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