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

Figure 6

Inhibiting enlargeosome release attenuates HMW-HA-mediated sustained human EC barrier enhancement. Panel (a): inhibition of annexin II expression using siRNA in HPMVEC. Human EC were plated at ~50% confluence and treated with either no siRNA (control), siControl, or siAnnexin II with or without 100 nM LMW-HA or 100 nM HMW-HA for 48 hours. EC lysates were then obtained, run on SDS-PAGE, and immunoblotted with anti-annexin II (a) or anti-actin (b) antibodies. Panel (b): graphical representation of the role of annexin II inhibition in HA-mediated EV production from human EC. HPMVEC were treated as described in Panel (a) and EVs were analyzed utilizing nanosight nanoparticle tracking analysis (NTA). Silencing of annexin II, which has previously been reported to be crucial for enlargeosome exocytosis [23], significantly reduces HMW-HA-, but not LMW-HA-, mediated EV secretion from human EC with per group and error bars = standard deviation. Panel (c): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM LMW-HA was then added. Silencing of annexin II did not affect the sustained human EC barrier disruptive effects of LMW-HA with per group and error bars = standard deviation. Panel (d): HPMVEC previously treated with either no siRNA (control), siControl, or annexin II siRNA were plated on transendothelial electrical resistance (TER) electrodes, grown to confluence, and switched to serum-free media and 100 nM HMW-HA was then added. In contrast to LMW-HA, silencing annexin II almost completely inhibited the sustained barrier enhancing effects of HMW-HA with per group and error bars = standard deviation.

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