Matrix metalloproteinase-2 governs lymphatic vessel formation as an interstitial collagenase.
Detry, Benoît ; Erpicum, Charlotte ; Paupert, Jenny et al
in Blood (2012), 119(21), 5048-56
Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to ... [more ▼]
Lymphatic dysfunctions are associated with several human diseases, including lymphedema and metastatic spread of cancer. Although it is well recognized that lymphatic capillaries attach directly to interstitial matrix mainly composed of fibrillar type I collagen, the interactions occurring between lymphatics and their surrounding matrix have been overlooked. In this study, we demonstrate how matrix metalloproteinase (MMP)–2 drives lymphatic morphogenesis through Mmp2-gene ablation in mice, mmp2 knockdown in zebrafish and in 3D-culture systems, and through MMP2 inhibition. In all models used in vivo (3 murine models and thoracic duct development in zebrafish) and in vitro (lymphatic ring and spheroid assays), MMP2 blockage or down-regulation leads to reduced lymphangiogenesis or altered vessel branching. Our data show that lymphatic endothelial cell (LEC) migration through collagen fibers is affected by physical matrix constraints (matrix composition, density and cross-linking). Transmission electron microscopy (TEM) and confocal reflection microscopy using DQ-collagen highlight the contribution of MMP2 to mesenchymal-like migration of LEC associated with collagen fiber remodeling. Our findings provide new mechanistic insight into how LEC negotiate an interstitial type I collagen barrier and reveal an unexpected MMP2-driven collagenolytic pathway for lymphatic vessel formation and morphogenesis. [less ▲]Detailed reference viewed: 119 (55 ULg)
Dre-miR-2188 Targets Nrp2a and Mediates Proper Intersegmental Vessel Development in Zebrafish Embryos.
; ; et al
in PLoS ONE (2012), 7(6), 39417
BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed ... [more ▼]
BACKGROUND: MicroRNAs (miRNAs) are a class of small RNAs that are implicated in the control of eukaryotic gene expression by binding to the 3'UTR of target mRNAs. Several algorithms have been developed for miRNA target prediction however, experimental validation is still essential for the correct identification of miRNA targets. We have recently predicted that Neuropilin2a (Nrp2a), a vascular endothelial growth factor receptor which is essential for normal developmental angiogenesis in zebrafish, is a dre-miR-2188 target. METHODOLOGY: Here we show that dre-miR-2188 targets the 3'-untranslated region (3'UTR) of Nrp2a mRNA and is implicated in proper intersegmental vessel development in vivo. Over expression of miR-2188 in zebrafish embryos down regulates Nrp2a expression and results in intersegmental vessel disruption, while its silencing increases Nrp2a expression and intersegmental vessel sprouting. An in vivo GFP sensor assay based on a fusion between the GFP coding region and the Nrp2a 3'UTR confirms that miR-2188 binds to the 3'UTR of Nrp2a and inhibits protein translation. CONCLUSIONS: We demonstrate that miR-2188 targets Nrp2a and affects intersegmental vessel development in zebrafish embryos. [less ▲]Detailed reference viewed: 10 (1 ULg)
Novel HDAC/DNMT Twin Inhibitors Interfere with Angiogenesis
Shiva Shankar, Thammadihalli Veerasangaiah ; Sulka, Béatrice ; Blacher, Silvia et al
DNA methylation and histone deacetylation are two key epigenetic modifications that play central role in regulation of gene expression. Several studies have shown that histone deacetylases (HDAC) and DNA ... [more ▼]
DNA methylation and histone deacetylation are two key epigenetic modifications that play central role in regulation of gene expression. Several studies have shown that histone deacetylases (HDAC) and DNA methyltransferases (DNMT) inhibitors are potent anti-angiogenic compounds. Though combination of HDAC and DNMT inhibitors are now being examined in clinical trials of hematological malignancies, little work has been done to understand the effect of this combination on physiological and tumoral angiogenesis. We have designed and tested a family of twin drugs with intrinsic HDAC and DNMT inhibitory activities in relevant models of angiogenesis in vitro (Human Umbilical Vein Endothelial Cells – HUVEC and aortic ring) and in vivo (chick chorioallantoic membrane and Zebrafish). We have identified a lead compound having quantifiable anti-angiogenic effect without cytotoxicity affecting global histone acetylation and DNA methylation levels. In order to elucidate its anti-angiogenic mechanism, we characterized gene expression pattern simultaneously with the methylation profile of HUVEC cells treated with the lead compound and reference epigenetic modulators. This approach based on parallel microarray analyses permitted us to underscore a list of genes exclusively affected by the lead compound but not by other HDAC or DNMT inhibitors. These genes were then analyzed using the Ingenuity Pathway software revealing potential involvement of a subset of genes in angiogenesis. Our present aim is to validate the expression levels of a series of genes with respect to epigenetic mechanisms (histone modifications and DNA methylation). Finally, the biological relevance of the target genes will be explored by RNA silencing. Hence, we are using these novel epigenetic modulators as a tool to understand the regulatory mechanism of angiogenesis and to develop effective approaches to treat cancer. [less ▲]Detailed reference viewed: 157 (51 ULg)
Involvement of miR-125b in in vitro and in vivo angiogenesis
; ; et al
Poster (2010, May 21)Detailed reference viewed: 7 (0 ULg)
Involvement of miR-125b in in vitro and in vivo angiogenesis
; ; et al
Poster (2010, March)Detailed reference viewed: 5 (0 ULg)
Study of the role of microRNAs in angiogenesis
; ; et al
Poster (2009, May 29)Detailed reference viewed: 2 (0 ULg)
Cloning and embryonic expression of zebrafish PLAG genes.
; Peers, Bernard ; et al
in Gene Expression Patterns (2006), 6(3), 267-76
PLAG transcription factors play important roles in oncogenesis. To date three members of this subfamily of zinc finger proteins have been identified in humans and mice: PLAG1, PLAGL1 and PLAGL2. In this ... [more ▼]
PLAG transcription factors play important roles in oncogenesis. To date three members of this subfamily of zinc finger proteins have been identified in humans and mice: PLAG1, PLAGL1 and PLAGL2. In this study, we identified zebrafish orthologs of PLAG1 and PLAGL2 and a novel member of this family, PLAGX. We examined the temporal expression of these three genes by quantitative real time RT-PCR and found that all three genes are maternally provided, expressed at low level during early somitogenesis and, during late somitogenesis and beyond, PLAG expression increases to reach a plateau level around 5 dpf. Whole mount in situ experiments revealed that PLAG1, PLAGL2 and PLAGX display a similar pattern of expression characterized by a low ubiquitous expression overcame by high expression in some restricted compartments such as the ventricular zone of the brain, the pectoral fin buds, the developing pharyngeal arches and the axial vasculature. We show that this pattern resembles the one observed for the proliferative marker PCNA, suggesting that the PLAG genes are expressed more strongly in zones of active proliferation. This hypothesis was proven for the ventricular zone shown to be a highly proliferative zone using the anti-phosphohistone H3 antibody that detects cells in mitosis. [less ▲]Detailed reference viewed: 10 (4 ULg)
The Na+/PO4 cotransporter SLC20A1 gene labels distinct restricted subdomains of the developing pronephros in Xenopus and zebrafish embryos.
; ; et al
in Gene Expression Patterns (2006), 6(7), 667-72
The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the ... [more ▼]
The embryonic pronephric kidneys of Xenopus and zebrafish serve as models to study vertebrate nephrogenesis. Recently, multiple subdomains within the Xenopus pronephros have been defined based on the expression of several transport proteins. In contrast, very few studies on the expression of renal transporters have been conducted in zebrafish. We have recently shown that the anterior and posterior segments of the zebrafish pronephric duct may correspond to the proximal tubule and distal tubule/duct compartments of the Xenopus and higher vertebrate pronephros, respectively. Here, we report the embryonic expression pattern of the Na(+)/PO(4) cotransporter SLC20A1 (PiT1/Glvr-1) gene encoding a type III sodium-dependent phosphate cotransporter in Xenopus and zebrafish. In Xenopus, SLC20A1 mRNA is expressed in the somitic mesoderm and lower level of expression is detected in the neural tube, eye, and neural crest cells. From stage 25, SLC20A1 is also detectable in the developing pronephros where expression is restricted to the late portion of the distal pronephric tubules. In zebrafish, SLC20A1 is transcribed from mid-somitogenesis in the anterior part of the pronephros where its expression corresponds to the rostral portion of the expression of other proximal tubule-specific markers. Outside the pronephros, lower level of SLC20A1 expression is also observed in the posterior cardinal and caudal veins. Based on the SLC20A1 expression domain and that of other transporters, four segments have been defined within the zebrafish pronephros. Together, our data reveal that the zebrafish and Xenopus pronephros have non-identical proximo-distal organizations. [less ▲]Detailed reference viewed: 26 (4 ULg)
Evi1 is specifically expressed in the distal tubule and duct of the Xenopus pronephros and plays a role in its formation.
; ; et al
in Developmental Biology (2006), 294(1), 203-19
The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus ... [more ▼]
The ecotropic viral integration site 1 (Evi1) and related MEL1 (MDS1/Evi1-like gene 1) genes are zinc finger oncogenic transcription factors involved in myeloid leukaemia. Here, we show that in Xenopus, Evi1 and MEL1 have partially overlapping restricted embryonic expression profiles. Within the pronephros, Evi1 and MEL1 are sequentially expressed within the distal tubule and duct compartments, Evi1 transcription being detected prior to any sign of pronephric morphogenesis. In the pronephros of zebrafish embryos, Evi1 expression is restricted to the posterior portion of the duct, the anterior portion having characteristics of proximal tubules. In the Xenopus pronephros, Evi1 expression is upregulated by retinoid signaling and repressed by overexpression of xWT1 and by Notch signaling. Overexpression of Evi1 from late neurula stage specifically inhibits the expression of proximal tubule and glomus pronephric markers. We show that the first zinc finger and CtBP interaction domains are required for this activity. Overexpression of a hormone-inducible Evi1-VP16 antimorphic fusion with activation at neurula stage disrupts distal tubule and duct formation and expands the expression of glomus markers. Although overexpression of this construct also causes in many embryos a reduction of proximal tubule markers, embryos with expanded and ectopic staining have been also observed. Together, these data indicate that Evi1 plays a role in the proximo-distal patterning of the pronephros and suggest that it may do so by functioning as a CtBP dependent repressor. [less ▲]Detailed reference viewed: 19 (1 ULg)
Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.
; Voz, Marianne ; et al
in Human Molecular Genetics (2006), 15(22), 3369-77
Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans ... [more ▼]
Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling. [less ▲]Detailed reference viewed: 18 (5 ULg)
Microarray screening for target genes of the proto-oncogene PLAG1.
Voz, Marianne ; ; et al
in Oncogene (2004), 23(1), 179-91
PLAG1 is a proto-oncogene whose ectopic expression can trigger the development of pleomorphic adenomas of the salivary glands and of lipoblastomas. As PLAG1 is a transcription factor, able to activate ... [more ▼]
PLAG1 is a proto-oncogene whose ectopic expression can trigger the development of pleomorphic adenomas of the salivary glands and of lipoblastomas. As PLAG1 is a transcription factor, able to activate transcription through the binding to the consensus sequence GRGGC(N)(6-8)GGG, its ectopic expression presumably results in the deregulation of target genes, leading to uncontrolled cell proliferation. The identification of PLAG1 target genes is therefore a crucial step in understanding the molecular mechanisms involved in PLAG1-induced tumorigenesis. To this end, we analysed the changes in gene expression caused by the conditional induction of PLAG1 expression in fetal kidney 293 cell lines. Using oligonucleotide microarray analyses of about 12 000 genes, we consistently identified 47 genes induced and 12 genes repressed by PLAG1. One of the largest classes identified as upregulated PLAG1 targets consists of growth factors such as the insulin-like growth factor II and the cytokine-like factor 1. The in silico search for PLAG1 consensus sequences in the promoter of the upregulated genes reveals that a large proportion of them harbor several copies of the PLAG1-binding motif, suggesting that they represent direct PLAG1 targets. Our approach was complemented by the comparison of the expression profiles of pleomorphic adenomas induced by PLAG1 versus normal salivary glands. Concordance between these two sets of experiments pinpointed 12 genes that were significantly and consistently upregulated in pleomorphic adenomas and in PLAG1-expressing cells, identifying them as putative PLAG1 targets in these tumors. [less ▲]Detailed reference viewed: 12 (1 ULg)