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》

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 ... -  《-》

Heme Oxygenase-1 Is Not Decreased in Preeclamptic Placenta and Does Not Negatively Regulate Placental Soluble fms-Like Tyrosine Kinase-1 or Soluble Endoglin Secretion.

Elevated placental release of the antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sENG), is central to the pathophysiology of preeclampsia. It is widely accepted that heme oxygenase-1 (HO-1) is decreased in preeclamptic placenta and negatively regulates sFlt-1 and sENG production. We set out to verify these contentions. There was no difference in HO-1 mRNA or protein levels in preterm preeclamptic placentas (n=17) compared with gestationally matched controls (n=27). In silico analysis of microarray studies did not identify decreased placental HO-1 expression in preeclamptic placenta. Silencing HO-1 in primary trophoblasts did not affect sFlt-1 protein secretion after 24 or 48 hours. Silencing nuclear factor (erythroid-derived 2)-like 2 (transcription factor that upregulates HO-1) in trophoblasts also did not affect sFlt-1 secretion. Administering tin protoporphyrin IX dichloride (HO-1 inhibitor) or cobalt protoporphyrin (HO-1 inducer) into placental explants did not affect sFlt-1 or sENG secretion. Silencing HO-1 in 2 types of primary endothelial cells (human umbilical vein endothelial and uterine microvascular endothelial cells) significantly increased sFlt-1 secretion but not sENG secretion. However, HO-1 silencing selectively increased mRNA expression of sFlt-1 i13 (generically expressed sFlt-1 variant) but not of sFlt-1 e15a (sFlt-1 variant mainly expressed in placenta). Furthermore, adding tin protoporphyrin IX dichloride decreased sFlt-1, whereas adding HO-1 inducers (cobalt protoporphyrin, dimethyl fumarate, and rosiglitazone) either had no effect or increased sFlt-1 or sENG secretion (these trends are opposite to what is expected). We conclude that HO-1 expression is not decreased in preeclamptic placenta and HO-1 does not negatively regulate placental sFlt-1 and sENG secretion in placental or endothelial cells.

Tong S ，Kaitu'u-Lino TJ ，Onda K ，Beard S ，Hastie R ，Binder NK ，Cluver C ，Tuohey L ，Whitehead C ，Brownfoot F ，De Silva M ，Hannan NJ ... -  《-》

Effects of simvastatin, rosuvastatin and pravastatin on soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG) secretion from human umbilical vein endothelial cells, primary trophoblast cells and placenta.

Brownfoot FC ，Tong S ，Hannan NJ ，Hastie R ，Cannon P ，Kaitu'u-Lino TJ ... -  《BMC Pregnancy and Childbirth》

Sofalcone upregulates the nuclear factor (erythroid-derived 2)-like 2/heme oxygenase-1 pathway, reduces soluble fms-like tyrosine kinase-1, and quenches endothelial dysfunction: potential therapeutic for preeclampsia.

Preeclampsia is a severe complication of pregnancy, characterized by hypertension, oxidative stress, and severe endothelial dysfunction. Antiangiogenic factors, soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin, play key pathophysiological roles in preeclampsia. Heme oxygenase-1 (HO-1) is a cytoprotective, antioxidant enzyme reported to be downregulated in preeclampsia. Studies propose that inducing HO-1 may also decrease sFlt-1 production. Sofalcone, a gastric antiulcer agent in clinical use, is known to induce HO-1 in gastric epithelium. We aimed to investigate whether sofalcone induces HO-1 and reduces sFlt-1 release from primary human placental and endothelial cells and blocks endothelial dysfunction in vitro. We isolated human trophoblasts and endothelial cells (human umbilical vein endothelial cells) and also used uterine microvascular cells. We investigated the effects of sofalcone on (1) HO-1 production, (2) activation of the nuclear factor (erythroid-derived 2)-like 2 pathway, (3) sFlt-1 and soluble endoglin release, (4) tumor necrosis factor α-induced monocyte adhesion and vascular cell adhesion molecule upregulation, and (5) endothelial tubule formation. Sofalcone potently increased HO-1 mRNA and protein in both primary trophoblasts and human umbilical vein endothelial cells. Furthermore, sofalcone treatment caused nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 and transactivation of other nuclear factor (erythroid-derived 2)-like 2 responsive genes (NQO1, TXN, and GCLC). Importantly, sofalcone significantly decreased the secretion of sFlt-1 from primary human trophoblasts. Sofalcone potently suppressed endothelial dysfunction in 2 in vitro models, blocking tumor necrosis factor α-induced monocyte adhesion and vascular cell adhesion molecule 1 expression in human umbilical vein endothelial cells. These results indicate that in primary human tissues, sofalcone can potently activate antioxidant nuclear factor (erythroid-derived 2)-like 2/HO-1 pathway, decrease sFlt-1 production, and ameliorate endothelial dysfunction. We propose that sofalcone is a novel therapeutic candidate for preeclampsia.

Onda K ，Tong S ，Nakahara A ，Kondo M ，Monchusho H ，Hirano T ，Kaitu'u-Lino T ，Beard S ，Binder N ，Tuohey L ，Brownfoot F ，Hannan NJ ... -  《-》