Patient population

We performed functional experiments in which we administered metformin to human tissues and assessed its effects on sFlt-1 and sENG secretion, mitochondrial electron transport chain function, and endothelial dysfunction. To perform our experiments, we examined tissues from placenta and blood vessels.

We collected several types of placental tissues. We isolated human umbilical vein endothelial cells (HUVECs) and primary villous cytotrophoblast cells from the placenta and umbilical cord that had been collected from patients at term. We also collected placental villous explants from patients with severe early onset proteinuric preeclampsia (delivered at ≤34 weeks gestation by cesarean delivery) as defined by the American College of Obstetricians and Gynecologists (ACOG) guidelines. Placental biopsy specimens were taken from 4 random placental sites as recommended by the Co-Lab consortium. There was hypertension and proteinuria (>300 mg of protein in a 24-hour urine collection) in all cases. The placental villous explants were prepared, as previously described.

To assess mitochondrial electron transport chain activity, we obtained a placental biopsy specimen from 23 women with severe preterm proteinuric preeclampsia (defined by ACOG 2013 guidelines ) and from 25 gestationally matched normotensive control subjects who had a preterm delivery without evidence of significant chorioamnionitis or maternal comorbidities ( Supplementary Table ). All the women who were diagnosed with preeclampsia had proteinuria. All placental samples were collected from women who underwent a cesarean delivery. Mitochondrial electron transport chain activity studies were performed, as previously described.

We also collected omental tissue from patients who had elective term cesarean delivery and dissected omental arteries, as previously described. These omental arteries were used to assess the effects of metformin on vasodilation (by pressure myography) and angiogenesis (omental artery explant assay), as described later.

This study was approved by The Mercy Health Human Research Ethics Committee (Institutional review board number R11/34; approved on the November 12, 2014); all women gave written informed consent.

In vitro experiments that assessed metformin and mitochondrial electron transport chain activity inhibitors and substrates on sFlt-1 and sENG production and endothelial dysfunction

HUVECs were plated at 24,000 cells/cm ² between passages 2 and 4 and cultured at 37°C in 20% O ₂ and treated for 24 hours. Primary villous cytotrophoblast cells were plated at 24,000 cells/cm ² , incubated overnight to ensure larger viable villous cytotrophoblasts had adhered, washed to remove apoptotic mononuclear syncytiotrophoblast fragments, then treated for 24 hours at 500,000 cells/cm ² and treated for 48 hours at 37°C in 8% O ₂ to assess sFlt-1 and sENG secretion, respectively. Placental villous explants of approximately 20-mg tissue per well (dried weight) were treated for 72 hours at 37°C in 8% O ₂ .

HUVECs, primary villous cytotrophoblast cells, and placental villous explants were treated with 0, 1, 2, and 5 mmol/L metformin (Sigma Chemical Company, St. Louis, MO); HUVECs and primary villous cytotrophoblast cells were treated with 0, 0.5, and 1 mmol/L metformin ± 25 mmol/L succinate (mitochondrial electron transport chain substrate; Sigma Chemical Company). Primary villous cytotrophoblast cells were also treated with mitochondrial electron transport chain inhibitors rotenone (Sigma Chemical Company) at 0, 0.625, 1.25, 2.5, and 5 μmol/L or antimycin (Sigma Chemical Company) at 0, 0.156, 0.31, 0.63, and 1.25 μmol/L.

Endothelial dysfunction was induced in (1) HUVECs that used a constant dose of 10ng/mL tumor necrosis factor α (TNFα; Sigma Chemical Company), (2) whole omental arteries with the use of 25% conditioned placental villous explant media (this media is collected 24 hours after being cultured with placental villous explants that were obtained from normal pregnancies at term), and (3) omental artery explants that used 250 ng/mL sFlt-1 at 37°C at 20% O ₂ . Metformin was administered simultaneously to HUVECs at 0, 1, 2, or 5 mmol/L for 24 hours, to whole omental arteries at 0 and 5 mmol/L for 3 hours, and omental artery explants at 1 mmol/L for 120 hours.

Measurement of sFlt-1, sENG, and VCAM-1, sFlt-1 e15a and i13

Conditioned media were collected, and RNA was extracted with the RNeasy mini kit (Qiagen, Valencia, CA) from functional experiments and HUVEC endothelial dysfunction assay. Enzyme-linked immunosorbent assay for sFlt-1 and sENG was performed with the DuoSet VEGF R1/Flt-1 kit (R&D Systems by Bioscience, Waterloo, Australia) and a DuoSet Human Endoglin CD/105 ELISA kit (R&D Systems), respectively. RNA was quantified with the Nanodrop ND 1000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE). RNA (0.2 μg) was converted to complementary DNA with the use of Applied Biosystems high capacity cDNA reverse transcriptase kit (Life Technologies, Mulgrave, Australia). Sybr gene expression assay for sFlt-1 e15a and sFlt-1 i13 (Geneworks, South Australia, Australia) was performed, and a taqman gene expression assay was used for VCAM-1 (Life Technologies).

Assessment of the effect of metformin on whole omental artery vasodilation

Treated whole omental arteries were mounted on a pressure myograph organ bath (Living Systems Instrumentation, Burlington, VT). Incremental doses of 0.01 nmol/L to 1μmol/L bradykinin (Auspep, West Melbourne, Australia) were infused, and vasodilation was assessed with video microscopy (Diamtrak Software, Adelaide, SA, Australia).

Assessment of the effect of metformin on angiogenesis with the use of omental artery explants

Omental artery explants (0.5-mm rings) were stained with calcein AM (Merck Millipore, Darmstadt, Germany) and imaged at ×40 magnification with the EVOS FL microscope (Life Technologies); outgrowth was assessed with image J ( http://imagej.nih.gov/ij/ ).

Statistical analysis

Technical triplicates were performed for each experiment, with a minimum of 3 biologic replicates for each in vitro study (samples from different patients were used for each biologic replicate). When 2 groups were analyzed, a t -test (parametric) or a Mann-Whitney test (nonparametric data) was used. When ≥3 groups were compared, a 1-way analysis of variance test (parametric) or a Kruskal-Wallis test (non-parametric) was used. Statistical analysis was done with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). All data are expressed as mean ± SEM; probability values of <.05 were considered significant.

Detailed methods are included in the supplementary “Methods” section.

Patient population

We performed functional experiments in which we administered metformin to human tissues and assessed its effects on sFlt-1 and sENG secretion, mitochondrial electron transport chain function, and endothelial dysfunction. To perform our experiments, we examined tissues from placenta and blood vessels.

We collected several types of placental tissues. We isolated human umbilical vein endothelial cells (HUVECs) and primary villous cytotrophoblast cells from the placenta and umbilical cord that had been collected from patients at term. We also collected placental villous explants from patients with severe early onset proteinuric preeclampsia (delivered at ≤34 weeks gestation by cesarean delivery) as defined by the American College of Obstetricians and Gynecologists (ACOG) guidelines. Placental biopsy specimens were taken from 4 random placental sites as recommended by the Co-Lab consortium. There was hypertension and proteinuria (>300 mg of protein in a 24-hour urine collection) in all cases. The placental villous explants were prepared, as previously described.

To assess mitochondrial electron transport chain activity, we obtained a placental biopsy specimen from 23 women with severe preterm proteinuric preeclampsia (defined by ACOG 2013 guidelines ) and from 25 gestationally matched normotensive control subjects who had a preterm delivery without evidence of significant chorioamnionitis or maternal comorbidities ( Supplementary Table ). All the women who were diagnosed with preeclampsia had proteinuria. All placental samples were collected from women who underwent a cesarean delivery. Mitochondrial electron transport chain activity studies were performed, as previously described.

We also collected omental tissue from patients who had elective term cesarean delivery and dissected omental arteries, as previously described. These omental arteries were used to assess the effects of metformin on vasodilation (by pressure myography) and angiogenesis (omental artery explant assay), as described later.

This study was approved by The Mercy Health Human Research Ethics Committee (Institutional review board number R11/34; approved on the November 12, 2014); all women gave written informed consent.

In vitro experiments that assessed metformin and mitochondrial electron transport chain activity inhibitors and substrates on sFlt-1 and sENG production and endothelial dysfunction

HUVECs were plated at 24,000 cells/cm ² between passages 2 and 4 and cultured at 37°C in 20% O ₂ and treated for 24 hours. Primary villous cytotrophoblast cells were plated at 24,000 cells/cm ² , incubated overnight to ensure larger viable villous cytotrophoblasts had adhered, washed to remove apoptotic mononuclear syncytiotrophoblast fragments, then treated for 24 hours at 500,000 cells/cm ² and treated for 48 hours at 37°C in 8% O ₂ to assess sFlt-1 and sENG secretion, respectively. Placental villous explants of approximately 20-mg tissue per well (dried weight) were treated for 72 hours at 37°C in 8% O ₂ .

HUVECs, primary villous cytotrophoblast cells, and placental villous explants were treated with 0, 1, 2, and 5 mmol/L metformin (Sigma Chemical Company, St. Louis, MO); HUVECs and primary villous cytotrophoblast cells were treated with 0, 0.5, and 1 mmol/L metformin ± 25 mmol/L succinate (mitochondrial electron transport chain substrate; Sigma Chemical Company). Primary villous cytotrophoblast cells were also treated with mitochondrial electron transport chain inhibitors rotenone (Sigma Chemical Company) at 0, 0.625, 1.25, 2.5, and 5 μmol/L or antimycin (Sigma Chemical Company) at 0, 0.156, 0.31, 0.63, and 1.25 μmol/L.

Endothelial dysfunction was induced in (1) HUVECs that used a constant dose of 10ng/mL tumor necrosis factor α (TNFα; Sigma Chemical Company), (2) whole omental arteries with the use of 25% conditioned placental villous explant media (this media is collected 24 hours after being cultured with placental villous explants that were obtained from normal pregnancies at term), and (3) omental artery explants that used 250 ng/mL sFlt-1 at 37°C at 20% O ₂ . Metformin was administered simultaneously to HUVECs at 0, 1, 2, or 5 mmol/L for 24 hours, to whole omental arteries at 0 and 5 mmol/L for 3 hours, and omental artery explants at 1 mmol/L for 120 hours.

Measurement of sFlt-1, sENG, and VCAM-1, sFlt-1 e15a and i13

Conditioned media were collected, and RNA was extracted with the RNeasy mini kit (Qiagen, Valencia, CA) from functional experiments and HUVEC endothelial dysfunction assay. Enzyme-linked immunosorbent assay for sFlt-1 and sENG was performed with the DuoSet VEGF R1/Flt-1 kit (R&D Systems by Bioscience, Waterloo, Australia) and a DuoSet Human Endoglin CD/105 ELISA kit (R&D Systems), respectively. RNA was quantified with the Nanodrop ND 1000 spectrophotometer (NanoDrop Technologies Inc, Wilmington, DE). RNA (0.2 μg) was converted to complementary DNA with the use of Applied Biosystems high capacity cDNA reverse transcriptase kit (Life Technologies, Mulgrave, Australia). Sybr gene expression assay for sFlt-1 e15a and sFlt-1 i13 (Geneworks, South Australia, Australia) was performed, and a taqman gene expression assay was used for VCAM-1 (Life Technologies).

Assessment of the effect of metformin on whole omental artery vasodilation

Treated whole omental arteries were mounted on a pressure myograph organ bath (Living Systems Instrumentation, Burlington, VT). Incremental doses of 0.01 nmol/L to 1μmol/L bradykinin (Auspep, West Melbourne, Australia) were infused, and vasodilation was assessed with video microscopy (Diamtrak Software, Adelaide, SA, Australia).

Assessment of the effect of metformin on angiogenesis with the use of omental artery explants

Omental artery explants (0.5-mm rings) were stained with calcein AM (Merck Millipore, Darmstadt, Germany) and imaged at ×40 magnification with the EVOS FL microscope (Life Technologies); outgrowth was assessed with image J ( http://imagej.nih.gov/ij/ ).

Statistical analysis

Technical triplicates were performed for each experiment, with a minimum of 3 biologic replicates for each in vitro study (samples from different patients were used for each biologic replicate). When 2 groups were analyzed, a t -test (parametric) or a Mann-Whitney test (nonparametric data) was used. When ≥3 groups were compared, a 1-way analysis of variance test (parametric) or a Kruskal-Wallis test (non-parametric) was used. Statistical analysis was done with GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). All data are expressed as mean ± SEM; probability values of <.05 were considered significant.

Detailed methods are included in the supplementary “Methods” section.

Metformin reduces sFlt-1 secretion from primary endothelial cells and placental tissues

We assessed the effects of metformin on sFlt-1 secretion from endothelial and placental tissues because they are its main tissue source. Administering metformin dose-dependently reduced sFlt-1 secretion from endothelial cells (HUVECs; Figure 1 , A) and primary cells that were isolated from placenta (villous cytotrophoblast cells; Figure 1 , B). At the highest doses, metformin reduced endothelial and placental cell secretion by 53% and 63%, respectively. Metformin also reduced sFlt-1 secretion from placental villous explants that were obtained from 4 women who had been diagnosed with preterm preeclampsia (delivery required <34 weeks gestation; Figure 1 , C).

Effect of metformin on soluble fms-like tyrosine kinase 1 secretion and isoforms e15a and i13 expression in endothelial cells and placental tissue

Metformin (0, 1, 2, 5 mmol/L) dose-dependently reduced soluble fms-like tyrosine kinase 1 secretion from A, endothelial cells, B, villous cytotrophoblast cells, and C, preterm preeclamptic placental villous explants. Metformin reduced endothelial cell expression of D, sFlt-1 i13 isoform, E, villous cytotrophoblast cells, and F, preterm preeclamptic placental villous explant messenger RNA expression of sFlt-1 e15a . The single asterisk indicates P < .05; the double asterisks indicate P < .01; the triple asterisks indicate P < .0001; the quadruple asterisks indicate P < .00001.

sFlt-1 , soluble fms-like tyrosine kinase 1.

Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016 .

We investigated the effect of metformin on messenger RNA (mRNA) expression of different sFlt-1 variants in the cells, or placental villous explant tissues. sFlt-1 i13 is the most abundant sFlt-1 variant in endothelial cells. Metformin dose-dependently reduced sFlt-1 i13 mRNA expression in endothelial cells ( Figure 1 , D). sFlt-1 e15a is the predominant variant expressed in human placenta. Metformin reduced sFlt-1 e15a mRNA expression in primary villous cytotrophoblasts cells ( Figure 1 , E) and placental villous explants ( Figure 1 , F) that were obtained from women with preterm preeclampsia. Thus, we conclude that metformin reduces sFlt-1 isoform expression and sFlt-1 secretion in endothelial and placental cells/tissues, including placental villous explants from patients diagnosed with preterm preeclampsia.

Metformin reduces sENG secretion from primary endothelial and placental tissues

We next investigated the effects of metformin on sENG secretion from primary endothelial cells and placental cells/tissues. Metformin dose-dependently reduced sENG secretion from HUVECs ( Figure 2 , A) and primary villous cytotrophoblast cells ( Figure 2 , B). Metformin induced a trend towards a reduction in sENG secretion from preterm preeclamptic placental villous explants at 3 doses, but none of these decreases were significant ( Figure 2 , C).

Effect of metformin on soluble endoglin secretion from endothelial cells and placental tissue

Metformin (0, 1, 2, and 5 mmol/L) reduced soluble endoglin secretion from A, endothelial cells and B, villous cytotrophoblast cells. Metformin did not change soluble endoglin secretion from C, preterm preeclamptic placental villous explants. The single asterisk indicates P < .05; the double asterisks indicate P < .01; the quadruple asterisks indicate P < .00001.

sENG , soluble endoglin.

Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016 .

Metformin reduces sFlt-1 and sENG secretion by inhibiting the mitochondrial electron transport chain

Given that metformin inhibits mitochondrial electron transport chain activity by blocking complex I, we examined whether the decrease in sFlt-1 secretion was mediated through mitochondrial electron transport chain inhibition. Succinate is a substrate for complex II of the mitochondria, downstream of the effect of metformin blockade. Thus, if metformin was reducing sFlt-1 secretion by directly blocking complex I, then succinate should restore electron flow and rescue this effect. Indeed, succinate rescued a reduction in sFlt-1 secretion that was induced by endothelial cells ( Figure 3 , A) and primary villous cytotrophoblasts cells ( Figure 3 , B). Succinate also rescued a decrease in sENG secretion that was induced by metformin in primary HUVECs ( Figure 3 , C) and villous cytotrophoblast cells ( Figure 3 , D). These data raise the possibility that the effects of metformin on sFlt-1 and sENG secretion are mediated through its effects on the mitochondria.

Effect of metformin and mitochondrial electron transport chain complex I activity on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from endothelial cells and villous cytotrophoblast cells

The effect of metformin (0.5 and 1 mmol/L) on soluble fms-like tyrosine kinase 1 secretion from A, endothelial cells and B, villous cytotrophoblast cells is likely mediated through mitochondrial electron transport chain complex 1 inhibition because the effect is rescued in the presence of the mitochondrial electron transport chain substrate, succinate (25 mmol/L). Furthermore, metformin also reduced soluble endoglin secretion likely through mitochondrial electron transport chain complex 1 inhibition from C, endothelial cells (0.5, 1 mmol/L) and D, villous cytotrophoblast cells (1 mmol/L) because succinate (25 mmol/L) rescued its effect on secretion. The single asterisk indicates P < .05; the double asterisks indicate P < .01; the triple asterisks indicate P < .0001; the quadruple asterisks indicate P < .00001.

sENG , soluble endoglin; sFlt-1 , soluble fms-like tyrosine kinase 1.

Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016 .

Inhibition of the mitochondrial electron transport chain reduces sFlt-1 secretion from primary villous cytotrophoblasts cells

To our knowledge, the concept that the mitochondria regulate sFlt-1 secretion is novel. To obtain further evidence that this is the case, we examined whether other mitochondrial electron transport chain inhibitors reduce sFlt-1 secretion. Administering rotenone, another complex I inhibitor, to primary villous cytotrophoblast cells reduced sFlt-1 secretion by 65% ( Figure 4 , A). Antimycin, a complex III inhibitor, also reduced sflt-1 secretion by 75% ( Figure 4 , B). These doses of rotenone and antimycin did not induce cell death (MTS assay and calcein stain; data not shown). These studies provide further evidence that the mitochondria is involved in the regulation of sFlt-1 secretion.

Mitochondrial electron transport chain inhibitors and sFlt-1 secretion

A, Rotenone (0, 0.62, 1.25, 2.5, and 5 mmol/L), a complex 1 mitochondrial electron transport chain inhibitor, and B, antimycin (0, 0.16, 0.31, 0.63, and 1.25 mmol/L), a complex III mitochondrial electron transport chain inhibitor, both significantly reduce soluble fms-like tyrosine kinase 1 secretion from villous cytotrophoblast cells. The single asterisk indicates P < .05; the double asterisks indicate P < .01; the triple asterisks indicate P < .0001.

sFlt-1 , soluble fms-like tyrosine kinase 1.

Brownfoot et al. Metformin decreases sFlt-1 and sENG, improves endothelial function, and is angiogenic. Am J Obstet Gynecol 2016 .

Mitochondrial electron transport chain activity is up-regulated in preterm preeclamptic placenta

Given the mitochondria appears to positively regulate sFlt-1 and sENG secretion, we hypothesized that preeclamptic placentas might have increased mitochondrial electron transport chain activity. We therefore compared mitochondrial electron transport chain activity in preterm preeclamptic placentas (n = 23) and normotensive gestation matched preterm control placentas (n = 25; Supplementary Table contains baseline characteristics). We observed an increase in mitochondrial electron transport chain activity in the preeclamptic placentas for all 4 complexes, and this was significant for complex II ( Figure 5 ). Therefore, mitochondrial electron transport chain activity may be increased in preterm preeclamptic placenta.

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