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Tumor progression and metastatic dissemination are driven by cell-intrinsic and biomechanical cues that favor the growth of life-threatening secondary tumors. We recently identified pro-metastatic vascular regions with blood flow profiles that are permissive for the arrest of circulating tumor cells. We have further established that such flow profiles also control endothelial remodeling, which favors extravasation of arrested CTCs. Yet, how shear forces control endothelial remodeling is unknown. In the present work, we aimed at dissecting the cellular and molecular mechanisms driving blood flow-dependent endothelial remodeling. Transcriptomic analysis of endothelial cells revealed that blood flow enhanced VEGFR signaling, among others. Using a combination of in vitro microfluidics and intravital imaging in zebrafish embryos, we now demonstrate that the early flow-driven endothelial response can be prevented upon specific inhibition of VEGFR tyrosine kinase and subsequent signaling. Inhibitory targeting of VEGFRs reduced endothelial remodeling and subsequent metastatic extravasation. These results confirm the importance of VEGFR-dependent endothelial remodeling as a driving force of CTC extravasation and metastatic dissemination. Furthermore, the present work suggests that therapies targeting endothelial remodeling might be a relevant clinical strategy in order to impede metastatic progression.

MicroRNAs (miRNAs) are single-stranded small non-coding RNAs, generally 18-25 nucleotides in length, that act as repressors of gene expression. miRNAs are encoded by independent genes or processed from a variety of different RNA species. So far, there is no evidence showing that the ribosomal DNA-hosted microRNA is implicated in vertebrate development. Currently, we found a highly expressed small RNA hosted in ribosomal DNA was predicted as a novel miRNA, named miR-ntu1, in zebrafish endothelial cells by deep sequencing analysis. The miRNA was validated by custom-designed Taqman PCR, Northern Blot, and in silico analysis. Furthermore, we demonstrated that miR-ntu1 played a crucial role in zebrafish angiogenesis via modulation of Notch signaling. Our findings provide a notable case that a miRNA hosted in ribosomal DNA is involved in vertebrate development.

Angiogenesis, the process of neovascularization, plays an important role in physiological and pathological conditions. ST104P is a soluble polysulfated-cyclo-tetrachromotropylene compound with anti-viral and anti-thrombotic activities. However, the functions of ST104P in angiogenesis have never been explored. In this study, we investigated the effects of ST104P in angiogenesis in vitro and in vivo. Application of ST104P potently suppressed the microvessels sprouting in aortic rings ex vivo. Furthermore, ST104P treatment significantly disrupted the vessels' development in transgenic zebrafish in vivo. Above all, repeated administration of ST104P resulted in delayed tumor growth and prolonged the life span of mice bearing Lewis lung carcinoma. Mechanistic studies revealed that ST104P potently inhibited the migration, tube formation and wound closure of human umbilical endothelial cells (HUVECs). Moreover, ST104P treatment inhibited the secretion and expression of matrix metalloproteinase-2 (MMP-2) in a dose-dependent manner. Together, these results suggest that ST104P is a potent angiogenesis inhibitor and may hold potential for treatment of diseases due to excessive angiogenesis including cancer.

Blood flow plays crucial roles in vascular development, remodeling and homeostasis, but the molecular pathways required for transducing flow signals are not well understood. In zebrafish embryos, arterial expression of activin receptor-like kinase 1 (alk1), which encodes a TGFβ family type I receptor, is dependent on blood flow, and loss of alk1 mimics lack of blood flow in terms of dysregulation of a subset of flow-responsive arterial genes and increased arterial endothelial cell number. These data suggest that blood flow activates Alk1 signaling to promote a flow-responsive gene expression program that limits nascent arterial caliber. Here, we demonstrate that restoration of endothelial alk1 expression to flow-deprived arteries fails to rescue Alk1 activity or normalize arterial endothelial cell gene expression or number, implying that blood flow may play an additional role in Alk1 signaling independent of alk1 induction. To this end, we define cardiac-derived Bmp10 as the crucial ligand for endothelial Alk1 in embryonic vascular development, and provide evidence that circulating Bmp10 acts through endothelial Alk1 to limit endothelial cell number in and thereby stabilize the caliber of nascent arteries. Thus, blood flow promotes Alk1 activity by concomitantly inducing alk1 expression and distributing Bmp10, thereby reinforcing this signaling pathway, which functions to limit arterial caliber at the onset of flow. Because mutations in ALK1 cause arteriovenous malformations (AVMs), our findings suggest that an impaired flow response initiates AVM development.

Viral nanoparticles are a novel class of biomolecular agents that take advantage of the natural circulatory and targeting properties of viruses to allow the development of therapeutics, vaccines and imaging tools. We have developed a multivalent nanoparticle platform based on the cowpea mosaic virus (CPMV) that facilitates particle labeling at high density with fluorescent dyes and other functional groups. Compared with other technologies, CPMV-based viral nanoparticles are particularly suited for long-term intravital vascular imaging because of their biocompatibility and retention in the endothelium with minimal side effects. The stable, long-term labeling of the endothelium allows the identification of vasculature undergoing active remodeling in real time. In this study, we describe the synthesis, purification and fluorescent labeling of CPMV nanoparticles, along with their use for imaging of vascular structure and for intravital vascular mapping in developmental and tumor angiogenesis models. Dye-labeled viral nanoparticles can be synthesized and purified in a single day, and imaging studies can be conducted over hours, days or weeks, depending on the application.

The peptide growth factor apelin is the high affinity ligand for the G-protein-coupled receptor APJ. During embryonic development of mouse and frog, APJ receptor is expressed at high levels in endothelial precursor cells and in nascent vascular structures. Characterization of Xenopus apelin shows that the sequence of the bioactive region of the peptide is perfectly conserved between frogs and mammals. Embryonic expression studies indicate that apelin is expressed in, or immediately adjacent to, a subset of the developing vascular structures, particularly the intersegmental vessels. Experimental inhibition of either apelin or APJ expression, using antisense morpholino oligos, results in elimination or disruption of intersegmental vessels in a majority of embryos. In gain of function experiments, apelin peptide is a potent angiogenic factor when tested using two in vivo angiogenesis assays, the frog embryo and the chicken chorioallantoic membrane. Furthermore, studies using the mouse brain microvascular cell line bEnd.3 show that apelin acts as a mitogenic, chemotactic and anti-apoptotic agent for endothelial cells in culture. Finally, we show that, similar to a number of other angiogenic factors, expression of the apelin gene is increased under conditions of hypoxia. Taken together, these studies indicate that apelin is required for normal vascular development in the frog embryo and has properties consistent with a role during normal and pathological angiogenesis.

Vascular endothelial growth factor (VEGF) is known to play an essential role in vascular development. We have overexpressed VEGF122 or VEGF170, which are equivalent to mouse VEGF120 and VEGF164, in developing Xenopus embryos. Overexpression of VEGF170 but not VEGF122 demonstrated an absence of expression of hematopoietic markers alpha-globin and GATA-1 but only in the posterior portion of the blood island. Interestingly, strong signals of endothelial markers, msr, fli-1, and tie-2, were detectable in those regions, instead of hematopoietic markers. These results suggested both that injection of VEGF170 resulted in disturbance of vasculogenesis in the posterior portion of the blood island, with excessive production of endothelial cells at the expense of blood cells, and that the anterior and posterior portions of the VBI may have distinct characteristics.

In order to sustain growth, differentiation, and organogenesis, vertebrate embryos must form a functional vascular system early in embryonic development. Intrinsic interest in this process as well as the promise of potential clinical applications has led to significant progress in understanding the mechanisms governing the formation of the vascular system, however the earliest stages of vascular development--the emergence of committed endothelial precursors from the mesoderm--remain unclear. A review of the current literature reveals an unexpected diversity and heterogeneity with respect to where vascular endothelial cells originate in the embryo, when they become committed and the mechanisms governing how endothelial cells acquire their identity. Spatially, a widespread region of the early mesoderm possesses the ability to give rise to vascular endothelial cells; temporally the process is not limited to a small window during embryogenesis, but rather, may continue throughout the lifespan of the organism. On the molecular level, recent findings point to several determinative pathways that regulate, modulate, and extend the scope of the Flk1/VEGF signaling system. An expanding array of novel gene products implicated in endothelial cell type determination appear to act synergistically, with different combinations of factors leading to diverse cellular responses, varying patterns of differentiation, and considerable heterogeneity of endothelial cell types during embryogenesis.

We report morphological and immunohistochemical evidence for a translocation of cells from the coelomic mesothelium to the aortic wall between the developmental stages HH16 and HH22 of the quail embryos. The coelomic mesothelial cells closest to the aorta showed, at these stages, increased mitotic activity, reduced intercellular adhesion, loss of tight junctions, and long basal cytoplasmic processes. Coinciding with these morphological traits, cytokeratin immunoreactivity was found in the mesothelium, in cells of the aortic wall and throughout the ventral periaortic mesenchyme (but not in the lateral and dorsal aortic regions). Vimentin immunoreactivity colocalized with cytokeratin in the mesothelial cells adjacent to the aorta. In the ventral aortic wall, cytokeratin colocalized with smooth muscle alpha-actin and with the 1E12 antigen (a smooth muscle-specific alpha-actinin isoform). We think that the morphological and immunolocalization data observed are compatible with an epithelial-mesenchymal transition by which mesothelial-derived cells contribute to the splanchnic mesoderm and aortic wall. The precise coincidence between the mesothelial contribution and the emergence of the aortic smooth muscle cells progenitors, as well as the immunolocalization data, suggest a potential relationship of the mesothelial-derived cells with this cell lineage. This may explain the observed ventrodorsal asymmetry in the distribution of smooth muscle cells progenitors in the aortic wall.

Required to supply nutrients and oxygen to the growing embryo, the vascular system is the first functional organ system to develop during vertebrate embryogenesis. Although there has been substantial progress in identifying the genetic cascade regulating vascular development, the initial stages of vasculogenesis, namely, the origin of vascular endothelial cells within the early embryo, remain unclear. To address this issue we constructed a fate map for specific vascular structures, including the aortic arches, endocardium, dorsal aorta, cardinal veins, and lateral abdominal veins, as well as for the red blood cells at the 16-cell stage and the 32-cell stage of Xenopus laevis. Using genetic markers to identify these cell types, our results suggest that vascular endothelial cells can arise from virtually every blastomere of the 16-cell-stage and the 32-cell-stage embryo, with different blastomeres preferentially, though not exclusively, giving rise to specific vascular structures. Similarly, but more surprisingly, every blastomere in the 16-cell-stage embryo and all but those in the most animal tier of the 32-cell-stage embryo serve as progenitors for red blood cells. Taken together, our results suggest that during normal development, both dorsal and ventral blastomeres contribute significantly to the vascular endothelial and red blood cell lineages., (Copyright 1999 Academic Press.)

The origin of endothelial cells and their subsequent assembly into the primary vascular system have been mostly analyzed in the avian embryo. Following the discovery of specific growth factors and their cognate receptors, the molecular mechanisms underlying these processes have been unraveled in both birds and mammals. In particular, experimental studies of the angiogenic vascular endothelial growth factor (VEGF) and its receptors, carried out in both vertebrate classes, have provided significant insight into the developmental biology of endothelial cells. The VEGF receptor VEGFR2 is the earliest marker known to be expressed by endothelial precursor cells of avian and mouse embryos. Based on the localization of VEGFR2+ cells in the avian embryo and on clonal culture experiments, two types of endothelial precursor cells can be distinguished from gastrulation stages onward: posterior mesodermal VEGFR2+ hemangioblasts, which have the capacity to differentiate into endothelial and hemopoietic cells, and anterior VEGFR2+ angioblasts, which can only give rise to endothelial cells.