Extracellular Vesicles from Caveolin-Enriched Microdomains Regulate Hyaluronan-Mediated Sustained Vascular Integrity

Figure 2

Identification of LMW-HA and HMW-HA-induced EV from human EC. Panel (a): biomarker analysis of HA-induced EV. HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, or 100 nM LMW-HA for 24 hours. EVs were then isolated, run on SDS-PAGE, and immunoblotted with anti-CD9 (a), anti-CD63 (b), anti-CD81 (c), anti-AHNAK (d), antiannexin II (e), or anti-caveolin-1 (f) antibodies. LMW-HA-induced EV expressed exosome markers while HMW-HA-induced EV expressed enlargeosome markers. EV also expressed caveolin-1, a crucial component of caveolin-enriched microdomains (CEM). Panel (b): topographical images of HA-induced EV using atomic force microscopy (AFM). Isolated HA-induced EVs as described in Panel (a) were plated on mica and subjected to AFM analysis (see Section 2). The vesicles were never dried and are shown as imaged under buffer. Control EV and exosomes were round in shape and had a relatively smooth surface. In contrast, enlargeosomes were relatively round in shape but had a rough uneven surface topology. Control EV and exosomes had a diameter of ~50 nm which is consistent with the accepted exosome size range of 30–100 nm [20]. HMW-HA-induced enlargeosomes appeared slightly larger in comparison to exosomes.

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