Objective
Soluble vascular endothelial growth factor receptor–1 (also know as soluble fms-like tyrosine kinase [sFlt]-1) is a key causative factor of preeclampsia. Resveratrol, a plant phytoalexin, has antiinflammatory and cardioprotective properties. We sought to determine the effect of resveratrol on sFlt-1 release.
Study Design
Human umbilical vein endothelial cells, transformed human trophoblast-8 (HTR/SVneo)-8/SVneo trophoblast cells, or placental explants were incubated with cytokines and/or resveratrol. Conditioned media were assayed for sFlt-1 by enzyme-linked immunosorbent assay and cell proteins used for Western blotting.
Results
Resveratrol inhibited cytokine-induced release of sFlt-1 from normal placental explants and from preeclamptic placental explants. Preincubation of human umbilical vein endothelial cells or HTR-8/SVneo cells with resveratrol abrogated sFlt-1 release. Resveratrol prevented the up-regulation of early growth response protein-1 (Egr-1), a transcription factor necessary for induction of the vascular endothelial growth factor receptor–1 gene and caused up-regulation of heme oxygenase–1, a cytoprotective enzyme found to be dysfunctional in preeclampsia.
Conclusion
In summary, resveratrol can inhibit sFlt-1 release and up-regulate heme oxygenase–1; thus, may offer therapeutic potential in preeclampsia.
Levels of soluble vascular endothelial growth factor (VEGF) receptor–1 (also known as soluble fms-like tyrosine kinase [sFlt]-1) are elevated in pregnancies complicated with preeclampsia and elevated sFlt-1 generated by preeclamptic placenta is responsible for suppressing angiogenesis. Adenoviral delivery of sFlt-1 to pregnant rats mimics the clinical manifestations of preeclampsia and introduction of sFlt-1 into pregnant mice caused hypertension and proteinuria, which could be reversed by VEGF. This latter study demonstrated that below a critical threshold level, sFlt-1 fails to elicit preeclamptic-like symptoms; thus, a therapy that can reduce the circulating levels of free sFlt-1 in women with preeclampsia should alleviate the clinical signs of the condition. Indeed, an elegant study by Thadhani et al recently showed that preeclamptic patients undergoing plasma apheresis resulted in approximately a 35% reduction of circulating sFlt-1 and was accompanied a reduction and stabilization of maternal blood pressure leading to an increase in gestational age.
Heme oxygenase (HO)-1 confers cytoprotection against tissue and cellular injury. Previous studies from our laboratory showed that the HO pathway inhibits cytokine-induced placental damage and the release of sFlt-1, and that HO-1 9 and HO-2 are dysregulated in preeclamptic placenta. Furthermore, HO-1 messenger RNA (mRNA) is decreased in the blood of preeclamptic women at term. Most strikingly, chorionic villous samples (fetal placental cells) from women at just 11 weeks’ gestation who went on to develop preeclampsia showed reduced HO-1 mRNA. Thus, agents capable of substituting for the deficiency or inducing the activity of the HO system and/or reducing the elevated sFlt-1 may have therapeutic potential for alleviating the severity of preeclampsia and, in turn, prolong pregnancy, thereby reducing the complication burden for both mother and neonate.
Resveratrol ( trans -3,4′,5-trihydroxystilbene) is an antimicrobial agent produced by plants found in a number of fruits, with the most abundant natural source being the skin of grapes. Resveratrol is a cardioprotective agent with antiinflammatory and antioxidant abilities. It can inhibit low-density lipoprotein oxidation and platelet aggregation, down-regulate tissue factor expression, and suppress agonist-induced monocyte adhesion to human umbilical vein endothelial cells (HUVEC). Moreover, resveratrol was shown to modulate nitric oxide (NO) production, up-regulate endothelial NO synthase, prevent generation of reactive oxygen species, and up-regulate HO-1 expression. As an anticancer agent, resveratrol can restrain tumor initiation and progression, and suppress metastasis.
Resveratrol has been safely administered to healthy volunteers at very high doses and caused no serious adverse effects. In rats, embryo-fetal toxicity studies showed no adverse effects of high-dose resveratrol, and daily administration to mice from gestational day 8 onward did not affect fetal/placental growth and even reversed dioxin-induced teratogenicity. In addition, in a murine model of diabetic pregnancy, resveratrol was found to prevent embryonic oxidative stress and apoptosis and improve the glucose and lipid profile of diabetic mothers. Thus, it is likely that resveratrol would be a safe and beneficial drug to give during pregnancy, but proof-of-concept studies and toxicology would evidently need to be rigorous before therapeutic use is initiated.
In this study, we investigated the effects of resveratrol on sFlt-1 and HO-1 expression in placental villous explants, trophoblasts, and endothelial cells. We show that resveratrol significantly inhibits sFlt-1 release and up-regulates HO-1, thus demonstrating that resveratrol has the potential to be investigated as a therapeutic agent for preeclampsia.
Materials and Methods
Reagents and antibodies
Recombinant VEGF was purchased from ReliaTech (Braunschweig, Germany). Antibodies for the sFlt-1 enzyme-linked immunosorbent assay (ELISA) were purchased from R and D Systems (Abingdon, UK). Early growth response protein-1 (Egr-1) antibody was from Autogen Bioclear Ltd (Wiltshire, UK). Polyclonal rabbit anti-HO-1 antibody was purchased from StressGen Biotechnologies Corp (Victoria, British Columbia, Canada). Angiotensin II, phorbol-12-myristate-13-acetate (PMA), resveratrol, tumor necrosis factor (TNF)-α, interferon-γ, mouse anti-β-actin, and all other cell culture reagents were purchased from Sigma-Aldrich Ltd (Dorset, UK).
Cell culture
HUVEC were isolated from umbilical cords, characterized, and cultured as previously described. The transformed human trophoblast-8 (HTR-8/SVneo) cell line were grown in Roswell Park Memorial Institute (RMPI) medium containing 10% fetal calf serum (FCS).
Placental tissue collection and explant culture
Human placental tissue was obtained from normal pregnancies and gestationally matched pregnancies complicated by preeclampsia. Preeclampsia was defined as blood pressure >140/90 mm Hg on at least 2 consecutive measurements and proteinuria of at least 300 mg per 24 hours. Informed consent was obtained from the patients and the study had the approval of the South Birmingham Ethical Committee (Birmingham, UK) and the Edinburgh Reproductive Tissue Bio-Bank (Edinburgh, UK). Villous explants were prepared as described previously and incubated in the presence or absence of resveratrol (100 μmol/L), VEGF (20 ng/mL), TNF-α (50 ng/mL), or under hypoxia (1% oxygen) and conditioned media assayed for sFlt-1 by ELISA.
ELISA for sFlt-1
sFlt-1 levels in culture supernatants were measured as previously described.
MTT assay for cell viability
HUVEC were seeded at a density of 5 × 10 4 cells/well in a 96-well plate, left to attach for 4 hours, and then rested overnight in medium containing 5% FCS. Cells were incubated with resveratrol for 24 hours, then 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL) was added and cells incubated in the dark at 37°C for 4 hours. MTT was aspirated and reconstituted in dimethyl sulfoxide (DMSO). Optical density values were measured at 540 and 690 nm.
Western blotting
Proteins were extracted from cells and subjected to Western blot analysis as previously described. HUVEC were either preincubated with resveratrol (100 μmol/L) for 30 minutes prior to addition of PMA (10 nmol/L) for 2 and 4 hours or incubated for 24 hours with resveratrol, and then lysates in radioimmunoprecipitation assay buffer were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 15% gels and Western blotted with rabbit anti-Egr-1 antibody (Autogen Bioclear Ltd) or rabbit anti-HO-1 antibody (StressGen Biotechnologies Corp).
Quantitative real-time polymerase chain reaction
Sample preparation and real-time polymerase chain reaction was performed as described previously. Briefly, mRNA was prepared using TRIzol and DNase-1 digestion/purification on RNAeasy columns (Qiagen, West Sussex, UK), and reverse transcribed with the complementary DNA (cDNA) synthesis kit (Promega, Hampshire, UK). Triplicate cDNA samples and standards were amplified in SensiMix containing SYBR green (Quantace, London, UK) with primers specific for HO-1 ( sense : 5′-GGG TGA TAG AAG AGG CCA AGA CT-3′ and anti-sense : 5′-GCA GAA TCT TGC ACT TTG TTG CT-3′) or β-actin. The mean threshold cycle (C T ) for each HO-1 was normalized to β-actin and expressed relative to control.
Statistical analysis
All data are expressed as mean ± SEM. For placental studies, data are from 3 normal and 3 preeclamptic placentas and experiments were performed in duplicate. For HUVEC, all experiments were performed on cells from 3 separate pooled preparations of HUVEC, and each stimulation was performed in triplicate on the cell preparations. For the cell line, HTR-8/SVneo, 3 experiments were performed in duplicate. Blots are representative of 3 experiments. Statistical comparisons were performed using 1-way analysis of variance followed by the Student-Newman-Keuls test as appropriate. Statistical significance was set at a value of P < .05.
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
