Quantification of p-VEGFR3 MFI in LECs of each treatment group (n = 7 experiments, 1-way ANOVA with Bonferroni posttest). The red horizontal line represents the mean. **P < 0.01; ***P < 0.001 versus control.

Figure 5. VEGFC from uNK cells is required for SAR.

(A and B) The decidua of Vegfc^fl/fl Vav1-Cre mouse E13.5 placentas have a similar uNK cell density compared with Vegfc^fl/fl placentas as determined by DBA lectin labeling (per genotype, n = 12–14 total placentas from 3 litters with 2–7 placentas from each litter, unpaired t test). (C and D) At E13.5, Vegfc^fl/fl Vav1-Cre SAs have increased smooth muscle coverage determined by αSMA staining (per genotype, n = 11–12 total placentas from 3 litters with 3–5 placentas from each litter; unpaired t test). (E–G) In H&E-stained placentas at E13.5, the lumen area of Vegfc^fl/fl Vav1-Cre SAs was reduced while the relative wall area was increased (per genotype, n = 15–22 total placentas from 3 litters with 4–10 placentas from each litter, unpaired t test). All scale bars: 50 μm. In all graphs the red horizontal line represents the mean. **P < 0.01; ***P < 0.001 versus control.

Figure 6. A model of VEGFR3 activation in SAs.

(A) Graphical representation of the mechanisms by which the mouse models examined in this study affect VEGFR3 signaling and SAR. (B) Mechanism of lymphatic mimicry to promote SAR. During SAR, endothelial cells acquire a hybrid vessel phenotype becoming more lymphatic-like; expression of certain lymphatic markers is upregulated, SMC layer is reduced, and luminal expansion occurs.