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

Figure 4

Characterization of potential bioactive components in HA-induced EV. Panel (a): HPMVEC were grown to confluence and switched to serum-free media and either no HA (control), 100 nM HMW-HA, 100 nM LMW-HA, or 500 ng/mL LPS for 24 hours. EVs were then isolated, run on 4–20% TBE gels, and stained with Alcian blue. Purified enlargeosomes contained HA of ~1 million Da (see arrow) consistent with HMW-HA which we have previously demonstrated to be barrier enhancing [4, 5, 7, 8]. In contrast, control, LMW-HA, or LPS-induced EV contained negligible HA. Human plasma was used as a control. Panel (b): 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-CD44 (IM-7) (a), anti-CD44v10 (b), anti-HABP2 (c), anti-CD63 (d), or anti-AHNAK (e) antibodies. HMW-HA-induced enlargeosomes expressed the EC barrier enhancing CD44 isoform, CD44s (standard form) [4, 7]. In contrast, LMW-HA-induced exosomes expressed the EC barrier disrupting HA binding proteins, CD44 isoform CD44v10, and the extracellular serine protease, HABP2 [7, 25]. Basally secreted EV (control) had low expression of these molecules. Panel (c): isolated EVs as described in Panel (b) were subjected to RNA isolation and analysis (see Section 2). Compared to control EV, LMW-HA-induced EV had less total RNA and microRNA while HMW-HA-induced enlargeosomes had ~2-fold higher levels of total RNA and microRNA.

(a)

(b)

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