High levels of HtrA4 observed in preeclamptic circulation drastically alter endothelial gene expression and induce inflammation in human umbilical vein endothelial cells

Abstract

IntroductionPreeclampsia (PE) is a life-threatening pregnancy disorder characterized by wide-spread endothelial dysfunction. Placental factors circulating in the maternal blood are believed to cause endothelial dysfunction. Our previous study identified HtrA4 as a placenta-specific serine protease that is released into the maternal circulation and significantly increased in early-onset PE. In this study, we examined the impact of HtrA4 on expression of endothelial genes related to vessel biology, using human umbilical vein endothelial cells (HUVECs) as a model.MethodsHUVECs were treated with 0 or 3 μg/ml HtrA4 (highest concentration seen in PE circulation) for 24 h and analysed by an endothelial cell biology PCR array containing 84 genes. HtrA4-induced changes were then validated by real-time RT-PCR and ELISA for time and dose dependency.ResultsHigh levels of HtrA4 significantly altered the expression of a range of genes related to inflammation, vaso-activity, angiogenesis, cell adhesion, platelet activation and coagulation. In particular, pro-inflammatory genes IL6, PTGS2 (COX2) and IL1B were significantly increased by HtrA4. IL6 protein in HUVEC media was also drastically increased. THBD, an anticoagulant factor reported to be increased in PE, was significantly up-regulated by HtrA4. In contrast, THBS1, which is involved in many regulatory processes of endothelial cell biology, was severely down-regulated by HtrA4.DiscussionHtrA4 significantly increased the inflammatory responses of HUVECs, and altered their expression of a number of genes important for vessel biology. These data suggest that placenta-derived HtrA4 that is increased in PE circulation is a potential causal factor of endothelial dysfunction.

High levels of HtrA4 detected in preeclamptic circulation may disrupt endothelial cell function by cleaving the main VEGFA receptor KDR

“…Placental HtrA4 expression and its circulating levels are significantly increased in early, but not late onset, PE at the time of disease presentation in the third trimester (21, 26–30). We have also demonstrated that high levels of HtrA4 detected in early onset PE inhibit tube formation and induce proinflammatory factors in endothelial cells (21, 31). These data suggest that HtrA4 may represent a placenta‐derived contributor of endothelial dysfunction in early‐onset PE development.…”

“…We have previously identified that serine protease HtrA4 is produced specifically by the placenta and significantly up‐regulated in early onset PE (21, 26–30). We have further reported that high levels of circulating HtrA4 in PE may be a potential causal factor of endothelial dysfunction (21, 31). Here, we demonstrated that high levels of circulating HtrA4 could cleave the main VEGFA receptor KDR to disrupt endothelial cell function and inhibit the VEGFA‐induced angiogenesis.…”

“…We used HUVECs as an endothelial cell model for this study. Because HUVECs have been widely used as a cell model to study endothelial cell functions and angiogenesis (40), they express KDR and respond well to VEGFA, and our previous studies showed that these cells respond to HtrA4 (21, 31). HUVECs (American Type Culture Collection, Rockville, MD, USA) were cultured at 37°C in a humidified atmosphere of 5% CO 2 in air in EGM‐2MV BulletKit (Lonza, Basel, Switzerland) medium supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% antibiotics (Thermo Fisher Scientific).…”

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“…The mechanisms underlying PE is still unclear but generally accepted that in PE, the factors released from placenta into the maternal circulation induce inflammation [3]. Significantly elevated circulating factors in PE include cytokines and antiangiogenic factors and these factors modulate trophoblast invasion [4]. The hypothesis produced by Ahmed and Ramma suggested that preeclampsia might be result of the imbalance between new-onset inflammatory state and endogenous protective pathways during pregnancy [5].…”

Maternal HtrA3 optimizes placental development to influence offspring birth weight and subsequent white fat gain in adulthood

“…To date, four mammalian HtrAs (HtrA1-4) have been identified [1][2][3][4][5][6][7] , and their dysregulation is associated with a number of diseases, including cancer, arthritis, neurodegenerative disorders, age-related macular degeneration, and pregnancy diseases [8][9][10][11][12][13][14][15][16][17] . In particular, HtrA1 and HtrA3 have been suggested as tumor suppressors, because they are down-regulated in a number of cancers and this reduction is suggested to promote tumorigenesis [18][19][20][21][22] .…”