Fig 2: Induced endothelial dysfunction in HUVECs has no effect on LGMN expression but decreases CST6 expression. Successful induction of endothelial dysfunction in HUVECs was confirmed by significant increase of: (A) VCAM1 (B) ET-1 and (C) ICAM1. (D) CST6 expression in HUVECs was significantly decreased following treatment with increased doses of TNFα. (E) LGMN mRNA expression was unchanged. mRNA expression was normalised to the appropriate housekeeping gene. To calculate significance, Kruskal Wallis test was used. Data expressed as mean ± SEM. Experiments repeated n = 5 in duplicate. ** p < 0.01, *p < 0.05.

Fig 3: Recombinant CST6 enhances induction of endothelial dysfunction and LGMN in HUVECs. Induction of endothelial dysfunction in HUVECs was assessed by induction of markers: ICAM1 (A), VCAM1 (B) and ET-1 (C). Recombinant CST6 enhanced induction of dysfunction markers and (D) LGMN in HUVECs under inflammatory conditions. mRNA expression was normalised to the appropriate housekeeping gene. To calculate significance, one-way ANOVA (parametric) or a Kruskal Wallis test (non-parametric) was used. Data expressed as mean ± SEM for experiments (A, C and D) or median ± IQR (B). Experiments repeated n = 5 in duplicate. *** p < 0.001, * p < 0.05.

Fig 4: Single-nuclei RNA sequencing (snRNA-seq) analysis of early-onset preeclamptic, healthy and preterm placenta. (A) and (B), Uniform manifold approximation and projection (UMAP) visualising and analysis resulted in 7 distinct transcriptomic cell identities obtained from the data: villous syncytiotrophoblast (vSTB1 and vSTB2), villous juvenile syncytiotrophoblast (vSTBjuv), villous pre-fusion cytotrophoblast (vCTBpf), villous proliferative cytotrophoblast (vCTBp), villous cytotrophoblast (vCTB) and villous cell column trophoblast (vCCT). Samples from preterm pregnancy, early-onset preeclampsia and term healthy pregnancy were integrated for visualisation. Each dot represents a nucleus with each colour representing a cell identity. (C) Violin plot of CST6 and LGMN to indicate levels of gene expression in cell proportions of specific cell identities for each condition. Code available at https://github.com/HiDiHlabs/preeclamspsia_Nonn_etal/, data available in supplementary methods of manuscript28.

Fig 5: Regulation of CST6 and LGMN expression in syncytiotrophoblast cells. (A) CST6 expression was significantly increased in syncytiotrophoblast cells incubated in a hypoxic environment (1% O2) compared to control normoxic environment (8% O2). (B) LGMN expression was unchanged when incubated in a hypoxic environment relatively to control. mRNA expression was normalised to the geometric mean of housekeeper genes or single housekeeping gene depending on the treatment. To calculate significance, one-way ANOVA or Unpaired t test (parametric) or a Kruskal Wallis test (non-parametric) was used. Data expressed as mean ± SEM for experiments. Experiments repeated n = 5 in triplicate. *** p < 0.001.