Placental Growth Factor Secretion Research Articles

Effects of sirtuin 1 deficiency on trophoblasts and its implications in the pathogenesis of pre-eclampsia

Background Sirtuin 1 (SIRT1) is mainly localised in syncytiotrophoblasts and cytotrophoblasts, and is involved in pregnancy regulation. However, data on the association between SIRT1 and pre-eclampsia (PE) remains limited. This study aimed to investigate the role of SIRT1 in PE pathophysiology. Methods Placental SIRT1 expression, as well as serum SIRT1, placental growth factor (PlGF), and soluble FMS-like tyrosine kinase 1 (sFlt-1) levels, were measured using quantitative real-time polymerase chain reaction (qRT-PCR), western blotting, and enzyme-linked immunosorbent assays in 40 healthy pregnant women (NP group) and 40 women with severe PE (PE group). Additionally, the effects of SIRT1 on the migration, invasion, PlGF, and sFlt-1 secretion of HTR-8/SVneo cells were analysed. Results SIRT1 expression was significantly reduced in the placenta of patients with severe PE compared with that in healthy pregnant women. Compared with the NP group, serum SIRT1 and PlGF expression was significantly lower in the PE group; however, the expression of serum sFlt-1 was significantly higher in the PE group. Correlation analysis showed that in the PE group, placental SIRT1 protein levels positively correlated with serum PlGF levels (r = 0.468, P = .002) and negatively correlated with serum sFlt-1 levels (r = −0.542, P < .001). Cells with downregulated SIRT1 had a significantly shorter migration distance and a prominently reduced number of invasive cells compared with the corresponding negative control group, suggesting that SIRT1 deficiency may inhibit the migration and invasive ability of HTR-8/SVneo cells. The opposite results were observed after transfection with lentivirus overexpressing SIRT1. Compared with the corresponding controls, cells with downregulated SIRT1 had significantly reduced PlGF levels and significantly increased sFlt-1 levels in the cell culture supernatants, whereas SIRT1 overexpression produced the opposite results. Conclusions SIRT1 deficiency may contribute to the pathogenesis of pre-eclampsia by reducing trophoblastic migration, invasion, and PlGF secretion and increasing sFlt-1 secretion.

Chemerin affects the expression of angiogenesis-related factors in the porcine endometrium during early pregnancy and the oestrous cycle: an in vitro study.

The appropriate course of angiogenesis in the endometrium is crucial for pregnancy establishment and maintenance. Very little is known about the factors linking vessel formation and immune system functioning. We hypothesised that chemerin, an adipokine known for its involvement in the regulation of energy balance and immunological functions, may act as a potent regulator of endometrial angiogenesis during early pregnancy in pigs. Porcine endometrial tissue explants were obtained from pregnant pigs on days 10-11, 12-13, 15-16 and 27-28, and on days 10-12 of the oestrous cycle. The explants were in vitro cultured for 24h in the presence of chemerin (100, 200ng/mL) or in medium alone (control). We evaluated the in vitro effect of chemerin on the secretion of vascular endothelial growth factors A-D (VEGF-A-D), placental growth factor (PlGF), basic fibroblast growth factor (bFGF) and angiopoietin 1 and 2 (ANG-1, ANG-2) with the ELISA method. The protein abundance of angiogenesis-related factor receptors, VEGF receptors 1-3 (VEGFR1-3), FGF receptors 1 and 2 (FGFR1-2) and ANG receptor (TIE2) was evaluated with the Western blot (WB) method. We also analysed the influence of chemerin on the phosphorylation of AMPK using WB. We found that in the studied endometrial samples, chemerin up-regulated the secretion of VEGF-A, VEGF-B and PlGF, and protein expression of VEGFR3. The adipokine caused a decrease in VEGF-C, VEGF-D and ANG-1 release. Chemerin effect on bFGF and ANG-2 secretion, and protein content of VEGFR1, VEGFR2, FGFR1, FGFR2 and TIE2 were dependent on the stage of pregnancy. Chemerin was found to down-regulate AMPK phosphorylation. The obtained in vitro results suggest that chemerin could be an important factor in the early pregnant uterus by its influence on angiogenic factors' secretion and signalling. The obtained results on the role of chemerin in the process of endometrial angiogenesis may, in the long term perspective, contribute to the elaboration of more effective methods of modifying reproductive processes and maintaining energy homeostasis in farm animals.

Monosomy X in isogenic human iPSC-derived trophoblast model impacts expression modules preserved in human placenta

Mammalian sex chromosomes encode homologous X/Y gene pairs that were retained on the Y chromosome in males and escape X chromosome inactivation (XCI) in females. Inferred to reflect X/Y pair dosage sensitivity, monosomy X is a leading cause of miscarriage in humans with near full penetrance. This phenotype is shared with many other mammals but not the mouse, which offers sophisticated genetic tools to generate sex chromosomal aneuploidy but also tolerates its developmental impact. To address this critical gap, we generated X-monosomic human induced pluripotent stem cells (hiPSCs) alongside otherwise isogenic euploid controls from male and female mosaic samples. Phased genomic variants in these hiPSC panels enable systematic investigation of X/Y dosage-sensitive features using invitro models of human development. Here, we demonstrate the utility of these validated hiPSC lines to test how X/Y-linked gene dosage impacts a widely used model for human syncytiotrophoblast development. While these isogenic panels trigger a GATA2/3- and TFAP2A/C-driven trophoblast gene circuit irrespective of karyotype, differential expression implicates monosomy X in altered levels of placental genes and in secretion of placental growth factor (PlGF) and human chorionic gonadotropin (hCG). Remarkably, weighted gene coexpression network modules that significantly reflect these changes are also preserved in first-trimester chorionic villi and term placenta. Our results suggest monosomy X may skew trophoblast cell type composition and function, and that the combined haploinsufficiency of the pseudoautosomal region likely plays a key role in these changes.

Polarized macrophages promote gestational beta cell growth through extracellular signal-regulated kinase 5 signalling.

To show that depletion of pancreatic macrophages impairs gestational beta cell proliferation and leads to glucose intolerance. Genetic animal models were applied to study the effects of depletion of pancreatic macrophges on gestational beta-cell proliferaiton and glucose response. The crosstalk between macrophages and beta-cells was studied in vivo using beta-cell-specific extracellular-signal-regulated kinase 5 (ERK5) knockout and epidermal growth receptor (EGFR) knockout mice, and in vitro using a co-culture system. Beta cell-derived placental growth factor (PlGF) recruited naïve macrophages and polarized them towards an M2-like phenotype. These macrophages then secreted epidermal growth factor (EGF), which activated extracellular signal-regulated kinase 5 (ERK5) signalling in beta cells to promote gestational beta cell proliferation. On the other hand, activation of ERK5 signalling in beta cells likely, in turn, enhanced the production and secretion of PlGF by beta cells. Our study shows a regulatory loop between macrophages and beta cells through PlGF/EGF/ERK5 signalling cascades to regulate gestational beta cell growth.

Hyperglycaemia up-regulates placental growth factor (PlGF) expression and secretion in endothelial cells via suppression of PI3 kinase-Akt signalling and activation of FOXO1

Placenta growth factor (PlGF) is a pro-inflammatory angiogenic mediator that promotes many pathologies including diabetic complications and atherosclerosis. Widespread endothelial dysfunction precedes the onset of these conditions. As very little is known of the mechanism(s) controlling PlGF expression in pathology we investigated the role of hyperglycaemia in the regulation of PlGF production in endothelial cells. Hyperglycaemia stimulated PlGF secretion in cultured primary endothelial cells, which was suppressed by IGF-1-mediated PI3K/Akt activation. Inhibition of PI3K activity resulted in significant PlGF mRNA up-regulation and protein secretion. Similarly, loss or inhibition of Akt activity significantly increased basal PlGF expression and prevented any further PlGF secretion in hyperglycaemia. Conversely, constitutive Akt activation blocked PlGF secretion irrespective of upstream PI3K activity demonstrating that Akt is a central regulator of PlGF expression. Knock-down of the Forkhead box O-1 (FOXO1) transcription factor, which is negatively regulated by Akt, suppressed both basal and hyperglycaemia-induced PlGF secretion, whilst FOXO1 gain-of-function up-regulated PlGF in vitro and in vivo. FOXO1 association to a FOXO binding sequence identified in the PlGF promoter also increased in hyperglycaemia. This study identifies the PI3K/Akt/FOXO1 signalling axis as a key regulator of PlGF expression and unifying pathway by which PlGF may contribute to common disorders characterised by endothelial dysfunction, providing a target for therapy.

Paracrine Placental Growth Factor Signaling in Response to Ionizing Radiation Is p53-Dependent and Contributes to Radioresistance.

Placental growth factor (PlGF) is a pro-angiogenic, N-glycosylated growth factor, which is secreted under pathologic situations. Here, we investigated the regulation of PlGF in response to ionizing radiation (IR) and its role for tumor angiogenesis and radiosensitivity. Secretion and expression of PlGF was induced in multiple tumor cell lines (medulloblastoma, colon and lung adenocarcinoma) in response to irradiation in a dose- and time-dependent manner. Early upregulation of PlGF expression and secretion in response to irradiation was primarily observed in p53 wild-type tumor cells, whereas tumor cells with mutated p53 only showed a minimal or delayed response. Mechanistic investigations with genetic and pharmacologic targeting of p53 corroborated regulation of PlGF by the tumor suppressor p53 in response to irradiation under normoxic and hypoxic conditions, but with so far unresolved mechanisms relevant for its minimal and delayed expression in tumor cells with a p53-mutated genetic background. Probing a paracrine role of IR-induced PlGF secretion in vitro, migration of endothelial cells was specifically increased towards irradiated PlGF wild type but not towards irradiated PlGF-knockout (PIGF-ko) medulloblastoma cells. Tumors derived from these PlGF-ko cells displayed a reduced growth rate, but similar tumor vasculature formation as in their wild-type counterparts. Interestingly though, high-dose irradiation strongly reduced microvessel density with a concomitant high rate of complete tumor regression only in the PlGF-ko tumors. IMPLICATIONS: Our study shows a strong paracrine vasculature-protective role of PlGF as part of a p53-regulated IR-induced resistance mechanism and suggest PlGF as a promising target for a combined treatment modality with RT.

Human Placental NADPH Oxidase Mediates sFlt-1 and PlGF Secretion in Early Pregnancy: Exploration of the TGF-β1/p38 MAPK Pathways.

Preeclampsia, a hypertensive disorder occurring during pregnancy, is characterized by excessive oxidative stress and trophoblast dysfunction with dysregulation of soluble Fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF) production. Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (Nox) is the major source of placental superoxide in early pregnancy and its activation with the subsequent formation of superoxide has been demonstrated for various agents including Transforming Growth Factor beta-1 (TGF-β1), a well-known p38 MAPK pathway activator. However, the bridge between Nox and sFlt-1 remains unknown. The purpose of this study was to explore the possible signaling pathway of TGF-β1/Nox/p38 induced sFlt-1 production in human chorionic villi (CV). Methods: Human chorionic villi from first trimester placenta (7–9 Gestational Weeks (GW)) were treated with TGF-β1 or preincubated with p38 inhibitor, SB203580. For NADPH oxidase inhibition, CV were treated with diphenyleneiodonium (DPI). The protein levels of phospho-p38, p38, phospho-Mothers Against Decapentaplegic homolog 2 (SMAD2), and SMAD2 were detected by Western blot. The secretion of sFlt-1 and PlGF by chorionic villi were measured with Electrochemiluminescence Immunologic Assays, and NADPH oxidase activity was monitored by lucigenin method. Results: We demonstrate for the first time that NADPH oxidase is involved in sFlt-1 and PlGF secretion in first trimester chorionic villi. Indeed, the inhibition of Nox by DPI decreases sFlt-1, and increases PlGF secretions. We also demonstrate the involvement of p38 MAPK in sFlt-1 secretion and Nox activation as blocking the p38 MAPK phosphorylation decreases both sFlt-1 secretion and superoxide production. Nevertheless, TGF-β1-mediated p38 activation do not seem to be involved in regulation of the first trimester placental angiogenic balance and no crosstalk was found between SMAD2 and p38 MAPK pathways. Conclusions: Thus, the placental NADPH oxidase play a major role in mediating the signal transduction cascade of sFlt-1 production. Furthermore, we highlight for the first time the involvement of p38 activation in first trimester placental Nox activity.

1724-P: MicroRNA Regulation of Endothelial Cell Secretion of Placental Growth Factor and Adipose Browning

Brown adipose tissue (BAT), a highly vascularized, thermogenic tissue, converts chemical energy into heat. The ability of BAT to expand based on the metabolic demand, known as plasticity, plays a critical role in the regulation of whole body energy balance. Endothelial cells (ECs) within the microvasculature of the adipose tissue play an important role in aiding BAT retain plasticity by actively modulating the angiogenic response. However, the mechanism of EC-adipocyte cross-talk during this process is not completely understood. In BAT ECs of obese mice, we have previously identified an anti-angiogenic microRNA, miR-409-3p, using a RNA-seq profiling approach. We now show that miR-409-3p targets MAP4K3 to confer its anti-angiogenic effects in ECs. Overexpression of miR-409-3p markedly decreased MAP4K3 expression in ECs, whereas inhibition of miR-409-3p had the opposite effects. SiRNA knockdown of MAP4K3 expression in ECs phenocopied the effects of miR-409-3p overexpression and significantly decreased cell proliferation and migration. Adipocytes co-cultured with conditioned media from ECs deficient in miR-409-3p showed increased expression of brown fat markers UCP1 and Cidea by RT-qPCR and Western. By multiplex ELISA, we identified a pro-angiogenic growth factor, Placental Growth Factor (PLGF), that is released from ECs in response to miR-409-3p deficiency. PLGF stimulation of 3T3-L1 adipocytes differentiated them into brown adipocytes. Furthermore, siRNA knockdown of MAP4K3 blocked the release of PLGF from ECs. Co-culturing conditioned media from ECs deficient in PLGF expression by siRNA silencing significantly decreased UCP1 expression in 3T3-L1 adipocytes, whereas conditioned media from ECs deficient both in miR-409-3p and PLGF further decreased UCP1 expression showing dependency for miR-409-3p regulated pathways. MiR-409-3p plays a key role in EC-BAT crosstalk by modulating PLGF, an effect that could be exploited for therapeutic intervention in obesity. Disclosure D. Pérez-Cremades: None. D.R. Becker-Greene: None. H. Li: None. W. Wu: None. C.O. Aydogan: None. D. Ozdemir: None. M.W. Feinberg: None. B. Icli: None. Funding American Diabetes Association (1-16-JDF-046 to B.I.)

Low molecular weight heparin promotes transcription and release of placental growth factor from endothelial cells.

Circulating levels of placental growth factor (PlGF) are significantly reduced in women who develop preeclampsia. Low molecular weight heparin (LMWH) has been shown to acutely elevate circulating PlGF levels in pregnant women at increased risk of preeclampsia. The objective of the current investigation was to determine the mechanisms by which LMWH mediates the extracellular release of PlGF from endothelial cells. Cultured human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) were exposed to LMWH; PlGF transcription, translation, mobilization, and secretion were then assessed. LMWH significantly increased the release of PlGF from both HAECs and HUVECs. LMWH treatment promoted a significant increase of PlGF-1 mRNA expression in HAECs, accompanied by the intracellular transport and release of PlGF into the conditioned media. LMWH-mediated release of PlGF from HAECs was not directly mediated by extracellular mobilization, synthesis, or stability of PlGF mRNA/protein. LMWH exposure promotes the release of PlGF from endothelial cells through the upregulation of PlGF-1 mRNA expression. Stimulation of circulating PlGF levels by LMWH may be an important mechanism by which LMWH could reduce the risk of preeclampsia or minimize disease severity.NEW & NOTEWORTHY There are few therapeutic options available for the prevention of preeclampsia, a serious hypertensive disorder of pregnancy. Women who subsequently develop preeclampsia exhibit significantly reduced circulating levels of the proangiogenic placental growth factor protein. Low molecular weight heparin (LMWH) has previously been investigated as a preventative therapy against the development of preeclampsia; however, its mechanism of action is not known. The current study determined that LMWH promotes the transcription and release of placental growth factor protein from endothelial cells, providing a mechanistic basis by which LMWH could reduce the risk of preeclampsia or minimize disease severity.

Apelin decreased placental hormone secretion by human trophoblast BeWo cells via apelin receptor, protein kinase A and extracellular signal-regulated kinases 1/2 activation.

Apelin was thought to be an adipocyte-specific hormone, but recent studies have indicated a link between apelin and placenta function e.g. cell proliferation. The aim of the study was investigating dose- and time-dependent effect of apelin on hormone secretion including steroids: progesterone (P4) and estradiol (E2) and proteins: chorionic gonadotropin (hCG), human placental lactogen (hPL), placental growth factor (PLGF), as well as protein expression of steroid enzymes (3βHSD, CYP19) and protein hormones (hCG, hPL and PLGF) in placental cells. Syncytiotrophoblast BeWo cells, as human trophoblast models, were treated for 24, 48, and 72 hours with the human recombinant apelin at doses 0.02, 0.2, 2.0, 20 and 200 ng/ml followed by culture medium. Concentrations of the above hormones were studied by ELISA kits. Furthermore, protein expression of steroid enzymes and protein hormones were measured using Western blot. Our results showed that apelin significantly decreased both steroid and protein hormones by inhibiting steroid enzymes or protein hormone expression. Moreover, we demonstrated that apelin at dose 2.0 ng/ml increased phosphorylation of protein kinase A (PKA) from 1 to 60 min of BeWo cell incubation. Inhibitory effect of apelin on P4, E2 and PLGF secretion were abolished when BeWo cells were cultured in the presence of ML221, an apelin receptor antagonist, PD98059, an extracellular signal-regulated kinases (ERK1/2) antagonist and KT5720, a PKA antagonist. In turn, secretion of hCG and hPL occurs only in the presence of ML221 and PD98059. In conclusion, our results indicate that apelin can be considered as a gestational hormone implied in the endocrine function of the human placenta, with an important role in controlling the production of steroid and protein hormones in placental BeWo cells.

PlGF signaling and macrophage repolarization contribute to the anti-neoplastic effect of metformin

Placental growth factor (PlGF) related signaling pathway has been shown to have close relationship with the progression of cancers. Metformin has been reported to have an inhibitory effect on PlGF expression in a breast cancer model. However, little is known about whether the anti-neoplastic activity of metformin is contributed by its inhibitory effect on PlGF expression. Protein, mRNA and secretion levels of PlGF were tested and the proliferation of cancer cells was determined. After treatment of metformin, BALB/c mice bearing 4T1 tumors were sacrificed and immunohistochemistry staining of the tumor sections was obtained. Baseline expression of autocrine PlGF varied between different breast cancer cell lines, while the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) was comparable between cell lines. Other clinical data showed that the expression of PlGF other than VEGFR-1 had a prognostic value for patients with breast cancers. Metformin significantly decreased the secretion and mRNA levels of PlGF, which greatly contributed to its inhibitory effect on the proliferation of breast cancer cells with high P1GF expression. The unresponsiveness of tumor cells with low PlGF expression to genetic silencing was reversed by the supplementation of exogenous PlGF. Systemic metformin administration apparently inhibited the in vivo growth of 4T1 carcinoma, which was accompanied by the repolarization of macrophages from M2 to M1. These findings indicated that both autocrine and paracrine PlGF signaling and macrophage repolarization are involved in the progression of breast cancer, which could be targeted by metformin.

Abstract 150: Obese Hypertensive Pregnant Rats Have Reduced Placental Growth Factor Whereas Increasing It's Levels Decreases Their Blood Pressure

While epidemiological studies have repeatedly shown that overweight and obesity are associated with hypertension during pregnancy, the mechanisms linking these co-morbidities are not as explored. Reports show reduced levels of the anti-hypertensive and pregnancy-related factor, placental growth factor (PlGF), in obese hypertensive pregnant humans. We tested the hypothesis that obese hypertensive pregnant rats have reduced PlGF whereas increasing its levels reduces their blood pressure. We utilized a genetic model of obesity in rats having heterozygous deficiency of the melanocortin-4 receptor (MC4R-def) compared to wild-type lean controls. By gestational day 19, body mass (341±8 vs 311±7 g), periaortic adipose tissue (10.9±1.1 vs 8.2±0.5 mg), retroperitoneal adipose tissue (4.4±0.4 vs 2.5±0.2 g), circulating levels of leptin (10±1 vs 6±1 ng/mL), and mean arterial blood pressure (116±2 vs 105±2 mmHg) were greater (all P&lt;0.05) in obese (N=17) compared to lean (N=14) pregnant rats. PlGF levels were lower (P&lt;0.05) in serum (61±6 vs. 78±7 pg/mL) and secreted from placental (58±3 vs 71±3 pg/mg) and periaortic adipose tissue (7±1 vs 15±2 pg/mL) explants in obese compared to lean groups. PlGF secretion from retroperitoneal adipose tissue was similar between obese (78±10 pg/mL) and lean (62±12 pg/mL) groups. Vascular expression of the PlGF receptor, vascular endothelial growth factor receptor 1 (VEGFR1), was lower (P&lt;0.05) in aortic tissue (832±60 vs 1106±91 pg/mg) but higher (P&lt;0.05) in small mesenteric arteries (214±23 vs. 151±17 pg/mg) from the obese pregnant rats. Chronic administration of recombinant human (rh)PlGF (180 ug/kg per day) from gestational day 13-19 produced detectable levels of human PlGF in serum from both lean (33±4 pg/mL) and obese (43±4 pg/mL) pregnant rats and reduced (P&lt;0.05) blood pressure in obese (102±3 mmHg) but not in lean (101±3 mmHg) groups. The treatment did not alter maternal body or fat masses or circulating leptin. In summary, PlGF is reduced in MC4R-def obese hypertensive pregnant rats, similar to findings in obese pregnant humans, whereas increasing the levels of PlGF reduced their hypertensive phenotype. Thus, treatment with rhPlGF may prove beneficial in attenuating blood pressure in obese hypertensive pregnancies.

Sulfasalazine reduces placental secretion of antiangiogenic factors, up-regulates the secretion of placental growth factor and rescues endothelial dysfunction.

BackgroundPreeclampsia is a major complication of pregnancy with no medical treatment. It is associated with placental oxidative stress, hypoxia and inflammation leading to soluble fms-like tyrosine kinase 1 (sFlt-1) and soluble endoglin (sENG) secretion and reduced placental growth factor (PlGF). This results in widespread endothelial dysfunction causing hypertension and multisystem organ injury. Sulfasalazine is an anti-inflammatory and antioxidant medication used to treat autoimmune disease. Importantly, it is safe in pregnancy. We examined the potential of sulfasalazine to quench antiangiogenic factors and endothelial dysfunction and increase angiogenic factor secretion.MethodsWe performed functional experiments using primary human pregnancy tissues to examine the effects of sulfasalazine on sFlt-1, sENG and PlGF secretion. Sulfasalazine is known to inhibit nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) and upregulate heme-oxygenase 1 (HO-1) thus we explored the effect of these transcription factors on sFlt-1 secretion from human cytotrophoblasts. We examined the ability of sulfasalazine to reduce key markers of endothelial dysfunction and dilate whole blood vessels.FindingsWe demonstrate sulfasalazine administration reduces sFlt-1 and sENG and upregulates PlGF secretion from human placental tissues. Furthermore sulfasalazine mitigates endothelial dysfunction in several in vitro/ex vivo assays. It enhanced endothelial cell migration and proliferation, promoted blood vessel dilation (vessels obtained from women at caesarean section) and angiogenic sprouting from whole blood vessel rings. The effect of sulfasalazine on the secretion of sFlt-1 was not mediated through either the NFkB or HO-1 pathways.InterpretationWe conclude that sulfasalazine reduces sFlt-1 and sENG secretion and endothelial dysfunction and upregulates PlGF. Sulfasalazine has potential to treat or prevent preeclampsia and warrants investigation in clinical trials.FundingThis work was funded by The National Health and Medical Research Council of Australia (NHMRC; #1048707, #1046484. #1101871, #1064845), an Arthur Wilson RANZCOG scholarship and a Norman Beischer Medical Research Foundation grant. FB was supported by a NHMRC Early Career Fellowship (NHMRC #1142636). NJH was supported by a CR Roper Research Fellowship. The NHMRC provided salary support (#1136418 to ST #1062418 to TKL, #1064845 to SS). The funders had no role in study design, data collection, analysis, decision to publish or the preparation of the manuscript.

Co-inhibition of PGF and VEGF blocks their expression in mononuclear phagocytes and limits neovascularization and leakage in the murine retina

BackgroundAge-related macular degeneration (AMD) is a leading cause of visual impairment in the elderly. The neovascular (wet) form of AMD can be treated with intravitreal injections of different anti-vascular endothelial growth factor (VEGF) agents. Placental growth factor (PGF) is another member of the VEGF family of cytokines with pro-angiogenic and pro-inflammatory effects. Here, we aimed to compare single and combined inhibition of VEGF-A and PGF in the laser-induced mouse model of choroidal neovascularization (CNV) with a focus on the effects on retinal mononuclear phagocytes.MethodsCNV was induced in C57BL/6J mice using a YAG-Laser. Immediately after laser damage antibodies against VEGF-A (aVEGF), anti-PGF (aPGF), aVEGF combined with aPGF, aflibercept, or IgG control were injected intravitreally in both eyes. Three and 7 days after laser damage, the vascular leakage was determined by fluorescence angiography. Lectin staining of retinal and RPE/choroidal flat mounts was used to monitor CNV. In situ mRNA co-expression of Iba1, VEGF and PGF were quantified using in situ hybridization. Retinal and RPE/choroidal protein levels of VEGF and PGF as well as the pro-inflammatory cytokines IL-6, IL1-beta, and TNF were determined by ELISA.ResultsEarly (day 3) and intermediate (day 7) vascular leakage and CNV were significantly inhibited by PGF and VEGF-A co-inhibition, most effectively with the trap molecule aflibercept. While VEGF-A blockage alone had no effects, trapping PGF especially with aflibercept prevented the accumulation of reactive microglia and macrophages in laser lesions. The lesion-related mRNA expression and secretion of VEGF-A and PGF by mononuclear phagocytes were potently suppressed by PGF and partially by VEGF-A inhibition. Protein levels of IL-6 and IL1-beta were strongly reduced in all treatment groups.ConclusionsRetinal inhibition of PGF in combination with VEGF-A prevents vascular leakage and CNV possibly via modulating their own expression in mononuclear phagocytes. PGF-related, optimized strategies to target inflammation-mediated angiogenesis may help to increase efficacy and reduce non-responders in the treatment of wet AMD patients.

Efficient Nonviral Transfection of Human Bone Marrow Mesenchymal Stromal Cells Shown Using Placental Growth Factor Overexpression.

Background Human mesenchymal stromal/stem cells (hMSCs) hold great therapeutic potential due to their immunomodulatory and tissue regenerative properties. Enhancement of biological features of hMSCs by transfection has become a focus of investigation for cell- and gene-based therapies. However, many of the current transient transfection methods result in either low transfection efficiency or high cytotoxicity. Methods In order to find a transfection method that would address the current issues of low transfection efficiency and high cytotoxicity, 6 commercially available cationic lipid and polymer reagents were tested on human bone marrow-derived MSCs (hBM-MSCs) using GFP as a reporter gene. One transfection method using TransIT-2020 was selected and tested with an emphasis on cell quality (viability, identity, and yield), as well as efficacy with a human placental growth factor (PlGF) plasmid. Results TransIT-2020 yielded the highest fluorescence signal per cell out of the methods that did not decrease cell recovery. Transfecting GFP to 5 hBM-MSC donors using TransIT-2020 yielded 24–36% GFP-expressing cells with a viability of 85–96%. hBM-MSC identity was unaffected as CD90, CD105, and CD73 markers were retained (>95%+) after transfection. When this method was applied to PlGF expression, there was up to a 220-fold increase in secretion. Both growth and secretion of PlGF in overexpressing hBM-MSC were sustained over 7 days, confirming the sustainability and applicability of the TransIT-2020 transfection system. Discussion We report a simple and efficient method for transient transfection that has not been reported for hBM-MSCs, encompassing high levels of plasmid expression without significant changes to fundamental hBM-MSC characteristics.

190. Combination sulfasalazine and metformin are additive in reducing placental secretion of antiangiogenic factor sFlt

Introduction Preeclampsia is associated with increased placental secretion of antiangiogenic factors sFlt-1 and sENG and a reduction in angiogenic proteins placental growth factor secretion (PlGF) and vascular endothelial growth factor (VEGF). We have identified two medications, safe in pregnancy, that mitigate key aspects of preeclampsia in vitro. We have demonstrated metformin and sulfasalazine independently reduce placental sFlt-1 secretion and vessel dysfunction. In this study we investigate the possibility of combining these medications to reduce the dose required to mitigate key aspects of preeclampsia; sFlt-1 and sENG secretion and PlGF and VEGF expression, using primary human tissues. Methods Metformin and sulfasalazine, at low doses individually and in combination, were administered to primary trophoblasts and placental explants and sFlt-1 secretion assessed. At the mRNA level there are two sFlt-1 splice variants; the primate and placental specific isoform sFlt-1 e15a and the predominately vascular isoform sFlt-1 i13. Both were measured following combination treatment. In addition the expression of angiogenic factors PlGF and VEGFα were assessed. Results Combining low-dose metformin and sulfasalazine additively decreased sFlt-1 secretion from primary trophoblast compared to either drug alone. Similarly, an additive decrease in sFlt-1 e15a and i13 was identified following combination treatment. In placental explants, combination treatment also additively decreased sFlt-1 secretion. Low dose sulfasalazine potently upregulated PlGF expression in primary trophoblasts and this was maintained with the addition of metformin. Metformin and sulfasalazine individually increased VEGFα, and this effect was additive when the two drugs were combined. Conclusion Combination metformin and sulfasalazine additively reduced sFlt-1 secretion and the expression of its splice variants, sFlt-1 e15a and i13. Furthermore, in combination metformin and sulfasalazine upregulate angiogenic factor VEGFα. Therefore we conclude that they might be promising combination therapeutics that could reduce the burden of this devastating disease.

Placental growth factor (PlGF) is linked to inflammation and metabolic disorders in mice with diet-induced obesity.

Placental growth factor (PlGF), a member of the vascular endothelial growth factor (VEGF) sub-family, plays a major role in angiogenesis and vasculogenesis. Previous study demonstrated that PlGF-overexpressing transgenic (Tg) mice had gestational loss. In addition, PlGF secretion was up-regulated in isolated T lymphocytes (T-cell) upon CD3/CD28 stimulation, suggesting that PlGF could be a regulator of T-cell differentiation and development. T-cells are well known to play a critical role in obesity-induced inflammation. Therefore, to verify the possible link of diet-induced obesity (DIO) with inflammation and related metabolic disorders, such as insulin resistance, we fed high-fat diet (HFD) to Tg mice for 16 weeks. Adiposity and glucose intolerance significantly increase in Tg mice fed a HFD (Tg HFD) compared to wild-type (WT) mice fed HFD (WT HFD). In addition, macrophage infiltrations were significantly higher in the epididymal white adipose tissue (EWAT), liver, and pancreatic islets of Tg HFD mice compared to WT HFD mice. In the in vitro study, we showed that isolated CD4+ T-cells from Tg mice further differentiate into type 1 (Th1) and type 17 (Th17) helper T-cells via CD3/CD28 stimulation. Furthermore, we observed that the pro-inflammatory cytokines IL-6, IL-17, and TNFα, are remarkably increased in Tg mice compared to WT mice. These findings demonstrate that PlGF overexpression in T-cells might lead to inflammatory T-cell differentiation and accumulation in adipose tissue (AT) or metabolism-related tissues, contributing to the development of systemic metabolic disorders. Thus, PlGF may provide an effective therapeutic target in the management of obesity-induced inflammation and related metabolic disorders.

Enhanced HIF2\u03b1 expression during human trophoblast differentiation into syncytiotrophoblast suppresses transcription of placental growth factor

Placental growth factor (PlGF), abundantly produced from trophoblasts is involved in placental angiogenesis. The regulatory mechanism of its expression is poorly understood. Hypoxia inducible factors (HIFs) are centrally involved in the modulation of cellular function in response to low oxygen conditions. This study aimed to clarify HIF1α and HIF2α expression patterns during cytotrophoblast differentiation into syncytiotrophoblast and the impact of any changes on PlGF expression. HIF proteins were induced remarkably under low oxygen condition (2%). HIF1α expression decreased and HIF2α expression increased when syncytialization of cultured cytotrophoblasts is progressed. Those expression changes of HIF proteins in the process of in-vitro syncytialization was congruent with the immunohistochemical findings in preeclamptic placenta as well as uncomplicated placenta. Low oxygen condition was also associated with reduced PlGF production in syncytializing primary cells and BeWo choriocarcinoma cells. Small interfering RNA-mediated HIF2α knockdown in BeWo cells abrogated hypoxia-associated decreases in PlGF secretion; HIF1α silencing had no significant effect on PlGF secretion. In summary, HIF2α, rather than HIF1α, is most affected by reduced oxygen level during syncytialization and increases in HIF2α trigger a reduction of PlGF production. Our findings suggest new and important connections between HIF proteins and PlGF pathways in the regulation of placental angiogenesis.

P 7 Fluid shear stress promotes placental growth factor upregulation in human syncytiotrophoblast through the camp-pka signaling pathway

The effects of fluid shear stress (FSS) on the human syncytiotrophoblast and its biological functions have never been studied. During pregnancy, the syncytiotrophoblast is the main source of placental growth factor (PlGF), a proangiogenic factor involved in the placental angiogenesis and the vascular adaptation to pregnancy. The role of FSS in regulating PlGF expression in syncytiotrophoblasts is unknown. We investigated the impact of FSS on the production and secretion of the PlGF by the human syncytiotrophoblasts in primary cell culture. Laminar and continuous FSS (1 dyn cm−2) was applied to human syncytiotrophoblasts cultured in a parallel-plate flow chambers. Secreted levels of PlGF, sFlt-1 (soluble fms-like tyrosin kinase-1), and prostaglandin E2 were tested by immunologic assay. PlGF levels of mRNA and intracellular protein were examined by RT-PCR and Western blot, respectively. Intracellular cAMP levels were examined by time-resolved fluorescence resonance energy transfer cAMP accumulation assay. Production of cAMP and PlGF secretion was significantly increased in FSS conditions compared with static conditions. Western blot analysis of cell extracts exposed to FSS showed an increased phosphorylation of protein kinase A substrates and cAMP response element-binding protein on serine 133. FSS-induced phosphorylation of cAMP response element-binding protein and upregulation of PlGF were prevented by inhibition of protein kinase A with H89 (3 μmol/L). FSS also triggers intracellular calcium flux, which increases the synthesis and release of prostaglandin E2. The enhanced intracellular cAMP in FSS conditions was blocked by COX1/COX2 (cyclooxygenase) inhibitors, suggesting that the increase in prostaglandin E2 production could activate the cAMP/protein kinase A pathway in an autocrine/paracrine fashion. FSS activates the cAMP/protein kinase A pathway leading to upregulation of PlGF in human syncytiotrophoblast.

The effects of pravastatin on the normal human placenta: Lessons from ex-vivo models.

IntroductionResearch in animal models and preliminary clinical studies in humans support the use of pravastatin for the prevention of preeclampsia. However, its use during pregnancy is still controversial due to limited data about its effect on the human placenta and fetus.MethodsIn the present study, human placental cotyledons were perfused in the absence or presence of pravastatin in the maternal reservoir (PraM). In addition, placental explants were treated with pravastatin for 5, 24 and 72 h under normoxia and hypoxia. We monitored the secretion of placental growth factor (PlGF), soluble fms-like tyrosine kinase-1 (sFlt-1), soluble endoglin (sEng), endothelial nitric oxide synthase (eNOS) expression and activation and the fetal vasoconstriction response to angiotensin-II.ResultsThe concentrations of PlGF, sFlt-1 and sEng were not significantly altered by pravastatin in PraM cotyledons and in placental explants compared to control. Under hypoxic conditions, pravastatin decreased sFlt-1 concentrations. eNOS expression was significantly increased in PraM cotyledons but not in pravastatin-treated placental explants cultured under normoxia or hypoxia. eNOS phosphorylation was not significantly affected by pravastatin. The feto-placental vascular tone and the fetal vasoconstriction response to angiotensin-II, did not change following exposure of the maternal circulation to pravastatin.ConclusionWe found that pravastatin does not alter the essential physiological functions of the placenta investigated in the study. The relevance of the study lays in the fact that it expands the current knowledge obtained thus far regarding the effect of the drug on the normal human placenta. This data is reassuring and important for clinicians that consider the treatment of high-risk patients with pravastatin, a treatment that exposes some normal pregnancies to the drug.