Search

L’ARN interférence, mécanisme de régulation de l’expression des gènes, est médiée par les siARNs et les microARNs, ARN non-codants de 20 à 22 nucléotides affectant la régulation post-transcriptionnelle d’ARNm cibles avec lesquels ils s’apparient. La RNase DICER est une enzyme centrale de la biosynthèse des siARNs et microARNs. Les souris dont le gène dicer est invalidé ont un phénotype complexe, et meurent très tôt pendant le développement, notamment à cause d’un défaut d’angiogenèse. Afin d’étudier l’ARN interférence au cours de l’angiogenèse embryonnaire, des souris dont le gène dicer est floxé (mutant conditionnel) sont croisées avec des souris exprimant la recombinase Cre, de manière constitutive, sous le contrôle du promoteur du gène tie2, dirigeant ainsi son expression dans les cellules endothéliales (CE) et les cellules hématopiétiques. Nos résultats montrent que l’invalidation de dicer sous le contrôle du promoteur du gène tie2 entraine une mortalité embryonnaire suite à un œdème et des hémorragies au treizième jour du développement (E13,5). L’analyse histologique montre des vaisseaux lymphatiques remplis de sang, suggérant une mauvaise séparation du réseau sanguin et lymphatique. Cette hypothèse est étudiée par marquage des vaisseaux lymphatiques (LYVE-1) et des vaisseaux sanguins (PECAM) sur embryon entier et peaux isolées à différents stades précédant la mort. Ces embryons présentent également un problème de développement du foie, probablement dû à l’activité du promoteur tie2 dans les lignées hématopoiétiques. La mise en culture de ces foies fœtaux à E13,5 révèle une atteinte des précurseurs hématopoétiques. L’étude de ces précurseurs à des stades plus précoces (E8,5) est en cours au laboratoire. Nos résultats démontrent donc un rôle important de l’ARN interférence dans le contrôle épigénétique de l’angiogenèse et de la lymphangiogenèse embryonnaire mais également dans le développement de l’hématopoièse, suggérant son implication dans la différenciation veino-lymphangiogenèse, dont les mécanismes moléculaires seront discutés. [Copyright &y& Elsevier]

De nombreux facteurs épigénétiques, dont l’ARN interférence module l’expression génique au cours des pathologies cardiovasculaires. Bien que les micro-ARNs (miARN) soient exprimés dans le système cardiovasculaire, leur rôle est jusqu’alors peu connu. L’objectif de ce travail est d’étudier le rôle de l’ARN interférence dans la régulation de l’angiogenèse développementale et pathologique par invalidation de DICER in vivo spécifiquement dans les cellules musculaires lisses (CML). Nous disposons de souris dont le gène dicer est floxé (allèle dicer conditionnel) 1 [1] Harfe BD, McManus MT, Mansfield JH, Hornstein E, Tabin CJ: The RNaseIII enzyme Dicer is required for morphogenesis but not patterning of the vertebrate limb, Proc Natl Acad Sci U S A 2005, 102:10898-10903. , permettant d’invalider spécifiquement Dicer dans les cellules exprimant une recombinase Cre sous le contrôle de promoteurs spécifiques des cellules musculaires lisses (α-SMA ; SM-MHC ; sm22), Ces souris ont aussi été croisées avec des souris R26R permettant d’analyser les cellules recombinées 2 [2] Soriano P: Generalized lacZ expression with the ROSA26 Cre reporter strain, Nat Genet 1999, 21:70-71. . Nos premiers résultats montrent que les embryons dont le gène dicer sous le contrôle du promoteur sm22 présentent des anomalies de développement embryonnaire à E16,5 (figure 1). Les embryons mutants souffrent d’hémorragies importantes entraînant la mort. La mort embryonnaire ne correspond à priori pas à des défauts de vasculogenèse importants, mais est plutôt caractéristique de défauts de remodelage angiogénique tels qu’une perméabilité endothéliale exacerbée ou une mauvaise différenciation des péricytes, induisant des vaisseaux immatures et perméables. Nous analysons donc les défauts vasculaires responsables de la mort embryonnaire en étudiant la morphologie des vaisseaux sanguins des souris mutantes, au cours du développement, par marquage spécifique des veines, des artères, des CML ou des péricytes et des vaisseaux lymphatiques par marquages immunohistochimiques CD31, α-SMA, NG2, et VEGF-R3. Display Omitted [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Hypoxia stimulates angiogenesis during development, as well as in the course of ischemic cardiovascular diseases and tumor growth. In a hypoxic environment, endothelial cells (EC) are key players of the angiogenic response. Their activation leads to remodeling of the extracellular matrix (ECM): a degradation step generates a provisional ECM supporting EC proliferation and migration; assembly of a new basal lamina leads to pericyte recruitment and neovessel maturation. In order to identify endothelial ECM proteins regulated by hypoxia, 2D gel electrophoresis was performed on ECM samples prepared from cultured EC of micro or large vessels (HDMEC or HUVEC respectively). Mass spectrometry identified lysyl oxidase-like protein 2 (LOXL2) as a highly hypoxia-induced protein. Lysyl oxidases are secreted enzymes involved in ECM maturation through covalent cross-linking of its major components, collagens and elastin. The induction of LOXL2 expression was detected both at the mRNA and protein levels. Using siRNA, we demonstrated that LOXL2 is responsible for 65 % of lysyl oxidase total activity in hypoxic EC. In addition, LOXL2 protein was colocalised with type IV collagen in endothelial ECM. We further investigated the expression of LOXL2 in vivo. The enzyme was expressed in rat EC from retina during postnatal vascular development, and from adult skeletal muscle. In a murine model of hindlimb ischemia, LOXL2 was upregulated at the protein and mRNA levels. In situ hybridization revealed its expression in both EC and macrophages. Involvement of LOXL2 at different steps of the angiogenic process was further studied in vitro. We demonstrated that LOXL2 increases EC migration on fibronectin, as well as migration and tube formation in fibrin 3D gels. In addition, LOXL2 knock-down inhibited type IV collagen deposition in the ECM, suggesting a major role for LOXL2 in the organisation of endothelial basal lamina. Finally, whereas the efficiency of endothelial ECM for recruiting VSMC was increased by hypoxia, this effect was reduced upon knocking down endothelial LOXL2 expression. Altogether, these data suggest that LOXL2 plays a major role in EC migration, vascular basal lamina deposition and neovessel stabilisation during hypoxia-induced angiogenesis. [Copyright &y& Elsevier]

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. [Copyright &y& Elsevier]

Hypoxia is a potent inducer of angiogenesis during which endothelial cells (ECs) undergo modulations of cell-cell and cell-matrix interactions. ANGPTL4 expression is induced by hypoxia in ECs (1,2) and interacts with the extracellular matrix (3) modulating ECs adhesion, migration and sprouting through cytoskeleton reorganisation (4). Objective: Analyse whether in vivo ANGPTL4 plays a role in modulating hypoxia-driven angiogenesis in the retina, both in development and in vascular retinal pathologies. Materials: C57/Bl6 angptl4 KO mice and WT littermates were used. An hyperoxia chamber was used to establish the oxygen-induced retinopathy (OIR). Mice were exposed to 75 % oxygen from P7 to P12 leading to retinal vessel loss. After returning to room air, hypoxia induces neovascularisation, evaluated at P17 when greatest. Results: Developmental angiogenesis of the retina in P5-P7 pups was analysed. Angptl4 is expressed by ECs and angptl4 -/- retinas show a decreased number of branchpoints (p<0,005) as well as an increased vascular density (p<0,001). Angptl4 -/- veins were dilated compared to angptl4+/- (p<0,001). Altogether, loss of angptl4 expression in the developing retina lead to an immature vascular plexus. Moreover, angptl4 -/- retinas showed an increased basal vascular leakage (p<0,0,05). Confocal analysis also showed that pericyte coverage might be involved. We also show that ANGPTL4 is expressed during OIR by ECs. Retinal wholemount preparations stained with isolectin B4 were performed. At P12, the retinal vaso-obliterated area was not affected in angptl4 -/- mice, suggesting that ANGPTL4 has no effect in the hyperoxia-induced vaso-obliteration. In contrast, the neovascular angiogenic response was significantly alteredin angptl4 -/- mice at P17 (p<0,001). Thus, ANGPTL4 is essential for regulating the growth of new retinal vessels in an ischemic environment. Conclusions: Altogether, this data demonstrate that ANGPTL4 plays a critical role in hypoxia-induced angiogenesis, both in developmental and pathological conditions, by affecting 1) vascular density, 2) basal vascular permeability, and 3) pericyte coverage. (1)Le Jan S, Am J Path. 2003 (2)Galaup A, Proc Natl Acad Sci U S A. 2006 (3)Cazes A, Circ Res. 2006 (4)Chomel C, FASEB J. 2008 [Copyright &y& Elsevier]

Abstract: Renin plays a critical role in fluid and electrolyte homeostasis by cleaving angiotensinogen to produce Ang peptides. Whilst it has been demonstrated that renin mRNA is expressed in the brain, the distribution of cells responsible for this expression remains uncertain. We have used a transgenic mouse approach in an attempt to address this question. A transgenic mouse, in which a 12.2 kb fragment of the human renin promoter was used to drive expression of Cre-recombinase, was crossed with the ROSA26-lac Z reporter mouse strain. Cre-recombinase mediated excision of the floxed stop cassette resulted in expression of the reporter protein, β-galactosidase. This study describes the distribution of β-galactosidase in the brain of the crossed transgenic mouse. In all cases where it was examined the reporter protein was co-localized with the neuronal marker NeuN. An extensive distribution was observed with numerous cells labeled in the somatosensory, insular, piriform and retrosplenial cortices. The motor cortex was devoid of labeled cells. Several other regions were labeled including the parts of the amygdala, periaqueductal gray, lateral parabrachial nucleus and deep cerebellar nuclei. Overall the distribution shows little overlap with those regions that are known to express receptors for the renin–angiotensin system in the adult brain. This transgenic approach, which demonstrates the distribution of cells which have activated the human renin promoter at any time throughout development, yields a unique and extensive distribution of putative renin-expressing neurons. Our observations suggest that renin may have broader actions in the brain and may indicate a potential for interaction with the (pro)renin receptor or production of a ligand for non-AT1/AT2 receptors. [Copyright &y& Elsevier]