Elsevier

Placenta

Volume 95, June 2020, Pages 78-83
Placenta

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

https://doi.org/10.1016/j.placenta.2020.04.010Get rights and content

Highlights

  • Combination treatment reduced cytotrophoblast sFlt-1 and sENG secretion.

  • Combination treatment increased VEGFα expression.

  • Combination treatment reduced endothelin-1 expression.

Abstract

Introduction

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.

Methods

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).

Results

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.

Discussion

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.

Introduction

Preeclampsia is a common complication of pregnancy characterized by hypertension and multisystem organ involvement [1,2]. An increase in antiangiogenic factors FMS-like tyrosine kinase-1 (sFlt-1) [[3], [4], [5]] and soluble endoglin (sENG) [6] and reduced angiogenic markers placental growth factor (PlGF) and VEGF resulting in endothelial dysfunction are strongly implicated in its pathogenesis [[7], [8], [9]]. Repurposing medications that target the production or secretion of these proteins has been used to identify potential treatments for preeclampsia.

We have identified esomeprazole [10], metformin [16] and sulfasalazine [11] as medications that individually reduce sFlt-1 secretion with varying effects on other cellular processes altered in preeclampsia. We are pursuing clinical trials for each of these medications with the clinical trial for metformin (trial number PACTR201608001752102) [12] and sulfasalazine (trial number ACTRN12617000226303) currently in progress. We demonstrated in a randomized controlled trial that esomeprazole was not an effective treatment for preeclampsia [13]. Perhaps this may have resulted from insufficient drug concentrations reaching the placenta and vascular tissues.

An effective strategy used clinically to improve drug efficacy is to combine agents to obtain an additive or synergist effect. We have investigated the effect of combining esomeprazole and metformin at doses that individually had no effect on sFlt-1 secretion and demonstrated a significant reduction in antiangiogenic factor secretion and endothelial dysfunction [14]. In this study we examined whether combining metformin and sulfasalazine at doses that individually did not reduce sFlt-1 secretion might be additive or indeed synergistic in reducing sFlt-1 and sEng secretion, enhancing VEGF or PlGF, and reducing endothelial dysfunction, compared to either alone.

Section snippets

Materials and methods

We performed functional experiments to investigate whether sulfasalazine and metformin were additive in their ability to reduce antiangiogenic or increase angiogenic factor expression and/or secretion from primary human placental tissues. Specifically, we examined the effect of metformin and sulfasalazine independently and in combination on the placental secretion or expression of sFlt-1, sFlt-1 variants e15a and i13, sENG, PlGF and VEGFα. We also explored whether this combination additively

Isolating and treating primary human cytotrophoblast cells

Term placental tissue were collected from women having elective caesarean sections on the background of uncomplicated pregnancies and human cytotrophoblasts were isolated as previously described [15,16]. Cells were plated at 24,000 cells/cm2 (n = 3–4) and incubated overnight to ensure viable cytotrophoblasts had adhered, washed to remove cell debris and treated. The cells were treated with 200 μM metformin or 200 μM sulfasalazine alone or in combination under 8% O2 and 5% CO2 at 37 °C. After

Combining metformin and sulfasalazine was additive in reducing sFlt-1 secretion from placental cells/tissues

We have previously demonstrated that metformin [16] and sulfasalazine [11] reduce placental sFlt-1 secretion. We examined whether combining them would additively reduce sFlt-1 secretion. At lower doses than previously published metformin did not change sFlt-1 secretion whilst sulfasalazine had a significant but modest effect (Fig. 1A) on sFlt-1 secretion from primary cytotrophoblasts. However, combining these low doses of metformin and sulfasalazine additively reduced sFlt-1 secretion beyond

Discussion

Preeclampsia is a serious complication of pregnancy with no medical treatment. Previously, we have demonstrated metformin [16] and sulfasalazine [11] independently reduce the secretion of key antiangiogenic factors, sFlt-1 and sENG and markers of endothelial dysfunction specific to preeclampsia [11,21]. In this study we examined the effect of combining metformin and sulfasalazine at doses previously shown too low to exert an effect on sFlt-1 secretion. Whilst combination therapy additively

Funding

This work was funded by a Norman Beischer Medical Research Foundation grant. FB was supported by a NHMRC Early Career Fellowship (NHMRC #1142636). The NHMRC provided salary support (#1136418 to ST #1159261 to TKL). NJH was supported by a CR Roper Research Fellowship. The funders had no role in study design, data collection, analysis, decision to publish or the preparation of the manuscript.

Author contributions

FCB, NJH, TKL, and ST designed the experiments and wrote the main manuscript. SB, PC, MD, and TN were involved in data generation. All authors reviewed the manuscript.

Declaration of competing interest

The authors report no conflict of interest.

Acknowledgements

We would like to thank the research midwives, Gabrielle Pell, Genevieve Christophers, Rachel Murdoch and Debra Jinks, and patients at Mercy Hospital for Women for participating in this research.

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