Inhibition of human placental endothelial cell proliferation and angiogenesis by netrin-4.

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.

Endocrine gland-derived endothelial growth factor (EG-VEGF) is a potential novel regulator of human parturition.

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.

Characterization of the adverse effects of nicotine on placental development: in vivo and in vitro studies.

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.

Serum miR-483-5p and miR-195 are predictive of recurrence risk in adrenocortical cancer patients.

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.

[Potential role of the angiogenic factor "EG-VEGF" in gestational trophoblastic diseases]. / Rôle potentiel du facteur angiogénique «EG-VEGF¼ dans les maladies gestationnelles trophoblastiques.

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.

[Tumor angiogenesis: recent progress and remaining challenges]. / L'angiogenèse tumorale : progrès récents et défis persistants.

Anti-angiogenic therapies of solid cancers aim at specifically destroying the tumor vasculature in order to "asphyxiate" the tumors. Since few years, they represent a novel therapeutic tool, which allowed to significantly improve the survival of patients suffering from colon, breast, kidney and lung cancers. However, these therapies are limited in their efficacy by the appearance of tumor resistance phenomena. In this review, I describe the molecular and cellular mechanisms of tumor angiogenesis with a special focus on the important roles played by hypoxia, the endothelial growth factor VEGF and the endothelial tip-cells located at the extremity of sprouting neo-vessels. I present the factors that respectively control the activation phase and the maturation phase of angiogenesis, as well as their mechanisms of action. In a second part, the efficacy and the limits of anti-angiogenic therapies presently available on the market are described, and the recent elucidation of some molecular mechanisms of tumor resistance to anti-angiogenic therapies is reviewed.

A novel concept in antiangiogenic and antitumoral therapy: multitarget destabilization of short-lived mRNAs by the zinc finger protein ZFP36L1.

Angiogenesis inhibitors have shown clinical benefits in patients with advanced cancer, but further therapeutic improvement is needed. We have previously shown that the zinc finger protein 36, C3H type-like 1 (ZFP36L1) enhances vascular endothelial growth factor (VEGF) mRNA decay through its interaction with AU-rich elements within VEGF 3'-untranslated region. In this study, we evaluated the possibility to develop an antiangiogenic and antitumoral strategy using the mRNA-destabilizing activity of ZFP36L1. We engineered a cell-penetrating ZFP36L1, by fusing it to the protein transduction domains (PTDs) TAT derived from HIV, or the polyarginine peptides R7 or R9. PTD-ZFP36L1 fusion proteins were expressed in bacterial cells and affinity-purified to homogeneity. TAT-, R7- and R9-ZFP36L1 were efficiently internalized into living cells and decreased both endogenous VEGF mRNA half-life and VEGF protein levels in vitro. Importantly, a single injection of R9-TIS11b fusion protein into a high-VEGF expressing tissue in vivo (in this study, the mouse adrenal gland) markedly decreased VEGF expression. We further evaluated the effect of R9-ZFP36L1 on tumor growth using Lewis Lung Carcinoma (LL/2) cells implanted subcutaneously into nude mice. Intratumoral injection of R9-ZFP36L1 significantly reduced tumor growth and markedly decreased the expression of multiple angiogenic and inflammatory cytokines, including VEGF, acidic fibroblast growth factor, tumor necrosis factor α, interleukin (IL)-1α and IL-6, with a concomitant obliteration of tumor vascularization. These findings indicate that R9-ZFP36L1 fusion protein may represent a novel antiangiogenic and antitumoral agent, and supports the emerging idea that modulation of mRNA stability represents a promising therapeutic approach to treat cancer.

Insights into the role of genetic alterations in adrenocortical tumorigenesis.

Whereas benign adrenocortical tumors are frequent in the population, adrenocortical carcinoma (ACC) is a rare cancer. Significant advances in the understanding of the pathogenesis of sporadic ACCs have been possible through the study of hereditary syndromes responsible for ACCs. The genetic alterations involved in these syndromes have also been found in sporadic ACCs. Several specific genes have been shown to be altered in sporadic ACCs. Despite these progresses, the underlying sequence(s) of events remains to be elucidated. Progressive transformation of a normal tissue into a benign tumor and ultimately into a carcinoma occurs via accumulation of genetic and epigenetic alterations. Likewise, a multistage model has been proposed for the adrenal tumor development. This review summarizes the molecular alterations likely involved in the multistage tumorigenesis and describes a mouse model which allows us to evaluate the effect of individual genes or combination of genes in the development of adrenocortical tumors.

Placental expression of EG-VEGF and its receptors PKR1 (prokineticin receptor-1) and PKR2 throughout mouse gestation.

Compelling evidence indicates that vascular endothelial growth factor (VEGF) is an important mediator of placental angiogenesis and appears to be disregulated in pre-eclampsia (PE). Recently, we characterised the expression of EG-VEGF (endocrine gland-derived vascular endothelial growth factor), also known as prokineticin 1 (PK1) in human placenta during the first trimester of pregnancy and showed that this factor is likely to play an important role in human placentation. However, because it is impossible to prospectively study placentation in humans, it has been impossible to further characterise EG-VEGF expression throughout complete gestation and especially at critical gestational ages for PE development. In the present study, we used mouse placenta to further characterise EG-VEGF expression throughout gestation. We investigated the pattern of expression of EG-VEGF and its receptors, PKR1 and PKR2 at the mRNA and protein levels. Our results show that EG-VEGF and VEGF exhibit different patterns of expression and different localisations in the mouse placenta. EG-VEGF was mainly localised in the labyrinth whereas VEGF was mainly present in glycogen and giant cells. EG-VEGF mRNA and protein levels were highest before 10.5days post coitus (dpc) whereas those of VEGF showed stable expression throughout gestation. PKR1 protein was localised to the labyrinth layer and showed the same pattern of expression as EG-VEGF whereas PKR2 expression was maintained over 10.5dpc with both trophoblastic and endothelial cell localisations. Altogether these findings suggest that EG-VEGF may have a direct effect on both endothelial and trophoblastic cells and is likely to play an important role in mouse placentation.

Aberrant GPCR expression is a sufficient genetic event to trigger adrenocortical tumorigenesis.

Aberrant expression of G protein-coupled receptors (GPCR) in the adrenal cortex is observed in some cases of ACTH-independent macronodular adrenal hyperplasias and adenomas associated with Cushing syndrome (CS). Although there is clinical evidence for the implication of these receptors in abnormal regulation of cortisol secretion, whether this aberrant expression also directly causes the development of a benign adrenocortical tumor is an open question. Cell transplantation provides a way to study genes that may be important in human tumor development. The system we developed uses genetically modified adrenocortical cells transplanted into adrenalectomized immunodeficient mice, which form a functional tissue structure. We observed that enforcing expression of the gastric inhibitory polypeptide (GIP) receptor or the luteinizing hormone (LH) receptor genes (taken as canonical examples of aberrantly expressed GPCRs) in adrenocortical cells resulted in the formation of hyperplastic tissues and the development of Cushing syndrome features in transplanted mice.

Transforming growth factor beta1 inhibits aldosterone and cortisol production in the human adrenocortical cell line NCI-H295R through inhibition of CYP11B1 and CYP11B2 expression.

Transforming growth factor beta1 (TGFbeta1) has been shown to exert strong inhibitory effects on adrenocortical cell steroidogenesis. However, the molecular targets of TGFbeta1 in adrenocortical cells appear to differ between species. Here, we report the first characterization of the regulatory effects of TGFbeta1 on the steroidogenic functions of the human adrenocortical tumor cell line NCI-H295R. After treatment with 2 ng/ml TGFbeta1 for 24 h, basal production of corticosterone, cortisol and androstenedione was dramatically decreased. When TGFbeta1 was added simultaneously with forskolin, the production of cortisol and 11-hydroxyandrostenedione was decreased by 85% whereas that of deoxycortisol was increased. When TGFbeta1 was added simultaneously with angiotensin II, aldosterone production was reduced by 80%. We observed that TGFbeta1 strongly inhibits forskolin-induced steroid 11beta-hydroxylase activity and CYP11B1 mRNA levels, as well as angiotensin II-induced aldosterone synthase activity and CYP11B2 mRNA levels. CYP11B1 and CYP11B2 gene products thus appear as the major steroidogenic enzymes down-regulated by TGFbeta1 in the human adrenocortical tumor cell line NCI-H295R.

Thrombospondins and tumor angiogenesis.

The thrombospondins (TSPs) are a family of five secreted proteins that are widely distributed in the extracellular matrix of numerous tissues. TSPs are multimodular and each domain specifies a distinct biological function through interaction with a specific receptor. TSP1 and TSP2 have anti-angiogenic activity, which, at least for TSP1, involves interaction with the microvascular endothelial cell receptor CD36. Expression of TSP1 and TSP2 is modulated by hypoxia and by oncogenes. In several tumors (thyroid, colon, bladder carcinomas), TSP1 expression is inversely correlated with tumor grade and survival rate, whereas in others (e.g. breast carcinomas), it is correlated with the stromal response and is of little prognostic value. Recent studies suggest that TSPs or TSP-derived peptides retaining biological activity could be developed into promising new therapeutic strategies for the anti-angiogenic treatment of solid tumors.

ACTH-regulated expression of vascular endothelial growth factor in the adult bovine adrenal cortex: a possible role in the maintenance of the microvasculature.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

[Molecular bases of angiogenesis]. / Bases moléculaires de l'angiogenèse.

Angiogenesis is a multistep process that drives the formation of new blood capillaries from the pre-existing vasculature. It is usual to distinguish two phases in this process: a morphogenic phase that requires endothelial cell proliferation and migration followed by a maturation phase characterized by the reconstitution of extracellular matrix and the recruitment of pericytes around the newly formed microvessel. Angiogenesis is actively involved in embryonic organogenesis as well as in cancer tumor growth. Although the properties of neovessels formed under these two situations are different, the molecules and the mechanisms implicated are common. VEGF and its receptors flk-1 and flt-1 are the main actors of the morphogenic phase whereas PDGF-BB and its receptor PDGF-R beta, TGF beta 1 and its receptors ALK-5 and ALK-1, angiopoietin-1 and its receptor tie-2 are implied in the vascular maturation phase.

Fibroblast growth factor-2 inhibits the maturation of pro-insulin-like growth factor-II (Pro-IGF-II) and the expression of insulin-like growth factor binding protein-2 (IGFBP-2) in the human adrenocortical tumor cell line NCI-H295R.

The IGF system is thought to play a major role in adrenocortical tumorigenesis. In this study, we used the NCI H295R cell line as a model to investigate the effects of fibroblast growth factor-2 (FGF-2), a potent mitogen for normal adrenal cells, on the proliferation and on the expression of the IGF system in cultured adrenocortical tumor cells. Three immunoreactive FGF-2 isoforms of molecular masses 18, 22, and 24 kDa were detected in H295R cell extracts. Recombinant human FGF-2 stimulated the proliferation of adrenocortical tumor cells in a dose- and time-dependent manner, with a maximal effect at a concentration of about 1 ng/ml. Treatment of H295R cells with 10 ng/ml FGF-2 for 7 days had no significant effect on IGF-II messenger RNA levels. However, a marked increase in levels of intracellular IGF-II protein was detected by immunoblotting. In contrast, FGF-2 induced a marked decrease in the amount of IGF-II protein secreted, with the disappearance of mature IGF-II and secretion of higher molecular forms of the growth factor, suggesting modifications of IGF-II processing. Cell cultures in the presence of brefeldin A (1 microg/ml), a specific inhibitor of protein secretion, suggested that FGF-2 did not increase IGF-II synthesis but instead inhibited the secretion of pro-IGF-II from H295R cells, thereby impairing the final steps of IGF-II processing to the mature 7.5-kDa peptide. At the same concentrations, FGF-2 also decreased both IGFBP-2 messenger RNA and secreted protein, which might increase IGF-II bioavailability. No proteolysis of IGFBP-2 was detected in FGF-2-conditioned medium. Altogether, these results indicate that FGF-2 is mitogenic for NCI H295R tumor cells and regulates the expression of both IGF-II and IGFBP-2 in this tumor model. Moreover, this study shows a novel effect of FGF-2, by which this growth factor modulates the processing of pro-IGF-II.

Thrombospondin-1 is downregulated by anoxia and suppresses tumorigenicity of human glioblastoma cells.

Angiogenesis, the sprouting of new capillaries from preexisting blood vessels, results from a disruption of the balance between stimulatory and inhibitory factors. Here, we show that anoxia reduces expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis, in glioblastoma cells. This suggests that reduced oxygen tension can promote angiogenesis not only by stimulating the production of inducers, such as vascular endothelial growth factor, but also by reducing the production of inhibitors. This downregulation may significantly contribute to glioblastoma development, since we show that an increase in TSP-1 expression is sufficient to strongly suppress glioblastoma cell tumorigenicity in vivo.

Expression of the angiogenesis markers vascular endothelial growth factor-A, thrombospondin-1, and platelet-derived endothelial cell growth factor in human sporadic adrenocortical tumors: correlation with genotypic alterations.

Several studies have supported the hypothesis that adrenocortical tumor formation is the result of a multistep process. The angiogenic switch has been proposed to be a key step in tumor progression from adenoma to carcinoma. In this study we measured the cytosolic concentrations of three proteins involved in angiogenesis [namely platelet-derived endothelial cell growth factor vascular endothelial cell growth factor A (VEGF-A), and thrombospondin-1 (TSP1)] in a series of 43 human sporadic adrenocortical tumors. The tumors were classified as adenomas (n = 18), transitional tumors (n = 12), or carcinomas (n = 13) according to the histological criteria defined by Weiss. Platelet-derived endothelial cell growth factor/thymidine phosphorylase levels were not significantly different among these three groups. One hundred percent of the adenomas and 73% of the transitional tumors showed VEGF-A concentrations under the threshold value of 107 ng/g protein, whereas 75% of the carcinomas had VEGF-A concentrations above this threshold value. Similarly, 89% of the adenomas showed TSP1 concentrations above the threshold value of 57 microg/g protein, whereas only 25% of the carcinomas and 33% of the transitional tumor samples did so. Insulin-like growth factor II overexpression, a common genetic alteration of adrenocortical carcinomas, was significantly correlated with higher VEGF-A and lower TSP1 concentrations. The tumors from the 6 patients with tumor recurrence after surgical ablation showed significantly higher VEGF-A values than the carcinomas and the transitional tumors from patients that did not relapse. Taken together, these data suggest that a decrease in TSP1 expression is an event that precedes an increase in VEGF-A expression during adrenocortical tumor progression. The population of premalignant tumors with low TSP1 and normal VEGF-A levels could represent a selective target for antiangiogenic therapies.

Transcriptional regulation of the gene encoding the StAR protein in the human adrenocortical cell line, H295R by cAMP and TGFbeta1.

In the present work, we have analyzed the transcriptional regulation of StAR expression in the human adrenocortical cell line H295R. We observed that StAR mRNA levels rapidly increased in response to forskolin, starting after 2 h of treatment and reaching a maximum by 12 h. Deletion analysis of the human StAR promoter demonstrated that the first 150 bp upstream of the transcription start were sufficient for both basal and cAMP-induced expression of a reporter gene. We demonstrate that out of the three binding sequences for the steroidogenic factor 1 (SF-1) only the central one (-105 to -96) is implicated in both basal and cAMP-induced StAR expression. In addition, another important regulatory element for both basal and cAMP-induced StAR expression is present between -62 and -24 upstream of the transcription start. We also show that TGF1beta1 inhibits StAR expression at the transcriptional level. We found that the TGFbeta-inhibitory element lies between -150 and -85 upstream of the transcription start and that the SF-1 binding sites are not implicated in TGFbeta1 regulation.