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Dynamic control of endothelial cell junctions is essential for vascular homeostasis and angiogenesis. We recently provided genetic evidence that ANGPTL4 is a key regulator of vascular integrity both during developmental and in hypoxia-induced pathological conditions. The purpose of the present study was to decipher the molecular mechanisms through which ANGPTL4 regulates vascular integrity. Using surface plasmon resonance and proximity ligation assays, we show that ANGPTL4 binds integrin αvβ3. In vitro and in vivo functional assays with Angptl4-deficient mice demonstrate that ANGPTL4-αvβ3 interaction is necessary to mediate ANGPTL4 vasoprotective effects. Mechanistically, ANGPTL4-αvβ3 interaction enhances Src recruitment to integrin αvβ3 and inhibits Src signalling downstream of vascular endothelial growth factor receptor 2 (VEFGR2), thereby repressing hypoxia-induced breakdown of VEGFR2-VE-cadherin and VEGFR2-αvβ3 complexes. We further demonstrate that intravitreal injection of recombinant human ANGPTL4 limits vascular permeability and leads to increased adherens junction and tight junction integrity. These findings identify a novel mechanism by which ANGPTL4 counteracts hypoxia-driven vascular permeability through integrin αvβ3 binding, modulation of VEGFR2-Src kinase signalling, and endothelial junction stabilization. We further demonstrate that Angptl4-deficient mice show increased vascular leakage in vivo in a model of laser-induced choroidal neovascularization, indicating that this newly identified ANGPTL4-αvβ3 axis might be a target for pharmaceutical intervention in pathological conditions. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: No-reflow in ST-segment elevation acute myocardial infarction (STEMI) is associated with a poor clinical prognosis. Its pathophysiological mechanisms are not fully elucidated yet but enhanced vascular permeability plays a key role in this phenomenon. Angiopoietin-like 4 (ANGPTL4) has been implicated in vascular permeability in experimental models of acute myocardial infarction (AMI). We therefore sought to investigate whether baseline ANGPTL4 serum levels are associated with no-reflow after primary percutaneous coronary intervention (PPCI)., Methods: We studied a group of 41 patients presenting with a first STEMI within 12h of onset of symptoms and who underwent successful PPCI. Blood samples were obtained from all patients on admission before the start of the procedure, for ANGPTL4 level measurement. No-reflow was assessed by cardiac magnetic resonance imaging (MRI), the reference method., Results: MRI-detected no-reflow was observed in 20 patients (48.8%). Variables independently associated with no-reflow on multivariate logistic regression analysis were: lower ANGPTL4 serum levels (odds ratio 0.82, 95% CI 0.70-0.98, P=0.02), higher troponin T peak (odds ratio 1.03, 95% CI 1.00-1.05, P=0.03), higher incidence of left anterior descending coronary artery (LAD) as culprit artery (odds ratio 14.61, 95% CI 1.24-172.49, P=0.03), and higher C-reactive protein levels (odds ratio 1.18, 95% CI 1.00-1.39, P=0.05)., Conclusion: ANGPTL4 serum levels predict MRI-detected no-reflow after successful PPCI in STEMI patients. Given the recently demonstrated therapeutic role of ANGPTL4 in diminishing no-reflow and limiting infarct size in pre-clinical animal models, these findings in humans may open up new possibilities in the field of research., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Aims: Given 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 Results: Using 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.

Background: 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., Methods and Results: 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., Conclusions: 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.

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.