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Background— Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell–cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI. Methods and Results— We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4 -deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4 -deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4 -deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits. Conclusions— These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.

The production of renin, which catalyzes the rate-limiting step of the renin-angiotensin system, is tightly regulated by intracellular second messengers. Among them, an increase of intracellular calcium represses renin gene expression. This inhibition of gene expression by intracellular calcium is exceptional, and the molecular mechanism supporting this phenomenon has not yet been identified. As the renin gene is negatively regulated by calcium in the same way as the parathormone (PTH) gene, we hypothesized that a similar molecular transcriptional mechanism could be involved.Analysis of the human renin proximal promoter led to the identification of a negative calcium response element (nCaRE), which is identical to the region of the PTH promoter and is involved in its repression by calcium. Transfection experiments in renin-expressing chorio-decidual cells demonstrated the transcriptional functionality of the human renin promoter nCaRE. In addition, mutation of nCaRE suppressed the sensitivity of the renin promoter to the increase in intracellular calcium. Gel shift assays demonstrated that Redox factor 1, a multifunctional protein involved in the repair of damaged DNA and the redox activation of AP-1 transcriptional factors, binds specifically to nCaRE. Immunostaining showed that this factor is translocated from the cytoplasm to the nucleus in response to an increase in the intracellular calcium concentration.Thus, the repression of renin expression by intracellular calcium may be mediated by the calcium-induced translocation of Ref-1 to the nucleus, where it binds to the renin promoter nCaRE, to repress the transcription of the renin gene.

International audience; Renin plays a central role in controlling blood pressure as it catalyzes the first step in the production of angiotensin II. The aim of this study was to isolate fragments of the human renin (hREN) promoter able to direct tissue-specific and regulated expression of a LacZ reporter gene mimicking endogenous renin. We screened several hREN promoter/LacZ constructs for transgene expression in transient embryos at E15 when renin expression begins. We found that a 12-kb hREN promoter conferred high expression in the kidney at both embryonic and adult stages and that the transgene was expressed in the same cells as endogenous renin. We explored two pathophysiological models in which renin is stimulated and showed concomitant increases in beta-galactosidase and renin activities. In situ beta-galactosidase staining showed renin/transgene-expressing cells are recruited in the juxtaglomerular apparatus and in the afferent arterioles as well as in larger arteries outside the kidney. Using our model, renin expression in interlobular arteries was confirmed as being striped and, for the first time, expression of renin in larger arteries outside the kidney was shown. Therefore, this strain is a suitable model to investigate renin gene pathophysiological regulations in vivo.

International audience; 1. The renin-angiotensin system plays a major role in blood pressure regulation and electrolyte homeostasis through the action of angiotensin (Ang) II. The first and rate-limiting step in the production of AngII is the conversion of angiotensinogen into AngI, which is catalysed by the aspartyl protease renin (EC 3.4.23.15). Circulating active renin is mainly synthesized, processed and secreted by the juxtaglomerular cells within the kidney. 2. To determine the renin 5'-flanking sequences involved in cell and tissue specificity, ex vivo and in vivo studies were performed. Several constructs of various lengths of renin promoter linked to the luciferase gene were first tested ex vivo by transfection in primary cultures of human chorionic cells. The constructs giving a high and specific expression in renin-producing cells were then tested in vivo in a transgenic mice model. 3. The reporter gene chosen to generate transgenic mice was LacZ and the screening was performed in embryos at the embryonic day (E) 15 stage, at which mouse renin is expressed in the developing vessels of the kidney. 4. Only constructs containing more than 5.7 kb of the human renin promoter lead to specific expression of beta-galactosidase in the kidney. 5. Our results demonstrate that the human renin distal promoter region allows a more restricted expression of LacZ in the renin-expressing cells in transgenic mice.

International audience; Renin catalyzes the rate-limiting step of the renin-angiotensin system, which regulates blood pressure and electrolyte homeostasis. To determine cell-specific human renin gene control elements, the transcriptional activity of promoter regions up to position -8876 was studied in renin-expressing cells. A positive regulatory region conferring approximately 57-fold higher transcriptional activity to the human renin gene promoter in chorionic cells was identified between nucleotides -5777 and -5552. It had the orientation-independent activity typical of classical enhancers. It also conferred approximately 59-fold higher transcriptional levels from the heterologous simian virus 40 (SV40) promoter in chorionic cells and approximately 6-fold higher transcriptional levels in Calu-6 and As4.1 cells, whereas no effect was measured in non-renin-expressing cells. DNase I footprinting showed that this enhancer contains three binding sites for chorionic cell nuclear extracts. Functional analysis suggested that the activity of the enhancer is regulated by differential mechanisms in the three renin-expressing cells involving a complex arrangement of AP-1 motifs binding cell-specific members of the basic leucine zipper family of transcription factors. Thus, our results demonstrate that this enhancer plays a key role in the expression of the human renin gene in the chorion and may also be involved in its regulated expression in other tissues.

International audience; BACKGROUND: Dicer is an RNase III enzyme that cleaves double stranded RNA and generates functional interfering RNAs that act as important regulators of gene and protein expression. Dicer plays an essential role during mouse development because the deletion of the dicer gene leads to embryonic death. In addition, dicer-dependent interfering RNAs regulate postnatal angiogenesis. However, the role of dicer is not yet fully elucidated during vascular development. METHODS: In order to explore the functional roles of the RNA interference in vascular biology, we developed a new constitutive Cre/loxP-mediated inactivation of dicer in tie2 expressing cells. RESULTS: We show that cell-specific inactivation of dicer in Tie2 expressing cells does not perturb early blood vessel development and patterning. Tie2-Cre; dicerfl/fl mutant embryos do not show any blood vascular defects until embryonic day (E)12.5, a time at which hemorrhages and edema appear. Then, midgestational lethality occurs at E14.5 in mutant embryos. The developing lymphatic vessels of dicer-mutant embryos are filled with circulating red blood cells, revealing an impaired separation of blood and lymphatic vasculature. CONCLUSION: Thus, these results show that RNA interference perturbs neither vasculogenesis and developmental angiogenesis, nor lymphatic specification from venous endothelial cells but actually provides evidence for an epigenetic control of separation of blood and lymphatic vasculature.

Evidence that renal and chorionic tissues contain similar nuclear binding proteins that recognize the human renin promoter. This study examines whether the human renal cortex, the major renin producing site, contains nuclear factors that bind to the human renin proximal promoter. Footprint analysis of the human renin promoter region showed that human renal cortex cell nuclear extracts interacted with 6 putative cis -elements (the Ets domain-protein, a Pit-1 like binding site, a CRE sequence, an ARP-1 like binding site, an AGE3 like region, and a unknown consensus region, designated element C). Transient DNA transfection studies on chorionic cells implicated the CRE and Pit-1 consensus sites in the regulation of renin gene transcription by cAMP. Electromobility shift assays showed that renal proteins bind specifically to these sequences, and that one of them is CREB; two others seem to be Ets-1 and ARP-1. These results raise the possibility that the human renal cortex and human chorionic cells have the same trans -acting factors that bind to the proximalhuman renin promoter.

Proper vessel maturation, remodeling of endothelial junctions, and recruitment of perivascular cells is crucial for establishing and maintaining vessel functions. In proliferative retinopathies, hypoxia-induced angiogenesis is associated with disruption of the vascular barrier, edema, and vision loss. Therefore, identifying factors that regulate vascular maturation is critical to target pathological angiogenesis. Given the conflicting role of angiopoietin-like-4 (ANGPTL4) reported in the current literature using gain of function systems both in vitro and in vivo, the goal of this study was to characterize angiogenesis, focusing on perinatal retinal vascularization and pathological circumstances in angpl4-deficient mice. We report altered organization of endothelial junctions and pericyte coverage, both leading to impaired angiogenesis and increased vascular leakage that were eventually caught up, suggesting a delay in vessel maturation. In a model of oxygen-induced retinopathy, pathological neovascularization, which results from tissue hypoxia, was also strongly inhibited in angptl4-deficient mice. This study therefore shows that ANGPTL4 tunes endothelial cell junction organization and pericyte coverage and controls vascular permeability and angiogenesis, both during development and in pathological conditions.

A quantitative PCR was developed to measure human renin mRNA in chorionic cells under various stimuli. An internal standard consisting of a mutated renin mRNA with an insertion of 60 bp was designed to quantify the reaction. Quantitative PCR is a suitable tool for studying human renin gene transcription from a low number of cells. Forskolin alone had no effect on either renin mRNA levels or renin production whereas 10(-7)M PMA stimulated renin mRNA levels and renin production 5- and 2.6-fold, respectively, after 24 hours incubation. PMA and forskolin acted synergistically to increase both renin mRNA levels and renin production in a dose-dependent manner.

International audience; BACKGROUND: We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors. METHODOLOGY/PRINCIPAL FINDINGS: Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status. CONCLUSIONS/SIGNIFICANCE: Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10(-49), Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction.

L’ARN interference, mecanisme de regulation de l’expression des genes, est mediee par les siARNs et les microARNs, ARN non-codants de 20 a 22 nucleotides affectant la regulation post-transcriptionnelle d’ARNm cibles avec lesquels ils s’apparient. La RNase DICER est une enzyme centrale de la biosynthese des siARNs et microARNs. Les souris dont le gene dicer est invalide ont un phenotype complexe, et meurent tres tot pendant le developpement, notamment a cause d’un defaut d’angiogenese. Afin d’etudier l’ARN interference au cours de l’angiogenese embryonnaire, des souris dont le gene dicer est floxe (mutant conditionnel) sont croisees avec des souris exprimant la recombinase Cre, de maniere constitutive, sous le controle du promoteur du gene tie2, dirigeant ainsi son expression dans les cellules endotheliales (CE) et les cellules hematopietiques. Nos resultats montrent que l’invalidation de dicer sous le controle du promoteur du gene tie2 entraine une mortalite embryonnaire suite a un œdeme et des hemorragies au treizieme jour du developpement (E13,5). L’analyse histologique montre des vaisseaux lymphatiques remplis de sang, suggerant une mauvaise separation du reseau sanguin et lymphatique. Cette hypothese est etudiee par marquage des vaisseaux lymphatiques (LYVE-1) et des vaisseaux sanguins (PECAM) sur embryon entier et peaux isolees a differents stades precedant la mort. Ces embryons presentent egalement un probleme de developpement du foie, probablement du a l’activite du promoteur tie2 dans les lignees hematopoietiques. La mise en culture de ces foies fœtaux a E13,5 revele une atteinte des precurseurs hematopoetiques. L’etude de ces precurseurs a des stades plus precoces (E8,5) est en cours au laboratoire. Nos resultats demontrent donc un role important de l’ARN interference dans le controle epigenetique de l’angiogenese et de la lymphangiogenese embryonnaire mais egalement dans le developpement de l’hematopoiese, suggerant son implication dans la differenciation veino-lymphangiogenese, dont les mecanismes moleculaires seront discutes.

Backround Angiopoietin-like 4 (ANGPTL4) is a secreted protein of the Angiopoietins family involved in angiogenesis and vessel maintenance. We identified angptl4 as a hypoxia induced gene in endothelial cells in vitro. In humans, ANGPTL4 is expressed in hypoxic areas such as vascular cells of human critical leg ischemia samples. In the heart, focal expression in myocytes, vessels and infiltrating monocytes was also evidenced in infarcted areas. Material & Methods We used C57/Bl6 angptl4 knock-out mice (Regeneron pharmaceuticals, Tarrytown). The pattern of expression of angptl4 was determined during development using Lac Z staining allowed by the insertion of a beta-galactosidase cassette. At the adult stage, myocardial infarction was induced by ligation of the left coronary artery for 45mn. The hearts were analyzed 24 h, 48 h and 2 weeks after reperfusion. Results During embryonic development, angptl4 is expressed in endothelial cells from intersomitic, limb bud (E10,5-E11,5) and heart vessels (E12,5-E14,5). At later stages (from E15,5), angptl4 is also expressed in skin vessels. Angptl4 knock-out mice showed an increased infarcted area compared to wild type littermates mice (+31 %, p=0.01) 48h after reperfusion, suggesting a cardioprotective role of ANGPTL4. Immunohistological analyzes revealed dramatic tissue damages in the angptl4 knock-out mice in term of oedema, hemorragies and necrosis correlated with an increased circulating monocytes infiltration (+36 %, p=0.004). PECAM-1 stainings revealed morphologically modified angiogenic capillaries in angptl4 KO mice compared to controls suggesting a modulated vascular permeability. No difference was observed between isolated cardiomyocytes from both groups submitted to a survival assay under hypoxia. Conclusion Angptl4 is expressed by endothelial cells during embryonic and during adult stage following hypoxic stress. Experiments performed using the ischemia-reperfusion model show that ANGPTL4 has a cardioprotective role modulating both endothelium integrity and post-ischaemic inflammation.

Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells as well as in hypoxic perinecrotic areas of numerous cancers. Here, we investigated whether ANGPTL4 might affect tumor growth as well as metastasis. Metastatic 3LL cells were therefore xenografted into control mice and mice in which ANGPTL4 was expressed by using in vivo DNA electrotransfer. Whereas primary tumors grew at a similar rate in both groups, 3LL cells metastasized less efficiently to the lungs of mice that expressed ANGPTL4. Fewer 3LL emboli were observed in primary tumors, suggesting that intravasation of 3LL cells was inhibited by ANGPTL4. Furthermore, melanoma B16F0 cells injected into the retro-orbital sinus also metastasized less efficiently in mice expressing ANGPTL4. Although B16F0 cells were observed in lung vessels, they rarely invaded the parenchyma, suggesting that ANGPTL4 affects extravasation. In addition, recombinant B16F0 cells that overexpress ANGPTL4 were generated, showing a lower capacity for in vitro migration, invasion, and adhesion than control cells. Expression of ANGPTL4 induced reorganization of the actin cytoskeleton through inhibition of actin stress fiber formation and vinculin localization at focal contacts. Together, these results show that ANGPTL4, through its action on both vascular and tumor compartments, prevents the metastatic process by inhibiting vascular activity as well as tumor cell motility and invasiveness.

International audience; By triggering an adaptive response to hypoxia which is a common feature of tumor microenvironments, endothelial cells contribute to the onset of angiogenic responses involved in tumor growth. Therefore, identifying hypoxic markers represent a challenge for a better understanding of tumor angiogenesis and for the optimization of anti-angiogenic therapeutic strategy. Using representational difference analysis combined with microarray, we here report the identification of 133 hypoxia-induced transcripts in human microendothelial cells (HMEC-1). By Northern blot, we confirm hypoxia-induced expression of insulin-like growth factor binding protein 3 (igfbp3), thioredoxin-interacting protein (txnip), neuritin (nrn1). Finally, by performing in situ hybridization on several types of human tumors, we provide evidence for nrn1 and txnip as hypoxic perinecrotic markers and for igfbp3 as a tumor endothelial marker. We propose these hypoxia-induced genes could represent relevant prognostic tools and targets for therapeutic intervention in cancers.

BackgroundThe lipodystrophic syndrome (LD) is a disorder resulting from selective damage of adipose tissue by antiretroviral drugs included in therapy controlling human-immunodeficiency-virus-1. In the therapy cocktail the nucleoside reverse transcriptase inhibitors (NRTI) contribute to the development of this syndrome. Cellular target of NRTI was identified as the mitochondrial polymerase-gamma and their toxicity described as a mitochondrial DNA (mtDNA) depletion resulting in a mitochondrial cytopathy and involved in fat redistribution. No mechanisms offer explanation whatsoever for the lipo-atrophic and lipo-hypertrophic phenotype of LD. To understand the occurrence we proposed that the pO2 (oxygen partial pressure) could be a key factor in the development of the LD. For the first time, we report here differential effects of NRTIs on human adipose cells depending on pO2 conditions.Results and discussionWe showed that the hypoxia conditions could alter adipogenesis process by modifying expression of adipocyte makers as leptin and the peroxisome proliferator-activated receptor PPARgamma and inhibiting triglyceride (TG) accumulation in adipocytes. Toxicity of NRTI followed on adipose cells in culture under normoxia versus hypoxia conditions showed, differential effects of drugs on mtDNA of these cells depending on pO2 conditions. Moreover, NRTI-treated adipocytes were refractory to the inhibition of adipogenesis under hypoxia. Finally, our hypothesis that variations of pO2 could exist between adipose tissue from anatomical origins was supported by staining of the hypoxic-induced angiopoietin ANGPTL4 depended on the location of fat.ConclusionToxicity of NRTIs have been shown to be opposite on human adipose cells depending on the oxygen availability. These data suggest that the LD phenotype may be a differential consequence of NRTI effects, depending on the metabolic status of the targeted adipose tissues and provide new insights into the opposite effects of antiretroviral treatment, as observed for the lipo-atrophic and lipo-hypertrophic phenotype characteristic of LD.

Ischemic and solid tumor tissues are less well perfused than normal tissue, leading to metabolic changes and chronic hypoxia, which in turn promotes angiogenesis. We identified human angiopoietin-like 4 (angptl4) as a gene with hypoxia-induced expression in endothelial cells. We showed that the levels of both mRNA and protein for ANGPTL4 increased in response to hypoxia. When tested in the chicken chorioallantoic membrane assay, ANGPTL4 induced a strong proangiogenic response, independently of vascular endothelial growth factor. In human pathology, ANGPTL4 mRNA is produced in ischemic tissues, in conditions such as critical leg ischemia. In tumors, ANGPTL4 is produced in the hypoxic areas surrounding necrotic regions. We observed particularly high levels of ANGPTL4 mRNA in tumor cells of conventional renal cell carcinoma. Other benign and malignant renal tumor cells do not produce ANGPTL4 mRNA. This molecule therefore seems to be a marker of conventional renal cell carcinoma. ANGPTL4, originally identified as a peroxisome proliferator-activated receptor alpha and gamma target gene, has potential for use as a new diagnostic tool and a potential therapeutic target, modulating angiogenesis both in tumors and in ischemic tissues. This study also suggests that ANGPTL4 may provide a link between metabolic disorders and hypoxia-induced angiogenesis.

International audience; BACKGROUND The production of renin, which catalyses the rate-limiting step of the renin-angiotensin system, is strongly stimulated by a 225 bp enhancer element in the distal region of the promoter of the human gene (-5777 to -5552). OBJECTIVE To demonstrate the major role played by this enhancer in decoy experiments, to identify variants in this region, and to determine their effects on renin gene transcription. METHODS AND RESULTS We used this element as a decoy for transcription factors in human choriodecidual cells. The activity of the renin gene promoter was inhibited by 95% in the presence of this 225 bp enhancer element. This confirmation of the key role of this element suggested that changes in this region would be likely to affect renin gene expression. We therefore sequenced 70 genomic DNAs to identify variations in this region. We identified two new single nucleotide polymorphisms (SNPs) downstream from the 225 bp enhancer element at positions -5434 and -5312. We transfected choriodecidual cells with the four variants and found that a 592 bp region (-5870 to -5312) including the 225 bp element and the two SNPs had stronger enhancer activity than the 225 bp element alone, and that levels of transcription were 45% greater with the -5312T variant than with the -5312C variant, whereas none of the -5434 variants had an effect on renin transcription. Cis-regulatory elements close to the -5312 variant were identified in gel mobility shift assays on the basis of specific interactions between human choriodecidual cell nuclear extracts and an oligonucleotide including this polymorphism. CONCLUSION This study demonstrates that the human renin enhancer not only comprises the 225 bp element, but also extends to the region containing the -5312 SNP.

Background Macrophages, key regulators of healing/regeneration processes, strongly infiltrate ischemic tissues from patients suffering from critical limb ischemia (CLI). However pro-inflammatory markers correlate with disease progression and risk of amputation, suggesting that modulating macrophage activation state might be beneficial. We previously reported that thrombospondin-1 (TSP-1) is highly expressed in ischemic tissues during CLI in humans. TSP-1 is a matricellular protein that displays well-known angiostatic properties in cancer, and regulates inflammation in vivo and macrophages properties in vitro. We therefore sought to investigate its function in a mouse model of CLI. Methods and findings Using a genetic model of tsp-1-/- mice subjected to femoral artery excision, we report that tsp-1-/- mice were clinically and histologically protected from necrosis compared to controls. Tissue protection was associated with increased postischemic angiogenesis and muscle regeneration. We next showed that macrophages present in ischemic tissues exhibited distinct phenotypes in tsp-1-/- and wt mice. A strong reduction of necrotic myofibers phagocytosis was observed in tsp-1-/- mice. We next demonstrated that phagocytosis of muscle cell debris is a potent pro-inflammatory signal for macrophages in vitro. Consistently with these findings, macrophages that infiltrated ischemic tissues exhibited a reduced postischemic pro-inflammatory activation state in tsp-1-/- mice, characterized by a reduced Ly-6C expression and a less pro-inflammatory cytokine expression profile. Finally, we showed that monocyte depletion reversed clinical and histological protection from necrosis observed in tsp-1-/- mice, thereby demonstrating that macrophages mediated tissue protection in these mice. Conclusion This study defines targeting postischemic macrophage activation state as a new potential therapeutic approach to protect tissues from necrosis and promote tissue repair during CLI. Furthermore, our data suggest that phagocytosis plays a crucial role in promoting a deleterious intra-tissular pro-inflammatory macrophage activation state during critical injuries. Finally, our results describe TSP-1 as a new relevant physiological target during critical leg ischemia.

International audience; OBJECTIVE A silencer within the renin first intron (intron A) was identified using Calu-6 cells, a pulmonary carcinoma cell line which produced renin. In the present study, a dissection of the first intron was performed to determine precisely the cis-regulatory elements involved in the silencer transcriptional effects. MATERIALS AND METHODS Intron A was completely sequenced to characterize potential binding sites for known transcription factors. Partial portions of intron A were subcloned upstream the 892 bp of the renin promoter and transfected in different models of renin-producing cells: primary culture of human chorionic cells, human Calu-6 cells and mouse As4.1 cells. RESULTS There is significant DNA homology (67%) between the 3' and 5' ends of the human and rat renin first intron. Several transcription factor binding sites identified in human first intron, but not in rat intron, do not contribute to the reported silencer activity. Transfections of renin/ luciferase constructs containing partial portions of first intron inserted upstream of the 892 bp in both renin-producing cells do not allow the precise characterization of cis-elements involved in the silencer effect. CONCLUSIONS The silencer located renin intron A is cell specific. The integrity of the human first intron seems necessary for its repressor activity on renin proximal promoter in renin-producing cells.

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International audience; To delineate the cis-acting elements of the proximal promoter responsible for cAMP-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4 fold when the reporter plasmid contained the promoter region (-582 to +16). Mutation or deletion of the CRE diminished (1.7 fold) but did not abolish cAMP-induced transcription, demonstrating that region containing the CRE and region containing a Pit-1 site were both necessary for cAMP maximal induction. Taken together, these results show that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors (from chorionic and kidney cell origin), different from Pit-1, that interact with the Pit-1 response DNA elements.

International audience; Calu-6 cells were characterized for studying the transcriptional regulation of the human renin gene. Analysis of cis-acting elements of the renin promoter showed the highest activity within the first 582 bp in serum-free conditions and of the 892 bp in the presence of serum. cAMP activates renin mRNA synthesis parallel to renin production (20-fold increase) as well renin promoter activity (2-fold). cAMP response element and the (-77 to -67) element are both necessary for activation of the renin promoter but do not act independently. Functional analysis of Intron A revealed the presence of a silencer specific to renin-producing cells.

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International audience; Much knowledge was accumulated in the regulation of plasma renin activity and renin secretion during recent years. However, the mechanisms of renin gene transcription, especially for the human gene, have been poorly studied because of the lack of cell lines expressing renin. Cells derived from chorion tissue were used to study renin gene transcription because these cells express renin and regulate renin secretion in a similar way to JG cells. The present study was performed to determine the cis-regulatory elements and the trans-acting factors involved in human renin gene expression using chorionic cells. Transient DNA transfections were performed with various constructs containing the 5'-flanking region of the human renin gene. 5'-Deletion analysis of the human renin promoter (from -2616 to -67 bp) revealed the presence of two proximal negative cis-regulatory elements between -374 and -273 bp and between -273 and -137 bp. These elements were not present in a non-renin-producing cell line, JEG-3 cells. DNase I footprinting revealed that two sequences located within these regions bind trans-factors present in chorionic cellular nuclear extract: AGE3-like sequence (-293/-273) and apolipoprotein A1 regulatory protein-1-like sequence (-259/-245). The first 110 bp of the renin promoter were sufficient to direct specific expression in chorionic cells and contained two footprints sharing homology with ets (-29/-6) and pituitary-specific factor (Pit-1) (-70/-62) sequences. Furthermore, one footprint (-234/-214) contained the sequence TAGCGTCA, which shares strong homology to the cAMP-responsive element (CRE) binding site. Gel shift analysis showed specific DNA/protein complexes within this region, which were displaced by the somatostatin consensus CRE. Finally, luciferase analysis of 5'-deletion mutant revealed that -273 to +16 bp of the renin promoter was sufficient to confer complete forskolin stimulation, whereas deletion to -130 (deletion of the CRE) decreased cAMP responsiveness by 50% and those to -67 bp (deletion of the CRE and Pit-1-like sequences) suppressed it. Thus, these latter two sequences probably act together to confer complete cAMP responsiveness.

De nombreux facteurs epigenetiques, dont l’ARN interference module l’expression genique au cours des pathologies cardiovasculaires. Bien que les micro-ARNs (miARN) soient exprimes dans le systeme cardiovasculaire, leur role est jusqu’alors peu connu. L’objectif de ce travail est d’etudier le role de l’ARN interference dans la regulation de l’angiogenese developpementale et pathologique par invalidation de DICER in vivo specifiquement dans les cellules musculaires lisses (CML). Nous disposons de souris dont le gene dicer est floxe (allele dicer conditionnel) 1 , permettant d’invalider specifiquement Dicer dans les cellules exprimant une recombinase Cre sous le controle de promoteurs specifiques des cellules musculaires lisses (α-SMA ; SM-MHC ; sm22), Ces souris ont aussi ete croisees avec des souris R26R permettant d’analyser les cellules recombinees 2 . Nos premiers resultats montrent que les embryons dont le gene dicer sous le controle du promoteur sm22 presentent des anomalies de developpement embryonnaire a E16,5 (figure 1). Les embryons mutants souffrent d’hemorragies importantes entrainant la mort. La mort embryonnaire ne correspond a priori pas a des defauts de vasculogenese importants, mais est plutot caracteristique de defauts de remodelage angiogenique tels qu’une permeabilite endotheliale exacerbee ou une mauvaise differenciation des pericytes, induisant des vaisseaux immatures et permeables. Nous analysons donc les defauts vasculaires responsables de la mort embryonnaire en etudiant la morphologie des vaisseaux sanguins des souris mutantes, au cours du developpement, par marquage specifique des veines, des arteres, des CML ou des pericytes et des vaisseaux lymphatiques par marquages immunohistochimiques CD31, α-SMA, NG2, et VEGF-R3. Download : Download full-size image

International audience; To delineate the cis-acting elements of the proximal promoter responsible for cyclic AMP (cAMP)-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4-fold when the reporter plasmid contained the promoter region (-582 to + 16). Mutation or deletion of the cAMP response element (CRE) diminished (1.7-fold) but did not abolish cAMP-induced transcription, demonstrating that the (-582 to -145) region containing the CRE and the region (-145 to -38) containing a Pit-1 (pituitary-specific trans-acting factor) site were both necessary for cAMP maximal induction. To study the molecular events mediating the cAMP induction, DNase I footprinting and electromobility shift assays (EMSAs) were performed with renin-producing chorionic cell and kidney cortex cell nuclear extracts, showing that the CRE-binding protein (CREB) interacts with the CRE and that tissue-specific factors, distinct from Pit-1, specifically bind the renin Pit-1 motif. Taken together, these results demonstrate that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors, different from Pit-1, that interact with the Pit-1 response DNA elements.

Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells. It is highly expressed in tumor cells from conventional renal carcinoma and in hypoxic perinecrotic areas of numerous types of human tumors (1). We previously showed that ANGPTL4, through its action on both vascular and tumor cells, prevents metastastes through inhibition of vascular permeability as well as tumor cell motility and invasiveness. In vivo, using both Lewis Lung carcinoma cells as well as melanoma B16 cells, we showed ANGPTL4 inhibits both intravasation extravasation of tumor cells. Using Miles assay, ANGPTL4 inhibited the histamine-induced vascular permeability in electrotransferred mice overexpressing ANGPTL4 compared to control mice (2). More recently, Padua et al. revealed a clinical association between TGFbeta activity in primary tumors and risk of distant recurrence, specifically for estrogen receptor negative breast tumors with lung metastasis but not bone metastasis. In vivo, lung metastasis seeding from mammary tumors depends on TGFbeta receptors, Smad function and ANGPTL4 expression (3). Both groups therefore report ANGPTL4 as a key regulator of vascular permeability. Nevertheless, better insights are needed in order to precisely characterize its role during tumor angiogenesis and subsequent metastases in various organs, namely lungs and bones. The aim of the present study is to address the in vivo role of ANGPTL4 in modulating vascular integrity, using Lewis Lung carcinoma cells xenografted in ANGPTL4 KO mice. Inhibition of tumor growth is observed in KO mice compared to WT mice (1207+/-105 mm3 versus 3053+/-569 mm3 at day 26, p=0.002). At day 33, 60 % of the angptl4 KO mice show large lung macrometastases whereas only 28 % of wild type lungs have been invaded by small micrometastases. Further experiments using different metastatic models are ongoing in order to perform imaging analysis of blood vessels and endothelium integrity the during the metastatic process in angptl4 knock-out and WT mice.1. Le Jan, S. (2003) Am J Pathol 162, 1521-8.2. Galaup, A. (2006) Proc Natl Acad Sci U S A 103, 18721-6.3. Padua, D. (2008) Cell 133, 66-77.