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SummaryBackgroundPancreatic ductal adenocarcinoma (PDAC) is categorized as the seventh leading cause of cancer mortality worldwide. Its predictive markers for long-term survival are not well known. Therefore, it is interesting to delineate individual-specific perturbed genes when comparing long-term (LT) and short-term (ST) PDAC survivors, and to exploit the integrative individual- and group-based transcriptome profiling.MethodUsing a discovery cohort of 19 PDAC patients from CHU-Liège (Belgium), we first performed differential gene expression (DGE) analysis comparing LT to ST survivor. Second, we adopted unsupervised systems biology approaches to obtain gene modules linked to clinical features. Third, we created individual-specific perturbation profiles and identified key regulators across the LT patients. Furthermore, we applied two gene prioritization approaches: random walk-based Degree-Aware disease gene prioritizing (DADA) method to develop PDAC disease modules; Network-based Integration of Multi-omics Data (NetICS) to integrate group-based and individual-specific perturbed genes in relation to PDAC LT survival.FindingsWe identified 173 differentially expressed genes (DEGs) in ST and LT survivors and five modules (including 38 DEGs) showing associations to clinical traits such as tumor size and chemotherapy. DGE analysis identified differences in genes involved in metabolic and cell cycle activity. Validation of DEGs in the molecular lab suggested a role of REG4 and TSPAN8 in PDAC survival. Individual-specific omics changes across LT survivors revealed biological signatures such as focal adhesion and extracellular matrix receptors, implying a potential role in molecular-level heterogeneity of LT PDAC survivors. Via NetICS and DADA we not only identified various known oncogenes such as CUL1, SCF62, EGF, FOSL1, MMP9, and TGFB1, but also highlighted novel genes (TAC1, KCNH7, IRS4, DKK4).InterpretationOur proposed analytic workflow shows the advantages of combining clinical and omics data as well as individual- and group-level transcriptome profiling. It suggested novel potential transcriptome marks of LT survival heterogeneity in PDAC.FundingTélévie-FRS-FNRS

IntroductionIdiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology and cure. Recent studies have reported a dysregulation of exosomal microRNAs (miRs) in the IPF context. However, the impact of IPF-related exosomal miRs on the progression of pulmonary fibrosis is unknown.MethodsTwo independent cohorts were enrolled at the ambulatory care polyclinic of Liège University. Exosomes from sputum were obtained from 19 patients with IPF and 23 healthy subjects (HSs) (cohort 1), and the ones from plasma derived from 14 patients with IPF and 14 HSs (cohort 2). Exosomal miR expression was performed by quantitative reverse transcription–PCR. The functional role of exosomal miRs was assessed in vitro by transfecting miR mimics in human alveolar epithelial cells and lung fibroblasts.ResultsExosomal miR analysis showed that miR-142-3p was significantly upregulated in sputum and plasma of patients with IPF (8.06-fold, pDiscussionOur results suggest that macrophage-derived exosomes may fight against pulmonary fibrosis progression via the delivery of antifibrotic miR-142–3 p to alveolar epithelial cells and lung fibroblasts.

SSc is a rare disease of unknown origin associated with multiple organ involvement. One of the major complications that drives the mortality of SSc patients is interstitial lung disease. The course of SSc-interstitial lung disease progression has a wide spectrum. Since the treatment is based on aggressive immunosuppression it should not be given to stable or non-progressing disease. The correct identification of disease with high risk of progression remains a challenge for early therapeutic intervention, and biomarkers remain urgently needed. In fact, eight categories of biomarkers have been identified and classified according to the different biological pathways involved. The purpose of this article is to describe the main biomarkers thought to be of interest with clinical value in the diagnosis and prognosis of SSc-interstitial lung disease.

Background Extracellular vesicles (EVs) are released by nearly every cell type and have attracted much attention for their ability to transfer protein and diverse RNA species from donor to recipient cells. Much attention has been given so far to the features of EV short RNAs such as miRNAs. However, while the presence of mRNA and long noncoding RNA (lncRNA) transcripts in EVs has also been reported by multiple different groups, the properties and function of these longer transcripts have been less thoroughly explored than EV miRNA. Additionally, the impact of EV export on the transcriptome of exporting cells has remained almost completely unexamined. Here, we globally investigate mRNA and lncRNA transcripts in endothelial EVs in multiple different conditions. Results In basal conditions, long RNA transcripts enriched in EVs have longer than average half-lives and distinctive stability-related sequence and structure characteristics including shorter transcript length, higher exon density, and fewer 3′ UTR A/U-rich elements. EV-enriched long RNA transcripts are also enriched in HNRNPA2B1 binding motifs and are impacted by HNRNPA2B1 depletion, implicating this RNA-binding protein in the sorting of long RNA to EVs. After signaling-dependent modification of the cellular transcriptome, we observed that, unexpectedly, the rate of EV enrichment relative to cells was altered for many mRNA and lncRNA transcripts. This change in EV enrichment was negatively correlated with intracellular abundance, with transcripts whose export to EVs increased showing decreased abundance in cells and vice versa. Correspondingly, after treatment with inhibitors of EV secretion, levels of mRNA and lncRNA transcripts that are normally highly exported to EVs increased in cells, indicating a measurable impact of EV export on the long RNA transcriptome of the exporting cells. Compounds with different mechanisms of inhibition of EV secretion affected the cellular transcriptome differently, suggesting the existence of multiple EV subtypes with different long RNA profiles. Conclusions We present evidence for an impact of EV physiology on the characteristics of EV-producing cell transcriptomes. Our work suggests a new paradigm in which the sorting and packaging of transcripts into EVs participate, together with transcription and RNA decay, in controlling RNA homeostasis and shape the cellular long RNA abundance profile.

The chemotherapeutic drug epirubicin increases the exosomal export of miR-503 in endothelial cells. To understand the mechanisms behind this process, we transfected endothelial cells with miR-503 carrying a biotin tag. Then, we pulled-down the proteins interacting with miR-503 and studied their role in microRNA exosomal export. A total of four different binding partners were identified by mass spectrometry and validated by western blotting and negative controls, among them ANXA2 and hnRNPA2B1. Using knock-down systems combined with pull-down analysis, we determined that epirubicin mediates the export of miR-503 by disrupting the interaction between hnRNPA2B1 and miR-503. Then, both ANXA2 and miR-503 are sorted into exosomes while hnRNPA2B1 is relocated into the nucleus. The combination of these processes culminates in the increased export of miR-503. These results suggest, for the first time, that RNA-binding proteins can negatively regulate the exosomal sorting of microRNAs.

Background: IPF is a progressive fibrosing interstitial lung disease of unknown etiology and cure. Recent studies have reported a dysregulation of exosomal microRNAs (miRs) in IPF. However, the impact of IPF-related exosomal miRs on the progression of pulmonary fibrosis is unknown. Methods: Two independent cohorts were enrolled at the ambulatory care polyclinic of Liege University. Exosomes from sputum were obtained from 16 IPF patients and 14 healthy subjects (HS) (cohort 1), and the ones from plasma derived from 9 IPF patients and 9 HS (cohort 2). Exosomal miR expression was performed by quantitative RT-PCR. The functional role of exosomal miRs was assessed in vitro by transfecting miR mimics in human alveolar epithelial cells and lung fibroblasts. Results: Exosomal miR analysis showed that miR-142-3p was significantly upregulated in sputum and plasma of IPF patients. Correlation analysis revealed that the expression of sputum exosomal miR-142-3p was positively associated with sputum macrophages of IPF patients (r = 0.585; p=0.028), suggesting the macrophage origin of this miR. Overexpression of miR-142-3p in A549 alveolar epithelial cells and MRC5 lung fibroblasts revealed that this miR downregulates the expression of Transforming Growth Factor β Receptor 1 (TGFβ-R1) and pro-fibrotic genes. Furthermore, exosomes isolated from THP-1 macrophages also present anti-fibrotic properties due in part to the transfer of miR-142-3p to alveolar epithelial cells and lung fibroblasts. Conclusion: Our results suggest that macrophage-derived exosomes may exert protective effect against pulmonary fibrosis progression via the delivery of anti-fibrotic miR-142-3p.

Exosomes are small extracellular vesicles of around 100 nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids, and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes are still not completely elucidated. In this study, by the use of next-generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and noncoding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis for studying the involvement of RNA-binding proteins capable of recognizing RNA sequences and their role in the export of RNAs into exosomes.

Increasing evidence suggests the potential role of extracellular vesicles (EVs) in many lung diseases. According to their subcellular origin, secretion mechanism, and size, EVs are currently classified into three subpopulations: exosomes, microvesicles, and apoptotic bodies. Exosomes are released in most biofluids, including airway fluids, and play a key role in intercellular communication via the delivery of their cargo (e.g., microRNAs (miRNAs)) to target cell. In a physiological context, lung exosomes present protective effects against stress signals which allow them to participate in the maintenance of lung homeostasis. The presence of air pollution alters the composition of lung exosomes (dysregulation of exosomal miRNAs) and their homeostatic property. Indeed, besides their potential as diagnostic biomarkers for lung diseases, lung exosomes are functional units capable of dysregulating numerous pathophysiological processes (including inflammation or fibrosis), resulting in the promotion of lung disease progression. Here, we review recent studies on the known and potential role of lung exosomes/exosomal miRNAs, in the maintaining of lung homeostasis on one hand, and in promoting lung disease progression on the other. We will also discuss using exosomes as prognostic/diagnostic biomarkers as well as therapeutic tools for lung diseases.

Epithelial–mesenchymal transitions (EMTs) are high-profile in the field of circulating tumor cells (CTCs). EMT-shifted CTCs are considered to encompass pre-metastatic subpopulations though underlying molecular mechanisms remain elusive. Our previous work identified tissue factor (TF) as an EMT-induced gene providing tumor cells with coagulant properties and supporting metastatic colonization by CTCs. We here report that vimentin, the type III intermediate filament considered a canonical EMT marker, contributes to TF regulation and positively supports coagulant properties and early metastasis. Different evidence further pointed to a new post-transcriptional regulatory mechanism of TF mRNA by vimentin: (1) vimentin silencing accelerated TF mRNA decay after actinomycin D treatment, reflecting TF mRNA stabilization, (2) RNA immunoprecipitation revealed enriched levels of TF mRNA in vimentin immunoprecipitate, (3) TF 3′-UTR-luciferase reporter vector assays implicated the 3′-UTR of TF mRNA in vimentin-dependent TF regulation, and (4) using different TF 3′UTR-luciferase reporter vectors mutated for potential miR binding sites and specific Target Site Blockers identified a key miR binding site in vimentin-dependent TF mRNA regulation. All together, these data support a novel mechanism by which vimentin interferes with a miR-dependent negative regulation of TF mRNA, thereby promoting coagulant activity and early metastasis of vimentin-expressing CTCs.

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrosing interstitial lung disease of unknown aetiology which leads rapidly to death. As diagnosis of IPF is complex, we aimed to characterise microRNA (miRNA) content of exosomes from sputum of patients with IPF. Using miRNA quantitative PCR array, we found a substantial dysregulation of sputum exosomal miRNA levels between patients with IPF and healthy subjects and identified a unique signature of three miRNAs. Interestingly, we found a negative correlation between miR-142-3p and diffusing capacity of the lungs for carbon monoxide/alveolar volume. This is the first characterisation of miRNA content of sputum-derived exosomes in IPF that identified promising biomarkers for diagnosis and disease severity.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive, fatal lung disorder of unknown origin with a highly variable and unpredictable clinical course. Polymorphisms and environmentally induced epigenetic variations seem to determine individual susceptibility to the development of lung fibrosis. Methods: We have studied circulating epitopes on cell-free nucleosomes (cfnucleosomes) in 50 IPF patients. We have compared untreated IPF (n=23) with IPF receiving antifibrotic therapy (n=27) and healthy subjects (HS) (n=27). We analyzed serum levels of five cfnucleosomes including bound HMGB1 (nucleosomes adducted to high-mobility growth protein B1), mH2A1.1 (nucleosomes containing the histone variant mH2A1.1), 5mC (nucleosomes associated with methylated DNA), and H3K9Ac and H3K27Ac (nucleosomes associated with histone H3 acetylated at lysine 9 or 27 residue). Results: Our findings showed that serum levels of bound HMGB1, mH2A1.1, 5mC, H3K9Ac, and H3K27Ac were significantly lower in IPF patients than in HS (p, SCOPUS: ar.j, info:eu-repo/semantics/published

Circulating microRNAs (miRNAs) have been extensively studied in cancer as biomarkers but little is known regarding the influence of anti-cancer drugs on their expression levels. In this article, we describe the modifications of circulating miRNAs profile after neoadjuvant chemotherapy (NAC) for breast cancer. The expression of 188 circulating miRNAs was assessed in the plasma of 25 patients before and after NAC by RT-qPCR. Two miRNAs, miR-34a and miR-122, that were significantly increased after NAC, were measured in tumor tissue before and after chemotherapy in 7 patients with pathological partial response (pPR) to NAC. These two chemotherapy-induced miRNAs were further studied in the plasma of 22 patients with adjuvant chemotherapy (AC) as well as in 12 patients who did not receive any chemotherapy. Twenty-five plasma miRNAs were modified by NAC. Among these miRNAs, miR-34a and miR-122 were highly upregulated, notably in pPR patients with aggressive breast cancer. Furthermore, miR-34a level was elevated in the remaining tumor tissue after NAC treatment. Studying the kinetics of circulating miR-34a and miR-122 expression during NAC revealed that their levels were especially increased after anthracycline-based chemotherapy. Comparisons of the plasma miRNA profiles after NAC and AC suggested that chemotherapy-induced miRNAs originated from both tumoral and non-tumoral compartments. This study is the first to demonstrate that NAC specifically induces miRNA expression in plasma and tumor tissue, which might be involved in the anti-tumor effects of chemotherapy in breast cancer patients. J. Cell. Physiol. 230: 473–481, 2015. © 2014 Wiley Periodicals, Inc.

Peripartum cardiomyopathy (PPCM) is a life-threatening pregnancy-associated cardiomyopathy in previously healthy women. Although PPCM is driven in part by the 16-kDa N-terminal prolactin fragment (16K PRL), the underlying molecular mechanisms are poorly understood. We found that 16K PRL induced microRNA-146a (miR-146a) expression in ECs, which attenuated angiogenesis through downregulation of NRAS. 16K PRL stimulated the release of miR-146a-loaded exosomes from ECs. The exosomes were absorbed by cardiomyocytes, increasing miR-146a levels, which resulted in a subsequent decrease in metabolic activity and decreased expression of Erbb4, Notch1, and Irak1. Mice with cardiomyocyte-restricted Stat3 knockout (CKO mice) exhibited a PPCM-like phenotype and displayed increased cardiac miR-146a expression with coincident downregulation of Erbb4, Nras, Notch1, and Irak1. Blocking miR-146a with locked nucleic acids or antago-miRs attenuated PPCM in CKO mice without interrupting full-length prolactin signaling, as indicated by normal nursing activities. Finally, miR-146a was elevated in the plasma and hearts of PPCM patients, but not in patients with dilated cardiomyopathy. These results demonstrate that miR-146a is a downstream-mediator of 16K PRL that could potentially serve as a biomarker and therapeutic target for PPCM.

Control of mRNA levels, a fundamental aspect in the regulation of gene expression, is achieved through a balance between mRNA synthesis and decay. E26-related gene (Erg) proteins are canonical transcription factors whose previously described functions are confined to the control of mRNA synthesis. Here, we report that ERG also regulates gene expression by affecting mRNA stability and identify the molecular mechanisms underlying this function in human cells. ERG is recruited to mRNAs via interaction with the RNA-binding protein RBPMS, and it promotes mRNA decay by binding CNOT2, a component of the CCR4-NOT deadenylation complex. Transcriptome-wide mRNA stability analysis revealed that ERG controls the degradation of a subset of mRNAs highly connected to Aurora signaling, whose decay during S phase is necessary for mitotic progression. Our data indicate that control of gene expression by mammalian transcription factors may follow a more complex scheme than previously anticipated, integrating mRNA synthesis and degradation.

An error in the orientation of the proteins in an intracellular vesicle is corrected.

The 16-kDa angiostatic N-terminal fragment of human prolactin (16K hPRL) has been reported to be a new potent anticancer compound. This protein has already proven its efficiency in several mouse tumor models in which it prevented tumor-induced angiogenesis and delayed tumor growth. In addition to angiogenesis, tumors also stimulate the formation of lymphatic vessels, which contribute to tumor cell dissemination and metastasis. However, the role of 16K hPRL in tumor-induced lymphangiogenesis has never been investigated. We establish in vitro that 16K hPRL induces apoptosis and inhibits proliferation, migration, and tube formation of human dermal lymphatic microvascular endothelial cells. In addition, in a B16F10 melanoma mouse model, we found a decreased number of lymphatic vessels in the primary tumor and in the sentinel lymph nodes after 16K hPRL treatment. This decrease is accompanied by a significant diminished expression of lymphangiogenic markers in primary tumors and sentinel lymph nodes as determined by quantitative RT-PCR. These results suggest, for the first time, that 16K hPRL is a lymphangiostatic as well as an angiostatic agent with antitumor properties.

Sox7 and Sox18 are members of the F-subgroup of Sox transcription factors family and are mostly expressed in endothelial compartments. In humans, dominant mutations in Sox18 are the underlying cause of the severe hypotrichosis–lymphedema–telangiectasia disorder characterized by vascular defects. However little is known about which vasculogenic processes Sox7 and Sox18 regulate in vivo. We cloned the orthologs of Sox7 and Sox18 in zebrafish, analysed their expression pattern and performed functional analyses. Both genes are expressed in the lateral plate mesoderm during somitogenesis. At later stages, Sox18 is expressed in all axial vessels whereas Sox7 expression is mainly restricted to the dorsal aorta. Knockdown of Sox7 or Sox18 alone failed to reveal any phenotype. In contrast, blocking the two genes simultaneously led to embryos displaying dysmorphogenesis of the proximal aorta and arteriovenous shunts, all of which can account for the lack of circulation observed in the trunk and tail. Gene expression analyses performed with general endothelial markers on double morphants revealed that Sox7 and Sox18 are dispensable for the initial specification and positioning of the major trunk vessels. However, morphants display ectopic expression of the venous Flt4 marker in the dorsal aorta and a concomitant reduction of the artery-specific markers EphrinB2a and Gridlock. The striking similarities between the phenotype of Sox7/Sox18 morphants and Gridlock mutants strongly suggest that Sox7 and Sox18 control arterial–venous identity by regulating Gridlock expression.

Tumor metastases, the most fearsome aspect of cancer, are generally resistant to conventional therapies. Angiogenesis is a crucial aspect of tumor growth and metastatic dissemination. Antiangiogenic therapy, therefore, holds potential as an attractive strategy for inhibiting metastasis development. Human 16K PRL (16K hPRL), a potent inhibitor of angiogenesis, has been demonstrated to prevent tumor growth in two xenograft mouse models, but whether it also affects tumor metastasis is unknown. In this study we will investigate the ability of 16K hPRL to prevent the establishment of metastasis. We demonstrate that 16K hPRL administered via adenovirus-mediated gene transfer, inhibits tumor growth by 86% in a subcutaneous (SC) B16-F10 mouse melanoma model. Computer-assisted image analysis shows that 16K hPRL treatment results in a reduction of tumor-vessel length and width, leading to a 57% reduction of average vessel size. In a pre-established tumor model, moreover, 16K hPRL can significantly delay tumor development. Finally, for the first time, we provide evidence that 16K hPRL considerably reduces the establishment of B16-F10 metastasis in an experimental lung metastasis model. Both the number and size of metastases are reduced by 50% in 16K hPRL-treated mice. These results highlight a potential role for 16K hPRL in anticancer therapy for both primary tumors and metastases.

In the corpus luteum (CL), blood vessels develop, stabilize, and regress. This process depends on the ratio of pro- and antiangiogenic factors, which change during the ovarian cycle. The present study focuses on the possible roles of 23,000 (23K) prolactin (PRL) in the bovine CL and its antiangiogenic NH2-terminal fragments after extracellular cleavage by cathepsin D (Cath D). PRL RNA and protein were demonstrated in the CL tissue, in luteal endothelial cells, and in steroidogenic cells. Cath D was detected in CL tissue, cell extracts, and corresponding cell supernatants. In the intact CL, 23K PRL levels decreased gradually, whereas Cath D levels concomitantly increased between early and late luteal stages. In vitro, PRL cleavage occurred in the presence of acidified homogenates of CL tissue, cells, and corresponding cell supernatants. Similar fragments were obtained with purified Cath D, and their appearance was inhibited by pepstatin A. The aspartic protease specific substrate MOCAc-GKPILF∼FRLK(Dnp)-d- R-NH2was cleaved by CL cell supernatants, providing further evidence for Cath D activity. The 16,000 PRL inhibited proliferation of luteal endothelial cells accompanied by an increase in cleaved caspase-3. In conclusion, 1) the bovine CL is able to produce PRL and to process it into antiangiogenic fragments by Cath D activity and 2) PRL cleavage might mediate angioregression during luteolysis.

In endothelial cells, binding of vascular endothelial growth factor (VEGF) to the receptor VEGFR2 activates multiple signaling pathways that trigger processes such as proliferation, survival, and migration that are necessary for angiogenesis. VEGF-bound VEGFR2 becomes internalized, which is a key step in the proangiogenic signal. We showed that the urokinase plasminogen activator receptor (uPAR) interacted with VEGFR2 and described the mechanism by which this interaction mediated VEGF signaling and promoted angiogenesis. Knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) impaired VEGFR2 signaling, and uPAR deficiency in mice prevented VEGF-induced angiogenesis. Upon exposure of HUVECs to VEGF, uPAR recruited the low-density lipoprotein receptor-related protein 1 (LRP-1) to VEGFR2, which induced VEGFR2 internalization. Thus, the uPAR-VEGFR2 interaction is crucial for VEGF signaling in endothelial cells.

Circulating microRNAs (miRNAs) are increasingly recognized as powerful biomarkers in several pathologies, including breast cancer. Here, their plasmatic levels were measured to be used as an alternative screening procedure to mammography for breast cancer diagnosis. A plasma miRNA profile was determined by RT-qPCR in a cohort of 378 women. A diagnostic model was designed based on the expression of 8 miRNAs measured first in a profiling cohort composed of 41 primary breast cancers and 45 controls, and further validated in diverse cohorts composed of 108 primary breast cancers, 88 controls, 35 breast cancers in remission, 31 metastatic breast cancers and 30 gynecologic tumors. A receiver operating characteristic curve derived from the 8-miRNA random forest based diagnostic tool exhibited an area under the curve of 0.81. The accuracy of the diagnostic tool remained unchanged considering age and tumor stage. The miRNA signature correctly identified patients with metastatic breast cancer. The use of the classification model on cohorts of patients with breast cancers in remission and with gynecologic cancers yielded prediction distributions similar to that of the control group. Using a multivariate supervised learning method and a set of 8 circulating miRNAs, we designed an accurate, minimally invasive screening tool for breast cancer.

The interaction between tumor cells and their microenvironment is an essential aspect of tumor development. Therefore, understanding how this microenvironment communicates with tumor cells is crucial for the development of new anti-cancer therapies. MicroRNAs (miRNAs) are small non-coding RNAs that inhibit gene expression. They are secreted into the extracellular medium in vesicles called exosomes, which allow communication between cells via the transfer of their cargo. Consequently, we hypothesized that circulating endothelial miRNAs could be transferred to tumor cells and modify their phenotype. Using exogenous miRNA, we demonstrated that endothelial cells can transfer miRNA to tumor cells via exosomes. Using miRNA profiling, we identified miR-503, which exhibited downregulated levels in exosomes released from endothelial cells cultured under tumoral conditions. The modulation of miR-503 in breast cancer cells altered their proliferative and invasive capacities. We then identified two targets of miR-503, CCND2 and CCND3. Moreover, we measured increased plasmatic miR-503 in breast cancer patients after neoadjuvant chemotherapy, which could be partly due to increased miRNA secretion by endothelial cells. Taken together, our data are the first to reveal the involvement of the endothelium in the modulation of tumor development via the secretion of circulating miR-503 in response to chemotherapy treatment.

Angiogenesis is a crucial step in many pathologies, including tumor growth and metastasis. Here, we show that tilted peptides exert antiangiogenic activity. Tilted (or oblique-oriented) peptides are short peptides known to destabilize membranes and lipid cores and characterized by an asymmetric distribution of hydrophobic residues along the axis when helical. We have previously shown that 16-kDa fragments of the human prolactin/growth hormone (PRL/GH) family members are potent angiogenesis inhibitors. Here, we demonstrate that all these fragments possess a 14-aa sequence having the characteristics of a tilted peptide. The tilted peptides of human prolactin and human growth hormone induce endothelial cell apoptosis, inhibit endothelial cell proliferation, and inhibit capillary formation both in vitro and in vivo . These antiangiogenic effects are abolished when the peptides' hydrophobicity gradient is altered by mutation. We further demonstrate that the well known tilted peptides of simian immunodeficiency virus gp32 and Alzheimer's β-amyloid peptide are also angiogenesis inhibitors. Taken together, these results point to a potential new role for tilted peptides in regulating angiogenesis.

The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent antiangiogenic factor that has been shown to prevent tumor growth in a xenograph mouse model. In this paper we first demonstrate that 16K hPRL inhibits serum-induced DNA synthesis in adult bovine aortic endothelial cells. This inhibition is associated with cell cycle arrest at both the G0–G1 and the G2–M phase. Western blot analysis revealed that 16K hPRL strongly decreases levels of cyclin D1 and cyclin B1, but not cyclin E. The effect on cyclin D1 is at least partially transcriptional, because treatment with 16K hPRL both reduces the cyclin D1 mRNA level and down-regulates cyclin D1 promoter activity. This regulation may be due to inhibition of the MAPK pathway, but it is independent of the glycogen synthase kinase-3β pathway. Lastly, 16K hPRL induces the expression of negative cell cycle regulators, the cyclin-dependent kinase inhibitors p21(cip1) and p27(kip1). In summary, 16K hPRL inhibits serum-induced proliferation of endothelial cells through combined effects on positive and negative regulators of cell cycle progression.

We have previously shown that the 16-kDa N-terminal fragment of human prolactin (16K hPRL) has antiangiogenic properties, including the ability to induce apoptosis in vascular endothelial cells. Here, we examined whether the nuclear factor-kappaB (NF-kappaB) signaling pathway was involved in mediating the apoptotic action of 16K hPRL in bovine adrenal cortex capillary endothelial cells. In a dose-dependent manner, treatment with 16K hPRL induced inhibitor kappaB-alpha degradation permitting translocation of NF-kappaB to the nucleus and reporter gene activation. Inhibition of NF-kappaB activation by overexpression of a nondegradable inhibitor kappaB-alpha mutant or treatment with NF-kappaB inhibitors blocked 16K hPRL-induced apoptosis. Treatment with 16K hPRL activated the initiator caspases-8 and -9 and the effector caspase-3, all of which were essential for stimulation of DNA fragmentation. This activation of the caspase cascade by 16K hPRL was also NF-kappaB dependent. These findings support the conclusion that NF-kappaB signaling plays a central role in 16K hPRL-induced apoptosis in vascular endothelial cells.

Circulating microRNAs (miRNAs) have been extensively studied in cancer as biomarkers but little is known regarding the influence of anti-cancer drugs on their expression levels. In this article, we describe the modifications of circulating miRNAs profile after neoadjuvant chemotherapy (NAC) for breast cancer. The expression of 188 circulating miRNAs was assessed in the plasma of 25 patients before and after NAC by RT-qPCR. Two miRNAs, miR-34a and miR-122, that were significantly increased after NAC, were measured in tumor tissue before and after chemotherapy in 7 patients with pathological partial response (pPR) to NAC. These two chemotherapy-induced miRNAs were further studied in the plasma of 22 patients with adjuvant chemotherapy (AC) as well as in 12 patients who did not receive any chemotherapy. Twenty-five plasma miRNAs were modified by NAC. Among these miRNAs, miR-34a and miR-122 were highly upregulated, notably in pPR patients with aggressive breast cancer. Furthermore, miR-34a level was elevated in the remaining tumor tissue after NAC treatment. Studying the kinetics of circulating miR-34a and miR-122 expression during NAC revealed that their levels were especially increased after anthracycline-based chemotherapy. Comparisons of the plasma miRNA profiles after NAC and AC suggested that chemotherapy-induced miRNAs originated from both tumoral and non-tumoral compartments. This study is the first to demonstrate that NAC specifically induces miRNA expression in plasma and tumor tissue, which might be involved in the anti-tumor effects of chemotherapy in breast cancer patients.

et al., Bone metastasis represents one of the most deleterious clinical consequences arising in the context of many solid tumors. Severe osteolysis results from tumor cell colonization of the bone compartment, a process which entails reciprocal exchange of soluble signals between tumor cells and their osseous microenvironment. Recent evidence indicates that tumor-intrinsic miRNAs are pleiotropic regulators of gene expression. But they are also frequently released in exosome-like vesicles (ELV). Yet the functional relevance of the transference of tumor-derived ELV and their miRNA cargo to the extracellular milieu during osseous colonization is unknown. Comparative transcriptomic profiling using an in vivo murine model of bone metastasis identified a repressed miRNA signature associated with high prometastatic activity. Forced expression of single miRNAs identified miR-192 that markedly appeased osseous metastasis in vivo, as shown by X-ray, bioluminescence imaging and microCT scans. Histological examination of metastatic lesions revealed impaired tumor-induced angiogenesis in vivo, an effect that was associated in vitro with decreased hallmarks of angiogenesis. Isolation and characterization of ELV by flow cytometry, Western blot analysis, transmission electron microscopy and nanoparticle tracking analysis revealed the ELV cargo enrichment in miR-192. Consistent with these findings, fluorescent labeled miR-192-enriched-ELV showed the in vitro transfer and release of miR-192 in target endothelial cells and abrogation of the angiogenic program by repression of proangiogenic IL-8, ICAM and CXCL1. Moreover, in vivo infusion of fluorescent labeled ELV efficiently targeted cells of the osseous compartment. Furthermore, treatment with miR-192 enriched ELV in a model of in vivo bone metastasis pre-conditioned osseous milieu and impaired tumor-induced angiogenesis, thereby reducing the metastatic burden and tumor colonization. Changes in the miRNA-cargo content within ELV represent a novel mechanism heavily influencing bone metastatic colonization, which is most likely relevant in other target organs. Mechanistic mimicry of this phenomenon by synthetic nanoparticles could eventually emerge as a novel therapeutic approach., This work was supported by “UTE project FIMA” agreement, the Cancer Research Thematic Network of the Health Institute Carlos III (RTICC RD12/0036/0066), European Regional Development Fund (ERDF) “Una manera de hacer Europa”, SAF-2009-11280, SAF2012-40056, the Government of Navarra (09/2009) awards to F.L.. K.V. and D.L-R were funded by the FIMA and FPU. K.V. was the recipient of a Young Investigator Award at the ASBMR 2012. I.A. was funded by the Basque Government. F.L. is an investigator from the I3 Program.

The N-terminal fragment of prolactin (16K PRL) inhibits tumor growth by impairing angiogenesis, but the underlying mechanisms are unknown. Here, we found that 16K PRL binds the fibrinolytic inhibitor plasminogen activator inhibitor-1 (PAI-1), which is known to contextually promote tumor angiogenesis and growth. Loss of PAI-1 abrogated the antitumoral and antiangiogenic effects of 16K PRL. PAI-1 bound the ternary complex PAI-1-urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR), thereby exerting antiangiogenic effects. By inhibiting the antifibrinolytic activity of PAI-1, 16K PRL also protected mice against thromboembolism and promoted arterial clot lysis. Thus, by signaling through the PAI-1-uPA-uPAR complex, 16K PRL impairs tumor vascularization and growth and, by inhibiting the antifibrinolytic activity of PAI-1, promotes thrombolysis.

The N-terminal fragment of PRL (16K PRL) is an antiangiogenic factor that, in vitro, inhibits several components of angiogenesis including basic fibroblast growth factor (bFGF)-induced cell division, migration, and organization of capillary endothelial cells. An essential step in the regulation of angiogenesis is the activation of urokinase (urokinase type plasminogen activator, uPA), which in turn activates a cascade of proteases that play essential roles in endothelial cell migration and tissue remodeling. Treatment of bovine capillary endothelial cells (BBEC) with 16K PRL inhibited bFGF-stimulated urokinase activity in BBEC as detected by plasminogen substrate gel assay. 16K PRL did not appear to be acting via an effect on uPA expression because no change in messenger RNA levels were observed. However, protein levels of plasminogen activator inhibitor-1 (PAI-1), a specific inhibitor of urokinase, were increased by 16K PRL independent of the action of bFGF. The 16K PRL-induced increase in PAI-1 protein levels appear to be the result of increased expression of the PAI-1 gene. Increased production of PAI-1 induced by 16K PRL results in the formation of inactive PAI-1/uPA complexes, consistent with the observed decrease in uPA activity.

The mouse model of laser-induced choroidal neovascularization (CNV) has been used extensively in studies of the exudative form of age-related macular degeneration (AMD). This experimental in vivo model relies on laser injury to perforate Bruch's membrane, resulting in subretinal blood vessel recruitment from the choroid. By recapitulating the main features of the exudative form of human AMD, this assay has served as the backbone for testing antiangiogenic therapies. This standardized protocol can be applied to transgenic mice and can include treatments with drugs, recombinant proteins, antibodies, adenoviruses and pre-microRNAs to aid in the search for new molecular regulators and the identification of novel targets for innovative treatments. This robust assay requires 7-14 d to complete, depending on the treatment applied and whether immunostaining is performed. This protocol includes details of how to induce CNV, including laser induction, lesion excision, processing and different approaches to quantify neoformed vasculature.

Peripartum cardiomyopathy (PPCM) is a life-threatening heart disease developing towards the end of pregnancy or in the months following delivery in previously healthy women in terms of cardiac disease. Enhanced oxidative stress and the subsequent cleavage of the nursing hormone Prolactin into an anti-angiogenic 16 kDa subfragment emerged as a potential causal factor of the disease. We established a prospective registry with confirmed PPCM present in 115 patients (mean baseline left ventricular ejection fraction, LVEF: 27 ± 9 %). Follow-up data (6 ± 3 months) showed LVEF improvement in 85 % and full recovery in 47 % while 15 % failed to recover with death in 2 % of patients. A positive family history of cardiomyopathy was present in 16.5 %. Pregnancy-associated hypertension was associated with a better outcome while a baseline LVEF ≤ 25 % was associated with a worse outcome. A high recovery rate (96 %) was observed in patients obtaining combination therapy with beta-blocker, angiotensin-converting enzyme (ACE) inhibitors/angiotensin-receptor-blockers (ARBs) and bromocriptine. Increased serum levels of Cathepsin D, the enzyme that generates 16 kDa Prolactin, miR-146a, a direct target of 16 kDa Prolactin, N-terminal-pro-brain-natriuretic peptide (NT-proBNP) and asymmetric dimethylarginine (ADMA) emerged as biomarkers for PPCM. In conclusion, low baseline LVEF is a predictor for poor outcome while pregnancy-induced hypertensive disorders are associated with a better outcome in this European PPCM cohort. The high recovery rate in this collective is associated with a treatment concept using beta-blockers, ACE inhibitors/ARBs and bromocriptine. Increased levels of Cathepsin D activity, miR-146a and ADMA in serum of PPCM patients support the pathophysiological role of 16 kDa Prolactin for PPCM and may be used as a specific diagnostic marker profile. Electronic supplementary material The online version of this article (doi:10.1007/s00395-013-0366-9) contains supplementary material, which is available to authorized users.

20 p.-6 fig., Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by arteriovenous malformations and hemorrhages. This vascular disease results mainly from mutations in 2 genes involved in the TGF-β pathway (ENG and ALK1) that are exclusively expressed by endothelial cells. The present study identified miR-27a and miR-205 as two circulating miRNAs differentially expressed in HHT patients. The plasma levels of miR-27a are elevated while those of miR-205 are reduced in both HHT1 and HHT2 patients compared to healthy controls. The role of miR-205 in endothelial cells was further investigated. Our data indicates that miR-205 expression displaces the TGF-β balance towards the anti-angiogenic side by targeting Smad1 and Smad4. In line, overexpression of miR-205 in endothelial cells reduces proliferation, migration and tube formation while its inhibition shows opposite effects. This study not only suggests that detection of circulating miRNA (miR-27a and miR-205) could help for the screening of HHT patients but also provides a functional link between the deregulated expression of miR-205 and the HHT phenotype. © 2013 Springer Science+Business Media Dordrecht., This study was supported by the University of Liège (Fonds Speciaux), le centre anti-cancéreux (CAC ULg), the FRIA (Fonds pour la Recherche Industrielle et Agricole, Belgium), the FNRS (Fonds National de la Recherche Scientifique, Belgium), the Neoangio program #616476 of the “Service Public de Wallonie”. This work was supported by the Ministerio de Ciencia e Innovacion: Grants SAF2008–01218, SAF2011-23475, SAF2007–61827, and SAF2010–19222, and Fundación Ramón Areces of Spain (Rare and Emergent Diseases).

Peripartum cardiomyopathy (PPCM) is a potentially life-threatening heart disease emerging toward the end of pregnancy or in the first postpartal months in previously healthy women. Recent data suggest a central role of unbalanced peri-/postpartum oxidative stress that triggers the proteolytic cleavage of the nursing hormone prolactin (PRL) into a potent antiangiogenic, proapoptotic, and proinflammatory 16-kDa PRL fragment. This notion is supported by the observation that inhibition of PRL secretion by bromocriptine, a dopamine D2-receptor agonist, prevented the onset of disease in an animal model of PPCM and by first clinical experiences where bromocriptine seem to exert positive effects with respect to prevention or treatment of PPCM patients. Here, we highlight the current state of knowledge on diagnosis of PPCM, provide insights into the biology and pathophysiology of 16-kDa PRL and bromocriptine, and outline potential consequences for the clinical management and treatment options for PPCM patients.

Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)-2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density, and cross-linking). Transmission electron microscopy and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LECs associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LECs negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis., info:eu-repo/semantics/published

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of plasminogen activators (uPA and tPA) and thus plays a central role in fibrinolysis. The spontaneous insertion of its reactive centre loop (RCL) into β-sheet A is responsible for its irreversible conversion into the inactive latent form. In this study, we used two peptides mimicking residues P14–P9 and P8–P3 of the RCL so as to understand this dynamic process. We show that both peptides inhibit the formation of PAI-1/uPA and PAI-1/tPA complexes via two different mechanisms. Targeting the N-terminal part of the loop induces the cleavage of PAI-1 by the proteases uPA/tPA while targeting its C-terminal part greatly favors the irreversible formation of latent PAI-1. Structured summary of protein interactions PAI-1 and uPA bind by comigration in gel electrophoresis ( View interaction ) tPA and PAI-1 bind by comigration in gel electrophoresis ( View interaction )