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

Figure 3

Analysis of HMW-HA-induced AHNAK redistribution in human EC. HPMVEC were grown to confluence on glass coverslips and treated with 100 nM HMW-HA for 6 hours. 50 μM ATP (six hours) and 3 μM ionomycin (10 minutes) were used as controls due to their previously reported ability to stimulate enlargeosome exocytosis [21–24]. The samples were then fixed in 4% paraformaldehyde and subjected to immunocytochemical analysis using AHNAK antibodies (green). Plasma membranes were labeled with Alexa Fluor 594 wheat germ agglutinin (red) and nuclei were stained with Hoechst 33342 (blue). Control HPMVEC exhibited diffuse cytoplasmic AHNAK staining. When stimulated with HMW-HA, intracellular AHNAK redistributed to a “vesicular” pattern which is similar to treatment with ATP and ionomycin.