Sprouting angiogenesis is associated with extensive extracellular matrix (ECM) remodeling. The molecular mechanisms involved in building the vascular microenvironment and its impact on capillary formation remain elusive. We therefore performed a proteomic analysis of ECM from endothelial cells maintained in hypoxia, a major stimulator of angiogenesis. Here, we report the characterization of lysyl oxidase-like protein-2 (LOXL2) as a hypoxia-target expressed in neovessels and accumulated in the endothelial ECM. IntroductionAngiogenesis occurs during development and tissue remodeling, and in the pathologic context of cardiovascular ischemic diseases or tumor growth. Sprouting of new vessels is initiated by stimulation of endothelial cells (ECs) by a combination of signals from the microenvironment that includes oxygen tension and growth factors. Cells from not yet vascularized tissue and ECs that invade this microenvironment are both hypoxia targets through the Hypoxia Inducible Factor (HIF) pathway. HIF activates transcription of genes coding for autocrine/paracrine factors like vascular endothelial growth factor (VEGF) and extracellular matrix (ECM) components. 1 Synergy between these responses is assumed through local concentration of growth factors in the ECM where they function as attractant for ECs. Specialized ECs, called tip cells, lead vascular growth by sending out filopodia to explore the hypoxic microenvironment. 2 Tip cells are thus continuously exposed to low oxygen concentration. 3 Stalk cells, located behind the tip cells, serve to vessel growth by proliferation, lumen formation and junction establishment. 4 Vascular ECM undergoes major remodeling during angiogenesis, consisting in ECM/basement membrane degradation, provisional ECM generation and assembly of a new basement membrane. ECM-mediated mechanotransductive signaling regulates 3D multicellular organization, including lumen formation and tubulogenesis. Thus, in addition to storing angiogenic factors and providing structural features, ECM is a dynamic promoter of angiogenesis. 5 Subendothelial basement membrane is composed of nonfibrillar collagen IV, laminin, perlecan and nidogens. Other collagens (VIII, XV, and XVIII), fibronectin and matricellular proteins (thrombospondin-1, Cyr61) are associated with the basement membrane. There is only little data concerning the assembly of the vascular microenvironment and its impact on angiogenesis. Genes encoding ECM components and enzymes involved in their assembly and stabilization are targets of hypoxia in ECs. 1 To identify key regulators of the angiogenesis-associated ECM remodeling, we performed a proteomic analysis of endothelial ECM generated in vitro under hypoxic conditions. We found that the ECM cross-linking enzyme, lysyl oxidase-like protein-2 (LOXL2) is a major target of hypoxia. Lysyl oxidases, consisting in lysyl oxidase (LOX) and 4 lysyl oxidase-like proteins (LOXL 1 to 4), catalyze the deamination of lysines and hydroxylysines, generating aldehydes that spontaneously react to form co...

Loss of CCM1/2 leads to destabilization of ICAP-1 and up-regulation of β1 integrin, resulting in the destabilization of intercellular junctions due to increased cell contractility and aberrant extracellular matrix remodeling.

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The myotendinous junction (MTJ) is the major site of force transfer in skeletal muscle, and defects in its structure correlate with a subset of muscular dystrophies. Col22a1 encodes the MTJ component collagen XXII, the function of which remains unknown. Here, we have cloned and characterized the zebrafish col22a1 gene and conducted morpholino-based loss-of-function studies in developing embryos. We showed that col22a1 transcripts localize at muscle ends when the MTJ forms and that COLXXII protein integrates the junctional extracellular matrix. Knockdown of COLXXII expression resulted in muscular dystrophy-like phenotype, including swimming impairment, curvature of embryo trunk/tail, strong reduction of twitch-contraction amplitude and contraction-induced muscle fiber detachment, and provoked significant activation of the survival factor Akt. Electron microscopy and immunofluorescence studies revealed that absence of COLXXII caused a strong reduction of MTJ folds and defects in myoseptal structure. These defects resulted in reduced contractile force and susceptibility of junctional extracellular matrix to rupture when subjected to repeated mechanical stress. Co-injection of subphenotypic doses of morpholinos against col22a1 and genes of the major muscle linkage systems showed a synergistic gene interaction between col22a1 and itga7 (α7β1 integrin) that was not observed with dag1 (dystroglycan). Finally, pertinent to a conserved role in humans, the dystrophic phenotype was rescued by microinjection of recombinant human COLXXII. Our findings indicate that COLXXII contributes to the stabilization of myotendinous junctions and strengthens skeletal muscle attachments during contractile activity.

The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility.

The extracellular matrix plays an essential role for stem cell differentiation and niche homeostasis. Yet, the origin and mechanism of assembly of the stem cell niche microenvironment remain poorly characterized. Here, we uncover an association between the niche and blood cells, leading to the formation of the Drosophila ovarian germline stem cell niche basement membrane. We identify a distinct pool of plasmatocytes tightly associated with the developing ovaries from larval stages onward. Expressing tagged collagen IV tissue specifically, we show that the germline stem cell niche basement membrane is produced by these "companion plasmatocytes" in the larval gonad and persists throughout adulthood, including the reproductive period. Eliminating companion plasmatocytes or specifically blocking their collagen IV expression during larval stages results in abnormal adult niches with excess stem cells, a phenotype due to aberrant BMP signaling. Thus, local interactions between the niche and blood cells during gonad development are essential for adult germline stem cell niche microenvironment assembly and homeostasis.

Collagen V is a minor component of the heterotypic I/III/V collagen fibrils and the defective product in most cases of classical Ehlers Danlos syndrome (EDS). The present study was undertaken to elucidate the impact of collagen V mutations on skin development, the most severely affected EDS tissues, using mice harboring a targeted deletion of the ␣2(V) collagen gene (Col5a2). Contrary to the original report, our studies indicate that the Col5a2 deletion (a.k.a. the pN allele) represents a functionally null mutation that affects matrix assembly through a complex sequence of events. First the mutation impairs assembly and/or secretion of the ␣1(V) 2 ␣2(V) heterotrimer with the result that the ␣1(V) homotrimer is the predominant species deposited into the matrix. Second, the ␣1(V) homotrimer is excluded from incorporation into the heterotypic collagen fibrils and this in turn severely impairs matrix organization. Third, the mutant matrix stimulates a compensatory loop by the ␣1(V) collagen gene that leads to additional deposition of ␣1(V) homotrimers. These data therefore underscore the importance of the collagen V heterotrimer in dermal fibrillogenesis. Furthermore, reduced thickness of the basement membranes underlying the epidermis and increased apoptosis of the stromal fibroblasts in pN/pN skin strongly indicate additional roles of collagen V in the development of a functional skin matrix.The physiological and biomechanical properties of different extracellular matrices depend in large part on the timely and tissue-specific deposition of collagen trimers (or types) that assemble into unique macromolecular aggregates. Among them, the banded fibrils represent the most abundant and widely distributed class of collagen assemblies. Fibril-forming collagens include five distinct types (I to III, V, and XI), which are found in most connective tissues (I, III, and V) or only in cartilage and vitreous (II and XI). Genetic studies have underscored the critical contribution of fibrillar collagens to organismal function by correlating mutations in the ␣ chain subunits with the genesis of several connective tissue disorders (17). Relevant to the present study, they have established the role of collagen V in regulating collagen I fibrillogenesis and in maintaining tissue integrity.Collagen V is a quantitatively minor component of tissues rich in collagen I, such as dermis, tendons/ligaments, bones, blood vessels, and cornea. Unlike other tissues, where collagen V represents only 1 to 3% of the total collagen fiber content, the relative concentration of this collagen type in cornea is significantly higher, 20 to 25% (3). Collagen V copolymerizes with collagens I and III to form heterotypic I/III/V fibrils in which the triple helical portion of the molecule is embedded and the amino-terminal globular domain projects onto the surface (4, 19). Very thin (5 to 10 nm in diameter) fibrils of collagen V have also been reported immediately near basement membranes and extending into the adjacent interstitial matrix (5,12,14,24). There...

Zebrafish myosepta connect two adjacent muscle cells and transmit muscular forces to axial structures during swimming via the myotendinous junction (MTJ). The MTJ establishes transmembrane linkages system consisting of extracellular matrix molecules (ECM) surrounding the basement membrane, cytoskeletal elements anchored to sarcolema, and all intermediate proteins that link ECM to actin filaments. Using a series of zebrafish specimens aged between 24 h post-fertilization and 2 years old, the present paper describes at the transmission electron microscope level the development of extracellular and intracellular elements of the MTJ. The transverse myoseptum development starts during the segmentation period by deposition of sparse and loosely organized collagen fibrils. During the hatching period, a link between actin filaments and sarcolemma is established. The basal lamina underlining sarcolemma is well differentiated. Later, collagen fibrils display an orthogonal orientation and fibroblast-like cells invade the myoseptal stroma. A dense network of collagen fibrils is progressively formed that both anchor myoseptal fibroblasts and sarcolemmal basement membrane. The differentiation of a functional MTJ is achieved when sarcolemma interacts with both cytoskeletal filaments and extracellular components. This solid structural link between contractile apparatus and ECM leads to sarcolemma deformations resulting in the formation of regular invaginations, and allows force transmission during muscle contraction. This paper presents the first ultrastructural atlas of the zebrafish MTJ development, which represents an useful tool to analyse the mechanisms of the myotendinous system formation and their disruption in muscle disorders.