title: Evidence for the Involvement of MMP14 in MMP2 Processing and Recruitment in Exosomes of Corneal Fibroblasts.
abstract: OBJECTIVE
  Matrix metalloproteinase (MMP) 14 has been shown to promote angiogenesis, but the underlying mechanisms are poorly understood. In this study, we investigated exosomal transport of MMP14 and its target, MMP2, from corneal fibroblasts to vascular endothelial cells as a possible mechanism governing MMP14 activity in corneal angiogenesis.
  
  
  METHODS
  We isolated MMP14-containing exosomes from corneal fibroblasts by sucrose density gradient and evaluated exosome content and purity by Western blot analysis. We then investigated exosome transport in vitro from corneal fibroblasts to two populations of vascular endothelial cells, human umbilical vein endothelial cells (HUVECs) and calf pulmonary artery endothelial cells (CPAECs). Western blot analysis and gelatin zymography were used to determine levels of MMP14 and MMP2, respectively, in exosomal fractions derived from cultured wild-type, MMP14 enzymatic domain-deficient (MMP14Δexon4), and MMP14-null corneal fibroblasts.
  
  
  RESULTS
  Matrix metalloproteinase 14-containing exosomes isolated from corneal fibroblasts were readily taken up in vitro by HUVECs and CPAECs. We found that MMP14 was enriched in exosomal fractions of cultured corneal fibroblasts. Moreover, loss of the MMP14 enzymatic domain resulted in accumulation of pro-MMP2 protein in exosomes, whereas MMP2 was nearly undetectable in exosomes of MMP14-null fibroblasts.
  
  
  CONCLUSIONS
  Our results indicate that exosomes secreted by corneal fibroblasts can transport proteins, including MMP14, to vascular endothelial cells. In addition, recruitment of MMP2 into corneal fibroblast exosomes is an active process that depends, at least in part, on the presence of MMP14. The role of exosomal MMP14 transport in corneal angiogenesis has important implications for therapeutic applications targeting angiogenic processes in the cornea.
authors.affiliation: Department of Ophthalmology and Visual Sciences Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, United States.
authors.forename: Kyu-Yeon
authors.initials: KY
authors.lastname: Han
authors.affiliation: Department of Ophthalmology and Visual Sciences Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, United States.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Dugas-Ford
authors.affiliation: Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo, Japan.
authors.forename: Motoharu
authors.initials: M
authors.lastname: Seiki
authors.affiliation: Department of Ophthalmology and Visual Sciences Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, United States.
authors.forename: Jin-Hong
authors.initials: JH
authors.lastname: Chang
authors.affiliation: Department of Ophthalmology and Visual Sciences Illinois Eye and Ear Infirmary, University of Illinois at Chicago, Chicago, United States.
authors.forename: Dimitri T
authors.initials: DT
authors.lastname: Azar
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D003315:Cornea / Q000166:cytology / Q000201:enzymology*; D016510:Corneal Neovascularization / Q000201:enzymology*; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000201:enzymology*; D055354:Exosomes / Q000201:enzymology*; D005347:Fibroblasts / Q000201:enzymology*; D061307:Human Umbilical Vein Endothelial Cells / Q000201:enzymology; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000502:physiology*; D020778:Matrix Metalloproteinase 2 / Q000502:physiology*; D051379:Mice
chemical_list: D015415:Biomarkers; D020778:Matrix Metalloproteinase 2; D053511:Matrix Metalloproteinase 14