Mitochondrial Ribosomal Protein MRPS15 Is a Component of Cytosolic Ribosomes and Regulates Translation in Stressed Cardiomyocytes.

Regulation of mRNA translation is a crucial step in controlling gene expression in stressed cells, impacting many pathologies, including heart ischemia. In recent years, ribosome heterogeneity has emerged as a key control mechanism driving the translation of subsets of mRNAs. In this study, we investigated variations in ribosome composition in human cardiomyocytes subjected to endoplasmic reticulum stress induced by tunicamycin treatment. Our findings demonstrate that this stress inhibits global translation in cardiomyocytes while activating internal ribosome entry site (IRES)-dependent translation. Analysis of translating ribosome composition in stressed and unstressed cardiomyocytes was conducted using mass spectrometry. We observed no significant changes in ribosomal protein composition, but several mitochondrial ribosomal proteins (MRPs) were identified in cytosolic polysomes, showing drastic variations between stressed and unstressed cells. The most notable increase in polysomes of stressed cells was observed in MRPS15. Its interaction with ribosomal proteins was confirmed by proximity ligation assay (PLA) and immunoprecipitation, suggesting its intrinsic role as a ribosomal component during stress. Knock-down or overexpression experiments of MRPS15 revealed its role as an activator of IRES-dependent translation. Furthermore, polysome profiling after immunoprecipitation with anti-MRPS15 antibody revealed that the "MRPS15 ribosome" is specialized in translating mRNAs involved in the unfolded protein response.

Long non-coding RNA Neat1 and paraspeckle components are translational regulators in hypoxia.

Internal ribosome entry sites (IRESs) drive translation initiation during stress. In response to hypoxia, (lymph)angiogenic factors responsible for tissue revascularization in ischemic diseases are induced by the IRES-dependent mechanism. Here, we searched for IRES trans-acting factors (ITAFs) active in early hypoxia in mouse cardiomyocytes. Using knock-down and proteomics approaches, we show a link between a stressed-induced nuclear body, the paraspeckle, and IRES-dependent translation. Furthermore, smiFISH experiments demonstrate the recruitment of IRES-containing mRNA into paraspeckle during hypoxia. Our data reveal that the long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator, active on IRESs of (lymph)angiogenic and cardioprotective factor mRNAs. In addition, paraspeckle proteins p54nrb and PSPC1 as well as nucleolin and RPS2, two p54nrb-interacting proteins identified by mass spectrometry, are ITAFs for IRES subgroups. Paraspeckle thus appears as a platform to recruit IRES-containing mRNAs and possibly host IRESome assembly. Polysome PCR array shows that Neat1 isoforms regulate IRES-dependent translation and, more widely, translation of mRNAs involved in stress response.

Coordinating Effect of VEGFC and Oleic Acid Participates to Tumor Lymphangiogenesis.

In cancer, the lymphatic system is hijacked by tumor cells that escape from primary tumor and metastasize to the sentinel lymph nodes. Tumor lymphangiogenesis is stimulated by the vascular endothelial growth factors-C (VEGFC) after binding to its receptor VEGFR-3. However, how VEGFC cooperates with other molecules to promote lymphatics growth has not been fully determined. We showed that lymphangiogenesis developed in tumoral lesions and in surrounding adipose tissue (AT). Interestingly, lymphatic vessel density correlated with an increase in circulating free fatty acids (FFA) in the lymph from tumor-bearing mice. We showed that adipocyte-released FFA are uploaded by lymphatic endothelial cells (LEC) to stimulate their sprouting. Lipidomic analysis identified the monounsaturated oleic acid (OA) as the major circulating FFA in the lymph in a tumoral context. OA transporters FATP-3, -6 and CD36 were only upregulated on LEC in the presence of VEGFC showing a collaborative effect of these molecules. OA stimulates fatty acid ß-oxidation in LECs, leading to increased AT lymphangiogenesis. Our results provide new insights on the dialogue between tumors and adipocytes via the lymphatic system and identify a key role for adipocyte-derived FFA in the promotion of lymphangiogenesis, revealing novel therapeutic opportunities for inhibitors of lymphangiogenesis in cancer.

Sex Hormones in Lymphedema.

Lymphedema is a disorder of the lymphatic vascular system characterized by impaired lymphatic return resulting in swelling of the extremities and accumulation of undrained interstitial fluid/lymph that results in fibrosis and adipose tissue deposition in the limb. Whereas it is clearly established that primary lymphedema is sex-linked with an average ratio of one male for three females, the role of female hormones, in particular estrogens, has been poorly explored. In addition, secondary lymphedema in Western countries affects mainly women who developed the pathology after breast cancer and undergo through hormone therapy up to five years after cancer surgery. Although lymphadenectomy is identified as a trigger factor, the effect of co-morbidities associated to lymphedema remains elusive, in particular, estrogen receptor antagonists or aromatase inhibitors. In addition, the role of sex hormones and gender has been poorly investigated in the etiology of the pathology. Therefore, this review aims to recapitulate the effect of sex hormones on the physiology of the lymphatic system and to investigate whetherhormone therapy could promote a lymphatic dysfunction leading to lymphedema.

High Level of Staufen1 Expression Confers Longer Recurrence Free Survival to Non-Small Cell Lung Cancer Patients by Promoting THBS1 mRNA Degradation.

Stau1 is a pluripotent RNA-binding protein that is responsible for the post-transcriptional regulation of a multitude of transcripts. Here, we observed that lung cancer patients with a high Stau1 expression have a longer recurrence free survival. Strikingly, Stau1 did not impair cell proliferation in vitro, but rather cell migration and cell adhesion. In vivo, Stau1 depletion favored tumor progression and metastases development. In addition, Stau1 depletion strongly impaired vessel maturation. Among a panel of candidate genes, we specifically identified the mRNA encoding the cell adhesion molecule Thrombospondin 1 (THBS1) as a new target for Staufen-mediated mRNA decay. Altogether, our results suggest that regulation of THBS1 expression by Stau1 may be a key process involved in lung cancer progression.

Circular RNA, the Key for Translation.

It was thought until the 1990s that the eukaryotic translation machinery was unable to translate a circular RNA. However internal ribosome entry sites (IRESs) and m6A-induced ribosome engagement sites (MIRESs) were discovered, promoting 5' end-independent translation initiation. Today a new family of so-called "noncoding" circular RNAs (circRNAs) has emerged, revealing the pivotal role of 5' end-independent translation. CircRNAs have a strong impact on translational control via their sponge function, and form a new mRNA family as they are translated into proteins with pathophysiological roles. While there is no more doubt about translation of covalently closed circRNA, the linearity of canonical mRNA is only theoretical: it has been shown for more than thirty years that polysomes exhibit a circular form and mRNA functional circularization has been demonstrated in the 1990s by the interaction of initiation factor eIF4G with poly(A) binding protein. More recently, additional mechanisms of 3'-5' interaction have been reported, including m6A modification. Functional circularization enhances translation via ribosome recycling and acceleration of the translation initiation rate. This update of covalently and noncovalently closed circular mRNA translation landscape shows that RNA with circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

The Impact of Estrogen Receptor in Arterial and Lymphatic Vascular Diseases.

The lower incidence of cardiovascular diseases in pre-menopausal women compared to men is well-known documented. This protection has been largely attributed to the protective effect of estrogens, which exert many beneficial effects against arterial diseases, including vasodilatation, acceleration of healing in response to arterial injury, arterial collateral growth and atheroprotection. More recently, with the visualization of the lymphatic vessels, the impact of estrogens on lymphedema and lymphatic diseases started to be elucidated. These estrogenic effects are mediated not only by the classic nuclear/genomic actions via the specific estrogen receptor (ER) α and ß, but also by rapid extra-nuclear membrane-initiated steroid signaling (MISS). The ERs are expressed by endothelial, lymphatic and smooth muscle cells in the different vessels. In this review, we will summarize the complex vascular effects of estrogens and selective estrogen receptor modulators (SERMs) that have been described using different transgenic mouse models with selective loss of ERα function and numerous animal models of vascular and lymphatic diseases.

[Do circular RNAs play tricks on us?] / L'ARN circulaire nous joue-t-il des tours ?

RNA has not said its last word with the rise of a new RNA family, circular RNAs (circRNAs). Discovered 25 years ago, circRNAs were initially considered as splicing byproducts. Today it appears that 14% of human genes produce circRNAs, whereas more than 100 000 different circRNAs are expressed. They are produced from coding genes through an alternative splicing mechanism called backsplicing, where an acceptor site is linked with a donor site located downstream. Nuclear circRNAs regulate transcription and splicing of their linear isoform. Cytoplasmic circRNAs, which are predominant, either sequester miRNAs or RNA binding proteins, or are translated via internal initiation mechanisms. CircRNAs may constitute a powerful biotechnogical tool for protein synthesis, as their translation is stable over time. In addition, exogenous circRNAs generate less immune response than their linear counterparts. We will also discuss in this review their biotechnological potential and their roles in pathological processes.

TITLE: L'ARN circulaire nous joue-t-il des tours ? ABSTRACT: L'ARN n'a pas dit son dernier mot… avec l'émergence des ARN circulaires (circARN). Quatorze pour cent des gènes humains produisent en effet des circARN par un mécanisme d'épissage alternatif : le rétro-épissage. Chez l'homme, plus de 100 000 circARN différents ont ainsi été répertoriés. Dans le noyau, ils régulent la transcription ou l'épissage des ARNm, alors que, dans le cytoplasme, ils séquestrent des miARN et des protéines, ou sont traduits par un mécanisme d'initiation interne de la traduction. Ces circARN constituent en fait un outil biotechnologique performant car leur traduction est très stable dans le temps, et les circARN exogènes induisent moins de réponses immunitaires que les ARNm linéaires. Dans cette revue, nous discuterons, après les avoir décrits, du rôle des circARN dans différents processus pathologiques et de leur utilisation en biotechnologie.

Vasohibin1, a new mouse cardiomyocyte IRES trans-acting factor that regulates translation in early hypoxia.

Hypoxia, a major inducer of angiogenesis, triggers major changes in gene expression at the transcriptional level. Furthermore, under hypoxia, global protein synthesis is blocked while internal ribosome entry sites (IRES) allow specific mRNAs to be translated. Here, we report the transcriptome and translatome signatures of (lymph)angiogenic genes in hypoxic HL-1 mouse cardiomyocytes: most genes are induced at the translatome level, including all IRES-containing mRNAs. Our data reveal activation of (lymph)angiogenic factor mRNA IRESs in early hypoxia. We identify vasohibin1 (VASH1) as an IRES trans-acting factor (ITAF) that is able to bind RNA and to activate the FGF1 IRES in hypoxia, but which tends to inhibit several IRESs in normoxia. VASH1 depletion has a wide impact on the translatome of (lymph)angiogenesis genes, suggesting that this protein can regulate translation positively or negatively in early hypoxia. Translational control thus appears as a pivotal process triggering new vessel formation in ischemic heart.

Paclitaxel induces lymphatic endothelial cells autophagy to promote metastasis.

Cytotoxic therapy for breast cancer inhibits the growth of primary tumors, but promotes metastasis to the sentinel lymph nodes through the lymphatic system. However, the effect of first-line chemotherapy on the lymphatic endothelium has been poorly investigated. In this study, we determined that paclitaxel, the anti-cancer drug approved for the treatment of metastatic or locally advanced breast cancer, induces lymphatic endothelial cell (LEC) autophagy to increase metastases. While paclitaxel treatment was largely efficacious in inhibiting LEC adhesion, it had no effect on cell survival. Paclitaxel inhibited LEC migration and branch point formation by inducing an autophagy mechanism independent of Akt phosphorylation. In vivo, paclitaxel mediated a higher permeability of lymphatic endothelium to tumor cells and this effect was reversed by chloroquine, an autophagy-lysosome inhibitor. Despite a strong effect on reducing tumor size, paclitaxel significantly increased metastasis to the sentinel lymph nodes. This effect was restricted to a lymphatic dissemination, as chemotherapy did not affect the blood endothelium. Taken together, our findings suggest that the lymphatic system resists to chemotherapy through an autophagy mechanism to promote malignant progression and metastatic lesions. This study paves the way for new combinative therapies aimed at reducing the number of metastases.

How are circRNAs translated by non-canonical initiation mechanisms?

Circular RNAs (circRNAs) are covalently closed RNA loops produced by a very large number of expressed eukaryotic genes. Initially considered as splicing background and/or splicing side products, recent studies have shown that they are evolutionary conserved and abundant in cells. Yet, their functions remain largely unknown. Because of their circular shape, they were initially categorized as non-coding RNAs. However, recent studies based on mass spectrometry analysis indicate that some cytoplasmic circRNAs are effectively translated into detectable peptides. This raises the interesting question of which mechanisms regulate the translation initiation of those circular transcripts, i.e. unable to recruit the small ribosome subunit through the 5' cap. A possible mechanism for alternative translation initiation is the presence of an IRES (Internal Ribosome Entry Site) that allows direct recruitment of initiation factors and ribosomes on the RNA independently from the cap. This is the case for several circRNAs that exhibit IRESs upstream from the start codon. Yet, another process seems to be involved in initiating the translation of circRNAs: the presence of N6-methyladenosine (m6A) residues. These m6A can promote cap-independent translation and have been shown to be enriched in circRNAs. Interestingly, these two alternative translation initiation processes are generally activated under cellular stress to allow expression of specific stress response genes. These discoveries therefore link circRNA translation to cellular response to stress conditions, raising new enquiries about the regulation of circRNA expression under stress conditions and their functions. This review provides a state of the art on this emerging area.

IRES Trans-Acting Factors, Key Actors of the Stress Response.

The cellular stress response corresponds to the molecular changes that a cell undergoes in response to various environmental stimuli. It induces drastic changes in the regulation of gene expression at transcriptional and posttranscriptional levels. Actually, translation is strongly affected with a blockade of the classical cap-dependent mechanism, whereas alternative mechanisms are activated to support the translation of specific mRNAs. A major mechanism involved in stress-activated translation is the internal ribosome entry site (IRES)-driven initiation. IRESs, first discovered in viral mRNAs, are present in cellular mRNAs coding for master regulators of cell responses, whose expression must be tightly controlled. IRESs allow the translation of these mRNAs in response to different stresses, including DNA damage, amino-acid starvation, hypoxia or endoplasmic reticulum stress, as well as to physiological stimuli such as cell differentiation or synapse network formation. Most IRESs are regulated by IRES trans-acting factor (ITAFs), exerting their action by at least nine different mechanisms. This review presents the history of viral and cellular IRES discovery as well as an update of the reported ITAFs regulating cellular mRNA translation and of their different mechanisms of action. The impact of ITAFs on the coordinated expression of mRNA families and consequences in cell physiology and diseases are also highlighted.

Lymphatic Vasculature Requires Estrogen Receptor-α Signaling to Protect From Lymphedema.

OBJECTIVE: Estrogens exert beneficial effect on the blood vascular system. However, their role on the lymphatic system has been poorly investigated. We studied the protective effect of the 17ß estradiol-the most potent endogenous estrogen-in lymphedema-a lymphatic dysfunction, which results in a massive fluid and fat accumulation in the limb. APPROACH AND RESULTS: Screening of DNA motifs able to mobilize ERs (estrogen receptors) and quantitative real-time polymerase chain reaction analysis revealed that estradiol promotes transcriptional activation of lymphangiogenesis-related gene expression including VEGF (vascular endothelial growth factor)-D, VEGFR (VEGF receptor)-3, lyve-1, and HASs (hyaluronan synthases). Using an original model of secondary lymphedema, we observed a protective effect of estradiol on lymphedema by reducing dermal backflow-a representative feature of the pathology. Blocking ERα by tamoxifen-the selective estrogen modulator-led to a remodeling of the lymphatic network associated with a strong lymphatic leakage. Moreover, the protection of lymphedema by estradiol treatment was abrogated by the endothelial deletion of the receptor ERα in Tie2-Cre; ERαlox/lox mice, which exhibit dilated lymphatic vessels. This remodeling correlated with a decrease in lymphangiogenic gene expression. In vitro, blocking ERα by tamoxifen in lymphatic endothelial cells decreased cell-cell junctions, inhibited migration and sprouting, and resulted in an inhibition of Erk but not of Akt phosphorylation. CONCLUSIONS: Estradiol protection from developing lymphedema is mediated by an activation of its receptor ERα and is antagonized by tamoxifen. These findings reveal a new facet of the estrogen influence in the management of the lymphatic system and provide more evidence that secondary lymphedema is worsened by hormone therapy.

Therapeutic Benefit and Gene Network Regulation by Combined Gene Transfer of Apelin, FGF2, and SERCA2a into Ischemic Heart.

Despite considerable advances in cardiovascular disease treatment, heart failure remains a public health challenge. In this context, gene therapy appears as an attractive approach, but clinical trials using single therapeutic molecules result in moderate benefit. With the objective of improving ischemic heart failure therapy, we designed a combined treatment, aimed to simultaneously stimulate angiogenesis, prevent cardiac remodeling, and restore contractile function. We have previously validated IRES-based vectors as powerful tools to co-express genes of interest. Mono- and multicistronic lentivectors expressing fibroblast growth factor 2 (angiogenesis), apelin (cardioprotection), and/or SERCA2a (contractile function) were produced and administrated by intramyocardial injection into a mouse model of myocardial infarction. Data reveal that combined treatment simultaneously improves vessel number, heart function parameters, and fibrosis prevention, due to FGF2, SERCA2a, and apelin, respectively. Furthermore, addition of SERCA2a in the combination decreases cardiomyocyte hypertrophy. Large-scale transcriptome analysis reveals that the triple treatment is the most efficient in restoring angiogenic balance as well as expression of genes involved in cardiac function and remodeling. Our study validates the concept of combined treatment of ischemic heart disease with apelin, FGF2, and SERCA2a and shows that such therapeutic benefit is mediated by a more effective recovery of gene network regulation.

Dachsous1-Fat4 Signaling Controls Endothelial Cell Polarization During Lymphatic Valve Morphogenesis-Brief Report.

OBJECTIVE: The purpose of this study was to investigate the role of Fat4 and Dachsous1 signaling in the lymphatic vasculature. APPROACH AND RESULTS: Phenotypic analysis of the lymphatic vasculature was performed in mice lacking functional Fat4 or Dachsous1. The overall architecture of lymphatic vasculature is unaltered, yet both genes are specifically required for lymphatic valve morphogenesis. Valve endothelial cells (Prox1high [prospero homeobox protein 1] cells) are disoriented and failed to form proper valve leaflets. Using Lifeact-GFP (green fluorescent protein) mice, we revealed that valve endothelial cells display prominent actin polymerization. Finally, we showed the polarized recruitment of Dachsous1 to membrane protrusions and cellular junctions of valve endothelial cells in vivo and in vitro. CONCLUSIONS: Our data demonstrate that Fat4 and Dachsous1 are critical regulators of valve morphogenesis. This study highlights that valve defects may contribute to lymphedema in Hennekam syndrome caused by Fat4 mutations.

Apelin modulates pathological remodeling of lymphatic endothelium after myocardial infarction.

Lymphatic endothelium serves as a barrier to control fluid balance and immune cell trafficking to maintain tissue homeostasis. Long-term alteration of lymphatic vasculature promotes edema and fibrosis, which is an aggravating factor in the onset of cardiovascular diseases such as myocardial infarction. Apelin is a bioactive peptide that plays a central role in angiogenesis and cardiac contractility. Despite an established role of apelin in lymphangiogenesis, little is known about its function in the cardiac lymphatic endothelium. Here, we show that apelin and its receptor APJ were exclusively expressed on newly formed lymphatic vasculature in a pathological model of myocardial infarction. Using an apelin-knockout mouse model, we identified morphological and functional defects in lymphatic vasculature associated with a proinflammatory status. Surprisingly, apelin deficiency increased the expression of lymphangiogenic growth factors VEGF-C and VEGF-D and exacerbated lymphangiogenesis after myocardial infarction. Conversely, the overexpression of apelin in ischemic heart was sufficient to restore a functional lymphatic vasculature and to reduce matrix remodeling and inflammation. In vitro, the expression of apelin prevented the alteration of cellular junctions in lymphatic endothelial cells induced by hypoxia. In addition, we demonstrated that apelin controls the secretion of the lipid mediator sphingosine-1-phosphate in lymphatic endothelial cells by regulating the level of expression of sphingosine kinase 2 and the transporter SPNS2. Taken together, our results show that apelin plays a key role in lymphatic vessel maturation and stability in pathological settings. Thus, apelin may represent a novel candidate to prevent pathological lymphatic remodeling in diseases.

Nucleolin Promotes Heat Shock-Associated Translation of VEGF-D to Promote Tumor Lymphangiogenesis.

The vascular endothelial growth factor VEGF-D promotes metastasis by inducing lymphangiogenesis and dilatation of the lymphatic vasculature, facilitating tumor cell extravasion. Here we report a novel level of control for VEGF-D expression at the level of protein translation. In human tumor cells, VEGF-D colocalized with eIF4GI and 4E-BP1, which can program increased initiation at IRES motifs on mRNA by the translational initiation complex. In murine tumors, the steady-state level of VEGF-D protein was increased despite the overexpression and dephosphorylation of 4E-BP1, which downregulates protein synthesis, suggesting the presence of an internal ribosome entry site (IRES) in the 5' UTR of VEGF-D mRNA. We found that nucleolin, a nucleolar protein involved in ribosomal maturation, bound directly to the 5'UTR of VEGF-D mRNA, thereby improving its translation following heat shock stress via IRES activation. Nucleolin blockade by RNAi-mediated silencing or pharmacologic inhibition reduced VEGF-D translation along with a subsequent constriction of lymphatic vessels in tumors. Our results identify nucleolin as a key regulator of VEGF-D expression, deepening understanding of lymphangiogenesis control during tumor formation. Cancer Res; 76(15); 4394-405. ©2016 AACR.

Nonvenous origin of dermal lymphatic vasculature.

RATIONALE: The formation of the blood vasculature is achieved via 2 fundamentally different mechanisms, de novo formation of vessels from endothelial progenitors (vasculogenesis) and sprouting of vessels from pre-existing ones (angiogenesis). In contrast, mammalian lymphatic vasculature is thought to form exclusively by sprouting from embryonic veins (lymphangiogenesis). Alternative nonvenous sources of lymphatic endothelial cells have been suggested in chicken and Xenopus, but it is unclear whether they exist in mammals. OBJECTIVE: We aimed to clarify the origin of the murine dermal lymphatic vasculature. METHODS AND RESULTS: We performed lineage tracing experiments and analyzed mutants lacking the Prox1 transcription factor, a master regulator of lymphatic endothelial cell identity, in Tie2 lineage venous-derived lymphatic endothelial cells. We show that, contrary to current dogma, a significant part of the dermal lymphatic vasculature forms independently of sprouting from veins. Although lymphatic vessels of cervical and thoracic skin develop via sprouting from venous-derived lymph sacs, vessels of lumbar and dorsal midline skin form via assembly of non-Tie2-lineage cells into clusters and vessels through a process defined as lymphvasculogenesis. CONCLUSIONS: Our results demonstrate a significant contribution of nonvenous-derived cells to the dermal lymphatic vasculature. Demonstration of a previously unknown lymphatic endothelial cell progenitor population will now allow further characterization of their origin, identity, and functions during normal lymphatic development and in pathology, as well as their potential therapeutic use for lymphatic regeneration.

Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis.

Planar cell polarity (PCP) signaling controls tissue morphogenesis by coordinating collective cell behaviors. We show a critical role for the core PCP proteins Celsr1 and Vangl2 in the complex morphogenetic process of intraluminal valve formation in lymphatic vessels. We found that valve-forming endothelial cells undergo elongation, reorientation, and collective migration into the vessel lumen as they initiate valve leaflet formation. During this process, Celsr1 and Vangl2 are recruited from endothelial filopodia to discrete membrane domains at cell-cell contacts. Celsr1- or Vangl2-deficient mice show valve aplasia due to failure of endothelial cells to undergo rearrangements and adopt perpendicular orientation at valve initiation sites. Mechanistically, we show that Celsr1 regulates dynamic cell movements by inhibiting stabilization of VE-cadherin and maturation of adherens junctions. These findings reveal a role for PCP signaling in regulating adherens junctions and directed cell rearrangements during vascular development.