Nicotine and cotinine affect the release of vasoactive factors by trophoblast cells and human umbilical vein endothelial cells

Objective

To examine nicotine (N) and cotinine (C) effects on trophoblast cells (TCs) and human umbilical vein endothelial cells (HUVEC) secretion of soluble fms-like tyrosine kinase (sFlt-1), soluble endoglin (sENG), placental growth factor (PlGF), transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF).

Study design

Human placentas and umbilical cords were collected from uncomplicated pregnancies at term from a total of 24 non-smoking women with a history of normal blood pressure. TCs and HUVEC were cultured for 24 h with C or N (from 10⁻¹² to 10⁻⁷ M).

Main outcome measures

sFlt-1, sENG, PlGF, TGF-beta and VEGF release and messenger RNA (mRNA) expression were evaluated by ELISA and real-time polymerase chain reaction (PCR), respectively.

Results

N and C reduced sFlt-1, sENG and PlGF release by TCs and TGF-beta release by HUVEC. Conversely, N and C increased PlGF secretion, while N alone increased sFlt-1 release by HUVEC. N and C were able to modulate VEGF mRNA expression in HUVEC.

Conclusions

Our results suggest that N and C affect the balance of some important vasoactive factors released by TCs and HUVEC. This might be one of the possible mechanism through which smoke reduces the risk of hypertensive disorders during pregnancy as well as contributes to the well known detrimental effects of smoking on fetal development.     

Edaravone inhibits hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 expression: A possible therapeutic approach to preeclampsia

Objective

To investigate the effects of edaravone, a potent free radical scavenger used clinically, on hypoxia-induced trophoblast-soluble Fms-like tyrosine kinase 1 (sFlt-1) expression.

Methods

A trophoblast cell line (HRT-8/SVneo) impaired by cobalt chloride (CoCl₂) was used as the cell model under hypoxic conditions. 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) was used to measure the viability of cells exposed to CoCl₂ and edaravone. The levels of intracellular reactive oxygen species (ROS) were analyzed by flow cytometry. mRNA expression of sFlt-1, vascular endothelial growth factor (VEGF), and placental growth factor (PlGF) in trophoblasts was measured by real-time polymerase chain reaction, and the secretion of sFlt-1, VEGF, and PlGF proteins was analyzed by enzyme-linked immunosorbent assays (ELISAs). A human umbilical vein endothelial cell (HUVEC) tube-formation assay was performed to identify the effects of CoCl₂ and edaravone on vascular development.

Results

CoCl₂ treatment caused the loss of trophoblast viability, the formation of ROS, and sFlt-1 mRNA and protein expression in a dose-dependent manner. Pretreatment with edaravone significantly inhibited hypoxia-induced oxidative stress formation and sFlt-1 expression in trophoblasts. Neither PlGF nor VEGF mRNA or protein expression was increased by CoCl₂. In the in vitro tube formation assay, edaravone showed a protective role in vascular development under hypoxic conditions.

Conclusion

This study demonstrated that hypoxia leading to increased sFlt-1 release in trophoblasts may contribute to the placental vascular formation abnormalities observed in preeclampsia and suggested that the free radical scavenger edaravone could be a candidate for the effective treatment of preeclampsia.     

Effects of sFlt-1 and alpha 2-macroglobulin on vascular endothelial growth factor-induced endothelin-1 upregulation in human microvascular endothelial cells

Introduction

Soluble fms-like tyrosine kinase-1 (sFlt-1) is a vascular endothelial growth factor (VEGF) binding protein and potent antagonist of VEGF. Alpha 2 macroglobulin (α₂M) is another major binding protein for circulating VEGF, which is present in human plasma at higher concentration (2–4 mg/mL) than sFlt-1. This study investigated the effects of sFlt-1 and α₂M on VEGF-induced endothelin-1 (ET-1) upregulation in human microvascular endothelial cell-1 (HMEC-1).

Methods

HMEC-1 was cultured and incubated with varying concentrations of sFlt-1 and α₂M in combination with VEGF. ET-1 mRNA expression in the cells was measured by real time RT-PCR and ET-1 protein by western blot analysis.

Results

ET-1 expression in HMEC-1 incubated with VEGF significantly increased in time- and dose-dependent manners. Next, HMEC-1 was treated with the sFlt-1 (10–1000 ng/mL) or α₂M (10–10000 ng/mL) in the presence of VEGF (10 ng/mL). We found that sFlt-1 induced a significant decrease of ET-1 expression upregulated by VEGF, while α₂M did not affect the VEGF-induced ET-1 expression.

Conclusions

sFLT-1 suppressed the VEGF-induced the ET-1 expression of HMEC-1. However, α₂M did not show a significant effect on the ET-1 expression that was induced by VEGF. The results suggest that a certain proportion of the bound form α₂M-VEGF have a biological action involved in the pathophysiology of preeclampsia.     

Soluble fms-like tyrosine kinase-1 in HIV infected pre-eclamptic South African Black women

Introduction

Hypertensive disorders of pregnancy are the commonest direct cause of maternal deaths in South Africa, 83% being attributed to pre-eclampsia. Elevated placental sFlt-1 levels are linked with angiogenic disruption and subsequent pre-eclampsia development. The impact of HIV infection on pre-eclampsia is controversial. Its effect on angiogenic imbalance in both normotensive and pre-eclamptic pregnancies remains unknown.

Methods

We examined the immunolocalisation of both membrane bound and soluble forms of Flt-1, within placentae of HIV negative and positive normotensive and pre-eclamptic pregnancies at term using immunohistochemistry and immuno-electron microscopy.

Results

Strong Flt-1 and sFlt-1 immunoreactivity was observed within endothelial, syncytio and cytotrophoblast cells. Subcellularly, gold particles were localised predominantly within the endoplasmic reticulum and mitochondria and occurring free within the cytoplasm. There was no significant effect of HIV on Flt-1 and sFlt-1 immunoexpression in both exchange and stem villi. A significant effect of type of pregnancy (normotensive vs pre-eclamptic) on Flt-1 and sFlt-1 immunoexpression (p = 0.003) within exchange rather than stem villi, indicated that the pre-eclamptic had elevated Flt-1 and sFlt-1 expressions compared to the normotensive pregnant women. There was no interaction between HIV and pregnancy type (normotensive vs pre-eclampsia) for Flt-1 and sFlt-1 expressions in both exchange and stem villi. A weak correlation of Flt-1 and sFlt-1 intensity between the exchange and stem villi was noted.

Discussion

Elevated immunoexpression of Flt-1 and sFlt-1 within trophoblasts suggests an autocrine mode of action on trophoblast invasion and differentiation thereby contributing to abnormal placentation with consequential endothelial dysfunction in pre-eclampsia.

Conclusion

Irrespective of the HIV status, placental Flt-1 and sFlt-1 expressions remain elevated in pre-eclampsia compared to normotensive pregnancies.     

Effect of laparoscopic ovarian drilling on vascular endothelial growth factor and ovarian stromal blood flow using 3-dimensional power Doppler

Objective

To determine, by using 3-dimensional power Doppler ultrasonography, the effect of laparoscopic ovarian drilling (LOD) on the serum level of vascular endothelial growth factor (VEGF) and ovarian stromal blood flow changes in polycystic ovary syndrome (PCOS).

Methods

A prospective controlled clinical study was conducted on 26 clomiphene-resistant women with PCOS who were scheduled for LOD and a control group of 22 fertile regularly menstruating women. VEGF and 3 ovarian Doppler indices—vascularization index, flow index, and vascularization flow index—were measured and compared between the 2 groups, and before and after LOD in the PCOS group.

Results

Serum VEGF and the Doppler indices of ovarian stromal blood flow were significantly higher in the PCOS group than in the control group. Serum VEGF and the ovarian stromal blood flow Doppler indices were significantly reduced in the PCOS group after LOD.

Conclusion

Increased vascularity in PCOS demonstrated by Doppler blood flow measurements might be explained by the high level of VEGF. LOD reduced ovarian vascularization and serum VEGF.     

EFEMP1 expression promotes angiogenesis and accelerates the growth of cervical cancer in vivo

Objective

The study was to investigate the role of EFEMP1 in angiogenesis and growth of cervical carcinoma in vivo.

Methods

Effects of EFEMP1 on proliferation of Hela cells and HUVECs, invasion of Hela cells and migration of HUVECs, and adhesion of Hela cells to HUVECs were evaluated by MTT, Transwell chamber assay and adhesion assay, respectively. EFEMP1 overexpression in Hela cells was achieved by stable EFEMP1 gene transfection into Hela cells by Lipofectamin™ 2000 and the effectiveness of transfection was verified with western-blotting. The effect of EFEMP1 transfection upon the VEGF expression of Hela cells was detected with ELISA. The nude mouse models bearing cervical cancer were established with Hela cells transfected with EFEMP1 gene to observe the role of EFEMP1 in angiogenesis and growth of cervical cancer in vivo. VEGF expression and microvascular density of cervical cancer tissues were detected with immunohistochemistry and CD34 labeling respectively to elucidate the pathway by which EFEMP1 influences the growth of cervical cancer.

Results

Proliferation and invasion of Hela cells were promoted by the EFEMP1 protein. The EFEMP1 gene transfection into Hela cells was successful and EFEMP1 gene obtained stable high expression in Hela cells. Compared to the control, the tumors with EFEMP1 overexpression showed a faster growth rate and had a higher level of VEGF expression and microvascular density. EFEMP1 gene transfection elevated the VEGF protein level in Hela cells and EFEMP1 protein facilitated the adhesion of Hela cells to HUVECs. However, no direct effect of EFEMP1 was observed on proliferation, migration and tube formation of HUVECs.

Conclusions

EFEMP1 promoted the angiogenesis and accelerated the growth of cervical carcinoma in vivo through a VEGF up-regulation pathway.     

sFlt-1及PLGF与子痫前期发病的关系

可溶性血管内皮生长因子受体1 (sFlt-1)与Flt-1均属于血管内皮生长因子受体(VEGFR),其与Flt-1竞争结合VEGF,完全阻断VEGF的生物学活性,引起血管生成障碍并影响血管壁的完整性和通透性.胎盘生长因子(PLGF)是VEGF家族成员之一,PLGF与Flt-1受体结合时,发挥促血管生成和促绒毛滋养细胞增...     

Is endometrial polyp formation associated with increased expression of vascular endothelial growth factor and transforming growth factor-beta1?

Objective

Endometrial polyp is a common cause of abnormal uterine bleeding, but the etiology and pathogenesis remain unclear. Vascular endothelial growth factor (VEGF) is angiogenic, related to thick walled vessels and transforming growth factor-beta1 (TGF-β1) is related to fibrotic tissue, which are characteristics of endometrial polyps. The primary objective of this study was to find out if endometrial polyp formation is associated with increased expression of VEGF or TGF-β1, or both. A secondary objective is to determine if the changes are related to steroid receptor expression.

Study design

This prospective study compared VEGF and TGF-β1 expression of endometrial polyps and adjacent endometrial tissue in 70 premenopausal women. The comparison of results was separately made for endometrium specimens obtained in the proliferative and secretory phases. The results were correlated with the steroid receptors (estrogen receptor and progesterone receptor) expression.

Results

The score of VEGF in glandular cells of endometrial polyps was significantly higher than the score in adjacent endometrium, both in the proliferative phase (P < 0.001) and the secretory phase (P = 0.03); the score of VEGF in stromal cells of endometrial polyps was significantly higher than the score in adjacent endometrium only in proliferative phase (P = 0.006). The score of TGF-β1 in glandular cells of endometrial polyps was significantly higher than the score in adjacent endometrium in proliferative phase (P = 0.02); whereas the score of TGF-β1 in stromal cells of endometrial polyps was significantly higher than the score in adjacent endometrium, both in the proliferative phase (P = 0.006) and the secretory phase (P = 0.008). There was a significant correlation between the expression of steroid receptors and VEGF and TGF-β1 (Spearman's correlation P < 0.001 and P < 0.05, respectively).

Conclusions

There was increased expression of TGF-β1 and VEGF in polyps compared to adjacent normal endometrial tissue. It suggested that these cytokines might play a role in endometrial polyp formation. In addition, there was a significant correlation between steroid receptor expression and VEGF and TGF-β1 expression.     

Adrenomedullin interacts with VEGF in endometrial cancer and has varied modulation in tumours of different grades

Objective

Endometrial cancer, in developed countries, is the most common malignancy of the female genital tract. Surgery and radiotherapy are successful in many patients but systemic and recurrent diseases have no consistently effective treatments, and for high grade advanced disease the prognosis is poor. The study investigated characteristics of adrenomedullin in endometrial cancer to assist in identifying targets for developing treatments.

Methods

Endometrial samples of women with and without cancer, and the Ishikawa cell line were used to investigate adrenomedullin mRNA regulation, peptide expression, adrenomedullin secretion and effects of adrenomedullin on VEGF secretion.

Results

Expression of adrenomedullin mRNA was upregulated compared to that in healthy post-menopausal endometria. Adrenomedullin secretion was increased by cobalt chloride in this study. Secretion was reduced by the naturally-occurring compounds, (−)-epigallocatechin gallate (EGCG) and 3,4′,5-trihydroxystilbene (resveratrol), which we have previously demonstrated to also suppress VEGF secretion in endometrial tumour tissue. We noted, for the first time, that adrenomedullin enhanced VEGF secretion from tumour cells.

Conclusions

Increased adrenomedullin expression may result in amplifying both tumorigenic and angiogenic activities. A substantial impact on growth of tumours may result in vivo as a consequence of the synergism between adrenomedullin and VEGF. Adrenomedullin, which has altered cellular characteristics in tumour compared to healthy tissue, offers an understudied target with potential to modify endometrial cancer behaviour, complementing other treatments.     

Increased placental sFlt-1 but unchanged PlGF expression in late-onset preeclampsia

Objective: To investigate whether differences between late-onset preeclampsia (PE) and intrauterine growth restriction (IUGR) can be explained by differential placental expression patters of sFlt-1, Flt-1, and placental growth factor (PlGF).

Methods: Placental tissues and maternal blood samples from seven patients with PE, five IUGR, and seven age-matched controls were studied for mRNA and protein levels as well as protein localization and expression intensity.

Results: Placental PlGF mRNA and protein expression were not altered by placental dysfunction while placental villous trophoblast expression intensity of PlGF was increased.

Conclusion: High sFlt-1 concentrations may account for diminished maternal serum PlGF levels.     

Nitric oxide (NO) reversed TNF-α inhibition of trophoblast interaction with endothelial cellular networks

Introduction

The interaction between trophoblast cells and maternal uterine endothelium is important for placental vascular modelling. Nitric oxide (NO) is a potent vasorelaxant that regulates systemic blood pressure and is reduced in preeclampsia. NO may affect placental cell interaction.

Objectives

This study was to examine whether NO plays a role in regulating TNF-α induced inhibition of trophoblast cell integration into endothelial cellular networks in-vitro.

Methods

Red fluorescent-labelled human uterine myometrial microvascular endothelial cells (UtMVECs) were seeded on Matrigel. After endothelial cellular networks formed, green fluorescent-labelled HTR-8/SVneo trophoblast cells were co-cultured with endothelial cells, together with/without TNF-α (0.5 ng/ml) and/or NO donor, sodium nitroprusside dihydrate (SNP) (100 μM). Images were captured after 24 h. The effects on HTR-8/SVneo cell integration were quantified by Image Analysis software. The cells were then recovered from Matrigel to extract mRNA. Quantitative PCR was performed to evaluate the expression of eNOS, VCAM-1 and E-selectin. The concentrations of sVCAM-1 and sE-selectin in the conditioned medium were measured by ELISA.

Results

TNF-α inhibited HTR-8/SVneo trophoblast cell integration into endothelial cellular networks, as well as decreased eNOS mRNA expression. Increases in VCAM-1 and E-selectin in cellular mRNA and protein concentrations in the conditioned medium were also seen. The NO donor reversed the inhibitory effect of TNF-α on trophoblast integration and increased eNOS mRNA expression. SNP also reduced sE-selectin and sVCAM-1 expressions which were increased by TNF-α in the conditioned medium.

Conclusion

Our data suggest the inhibitory effect of TNF-α on trophoblast integration may be mediated by NO, via reducing endothelial cell activation.     

Expression of transforming growth factor beta1 in nerve fibers is related to dysmenorrhea and laparoscopic appearance of endometriotic implants

Objective

To quantify the expression of transforming growth factor β1 in nerve fibers in endometriotic lesions and to correlate it with dysmenorrhea and appearance of endometriotic implants.

Design

Prospective comparative study.

Setting

University hospital.

Patient(s)

Peritoneal endometriotic specimens obtained from 35 patients diagnosed with endometriosis were compared with biopsies of normal peritoneum from 10 patients without endometriosis.

Intervention(s)

Endometriosis-associated dysmenorrhea for each patient was evaluated before surgery using a 10-point visual analog scale, which was followed by a laparoscopic staging of the patient's endometriosis.

Main outcome measure(s)

Immunohistochemical analysis of the peritoneal endometriotic specimens evaluated the maximal intensity of staining (INTMMAX) of TGFβ1, defined as higher staining intensity found within a selected structure.

Result(s)

When the nerve fibers of endometriotic lesions were compared with those of normal peritoneum, statistically significant differences were found in the INTMMAX of TGFβ1. Greater TGFβ1 INTMMAX was found in red lesions and deep endometriotic foci than in black lesions and normal peritoneum. A statistically significant relationship was found between the TGFβ1 INTMMAX score and dysmenorrhea; a relationship also was found to the color of the lesions.

Conclusion(s)

The physical appearance of endometriotic implants and the severity of dysmenorrhea appear to be related to the expression of TGFβ1 in nerve fibers.     

Modulation of endocrine and transport functions in human trophoblasts by saquinavir and nelfinavir

Objectives

The distribution of drugs to the maternal-fetal interface is influenced by the expression of various efflux transporters. Among these transporters, P-glycoprotein (P-gp) is responsible for the efflux of a great number of drugs such as protease inhibitors of the human immunodeficiency virus, thus reducing the chemical exposure of the fetus.

Study design

The effects of saquinavir and nelfinavir were evaluated on human trophoblast functions and integrity by investigating their effect on human chorionic gonadotropin (hCG) secretion and on P-gp expression and functionality.

Results

Nelfinavir significantly reduced hCG secretion by 30% after a 48-h treatment but it had no effect on syncytia formation. Saquinavir had no effect on hCG secretion but significantly increased both expression (to a 2-fold extent) and functionality (by 17.9%) of P-gp, whereas nelfinavir only increased functionality (by 23.1%) with a dissociation of P-gp from caveolin-1.

Conclusion

These results suggest that the effects of saquinavir and nelfinavir differ on trophoblast functions.     

Differential in vitro actions of nitric oxide on human endometrial cell survival

Objective

To investigate the presence of caspase-3 and Bcl-2 concentration in human endometrial tissue throughout the menstrual cycle, and study the effect of nitric oxide (NO) on cell proliferation and apoptosis during culture.

Design

Expression of caspase-3 and Bcl-2 concentration in endometrial explants, and examination of L-arginine (L-Arg) effect on epithelial and stromal cell proliferation and apoptosis in vitro.

Setting

Prospective study.

Patient(s)

Twenty-seven eumenorrheic women (37 ± 1.2 years).

Intervention(s)

Endometrial samples were obtained with Pipelle suction curette from the corpus of the uterus.

Main outcome measure(s)

Apoptosis (annexin V-FITC binding), Bcl-2 concentration (ELISA), caspase-3 (immunohistochemistry), cell proliferation (spectrophotometric assay), and gene expression (RT-PCR).

Result(s)

Caspase-3 was detected by immunoassay in epithelial tissue throughout the menstrual cycle and in stroma during secretory phase. The Bcl-2 concentration was similar in endometrial homogenates obtained throughout the menstrual cycle, but L-Arg decreased Bcl-2 only in endometrium from the proliferative phase. In epithelial cells, NO increased apoptosis by 2.1 ± 0.2-fold, augmented mRNA expression of Bax, and reduced expression of Bcl-2 compared with basal cultures. In stromal cells, NO increased cell proliferation in a dose-dependent manner, an effect that was blocked by a NO synthase inhibitor.

Conclusion(s)

These data indicate that NO has a differential regulatory function on endometrial cell survival, as indicated by the results on stromal cell proliferation and epithelial cell apoptosis during culture, which suggests paracrine interactions between both cell types.     

Placental expression of sFlt-1 and PlGF in early preeclampsia vs. early IUGR vs. age-matched healthy pregnancies

Objective: To investigate whether differences between early preeclampsia and early fetal growth restriction can be explained by differential placental expression patterns of sFlt-1, Flt-1, and PlGF. Methods: Placental tissues and maternal blood samples from six cases of preeclampsia, seven IUGR, and six age-matched controls were studied for mRNA and protein levels as well as protein localization and expression intensity. Results: Neither placental PlGF mRNA and protein expression nor placental villous trophoblast expression intensity of PlGF was altered by placental dysfunction. Conclusion: High sFlt-1 concentrations may account for diminished maternal serum PlGF levels.     

缺氧对滋养细胞sFlt-1及VEGF基因及蛋白表达的影响

目的:探讨缺氧对滋养细胞可溶性血管内皮生长因子受体1(soluble fims-like tyrosine kinase receptor-1,sFlt-1)及血管内皮生长因子(vascular epithelial growth factor,VEGF)表达的影响。方法:CoCl2诱导人早孕绒毛滋养细胞系TEV-1化学缺氧,分别于0h、24h、48h、72h、96h、120h用Real-time RT-PCR法检测滋养细胞中sFlt-1及VEGF mRNA表达,ELISA法检测细胞培养上清液中sFlt-1及VEGF蛋白表达。结果:滋养细胞缺氧72h后,胞质内可见明显空泡。滋养细胞sFlt-1mRNA及蛋白表达随缺氧时间的延长逐渐升高,且与缺氧72h、96h、120h间差异有显著性(P<0.05)。而滋养细胞VEGF mRNA及蛋白表达随缺氧时间的延长呈现出先上升后下降的趋势,并于缺氧48h升高达到峰值(P<0.05)。结论:不同的缺氧时间对滋养细胞VEGF及sFlt-1表达具有重要的调控作用。慢性缺氧可能导致滋养细胞sFlt-1及VEGF表达平衡失调,从而参与子痫前期的发生、发展。     

可溶性血管内皮生长因子受体-1与胎盘生长因子在子痫前期

归纳子痫前期的病理生理过程以及可溶性血管内皮生长因子受体-l（sFlt-1）、胎盘生长因子（PLGF）与子痫前期的研究情况,PLGF是血管内皮生长因子（VEGF）家族成员之一,PLGF与Flt-1受体结合时,发挥促血管生成和促绒毛滋养细胞增殖、浸润的效应,而这些生物学功能均可被sFlt-1阻断,且sFlt-1对PLGF具有强效的拮抗作用,调节PLGF的功能。在子痫前期妊娠妇女血清中sFlt-1的水平升高,早于疾病的发生。子痫前期患者血浆中PLGF的水平降低。总结sFlt-1、PLGF预测诊断子痫前期发生及病情严重程度的研究进展,探讨sFlt-1、PLGF在子痫前期临床诊断中的意义。     

Leptin induces tube formation in first-trimester extravillous trophoblast cells

Objectives

To study the roles of leptin on tube formation (as a measure of cellular angiogenesis) and expression of associated genes in first-trimester human extravillous trophoblast cells.

Study design

The effects of leptin on tube formation and fatty acid uptake in first trimester extravillous placental trophoblast cells, HTR8/SVneo, were investigated. We also investigated the effects of leptin on the expression of genes involved in angiogenesis and lipid metabolism in these cells.

Results

Leptin at 25 ng/ml maximally stimulated tube formation in the first trimester placental trophoblast cells, HTR8/SVneo, by increasing tube length as well as numbers (10,100 ± 150 pixels) compared with those of control cells (2900 ± 50 pixels) p > 0.05. Leptin-induced tube formation was not inhibited by the selective inhibitor of VEGF, indicating that its action was independent of VEGF. Leptin, however, significantly increased the expression of genes those are involved in angiogenesis pathways such as PECAM1, JAG1, CDH5, IL8, NRP1, SPHK1, S1PR1, CXCL 1 and 6, FGF1, EFNA3 and AKT1, as determined by PCR array. Leptin did not, however, stimulate expression of the primary angiogenic factors known in placenta such as VEGF or ANGPTL4, as determined by both qRTPCR and PCR array assays. Leptin increased 7-fold expression of FABP4, which is known to be involved in VEGF-mediated angiogenesis in endothelial cells. In addition, leptin treatment resulted a 48% increase in the uptake of docosahexaenoic acid, 22:6n-3 (DHA) which also stimulates tube formation in these cells.

Conclusions

Leptin may play an important role in early placentation by stimulating several genes involved in angiogenic signalling pathway and fatty acid metabolism.     

Activation of adenosine A2B receptor impairs properties of trophoblast cells and involves mitogen-activated protein (MAP) kinase signaling

Introduction

Shallow trophoblast invasion of the maternal spiral arteries contributes to impaired placental perfusion and is hypothesized to be involved in the pathophysiology of preeclampsia. Hypoxia is a potent stimulus for the release of adenosine.

Methods

We investigated the effects of hypoxia and A_2B adenosine receptor signaling on migration, invasion, proteolytic activity of matrix metalloproteinase (MMP)-2, expression of MMP-2 and vascular endothelial growth factor (VEGF) mRNA, and production of human chorionic gonadotropin (hCG) in trophoblast cells (HTR-8/SVneo, BeWo).

Results

The adenosine A_2B receptor agonist 5-N-ethylcarboxamidoadenosine (NECA) reduced trophoblast (HTR-8/SVneo and BeWo) migration at 2%, 8% and 21% O₂ compared to untreated control cells. A_2B adenosine receptor stimulation decreased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and stress-activated protein kinase/Jun-amino-terminal kinase (SAPK/JNK) at all three O₂ concentrations. ProMMP-2 activity, MMP-2 mRNA levels and hCG levels were markedly decreased after A_2B adenosine receptor activation in trophoblast cells. Adenosine receptor A_2B stimulation decreased VEGF expression at 2% and 8% O₂ but led to increased levels at 21% O₂.

Conclusions

These data indicate A_2B receptor activation blunts trophoblast migration possibly as a result of reduced activation of the MAPK signaling pathway and lower proMMP-2 levels. These data suggest a role for adenosine receptor A_2B in placental development and possibly in the pathophysiology of preeclampsia.