Excess glucose induce trophoblast inflammation and limit cell migration through HMGB1 activation of Toll-Like receptor 4.

Hyperglycemia increases the risk of preeclampsia. Hyperglycemia induces a placental trophoblast inflammatory (IL-1β, IL-6, IL-8), antiangiogenic (sFlt-1, sEndoglin), and anti-migratory profile. The IL-1β response is mediated via inflammasome activation by the damage-associated molecular pattern (DAMP), uric acid. The objective of this study was to determine the role of high-mobility group box-1 (HMGB1), a DAMP that activates Toll-like receptor 4 (TLR4), in human first trimester trophoblast responses to hyperglycemia. The trophoblast response to excess glucose under different oxygen tensions was also investigated. The human first trimester trophoblast cell line (Sw.71) was exposed to glucose mimicking normoglycemia (5 mmol/L) and hyperglycemia (10 mmol/L), either alone or with the TLR4 antagonist, LPS-RS; or the HMGB1 inhibitor, glycyrrhizin. Cells were also treated with glucose under hyperoxic (21% O2 ), normoxic (8% O2 ), and hypoxic (2% O2 ) conditions. Cell-free supernatants were assayed by ELISA and bioassays for inflammatory: IL-1β, IL-6, and IL-8; inflammasome-associated: uric acid and caspase-1; angiogenic: sEndoglin, sFlt-1, and PlGF; and the DAMP, HMGB1. Cell migration was measured using a two-chamber colormetric assay. Excess glucose triggered a trophoblast sterile inflammatory IL-8 and antimigratory response through HMGB1 activation of TLR4. The IL-1β and sFlt-1/PlGF response was TLR4-mediated, but HMGB1-independent, suggesting another DAMP may be involved. Hyperoxia rather than normoxia or hypoxia was a major driver of trophoblast dysfunction in response to excess glucose. The findings from this study indicate a novel mechanism by which hyperglycemia may impact trophoblast function, early placentation, and ultimately, pregnancy outcome.

Heim KR ，Mulla MJ ，Potter JA ，Han CS ，Guller S ，Abrahams VM ... -  《-》

Modulation of trophoblast function by concurrent hyperglycemia and antiphospholipid antibodies is in part TLR4-dependent.

While diabetes and APS are individually associated with increased risk of poor perinatal outcomes, in particular preeclampsia, recent studies have demonstrated an association between concurrent aPL and diabetes leading to an increased risk of pregnancy morbidity. Hyperglycemia and aPL have independently been shown to alter human trophoblast function by inducing a pro-inflammatory, anti-angiogenic, and antimigratory response. However, little is known about the effects of concurrent hyperglycemia and aPL on trophoblast function. A human first-trimester extravillous trophoblast cell line was exposed to glucose at 5 mmol/L (normoglycemia) or 25 mmol/L (hyperglycemia), all in the presence or absence of low-dose aPL or control IgG. For some experiments, the TLR4 antagonist, LPS-RS, was included. Cell culture supernatants were measured for inflammatory IL-1β and IL-8, and angiogenic PlGF, sFlt-1, and sEndoglin by ELISA. Inflammasome-associated uric acid was measured using a bioassay; caspase-1 was measured using an activity assay. Trophoblast migration was quantified using a two-chamber colorimetric assay. Compared to excess glucose alone, combination excess glucose and low-dose aPL (a) further augmented trophoblast inflammatory IL-1β, inflammasome-associated uric acid and caspase-1, and pro-angiogenic PlGF; (b) dampened trophoblast inflammatory IL-8, anti-angiogenic sEndoglin, and sFlt-1; and (c) further reduced trophoblast migration. Our findings indicate that while concurrent aPL and hyperglycemia are overall detrimental to trophoblast function, the presence of two simultaneous insults triggers some protective effects.

Leon-Martinez D ，Mulla MJ ，Han CS ，Chamley LW ，Abrahams VM ... -  《-》

Glucose and metformin modulate human first trimester trophoblast function: a model and potential therapy for diabetes-associated uteroplacental insufficiency.

Diabetes confers an increased risk of preeclampsia, but its pathogenic role in preeclampsia is poorly understood. The objective of this study was to elucidate the effects of excess glucose on trophoblast function and whether any changes could be reversed by metformin. The human first trimester trophoblast cell line (Sw.71) was treated with glucose at 5, 10, 25, and 50 mm, in the presence and absence of metformin. Trophoblast migration was quantified and supernatant cytokine, chemokine, and angiogenic factors measured. Increasing concentrations of glucose significantly increased trophoblast secretion of the inflammatory cytokines/chemokines: IL-1β, IL-6, IL-8, GRO-α, RANTES, and G-CSF; significantly increased trophoblast secretion of the anti-angiogenic factors sFlt-1 and sEndoglin; and significantly decreased trophoblast migration. Excess glucose-induced trophoblast IL-1β production was inhibited by disabling the Nalp3/ASC inflammasome. Metformin partially reduced the glucose-induced inflammatory response, but had no effect on the anti-angiogenic or antimigratory response. Excess glucose induced a pro-inflammatory, anti-angiogenic, and antimigratory state in first trimester trophoblast cells. Glucose-induced trophoblast IL-1β secretion was mediated by the inflammasome. Glucose-induced inflammation was partially reversed by metformin. These findings demonstrate the pleiotropic effects of hyperglycaemia on the trophoblast, providing potential explanations for the strong link between diabetes and preeclampsia.

Han CS ，Herrin MA ，Pitruzzello MC ，Mulla MJ ，Werner EF ，Pettker CM ，Flannery CA ，Abrahams VM ... -  《-》

Allopurinol inhibits excess glucose-induced trophoblast IL-1β and ROS production.

Negi M ，Mulla MJ ，Han CS ，Abrahams VM ... -  《-》

Hypoxic trophoblast HMGB1 induces endothelial cell hyperpermeability via the TRL-4/caveolin-1 pathway.

High mobility group box 1 (HMGB1) plays an important role in the pathologic processes of endothelial permeability under oxidative stress. Trophoblast oxidative stress has been implicated in the pathophysiology of preeclampsia (PE). HMGB1 serum levels are increased in PE. However, the potential roles of HMGB1 in endothelial permeability in PE remain unclear. We assessed the effects of the hypoxic trophoblast on the permeability of the endothelial monolayer. Our results showed that the hypoxic trophoblast displayed higher HMGB1 mRNA, intracellular HMGB1 protein, and HMGB1 in conditioned medium than those of the normoxic trophoblast did. The hypoxic trophoblast conditioned medium increased the endothelial monolayer permeability and increased TLR 4 and caveolin-1 (CAV-1) protein expression in endothelial cells, which was inhibited by glycyrrhizic acid and HMGB1 small interfering RNA transfection to trophoblasts before hypoxia. The increased endothelial permeability induced by hypoxic trophoblast conditioned medium could be inhibited with TLR4 or CAV-1 gene silencing in endothelial cells. Immunoprecipitation showed that CAV-1 and TLR4 are colocalized in HUVECs and C57BL/6 mouse kidney. TLR4 small interfering RNA suppressed CAV-1 protein expression in endothelial cells upon stimulation of hypoxic trophoblast conditioned medium or HMGB1. We conclude that hypoxic trophoblasts play an important role in the mechanism of general edema (including protein urine) in PE via increasing endothelial monolayer permeability through the HMGB1/TLR4/CAV-1 pathway.

Jiang R ，Cai J ，Zhu Z ，Chen D ，Wang J ，Wang Q ，Teng Y ，Huang Y ，Tao M ，Xia A ，Xue M ，Zhou S ，Chen AF ... -  《-》