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Figure S2. Expression of endogenous VEGF-D in mice tissues

Figure S5. VEGF-D 5'UTR mRNA exhibits two structural forms that allow the binding of different proteins

Figure S7. Tumor growth after NSAID treatment

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.

Figure S8. Schematic representation of VEGF-D synthesis in tumor cells

Supplementary figure legend

Figure S6. Polysome profiling of 4T1 cells

Figure S4. Lymphangiogenesis and tumor gowth are not affected by 4T1 or 67NR lentiviral transduction

Figure S3. Podoplanin staining of lymphatic vessels

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.

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.

In cancer, the lymphatic system is hijacked by tumor cells to escape from primary tumor and to 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 lymphatic neovessels growth is not fully determined. Here, we found that tumor lymphangiogenesis developed in tumoral lesions and in their surrounding adipose tissue (AT). Interestingly, lymphatic vessel density correlated with an increase of circulating free fatty acids (FFA) in the lymph from tumor-bearing mice. We found 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 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 released from adipocytes is taken up by LECs to stimulate the fatty acid β-oxidation, leading to increase adipose tissue 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.

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

SUMMARYInternal 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.HighlightsParaspeckle formation correlates with activation of translation via internal ribosome entry sites (IRES) in mouse hypoxic cardiomyocytes as well as in tumoral cells.The long non-coding RNA Neat1, an essential paraspeckle component, is a key translational regulator of (lymph)angiogenic and cardioprotective factor expression in this process.IRES-containing mRNA is recruited into paraspeckles during hypoxia.Paraspeckle proteins p54nrb and PSPC1 as well as two p54nrb-interacting proteins, nucleolin and RPS2, contribute to this process.Paraspeckle appears as a platform for IRESome formation in the nucleus.The Neat1 isoforms widely regulate the translation of mRNAs containing IRESs and of genes involved in the stress response.GRAPHICAL ABSTRACT

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 “non-coding” 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 non-covalently circular mRNA translation landscape shows that RNA circular shape might be the rule for translation with an important impact on disease development and biotechnological applications.

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.L’ARN circulaire nous joue-t-il des tours ?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.

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 (Prox1 high [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.

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.

The cellular stress response corresponds to the molecular changes that cell undergoes in response to various environmental stimuli. It induces drastic changes in the regulation of gene expression, at transcriptional and post-transcriptional levels. Actually, translation is strongly affected with a blockade of the classical cap-dependent mechanism, whereas alternative mechanisms are activated to support translation of specific mRNAs. One of the major mechanisms 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 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. Importantly, cellular mRNA IRESs are regulated by IRES trans-acting factor (ITAFs), exerting their action by at least nine different mechanisms. This review presents an update of the reported ITAFs regulating cellular mRNA translation and of the different mechanisms allowing them to control translation initiation in specific conditions. The impact of ITAFs on coordinated expression of mRNA families and consequences in cell physiology and diseases are also highlighted.

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.

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 17β 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; ERα lox/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.

International audience; 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.

Hypoxia, a major inducer of angiogenesis, is known to trigger major changes of gene expression at the transcriptional level. Furthermore, 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 cardiomyocytes: most genes are not induced at the transcriptome-, but at the translatome level, including all IRES-containing mRNAs. Our data reveal activation of (lymph)angiogenic mRNA IRESs in early hypoxia. We identify vasohibin1 (VASH1) as an IRES trans-acting factor (ITAF) able to activate FGF1 and VEGFD IRESs in hypoxia while it inhibits several IRESs in normoxia. Thus this new ITAF may have opposite effects on IRES activities. These data suggest a generalized process of IRES-dependent translational induction of (lymph)angiogenic growth factors expression in early hypoxia, whose pathophysiological relevance is to trigger formation of new functional vessels in ischemic heart. VASH1 is not always required, indicating that the IRESome composition is variable, thus allowing subgroups of IRESs to be activated under the control of different ITAFs.

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.

SummaryPlanar 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.

Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels and promotes smooth muscle cell recruitment, which is necessary for lymphatic vessel morphogenesis and function., Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels—smooth muscle and endothelial cells—and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.

Podosomes are punctate actin-rich adhesion structures which spontaneously form in cells of the myelomonocytic lineage. Their formation is dependent on Src and RhoGTPases. Recently, podosomes have also been described in vascular cells. These podosomes differ from the former by the fact that they are inducible. In endothelial cells, such a signal can be provided by either constitutively active Cdc42, the PKC activator PMA or TGFbeta, depending on the model. Consequently, other regulatory pathways have been reported to contribute to podosome formation. To get more insight into the mechanisms by which podosomes form in endothelial cells, we have explored the respective contribution of signal transducers such as Cdc42-related GTPases, Smads and PKCs in three endothelial cell models. Results presented demonstrate that, in addition to Cdc42, TC10 and TCL GTPases can also promote podosome formation in endothelial cells. We also show that PKCalpha can be either necessary or entirely dispensable, depending on the cell model. In contrast, PKCdelta is essential for podosome formation in endothelial cells but not smooth muscle cells. Finally, although podosomes vary very little in their molecular composition, the signalling pathways involved in their assembly appear very diverse.

The two isoforms of p190 RhoGAP (p190A and p190B) are important regulators of RhoGTPase activity in mammalian cells. Both proteins are ubiquitously expressed, are involved in the same signalling pathways and interact with the same identified binding partners. In search of isoform functional specificity, we knocked down the expression of each p190 protein using siRNA and examined the resulting phenotypic changes in human umbilical vein endothelial cells (HUVECs). We provide evidence that p190B plays a crucial role in the regulation of MT1-MMP expression and cell-surface presentation, as well as subsequent MMP2 activation. p190B is involved in both local extracellular matrix degradation at podosomes and endothelial cell assembly into tube-like structures in Matrigel. In addition, whereas p190B knockdown does not affect podosome formation, p190A knockdown increases the number of cells showing podosome structures in HUVECs. We conclude that the two p190 RhoGAP isoforms play distinct roles in endothelial cells. In addition, our data reveal an unsuspected role for p190B in the expression of the two collaborative proteases MT1-MMP and MMP2, thereby affecting matrix remodelling and angiogenesis.

The lymphatic vasculature has a major role in physiology. It drains the interstitial fluids and performs immune surveillance by transporting immunity cells. The lymphatic vessels are also involved in pathological conditions such as chronic inflammatory diseases, lymphedema and cancer. Lymphangiogenesis, the growth of new lymphatic vessels, is induced by the lymphangiogenic growth factors VEGF-C and VEGF-D. Recent studies suggest that VEGF-D is also involved in lymphatic dilatation through prostaglandin signaling pathways. In this study, we demonstrated the molecular mechanism involved in lymphatic dilatation. We identified a stress-induced translational regulation of VEGF-D expression through an IRES activation under heat shock conditions. We demonstrated that VEGF-D IRES activity was dependent of prostaglandin pathway as Cox-2 inhibitors are able to abolish the IRES-promoted VEGF-D protein synthesis, and lymphatic vessels dilatation. Using plasmon surface resonance on biotinylated VEGF-D mRNA coupled to mass spectrometry, we identified the IRES transacting factor (ITAF) that allows the recruitment of the ribosome to promote VEGF-D translational initiation: the Nucleolin. Our results bring new insights on the VEGF-D regulation and on the lymphatic vessels plasticity. As lymphatic vessels play a crucial role in inflammation by performing immune cells tissue trafficking, understanding the regulations of lymphatic vasodilatation is a crucial step toward an innovative inflammatory disease therapy.

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.

Lymphatic vessels participate in tissue homeostasis and immune surveillance by draining excess fluid and immune cells from tissues to blood circulation. Impaired lymphatic function can lead to tissue swelling, or lymphoedema, and associated complications, such as chronic inflammation and fat accumulation. The critical role of lymphatic vessels in a number of pathological conditions, including tumour metastasis, has led to an interest in identifying signalling pathways regulating lymphatic vessel development and growth. Here, we review the current knowledge on the molecular mechanisms of lymphatic development and how lymphatic vasculature contributes to diseases.

Background information. Fluoride is a well-known G-protein activator. Exposure of cultured cells to its derivatives results in actin cytoskeleton remodelling. Podosomes are actin-based structures endowed with adhesion and matrix-degradation functions. This study investigates actin cytoskeleton reorganization induced by fluoride in endothelial cells. Results. Treatment of cultured endothelial cells with sodium fluoride (NaF) results in a rapid and potent stimulation of podosome formation. Furthermore, we show that Cdc42 (cell-division cycle 42), Rac1 and RhoA activities are stimulated in NaF-treated cells. However, podosome assembly is dependent on Cdc42 and Rac1, but not RhoA. Although the sole activation of Cdc42 is sufficient to induce individual podosomes, a balance between RhoGTPase activities regulates podosome formation in response to NaF, which in this case are often found in groups or rosettes. As in other models, podosome formation in endothelial cells exposed to NaF also involves Src. Finally, we demonstrate that NaF-induced podosomes are fully competent for matrix protein degradation. Conclusions. Taken together, our findings establish NaF as a novel inducer of podosomes in endothelial cells in vitro.

International audience; Cytoskeletal rearrangements are central to endothelial cell physiology and are controlled by soluble factors, matrix proteins, cell-cell interactions, and mechanical forces. We previously reported that aortic endothelial cells can rearrange their cytoskeletons into complex actin-based structures called podosomes when a constitutively active mutant of Cdc42 is expressed. We now report that transforming growth factor beta (TGF-beta) promotes podosome formation in primary aortic endothelial cells. TGF-beta-induced podosomes assembled together into large ring- or crescent-shaped structures. Their formation was dependent on protein synthesis and required functional Src, phosphatidylinositide 3-kinase, Cdc42, RhoA, and Smad signaling. MT1-MMP and metalloprotease 9 (MMP9), both upregulated by TGF-beta, were detected at sites of podosome formation, and MT1-MMP was found to be involved in the local degradation of extracellular matrix proteins beneath the podosomes and required for the invasion of collagen gels by endothelial cells. We propose that TGF-beta plays an important role in endothelial cell physiology by inducing the formation of podosomal structures endowed with metalloprotease activity that may contribute to arterial remodeling.

Ectopic expression of a constitutive active mutant of the GTPase Cdc42 (V12Cdc42) in vascular endothelial cells triggers the dissolution of stress fibres and focal adhesion contacts and causes the repolymerisation of actin into dots. Each punctate structure consists of an F-actin core surrounded by a vinculin ring, consistent with the definition of podosomes. We now report further analysis of these complexes and show the presence of established podosomal markers such as cortactin, gelsolin, dynamin, N-WASP, and Arp2/3 which are absent in focal adhesions. Endothelial podosomes appear as randomly distributed conical structures, distributed on, but restricted to, the ventral membrane and confined to contact sites between cells and their substratum. The nature of the extracellular matrix does not influence podosome formation nor their spatial organisation. Induction of podosomes in response to V12Cdc42 is not associated with a migratory nor with a proliferative phenotype. These results add endothelial cells to the list of cell types endowed with the ability to form podosomes in vitro and raise the possibility that endothelial cells could form such structures under certain physiological or pathological conditions.

The involvement of Src, Cdc42, RhoA and PKC in the regulation of podosome assembly has been identified in various cell models. In endothelial cells, the ectopic expression of constitutively active mutants of Src or Cdc42, but not RhoA, induced the formation of podosomes. Short-term exposure to phorbol-12-myristate-13-acetate (PMA) induced the appearance of podosomes and rosettes after initial disruption of stress fibres. Molecular analysis of PMA-induced podosomes and rosettes revealed that their composition was identical to that of podosomes described in other models. Pharmacological inhibition and siRNA knock-down experiments revealed that both PKCα and PKCδ isotypes were necessary for podosome assembly. However, only constitutively active PKCα could mimic PMA in podosome formation. Src, Cdc42 and RhoA were required downstream of PKCs in this process. Src could be positioned between PKC and Cdc42 in a linear cascade leading to podosome assembly. Using in vitro matrix degradation assays, we demonstrated that PMA-induced podosomes are endowed with proteolytic activities involving MT1-MMP-mediated activation of MMP2. Endothelial podosomes may be involved in subendothelial matrix degradation during endothelium remodelling in pathophysiological processes.

Members of the Rho GTPase family play a central role in the orchestration of cytoskeletal rearrangements, which are of prime importance in endothelial cell physiology. To explore their role in this specialized cell type, we used the bacterial toxin cytotoxic necrotizing factor 1 (CNF1) as a Rho GTPase activator. Punctate filamentous actin structures appeared along the ventral plasma membrane of endothelial cells and were identified as the core of podosomes by the distinctive vinculin ring around the F-actin. Rho, Rac, and Cdc42 were all identified as targets of CNF1, but only a constitutively active mutant of Cdc42 could substitute for CNF1 in podosome induction. Accordingly, organization of F-actin in these structures was highly dependent on the main Cdc42 cytoskeletal effector N-Wiskott-Aldrich syndrome protein. Other components of the actin machinery such as Arp2/3 and for the first time WIP also colocalized at these sites. Like CNF1 treatment, sustained Cdc42 activity induced a time-dependent F-actin-vinculin reorganization, prevented cytokinesis, and downregulated Rho activity. Finally, podosomes were also detected on endothelial cells explanted from patients undergoing cardiac surgery. These data provide the first description of podosomes in endothelial cells. The identification of such specialized structures opens up a new field of investigation in terms of endothelium pathophysiology.

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 β, 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.