title: Angiostatin inhibits activation and migration of neutrophils.
abstract: There is a critical need to identify molecules that modulate the biology of neutrophils because activated neutrophils, though necessary for host defense, cause exuberant tissue damage through production of reactive oxygen species and increased lifespan. Angiostatin, an endogenous anti-angiogenic cleavage product of plasminogen, binds to integrin αvβ3, ATP synthase and angiomotin and its expression is increased in inflammatory conditions. We test the hypothesis that angiostatin inhibits neutrophil activation, induces apoptosis and blocks recruitment in vivo and in vitro. The data show immuno-reactivity for plasminogen/angiostatin in resting neutrophils. Angiostatin conjugated to FITC revealed that angiostatin was endocytozed by activated mouse and human neutrophils in a lipid raft-dependent fashion. Co-immunoprecipitation of human neutrophil lysates, confocal microscopy of isolated mouse and human neutrophils and functional blocking experiments showed that angiostatin complexes with flotillin-1 along with integrin αvβ3 and ATP synthase. Angiostatin inhibited fMLP-induced neutrophil polarization, as well as caused inhibition of hsp-27 phosphorylation and stabilization of microtubules. Angiostatin treatment, before or after LPS-induced neutrophil activation, inhibited phosphorylation of p38 and p44/42 MAPKs, abolished reactive oxygen species production and released the neutrophils from suppressed apoptosis, as indicated by expression of activated caspase-3 and morphological evidence of apoptosis. Finally, intravital microscopy and myeloperoxidase assay showed inhibition of neutrophil recruitment in post-capillary venules of TNFα-treated cremaster muscle in mouse. These in vitro and in vivo data demonstrate angiostatin as a broad deactivator and silencer of neutrophils and an inhibitor of their migration. These data potentially open new avenues for the development of anti-inflammatory drugs.
authors.affiliation: Veterinary Biomedical Sciences, Western College of Veterinary Medicine, University of Saskatchewan, 52 Campus Drive, Saskatoon, SK, S7N 5B4, Canada.
authors.forename: Gurpreet K
authors.initials: GK
authors.lastname: Aulakh
authors.forename: Yadu
authors.initials: Y
authors.lastname: Balachandran
authors.forename: Lixin
authors.initials: L
authors.lastname: Liu
authors.forename: Baljit
authors.initials: B
authors.lastname: Singh
mesh_terms: D043168:Angiostatins / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D018821:CD18 Antigens / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D016764:Cell Polarity / Q000187:drug effects; D004705:Endocytosis / Q000187:drug effects; D006801:Humans; D007249:Inflammation / Q000473:pathology; D008070:Lipopolysaccharides / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009132:Muscles / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects*; D020556:Neutrophil Infiltration / Q000187:drug effects; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D010084:Oxidation-Reduction / Q000187:drug effects; D021122:Protein Subunits / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D018821:CD18 Antigens; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D021122:Protein Subunits; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; C106354:flotillins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D043168:Angiostatins; D006180:Proton-Translocating ATPases