Human Uterine Microvascular Endothelial Cells Research Articles

The soluble (pro)renin receptor promotes a preeclampsia-like phenotype both in vitro and in vivo.

Preeclampsia is classified as new-onset hypertension coupled with gross endothelial dysfunction. Placental (pro)renin receptor ((P)RR) and plasma soluble (P)RR (s(P)RR) are elevated in patients with preeclampsia. Thus, we aimed to interrogate the role (P)RR may play in the pathogenesis of preeclampsia. Human uterine microvascular endothelial cells (HUtMECs, n = 4) were cultured with either; vehicle (PBS), 25-100 nM recombinant s(P)RR, or 10 ng/ml TNF-a (positive control) for 24 h. Conditioned media and cells were assessed for endothelial dysfunction markers via qPCR, ELISA, and immunoblot. Angiogenic capacity was assessed through tube formation and adhesion assays. Additionally, pregnant rats were injected with an adenovirus overexpressing s(P)RR from mid-pregnancy (day 8.5), until term (n = 6-7 dams/treatment). Maternal and fetal tissues were assessed. HUtMECs treated with recombinant s(P)RR displayed increased expression of endothelial dysfunction makers including vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and endothelin-1 mRNA expression (P = 0.003, P = 0.001, P = 0.009, respectively), along with elevated endothelin-1 protein secretion (P < 0.001) compared with controls. Recombinant s(P)RR impaired angiogenic capacity decreasing the number of branches, total branch length, and mesh area (P < 0.001, P = 0.004, and P = 0.009, respectively), while also increasing vascular adhesion (P = 0.032). +ADV rats exhibited increased systolic (P = 0.001), diastolic (P = 0.010), and mean arterial pressures (P = 0.012), compared with -ADV pregnancies. Renal arteries from +ADV-treated rats had decreased sensitivity to acetylcholine-induced relaxation (P = 0.030), compared with -ADV pregnancies. Our data show that treatment with s(P)RR caused hypertension and growth restriction in vivo and caused marked endothelial dysfunction in vitro. These findings demonstrate the significant adverse actions of s(P)RR on vascular dysfunction that is characteristic of the preeclamptic phenotype.

The involvement of long noncoding RNA APOA1-AS in the pathogenesis of preeclampsia.

Long noncoding RNAs (lncRNAs) are involved in preeclampsia (PE), and apolipoprotein A-1 antisense RNA (APOA1-AS) and has been found to be associated with a number of diseases. Our study aims to understand the involvement of APOA1-AS in PE. Clinically, APOA1-AS expression in early-onset severe PE (EOSPE) patients and healthy controls was detected by real-time quantitative polymerase chain reaction. In vitro experiments were divided into control [coculturing trophoblasts with human uterine microvascular endothelial cells (UtMVECs)], TNF-α [coculturing trophoblasts with UtMVECs treated with tumor necrosis factor-α (TNF-α)], and TNF-α + control siRNA/APOA1-AS siRNA groups (UtMVECs transfected with control siRNA/APOA1-AS siRNA were cocultured with trophoblasts in the presence of TNF-α). The animals were divided into normal group, PE group (PE model was established by administrating nitro-L-arginine methyl ester (L-NAME) in rats), PE + control siRNA group (PE rats were treated with control siRNA), and PE + APOA1-AS siRNA group (PE rats were treated with APOA1-AS siRNA). Increased APOA1-AS was found in the placental tissues of EOSPE patients. APOA1-AS siRNA abolished the decreased integration of trophoblasts into UtMVEC networks induced by TNF-α. Furthermore, APOA1-AS siRNA improved pregnancy outcomes in PE rats with increased expression of vascular endothelial growth factor, placental growth factor, and fms-like tyrosine kinase receptor (Flt-1) but decreased expression of E-cadherin, intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Downregulation of APOA1-AS protected against TNF-α-induced inhibition of trophoblast integration into endothelial networks, thus exerting protective effects against PE rats.

Decidual Inflammation Drives Chemokine-Mediated Immune Infiltration Contributing to Term Labor.

Infiltration of maternal peripheral leukocytes into the uterine tissues is a critical event occurring before, during, and after term labor (TL). In this article, we investigate the contribution of uterine smooth muscle (myometrium) and pregnant endometrium (decidua) to the inflammatory process during human TL. We hypothesize that labor-related physiological inflammation is orchestrated by uterine-secreted cytokines, which dually activate the uterine vascular endothelium and maternal leukocytes to promote their adhesion and infiltration into the uterus. Using Luminex and ELISA assays, we examine a full range of cytokines (45 proteins) in media conditioned by primary decidual and myometrial cells from TL and term not in labor (TNL) women. The effect of conditioned media on the activation of human uterine microvascular endothelial cells was measured by qPCR and on peripheral leukocytes by flow cytometry. Transendothelial migration of calcein-labeled primary leukocytes toward media was assessed by fluorometry. Stromal decidual cells secrete significantly higher levels of multiple cytokines compared with myometrial cells (p < 0.05) and significantly more cytokines during TL than TNL. These cytokines activate uterine microvascular endothelial cells through the upregulation of cell adhesion molecule VCAM-1 and peripheral leukocytes by upregulation of CD11b. Furthermore, multiple cytokines secreted from the TL decidua and myometrium significantly increase migration of granulocytes, monocytes, and lymphocytes compared with TNL (p < 0.05), which was blocked by a broad-spectrum chemokine inhibitor (FX125L). These data reveal the critical role for decidual- and myometrial-secreted cytokines in the activation of inflammatory pathways leading to labor. We suggest that these pathways represent targets for therapeutic intervention during preterm labor.

Angiopoietin-2 regulated by progesterone induces uterine vascular remodeling during pregnancy.

During pregnancy, the uterus undergoes intense neovascularization and vascular remodeling to supply oxygen and nutrients to the embryo. During this period, progesterone secreted from the ovary has effects on vascular remodeling in the endometrium and interacts with angiogenic factors. However, the exact mechanism of uterine vascular remodeling during pregnancy is poorly understood. Therefore, the aim of the present study was to investigate the association between angiopoietin-2 (Ang-2), one of the angiopoietins, and intrauterine vessel remodeling during pregnancy, and to determine the effect of progesterone on Ang-2 levels. Changes in Ang-2 expression were observed according to quantitative modification of progesterone using pregnant mice and human uterine microvascular endothelial cells. As a result, Ang-2 was observed mainly in the mesometrial region (MR) of the uterus during the period between implantation and placentation. Furthermore, a substantial amount of Ang-2 also appeared in endothelial cells, particularly of the venous sinus region (VSR). Interestingly, Ang-2 expression was increased by progesterone, whereas estrogen had limited effects. To confirm the association between Ang-2 and progesterone, the function of the progesterone receptor (PR) was inhibited using RU486, a blocker of PR. Ang-2 expression and vascular remodeling of the VSR in the uterus were decreased when the functions of progesterone were inhibited. Overall, the regulation of Ang-2 by progesterone/PR was associated with vascular remodeling in the VSR during pregnancy. The present study proposed a solution to prevent pregnancy failure due to a lack of vascularity in the uterus in advance.

Effect of placental growth factor in models of experimental pre-eclampsia and trophoblast invasion.

Placental growth factor (PlGF) is decreased in early gestation of pregnant women who subsequently develop pre-eclampsia. In this study, pre-emptive treatment with PlGF to prevent pre-eclampsia was evaluated in an in vivo rodent model of experimental pre-eclampsia (EPE) induced by TNF-α and in an in vitro model of human first-trimester trophoblast invasion. Pregnant C57/BL6 mice were treated with recombinant mouse placental growth factor-2 (rmPlGF-2) 100μg/kg/day IP from gestational day (gd) 10. Animals had EPE induced by continuous TNF-α infusion on gd 13 and were subject to either continuous blood pressure monitoring by radiotelemetry throughout pregnancy or live placenta T2 -weighted magnetic resonance imaging (MRI) to demonstrate placental function on gd 17. There was no difference in BP (P>.99), proteinuria (P=.9) or T2 values on MRI (P=.9) between control and rmPlGF-2-treated animals. On gd 13, animals treated with rmPlGF-2 demonstrated increased placenta PlGF (P=.01) and Toll-like receptor-3 (P=.03) mRNA expression as compared with controls. Fluorescent-labelled human uterine microvascular endothelial cells and HTR8/SVNeo cells were co-cultured on Matrigel™ and treated with recombinant human PlGF (rhPlGF) (10ng/mL) and/or TNF-α (0.5ng/mL). Trophoblast integration into endothelial networks was reduced by added TNF-α (P=.006), as was rhPlGF concentration in conditioned media (P<.0001). Cell integration was not ameliorated by addition of rhPlGF (P>.9). Although TNF-α-induced EPE was not reversed with pre-emptive rmPlGF-2, a further trial of pre-emptive rhPlGF in vivo is required to determine whether the absence of effect of rhPlGF demonstrated in vitro precludes PlGF as a preventative therapy for pre-eclampsia.

88. Integrins (α1, β1 and α6, β4) differentially modulate trophoblast-endothelial interaction in vitro

Introduction During normal trophoblast invasion, integrin α6β4 are downregulated and α1β1 are upregulated in invasive cytotrophoblast cells. In preeclampsia, both interstitial and endovascular invasion are shallow and cytotrophoblast fail to upregulate α1β1 and downregulate α6β4. Objective This study aims to investigate role of integrin α1β1 and integrin α6β4 during trophoblast integration into endothelial cellular networks in vitro. Methods Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel to form endothelial networks. Green fluorescent-labeled trophoblast HTR-8/SVneo cells pre-incubated with 20 μg/ml of neutralizing antibodies (anti-α1, β1, α6, β4, α1β1 or α6β4) for 1 h were then co-cultured with the endothelial networks with the neutralizing antibody for 24 h. Fluorescent images were captured and quantified by image J. Free soluble fms-like tyrosine kinase-1 (sFlt-1), placenta growth factor (PlGF), matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) from conditioned media were measured by ELISA. Cells were retrieved to examine mRNA expression of invasion markers of tissue inhibitor of metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor type 1 (PAI-1) by quantitative PCR. Results The integration of trophoblast cells into endothelial cellular networks was significantly inhibited by anti-β1 (72 ± 3%, p Discussion We have shown that anti-integrin β1, not anti-integrin a1 inhibited and anti-integrin a6, not anti-integrin β4 increased endothelial-trophoblast cellular integration in vitro. The matrix degradation enzymes (MMP-2 and MMP-9) and their associated inhibitors (TIMP-1 and PAI-1) may involve in this process.

Establishment of a three-dimensional model to study human uterine angiogenesis.

Can primary human uterine microvascular endothelial cells (UtMVECs) be used as a model to study uterine angiogenic responses in vitro that are relevant in pregnancy? UtMVECs demonstrated angiogenic responses when stimulated with proangiogenic factors, including sphingosine 1-phosphate (S1P), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), physiological levels of wall shear stress (WSS), human chorionic gonadotropin (hCG) and various combinations of estrogen and progesterone. During sprouting angiogenesis, signaling from growth factors and cytokines induces a monolayer of quiescent endothelial cells (ECs) lining the vasculature to degrade the extracellular matrix and invade the surrounding tissue to form new capillaries. During pregnancy and the female reproductive cycle, the uterine endothelium becomes activated and undergoes sprouting angiogenesis to increase the size and number of blood vessels in the endometrium. The study was designed to examine the angiogenic potential of primary human UtMVECs using the well-characterized human umbilical vein EC (HUVEC) line as a control to compare angiogenic potential. ECs were seeded onto three-dimensional (3D) collagen matrices, supplemented with known proangiogenic stimuli relevant to pregnancy and allowed to invade for 24 h. Sprouting responses were analyzed using manual and automated methods for quantification. RT-PCR, Western blot analysis and immunostaining were used to characterize UtMVECs. Angiogenic responses were examined using 3D invasion assays. Western blotting was used to confirm signaling responses after proangiogenic lipid, pharmacological inhibitor, and recombinant lentiviral treatments. All experiments were repeated at least three times. After ensuring that UtMVECs expressed the proper endothelial markers, we found that UtMVECs invade 3D collagen matrices dose-dependently in response to known proangiogenic stimuli (e.g. S1P, VEGF, bFGF, hCG, estrogen, progesterone and WSS) present during early pregnancy. Invasion responses were positively correlated with phosphorylation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) and p42/p44 mitogen-activated protein kinase (ERK). Inhibition of these second messengers significantly impaired sprouting (P < 0.01). Gene silencing of membrane type 1-matrix metalloproteinase using multiple approaches completely abrogated sprouting (P < 0.001). Finally, UtMVECs displayed a unique ability to undergo sprouting in response to hCG, and combined estrogen and progesterone treatment. Not applicable. The study of uterine angiogenesis in vitro has limitations and any findings many not fully represent the in vivo state. However, these experiments do provide evidence for the ability of UtMVECs to be used in functional sprouting assays in a 3D environment, stimulated by physiological factors that are produced locally within the uterus during early pregnancy. We show that UtMVECs can be used reliably to investigate how growth factors, hormones, lipids and other factors, such as flow, affect angiogenesis in the uterus. This work was supported by NIH award HL095786 to K.J.B. The authors have no conflicts of interest.

Fluid shear stress regulates vascular remodeling via VEGFR-3 activation, although independently of its ligand, VEGF-C, in the uterus during pregnancy

Early pregnancy is characterized by an increase in the blood volume of the uterus for embryonic development, thereby exerting fluid shear stress (FSS) on the vascular walls. The uterus experiences vascular remodeling to accommodate the increased blood flow. The blood flow-induced FSS elevates the expression of vascular endothelial growth factors (VEGFs) and their receptors, and regulates vascular remodeling through the activation of VEGF receptor-3 (VEGFR-3). However, the mechanisms responsible for FSS-induced VEGFR-3 expression in the uterus during pregnancy are unclear. In this study, we demonstrate that vascular remodeling in the uterus during pregnancy is regulated by FSS-induced VEGFR-3 expression. We examined the association between VEGFR-3 and FSS through in vivo and in vitro experiments. In vivo experiments revealed VEGFR-3 expression in the CD31-positive region of the uterus of pregnant mice; VEGF-C (ligand for VEGFR-3) was undetected in the uterus. These results confirmed that VEGFR-3 expression in the endometrium is independent of its ligand. In vitro studies experiments revealed that FSS induced morphological changes and increased VEGFR-3 expression in human uterine microvascular endothelial cells. Thus, VEGFR-3 activation by FSS is associated with vascular remodeling to allow increased blood flow in the uterus during pregnancy.

51 Acetyl Salicylic Acid (aspirin) reversed TNF- α inhibition of trophoblast–endothelial interaction

Introduction Early administration of low dose Acetyl Salicylic Acid (aspirin, ASA) has demonstrated a reduction in preterm preeclampsia in some clinical trials. However, whether the mechanism of this preventive strategy of aspirin is via placental function and the pathways involved have not been investigated. Objectives The aim of this study is to investigate the effect of ASA on trophoblast integration and related cytokine or angiogenic/invasive pathways in an in vitro model of preeclampsia. Methods Red fluorescent-labeled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. The endothelial cellular networks formed within 4 h. Green fluorescent-labeled trophoblastic HTR-8/SVneo cells were then co-cultured with the endothelial networks in the presence/absence of TNF- α (0.5 ng/ml) and/or ASA (0.1 mM) for 24 h. Red and green fluorescent images were captured. The effects of TNF- α and ASA on HTR-8/SVneo cells integration into endothelial cellular networks was quantified by Image Analysis software (Image J). The conditioned media were collected to measure IL-6, the free VEGF and PLGF by ELISA. Enzyme immunoassays (EIA) were performed to measure PGF1 α , the stable metabolite of vasodilator PGI2. Cells were also retrieved from Matrigel to extract mRNA. The mRNA expression of cytokine angiogenic factors (VEGF, and PlGF), invasion markers (MMP-2 and PAI-1), endothelial cell activation markers (E-selectin), eNOS and cyclooxygenase (COX)-2 were examined by quantitative PCR. Results ASA reversed the inhibitory effect of TNF- α on trophoblast cells integration into endothelial cellular networks. In the conditioned medium, TNF- α increased PGF1 α production (128%, p α production (19%, p α effect on PGF1 α production (19%, p α decreased PlGF and increased VEGF and IL-6 production. TNF- α also inhibited the mRNA expression of eNOS, MMP-2 and stimulated COX2, PAI-1 and E-selectin, however, ASA did not reverse TNF- α effects on these molecules. Conclusion The results showed that ASA may have a beneficial effect on trophoblast cells integration into endothelial cellular networks via inhibiting the metabolism of vasodilator PGI2, not the known factors of cytokine, angiogenic, invasion markers and endothelial activation markers.

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.

HCG stimulates angiogenic signals in lymphatic endothelial and circulating angiogenic cells

Human chorionic gonadotropin (hCG) has long been associated with the initiation and maintenance of pregnancy, where angiogenesis plays an important role. However, the function of hCG in angiogenesis and the recruitment of vascular active cells are not fully understood. In this study, the role of hCG and its receptor in circulating angiogenic and human endothelial cells, including lymphatic, uterine microvascular, and umbilical vein endothelial cells, was examined. Immunohistochemistry and immunoblot analysis were used to detect LH/hCG receptor expression and the expression of hCG-induced angiogenic molecules. HIF-1α was determined via ELISA and downstream molecules, such as CXCL12 and CXCR4, via real-time PCR. Chemotaxis was analyzed using Boyden chambers. Our results show that the LH/hCG receptor was present in all tested cells. Furthermore, hCG was able to stimulate LH/hCG-receptor-specific migration in a dose-dependent fashion and induce key angiogenic molecules, including HIF-1α, CXCL12, and CXCR4. In conclusion, our findings underscore the importance of hCG as one of the first angiogenic molecules produced by the conceptus. hCG itself alters endothelial motility, recruitment, and expression of pro-angiogenic molecules and may therefore play an important role in vascular adaption during implantation and early placental formation.

Umbilical Cord Stem Cells Induce Glial Fate in Neural Progenitor Cells

s / Placenta 35 (2014) A1eA112 A93 Objectives: The Wharton’s jelly (WJ) of the umbilical cord is derived from the mesoderm of the primitive streak which invades the connecting stalk and primary chorionic villi. Mesenchymal stem cells in the mesoderm differentiate and participate in neo-vasculogenesis of the umbilical and placental circulation. Whether these cells have a continued role in placental/umbilical vascular growth and maturity is not known. Fetal stem cells have been shown to traffic and repair injured myocardial endothelium in mice (Kara et al. 2012). Their ability to interact with the uterine endothelium has not been investigated. The aim of this study was to investigate whether umbilical stem cells (WJMSC) can interact and influence umbilical vein and uterine endothelial monolayers. Methods: Human umbilical vein (HUVEC) and uterine microvascular endothelial cells (HUtMEC) were grown to 70% confluence in endothelial media and to full confluence in mixed media (endothelial: stem cell media). Isolated WJMSC, labelled with the fluorescent tracker, PKH26 was added on top. Cell-cell interactions were monitored with fluorescence time-lapse microscopy. Key events were analysed by confocal microscopy after vascular endothelial (VE) cadherin immunocytochemistry. Results: WJMSC displayed non-apoptotic blebbing, amoeboid movement and shedding of exosomes as early events. There was a time lag of 60 min before transmigration of WJMSC via the paracellular pathway to subendothelial positions. This was concomitant with disruption of VE-cadherin (statistically significant reduction of continuous junctional profiles). By 16h, a majority of WJMSC were resident underneath the endothelial monolayers; junctional profiles returned to normal values. At 22h, the cocultures showed a higher % of paracellular clefts with continuous VEcadherin labelling, indicative of increased junctional maturity. Conclusion: Our data suggests that umbilical mesenchymal stem cells can interact with and influence junctional organisation of both fetal and uterine endothelial cells. Their role, if any, in uterine vascular re-modelling during pregnancy requires investigation. P2.102-N. UMBILICAL CORD STEM CELLS INDUCE GLIAL FATE IN NEURAL PROGENITOR CELLS Byron Oppliger , Marianne J€ orger-Messerli , Camilla Marini , Ursula Reinhart , Daniel V. Surbek , Andreina Schoeberlein a Department of Obstetrics and Gynecology, University Hospital, Department of Clinical Research, University of Bern, Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern, Bern, Bern,

Stretch-induced human myometrial cytokines enhance immune cell recruitment via endothelial activation.

Spontaneous term labour is associated with amplified inflammatory events in the myometrium including cytokine production and leukocyte infiltration; however, potential mechanisms regulating such events are not fully understood. We hypothesized that mechanical stretch of the uterine wall by the growing fetus facilitates peripheral leukocyte extravasation into the term myometrium through the release of various cytokines by uterine myocytes. Human myometrial cells (hTERT-HM) were subjected to static mechanical stretch; stretch-conditioned media was collected and analysed using 48-plex Luminex assay and ELISA. Effect of stretch-conditioned media on cell adhesion molecule expression of human uterine microvascular endothelial cells (UtMVEC-Myo) was detected by quantitative polymerase chain reaction (qPCR) and flow cytometry; functional assays testing leukocyte-endothelial interactions: adhesion of leukocytes to endothelial cells and transendothelial migration of calcein-labelled primary human neutrophils as well as migration of THP-1 monocytic cells were assessed by fluorometry. The current in vitro study demonstrated that mechanical stretch (i) directly induces secretion of multiple cytokines and chemokines by hTERT-HM cells (IL-6, CXCL8, CXCL1, migration inhibitory factor (MIF), VEGF, G-CSF, IL-12p70, bFGF and platelet-derived growth factor subunit B (PDGF-bb), P<0.05); stretch-induced cytokines (ii) enhance leukocyte adhesion to the endothelium of the surrounding uterine microvasculature by (iii) inducing the expression of endothelial cell adhesion molecules and (iv) directing the transendothelial migration of peripheral leukocytes. (vi) Chemokine-neutralizing antibodies and broad-spectrum chemokine inhibitor block leukocyte migration. Our data provide a proof of mechanical regulation for leukocyte recruitment from the uterine blood vessels to the myometrium, suggesting a putative mechanism for the leukocyte infiltrate into the uterus during labour and postpartum involution.

Adenosine A2Breceptors induce proliferation, invasion and activation of cAMP response element binding protein (CREB) in trophoblast cells

BackgroundPlacental hypoxia is a result of abnormal and shallow trophoblast invasion and involved in the pathophysiology of preeclampsia. Hypoxia increases extracellular adenosine levels and plays an important role in the regulation of angiogenesis, proliferation, vascular tone, endothelial permeability and inflammation. It was shown that adenosine concentrations are higher in preeclamptic patients. We tested the hypothesis that hypoxia and A2B adenosine receptor activation influence cyclic adenosine monophosphate (cAMP) production, proliferation, invasion and cAMP-PKA-CREB signaling in trophoblast cells (HTR-8/SVneo).MethodsHTR-8/SVneo and human uterine microvascular endothelial cells (HUtMVEC) were used as model for experiments. We employed a cAMP assay, invasion assay, proliferation, RT-PCR and Western Blot. Statistical analyses were performed with ANOVA, Kruskal-Wallis-, Wilcoxon signed rank- or Mann–Whitney Test, as appropriate.ResultsHypoxia (2% O2) in comparison to normoxia (21% O2) led to increased A2B mRNA levels (1.21 ± 0.06 fold, 1 h 2% O2; 1.66 ± 0.2 fold, 4 h 2% O2 and 1.2 ± 0.04 fold, 24 h 2% O2). A2B adenosine receptor activation (NECA) stimulated trophoblast proliferation at 2% O2 (1.27 ± 0.06 fold) and 8% O2 (1.17 ± 0.07 fold) after 24 h and at 2% O2 (1.22 ± 0.05 fold), 8% O2 (1.23 ± 0.09 fold) and 21% O2 (1.15 ± 0.04 fold) after 48 h of incubation. Trophoblast invasion into an endothelial monolayer was significantly expanded by activation of the receptor (NECA) at 8% O2 (1.20 ± 0.07 fold) and 21% O2 (1.22 ± 0.006 fold). A2B adenosine receptor stimulation (NECA) additionally led to increased CREB phosphorylation in trophoblast cells at 2% O2 (2.13 ± 0.45 fold), 8% O2 (1.55 ± 0.13 fold) and 21% O2 (1.71 ± 0.34 fold). Blocking of CREB signaling resulted in reduced proliferation and CREB phosphorylation.ConclusionThese data expand the recent knowledge regarding the role of adenosine receptor A2B in human placental development, and may provide insight in mechanisms associated with pregnancy complications linked to impaired trophoblast invasion such as preeclampsia.

Regulatory Role of Membrane-Bound Form Interleukin-15 on Human Uterine Microvascular Endothelial Cells in Circulating CD16(−) Natural Killer Cell Extravasation into Human Endometrium1

Interleukin (IL)-15 plays a major role in accumulation of unique CD16(-) natural killer (NK) cells in the human endometrium, partly via selective extravasation of peripheral blood (PB) counterparts from local microvascular circulation. While IL-15 exhibits a chemotactic activity for PB CD16(-) NK cells, IL-15 attenuates their binding capacity to dermatan sulfate, the major CD62L ligand expressed on human uterine microvascular endothelial cells (HUtMVECs). These findings suggest that premature action of IL-15 interferes with CD62L-dependent tethering/rolling of PB CD16(-) NK cells on HUtMVECs, which is an early critical process of leukocyte extravasation. In this study, we investigated the mechanisms underlying the IL-15 regulation in the initial CD62L-dependent contact between PB CD16(-) NK cells and HUtMVECs. Unlike other candidate molecules, recombinant IL-15 downregulated CD62L expression on freshly isolated PB CD16(-) NK cells. IL-12 and IL-10, the two known upregulators of CD62L on CD16(-) NK cells, were not detectable in HUtMVECs and endometrial perivascular stromal cells. Binding to immobilized dermatan sulfate increased surface IL-15 receptor-alpha chain expression on CD16(-) NK cells. Under ovarian steroid stimulation, IL-15 was detectable on the surface, but not in the supernatant, of cultured HUtMVECs. Ovarian steroid-induced IL-15 expression on HUtMVECs was not attenuated by chondroitinase ABC (which degrades chondroitin sulfate-A and -C and dermatan sulfate) or sodium acetate buffer (which dissociates cytokines from their cognate receptors). These results suggest that HUtMVECs secrete a less soluble form of IL-15 into local microcirculation. Instead, HUtMVECs bear a membrane-bound form IL-15 under the influence of ovarian steroids, which may be favorable for preventing downregulation of CD62L on PB CD16(-) NK cells and facilitating their initial contact with HUtMVECs.

NADPH oxidase 2-derived superoxide downregulates endothelial KCa3.1 in preeclampsia

Endothelial dysfunction is associated with KCa3.1 dysfunction and contributes to the development of hypertension in preeclampsia. However, evidence of endothelial KCa3.1 dysfunction in the vascular system from women with preeclampsia is still lacking. Therefore, we examined whether endothelial KCa3.1 dysfunction occurs in vessels from women with preeclampsia. We compared KCa3.1 and NADPH oxidase (NOX) expression in umbilical vessels and primary cultured human umbilical vein endothelial cells (HUVECs) from normal (NP; n=17) and preeclamptic pregnancy (PE; n=19) and examined the effects of plasma from NP or PE on KCa3.1 and NOX2 expression in primary cultured HUVECs from NP or human uterine microvascular endothelial cells. The endothelial KCa3.1 was downregulated, and NOX2 was upregulated, in umbilical vessels and HUVECs from PE, compared with those from NP. In addition, HUVECs from PE showed a significant decrease in KCa3.1 current. Plasma from PE induced KCa3.1 down regulation, NOX2 upregulation, phosphorylated-p38 mitogen-activated protein kinase downregulation, and superoxide generation, and these effects were prevented by antioxidants (tempol or tiron), NOX2 inhibition, or anti-lectin-like oxidized low-density lipoprotein (LDL) receptor 1 (LOX1) antibody. Oxidized LDL and the superoxide donor xanthine/xanthine oxidase mixture induced KCa3.1 downregulation. In contrast, plasma from PE did not generate hydrogen peroxide, and the hydrogen peroxide donor tert-butylhydroperoxide induced KCa3.1 upregulation. These results provide the first evidence that plasma from PE generates superoxide via a LOX1–NOX2-mediated pathway and downregulates endothelial KCa3.1, which may contribute to endothelial dysfunction and vasculopathy in preeclampsia. This suggests KCa3.1as a novel target for patients with preeclampsia.

Chymotrypsin-Like Protease (Chymase) Mediates Endothelial Activation by Factors Derived From Preeclamptic Placentas

Endothelial cells (EC) activation is an important inflammatory phenotypic change in the vascular system in women with preeclampsia (PE). In PE, maternal vessel chymotrypsin-like protease (CLP)/chymase expression was increased. Chymase is an inflammatory protease. In this study, we specifically examined whether placental-derived CLP could induce EC activation and whether EC activation is associated with increased cellular protease expression. Human uterine microvascular endothelial cells (UtMVECs) were used. Endothelial activation was determined by endothelial adhesion molecule P-selectin, E-selectin, inter-cellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) expressions and by extracellular regulated kinase (ERK) activity. Activation of endogenous CLP/chymase associated with ERK phosphorylation was further examined by CLP/chymase short interfering RNA (siRNA). Our results showed that cells treated with PE placental conditioned medium revealed increased P-selectin, E-selectin, and VCAM-1 expressions and increased ERK phosphorylation. Increased endothelial adhesion molecule expression and phosphorylated ERK (pERK) induction could be attenuated or abolished by depletion of CLP in the conditioned medium or by transfecting ECs with CLP/chymase siRNA. These observations suggest that placental-derived CLP/chymase is responsible for inducing endothelial inflammatory phenotypic changes possibly by upregulation of cell adhesion molecule expressions, activation of cellular protease, and induction of ERK phosphorylation. We speculate that activation of endothelial CLP/chymase may directly relate to the increased inflammatory phenotypic changes in the vascular system in women with PE.

Dermatan sulfate proteoglycan biglycan as a potential selectin L/CD44 ligand involved in selective recruitment of peripheral blood CD16(−) natural killer cells into human endometrium

Unique CD16(-) NK cells acutely increase in the human uterine endometrium after ovulation. The origin of these NK cells remains unknown, but they may be recruited selectively from the circulation. Proteoglycans and their glycosaminoglycan side-chains expressed on endometrial microvascular endothelial cells play a key role in lymphocyte tethering/rolling, the initial step of lymphocyte extravasation. In this study, we sought for the potential proteoglycans involved in tethering/rolling of peripheral blood CD16(-) NK cells on endometrial microvascular endothelial cells. As compared with CD16(+) NK cells and non-NK cells, enriched peripheral blood CD16(-) NK cells bound preferably to immobilized glycosaminoglycans except for keratan sulfate. CD16(-) NK cells bound maximally to dermatan sulfate (DS), which was diminished by enzymatic pretreatment with dermatanase and chondroitinase ABC, but not with chondroitinase ACII. The binding capacity of CD16(-) NK cells to DS was attenuated by blocking antibodies against selectin L and CD44 or pretreatment of CD16(-) NK cells with IL-15. Of three known DS proteoglycans, biglycan and decorin but not epiphycan were expressed in the human cycling endometrium. In the endometrial microvessels, the immunoreactivity for biglycan was greater in the secretory phase than in the proliferative phase, and there was little, if any, immunoreactivity for decorin throughout the menstrual cycle. The ovarian steroid progesterone enhanced biglycan expression in cultured human uterine microvascular endothelial cells. These findings demonstrated that DS proteoglycan biglycan is a potential selectin L/CD44 ligand involved in tethering/rolling of peripheral blood CD16(-) NK cells on endometrial microvascular endothelial cells.

Effect of ovarian steroids on gene expression profile in human uterine microvascular endothelial cells

To examine the effect of the ovarian steroids on the gene expression profile in human uterine microvascular endothelial cells. An experimental study. University hospital and research laboratory. Eighteen premenopausal women with proven fertility undergoing hysterectomy for cervical carcinoma in situ or cervical dysplasia. None. The effect of E(2) and P on gene expression profile in pooled human uterine microvascular endothelial cells was examined with cDNA microarray analysis and confirmed with quantitative real-time reverse transcriptase polymerase chain reaction. In vivo endometrial endothelial expression of the proteins encoded by the genes significantly regulated by E(2) and/or P was examined with Western blotting analysis and immunohistochemistry. The genes steadily up-regulated by E(2) and/or P included angiogenic factors, water transporters, immunomodulators, binding proteins, electron transporters, amino acid transporter, signal transduction proteins, and blood pressure regulation factors. The proteins encoded by IL1RL1, WAS, and NPPA were detected in endometrial microvascular endothelial cells. Progesterone (alone or in combination with E(2)) can induce the genes involved in angiogenesis, edematous change, and leukocyte recruitment in human uterine microvascular endothelial cells. Ovarian steroids may contribute to endometrial differentiation via the action on local microvessels.

Oxygen and the liberation of placental factors responsible for vascular compromise

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.