[Show abstract][Hide abstract] ABSTRACT: Hypoxia regulates composition of both the vascular basement membrane (BM) and the extracellular matrix (ECM) by modulating deposition, cross-linking, posttranslational modifications, and rearrangement events but also degradation. Hypoxia-driven remodeling of the ECM includes highly temporally and spatially coordinated processes that eventually affect angiogenesis leading to blood vessel formation from existing blood vessels. Hypoxia thereby affects the mechanical properties of the vascular milieu as well as matricellular proteins expression and function and availability of angiogenesis-regulating growth factors such as vascular endothelial growth factor (VEGF). ECM composition and stiffness may be required for optimal VEGFR2 expression and vascular development in vitro and in vivo (Mammoto et al. Nature 2009), but how it might control signaling pathways such as VEGFR2 signaling is not fully appreciated yet. Thus, vascular BM and ECM composition affects vascular microenvironment architecture and interaction with angiogenic growth factors but also exerts mechanical forces controlled by physical interactions between vascular cells and the ECM that cooperate in regulating angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: 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 sub-phenotypic 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.
[Show abstract][Hide abstract] ABSTRACT: AimsGiven the impact of vascular injuries and oedema on brain damage caused during stroke, vascular protection represents a major medical need. We hypothesized that angiopoietin-like 4 (ANGPTL4), a regulator of endothelial barrier integrity, might exert a protective effect during ischaemic stroke.Methods and resultsUsing a murine transient ischaemic stroke model, treatment with recombinant ANGPTL4 led to significantly decreased infarct size and improved behaviour. Quantitative characteristics of the vascular network (density and branchpoints) were preserved in ANGPTL4-treated mice. Integrity of tight and adherens junctions was also quantified and ANGPTL4-treated mice displayed increased VE-cadherin and claudin-5-positive areas. Brain oedema was thus significantly decreased in ANGPTL4-treated mice. In accordance, vascular damage and infarct severity were increased in angptl4-deficient mice thus providing genetic evidence that ANGPTL4 preserves brain tissue from ischaemia-induced alterations. Altogether, these data show that ANGPTL4 protects not only the global vascular network, but also interendothelial junctions and controls both deleterious inflammatory response and oedema.Mechanistically, ANGPTL4 counteracted VEGF signalling and thereby diminished Src-signalling downstream from VEGFR2. This led to decreased VEGFR2-VE-cadherin complex disruption, increased stability of junctions and thus increased endothelial cell barrier integrity of the cerebral microcirculation. In addition, ANGPTL4 prevented neuronal loss in the ischaemic area.Conclusion
These results, therefore, show ANGPTL4 counteracts the loss of vascular integrity in ischaemic stroke, by restricting Src kinase signalling downstream from VEGFR2. ANGPTL4 treatment thus reduces oedema, infarct size, neuronal loss, and improves mice behaviour. These results suggest that ANGPTL4 constitutes a relevant target for vasculoprotection and cerebral protection during stroke.
Full-text · Article · May 2013 · European Heart Journal
[Show abstract][Hide abstract] ABSTRACT: Increased permeability, predominantly controlled by endothelial junction stability, is an early event in the deterioration of vascular integrity in ischemic disorders. Hemorrhage, edema, and inflammation are the main features of reperfusion injuries, as observed in acute myocardial infarction (AMI). Thus, preservation of vascular integrity is fundamental in ischemic heart disease. Angiopoietins are pivotal modulators of cell-cell junctions and vascular integrity. We hypothesized that hypoxic induction of angiopoietin-like protein 4 (ANGPTL4) might modulate vascular damage, infarct size, and no-reflow during AMI.
We showed that vascular permeability, hemorrhage, edema, inflammation, and infarct severity were increased in angptl4-deficient mice. We determined that decrease in vascular endothelial growth factor receptor 2 (VEGFR2) and VE-cadherin expression and increase in Src kinase phosphorylation downstream of VEGFR2 were accentuated after ischemia-reperfusion in the coronary microcirculation of angptl4-deficient mice. Both events led to altered VEGFR2/VE-cadherin complexes and to disrupted adherens junctions in the endothelial cells of angptl4-deficient mice that correlated with increased no-reflow. In vivo injection of recombinant human ANGPTL4 protected VEGF-driven dissociation of the VEGFR2/VE-cadherin complex, reduced myocardial infarct size, and the extent of no-reflow in mice and rabbits.
These data showed that ANGPTL4 might constitute a relevant target for therapeutic vasculoprotection aimed at counteracting the effects of VEGF, thus being crucial for preventing no-reflow and conferring secondary cardioprotection during AMI.
[Show abstract][Hide abstract] ABSTRACT: 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. LOXL2 belongs to the lysyl oxidase family of secreted enzymes involved in ECM crosslinking. Knockdown experiments in Tg(fli1:egfp)y1 zebrafish embryos resulted in lack of intersegmental vessel circulation and demonstrated LOXL2 involvement in proper capillary formation. Further investigation in vitro by loss and gain of function experiments confirmed that LOXL2 was required for tubulogenesis in 3D fibrin gels and demonstrated that this enzyme was required for collagen IV assembly in the ECM. In addition, LOXL2 depletion down-regulated cell migration and proliferation. These data suggest a major role for LOXL2 in the organization of endothelial basal lamina and in the downstream mechanotransductive signaling. Altogether, our study provides the first evidence for the role of LOXL2 in regulating angiogenesis through collagen IV scaffolding.
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis is a highly coordinated tissue remodeling process leading to blood vessel formation. Hypoxia triggers angiogenesis via induction of expression of growth factors such as vascular endothelial growth factor (VEGF). VEGF instructs endothelial cells to form tip cells, which lead outgrowing capillary sprouts, whereas Notch signaling inhibits sprout formation. Basement membrane deposition and mechanical cues from the extracellular matrix (ECM) induced by hypoxia may participate to coordinated vessel sprouting in conjunction with the VEGF and Notch signaling pathways.
Hypoxia regulates ECM composition, deposition, posttranslational modifications and rearrangement. In particular, hypoxia-driven vascular remodeling is dynamically regulated through modulation of ECM-modifying enzyme activities that eventually affect both matricellular proteins and growth factor availability.
Better understanding of the complex interplay between endothelial cells and soluble growth factors and mechanical factors from the ECM will certainly have significant implications for understanding the regulation of developmental and pathological angiogenesis driven by hypoxia.
No preview · Article · Mar 2010 · Current opinion in hematology
[Show abstract][Hide abstract] ABSTRACT: Angiopoietin-like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled-coil domain (CCD) and fibrinogen-like domain (FLD) fragments. The full-length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM-bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure-function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full-length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule-like formation. In endothelial cells, ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor alpha1-PDX and abolished by the mutation of the (161)RRKR(164) cleavage site without modification of the ECM binding and release. These data suggest that the full-length form, which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.
Full-text · Article · Dec 2008 · The FASEB Journal
[Show abstract][Hide abstract] ABSTRACT: Angiopoietin-like 4 (ANGPTL4), a secreted protein of the angiopoietin-like family, is induced by hypoxia in both tumor and endothelial cells as well as in hypoxic perinecrotic areas of numerous cancers. Here, we investigated whether ANGPTL4 might affect tumor growth as well as metastasis. Metastatic 3LL cells were therefore xenografted into control mice and mice in which ANGPTL4 was expressed by using in vivo DNA electrotransfer. Whereas primary tumors grew at a similar rate in both groups, 3LL cells metastasized less efficiently to the lungs of mice that expressed ANGPTL4. Fewer 3LL emboli were observed in primary tumors, suggesting that intravasation of 3LL cells was inhibited by ANGPTL4. Furthermore, melanoma B16F0 cells injected into the retro-orbital sinus also metastasized less efficiently in mice expressing ANGPTL4. Although B16F0 cells were observed in lung vessels, they rarely invaded the parenchyma, suggesting that ANGPTL4 affects extravasation. In addition, recombinant B16F0 cells that overexpress ANGPTL4 were generated, showing a lower capacity for in vitro migration, invasion, and adhesion than control cells. Expression of ANGPTL4 induced reorganization of the actin cytoskeleton through inhibition of actin stress fiber formation and vinculin localization at focal contacts. Together, these results show that ANGPTL4, through its action on both vascular and tumor compartments, prevents the metastatic process by inhibiting vascular activity as well as tumor cell motility and invasiveness.
Full-text · Article · Jan 2007 · Proceedings of the National Academy of Sciences
[Show abstract][Hide abstract] ABSTRACT: Angiopoietin-like 4 (ANGPTL4) is a secreted protein that belongs to the angiopoietin family and is involved in angiogenesis and metabolism regulation. We previously reported the induction of angptl4 by hypoxia in endothelial cells and in human ischemic tissues from peripheral artery disease. We here observed in a mouse model of hindlimb ischemia that the mRNA upregulation in the vessels correlates with the accumulation of the full-length protein in ischemic tissues. We then investigated its functions in endothelial cells. In response to hypoxia, endogenous ANGPTL4 accumulates in the subendothelial extracellular matrix (ECM). Although the secreted protein undergoes proteolysis leading to truncated fragments present in the medium, only full-length ANGPTL4 interacts with the ECM. Competition and direct binding assays indicate that the strong interaction of ANGPTL4 with the ECM is heparin/heparan sulfate proteoglycan dependent. The balance between matrix-associated and soluble forms of ANGPTL4 points to the role of the ECM in the regulation of its bioavailability. The angiogenic function of the ECM-bound full-length protein was investigated using either the form associated with the conditioned ECM from ANGPTL4-transfected HEK293 cells or the purified immobilized protein. We show that matrix-associated and immobilized ANGPTL4 limit the formation of actin stress fibers and focal contacts in the adhering endothelial cells and inhibit their adhesion. Immobilized ANGPTL4 also decreases motility of endothelial cells and inhibits the sprouting and tube formation. Altogether, these findings show that hypoxic endothelial cells accumulate ANGPTL4 in the ECM, which in turn negatively regulates their angiogenic capacities through an autocrine pathway.
Full-text · Article · Dec 2006 · Circulation Research
[Show abstract][Hide abstract] ABSTRACT: Angiotensin II (AngII) type 1 receptors (AT1) regulate cell growth through the extracellular signal-regulated kinase (ERK)1/2 and phosphatidylinositol 3-kinase (PI3K) pathways. ERK1/2 and Akt/protein kinase B, downstream of PI3K, are independently activated but both required for mediating AngII-induced proliferation when expressed at endogenous levels. We investigate the effect of an increase in the expression of wild-type Akt1 by using Chinese hamster ovary (CHO)-AT1 cells. Unexpectedly, Akt overexpression inhibits the AT1-mediated proliferation. This effect could be generated by a cross-talk between the PI3K and ERK1/2 pathways. A functional partner is the phosphoprotein enriched in astrocytes of 15 kDa (PEA-15), an Akt substrate known to bind ERK1/2 and to regulate their nuclear translocation. We report that Akt binds to PEA-15 and that Akt activation leads to PEA-15 stabilization, independently of PEA-15 interaction with ERK1/2. Akt cross-talk with PEA-15 does not affect ERK1/2 activation but decreases their nuclear activity as a result of the blockade of ERK1/2 nuclear accumulation. In response to AngII, PEA-15 overexpression displays the same functional consequences on ERK1/2 signaling as Akt overactivation. Thus, Akt overactivation prevents the nuclear translocation of ERK1/2 and the AngII-induced proliferation through interaction with and stabilization of endogenous PEA-15.
Full-text · Article · Oct 2006 · Molecular Biology of the Cell
[Show abstract][Hide abstract] ABSTRACT: Like most extracellular matrix (ECM) components, fibronectin (Fn) is proteolyzed generating specific activities. Fibronectin proteinase (Fn-proteinase) represents such a cryptic activity located in the gelatin-binding domain (GBD) of Fn and displays a zinc metalloproteinase activity. The migration-stimulating factor (MSF) is a truncated Fn isoform generated by alternative mRNA splicing and corresponds to the N-terminal part of Fn that comprises the GBD. We show that several human mammary epithelial cells express MSF and constitutively produce Fn-proteinase activity. Furthermore, recombinant MSF produced by HEK-293 and MCF-7 cells possesses a constitutive Fn-proteinase activity. Mutating the putative zinc-binding motif, HEXXH, of the protein abolishes its activity thereby demonstrating its specificity. Using PCR, we showed that MSF is barely expressed in normal breast tissues, whereas its expression is significantly increased in tumors. Furthermore, an association between MSF expression and invasive capacity is observed in various breast adenocarcinoma cell lines. Indeed, when stably transfected in non-invasive MCF-7 cells, MSF promotes cell migration in a mechanism mostly dependent on its Fn-proteinase activity. In summary, our study shows that: (i) MSF displays constitutive Fn-proteinase activity; (ii) MSF expression is induced in human breast cancer; and (iii) MSF confers pro-migratory activity that depends mostly on its Fn-proteinase activity. These results suggest that MSF may be involved in tumor progression.
Full-text · Article · Sep 2005 · International Journal of Cancer
[Show abstract][Hide abstract] ABSTRACT: Ischemic and solid tumor tissues are less well perfused than normal tissue, leading to metabolic changes and chronic hypoxia, which in turn promotes angiogenesis. We identified human angiopoietin-like 4 (angptl4) as a gene with hypoxia-induced expression in endothelial cells. We showed that the levels of both mRNA and protein for ANGPTL4 increased in response to hypoxia. When tested in the chicken chorioallantoic membrane assay, ANGPTL4 induced a strong proangiogenic response, independently of vascular endothelial growth factor. In human pathology, ANGPTL4 mRNA is produced in ischemic tissues, in conditions such as critical leg ischemia. In tumors, ANGPTL4 is produced in the hypoxic areas surrounding necrotic regions. We observed particularly high levels of ANGPTL4 mRNA in tumor cells of conventional renal cell carcinoma. Other benign and malignant renal tumor cells do not produce ANGPTL4 mRNA. This molecule therefore seems to be a marker of conventional renal cell carcinoma. ANGPTL4, originally identified as a peroxisome proliferator-activated receptor alpha and gamma target gene, has potential for use as a new diagnostic tool and a potential therapeutic target, modulating angiogenesis both in tumors and in ischemic tissues. This study also suggests that ANGPTL4 may provide a link between metabolic disorders and hypoxia-induced angiogenesis.
Full-text · Article · Jun 2003 · American Journal Of Pathology