Roxanne Hastie

Effects of Pravastatin on Human Placenta, Endothelium, and Women With Severe Preeclampsia

&NA;Preeclampsia is a major pregnancy complication where excess placental release of soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin causes maternal endothelial and multisystem organ injury. Clinical trials have commenced examining whether pravastatin can be used to treat preeclampsia. However, the preclinical evidence supporting pravastatin as a treatment is limited to animal models, with almost no studies in human tissues. Therefore, we examined whether pravastatin reduced sFlt-1 and soluble endoglin secretion and decreased endothelial dysfunction in primary human tissues. Pravastatin reduced sFlt-1 secretion from primary endothelial cells, purified cytotrophoblast cells, and placental explants obtained from women with preterm preeclampsia. It increased soluble endoglin secretion from endothelial cells but did not change secretion from placental explants. The regulation of sFlt-1 by pravastatin seemed to be mediated via the 3-hydroxy-3-methylglutaryl-coenzyme A reductase cholesterol synthesis pathway. Pravastatin also reduced markers of endothelial dysfunction, including vascular cell adhesion molecule-1 expression and leukocyte adhesion on endothelial cells and increased endothelial cell migration and invasion. We also treated 4 patients with preterm preeclampsia presenting at <30 weeks of gestation with daily pravastatin. Pravastatin seemed to stabilize blood pressure, proteinuria, and serum uric acid levels. Furthermore, serum sFlt-1 levels decreased. We collected the placentas at delivery and found that pravastatin reduced sFlt-1 secretion. These results indicate that pravastatin reduced sFlt-1 and soluble endoglin production and decreased endothelial dysfunction in primary human tissues. We also present pilot data, suggesting that pravastatin can stabilize clinical and biochemical features of preterm preeclampsia. Our data obtained in human tissues support the concept that pravastatin is a candidate therapeutic for preeclampsia. Clinical Trial Registration—URL: http://www.anzctr.org.au. Unique identifier: ACTRN12613000268741.

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

BACKGROUND 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. OBJECTIVE 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. STUDY DESIGN 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. RESULTS 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. CONCLUSION 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.

Placental-Specific sFLT-1 e15a Protein Is Increased in Preeclampsia, Antagonizes Vascular Endothelial Growth Factor Signaling, and Has Antiangiogenic Activity

In preeclampsia, the antiangiogenic factor soluble fms-like tyrosine kinase-1 (sFLT-1) is released from placenta into the maternal circulation, causing endothelial dysfunction and organ injury. A recently described splice variant, sFLT-1 e15a, is primate specific and the most abundant placentally derived sFLT-1. Therefore, it may be the major sFLT-1 isoform contributing to the pathophysiology of preeclampsia. sFLT-1 e15a protein remains poorly characterized: its bioactivity has not been comprehensively examined, and serum levels in normal and preeclamptic pregnancy have not been reported. We generated and validated an sFLT-1 e15a–specific ELISA to further characterize serum levels during pregnancy, and in the presence of preeclampsia. Furthermore, we performed assays to examine the bioactivity and antiangiogenic properties of sFLT-1 e15a protein. sFLT-1 e15a was expressed in the syncytiotrophoblast, and serum levels rose across pregnancy. Strikingly, serum levels were increased 10-fold in preterm preeclampsia compared with normotensive controls. We confirmed sFLT-1 e15a is bioactive and is able to inhibit vascular endothelial growth factor signaling of vascular endothelial growth factor receptor 2 and block downstream Akt phosphorylation. Furthermore, sFLT-1 e15a has antiangiogenic properties. sFLT-1 e15a decreased endothelial cell migration, invasion, and inhibited endothelial cell tube formation. Administering sFLT-1 e15a blocked vascular endothelial growth factor induced sprouts from mouse aortic rings ex vivo. We have demonstrated that sFLT-1 e15a is increased in preeclampsia, antagonizes vascular endothelial growth factor signaling, and has antiangiogenic activity. Future development of diagnostics and therapeutics for preeclampsia should consider targeting placentally derived sFLT-1 e15a.

Characterization of protocols for primary trophoblast purification, optimized for functional investigation of sFlt-1 and soluble endoglin

OBJECTIVES Soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng) are the most studied molecules in preeclampsia. However, most trophoblast cell lines do not secrete both these factors. Thus, we set out to characterize protocols to functionally investigate sFlt-1 and sEng from primary trophoblast. STUDY DESIGN Primary trophoblasts were isolated from term placenta by percoll gradient, then negative selection using a CD9 antibody. Purity was assessed by cytokeratin 7 immunostaining. We first examined the effects of CD9 negative selection on sFlt-1, sEng and hCG secretion and the ability of forskolin to enhance syncytialization. We then examined the effects of hypoxia on sFlt-1 production and assessed gene knockdown using siRNA. RESULTS CD9 negative selection produced a pure population of primary trophoblasts. Secretion of sEng was 5-fold lower when CD9-positive cells were removed, sFlt1 was unchanged, and hCG was significantly increased. hCG analysis of the purified population indicated spontaneous syncytialization, which was not enhanced by forskolin. Forskolin similarly did not alter sFlt-1 secretion. Hypoxia significantly increased sFlt-1 secretion as expected. Importantly, high gene silencing efficiencies were readily achieved. CONCLUSION In conclusion, we present a protocol that yields primary trophoblasts of high purity that produce abundant sFlt-1 and low but detectable levels of sEng. Furthermore, these cells are readily amenable to gene silencing by siRNAs and hence suitable for functional studies.

Proton Pump Inhibitors Decrease Soluble fms-Like Tyrosine Kinase-1 and Soluble Endoglin Secretion, Decrease Hypertension, and Rescue Endothelial Dysfunction.

Preeclampsia is a severe complication of pregnancy. Antiangiogenic factors soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin are secreted in excess from the placenta, causing hypertension, endothelial dysfunction, and multiorgan injury. Oxidative stress and vascular inflammation exacerbate the endothelial injury. A drug that can block these pathophysiological steps would be an attractive treatment option. Proton pump inhibitors (PPIs) are safe in pregnancy where they are prescribed for gastric reflux. We performed functional studies on primary human tissues and animal models to examine the effects of PPIs on sFlt-1 and soluble endoglin secretion, vessel dilatation, blood pressure, and endothelial dysfunction. PPIs decreased sFlt-1 and soluble endoglin secretion from trophoblast, placental explants from preeclamptic pregnancies, and endothelial cells. They also mitigated tumor necrosis factor-&agr;–induced endothelial dysfunction: PPIs blocked endothelial vascular cell adhesion molecule-1 expression, leukocyte adhesion to endothelium, and disruption of endothelial tube formation. PPIs decreased endothelin-1 secretion and enhanced endothelial cell migration. Interestingly, the PPI esomeprazole vasodilated maternal blood vessels from normal pregnancies and cases of preterm preeclampsia, but its vasodilatory effects were lost when the vessels were denuded of their endothelium. Esomeprazole decreased blood pressure in a transgenic mouse model where human sFlt-1 was overexpressed in placenta. PPIs upregulated endogenous antioxidant defenses and decreased cytokine secretion from placental tissue and endothelial cells. We have found that PPIs decrease sFlt-1 and soluble endoglin secretion and endothelial dysfunction, dilate blood vessels, decrease blood pressure, and have antioxidant and anti-inflammatory properties. They have therapeutic potential for preeclampsia and other diseases where endothelial dysfunction is involved.

YC-1 reduces placental sFlt-1 and soluble endoglin production and decreases endothelial dysfunction: A possible therapeutic for preeclampsia

Preeclampsia is a serious complication of pregnancy with no medical treatment. It is caused by intermittent placental hypoxia and release of sFlt-1 and soluble endoglin, leading to wide spread maternal endothelial dysfunction and multisystem organ injury. YC-1 is a guanylyl cyclase activator and HIF1α inhibitor developed for use in hypertension and atherosclerosis. We examined whether YC-1 reduces sFlt-1 and sENG secretion and reverses endothelial dysfunction in primary human tissues. YC-1 significantly reduced sFlt-1 and sENG secretion from human umbilical vein endothelial cells, purified primary trophoblast cells and placental explants taken from patients with preterm preeclampsia. This was concordant with reduced HIF1α expression. YC-1 also reversed TNFα induced endothelial dysfunction, including reduced vascular cell adhesion molecule 1 expression and monocyte adhesion to primary endothelial cells. We conclude YC-1 decreases placental production of sFlt-1 and sENG and decreases endothelial dysfunction. It is a novel therapeutic candidate for preeclampsia.

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.

Stability of absolute copy number of housekeeping genes in preeclamptic and normal placentas, as measured by digital PCR

Measuring mRNA expression is fundamental to placental research. Ideally, mRNA transcript numbers are directly quantified. However, PCR analysis using the ΔΔCT method relies on the stability of housekeeping genes and only reports relative expression. Digital PCR (dPCR) directly quantifies mRNA copy number and is more accurate than quantitative PCR. We quantified absolute mRNA copy number of housekeeping genes in normotensive pre-term (n = 20), severe preeclamptic (n = 11) and term (n = 12) placenta using dPCR. Whilst there was some variation, we confirm absolute mRNA copy number of GAPDH, TOP1, CYC1 and YWHAZ in placenta does not significantly alter between these cohorts, or across gestation.

Activating Transcription Factor 3 Is Reduced in Preeclamptic Placentas and Negatively Regulates sFlt-1 (Soluble fms-Like Tyrosine Kinase 1), Soluble Endoglin, and Proinflammatory Cytokines in PlacentaNovelty and Significance

Preeclampsia is a major pregnancy complication associated with poor placental perfusion and placental hypoxia. Systemic and placental inflammation and elevated placental secretion of the antiangiogenic factors sFlt-1 (soluble fms-like tyrosine kinase 1) and sEng (soluble endoglin) are hallmarks of preeclampsia, causing endothelial dysfunction and multiorgan injury. A molecule that links placental hypoxia, inflammation, and antiangiogenic factor release has not been described. ATF3 (activating transcription factor 3) is highly expressed in placenta. We assessed whether placental ATF3 is dysregulated in preterm preeclampsia, is altered by hypoxia, and regulates proinflammatory cytokine and antiangiogenic factor production. ATF3 mRNA and protein expression was significantly reduced in preterm preeclamptic placentas compared with gestation-matched controls. Hypoxia reduced ATF3 expression in primary cytotrophoblast and placental explants. Silencing ATF3 in primary cytotrophoblast increased proinflammatory cytokine (IL-6 [interleukin 6], TNF-&agr; [tumor necrosis factor &agr;]) and NF-&kgr;B (nuclear factor &kgr;B) expression. In silico analysis identified an ATF3–binding site in the promoter of Flt-1 (the transcript from which sFlt-1 is produced). Silencing ATF3 increased sFlt-1 and sEng secretion from primary cytotrophoblast possibly by increasing Rab11a and Arf1, cargo proteins that facilitate exosomal release of sFlt-1. ATF3 knockout mice did not have a preeclampsia phenotype, suggesting that these pathways may be specific to humans (preeclampsia is a uniquely human condition). To conclude, we have shown that ATF3 is decreased in preeclamptic placentas and that this decrease is likely to occur after prolonged hypoxia. We show that ATF3 is a regulator of placental proinflammatory cytokines and antiangiogenic factors sFlt-1 and sEng. Therefore, reduced ATF3 may be centrally involved in the pathology of preeclampsia.

Placental SEMA3B expression is not altered in severe early onset preeclampsia

Recent evidence suggests that Semaphorin 3B (SEMA3B) is upregulated in severe preeclampsia, and a major driver of cytotrophoblast aberrations in this disease. Here we independently assess whether SEMA3B expression is altered in a large cohort of severe early onset preeclamptic placentas. We demonstrate that SEMA3B relative mRNA expression and copy number are not changed in PE placentas. We confirm this at the protein level by western blot. Interestingly, exposure of term trophoblasts or explants to hypoxia induced a significant down regulation of SEMA3B mRNA, but a trend towards increased SEMA3B protein expression. We conclude that SEMA3B mRNA and protein is not altered in severe early onset preeclamptic placentas.