Evidence for the involvement of MMP14 in MMP2 processing and recruitment in exosomes of corneal fibroblasts

Abstract

PURPOSE. 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 (MMP14Dexon4), 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.

Roles of exosomes in the normal and diseased eye

“…A large number of MMPs, ADAMs and ADAMTSs have been identified on exosomes and have been shown to be catalytically active (Shimoda and Khokha, 2013). Thus far in the eye, only two studies have identified MMPs as exosomal proteins, MMP-14 with corneal fibroblast exosomes (Han et al, 2015) and MMP-2 with trabecular meshwork exosomes (Stamer et al, 2011). …”

“…A critical component throughout this process is the transmembrane matrix metalloproteinase-14 (MMP-14). Corneal fibroblasts release exosomes with MMP-14, which are taken up by endothelial cells (Han et al, 2015). Exosomal MMP-14 activity is critical for the accumulation and activation of MMP-2 in the exosomes (Han et al, 2015).…”

“…Corneal fibroblasts release exosomes with MMP-14, which are taken up by endothelial cells (Han et al, 2015). Exosomal MMP-14 activity is critical for the accumulation and activation of MMP-2 in the exosomes (Han et al, 2015). This process likely plays a role in the multistep corneal wound healing and represents a therapeutic target to prevent or reverse corneal neovascularization.…”

Mechanism of Fibronectin Binding to Human Trabecular Meshwork Exosomes and Its Modulation by Dexamethasone

“…The exact role of exosomes in cell-matrix interactions and degradation is unclear; however exosomes from a number of sources have been shown to possess proteolytic activity crucial in degrading the ECM. The membrane bound MT1-MMP has been detected on exosomes [45] and was able to cleave collagen 1, gelatin and pro-MMP2 into its active form [46]. MMP activity is critically important for maintaining outflow facility and therefore TM function [11].…”

Exploring Packaged Microvesicle Proteome Composition of Chinese Hamster Ovary Secretome

“…Moreover, the amount of secreted microvesicles has also been correlated with the expression of phosphorylated protein kinase B (Ak) t and extracellular signal-regulated protein kinases (ERKs) which are crucial elements regulating apoptosis and cell differentiation processes. The microvesicles have also been shown to be enriched for multiple proteolytic enzymes, like MMP-2 and MMP-14 [12,13]. Though these enzymes are important for various cellular processes, they could also impact the quality and quantity of the product in culture.…”