title: Granzyme B releases vascular endothelial growth factor from extracellular matrix and induces vascular permeability.
abstract: The formation of unstable, leaky neovessels underlies the pathogenesis of many chronic inflammatory diseases. Granzyme B (GZMB) is an immune-derived serine protease that accumulates in the extracellular matrix (ECM) during chronic inflammation and is capable of cleaving fibronectin (FN). Vascular endothelial growth factor (VEGF) is a potent vascular permeabilizing agent that is sequestered in the ECM through its interaction with FN. As GZMB levels are elevated in chronic inflammatory diseases that are associated with increased vascular permeability, the role of GZMB in the regulation of VEGF bioavailability and vascular permeability were assessed. GZMB was added to either VEGF bound to FN or VEGF bound to endothelial cell (EC)-derived ECM. Supernatants containing released VEGF were assessed to determine VEGF activity by treating EC and evaluating VEGF receptor-2 (VEGFR2) phosphorylation. GZMB released VEGF from both FN and from EC-derived matrix, whereas GZMB inhibition prevented FN cleavage and VEGF release. GZMB-mediated VEGF release resulted in significant phosphorylation of VEGFR2. The role of GZMB-mediated VEGF release in altering vascular permeability was also assessed in vivo using Miles/Evans blue permeability assay. GZMB induced a significant VEGF-dependent increase in vascular permeability in vivo that was reduced in the presence of an anti-VEGF-neutralizing antibody. Inflammatory-mediated vascular leakage was also assessed in GZMB-KO mice using a delayed-type hypersensitivity model. GZMB-KO mice exhibited reduced microvascular leakage compared with C57\B6 controls. GZMB increases vascular permeability in part through the proteolytic release of ECM-sequestered VEGF, leading to VEGFR2 activation and increased vascular permeability in vivo. These findings present a novel role for GZMB as a modulator of vascular response during chronic inflammation.
authors.affiliation: 1] Centre for Heart Lung Innovation, St Paul's Hospital, Vancouver, BC, Canada [2] Departments of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Alon
authors.initials: A
authors.lastname: Hendel
authors.affiliation: 1] Centre for Heart Lung Innovation, St Paul's Hospital, Vancouver, BC, Canada [2] Departments of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Ivy
authors.initials: I
authors.lastname: Hsu
authors.affiliation: 1] Centre for Heart Lung Innovation, St Paul's Hospital, Vancouver, BC, Canada [2] Departments of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada.
authors.forename: David J
authors.initials: DJ
authors.lastname: Granville
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000494:pharmacology; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism*; D005353:Fibronectins / Q000097:blood / Q000378:metabolism; D053804:Granzymes / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D006968:Hypersensitivity, Delayed / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D007249:Inflammation / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008856:Microscopy, Fluorescence; D042461:Vascular Endothelial Growth Factor A / Q000276:immunology / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D005353:Fibronectins; D042461:Vascular Endothelial Growth Factor A; D040301:Vascular Endothelial Growth Factor Receptor-2; D053804:Granzymes