Preeclampsia (PE) is a pregnancy-related disorder characterized by hypertension and proteinuria that affects 3–10% of all pregnancies. Although its pathophysiology remains obscure, placental hypoxia-induced oxidative stress and alterations in vascular function, morphology, and endothelial barrier integrity are considered to play a key role in the development of preeclampsia. In this study, placental villous explants of noncomplicated placentae and BeWo cells were subjected to hypoxia. The effect of placental hypoxic-conditioned medium (HCM) on intraluminal-induced contraction and endothelial barrier integrity in chorionic arteries was investigated using pressure myography. The impact of BeWo cell HCM on endothelial cell viability, reactive oxygen species formation and inflammation was also determined. Alterations in arterial morphology and contractile responsiveness to the thromboxane A2 analog (U46619) after exposure to placental HCM were examined immunohistochemically and by wire myography, respectively. Intraluminal administration of placental HCM induced vasoconstriction and increased the endothelial permeability for KCl, which was concentration-dependently prevented by quercetin. Placental and BeWo cell HCMs decreased endothelial cell viability, increased the production of reactive oxygen species and enhanced the secretion of IL-6 and IL-8. The cross-sectional area of the arterial media was increased upon exposure to placental HCM, which was associated with increased vascular proliferation and contractile responsiveness to U46619, and all of these effects were prevented by the antioxidants quercetin and RRR-α-tocopherol. This study is the first to comprehensively demonstrate the link between factors secreted by placental cells in response to hypoxia and vascular abnormalities and paves the way for new diagnostic approaches and therapies to better protect the maternal vasculature during and after a preeclampsia-complicated pregnancy.
Preeclampsia (PE) is a pregnancy-related disorder that affects an estimated 3–10% of all pregnancies worldwide. PE is often associated with fetal intrauterine growth restriction and remains a leading cause of maternal death and perinatal morbidity . As its etiology and pathophysiology are not fully understood, aside from the delivery of the fetus, there is currently no available treatment.
One of the pathophysiological hallmarks of PE is severe maternal hypertension, which is occasionally associated with proteinuria and edema in the late second or third trimester . Importantly, there are two subtypes of PE: early- and late-onset PE. Early-onset PE, also known as severe PE, is widely acknowledged to involve a defect in placentation, while late-onset PE may center around interactions between senescence of the placenta and a maternal genetic predisposition to cardiovascular and metabolic diseases . The pathophysiological processes that have been proposed to underlie early-onset PE include defective placentation and apoptosis of invasive cytotrophoblasts, which cause inadequate remodeling of the spiral arteries and lead to a decrease in blood flow to the placenta [3,4,5]. Subsequent placental ischemia/hypoxia and the resultant oxidative stress  is believed to result in the production of a variety of placental secreted factors that collectively have profound effects on blood flow and arterial pressure regulation. These include pro- and anti-angiogenic factors such as soluble fms-like tyrosine kinase-1 (sFlt-1; an endogenous inhibitor of vascular endothelial growth factor), pro-inflammatory cytokines including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and vascular contractile compounds such as angiotensin II type 1 receptor agonistic autoantibody (AT1-AA) [1, 6,7,8,9,10]. In animal studies, reduced uterine perfusion (as demonstrated in PE) has been shown to induce hypertension [11,12,13]. In line with these studies, we previously demonstrated that factors released by a hypoxic placenta increase vasoconstriction via the angiotensin II receptor type 1 (AT1) and endothelin 1 (ET-1) receptors and increase vascular contractility to a thromboxane-A2 (TXA2) agonist analog (U46619) in chorionic arteries mounted in a wire myograph. When the exposure of placental explants to hypoxia was combined with the flavonoid quercetin, the acute and sustained vascular effects could be prevented in a concentration-dependent manner . Whether these effects on vascular contractility were endothelial-dependent remains to be elucidated, since the myograph setting used in our previous work is not intraluminal-specific .
Quercetin was selected for this study since it is the most abundant flavonoid found in our diet. To confirm whether the protective effect of quercetin is due to its antioxidant properties, RRR-α-tocopherol, which is the naturally occurring and most active form and has already been used as a fertility enhancement and to improve uterine artery blood flow, was tested in our study [14,15,16]. The parameters used in our study may serve as a tool to explore the benefits of dietary compounds on specific targets central to the pathogenesis of PE. There is growing attention being paid to the dietary-directed prevention of diseases, and consumption of these supplements with the diet appears to be safe [17, 18]. RRR-α-tocopherol is a fat-soluble nutrient that is extremely important from the time of conception to the postnatal development of the infant. The mechanisms involved in the placental uptake of RRR-α-tocopherol appear to be allowed by the presence of lipoprotein receptors (LDL-receptor, VLDL-receptor, scavenger receptor class B type I) together with lipoprotein lipase at the placental barriers . The ingestion of colostrum, which contains very high levels of RRR-α-tocopherol, is therefore of utmost importance to supply the newborn with an essential defense against oxygen toxicity . The use of RRR-α-tocopherol as a therapeutic agent in PE has been discussed in numerous papers . Our data will help in the selection of effective bioactives and the subsequent establishment of dietary recommendations to treat/prevent vascular consequences of PE.
In addition to the direct effects on contractility of the vessels during pregnancy, resulting in maternal hypertension, a preeclamptic pregnancy may also irreversibly modify the maternal vascular system, leaving the mother at increased risk for cardiovascular diseases in later life. Indeed, strikingly, women who have experienced PE are twice as likely to succumb to cardiovascular-related events in later life than women who have had normal pregnancies ; the reason for this remains unclear . Using laser Doppler and iontophoresis, impaired function of the endothelium and smooth muscle of the skin microvasculature in women who experienced PE was demonstrated 15–25 years postpartum . In the major blood vessel of the upper arm (brachial artery), endothelial function was found to be impaired in women in their early postpartum period (median, 3 years), and this condition was ameliorated by use of the systemically acting antioxidant ascorbic acid .
Understanding the link between a hypoxic placenta and its resultant vascular abnormalities could help in the identification of new serum biomarkers for the prediction of PE and in the design of new diagnostic approaches for better clinical management. Such research will also improve our knowledge of the sustained maternal vascular consequences, opening new postpartum treatment and follow-up strategies. Because of the difficulty of performing mechanistic studies on the complex interplay between the placenta and the maternal vascular system in pregnant women, an in vitro setting using human placentae and chorionic arteries (as used in our study) serves as a physiologically relevant tool. As recently recognized by Burton et al., the synergistic effect of placental secreted factors may explain why PE has proved so hard to treat, as it is likely that the peripheral aspects of the syndrome are caused by a complex mix of factors rather than any one mediator alone .
The aim of the present study, therefore, was to examine the effect of placental hypoxic-conditioned medium (HCM) on vascular function, morphology, and endothelial barrier integrity. To investigate whether the alterations in endothelial barrier integrity found were trophoblast specific, the effect of both placental and BeWo cells HCM on endothelial viability, inflammation, and oxidative stress was tested.
Materials and methods
Clinical subject characteristics
To generate placental-conditioned medium (CM), three term placentae were collected from cesarean deliveries by the Department of Obstetrics and Gynaecology at Maastricht University Medical Center+. The exclusion criteria were hypertension (pre-existing or exhibiting an onset during pregnancy) with or without proteinuria as defined by the International Society for the Study of Hypertension in Pregnancy (ISSHP), fetal chromosomal anomalies, multiple gestations, obesity and (pre)gestational diabetes. In addition, 66 placentae were used for the isolation of chorionic arteries. Chorionic arteries were chosen, since they contain AT-1, ET-1, serotonin (5-HT), and TXA2 receptors and thus are relevant with respect to identifying vasoactive compounds related to PE . The umbilical-placental vessels have the particular property of lacking autonomic innervation, which in fact permits the investigation of direct actions of vasoactive compounds and drugs . All experiments were approved by the Medical Ethics Committee Academic Hospital Maastricht and Maastricht University (METC 16-4-047).
Villous explant isolation and collection of placental CM
As described earlier by Vangrieken et al. , three placentae from noncomplicated cesarean deliveries were processed directly after delivery. For each collected placenta, five villous explants of 30 g were collected from the central region of the placenta at the maternal side. Subsequently, the basal plate of the specimens was removed, and the remaining tissue was rinsed in a HEPES solution (NaCl 143.3 mM, KCl 4.7 mM, MgSO4 1.2 mM, KH2PO4 1.2 mM, CaCl2 2.5 mM, glucose 5.5 mM, and HEPES 15 mM). Specimens (15 in total) were then separately transferred into a bottle containing 200 ml prewarmed (37 °C) HEPES buffer, kept in a warm water bath (37 °C) and aerated under standard culture conditions (21% O2, referred to as the control condition) or under hypoxic conditions (1% O2) in the presence/absence of 3 or 10 µM quercetin (which is comparable with the plasma levels of quercetin found in mice upon nutritional supplementation ) or 20 µM RRR-α-tocopherol (the relevant physiological plasma concentration upon nutritional supplementation of the naturally occurring and most active α-tocopherol stereoisomer ) for 3 h. Solutions were then centrifuged at 500 g for 10 min at 4 °C. The resultant placental CM was stored in aliquots of 3 ml at −80 °C until use.
Mounting of chorionic arteries into a pressure myograph
For pressure myography, freshly isolated chorionic arteries of 100–400 µm width and ≥1 cm length were used. A stainless-steel wire (diameter of 40 µm) was inserted through the lumen of the arterial segment. The isolated artery was cleaned by cutting away the connective perivascular tissue, stored in HEPES buffer (4 °C) and used within 24 h after isolation. Subsequently, a dual vessel chamber (CH-2, Living Systems Instrumentation, Vermont, USA) was used. The vessel chamber was filled with 7 ml HEPES buffer and kept at 37 °C by a temperature controller for the dual chamber (TC-09D Living Systems Instrumentation). The isolated artery was then mounted onto two glass cannulas of the vessel chamber. The artery was secured using two double knotted strings on both cannulas. Simultaneously, a pressure servo controller with a peristaltic pump (PS-200, Living Systems Instrumentations) was connected to the cannulas. The pressure pump was infused with HEPES buffer at 10 mmHg and subsequently increased to an intraluminal pressure of 50 mmHg, which was kept constant during the experiments. To infuse solutions of interest, a flow control peristaltic pump (FC, Living Systems Instrumentation) was used at a flow rate of 210 µl/min. A video camera (TVC, Living Systems Instrumentation) on the inverted microscope visualized the artery in the vessel chamber. A video dimension analyzer (VDA-10, Living Systems Instrumentation) connected to the monitor measured the lumen diameter. The diameter change was expressed as the delta diameter change (µm). Data measured by the VDA were transferred (Data acquisition transformer, Living System Instrumentation) and analyzed with the use of Wintech data acquisition (ASUS).
Testing vascular contractile responsiveness and endothelial barrier integrity upon intraluminal exposure to placental CM
To determine the maximal contraction, chorionic arteries were first extraluminally precontracted by KCl (62.5 mM). Subsequently, after complete relaxation, chorionic arteries were intraluminally exposed to KCl (62.5 mM) and flushed with HEPES for baseline conditions. Next, chorionic arteries were infused intraluminally for 2 h with undiluted placental CM of the control culture condition (21% O2, control-conditioned medium: CCM), the hypoxic condition (1% O2, hypoxic-conditioned medium: HCM) or the CM of placenta exposed to hypoxia in the presence of 3 or 10 µM quercetin (n = 8 for each condition). To determine the vascular contractile effect of placental CM, the arterial diameter was recorded after 2 h. Chorionic arteries were subsequently flushed again with HEPES for baseline conditions. Finally, the chorionic arteries were again intraluminally exposed to KCl (62.5 mM). Differences between intraluminal KCl-induced contraction before and after exposure to placental CM were used as a parameter for endothelial barrier integrity.
Chorionic artery isolation and culture exposure to placental CM
Areas from the chorionic plate containing chorionic arteries were macroscopically selected and excised in placentae from both vaginal and cesarean deliveries. The dissected specimens were immediately immersed in HEPES solution (4 °C) and pinned in a Sylgard dish with the fetal side oriented upwards. Chorionic arteries (second- and third-order branches of the umbilical cord) were dissected under a microscope. From each placenta, one isolated artery was cut into arterial segments of 2 mm. Arterial segments were first prewarmed (37 °C) in basal Dulbecco’s Modified Eagle Medium (DMEM; D-glucose and L-glutamine 1 g/L and HEPES and pyruvate 25 mM, GIBCO, Carlsbad, CA, USA) containing antibiotics (1000 U/ml penicillin and 1000 μg/ml streptomycin; both from GIBCO) for 2 × 5 min at 37 °C. Subsequently, arterial segments were cultured under a humidified atmosphere containing 21% O2 and 5% CO2 at 37 °C for 1, 3, or 7 days in a 24-well plate containing 400 µl placental CM and 600 µl basal medium containing antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin) (40% (vol/vol)). The diluted placental CM was refreshed daily.
Mounting and examination of cultured arterial segments in a wire myograph
Cultured arterial segments of 2 mm were mounted in a myograph for the recording of isometric tension development (DMT, Aarhus, Denmark). The organ chamber was filled with Krebs Ringer bicarbonate buffer (KRB) containing (in mM): NaCl 118.3, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0 and glucose 11.1 (pH = 7.4) and aerated with 95% O2 and 5% CO2. Two stainless steel wires (diameter of 40 µm) were inserted into the lumen of the arterial segments and were connected to a displacement device and an isometric force transducer. All arterial preparations were stretched to a diameter at which their individual mechanical performance was maximal. To achieve this, the arterial diameter was stepwise increased, and preparations were intermittently exposed to 65.5 mM potassium-KRB until maximal contractile responses were obtained. Contractile responses were expressed as the increase in wall tension (increase in force/twice the segment length; N/m) relative to the response to the maximal concentration of the thromboxane-A2 (TXA2) agonist analog (U46619, Enzo Life sciences, USA) of the control condition.
Testing vascular contractile responsiveness to U46619 after exposure to placental CM
Cultured arterial segments were contracted by U46619 in logarithmically increasing doses of 0.1 nM–1 µM. After washing and equilibration (±30 min), chorionic arteries were precontracted by U46619 (30 nM). Subsequently, vasodilation was induced by logarithmically increasing doses of sodium nitroprusside (0.01 nM–30 µM, SNP, Sigma Aldrich, Zwijndrecht, the Netherlands). After the experiments, arterial segments were formalin-fixed for paraffin embedding.
Immunohistochemistry on chorionic arteries exposed to placental CM
To identify the proliferative activity of the chorionic arteries exposed to the placental CM, following the myograph experiments, cultured arterial segments were immunohistochemically analyzed using commercially available antibodies following standard manual protocols on formalin-fixed, paraffin-embedded (FFPE) tissue. The slides were incubated with antibodies for mindbomb E3 ubiquitin protein ligase 1 (MIB-1, Ki-67, Dako, catalog no. M7240) (Agilent Technologies, Glostrup, Denmark) according to routine protocols. Briefly, 5 µm sections of paraffin-embedded tissue were first deparaffinized with absolute xylene, and endogenous peroxidase was blocked in 0.3% H2O2/methanol for 10 min. Sections were subsequently hydrated via a graded ethanol series (100–50% PBS (phosphate buffered saline)). Arterial sections were boiled in a sodium citrate solution (10 mM, pH 6.0) for 30 min, cooled at room temperature and washed three times in PBS. All subsequent steps were performed at room temperature. Sections were then incubated with PBST (phosphate buffer with Tween 20)/0.5% BSA (bovine serum albumin) for 30 min to minimize nonspecific antibody binding. Sections were incubated with Ki-67 1:40 in PBST/5% BSA) for 4 h. After washing three times with PBST, a goat anti-mouse secondary antibody (GAM-biotin Vector BA9200; 1:500 in PBST) was allowed to bind for 30 min at room temperature, followed by three washes in PBST. HRP-coupled avidin/streptavidin–biotin complex (ABC (avidin–biotin-complex) kit Elite, Vectastain PK6100, Vector Laboratories Inc., 30 Ingold Road, Burlingame, CA 94010) was used to amplify the signal and incubated for 30 min at room temperature, followed by three washes in PBST. Sections were then incubated with the substrate 3,3’-diaminobenzidine (DAB). Staining was stopped with water. In parallel, for the same placental chorionic arteries, a second 5 µm arterial section of the same artery was subjected to hematoxylin staining for the measurement of the cross-sectional area of the lumen, media, and adventitia. Finally, sections were dehydrated via a graded alcohol series (70%, 90%, 96%, 2 × 100%) and covered with Entellan (Merck KGaA, 64271 Darmstadt, Germany). For negative controls, the first antibody was replaced by a buffer.
Histological analysis of cultured chorionic arteries exposed to placental CM
All arterial cross-sections were examined at a magnification of ×10 using a light microscope (Leica DM4 B, Leica Microsystems GmbH, Ernst–Leitz–Strasse 17–37, 35578 Wetzlar, Germany). Ki-67-positive nuclei were counted in Ki-67-stained arterial cross-sections, and the areas of the lumen, media and adventitia were measured in hematoxylin-stained arterial cross-sections using Leica-QWin standard software.
Cell culture of BeWo cells and collection of CM
BeWo cells, a trophoblast cell line (ECACC, Porton Down, Salisbury, UK, No. 86082803), were cultured according to the manufacturer’s protocol. Briefly, BeWo cells were cultured in Ham’s F12 (Kaighn’s) growth medium (glutamine 2 mM and D-glucose 7 mM, GIBCO, Carlsbad, CA, USA) enriched with 10% (vol/vol) fetal bovine serum (FBS, SIGMA, St. Louis, MO, USA) and antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin; both from GIBCO). Cells were routinely maintained in 175-cm2 falcon flasks at pH 7.4 under 5% CO2 and 95% humidity at 37 °C and passaged when reaching confluency of 70–80%. For medium transfer experiments, 24 h after cell seeding in 6-well plates (3 × 105 cells/well in 2 ml growth medium), cells were cultured for 24 h under standard culture (control) conditions (21% O2, 5% CO2) or hypoxic conditions (1% O2, 5% CO2), whereafter CM was collected and stored at −80 °C until use.
Exposure of EA.hy926 cells to placental or BeWo cell CM
EA.hy926 cells, a vascular endothelial cell line derived by fusing primary human umbilical vein endothelial cells (HUVECs) with a thioguanine-resistant clone of A549 (ATCC CRL-2922, Manassas, Virginia, USA), were cultured according to the manufacturer’s protocol. Briefly, cells were cultured in DMEM (Dulbecco’s Modified Eagle Medium, 4 mM L-glutamine, 4500 mg/L glucose, 1 mM sodium pyruvate, and 1500 mg/L bicarbonate enriched with 10% (vol/vol) FBS, antibiotics (50 U/ml penicillin and 50 μg/ml streptomycin; both from GIBCO) and 1X HAT supplements (GIBCO). Cells were routinely maintained in 175 cm2 falcon flasks at pH 7.4 under 5% CO2 and 95% humidity at 37 °C and passaged when reaching confluency of 70–80%. The medium was refreshed every two days. For the CM exposure of EA.hy926 cells, CM of the control (CCM) and hypoxic condition (HCM) was first diluted 40% (vol/vol) with DMEM basal medium. Subsequently, 24 h after EA.hy926 cell seeding into six-well plates (1 × 105 cells/well in 2 ml growth medium), the medium was replaced by diluted placental or BeWo CCM or HCM and incubated for 24 h.
EA.hy926 cell viability upon exposure to placental or BeWo cell CM
Cell viability was assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. After 24 h of exposure of EA.hy926 cells to DMEM basal medium, which was used as a positive control or placental or BeWo CCM or HCM, the supernatant was removed, and 150 µl of MTT solution (0.5 mg/ml in DPBS) was added to the cells, after which the plate was incubated in the dark at 37 °C for 1 h. After 1 h, the MTT solution was removed from the wells, and 150 µl of DMSO (dimethylsulfoxide) was added to each well. After 10 min incubation at room temperature, the absorbance was measured at λ = 540 nm in a microplate reader (Bio-Rad, Hercules, California, USA).
Quantification of IL 6 and IL 8 secretion by EA.hy926 cells exposed to placental or BeWo cell CM
After 24 h of exposure of EA.hy926 cells to placental or BeWo cell CCM or HCM, the supernatant was removed and replaced by DMEM basal medium, which was collected after 24 h for cytokine detection. Human IL-8 (DY208) and human IL-6 (DY206) DuoSet ELISA kits (R&D Systems, Abingdon, UK) were used, and the analyses were carried out in accordance with the manufacturer’s instructions.
Intracellular ROS levels in EA.hy926 cells exposed to placental or BeWo cell CM
Intracellular reactive oxygen species (ROS) levels were quantified using the 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA)-assay. Seventy-two hours after cell seeding in black and clear bottom 96-well plates (Costar/Sigma Aldrich) (1 × 105 cells/well in 200 μl growth medium), cells were washed twice with 150 μl HBSS, incubated for 1 h with 100 μl H2DCFH-DA (50 μM) and washed twice again with 150 μl HBSS. Subsequently, cells were incubated with 200 μl of the diluted (40% (vol/vol)) placental or BeWo cell CCM or HCM for 24 h or with 100 µM H2O2, which was used as a positive control. Fluorescence intensity levels of DCF were measured at λexcitation = 485 nm and λemission = 525 nm and corrected for total protein content and expressed as percentage relative to the maximal (100%) intracellular ROS formation of the positive control group.
The results are expressed as the mean ± SEM, and n refers to the number of placentae from which blood vessels were isolated or the number of independent cell culture experiments. GraphPad Prism 8 Software, Inc., La Jolla, CA, USA, the maximal contractile response (Emax) and the negative logarithm of the half-maximal effective concentration (EC50) of various conditions were calculated. For each comparison, the D ‘Agostino and Pearson omnibus normality test was used to test normality. Subsequently, to compare the outcome of the control and hypoxic conditions, either an unpaired t-test or Mann–Whitney test was used accordingly. To compare the intervention conditions (quercetin or tocopherol) with the hypoxic condition, one-way analysis of variance was used followed by the multiple-comparison Tukey post hoc test. If not normally distributed, the Kruskal-Wallis test was used followed by Dunn’s multiple comparison post hoc test. A p value < 0.05 was considered to indicate a significant difference and is presented as follows: Ns: p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Intraluminal administration of placental HCM induces vasoconstriction
As depicted in Fig. 1, intraluminal administration of placental HCM significantly increased vascular contraction (+9753 ± 1183%, p < 0.001, n = 8) after 2 h compared to that after placental CCM administration (n = 8). Furthermore, when quercetin (3 or 10 µM) was present during the 3 h exposure of placental explants to hypoxia, the CM-induced vascular contraction showed a concentration-dependent reduction (−55 ± 13%, p < 0.001 and −131 ± 28%, p < 0.001, n = 8, respectively) compared to the HCM condition (Fig. 1).
Intraluminal administration of placental HCM increases endothelial permeability for KCl
To assess endothelial barrier integrity, intraluminal KCl-induced vascular contraction was tested before and after a 2 h intraluminal exposure to placental CCM or HCM. As presented in Fig. 2, 2 h intraluminal placental CCM did not change the endothelial permeability for KCl since it did not induce significant vascular contraction (+35 ± 75%, p = 0.2241, n = 8). After intraluminal exposure to placental HCM, intraluminal KCl induced significant vascular contraction (+1089 ± 276%, p < 0.001, n = 8) compared to the CCM condition, indicating increased endothelial permeability for KCl. Furthermore, when quercetin (3 or 10 µM) was present during the 3 h exposure of placental explants to hypoxia, the CM significantly reduced the intraluminal KCl-induced vascular contraction in a concentration-dependent manner (−30 ± 38%, p = 0.024 and −91 ± 21%, p < 0.001, n = 8, respectively) compared to the HCM condition.
Placental HCM decreases endothelial cell viability and increases intracellular ROS formation and inflammation
To determine the effect of placental CM on endothelial cell viability, ROS formation, and inflammatory status, EA.hy926 endothelial cells were incubated for 24 h with placental CCM or HCM. After 24 h of exposure to placental HCM, endothelial cell viability was significantly decreased (−68 ± 46%, p = 0.029, n = 3), whereas intracellular ROS formation was significantly increased (+493 ± 199%, p = 0.024, n = 3) compared to the CCM condition. Furthermore, 24 h after exposure of EA.hy926 endothelial cells to the placental HCM, both IL-6 (+470 ± 41%, p < 0.001, n = 3) and IL-8 (+566 ± 162%, p = 0.005, n = 3) levels in the replaced blank medium were significantly increased compared to the placental CCM condition (Fig. 3a–d).
BeWo cell HCM decreases endothelial cell viability and increases intracellular ROS formation and inflammation
To determine whether trophoblast cells play a significant role in the endothelial response to placental CM, EA.hy926 cells were incubated for 24 h with CCM or HCM from BeWo cells. In line with data obtained with placental HCM, 24 h exposure to BeWo HCM significantly decreased endothelial cell viability (−72 ± 30%, p = 0.027, n = 3) and increased intracellular ROS formation (+89 ± 27%, p = 0.029, n = 3) in cultured endothelial cells compared to the BeWo CCM condition. Furthermore, 24 h after exposure of EA.hy926 endothelial cells to BeWo HCM, both IL-6 (+100 ± 26%, p = 0.005, n = 3) and IL-8 (+393 ± 28%, p < 0.001, n = 3) levels in the replaced blank medium were significantly increased compared to the BeWo CCM condition (Fig. 3e–h).
Placental HCM increases the vascular cross-sectional area of the media
To determine whether placental HCM affects the arterial cross-sectional area, placental chorionic arteries were exposed to placental CCM or HCM for up to 7 days, after which the cross-sectional area of the lumen, media, and adventitia were assessed. Only the cross-sectional area of the media was significantly increased after 3 and 7 days of exposure to placental HCM (+44 ± 72%, p < 0.040, n = 10 and +65 ± 75%, p < 0.010, n = 10, respectively) compared to the placental CCM condition (n = 17 and n = 18, respectively) (Fig. 4 and Supplementary Fig. S1). Furthermore, when 10 µM quercetin was present during the 3 h exposure of placental explants to hypoxia, from day 3, the cross-sectional area of the media of chorionic arteries exposed to the CM was significantly lower (−44 ± 50%, p < 0.030, n = 9) than that with placental HCM. After 7 days of exposure to the placental CM, the 3 µM and 10 µM and 20 µM RRR-α-tocopherol conditions resulted in a significantly smaller cross-sectional area of the media (−43 ± 44%, p < 0.032, n = 12, −64 ± 56%, p < 0.041, n = 8 and −44 ± 45%, p < 0.004, n = 15, respectively) than that with placental HCM (Fig. 4). No changes in the cross-sectional area of the lumen and adventitia were found after exposure to placental CM between the placental CCM and HCM conditions (Supplementary Fig. S1).
Placental HCM increases vascular proliferation
To determine whether the increased cross-sectional area of the media after exposure to placental HCM was a result of hyperplasia, vascular proliferation was assessed by KI-67 staining. As presented in Fig. 5, after 1, 3 and 7 days of exposure to placental HCM, increased proliferating nuclei were found (+130 ± 110%, p = 0.010, n = 10, 131 ± 71%, p = 0.005, n = 11 and +114 ± 79%, p < 0.001, n = 13, respectively) compared to the placental CCM condition (n = 27, n = 17 and n = 30, respectively) (Fig. 5). Furthermore, when quercetin (3 µM) and RRR-α-tocopherol (20 µM) were present during the 3 h placental exposure to hypoxia, after 1 day of exposure, the resultant CM significantly reduced proliferating nuclei (−42 ± 44%, p = 0.028, n = 14 and −55 ± 48%, p = 0.003, n = 15, respectively) compared to the placental HCM condition. When quercetin (10 µM) was present during the 3 h placental exposure to hypoxia, after 3 days of exposure, the resultant CM significantly reduced the number of proliferating nuclei (−42 ± 27%, p = 0.027, n = 13) compared to the placental HCM condition. After 7 days of exposure, all three intervention conditions (quercetin 3 and 10 µM and RRR-α-tocopherol 20 µM) reduced the number of proliferating nuclei (−43 ± 38%, p = 0.003, n = 19, −41 ± 50%, p = 0.004, n = 11 and −49 ± 37%, p < 0.001, n = 18, respectively) compared to the placental HCM condition. See Supplementary Fig. S2 for a representative example of the light microscopy images of the Ki-67 immunohistochemistry-stained arterial cross-sections of placental chorionic arteries after 7 days of exposure to CCM or HCM.
Placental HCM increases the vascular contractile responsiveness to U46619
Finally, it was tested whether the increased cross-sectional area found in placental chorionic arteries exposed to placental HCM resulted in a different vascular contractile responsiveness to U46619. No differences were found for the LogEC50 calculated from the concentration-dependent contraction curves for U46619 (0.1 nM–1 µM) between the different conditions for each day (Supplementary Fig. S3). U46619 (1 µM), however, induced a significant increase in vascular contraction (+34 ± 20%, p = 0.003, n = 10) in placental chorionic arteries exposed to placental HCM for 3 days compared to the placental CCM condition (n = 21). Furthermore, after 3 days of exposure, the placental CM, which was produced in the presence of quercetin 3 or 10 µM induced a significantly lower vascular contraction against U46619 (1 µM) (−30 ± 22%, p = 0.018, n = 11 and −35 ± 31%, p = 0.016, n = 10, respectively) than that with placental HCM (n = 10) (Fig. 6). Placental CM did not affect the pEC50 of the concentration-dependent relaxation curves for the NO donor sodium nitroprusside (0.01 nM–30 µM) precontracted by U46619 (30 nM) between the conditions (Supplementary Fig. S4).
In the current study, we show for the first time that the intraluminal administration of placental HCM induces vasoconstriction and increased endothelial permeability for KCl in chorionic arteries. When the most abundant dietary flavonoid quercetin was present during placental exposure to hypoxia, these effects could be concentration-dependently prevented. Moreover, we show that exposure of cultured EA.hy926 endothelial cells to placental or BeWo HCM decreases cell viability and increases the endothelial production of ROS and pro-inflammatory cytokines IL-6 and IL-8. Furthermore, we observed that the cross-sectional area of the arterial media was increased after 3 days of exposure to placental HCM, which was associated with increased vascular proliferation and could be concentration-dependently prevented by the presence of quercetin as well as RRR-α-tocopherol during placental exposure to hypoxia. In addition to the effect of placental HCM on vascular contraction and morphology, vascular contractile responsiveness to U46619 was increased after 3 days of exposure, which could also be prevented by the presence of quercetin during placental exposure to hypoxia.
In PE pathophysiology, impaired uterine spiral artery remodeling by extravillous trophoblast cells is believed to lead to persistently hypoxic, dysfunctional placentae that release pathogenic molecules into the maternal circulation . It has been proposed that in response to the oxygenation shortage, placental cells release factors that affect endothelial function in the whole maternal circulation, induce vasoconstriction and increase the contractile responsiveness of the vasculature to vasopressors, which has been demonstrated to persist after delivery [28,29,30]. These mechanisms have been suggested to contribute to an increased risk for cardiovascular disease in later life in women with a history of PE. While several studies support the role of reduction in uteroplacental perfusion pressure in PE, the factors released in response to a reduction in uteroplacental perfusion pressure and its contribution to the development of generalized hypertension in PE pathogenesis are the subject of extensive research .
In previous work , where we mounted freshly isolated chorionic arteries into a wire myograph and used specific receptor antagonists, the AT1 and ET-1 receptors were identified as the main contributors to the acute vasoconstrictive activity of placental HCM . In this study, we show that in chorionic arteries mounted into a pressure myograph, the intraluminal administration of placental HCM also induced vascular contraction, suggesting that the contractile response might be endothelium-dependent. Interestingly, AT1-AA that are involved in the hypertensive state in PE are known to be induced by placental hypoxia and have been implicated as a major contributor to the activation of AT1 receptor-mediated hypertension in PE . Granger et al. have already proposed that in addition to activating the AT1 receptor, increased placental AT1-AA secretion upon hypoxia may stimulate endothelial cells to produce ET-1, explaining the significance of their receptors in the development of PE-induced hypertension . It is postulated that in severe PE, ET-1 production is increased to the extent that it loses its paracrine directionality and leads to increased circulating levels and, together with AT1-AA, induces the local and systemic release of oxidant substances [32,33,34]. Interestingly, the administration of ascorbic acid increases the flow-mediated vasodilation in large arteries of women who have formerly exhibited PE, further demonstrating the role of oxidative stress in maintaining vascular function in PE . Supporting these data, we were able to show that the presence of quercetin during the exposure of placental explants to hypoxia concentration-dependently protected the vascular contraction induced by placental HCM. Interestingly, plasma levels of antioxidants that counterbalance the increased ROS production were found to be lower in women with PE [36, 37], further supporting the role of oxidative stress in the release of vasoactive compounds that induce hypertension in the pathogenesis of PE.
Furthermore, animal studies on the PE phenotype have shown that antiangiogenic factors such as sFlt-1 released by the hypoxic placenta are associated with increased endothelial cell permeability, which causes proteinuria, cerebral edema and hypertension [27, 31, 38]. Moreover, electron microscopic studies of myometrial vessels and arteries isolated from subcutaneous fat in women with PE have revealed disrupted and enlarged intracellular junctions between endothelial cells . In support of these data, we found that vascular permeability for KCl was increased after intraluminal exposure to placental HCM compared to CCM. In addition, HCM from both placental explants and BeWo cells decreased the endothelial cell viability. TNF-α, a pro-inflammatory cytokine that is well known to be elevated in PE, modulates the immune response by increasing vascular permeability, lymphocyte activation, and IL-6 and IL-8 production upon hypoxic placental stress [40,41,42]. Consequently, these cytokines cause increased endothelial permeability by inducing the expression of adhesion molecules, e.g., E-selectin, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 [1, 43]. Furthermore, these cytokines are involved in the downregulation of endothelial nitric oxide synthase (eNOS), increased AT1-AA production via, e.g., T helper and B lymphocyte activation , and mitochondrial biogenesis, all leading to mitochondrial dysfunction and resulting in oxidative stress and increased ROS production [4, 45]. We found increased IL-6 and IL-8 cytokine expression and ROS production in endothelial cells exposed to HCM from both placental explants and BeWo cells. In accordance with the effect of placental HCM on EA.hy926 endothelial cells that was found in our study, exposing endothelial cells to serum from women exhibiting PE has been shown to induce endothelial damage . Remarkably, it has been demonstrated that in the earliest passage of cultured endothelial cells derived from women with PE, there is an increased endothelial monolayer permeability that is associated with alterations in the junctional adhesion molecule vascular endothelial cadherin and tight junction protein occludin [39, 46]. These alterations normalize again when these endothelial cells are continuously cultured in vitro, suggesting that the extracellular environment in PE contains factors that diminish endothelial barrier function in a reversible manner . Collectively, these findings may suggest that in addition to alterations found in endothelial cells upon exposure to placental HCM or blood plasma of women with PE, other vascular cell types are involved in the cardiovascular effects in PE.
By comparing cross-sectional areas of the lumen, media, and adventitia of chorionic arteries exposed to placental HCM for up to 7 days, the cross-sectional area of the media containing smooth muscle cells was increased after 3 days of exposure compared to the control condition. To determine whether the increased cross-sectional area of the media was a result of hyperplasia, vascular proliferation was assessed in the same chorionic arteries and was already significantly increased after 1 day of exposure to placental HCM. Both effects were concentration-dependently avoided by the presence of quercetin or RRR-α-tocopherol during placental exposure to hypoxia. AT1-AA in PE or in conditions of placental hypoxia not only has been proven to have a profound contribution to the increased vasoconstriction in PE but is also known to promote smooth muscle growth upon AT1 receptor activation [3, 47]. Together with pro-inflammatory cytokines, AT1-AA is known to promote the endothelial release of vasoactive factors such as ET-1 and TXA2, which stimulates smooth muscle proliferation through the ETB and thromboxane receptor, respectively [3, 34, 45]. Together with our findings, this may suggest that alterations in the morphology and/or function of not only endothelial cells but also smooth muscle cells may contribute significantly to cardiovascular problems during PE.
Three days of exposure to placental HCM caused a sustained increase in vascular contractile responsiveness to U46619. This finding is in line with a similar increase in the vascular contractility to TXA2, angiotensin II (AngII) and ET-1 of omental arteries observed in women exhibiting PE . In line with the protective effect on vascular morphology that was found, adding quercetin during the 3 h exposure of placental explants to hypoxia also normalized the increased vascular contractile responsiveness to U46619. A meta-analysis indicated a two-fold increase in cardiovascular risk in women with a history of PE . In addition, infants of preeclamptic mothers have higher blood pressure during young adulthood and an increased risk for stroke later in life . Furthermore, during pregnancy, the microvasculature of women exhibiting PE, compared to that of non-preeclamptic women, has a higher response to endothelial-dependent vasodilation (acetylcholine) and shows no differences in endothelial-independent vasodilation by SNP [50,51,52]. In line with this study, no changes were found in the effect of the smooth muscle dilator sodium nitroprusside on arterial relaxation after exposure to placental HCM for up to 7 days.
Limitations and future perspectives
Although the use of primary cells for this approach could be suggested, the endothelial EA.hy926 cell line was used to minimize senescence processes and morphological and functional changes during the cell cycle and to maximize the potential for self-renewal of the cells. By preventing genetic variation among donors, the conditioned medium was tested under similar and stable culture conditions. Placental villous explants were exposed to hypoxia for up to 3 h since previous studies using placental villous explants ex vivo showed that the viability of placental tissue in culture could only be maintained for 4 h . Longer exposure times have been shown to lead to pronounced tissue deterioration and substantial apoptosis of trophoblast cells in placental villous explants, indicating that this model is only suitable for studying acute effects in culture . To investigate the effects of long-term exposure to hypoxia and to determine if placental conditioned-induced changes are trophoblast-specific, the BeWo cell line was used. As shown in Fig. 6, placental HCM only showed a significant increase in vascular contractile responsiveness to U46619 at day 3, indicating that this parameter is less preserved over time compared to the corresponding effect on the cross-sectional area of the media and vascular proliferation in chorionic arteries. This finding, together with the variation within the test conditions, might be an explanation for the controversial effect of the intervention and underlines the need for alternative assessment options to examine long-term functional changes related to vascular responsiveness.
To investigate the long-term effects of placental HCM on vascular morphology, after exposing chorionic arteries, HCM could be replaced by placental CCM to investigate the recovery potential of the arteries. Furthermore, it should be of great interest to compare the proportion of the cross-sectional areal media in resistant blood vessels, such as omental blood vessels, in women with PE compared to controls. Even though atmospheric 21% O2 is generally considered a standard culture condition, determining dissolved O2 levels in culture medium and in cultured tissue remains difficult, since many factors, including the volume of the media and metabolic activity of the tissue, influence medium O2 levels. As recommended by Trenton et al., we constantly aerated our culture media and thereby improved the equilibration time, stabilized the pericellular oxygen tensions, and prevented gradual oxygen diffusion . To improve O2 monitoring during culture, we recommend measuring O2 levels in the medium throughout the experiment and assessing oxygen-dependent enzymes, including HIF-PHDs, which are well known as “oxygen sensors” in placental explants after culturing. For a better physiological approach, our experimental setting can now serve as a tool where the cell lines used in our study can be replaced by freshly isolated trophoblast and HUVECs, and the effect of the blood plasma of women with PE on alterations in vascular function, morphology, and endothelial barrier integrity can be examined.
In conclusion, this study shows that intraluminal administration of placental HCM induces vascular contraction and increases endothelial permeability. It also demonstrates that these placental factors induce endothelial cell death, ROS formation and inflammation, which are all known to be involved in the formation of vasoactive compounds such as ET-1 and thus indirectly contribute to vascular contraction. The increased contractility, cross-sectional area and vascular proliferation of the media observed in chorionic arteries upon exposure to placental HCM implies that vascular smooth muscle cells undergo long-lasting adaptations, which may contribute to the cardiovascular effects associated with PE. A better understanding of the complex interplay between the stressed placenta in PE and the maternal cardiovascular system may help in the identification of new serum biomarkers for the prediction of PE and in the design of new diagnostic approaches for better clinical management. Our work forms a first step to a better knowledge of the long-lasting maternal vascular consequences, opening new postpartum treatment and follow-up strategies.
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This study was funded by the NUTRIM Graduate Program and supported by the external source: “Fonds gezond geboren”. No additional external funding was received for this study.
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Vangrieken, P., Remels, A.H.V., Al-Nasiry, S. et al. Placental hypoxia-induced alterations in vascular function, morphology, and endothelial barrier integrity. Hypertens Res (2020). https://doi.org/10.1038/s41440-020-0528-8
- Placental hypoxia
- Vascular smooth muscle