The complex TIE between macrophages and angiogenesis

Macrophages are primarily known as phagocytic immune cells, but they also play a role in diverse processes, such as morphogenesis, homeostasis and regeneration. In this review, we discuss the influence of macrophages on angiogenesis, the process of new blood vessel formation from the pre-existing vasculature. Macrophages play crucial roles at each step of the angiogenic cascade, starting from new blood vessel sprouting to the remodelling of the vascular plexus and vessel maturation. Macrophages form promising targets for both pro- and anti-angiogenic treatments. However, to target macrophages, we will first need to understand the mechanisms that control the functional plasticity of macrophages during each of the steps of the angiogenic cascade. Here, we review recent insights in this topic. Special attention will be given to the TIE2-expressing macrophage (TEM), which is a subtype of highly angiogenic macrophages that is able to influence angiogenesis via the angiopoietin-TIE pathway

A Cryptic Frizzled Module in Cell Surface Collagen 18 Inhibits Wnt/β−Catenin Signaling

Collagens contain cryptic polypeptide modules that regulate major cell functions, such as cell proliferation or death. Collagen XVIII (C18) exists as three amino terminal end variants with specific amino terminal polypeptide modules. We investigated the function of the variant 3 of C18 (V3C18) containing a frizzled module (FZC18), which carries structural identity with the extracellular cysteine-rich domain of the frizzled receptors. We show that V3C18 is a cell surface heparan sulfate proteoglycan, its topology being mediated by the FZC18 module. V3C18 mRNA was expressed at low levels in 21 normal adult human tissues. Its expression was up-regulated in fibrogenesis and in small well-differentiated liver tumors, but decreased in advanced human liver cancers. Low FZC18 immunostaining in liver cancer nodules correlated with markers of high Wnt/β−catenin activity. V3C18 (Mr = 170 kD) was proteolytically processed into a cell surface FZC18-containing 50 kD glycoprotein precursor that bound Wnt3a in vitro through FZC18 and suppressed Wnt3a-induced stabilization of β−catenin. Ectopic expression of either FZC18 (35 kD) or its 50 kD precursor inhibited Wnt/β−catenin signaling in colorectal and liver cancer cell lines, thus downregulating major cell cycle checkpoint gatekeepers cyclin D1 and c-myc and reducing tumor cell growth. By contrast, full-length V3C18 was unable to inhibit Wnt signaling. In summary, we identified a cell-surface signaling pathway whereby FZC18 inhibits Wnt/β−catenin signaling. The signal, encrypted within cell-surface C18, is released by enzymatic processing as an active frizzled cysteine-rich domain (CRD) that reduces cancer cell growth. Thus, extracellular matrix controls Wnt signaling through a collagen-embedded CRD behaving as a cell-surface sensor of proteolysis, conveying feedback cues to control cancer cell fate

Inhibition of Wnt/β-Catenin Signaling by a Soluble Collagen-Derived Frizzled Domain Interacting with Wnt3a and the Receptors Frizzled 1 and 8

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth

Type XVIII collagen:characterization of the primary structure and expression pattern of different variants in <em>Xenopus laevis</em>, characterization of the human gene structure and analysis of transgenic mice expressing endostatin

Abstract In this work the type XVIII collagen has been studied by using several approaches, such as different animal models. The primary structure of frog, Xenopus laevis, type XVIII collagen and the expression pattern of its variants during early embryogenesis have been elucidated. The gene structure of human type XVIII collagen was characterized and the localization and processing of its longest variant was studied by generated antibodies. In addition, the function of the proteolytically released C-terminal part of type XVIII collagen, endostatin, was studied by generating transgenic mice expressing endostatin. The primary structure of X. laevis type XVIII collagen is comprised of three N-terminal variants resembling their mammalian counterparts. The sizes of the polypeptides are 1285, 1581, and 1886 residues. The most conserved regions are the C-terminal endostatin region and the cysteine-rich domain in the N-terminus. Whole-mount in situ hybridization reveals different expression patterns for variants during embryogenesis. The short variant is the most abundant, whereas the two longest variants exhibit more restricted expression. The gene structure of human type XVIII collagen reveals an exon-intron organization that is conserved with mouse. The length of the human gene is about 105 kb and contains 43 exons. The third variant of type XVIII collagen has a conserved cysteine-rich domain with homology to the extracellular part of frizzled proteins. This third variant is localized to developing muscle and lung, and is also found in serum. In cell culture, the proteolytic fragments of the N-terminus, including the cysteine-rich motif, are also detected. Endostatin function was studied by generating mouse lines expressing endostatin under the keratin-14 promoter, which drives the expression mainly in the skin. Three independent transgenic mouse lines were achieved with varied expression levels. The phenotype was seen in the eye with lens opacity and abnormal morphology of epithelial cells in the lens. In the skin, a broading of the basement membrane in the epidermis dermis junction was detected. Immunoelectron microscopy analysis revealed a polarized orientation of type XVIII collagen in the basement membrane. In transgenic mice, altered localization of endogenous type XVIII collagen was seen, suggesting displacement of the endogenous type XVIII collagen with transgenic endostatin leading to disorganized basement membrane

Cooperation of angiopoietin-2 and angiopoietin-4 in Schlemm’s canal maintenance

Abstract Purpose: Defects in the iridocorneal angle tissues, including the trabecular meshwork (TM) and Schlemm’s canal (SC), impair aqueous humor flow and increase the intraocular pressure (IOP), eventually resulting in glaucoma. Activation of endothelial tyrosine kinase receptor Tie2 by angiopoietin-1 (Angpt1) has been demonstrated to be essential for SC formation, but roles of the other two Tie2 ligands, Angpt2 and Angpt4, have been controversial or not yet characterized, respectively. Methods: Angpt4 expression was investigated using genetic cell fate mapping and reporter mice. Congenital deletion of Angpt2 and Angpt4 and tamoxifen-inducible deletion of Angpt1 in mice were used to study the effects of Angpt4 deletion alone and in combination with the other angiopoietins. SC morphology was examined with immunofluorescent staining. IOP measurements, electron microscopy, and histologic evaluation were used to study glaucomatous changes. Results: Angpt4 was postnatally expressed in the TM. While Angpt4 deletion alone did not affect SC and Angpt4 deletion did not aggravate Angpt1 deletion phenotype, absence of Angpt4 combined with Angpt2 deletion had detrimental effects on SC morphology in adult mice. Consequently, Angpt2−/−;Angpt4−/− mice displayed glaucomatous changes in the eye. Mice with Angpt2 deletion alone showed only moderate SC defects, but Angpt2 was necessary for proper limbal vasculature development. Mechanistically, analysis of Tie2 phosphorylation suggested that Angpt2 and Angpt4 cooperate as agonistic Tie2 ligands in maintaining SC integrity. Conclusions: Our results indicated an additive effect of Angpt4 in SC maintenance and Tie2 activation and a spatiotemporally regulated interplay between the angiopoietins in the mouse iridocorneal angle

Angiopoietin-4-dependent venous maturation and fluid drainage in the peripheral retina

Abstract The maintenance of fluid homeostasis is necessary for function of the neural retina; however, little is known about the significance of potential fluid management mechanisms. Here, we investigated angiopoietin-4 (Angpt4, also known as Ang3), a poorly characterized ligand for endothelial receptor tyrosine kinase Tie2, in mouse retina model. By using genetic reporter, fate mapping, and in situ hybridization, we found Angpt4 expression in a specific sub-population of astrocytes at the site where venous morphogenesis occurs and that lower oxygen tension, which distinguishes peripheral and venous locations, enhances Angpt4 expression. Correlating with its spatiotemporal expression, deletion of Angpt4 resulted in defective venous development causing impaired venous drainage and defects in neuronal cells. In vitro characterization of angiopoietin-4 proteins revealed both ligand-specific and redundant functions among the angiopoietins. Our study identifies Angpt4 as the first growth factor for venous-specific development and its importance in venous remodeling, retinal fluid clearance and neuronal function