Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

Olivier G De Jong,Bas W M Van Balkom,Hendrik Gremmels,Marianne C Verhaar

doi:10.1111/jcmm.12730

Olivier G De Jong, Bas W M Van Balkom + Show 2 more

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https://doi.org/10.1111/jcmm.12730

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Abstract

Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing.

Highlights

Lysyl oxidase-like 2 (LOXL2) is one of five members of the lysyl oxidase family, consisting of lysyl oxidase (LOX) and lysyl oxidase-like 1 – 4
To quantify the observed increase in exosomal LOXL2 abundance, HMEC-1 were cultured in control- and hypoxic conditions, as was confirmed by immunoblot analysis for hypoxia-inducible factor 1-alpha (HIF-1a) and LOXL2 (Fig. 1A), after which exosomes from these cells were isolated and immunoblotted for LOXL2, using b-actin, which we previously showed to remain unchanged upon hypoxic stimulation in endothelial cell (EC)-derived exosomes [8], as a loading control (Fig. 1B)
To further illustrate that LOXL2 is associated with exosomes, sucrose density gradient and subsequent immunoblot analysis were performed, showing that LOXL2 and the exosome marker Flotillin-1 are both detected at a density around 1.10 g/ml (Fig. 1D)

Summary

Introduction

Lysyl oxidase-like 2 (LOXL2) is one of five members of the lysyl oxidase family, consisting of lysyl oxidase (LOX) and lysyl oxidase-like 1 – 4. Like all LOX family members, LOXL2 facilitates crosslinking of collagens and elastin by catalysing oxidative deamination of lysine residues [1]. This process is crucial for giving the extracellular matrix (ECM) its tensile strength and load-bearing capabilities [2]. All LOX family members share a high degree of homology, only LOXL2, LOXL3 and LOXL4 possess four scavenger receptor cysteinerich domains at their N-termini [3]. Apart from its extracellular role, intracellular functions of LOXL2 have been reported in epithelial to mesenchymal transition (EMT) and breast cancer metastasis [1, 5]

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