Stefania Nicoli

MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis.

Within the circulatory system, blood flow regulates vascular remodelling, stimulates blood stem cell formation, and has a role in the pathology of vascular disease. During vertebrate embryogenesis, vascular patterning is initially guided by conserved genetic pathways that act before circulation. Subsequently, endothelial cells must incorporate the mechanosensory stimulus of blood flow with these early signals to shape the embryonic vascular system. However, few details are known about how these signals are integrated during development. To investigate this process, we focused on the aortic arch (AA) blood vessels, which are known to remodel in response to blood flow. By using two-photon imaging of live zebrafish embryos, we observe that flow is essential for angiogenesis during AA development. We further find that angiogenic sprouting of AA vessels requires a flow-induced genetic pathway in which the mechano-sensitive zinc finger transcription factor klf2a induces expression of an endothelial-specific microRNA, mir-126, to activate Vegf signalling. Taken together, our work describes a novel genetic mechanism in which a microRNA facilitates integration of a physiological stimulus with growth factor signalling in endothelial cells to guide angiogenesis.

Mammalian Tumor Xenografts Induce Neovascularization in Zebrafish Embryos

The zebrafish (Danio rerio)/tumor xenograft model represents a powerful new model system in cancer. Here, we describe a novel exploitation of the zebrafish model to investigate tumor angiogenesis, a pivotal step in cancer progression and target for antitumor therapies. Human and murine tumor cell lines that express the angiogenic fibroblast growth factor (FGF) 2 and/or vascular endothelial growth factor (VEGF) induce the rapid formation of a new microvasculature when grafted close to the developing subintestinal vessels of zebrafish embryos at 48 h postfertilization. Instead, no angiogenic response was exerted by related cell clones defective in the production of these angiogenic growth factors. The newly formed blood vessels sprout from the subintestinal plexus of the zebrafish embryo, penetrate the tumor graft, and express the transcripts for the zebrafish orthologues of the early endothelial markers Fli-1, VEGF receptor-2 (VEGFR2/KDR), and VE-cadherin. Accordingly, green fluorescent protein-positive neovessels infiltrate the graft when tumor cells are injected in transgenic VEGFR2:G-RCFP zebrafish embryos that express green fluorescent protein under the control of the VEGFR2/KDR promoter. Systemic exposure of zebrafish embryos immediately after tumor cell injection to prototypic antiangiogenic inhibitors, including the FGF receptor tyrosine kinase inhibitor SU5402 and the VEGFR2/KDR tyrosine kinase inhibitor SU5416, suppresses tumor-induced angiogenesis without affecting normal blood vessel development. Accordingly, VE-cadherin gene inactivation by antisense morpholino oligonucleotide injection inhibits tumor neovascularization without affecting the development of intersegmental and subintestinal vessels. These data show that the zebrafish/tumor xenograft model represents a novel tool for investigating the neovascularization process exploitable for drug discovery and gene targeting in tumor angiogenesis.

The Zebrafish/Tumor Xenograft Angiogenesis Assay

Here we describe a method to study tumor angiogenesis in zebrafish (Danio rerio) based on the injection of proangiogenic mammalian tumor cells into the perivitelline space of zebrafish embryos at 48 h post-fertilization. Within 24–48 h, proangiogenic tumor grafts induce a neovascular response originating from the developing subintestinal vessels. This can be observed at macroscopic and microscopic levels after whole-mount alkaline phosphatase staining of wild-type zebrafish embryos, or by fluorescence microscopy in transgenic VEGFR2:G-RCFP embryos in which endothelial cells express the green fluorescent protein under the control of the VEGFR2/KDR promoter. Angiogenesis inhibitors added to the injected cell suspension or to the fish water prevent tumor-induced neovascularization. The assay is rapid and inexpensive, representing a novel tool for investigating tumor angiogenesis and for antiangiogenic drug discovery. Also, gene inactivation by antisense morpholino oligonucleotides injection in zebrafish embryos may allow the identification of genes involved in tumor angiogenesis.

Fibroblast Growth Factor Receptor-1 Is Essential for In Vitro Cardiomyocyte Development

Abstract— Fibroblast growth factor (FGF)/FGF receptor (FGFR) signaling plays a crucial role in mesoderm formation and patterning. Heartless mutant studies in Drosophila suggest that FGFR1, among the different FGFRs, may play a role in cardiogenesis. However, fgfr1−/− mice die during gastrulation before heart formation. To establish the contribution of FGFR1 in cardiac development, we investigated the capacity of murine fgfr1+/− and fgfr1−/− embryonic stem (ES) cells to differentiate to cardiomyocytes in vitro. Clusters of pulsating cardiomyocytes were observed in >90% of 3-dimensional embryoid bodies (EBs) originated from fgfr1+/− ES cells at day 9 to 10 of differentiation. In contrast, 10% or less of fgfr1−/− EBs showed beating foci at day 16. Accordingly, fgfr1−/− EBs were characterized by impaired expression of early cardiac transcription factors Nkx2.5 and d-Hand and of late structural cardiac genes myosin heavy chain (MHC)-&agr;, MHC-&bgr;, and ventricular myosin light chain. Homozygous fgfr1 mutation resulted also in alterations of the expression of mesoderm-related early genes, including nodal, BMP2, BMP4, T (bra), and sonic hedgehog. Nevertheless, fgfr1+/− and fgfr1−/− EBs similarly express cardiogenic precursor, endothelial, hematopoietic, and skeletal muscle markers, indicating that fgfr1-null mutation exerts a selective effect on cardiomyocyte development in differentiating ES cells. Accordingly, inhibitors of FGFR signaling, including the FGFR1 tyrosine kinase inhibitor SU 5402, the MEK1/2 inhibitor U0126, and the protein kinase C inhibitor GF109 all prevented cardiomyocyte differentiation in fgfr1+/− EBs without affecting the expression of the hematopoietic/endothelial marker flk-1. In conclusion, the data point to a nonredundant role for FGFR1-mediated signaling in cardiomyocyte development.

A truncation allele in vascular endothelial growth factor c reveals distinct modes of signaling during lymphatic and vascular development

Vascular endothelial growth factor C (Vegfc) is a secreted protein that guides lymphatic development in vertebrate embryos. However, its role during developmental angiogenesis is not well characterized. Here, we identify a mutation in zebrafish vegfc that severely affects lymphatic development and leads to angiogenesis defects on sensitized genetic backgrounds. The um18 mutation prematurely truncated Vegfc, blocking its secretion and paracrine activity but not its ability to activate its receptor Flt4. When expressed in endothelial cells, vegfcum18 could not rescue lymphatic defects in mutant embryos, but induced ectopic blood vessel branching. Furthermore, vegfc-deficient endothelial cells did not efficiently contribute to tip cell positions in developing sprouts. Computational modeling together with assessment of endothelial cell dynamics by time-lapse analysis suggested that an autocrine Vegfc/Flt4 loop plays an important role in migratory persistence and filopodia stability during sprouting. Our results suggest that Vegfc acts in two distinct modes during development: as a paracrine factor secreted from arteries to guide closely associated lymphatic vasculature and as an autocrine factor to drive migratory persistence during angiogenesis.

Fibroblast growth factor 2-induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay

Angiogenesis plays a key role in tumour growth and metastasis. The teleost zebrafish (Danio rerio) represents a promising alternative model in cancer research. Here, we describe a zebrafish yolk membrane (ZFYM) angiogenesis assays based on the injection of 1–30 ng of human recombinant FGF2 (rFGF2) in the perivitelline space of zebrafish embryos in the proximity of developing subintestinal vein vessels (SIVs) at 48 hrs after fertilization. The rFGF2 induces a rapid and dose‐dependent angiogenic response from the SIV basket, characterized by the ectopic growth of newly formed, alkaline phosphatase‐positive blood vessels. These vessels are formed by proliferating cells that incorporate bromodeoxyuridine and express the endothelial cell markers vegfr2/kdr and fli1. Microangiography shows that rFGF2‐induced vessels are patent and connected to the systemic circulation of the embryo. In keeping with these observations, fli1:EGFP+ cells isolated from transgenic tg(fli1:EGFP)y1 zebrafish embryos express the tyrosine kinase (TK) FGF receptor‐1 (FGFR1) and activate extracellular signal‐regulated kinase signalling when stimulated in vitro by rFGF2. The low molecular weight TK‐FGFR1 inhibitor SU5402 and the high molecular weight FGF2 antagonist long‐pentraxin 3 inhibit the angiogenic activity of rFGF2 when added to fish water or when co‐injected with the growth factor, respectively. Moreover, similar to rFGF2, injection of the zebrafish form of vascular endothelial growth factor‐A (VEGF‐A) induces a significant angiogenic response in the ZFYM assay that is suppressed by the VEGF receptor‐2/KDR TK inhibitor SU5416. The ZFYM assay represents a novel tool for testing the activity of low and high molecular weight inhibitors targeting a defined angiogenic growth factor in zebrafish. The assay may offer significant advantages when compared to other animal models.

Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point

Vascular remodeling under conditions of growth or exercise, or during recovery from arterial restriction or blockage is essential for health, but mechanisms are poorly understood. It has been proposed that endothelial cells have a preferred level of fluid shear stress, or ‘set point’, that determines remodeling. We show that human umbilical vein endothelial cells respond optimally within a range of fluid shear stress that approximate physiological shear. Lymphatic endothelial cells, which experience much lower flow in vivo, show similar effects but at lower value of shear stress. VEGFR3 levels, a component of a junctional mechanosensory complex, mediate these differences. Experiments in mice and zebrafish demonstrate that changing levels of VEGFR3/Flt4 modulates aortic lumen diameter consistent with flow-dependent remodeling. These data provide direct evidence for a fluid shear stress set point, identify a mechanism for varying the set point, and demonstrate its relevance to vessel remodeling in vivo. DOI: http://dx.doi.org/10.7554/eLife.04645.001

Calcitonin receptor-like receptor guides arterial differentiation in zebrafish

The calcitonin receptor-like receptor (crlr) is a major endothelial cell receptor for adrenomedullin, a peptide vasodilator involved in cardiovascular development, homeostasis, and disease. Here, we used the zebrafish (Danio rerio) model to characterize the role of crlr in vascular development. Crlr is expressed within somites from the 4- to the 13-somite stage and by arterial progenitors and axial vessels during zebrafish development. Loss of crlr results in profound alterations in vascular development and angiogenesis, including atrophic trunk dorsal aorta and interruption of anterior aortic bifurcation, delay in intersomitic vessel development, and lack of blood circulation. Remarkably, crlr morphants are characterized by the loss of arterial endothelial cell identity in dorsal aorta, as shown by the lack of expression of the arterial markers ephrin-B2a, DeltaC, and notch5. Down-regulation of crlr affects vascular endothelial growth factor (vegf) expression, whereas vegf overexpression is sufficient to rescue arterial differentiation in crlr morphants. Finally, genetic and biochemical evidences indicate that somitic crlr expression is under the control of sonic hedgehog. These data demonstrate that crlr plays a nonredundant role in arterial differentiation, representing a novel element of the sonic hedgehog-vegf-notch signaling cascade that controls arterial/venous fate.

HIV-1 Tat and heparan sulfate proteoglycan interaction: a novel mechanism of lymphocyte adhesion and migration across the endothelium

The HIV-1 transactivating factor Tat accumulates on the surface of endothelium by interacting with heparan sulfate proteoglycans (HSPGs). Tat also interacts with B-lymphoid Namalwa cells but only when these overexpress HSPGs after syndecan-1 cDNA transfection (SYN-NCs). Accordingly, SYN-NCs, but not mock-transfected cells, adhere to endothelial cells (ECs) when Tat is bound to the surface of either one of the 2 cell types or when SYN-NCs are transfected with a Tat cDNA. Moreover, endogenously produced Tat bound to cell-surface HSPGs mediates cell adhesion of HIV(+) ACH-2 lymphocytes to the endothelium. This heterotypic lymphocyte-EC interaction is prevented by HSPG antagonist or heparinase treatment, but not by integrin antagonists and requires the homodimerization of Tat protein. Tat tethered to the surface of SYN-NCs or of peripheral blood monocytes from healthy donors promotes their transendothelial migration in vitro in response to CXCL12 or CCL5, respectively, and SYN-NC extravasation in vivo in a zebrafish embryo model of inflammation. In conclusion, Tat homodimers bind simultaneously to HSPGs expressed on lymphoid and EC surfaces, leading to HSPG/Tat-Tat/HSPG quaternary complexes that physically link HSPG-bearing lymphoid cells to the endothelium, promoting their extravasation. These data provide new insights about how lymphoid cells extravasate during HIV infection.

Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide

Fibroblast growth factor‐2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long‐pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2‐antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2‐binding region PTX3‐(97–110) were assessed for their FGF2‐binding capacity. Among them, the shortest pentapeptide Ac‐ARPCA‐NH2 (PTX3‐[100–104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2‐dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac‐ARPCA‐NH2 inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2‐overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine‐kinase FGF receptor‐1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac‐ARPSA‐NH2 peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF‐binding domain of the Ig‐like loop D2 of FGFR1, amino acid substitutions in Ac‐ARPCA‐NH2 and saturation transfer difference‐nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3‐derived anti‐angiogenic FGF2 antagonists.