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Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP-dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix. Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGF. In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGF, they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGF. The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser64 and Thr112, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr112), but instead is phosphorylated at the canonical PK-C site (Ser64). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Ser64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (Thr112), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGF. The results presented here demonstrate three novel features of basic FGF. First, they identify a functional effect of the binding of heparin to basic FGF. Second, they establish that the binding of heparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm.

Background and Purpose: Drugs targeting microtubules are commonly used for cancer treatment. However, the potency of microtubule inhibitors used clinically is limited by the emergence of resistance. We thus designed a strategy to find new cell-permeable microtubule-targeting agents.Experimental Approach: Using a cell-based assay designed to probe for microtubule polymerization status, we screened a chemical library and identified two azaindole derivatives, CM01 and CM02, as cell-permeable microtubule-depolymerizing agents. The mechanism of the anti-tumour effects of these two compounds was further investigated both in vivo and in vitro.Key Results: CM01 and CM02 induced G2/M cell cycle arrest and exerted potent cytostatic effects on several cancer cell lines including multidrug-resistant (MDR) cell lines. In vitro experiments revealed that the azaindole derivatives inhibited tubulin polymerization and competed with colchicines for this effect, strongly indicating that tubulin is the cellular target of these azaindole derivatives. In vivo experiments, using a chicken chorioallantoic xenograft tumour assay, established that these compounds exert a potent anti-tumour effect. Furthermore, an assay probing the growth of vessels out of endothelial cell spheroids showed that CM01 and CM02 exert anti-angiogenic activities.Conclusions and Implications: CM01 and CM02 are reversible microtubule-depolymerizing agents that exert potent cytostatic effects on human cancer cells of diverse origins, including MDR cells. They were also shown to inhibit angiogenesis and tumour growth in chorioallantoic breast cancer xenografts. Hence, these azaindole derivatives are attractive candidates for further preclinical investigations. [ABSTRACT FROM AUTHOR]

Normal fetal growth and placental development depend on active angiogenesis occurring at the fetomaternal interface throughout pregnancy. Nevertheless, reliable in vivo methods to assess placental angiogenesis are still missing. Here, we describe a quantitative and noninvasive in vivo method to specifically measure placental neovascularization in the gravid mouse. This method uses a technique based on the measurement of a fluorescent molecule Angiostamp700 that targets the alpha v beta 3 (α v β 3 ) integrin, a protein that is highly expressed by endothelial cells during the neovascularization and by trophoblast cells during invasion of the maternal decidua. Due to this noninvasive method, quantification of the fetomaternal angiogenic activity and information regarding the outcome of pregnancy are now possible., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Chronic Chagasic cardiomyopathy (CCC), a progressive inflammatory and fibrosing disease, is the most prominent clinical form of Chagas disease, a neglected tropical disease caused by Trypanosoma cruzi infection. During CCC, the parasite remains inside the cardiac cells, leading to tissue damage, involving extensive inflammatory response and irregular fibrosis. Among the fibrogenic factors is transforming growth factor-β (TGF-β), a key cytokine controlling extracellular matrix synthesis and degradation. TGF-β is involved in CCC onset and progression, with increased serum levels and activation of its signaling pathways in the cardiac tissue, which crucially contributes to fibrosis. Inhibition of the TGF-β signaling pathway attenuates T. cruzi infection and prevents cardiac damage in an experimental model of acute Chagas disease. The aim of this study was to investigate the effect of TGF-β neutralization on T. cruzi infection in both in vitro and in vivo pre-clinical models, using the 1D11 monoclonal antibody. To this end, primary cultures of cardiac cells were infected with T. cruzi trypomastigote forms and treated with 1D11. For in vivo studies, 1D11 was administered in different schemes for acute and chronic phase models (Swiss mice infected with 10 4 parasites from the Y strain and C57BL/6 mice infected with 10 2 parasites from the Colombian strain, respectively). Here we show that the addition of 1D11 to cardiac cells greatly reduces cardiomyocyte invasion by T. cruzi and the number of parasites per infected cell. In both acute and chronic experimental models, T. cruzi infection altered the electrical conduction, decreasing the heart rate, increasing the PR interval and the P wave duration. The treatment with 1D11 reduced cardiac fibrosis and reversed electrical abnormalities improving cardiac performance. Taken together, these data further support the major role of the TGF-β signaling pathways in T. cruzi -infection and their biological consequences on parasite/host interactions. The therapeutic effects of the 1D11 antibody are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-β neutralization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CC declared a shared affiliation with the authors RF, ES, WD, RA, and MW to the handling editor at the time of review., (Copyright © 2022 Ferreira, de Souza, Vilar-Pereira, Degrave, Abreu, Meuser-Batista, Ferreira, Ledbeter, Barker, Bailly, Feige, Lannes-Vieira, de Araújo-Jorge and Waghabi.)

Transforming growth factor beta (TGF-β) is deeply involved on the pathogenesis of Chagas disease. Our group has been investigating the participation of this pleiotropic cytokine in different aspects of Chagas disease over the last 20 years. Important observations have been made, such as: (i) the ability of Trypanosoma cruzi in activating latent TGF-β; (ii) the potential involvement of TGF-β pathway on T. cruzi invasion of host cells; (iii) association of TGF-β with parasite intracellular replication; (iv) cardiac fibrosis development and maintenance; (v) disruption of Connexin-43 plaque structures and (vi) inflammation and immune response. In this perspective article we intend to discuss the advances of the potential use of new therapies targeting TGF-β to treat the cardiac alterations of Chagas disease-affected patients.

The anti-inflammatory cytokine transforming growth factor beta (TGF-β) plays an important role in Chagas disease (CD), a potentially life-threatening illness caused by Trypanosoma cruzi . In this review we revisited clinical studies in CD patients combined with in vitro and in vivo experiments, presenting three main sections: an overview of epidemiological, economic, and clinical aspects of CD and the need for new biomarkers and treatment; a brief panorama of TGF-β roles and its intracellular signaling pathways, and an update of what is known about TGF-β and Chagas disease. In in vitro assays, TGF-β increases during T. cruzi infection and modulates heart cells invasion by the parasite fostering its intracellular parasite cycle. TGF-β modulates host immune response and inflammation, increases heart fibrosis, stimulates remodeling, and slows heart conduction via gap junction modulation. TGF-β signaling inhibitors reverts these effects opening a promising therapeutic approach in pre-clinical studies. CD patients with higher TGF-β1 serum level show a worse clinical outcome, implicating a predictive value of serum TGF-β as a surrogate biomarker of clinical relevance. Moreover, pre-clinical studies in chronic T. cruzi infected mice proved that inhibition of TGF-β pathway improved several cardiac electric parameters, reversed the loss of connexin-43 enriched intercellular plaques, reduced fibrosis of the cardiac tissue, restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Finally, TGF-β polymorphisms indicate that CD immunogenetics is at the base of this phenomenon. We searched in a Brazilian population five single-nucleotide polymorphisms (-800 G>A rs1800468, -509 C>T rs1800469, +10 T>C rs1800470, +25 G>C rs1800471, and +263 C>T rs1800472), showing that CD patients frequently express the TGF-β1 gene genotypes CT and TT at position -509, as compared to noninfected persons; similar results were observed with genotypes TC and CC at codon +10 of the TGF-β1 gene, leading to the conclusion that 509 C>T and +10 T>C TGF-β1 polymorphisms are associated with Chagas disease susceptibility. Studies in genetically different populations susceptible to CD will help to gather new insights and encourage the use of TGF-β as a CD biomarker., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ferreira, Waghabi, Bailly, Feige, Hasslocher-Moreno, Saraiva and Araujo-Jorge.)

Hereditary Hemorrhagic Telangiectasia (HHT), also known as Rendu-Osler syndrome, is a genetic vascular disorder affecting 1 in 5000-8000 individuals worldwide. This rare disease is characterized by various vascular defects including epistaxis, blood vessel dilations (telangiectasia) and arteriovenous malformations (AVM) in several organs. About 90% of the cases are associated with heterozygous mutations of ACVRL1 or ENG genes, that respectively encode a bone morphogenetic protein receptor (activin receptor-like kinase 1, ALK1) and a co-receptor named endoglin. Less frequent mutations found in the remaining 10% of patients also affect the gene SMAD4 which is part of the transcriptional complex directly activated by this pathway. Presently, the therapeutic treatments for HHT are intended to reduce the symptoms of the disease. However, recent progress has been made using drugs that target VEGF (vascular endothelial growth factor) and the angiogenic pathway with the use of bevacizumab (anti-VEGF antibody). Furthermore, several exciting high-throughput screenings and preclinical studies have identified new molecular targets directly related to the signaling pathways affected in the disease. These include FKBP12, PI3-kinase and angiopoietin-2. This review aims at reporting these recent developments that should soon allow a better care of HHT patients.

Bone morphogenetic protein 9 (BMP9) is a circulating factor produced by hepatic stellate cells that plays a critical role in vascular quiescence through its endothelial receptor activin receptor-like kinase 1 (ALK1). Mutations in the gene encoding ALK1 cause hereditary hemorrhagic telangiectasia type 2, a rare genetic disease presenting hepatic vessel malformations. Variations of both the circulating levels and the hepatic mRNA levels of BMP9 have been recently associated with various forms of hepatic fibrosis. However, the molecular mechanism that links BMP9 with liver diseases is still unknown. Here, we report that Bmp9 gene deletion in 129/Ola mice triggers hepatic perisinusoidal fibrosis that was detectable from 15 weeks of age. An inflammatory response appeared within the same time frame as fibrosis, whereas sinusoidal vessel dilation developed later on. Proteomic and mRNA analyses of primary liver sinusoidal endothelial cells (LSECs) both revealed that the expression of the LSEC-specifying transcription factor GATA-binding protein 4 was strongly reduced in Bmp9 gene knockout (Bmp9-KO) mice as compared with wild-type mice. LSECs from Bmp9-KO mice also lost the expression of several terminal differentiation markers (Lyve1, Stab1, Stab2, Ehd3, Cd209b, eNos, Maf, Plvap). They gained CD34 expression and deposited a basal lamina, indicating that they were capillarized. Another main characteristic of differentiated LSECs is the presence of permeable fenestrae. LSECs from Bmp9-KO mice had a significantly reduced number of fenestrae. This was already observable in 2-week-old pups. Moreover, we could show that addition of BMP9 to primary cultures of LSECs prevented the loss of their fenestrae and maintained the expression levels of Gata4 and Plvap. Conclusion: Taken together, our observations show that BMP9 is a key paracrine regulator of liver homeostasis, controlling LSEC fenestration and protecting against perivascular hepatic fibrosis., (© 2019 by the American Association for the Study of Liver Diseases.)

The aim of the present work was to address the role of BMP9 in different genetic backgrounds (C57BL/6, BALB/c, and 129/Ola) of mice deleted for Bmp9 . We found that Bmp9 deletion led to premature mortality only in the 129/Ola strain. We have previously shown that Bmp9 deletion led to liver sinusoidal endothelial cells (LSEC) capillarization and liver fibrosis in the 129/Ola background. Here, we showed that this is not the case in the C57BL/6 background. Analysis of LSEC from Wild-type (WT) versus Bmp9 -KO mice in the C57BL/6 background showed no difference in LSEC fenestration and in the expression of differentiation markers. Comparison of the mRNA expression of LSEC differentiation markers between WT C57BL/6 and 129/Ola mice showed a significant decrease in Stabilin2, Plvap, and CD209b, suggesting a more capillary-like phenotype in WT C57BL/6 LSECs. C57BL/6 mice also had lower BMP9 circulating concentrations and hepatic Vegfr2 mRNA levels, compared to the 129/Ola mice. Taken together, our observations support a role for BMP9 in liver endothelial cell fenestration and prevention of fibrosis that is dependent on genetic background. It also suggests that 129/Ola mice are a more suitable model than C57BL/6 for the study of liver fibrosis subsequent to LSEC capillarization.

TGF-β involvement in Chagas disease cardiomyopathy has been clearly demonstrated. The TGF-β signaling pathway is activated in the cardiac tissue of chronic phase patients and is associated with an increase in extracellular matrix protein expression. The aim of this study was to investigate the effect of GW788388, a selective inhibitor of TβR1/ALK5, on cardiac function in an experimental model of chronic Chagas' heart disease. To this end, C57BL/6 mice were infected with Trypanosoma cruzi (102 parasites from the Colombian strain) and treated orally with 3mg/kg GW788388 starting at 120 days post-infection (dpi), when 100% of the infected mice show cardiac damage, and following three distinct treatment schedules: i) single dose; ii) one dose per week; or iii) three doses per week during 30 days. The treatment with GW788388 improved several cardiac parameters: reduced the prolonged PR and QTc intervals, increased heart rate, and reversed sinus arrhythmia, and atrial and atrioventricular conduction disorders. At 180 dpi, 30 days after treatment interruption, the GW3x-treated group remained in a better cardiac functional condition. Further, GW788388 treatment reversed the loss of connexin-43 enriched intercellular plaques and reduced fibrosis of the cardiac tissue. Inhibition of the TGF-β signaling pathway reduced TGF-β/pSmad2/3, increased MMP-9 and Sca-1, reduced TIMP-1/TIMP-2/TIMP-4, and partially restored GATA-6 and Tbox-5 transcription, supporting cardiac recovery. Moreover, GW788388 administration did not modify cardiac parasite load during the infection but reduced the migration of CD3+ cells to the heart tissue. Altogether, our data suggested that the single dose schedule was not as effective as the others and treatment three times per week during 30 days seems to be the most effective strategy. The therapeutic effects of GW788388 are promising and suggest a new possibility to treat cardiac fibrosis in the chronic phase of Chagas' heart disease by TGF-β inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Altered expression of regulatory RNA-binding proteins (RBPs) in cancer leads to abnormal expression of mRNAs encoding many factors involved in cancer hallmarks. While conventional anticancer therapies usually target one pathway at a time, targeting key RBP would affect multiple genes and thus overcome drug resistance. Among the Tristetraprolin family of RBP, TIS11b/BRF1/ZFP36L1 mediates mRNA decay through binding to Adenylate/Uridylate (AU-rich elements) in mRNA 3'-untranslated region and recruitment of mRNA degradation enzymes. Here, we show that TIS11b is markedly underexpressed in three breast cancer cell lines, as well as in breast tumor samples. We hypothesized that restoring intracellular TIS11b levels could impair cancer cell phenotypic traits. We thus generated a derivative of TIS11b called R9-ZnC S334D , by combining N-terminal domain deletion, serine-to-aspartate substitution at position 334 to enhance the function of the protein and fusion to the cell-penetrating peptide polyarginine R9. R9-ZnC S334D not only blunted secretion of vascular endothelial growth factor (VEGF) but also inhibited proliferation, migration, invasion, and anchorage-independent growth of murine 4T1 or human MDA-MB-231 breast cancer cells. Moreover, R9-ZnC S334D prevented endothelial cell organization into vessel-like structures, suggesting that it could potentially target various cell types within the tumor microenvironment. In vivo, injection of R9-ZnC S334D in 4T1 tumors impaired tumor growth, decreased tumor hypoxia, and expression of the epithelial-to-mesenchymal transition (EMT) markers Snail, Vimentin, and N-cadherin. R9-ZnC S334D also hindered the expression of chemokines and proteins involved in cancer-related inflammation and invasion including Fractalkine (CX3CL1), SDF-1 (CXCL12), MCP-1 (CCL2), NOV (CCN3), and Pentraxin-3 (PTX3). Collectively, our data indicate that R9-ZnC S334D counteracts multiple traits of breast cancer cell aggressiveness and suggest that this novel protein could serve as the basis for innovative multi-target therapies in cancer.

Rationale: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized., Objective: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension., Method and Results: The absence of BMP9 in Bmp9 -/- mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1 ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9 -/- mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9 +/+ littermates., Conclusions: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.

We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFβ canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFβ1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.

Exogenous cues involved in the regulation of the initial steps of lymphatic endothelial development remain largely unknown. We have used an in vitro model based on the co-culture of vascular precursors derived from mouse embryonic stem cell (ESC) differentiation and OP9 stromal cells to examine the first steps of lymphatic specification and expansion. We found that bone morphogenetic protein 9 (BMP9) induced a dose-dependent biphasic effect on ESC-derived vascular precursors. At low concentrations, below 1 ng/mL, BMP9 expands the LYVE-1-positive lymphatic progeny and activates the calcineurin phosphatase/NFATc1 signaling pathway. In contrast, higher BMP9 concentrations preferentially enhance the formation of LYVE-1-negative endothelial cells. This effect results from an OP9 stromal cell-mediated VEGF-A secretion. RNA-silencing experiments indicate specific involvement of ALK1 and ALK2 receptors in these different BMP9 responses. BMP9 at low concentrations may be a useful tool to generate lymphatic endothelial cells from stem cells for cell-replacement strategies., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Adrenocortical carcinoma (ACC) is a tumor with poor prognosis in which overexpression of a panel of microRNAs has been associated with malignancy but a very limited number of investigations on their role in ACC pathogenesis have been conducted. We examined the involvement of miR-483-5p and miR-139-5p in adrenocortical cancer aggressiveness. Using bioinformatics predictions and mRNA/miRNA expression profiles, we performed an integrated analysis to identify inversely correlated miRNA-mRNA pairs in ACC. We identified N-myc downstream-regulated gene family members 2 and 4 (NDRG2 and NDRG4) as targets of miR-483-5p and miR-139-5p, respectively. NDRG2 and NDRG4 expressions were inversely correlated respectively with miR-483-5p and miR-139-5p levels in aggressive ACC samples from two independent cohorts of 20 and 44 ACC. Moreover, upregulation of miR-139-5p and downregulation of NDRG4 demonstrated a striking prognostic value. A direct interaction between miR-483-5p or miR-139-5p and their targets was demonstrated in reporter assays. Downregulation of miR-483-5p or miR-139-5p in the ACC cell lines NCI-H295R and SW13 increased NDRG2 or NDRG4 mRNA and protein expression, compromised adrenocortical cancer cell invasiveness and anchorage-independent growth. MiR-483-5p or miR-139-5p overexpression and NDRG2 or NDRG4 inhibition produce similar changes, which are rescued by NDRG2 or NDRG4 ectopic expression. We established that key factors mediating epithelial-to-mesenchymal transition are downstream effectors of miR-483-5p/NDRG2 and miR-139-5p/NDRG4 pathways. Collectively, our data show for the first time that miR-483-5p/NDRG2 and miR-139-5p/NDRG4 axes promote ACC aggressiveness, with potential implications for prognosis and therapeutic interventions in adrenocortical malignancies., (© 2018 UICC.)

Bone morphogenetic protein 9 (BMP9) and BMP10 are the two high-affinity ligands for the endothelial receptor activin receptor-like kinase 1 (ALK1) and are key regulators of vascular remodeling. They are both present in the blood, but their respective biological activities are still a matter of debate. The aim of the present work was to characterize their circulating forms to better understand how their activities are regulated in vivo First, by cotransfecting BMP9 and BMP10, we found that both can form a disulfide-bonded heterodimer in vitro and that this heterodimer is functional on endothelial cells via ALK1. Next, we developed an ELISA that could specifically recognize the BMP9-BMP10 heterodimer and which indicated its presence in both human and mouse plasma. In addition to using available Bmp9 -KO mice, we generated a conditional Bmp10 -KO mouse strain. The plasma from Bmp10 -KO mice, similarly to that of Bmp9 -KO mice, completely lacked the ability to activate ALK1-transfected 3T3 cells or phospho-Smad1-5 on endothelial cells, indicating that the circulating BMP activity is mostly due to the BMP9-BMP10 heterodimeric form. This result was confirmed in human plasma that had undergone affinity chromatography to remove BMP9 homodimer. Finally, we provide evidence that hepatic stellate cells in the liver could be the source of the BMP9-BMP10 heterodimer. Together, our findings demonstrate that BMP9 and BMP10 can heterodimerize and that this heterodimer is responsible for most of the biological BMP activity found in plasma., (© 2018 Tillet et al.)

Transforming growth factor β 1 (TGF- β 1) is an important mediator in Chagas disease. Furthermore, patients with higher TGF- β 1 serum levels show a worse clinical outcome. Gene polymorphism may account for differences in cytokine production during infectious diseases. We tested whether TGFB1 polymorphisms could be associated with Chagas disease susceptibility and severity in a Brazilian population. We investigated five single-nucleotide polymorphisms (-800 G>A, -509 C>T, +10 T>C, +25 G>C, and +263 C>T). 152 patients with Chagas disease (53 with the indeterminate form and 99 with the cardiac form) and 48 noninfected subjects were included. Genotypes CT and TT at position -509 of the TGFB1 gene were more frequent in Chagas disease patients than in noninfected subjects. Genotypes TC and CC at codon +10 of the TGFB1 gene were also more frequent in Chagas disease patients than in noninfected subjects. We found no significant differences in the distribution of the studied TGFB1 polymorphisms between patients with the indeterminate or cardiac form of Chagas disease. Therefore, -509 C>T and +10 T>C TGFB1 polymorphisms are associated with Chagas disease susceptibility in a Brazilian population.

We describe the design and optimization of polyfunctional scaffolds based on a fluorescent indolizine core derivatized with various orthogonal groups (amines, esters, oximes, alkynes, etc.). To show one application as tools in biology, the scaffold was used to prepare drug-biotin conjugates that were then immobilized onto avidin-agarose for affinity chromatography. More specifically, the antiangiogenic drug COB223, whose mechanism of action remained unclear, was chosen as a proof-of-concept drug. The drug-selective discrimination of proteins observed after elution of the cell lysates through the affinity columns, functionalized either with the biologically active COB223 or a structurally related inactive analogue (COB236), is a clear indication that the presence of the indolizine core does not limit drug-protein interaction and confirms the usefulness of the indolizine scaffold. Furthermore, the separation of COB223-interacting proteins from human placental extracts unveiled unanticipated protein targets belonging to the family of regulatory RNA-binding proteins, which opens the way to new hypotheses on the mode of action of this antiangiogenic drug., Competing Interests: The authors declare no competing financial interest.

Purpose: Choriocarcinoma (CC) is the most malignant gestational trophoblastic disease that often develops from complete hydatidiform moles (CHM). Neither the mechanism of CC development nor its progression is yet characterized. We recently identified endocrine gland-derived vascular endothelial growth factor (EG-VEGF) as a novel key placental growth factor that controls trophoblast proliferation and invasion. EG-VEGF acts via two receptors, PROKR1 and PROKR2. Here, we demonstrate that EG-VEGF receptors can be targeted for CC therapy. Experimental Design: Three approaches were used: (i) a clinical investigation comparing circulating EG-VEGF in control ( n = 20) and in distinctive CHM ( n = 38) and CC ( n = 9) cohorts, (ii) an in vitro study investigating EG-VEGF effects on the CC cell line JEG3, and (iii) an in vivo study including the development of a novel CC mouse model, through a direct injection of JEG3-luciferase into the placenta of gravid SCID-mice. Results: Both placental and circulating EG-VEGF levels were increased in CHM and CC (×5) patients. EG-VEGF increased JEG3 proliferation, migration, and invasion in two-dimensional (2D) and three-dimensional (3D) culture systems. JEG3 injection in the placenta caused CC development with large metastases compared with their injection into the uterine horn. Treatment of the animal model with EG-VEGF receptor's antagonists significantly reduced tumor development and progression and preserved pregnancy. Antibody-array and immunohistological analyses further deciphered the mechanism of the antagonist's actions. Conclusions: Our work describes a novel preclinical animal model of CC and presents evidence that EG-VEGF receptors can be targeted for CC therapy. This may provide safe and less toxic therapeutic options compared with the currently used multi-agent chemotherapies. Clin Cancer Res; 23(22); 7130-40. ©2017 AACR ., (©2017 American Association for Cancer Research.)

The regulation of mRNA stability has emerged as a critical control step in dynamic gene expression. This process occurs in response to modifications of the cellular environment, including hormonal variations, and regulates the expression of subsets of proteins whose levels need to be rapidly adjusted. Modulation of messenger RNA stability is usually mediated by stabilizing or destabilizing RNA-binding proteins (RNA-BP) that bind to the 3'-untranslated region regulatory motifs, such as AU-rich elements (AREs). Destabilizing ARE-binding proteins enhance the decay of their target transcripts by recruiting the mRNA decay machineries. Failure of such mechanisms, in particular misexpression of RNA-BP, has been linked to several human diseases. In the adrenal cortex, the expression and activity of mRNA stability regulatory proteins are still understudied. However, ACTH- or cAMP-elicited changes in the expression/phosphorylation status of the major mRNA-destabilizing protein TIS11b/BRF1 or in the subcellular localization of the stabilizing protein Human antigen R have been reported. They suggest that this level of regulation of gene expression is also important in endocrinology.

TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3'-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein-protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function., (© 2016 Rataj, Planel, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Pregnancy-induced hypertension diseases are classified as gestational hypertension, preeclampsia, or eclampsia. The mechanisms of their development and prediction are still to be discovered. Endocrine gland-derived vascular endothelial growth factor (EG-VEGF) is an angiogenic factor secreted by the placenta during the first trimester of human pregnancy that was shown to control trophoblast invasion, to be upregulated by hypoxia, and to be abnormally elevated in pathological pregnancies complicated with preeclampsia and intrauterine growth restriction. These findings suggested that sustaining EG-VEGF levels beyond the first trimester of pregnancy may contribute to pregnancy-induced hypertension. To test this hypothesis, osmotic minipumps delivering EG-VEGF were implanted subcutaneously into gravid OF1 (Oncins France 1) mice on day 11.5 post coitus, which is equivalent to the end of the first trimester of human pregnancy. Mice were euthanized at 15.5 and 18.5 days post coitus to assess (1) litter size, placental, and fetal weights; (2) placental histology and function; (3) maternal blood pressure; (4) renal histology and function; and (5) circulating soluble fms-like tyrosine kinase 1 and soluble endoglin. Increased EG-VEGF levels caused significant defects in placental organization and function. Both increased hypoxia and decreased trophoblast invasion were observed. Treated mice had elevated circulating soluble fms-like tyrosine kinase 1 and soluble endoglin and developed gestational hypertension with dysregulated maternal kidney function. EG-VEGF effect on the kidney function was secondary to its effects on the placenta as similarly treated male mice had normal kidney functions. Altogether, these data provide a strong evidence to confirm that sustained EG-VEGF beyond the first trimester of pregnancy contributes to the development of pregnancy-induced hypertension., (© 2016 American Heart Association, Inc.)

Studies developed by our group in the last years have shown the involvement of TGF-β in acute and chronic Chagas heart disease, with elevated plasma levels and activated TGF-β cell signaling pathway as remarkable features of patients in the advanced stages of this disease, when high levels of cardiac fibrosis is present. Imbalance in synthesis and degradation of extracellular matrix components is the basis of pathological fibrosis and TGF-β is considered as one of the key regulators of this process. In the present study, we investigated the activity of the TGF-β signaling pathway, including receptors and signaling proteins activation in the heart of animals experimentally infected with Trypanosoma cruzi during the period that mimics the acute phase of Chagas disease. We observed that T. cruzi-infected animals presented increased expression of TGF-β receptors. Overexpression of receptors was followed by an increased phosphorylation of Smad2/3, p38 and ERK. Furthermore, we correlated these activities with cellular factors involved in the fibrotic process induced by TGF-β. We observed that the expression of collagen I, fibronectin and CTGF were increased in the heart of infected animals on day 15 post-infection. Correlated with the increased TGF-β activity in the heart, we found that serum levels of total TGF-β were significantly higher during acute infection. Taken together, our data suggest that the commitment of the heart associates with increased activity of TGF-β pathway and expression of its main components. Our results, confirm the importance of this cytokine in the development and maintenance of cardiac damage caused by T. cruzi infection., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Context: Prokineticin 1 (PROK1), also called endocrine gland-derived vascular endothelial growth factor, is a well-established regulator of endometrial receptivity and placental development. However, its clinical usefulness as a noninvasive predictive biomarker of embryo implantation is yet to be validated., Objective: The main objective of this article was to determine the relationship between PROK1 levels in the follicular fluid (FF) and fertilization culture media (FCM) and the reproductive outcome in patients who received a first conventional in vitro fertilization-embryo transfer. The secondary objective was to characterize the expression of PROK1 and its receptors (PROKRs) in the human follicular microenvironment., Design and Setting: We conducted a prospective study between January 2013 and June 2015 at the University Hospital of Grenoble., Patients: A total of 135 infertile in vitro fertilization patients and 10 women undergoing ovarian tissue cryopreservation were included., Interventions: The PROK1 concentration was measured by ELISA in FF and FCM collected on the day of oocyte retrieval and the day of the oocyte denudation step, respectively. Follicular expression of the PROK1/PROKR system was determined by immunohistochemistry, RT-quantitative PCR, and ELISA., Main Outcome Measure: Assessment of the clinical pregnancy rates was the main outcome., Results: FF and FCM PROK1 levels were significantly higher in the embryo implantation group (P < .001) and were predictive of subsequent embryo implantation (area under the receiver operating characteristic curve, 0.91 [95% confidence interval, 0.81-1.00], P = .001; and 0.88 [0.72-1.00], P = .001, respectively). FF and FCM PROK1 levels remain similar irrespective of the embryo morphokinetic parameters (P = .71 and P = .83, respectively). The PROK1/PROKR system is expressed during human folliculogenesis., Conclusions: PROK1 levels in FF and FCM could constitute new predictive noninvasive markers of successful embryo implantation in conventional in vitro fertilization-embryo transfer.

Prokineticin 1 (PROK1) and (PROK2), are two closely related proteins that were identified as the mammalian homologs of their two amphibian homologs, mamba intestinal toxin (MIT-1) and Bv8. PROKs activate two G-protein linked receptors (prokineticin receptor 1 and 2, PROKR1 and PROKR2). Both PROK1 and PROK2 have been found to regulate a stunning array of biological functions. In particular, PROKs stimulate gastrointestinal motility, thus accounting for their family name "prokineticins". PROK1 acts as a potent angiogenic mitogen, thus earning its other name, endocrine gland-derived vascular endothelial factor. In contrast, PROK2 signaling pathway has been shown to be a critical regulator of olfactory bulb morphogenesis and sexual maturation. During the last decade, strong evidences established the key roles of prokineticins in the control of human central and peripheral reproductive processes. PROKs act as main regulators of the physiological functions of the ovary, uterus, placenta, and testis, with marked dysfunctions in various pathological conditions such as recurrent pregnancy loss, and preeclampsia. PROKs have also been associated to the tumor development of some of these organs. In the central system, prokineticins control the migration of GnRH neurons, a key process that controls reproductive functions. Importantly, mutations in PROK2 and PROKR2 are associated to the development of Kallmann syndrome, with direct consequences on the reproductive system. This review describes the finely tuned actions of prokineticins in the control of the central and peripheral reproductive processes. Also, it discusses future research directions for the use of these cytokines as diagnostic markers for several reproductive diseases.

Introduction: Netrin-4 is a secreted member of the laminin-related protein family, known to be involved in axonal guidance and endothelial cell survival, proliferation, and migration. We have recently reported the cellular localization of netrin-4 and its receptor neogenin in human first trimester and term placenta. A strong expression of netrin-4 was observed in trophoblast and in endothelial cells, suggesting a potential role of this protein in placental angiogenesis. In relation to human pregnancy, it has been reported that circulating netrin-4 were increased in fetal umbilical cord blood of intrauterine growth restriction IUGR compared to normal pregnancy suggesting an adverse effect of this protein on placental and fetal development. The aim of this study was to determine the role of netrin-4 in placental angiogenesis., Methods: The effects of netrin-4 on proliferation, migration, tube-like organization, and spheroid sprouting of human placental microvascular endothelial cells (HPEC) were studied., Results: We demonstrated that netrin-4 inhibits HPEC proliferation, tube-like formation, migration and spheroid sprouting, suggesting a direct role of netrin-4 in the regulation of intra-villus angiogenesis., Discussion: This is the first report of an anti-angiogenic activity of netrin-4 in human placenta. This study brings new insights into netrin-4 roles in placental angiogenesis and suggests possible involvements of netrin-4 in angiogenesis-related pathologies such as IUGR., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Idiopathic fetal growth restriction (FGR) is frequently associated with placental insufficiency. Previous reports have provided evidence that endocrine gland-derived vascular endothelial growth factor (EG-VEGF), a placental secreted protein, is expressed during the first trimester of pregnancy, controls both trophoblast proliferation and invasion, and its increased expression is associated with human FGR. In this study, we hypothesize that EG-VEGF-dependent changes in placental homeobox gene expressions contribute to trophoblast dysfunction in idiopathic FGR. The changes in EG-VEGF-dependent homeobox gene expressions were determined using a homeobox gene cDNA array on placental explants of 8-12 wks gestation after stimulation with EG-VEGF in vitro for 24 h. The homeobox gene array identified a greater-than-five-fold increase in HOXA9 , HOXC8 , HOXC10 , HOXD1 , HOXD8 , HOXD9 and HOXD11 , while NKX 3.1 showed a greater-than-two-fold decrease in mRNA expression compared with untreated controls. Homeobox gene NKX3.1 was selected as a candidate because it is a downstream target of EG-VEGF and its expression and functional roles are largely unknown in control and idiopathic FGR-affected placentae. Real-time PCR and immunoblotting showed a significant decrease in NKX3.1 mRNA and protein levels, respectively, in placentae from FGR compared with control pregnancies. Gene inactivation in vitro using short-interference RNA specific for NKX3.1 demonstrated an increase in BeWo cell differentiation and a decrease in HTR-8/SVneo proliferation. We conclude that the decreased expression of homeobox gene NKX3.1 downstream of EG-VEGF may contribute to the trophoblast dysfunction associated with idiopathic FGR pregnancies.

PPARγ-deficient mice die at E9.5 due to placental abnormalities. The mechanism by which this occurs is unknown. We demonstrated that the new endocrine factor EG-VEGF controls the same processes as those described for PPARγ, suggesting potential regulation of EG-VEGF by PPARγ. EG-VEGF exerts its functions via prokineticin receptor 1 (PROKR1) and 2 (PROKR2). This study sought to investigate whether EG-VEGF mediates part of PPARγ effects on placental development. Three approaches were used: 1) in vitro, using human primary isolated cytotrophoblasts and the extravillous trophoblast cell line (HTR-8/SVneo); 2) ex vivo, using human placental explants (n = 46 placentas); and 3) in vivo, using gravid wild-type PPARγ(+/-) and PPARγ(-/-) mice. Major processes of placental development that are known to be controlled by PPARγ, such as trophoblast proliferation, migration, and invasion, were assessed in the absence or presence of PROKR1 and PROKR2 antagonists. In both human trophoblast cell and placental explants, we demonstrated that rosiglitazone, a PPARγ agonist, 1) increased EG-VEGF secretion, 2) increased EG-VEGF and its receptors mRNA and protein expression, 3) increased placental vascularization via PROKR1 and PROKR2, and 4) inhibited trophoblast migration and invasion via PROKR2. In the PPARγ(-/-) mouse placentas, EG-VEGF levels were significantly decreased, supporting an in vivo control of EG-VEGF/PROKRs system during pregnancy. The present data reveal EG-VEGF as a new mediator of PPARγ effects during pregnancy and bring new insights into the fine mechanism of trophoblast invasion., (Copyright © 2015 the American Physiological Society.)

Enhanced lung angiogenesis has been reported in cystic fibrosis (CF). Recently, two highly homologous ligands, endocrine gland vascular endothelial growth factor (EG-VEGF) and mammalian Bv8, have been described as new angiogenic factors. Both ligands bind and activate two closely related G protein-coupled receptors, the prokineticin receptor (PROKR) 1 and 2. Yet, the expression, regulation, and potential role of EG-VEGF, BV8, and their receptors in normal and CF lung are still unknown. The expression of the receptors and their ligands was examined using molecular, biochemical, and immunocytochemistry analyses in lungs obtained from CF patients vs. control and in normal and CF bronchial epithelial cells. Cystic fibrosis transmembrane conductance regulator (CFTR) activity was evaluated in relation to both ligands, and concentrations of EG-VEGF were measured by ELISA. At the mRNA level, EG-VEGF, BV8, and PROKR2 gene expression was, respectively, approximately five, four, and two times higher in CF lungs compared with the controls. At the cellular level, both the ligands and their receptors showed elevated expressions in the CF condition. Similar results were observed at the protein level. The EG-VEGF secretion was apical and was approximately two times higher in CF compared with the normal epithelial cells. This secretion was increased following the inhibition of CFTR chloride channel activity. More importantly, EG-VEGF and BV8 increased the intracellular concentration of Ca(2+) and cAMP and stimulated CFTR-chloride channel activity. Altogether, these data suggest local roles for epithelial BV8 and EG-VEGF in the CF airway peribronchial vascular remodeling and highlighted the role of CFTR activity in both ligand biosynthesis and secretion., (Copyright © 2015 the American Physiological Society.)

The transition to pulmonary respiration after birth requires rapid alterations in the structure of the mammalian cardiovascular system. One dramatic change that occurs is the closure of the ductus arteriosus (DA), an arterial connection in the fetus that directs blood flow away from the pulmonary circulation. Two members of the TGFβ family, bone morphogenetic protein 9 (BMP9) and BMP10, have been recently involved in postnatal angiogenesis, both being necessary for remodeling of newly formed microvascular beds. The aim of the present work was to study whether BMP9 and BMP10 could be involved in closure of the DA. We found that Bmp9 knockout in mice led to an imperfect closure of the DA. Further, addition of a neutralizing anti-BMP10 antibody at postnatal day 1 (P1) and P3 in these pups exacerbated the remodeling defect and led to a reopening of the DA at P4. Transmission electron microscopy images and immunofluorescence stainings suggested that this effect could be due to a defect in intimal cell differentiation from endothelial to mesenchymal cells, associated with a lack of extracellular matrix deposition within the center of the DA. This result was supported by the identification of the regulation by BMP9 and BMP10 of several genes known to be involved in this process. The involvement of these BMPs was further supported by human genomic data because we could define a critical region in chromosome 2 encoding eight genes including BMP10 that correlated with the presence of a patent DA. Together, these data establish roles for BMP9 and BMP10 in DA closure.

Several studies indicate that the activity of cruzipain, the main lysosomal cysteine peptidase of Trypanosoma cruzi, contributes to parasite infectivity. In addition, the parasitic invasion process of mammalian host cells is described to be dependent on the activation of the host TGF-β signaling pathway by T. cruzi. Here, we tested the hypothesis that cruzipain could be an important activator of latent TGF-β and thereby trigger TGF-β-mediated events crucial for the development of Chagas disease. We found that live epimastigotes of T. cruzi, parasite lysates and purified cruzipain were able to activate latent TGF-β in vitro. This activation could be inhibited by the cysteine peptidase inhibitor Z-Phe-Ala-FMK. Moreover, transfected parasites overexpressing chagasin, a potent endogenous cruzipain inhibitor, prevented latent TGF-β activation. We also observed that T. cruzi invasion, as well as parasite intracellular growth, were inhibited by the administration of Z-Phe-Ala-FMK or anti-TGF-β neutralizing antibody to Vero cell cultures. We further demonstrated that addition of purified cruzipain enhanced the invasive activity of trypomastigotes and that this effect could be completely inhibited by addition of a neutralizing anti-TGF-β antibody. Taken together, these results demonstrate that the activities of cruzipain and TGF-β in the process of cell invasion are functionally linked. Our data suggest that cruzipain inhibition is an interesting chemotherapeutic approach for Chagas disease not only because of its trypanocidal activity, but also due to the inhibitory effect on TGF-β activation.

The efficacy of anti-angiogenic therapies on cancer patients is limited by the emergence of drug resistance, urging the search for second-generation drugs. In this study, we screened an academic chemical library (DCM, University of Grenoble-Alpes) and identified a leader molecule, COB223, that inhibits endothelial cell migration and proliferation. It inhibits also Lewis lung carcinoma (LLC/2) cell proliferation whereas it does not affect fibroblast proliferation. The anti-angiogenic activity of COB223 was confirmed using several in vitro and in vivo assays. In a mouse LLC/2 tumor model, ip administration of doses as low as 4 mg/kg COB223 efficiently reduced the tumor growth rate. We observed that COB223 inhibits endothelial cell ERK1/2 phosphorylation induced by VEGF, FGF-2 or serum and that it acts downstream of PKC and upstream of Ras. This molecule represents a novel anti-angiogenic and anti-tumorigenic agent with an original mechanism of action that deserves further development as an anti-cancer drug.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant inheritable vascular dysplasia caused by mutations in genes encoding either endoglin or activin receptor-like kinase-1 (ALK1). Functional significance of endoglin missense mutations remains largely unknown leading to a difficult discrimination between polymorphisms and pathogenic mutations. In order to study the functional significance of endoglin mutations and to help HHT1 diagnosis, we developed a cellular assay based on the ability of endoglin to enhance ALK1 response to bone morphogenetic protein 9 (BMP9). We generated and characterized 31 distinct ENG mutants reproducing human HHT1 missense mutations identified in patients of the Molecular Genetics Department in Lyon. We found that 16 mutants behaved like wild-type (WT) endoglin, and thus corresponded to benign rare variants. The 15 other variants showed defects in BMP9 response and were identified as pathogenic mutations. Interestingly, two mutants (S278P and F282V) had lost their ability to bind BMP9, identifying two crucial amino acids for BMP9 binding to endoglin. For all the others, the functional defect was correlated with a defective trafficking to the cell surface associated with retention in the endoplasmic reticulum. Further, we demonstrated that some intracellular mutants dimerized with WT endoglin and impaired its cell-surface expression thus acting as dominant-negatives. Taken together, we show that endoglin loss-of-function can result from different mechanisms in HHT1 patients. We also provide a diagnostic tool helping geneticists in screening for novel or conflicting ENG mutations., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

EG-VEGF is an angiogenic factor that we identified as a new placental growth factor during human pregnancy. EG-VEGF is also expressed in the mouse fetal membrane (FM) by the end of gestation, suggesting a local role for this protein in the mechanism of parturition. However, injection of EG-VEGF to gravid mice did not induce labor, suggesting a different role for EG-VEGF in parturition. Here, we searched for its role in the FM in relation to human parturition. Human pregnant sera and total FM, chorion, and amnion were collected during the second and third trimesters from preterm no labor, term no labor, and term labor patients. Primary human chorion trophoblast and FM explants cultures were also used. We demonstrate that circulating EG-VEGF increased toward term and significantly decreased at the time of labor. EG-VEGF production was higher in the FM compared to placentas matched for gestational age. Within the FM, the chorion was the main source of EG-VEGF. EG-VEGF receptors, PROKR1 and PROKR2, were differentially expressed within the FM with increased expression toward term and an abrupt decrease with the onset of labor. In chorion trophoblast and FM explants collected from nonlaboring patients, EG-VEGF decreased metalloproteinase-2 and -9 activities and increased PGDH (prostaglandin-metabolizing enzyme) expression. Altogether these data demonstrate that EG-VEGF is a new cytokine that acts locally to ensure FM protection in late pregnancy. Its fine contribution to the initiation of human labor is exhibited by the abrupt decrease in its levels as well as a reduction in its receptors., (© 2014 by the Society for the Study of Reproduction, Inc.)

During the last decade, there has been growing evidence for the involvement of prokineticins and their receptors (PROK/PROKR) in human reproduction, with multiple roles in the female and male reproductive systems. The PROK/PROKR signalling complex has been reported as a new actor in ovary, uterus, placenta, and testis physiology, with marked dysfunction in various pathological conditions such as polycystic ovary syndrome, recurrent pregnancy loss, preeclampsia, and ectopic pregnancy. Altogether, the results strongly suggest the involvement of prokineticins in spermatogenesis, oocyte competence, embryo implantation, pregnancy, and delivery, and argue for the clinical relevance of these cytokines and their receptors as diagnostic markers for several reproductive diseases., (© 2014 médecine/sciences – Inserm.)

In utero exposure to nicotine is associated with increased risk of numerous adverse fetal and neonatal outcomes, which suggests that it acts directly to affect placental development and the establishment of the fetomaternal circulation (FC). This study used both in vivo [Wistar rats treated with 1 mg/kg nicotine from 2 wk prior to mating until gestational day (GD) 15] and in vitro (RCHO-1 cell line; treated with 10(-9) to 10(-3)M nicotine) models to examine the effects of nicotine on these pathways. At GD 15, control and treated placentas were examined for the impact of nicotine on 1) trophoblast invasion, proliferation, and degree of hypoxia, 2) labyrinth vascularization, 3) expression of key genes of placental development, and 4) expression of placental angiogenic factors. The RCHO-1 cell line was used to determine the direct effects of nicotine on trophoblast differentiation. Our in vivo experiments show that nicotine inhibits trophoblast interstitial invasion, increases placental hypoxia, downregulates labyrinth vascularization as well as key transcription factors Hand1 and GCM1, and decreases local and circulating EG-VEGF, a key placental angiogenic factor. The in vitro experiments confirmed the inhibitory effects of nicotine on the trophoblast migration, invasion, and differentiation processes and demonstrated that those effects are most likely due to a dysregulation in the expression of nicotine receptors and a decrease in MMP9 activity. Taken together, these data suggest that adverse effects of maternal smoking on pregnancy outcome are due in part to direct and endocrine effects of nicotine on the main processes of placental development and establishment of FC.

Placentation is associated with several steps of vascular adaptations throughout pregnancy. These vascular changes occur both on the maternal and fetal sides, consisting of maternal uterine spiral arteries remodeling and placental vasculogenesis and angiogenesis, respectively. Placental angiogenesis is a pivotal process for efficient fetomaternal exchanges and placental development. This process is finely controlled throughout pregnancy, and it involves ubiquitous and pregnancy-specific angiogenic factors. In the last decade, endocrine gland derived vascular endothelial growth factor (EG-VEGF), also called prokineticin 1 (PROK1), has emerged as specific placental angiogenic factor that controls many aspects of normal and pathological placental angiogenesis such as recurrent pregnancy loss (RPL), gestational trophoblastic diseases (GTD), fetal growth restriction (FGR), and preeclampsia (PE). This review recapitulates EG-VEGF mediated-angiogenesis within the placenta and at the fetomaternal interface and proposes that its deregulation might contribute to the pathogenesis of several placental diseases including FGR and PE. More importantly this paper argues for EG-VEGF clinical relevance as a potential biomarker of the onset of pregnancy pathologies and discusses its potential usefulness for future therapeutic directions.

Study Question: To determine whether the umbilical cord insertion site of singleton pregnancies could be linked to the newborn birth weight at term and its individual growth potential achievement., Material and Methods: A cohort study including 528 records of term neonates was performed. Each neonate was assessed for growth adjusted for gestational age according to the infant's growth potential using the AUDIPOG module. We considered two categories of umbilical cord insertions: central and peripheral. Intrauterine growth restriction was defined as birth weight below the 10th percentile. Statistical analysis was performed using Chi-square, Student's t test, Wilcoxon test, ANOVA, and logistic regression., Results: We observed a total of 343 centrally inserted cords versus 185 peripheral cords. There were twice as many smokers in the mothers of the peripheral category compared to the centrally inserted ones. More importantly, we demonstrated that only 17/343 (5.0%) of infants with central cord insertion were growth restricted, compared to 37/185 (20.0%) of the infants born with a peripheral insertion. Neonates with centrally inserted cord were significantly heavier., Conclusion: The umbilical cord insertion site of singleton pregnancies is associated with the newborn's birth weight at term and its individual growth potential achievement.

Angiogenesis is a key process for proper placental development and for the success of pregnancy. Although numerous in vitro methods have been developed for the assessment of this process, relatively few reliable in vivo methods are available to evaluate this activity throughout gestation. Here we report an in vivo technique that specifically measures placental neovascularization. The technique is based on the measurement of a fluorescent alpha v beta 3 (αvβ3) integrin-targeting molecule called Angiolone-Alexa-Fluor 700. The αvβ3 integrin is highly expressed by endothelial cells during the neovascularization and by trophoblast cells during their invasion of the maternal decidua. Angiolone was injected to gravid mice at 6.5 and 11.5 days post coitus (dpc). The fluorescence was analyzed one day later at 7.5 and 12.5 dpc, respectively. We demonstrated that (i) Angiolone targets αvβ3 protein in the placenta with a strong specificity, (ii) this technique is quantitative as the measurement was correlated to the increase of the placental size observed with increasing gestational age, and (iii) information on the outcome is possible, as abnormal placentation could be detected early on during gestation. In conclusion, we report the validation of a new noninvasive and quantitative method to assess the placental angiogenic activity, in vivo.

Gestational trophoblastic disease (MGT) includes a wide spectrum of pathologies of the placenta, ranging from benign precancerous lesions, with gestational trophoblastic tumors. Metastases are the leading causes of death as a result of this tumor. They represent a major problem for obstetrics and for the public health system. To date, there is no predictor of the progression of molar pregnancies to gestational trophoblastic tumor (GTT). Only an unfavorable plasma hCG monitoring after evacuation of hydatidiform mole is used to diagnose a TTG. The causes of the development of this cancer are still poorly understood. Increasing data in the literature suggests a close association between the development of this tumor and poor placental vascularization during the first trimester of pregnancy. The development of the human placenta depends on a coordination between the trophoblast and endothelial cells. A disruption in the expression of angiogenic factors could contribute to uterine or extra-uterine tissue invasion by extravillous trophoblast, contributing to the development of TTG. This review sheds lights on the phenomenon of angiogenesis during normal and abnormal placentation, especially during the MGT and reports preliminary finding concerning, the variability of expression of "Endocrine Gland-Derived Vascular Endothelial Growth Factor" (EG-VEGF), a specific placental angiogenic factor, in normal and molar placentas, and the potential role of differentiated expressions of the main placental angiogenic factors in the scalability of hydatidiform moles towards a recovery or towards the development of gestational trophoblastic tumor. Deciphering the mechanisms by which the angiogenic factor influences these processes will help understand the pathophysiology of MGT and to create opportunities for early diagnosis and treatment of the latter., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

In spite of improvements in assisted reproductive technology (ART) during the last 30 years, the rate of pregnancy remains constrained, as only about 25 % of embryo transfer lead to successful pregnancies, even with an average of two embryos replaced. Embryo selection is currently based on the establishment of morphokinetic scores, a method that obviously exhibits limitations. Therefore, the assessment of embryo development potency by criteria of higher predictive value is mandatory in order to increase the rates of pregnancy. Nowadays, there is increasing evidence that angiogenic factors might contribute to the success of the implantation and to the pregnancy outcome. Among these factors, prokineticin 1 (PROK1) and its receptors (PROKRs) constitute new targets that showed over the last ten years strong biological features directly linked to ovarian physiology, endometrial receptivity, embryo implantation and thus successful pregnancies. In ART, the rates of circulating PROK1 were reported in 2012 as significantly linked to the quality of embryonic cohort, as well as to the rates of pregnancy. Our preliminary data suggest a high potential of this cytokine in the success of implantation and pregnancy, and strongly overtones the emergency to investigate the value of its measurement in conditioned media of oocytes and embryo cultures in ART., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Background: Transforming growth factor-β1 (TGF-β1) may be implicated in the development of Chagas heart disease. However, the clinical value of TGF-β1 measurement is yet to be determined., Methods: We retrospectively analyzed the outcome of 54 Chagas disease patients without heart failure and with left ventricular (LV) ejection fraction >45% whose TGF-β1 serum values were determined between January 1998 and December 1999. Primary end point was all-cause mortality and secondary end point was the combination of all-cause mortality or hospitalization due to worsening heart failure or cardiac arrhythmias., Results: TGF-β1 was independently associated with the occurrence of the primary and secondary end points. The optimal cutoff for TGF-β1 to identify the primary end point was 12.9 ng/ml (area under the curve = 0.82, p = 0.004, sensitivity 100%, and specificity 57%) and to identify the secondary end point was 30.8 ng/ml (area under the curve = 0.72, p = 0.03, sensitivity 60%, and specificity 86%). LV ejection fraction and LV end-diastolic diameter were also independent predictors of the primary and secondary endpoints, respectively., Conclusion: The described association between TGF-β1 and clinical outcome provides evidence towards the clinical value of TGF-β1 in Chagas disease.

Lymphatic vessels are critical for the maintenance of tissue fluid homeostasis and their dysfunction contributes to several human diseases. The activin receptor-like kinase 1 (ALK1) is a transforming growth factor-β family type 1 receptor that is expressed on both blood and lymphatic endothelial cells (LECs). Its high-affinity ligand, bone morphogenetic protein 9 (BMP9), has been shown to be critical for retinal angiogenesis. The aim of this work was to investigate whether BMP9 could play a role in lymphatic development. We found that Bmp9 deficiency in mice causes abnormal lymphatic development. Bmp9-knockout (KO) pups presented hyperplastic mesenteric collecting vessels that maintained LYVE-1 expression. In accordance with this result, we found that BMP9 inhibited LYVE-1 expression in LECs in an ALK1-dependent manner. Bmp9-KO pups also presented a significant reduction in the number and in the maturation of mesenteric lymphatic valves at embryonic day 18.5 and at postnatal days 0 and 4. Interestingly, the expression of several genes known to be involved in valve formation (Foxc2, Connexin37, EphrinB2, and Neuropilin1) was upregulated by BMP9 in LECS. Finally, we demonstrated that Bmp9-KO neonates and adult mice had decreased lymphatic draining efficiency. These data identify BMP9 as an important extracellular regulator in the maturation of the lymphatic vascular network affecting valve development and lymphatic vessel function.

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Local and distant recurrences occur in a subset of tumors classified as 'aggressive' ACC (aACC), as opposed to 'non-aggressive' ACC (naACC). In this study, we investigated whether tissue and serum microRNAs (miRNAs) are predictive of ACC prognosis. Tissue miRNA expression profiles were determined using microarrays in a test series of six adrenocortical adenomas (ACAs), six naACCs, and six aACCs. Eight miRNAs were selected for further validation by quantitative RT-PCR (ten ACAs, nine naACCs, nine aACCs, and three normal adrenals). Serum levels of five miRNAs were measured in samples from 56 subjects (19 healthy controls (HC), 14 ACA, nine naACC, and 14 aACC patients). MiR-195 and miR-335 levels were significantly decreased in both tumor and serum samples of ACC patients relative to ACA patients or HC. MiR-139-5p and miR-376a levels were significantly increased in aACC compared with naACC patients in tumor samples only. Tissue miR-483-5p was markedly upregulated in a majority of ACC compared with ACA patients or HC, but most importantly, serum miR-483-5p was detected only in aACC patients. High circulating levels of miR-483-5p or low circulating levels of miR-195 were associated with both shorter recurrence-free survival (P=0.0004 and P=0.0014 respectively) and shorter overall survival (P=0.0005 and P=0.0086 respectively). In conclusion, this study reports for the first time that circulating miR-483-5p and miR-195 are promising noninvasive biomarkers with a highly specific prognostic value for the clinical outcome of ACC patients.