Cystatin 6 (CST6) and Legumain (LGMN) are potential mediators in the pathogenesis of preeclampsia.

Preeclampsia results from placental insufficiency and causes maternal endothelial dysfunction and multi-organ damage. Our in-silico analysis identified Cystatin 6 (CST6), a cysteine protease inhibitor, as located on the placental surface where it might be released into maternal circulation. This study aimed to characterise CST6 and one of its high affinity targets, Legumain (LGMN), in preeclampsia and assess its biomarker potential by measuring levels in maternal circulation. Placental CST6 mRNA expression was significantly increased in 78 pregnancies complicated by early-onset preeclampsia (delivering at < 34 weeks' gestation) relative to 30 gestation matched controls (P < 0.0001). LGMN mRNA expression was significantly decreased (P = 0.0309). Circulating CST6 was increased in 35 pregnancies complicated by early-onset preeclampsia (< 34 weeks' gestation) relative to 27 gestation matched controls (P = 0.0261), and LGMN levels remained unchanged. At 36 weeks' gestation, circulating CST6 was significantly increased (P = 0.001), while LGMN was significantly decreased (P = 0.0135) in 21 pregnancies preceding diagnosis of preeclampsia at term, compared to 184 pregnancies that did not develop preeclampsia. Human trophoblast stem cells (hTSC) were differentiated into syncytiotrophoblast or extravillous trophoblast (EVT) to evaluate CST6 and LGMN expression in these trophoblast lineages. CST6 and LGMN mRNA expression were significantly increased across 96 h after syncytiotrophoblast (P = 0.0066 and P = 0.0010 respectively) and EVT differentiation (P = 0.0618 and P = 0.0016 respectively), with the highest expression in syncytiotrophoblast. Computational analysis of two publicly available single-cell and single-nuclei RNA sequencing datasets correlated with the expression pattern observed in vitro. When syncytiotrophoblast cells were exposed to hypoxia (1% O vs. 8% O), CST6 expression significantly increased (P = 0.0079), whilst LGMN expression was unchanged. The vascular endothelium may serve as an additional source of circulating CST6 and LGMN in preeclampsia.  Induction of dysfunction in endothelial cells by TNFα, caused reduced CST6 expression (P = 0.0036), whilst LGMN expression remained unchanged. Administering recombinant CST6 to endothelial cells enhanced markers of endothelial dysfunction and LGMN expression in the presence of TNFα. These findings indicate an inverse relationship between CST6 and LGMN in the placenta and maternal circulation in preeclampsia. We suggest elevated circulating levels of CST6 may be induced by placental hypoxia. This study provides novel insight into the dysregulation of CST6 and LGMN in preeclampsia and introduces their potential roles in human pregnancy and associated pathology.