title: Extracellular Vesicles Activate a CD36-Dependent Signaling Pathway to Inhibit Microvascular Endothelial Cell Migration and Tube Formation.
abstract: OBJECTIVE
  Literature on the effect of cell-derived extracellular vesicles (EV), ≤1 μm vesicles shed from various cell types during activation or apoptosis, on microvascular endothelial cell (MVEC) signaling is conflicting. Thrombospondin-1 and related proteins induce anti-angiogenic signals in MVEC via CD36. CD36 binds EV via phosphatidylserine exposed on their surface but the effects of this interaction on MVEC functions are not known. We hypothesized that EV would inhibit angiogenic MVEC functions via CD36.
  
  
  RESULTS
  EV generated in vitro from various cell types or isolated from plasma inhibited MVEC tube formation in in vitro matrigel assays and endothelial cell migration in Boyden chamber assays. Exosomes derived from the same cells did not have inhibitory activity. Inhibition of migration required endothelial cell expression of CD36. In mouse in vivo matrigel plug assays, EV inhibited cell migration into matrigel plugs in wild type but not in cd36 null animals. Annexin V, an anionic phospholipid binding protein, when incubated with EV partially reversed inhibition of migration, suggesting a phosphatidylserine-dependent effect. EV exposure induced reactive oxygen species generation in MVEC in a NADPH oxidase and Src family kinase-dependent manner, and their inhibition by apocynin and PP2, respectively, partially reversed the EV-mediated inhibition of migration. Annexin V partially reversed EV-induced reactive oxygen species generation in murine CD36 cDNA-transfected HVUEC but not in CD36-negative human umbilical vein endothelial cell.
  
  
  CONCLUSIONS
  These studies establish a general inhibitory effect of EV on endothelial cell proangiogenic responses and identify a CD36-mediated mechanistic pathway through which EV inhibit MVEC migration and tube formation.
authors.affiliation: From the Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH (D.P.R.); Laboratory of Vascular Pathobiology, Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI (D.P.R., Y.C., R.L.S.); Department of Rheumatologic and Immunologic Disease, Orthopedic and Rheumatologic Institute, Cleveland Clinic Foundation, Cleveland, OH (R.A.H.-A.); and Department of Medicine, Medical College of Wisconsin, Milwaukee, WI (R.L.S.).
authors.forename: Devi Prasadh
authors.initials: DP
authors.lastname: Ramakrishnan
authors.affiliation: From the Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH (D.P.R.); Laboratory of Vascular Pathobiology, Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI (D.P.R., Y.C., R.L.S.); Department of Rheumatologic and Immunologic Disease, Orthopedic and Rheumatologic Institute, Cleveland Clinic Foundation, Cleveland, OH (R.A.H.-A.); and Department of Medicine, Medical College of Wisconsin, Milwaukee, WI (R.L.S.).
authors.forename: Rula A
authors.initials: RA
authors.lastname: Hajj-Ali
authors.affiliation: From the Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH (D.P.R.); Laboratory of Vascular Pathobiology, Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI (D.P.R., Y.C., R.L.S.); Department of Rheumatologic and Immunologic Disease, Orthopedic and Rheumatologic Institute, Cleveland Clinic Foundation, Cleveland, OH (R.A.H.-A.); and Department of Medicine, Medical College of Wisconsin, Milwaukee, WI (R.L.S.).
authors.forename: Yiliang
authors.initials: Y
authors.lastname: Chen
authors.affiliation: From the Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, OH (D.P.R.); Laboratory of Vascular Pathobiology, Blood Research Institute, Blood Center of Wisconsin, Milwaukee, WI (D.P.R., Y.C., R.L.S.); Department of Rheumatologic and Immunologic Disease, Orthopedic and Rheumatologic Institute, Cleveland Clinic Foundation, Cleveland, OH (R.A.H.-A.); and Department of Medicine, Medical College of Wisconsin, Milwaukee, WI (R.L.S.). rsilverstein@mcw.edu.
authors.forename: Roy L
authors.initials: RL
authors.lastname: Silverstein
mesh_terms: D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D018955:CD36 Antigens / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000067128:Extracellular Vesicles / Q000378:metabolism*; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism*; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010718:Phosphatidylserines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D017304:Annexin A5; D018955:CD36 Antigens; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D017382:Reactive Oxygen Species; D019255:NADPH Oxidases; D019061:src-Family Kinases
keywords: CD36; angiogenesis; endothelial cell; extracellular vesicles
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