Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development.

After menstruation the uterine spiral arteries are repaired through angiogenesis. This process is tightly regulated by the paracrine communication between endometrial stromal cells (EnSCs) and endothelial cells. Any molecular aberration in these processes can lead to complications in pregnancy including miscarriage or preeclampsia (PE). Placental growth factor (PlGF) is a known contributing factor for pathological angiogenesis but the mechanisms remain poorly understood. In this study, we investigated whether PlGF contributes to pathological uterine angiogenesis by disrupting EnSCs and endothelial paracrine communication. We observed that PlGF mediates a tonicity-independent activation of nuclear factor of activated T cells 5 (NFAT5) in EnSCs. NFAT5 activated downstream targets including SGK1, HIF-1α and VEGF-A. In depth characterization of PlGF - conditioned medium (CM) from EnSCs using mass spectrometry and ELISA methods revealed low VEGF-A and an abundance of extracellular matrix organization associated proteins. Secreted factors in PlGF-CM impeded normal angiogenic cues in endothelial cells (HUVECs) by downregulating Notch-VEGF signaling. Interestingly, PlGF-CM failed to support human placental (BeWo) cell invasion through HUVEC monolayer. Inhibition of SGK1 in EnSCs improved angiogenic effects in HUVECs and promoted BeWo invasion, revealing SGK1 as a key intermediate player modulating PlGF mediated anti-angiogenic signaling. Taken together, perturbed PlGF-NFAT5-SGK1 signaling in the endometrium can contribute to pathological uterine angiogenesis by negatively regulating EnSCs-endothelial crosstalk resulting in poor quality vessels in the uterine microenvironment. Taken together the signaling may impact on normal trophoblast invasion and thus placentation and, may be associated with an increased risk of complications such as PE.

Eucommia granules activate Wnt/ß-catenin pathway, and improve oxidative stress, inflammation, and endothelial injury in preeclampsia rats.

PURPOSE: Pre-eclampsia (PE) is a pregnancy-related complication. Eucommia is effective in the treatment of hypertensive disorders in pregnancy, but the specific effects and possible mechanisms of Eucommia granules (EG) in PE remain unknown. The aim of this study was to investigate the effects and possible mechanisms of EG in PE rats. METHODS: Pregnant Sprague Dawley rats were divided into five groups (n = 6): the control group, the model group, the low-dose group, the medium-dose group, and the high-dose group of EG. The PE model was established by subcutaneous injection of levonitroarginine methyl ester. Saline was given to the blank and model groups, and the Eucommia granules were given by gavage to the remaining groups. Blood pressure and urinary protein were detected. The body length and weight of the pups and the weight of the placenta were recorded. Superoxide dismutase (SOD) activity and levels of malondialdehyde (MDA), placental growth factor (PIGF), and soluble vascular endothelial growth factor receptor-1 (sFIt-1) were measured in the placenta. Pathological changes were observed by hematoxylin-eosin staining. Wnt/ß-catenin pathway-related protein expression was detected using Western blot. RESULTS: Compared with the model group, the PE rats treated with EG had lower blood pressure and urinary protein. The length and weight of the pups and placental weight were increased. Inflammation and necrosis in the placental tissue was improved. SOD level increased, MDA content and sFIt-1/PIGF ratio decreased, and Wnt/ß-catenin pathway-related protein expression level increased. Moreover, the results of EG on PE rats increased with higher doses of EG. CONCLUSIONS: EG may activate the Wnt/ß-catenin pathway and inhibit oxidative stress, inflammation, and vascular endothelial injury in PE rats, thereby improving the perinatal prognosis of preeclamptic rats. EG may inhibit oxidative stress, inflammation, and vascular endothelial injury through activation of the Wnt/ß-catenin pathway in preeclampsia rats, thereby improving perinatal outcomes in PE rats.

Platelet indices and angiogenesis markers in hypertensive disorders of pregnancy.

INTRODUCTION: Activated platelets exert a key role in the pathogenesis of preeclampsia (PE). There is evidence of distinctive patterns of platelet indices in PE in comparison to healthy pregnancies, therefore these indices can be potential tools for PE detection, risk stratification, and management. Considering the vascular aspects of its pathophysiology, PE is characterized by the increased levels of soluble FMS-like tyrosine kinase-1 (sFlt-1) an antiangiogenic factor, and reduced placental growth factor (PlGF), a proangiogenic factor. This study aimed to assess the platelet indices in hypertensive disorders of pregnancy (HDP) and its correlation with angiogenesis-related biomarkers. METHODS: The groups for the study were: control (n = 114); gestational hypertension; (n = 112), and PE (n = 42). The platelet indices included were platelet counts (PLT-I and PLT-F), mean platelet volume (MPV), platelet distribution width (PDW), plateletcrit (PCT), platelet large cell ratio (P-LCR), and immature platelet fraction (IPF# and IPF%). Serum levels of sFlt-1 and PlGF were assessed. RESULTS: PLT-I, PLT-F, and PCT% were lower in PE, while MPV, PDW, P-LCR, IPF%, and IPF# were increased. The parameter MPV presented the best performance for the discrimination of PE. There was a moderate positive correlation between sFlt-1 levels and MPV, PDW, and P-LCR. CONCLUSION: Platelet indices can be potentially applied as additional tools for the diagnosis and management of HDP. Activated platelets may act as an extra source of sFlt-1 in PE.

Evaluation of the Effect of the Fibroblast Growth Factor Type 2 (FGF-2) Administration on Placental Gene Expression in a Murine Model of Preeclampsia Induced by L-NAME.

The abnormal implantation of the trophoblast during the first trimester of pregnancy precedes the appearance of the clinical manifestations of preeclampsia (PE), which is a hypertensive disorder of pregnancy. In a previous study, which was carried out in a murine model of PE that was induced by NG-nitro-L-arginine methyl ester (L-NAME), we observed that the intravenous administration of fibroblast growth factor 2 (FGF2) had a hypotensive effect, improved the placental weight gain and attenuated the fetal growth restriction, and the morphological findings that were induced by L-NAME in the evaluated tissues were less severe. In this study, we aimed to determine the effect of FGF2 administration on the placental gene expression of the vascular endothelial growth factor (VEGFA), VEGF receptor 2 (VEGFR2), placental growth factor, endoglin (ENG), superoxide dismutase 1 (SOD1), catalase (CAT), thioredoxin (TXN), tumor protein P53 (P53), BCL2 apoptosis regulator, Fas cell surface death receptor (FAS), and caspase 3, in a Sprague Dawley rat PE model, which was induced by L-NAME. The gene expression was determined by a real-time polymerase chain reaction using SYBR green. Taking the vehicle or the L-NAME group as a reference, there was an under expression of placental VEGFA, VEGFR2, ENG, P53, FAS, SOD1, CAT, and TXN genes in the group of L-NAME + FGF2 (p < 0.05). The administration of FGF2 in the murine PE-like model that was induced by L-NAME reduced the effects that were generated by proteinuria and the increased BP, as well as the response of the expression of genes that participate in angiogenesis, apoptosis, and OS. These results have generated valuable information regarding the identification of molecular targets for PE and provide new insights for understanding PE pathogenesis.

Obesity phenotype induced by high-fat diet leads to maternal-fetal constraint, placental inefficiency, and fetal growth restriction in mice.

The aim of this study was to investigate certain parameters regarding the maternal-fetal outcomes in a diet-induced obesity model. Obese, glucose-intolerant females who were exposed to a high-fat diet prior to pregnancy had lower placental efficiency and lower birth weight pups compared to the controls. Simple linear regression analyses showed that maternal obesity disrupts the proportionality between maternal and fetal outcomes during pregnancy. Maternal obesity is correlated with fetal outcomes, perhaps because of problems with hormonal signaling and exacerbation of inflammation in the maternal metabolic environment. The maternal obese phenotype altered the thickness of the placental layer, the transport of fatty acids, and the expression of growth factors. For example, lower expression of epidermal growth factor receptor (EGFR) mRNA in the obesity-prone group may have contributed to the rupture of the placental layers, leading to adverse fetal outcomes. Furthermore, maintenance of maternal glucose homeostasis and overexpression of placental growth factor (PGF) in the obesity-resistant group likely protected the placenta and fetuses from morphological and functional damage.

First trimester placental growth factor in maternal blood and placenta related disorders.

OBJECTIVE: To describe and compare the placental growth factor levels at first trimester in patients that developed preeclampsia, gestational hypertension, IUGR and in those patients without impaired placentation diseases. METHODS: Observational study based on a prospective cohort of 422 pregnant women. PlGF values were compared between the different groups (preeclampsia, intrauterine growth restriction (IUGR), gestational hypertension or normal group-patients without impaired placentation diseases). RESULTS: The 85.3% (n = 360, 95% CI = 81.9-88.7) had a normal pregnancy, 7.6% (n = 32, 95% CI = 5.1-10.1) had preeclampsia, 3.8% (n = 16, 95% CI = 2.0-5.6) had IUGR and 3.3% (n = 14, 95% CI = 1.6-5.0) had gestational hypertension. The median level of PlGF for preeclampsia (0.76) and IUGR (0.75) were lower than gestational hypertension (0.82) and normal group (1.02). The groups of preeclampsia >34 weeks (0.76), preeclampsia <37 weeks (0.73), and preeclampsia ≥37 weeks (0.77), were significantly lower than the normal group. The sensitivity and specificity of PlGF for impaired placentation diseases is 65% and 64.9%, respectively. CONCLUSION: It was found in this study that PlGF has significantly lower levels in gestational hypertension than normal pregnancies, in concordance with the other impaired placentation diseases. Additionally, a better comparison of the PlGF values was obtained when separating early onset of preeclampsia <37 weeks and late-onset of preeclampsia 37≥ weeks of gestations.

Anti-placental growth factor antibody ameliorates hyperoxia-mediated impairment of lung development in neonatal rats.

This study investigates the effect of the overexpression of the placental growth factor (PGF) and hyperoxia on lung development and determines whether anti-PGF antibody ameliorates hyperoxia-mediated impairment of lung development in newborn rats. After exposure to normoxic conditions for seven days, newborn rats subjected to normoxia were intraperitoneally or intratracheally injected with physiological saline, adenovirus-negative control (Ad-NC), or adenovirus-PGF (Ad-PGF) to create the Normoxia, Normoxia+Ad-NC, and Normoxia+Ad-PGF groups, respectively. Newborn rats subjected to hyperoxia were intraperitoneally injected with physiological saline or anti-PGF antibodies to create the Hyperoxia and Hyperoxia+anti-PGF groups, respectively. Our results revealed significant augmentation in the levels of PGF and its receptor Flt-1 in the lung tissues of newborn rats belonging to the Normoxia+Ad-PGF or Hyperoxia groups. PGF overexpression in these groups caused lung injury in newborn rats, while anti-PGF antibody treatment significantly cured the hyperoxia-induced lung injury. Moreover, PGF overexpression significantly increased TNF-α and Il-6 levels in the bronchoalveolar lavage (BAL) fluid of the Normoxia+Ad-PGF and Hyperoxia groups. However, their levels were significantly reduced in the BAL fluid of the Hyperoxia+anti-PGF group. Immunohistochemical analysis revealed that PGF overexpression and hyperoxia treatment significantly increased the expression of the angiogenesis marker, CD34. However, its expression was significantly decreased upon administration of anti-PGF antibodies (compared to the control group under hyperoxia). In conclusion, PGF overexpression impairs lung development in newborn rats while its inhibition using an anti-PGF antibody ameliorates the same. These results provided new insights for the clinical management of bronchopulmonary dysplasia in premature infants.

Anti-placental growth factor antibody ameliorates hyperoxia-mediated impairment of lung development in neonatal rats

This study investigates the effect of the overexpression of the placental growth factor (PGF) and hyperoxia on lung development and determines whether anti-PGF antibody ameliorates hyperoxia-mediated impairment of lung development in newborn rats. After exposure to normoxic conditions for seven days, newborn rats subjected to normoxia were intraperitoneally or intratracheally injected with physiological saline, adenovirus-negative control (Ad-NC), or adenovirus-PGF (Ad-PGF) to create the Normoxia, Normoxia+Ad-NC, and Normoxia+Ad-PGF groups, respectively. Newborn rats subjected to hyperoxia were intraperitoneally injected with physiological saline or anti-PGF antibodies to create the Hyperoxia and Hyperoxia+anti-PGF groups, respectively. Our results revealed significant augmentation in the levels of PGF and its receptor Flt-1 in the lung tissues of newborn rats belonging to the Normoxia+Ad-PGF or Hyperoxia groups. PGF overexpression in these groups caused lung injury in newborn rats, while anti-PGF antibody treatment significantly cured the hyperoxia-induced lung injury. Moreover, PGF overexpression significantly increased TNF-α and Il-6 levels in the bronchoalveolar lavage (BAL) fluid of the Normoxia+Ad-PGF and Hyperoxia groups. However, their levels were significantly reduced in the BAL fluid of the Hyperoxia+anti-PGF group. Immunohistochemical analysis revealed that PGF overexpression and hyperoxia treatment significantly increased the expression of the angiogenesis marker, CD34. However, its expression was significantly decreased upon administration of anti-PGF antibodies (compared to the control group under hyperoxia). In conclusion, PGF overexpression impairs lung development in newborn rats while its inhibition using an anti-PGF antibody ameliorates the same. These results provided new insights for the clinical management of bronchopulmonary dysplasia in premature infants.

The Impact of Immunosuppressive Drugs on Human Placental Explants.

The use of immunosuppressive drugs guarantees the vitality of the graft and allows gestation in spite of intercurrences such as prematurity and intrauterine growth restriction. However, little is known about the direct effects of immunosuppressive drugs on placental cells. We investigated the effects of immunosuppressive drugs in the chorionic villous explants from human term placentas of healthy gestations. Human placental explants from term gestations (37-39 week gestational age, n = 12) were exposed to cyclosporine A (CSA, 0, 62.5, 125, 1250 ng/mL) or azathioprine (AZA, 0, 5, 10, 100 ng/mL) separately or, in combination for up to 48 hours. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed a significant decrease in the explant metabolic activity between AZA and the control group (24 hours, 100 ng/mL, 48 hours, all concentrations, P < .005). Cyclosporin A (CsA) reduced cell activity when associated with AZA (48 hours, P < .005). Fibrinoid deposits increased in AZA-treated explants alone (5 ng/mL, 48 hours; 10 ng/mL, 24-48 hours; P < .005) or when associated with CsA (10 AZA/125 CsA, P < .05), whereas in CsA treatment alone, there was an augment in syncytial knots (24-48 hours, P < .005). The sFLT1 gene (24 hours, P < .05) and protein (P < .005) expression increased in AZA and CsA-treatments separately or in combination (P < .05). Placental growth factor increased in AZA (24 hours, 10 ng/mL) and CsA (125 ng/mL; P < .05). In conclusion, our data indicate that AZA primarily acts on the villous metabolism, perturbing placental homeostasis. Since these drugs may alter the balance of angiogenic factors in its selection for clinical application, their impact on the behavior of placental villous should be considered.

The impact of different torques for the insertion of immediately loaded implants on the peri-implant levels of angiogenesis- and bone-related markers.

The aim of this split-mouth, randomized, double-blind, controlled clinical trial was to evaluate the influence of different insertion torque values for dental implants on bone- and angiogenesis-related marker profiles. Eighteen edentulous patients received dental implants and fixed complete-arch mandibular prostheses. The implants (n=36) were assigned randomly to two groups: reduced torque (n=18), with insertion torque <30Ncm; and conventional torque (n=18), with insertion torque ≥30Ncm. Levels of vascular endothelial growth factor (VEGF), placental growth factor, bone morphogenetic protein 9 (BMP-9), periostin, osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) in the peri-implant fluid were quantified at 7, 14, 30, and 120days after implant placement. Inter-group comparisons showed that VEGF and OPG levels were higher in the low-level torque group than in the conventional torque group on days 7 and 30, respectively (P<0.05). BMP-9 and periostin levels were higher in the conventional group than in the low-level torque group on day 120, and TRAP was up-regulated around implants inserted with conventional torque when compared to those inserted with lower-level torque at all time points evaluated (P<0.05). In conclusion, the use of different levels of torque for implantation of immediately loaded implants significantly influenced the levels of bone- and angiogenesis-related markers during early peri-implant repair.

Association between Placental Lesions, Cytokines and Angiogenic Factors in Pregnant Women with Preeclampsia.

Preeclampsia (PE) is considered the leading cause of maternal and perinatal morbidity and mortality. The placenta seems to play an essential role in this disease, probably due to factors involved in its formation and development. The present study aimed to investigate the association between placental lesions, cytokines and angiogenic factors in pregnant women with preeclampsia (PE). We evaluated 20 normotensive pregnant women, 40 with early-onset PE and 80 with late-onset PE. Placental samples were analyzed for histopathology, immunohistochemistry and determination of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), transforming growth factor-beta 1 (TGF-ß1), tumor necrosis factor-alpha (TNF-α), placental growth factor (PlGF), vascular endothelial growth factor (VEGF), fms-like tyrosine-kinase-1 (Flt-1) and endoglin (Eng) levels. Higher percentages of increased syncytial knots and increased perivillous fibrin deposits, and greater levels of TNF-α, TGF-ß1and Flt-1 were detected in placentas from early-onset PE. Levels of IL-10, VEGF and PlGF were decreased in PE versus normotensive placentas. Both the TNF-α/IL-10 and sFlt-1/PlGF ratios were higher in placental homogenate of early-onset PE than late-onset PE and control groups. The more severe lesions and the imbalance between TNF-α/IL-10 and PlGF/sFlt-1 in placentas from early-onset PE allows differentiation of early and late-onset PE and suggests higher placental impairment in early-onset PE.

Placental biomarkers and angiogenic factors in oral fluids of patients with preeclampsia.

OBJECTIVES: The objectives of this study are to explore the feasibility of measuring endothelial and placental biomarkers in saliva and gingival crevicular fluid (GCF) and to determine if patients with preeclampsia (PE) have a different profile of these biomarkers in oral fluids. METHOD: A case-control study was conducted, including patients with PE (n = 10) and a control group with normal pregnancies randomly selected (n = 20) admitted at the Sótero del Río Hospital in Santiago, Chile. A complete periodontal and obstetric history that involved the collection of oral fluids was performed at the same gestational age. Levels of Cd63(+) extracellular vesicles, placental alkaline phosphatase (PLAP), placental growth factor (PlGF), and sFlt-1 levels were determined by ELISA assays. Data analysis was performed with chi-square or Fisher's exact test, and Mann-Whitney U-test for continuous variables. The association was assessed using a multiple logistic regression model. RESULTS: sFlt-1 concentrations in saliva and GCF were significantly higher in patients with PE (p = 0.045 and p = 0.033 respectively). Concentrations of PLAP were elevated in GCF of patients with PE (p = 0.049). The PLAP/CD63(+) ratio in GCF of patients with PE was significantly higher (p = 0.0008). No differences in PlGF levels were observed. CONCLUSION(S): GCF of patients with PE concentrates higher levels of biomarkers related with the PE development. © 2016 John Wiley & Sons, Ltd.