Submit Manuscript  

Article Details


Proteomics-Based Characterization of the Effects of MMP14 on the Protein Content of Exosomes from Corneal Fibroblasts

[ Vol. 27 , Issue. 10 ]

Author(s):

Kyu-Yeon Han*, Jin-Hong Chang and Dimitri T. Azar   Pages 979 - 988 ( 10 )

Abstract:


Background: Exosomes secreted by corneal fibroblasts contain matrix metalloproteinase (MMP) 14, which is known to influence pro-MMP2 accumulation on exosomes. Accordingly, we hypothesized that the enzymatic activity of MMP14 may alter the protein content of corneal fibroblast- secreted exosomes.

Objective: The aim of this study was to investigate the effects of MMP14 on the composition and biological activity of corneal fibroblast-derived exosomes.

Methods: Knock out of the catalytic domain (ΔExon4) of MMP14 in corneal fibroblasts was used to determine the effect of MMP14 expression on the characteristics of fibroblast-secreted exosomes. The amount of secreted proteins and their size distribution were measured using Nano Tracking Analysis. Proteins within exosomes from wild-type (WT) and ΔExon4-deficient fibroblasts were identified by liquid chromatography-tandem mass spectrometry (MS/MS) proteomics analysis. The proteolytic effects of MMP14 were evaluated in vitro via MS identification of eliminated proteins. The biological functions of MMP14-carrying exosomes were investigated via fusion to endothelial cells and flow cytometric assays.

Results: Exosomes isolated from WT and ΔExon4-deficient fibroblasts exhibited similar size distributions and morphologies, although WT fibroblasts secreted a greater amount of exosomes. The protein content, however, was higher in ΔExon4-deficient fibroblast-derived exosomes than in WT fibroblast-derived exosomes. Proteomics analysis revealed that WT-derived exosomes included proteins that regulated cell migration, and ΔExon4 fibroblast-derived exosomes contained additional proteins that were cleaved by MMP14.

Conclusion: Our findings suggest that MMP14 expression influences the protein composition of exosomes secreted by corneal fibroblasts, and through those biological components, MMP14 in corneal fibroblasts derived-exosomes may regulate corneal angiogenesis.

Keywords:

Extracellular vesicles, exosomes, metalloproteinase 14, corneal fibroblasts, angiogenesis, biological activity.

Affiliation:

Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, Department of Ophthalmology and Visual Sciences, Illinois Eye and Ear Infirmary, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612



Read Full-Text article