Combining metformin and sulfasalazine additively reduces the secretion of antiangiogenic factors from the placenta: Implications for the treatment of preeclampsia.

The antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG) are elevated in preeclampsia and have been implicated in its pathogenesis. We have previously demonstrated metformin and sulfasalazine independently reduce antiangiogenic factor secretion. Here we examined whether combining metformin and sulfasalazine may be more effective than either alone in reducing placental expression and secretion of antiangiogenic and angiogenic factors and the expression of markers of endothelial dysfunction. We performed functional experiments using primary human placenta to explore the effect of metformin and sulfasalazine, at lower doses than previously explored, individually and in combination, on sFlt-1 and sENG secretion and placental growth factor (PlGF) and vascular endothelial growth factor (VEGFα) expression. Using primary endothelial cells we induced dysfunction using cytokine tumor necrosis factor-α (TNF-α) and assessed the effect of low dose combination treatment on the expression of vascular cell adhesion molecule-1 (VCAM-1) and Endothelin-1 (a potent vasoconstrictor). We demonstrated combination metformin and sulfasalazine was additive in reducing sFlt-1 secretion from cytotrophoblasts and placental explants. Combination treatment was also additive in reducing sENG secretion from placental explants. Furthermore, combination treatment increased cytotrophoblast VEGFα mRNA expression. Whilst combination treatment increased PlGF mRNA expression this was similar to treatment with sulfasalazine alone. Combination therapy reduced TNFα induced endothelin-1 mRNA expression however did not change VCAM expression. Low dose combination metformin and sulfasalazine reduced cytotrophoblast sFlt-1 and sENG secretion, increased VEGFα expression and reduced TNFα induced endothelin-1 expression in primary endothelial cells. Combination therapy has potential to treat preeclampsia.

Brownfoot FC ，Hastie R ，Hannan NJ ，Cannon P ，Nguyen TV ，Tuohey L ，Cluver C ，Tong S ，Kaitu'u-Lino TJ ... -  《-》

Combining metformin and esomeprazole is additive in reducing sFlt-1 secretion and decreasing endothelial dysfunction - implications for treating preeclampsia.

Kaitu'u-Lino TJ ，Brownfoot FC ，Beard S ，Cannon P ，Hastie R ，Nguyen TV ，Binder NK ，Tong S ，Hannan NJ ... -  《PLoS One》

Sulfasalazine reduces placental secretion of antiangiogenic factors, up-regulates the secretion of placental growth factor and rescues endothelial dysfunction.

Preeclampsia is a major complication of pregnancy with no medical treatment. It is associated with placental oxidative stress, hypoxia and inflammation leading to soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG) secretion and reduced placental growth factor (PlGF). This results in widespread endothelial dysfunction causing hypertension and multisystem organ injury. Sulfasalazine is an anti-inflammatory and antioxidant medication used to treat autoimmune disease. Importantly, it is safe in pregnancy. We examined the potential of sulfasalazine to quench antiangiogenic factors and endothelial dysfunction and increase angiogenic factor secretion. We performed functional experiments using primary human pregnancy tissues to examine the effects of sulfasalazine on sFlt-1, sENG and PlGF secretion. Sulfasalazine is known to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and upregulate heme-oxygenase 1 (HO-1) thus we explored the effect of these transcription factors on sFlt-1 secretion from human cytotrophoblasts. We examined the ability of sulfasalazine to reduce key markers of endothelial dysfunction and dilate whole blood vessels. We demonstrate sulfasalazine administration reduces sFlt-1 and sENG and upregulates PlGF secretion from human placental tissues. Furthermore sulfasalazine mitigates endothelial dysfunction in several in vitro/ex vivo assays. It enhanced endothelial cell migration and proliferation, promoted blood vessel dilation (vessels obtained from women at caesarean section) and angiogenic sprouting from whole blood vessel rings. The effect of sulfasalazine on the secretion of sFlt-1 was not mediated through either the NFkB or HO-1 pathways. We conclude that sulfasalazine reduces sFlt-1 and sENG secretion and endothelial dysfunction and upregulates PlGF. Sulfasalazine has potential to treat or prevent preeclampsia and warrants investigation in clinical trials. This work was funded by The National Health and Medical Research Council of Australia (NHMRC; #1048707, #1046484. #1101871, #1064845), an Arthur Wilson RANZCOG scholarship and a Norman Beischer Medical Research Foundation grant. FB was supported by a NHMRC Early Career Fellowship (NHMRC #1142636). NJH was supported by a CR Roper Research Fellowship. The NHMRC provided salary support (#1136418 to ST #1062418 to TKL, #1064845 to SS). The funders had no role in study design, data collection, analysis, decision to publish or the preparation of the manuscript.

Brownfoot FC ，Hannan NJ ，Cannon P ，Nguyen V ，Hastie R ，Parry LJ ，Senadheera S ，Tuohey L ，Tong S ，Kaitu'u-Lino TJ ... -  《EBioMedicine》

Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

Preeclampsia is associated with placental ischemia/hypoxia and secretion of soluble fms-like tyrosine kinase 1 and soluble endoglin into the maternal circulation. This causes widespread endothelial dysfunction that manifests clinically as hypertension and multisystem organ injury. Recently, small molecule inhibitors of hypoxic inducible factor 1α have been found to reduce soluble fms-like tyrosine kinase 1 and soluble endoglin secretion. However, their safety profile in pregnancy is unknown. Metformin is safe in pregnancy and is also reported to inhibit hypoxic inducible factor 1α by reducing mitochondrial electron transport chain activity. The purposes of this study were to determine (1) the effects of metformin on placental soluble fms-like tyrosine kinase 1 and soluble endoglin secretion, (2) to investigate whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion are regulated through the mitochondrial electron transport chain, and (3) to examine its effects on endothelial dysfunction, maternal blood vessel vasodilation, and angiogenesis. We performed functional (in vitro and ex vivo) experiments using primary human tissues to examine the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from placenta, endothelial cells, and placental villous explants. We used succinate, mitochondrial complex II substrate, to examine whether the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion were mediated through the mitochondria. We also isolated mitochondria from preterm preeclamptic placentas and gestationally matched control subjects and measured mitochondrial electron transport chain activity using kinetic spectrophotometric assays. Endothelial cells or whole maternal vessels were incubated with metformin to determine whether it rescued endothelial dysfunction induced by either tumor necrosis factor-α (to endothelial cells) or placenta villous explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced angiogenesis. Metformin has potential to prevent or treat preeclampsia.

Brownfoot FC ，Hastie R ，Hannan NJ ，Cannon P ，Tuohey L ，Parry LJ ，Senadheera S ，Illanes SE ，Kaitu'u-Lino TJ ，Tong S ... -  《-》

Esomeprazole and sulfasalazine in combination additively reduce sFlt-1 secretion and diminish endothelial dysfunction: potential for a combination treatment for preeclampsia.

Binder NK ，Brownfoot FC ，Beard S ，Cannon P ，Nguyen TV ，Tong S ，Kaitu'u-Lino TJ ，Hannan NJ ... -  《-》