Figure 3. Analysis of ANXA1-containing EVs in ex vivo cultures of resealing mucosal wounds and functional effects of EVs on wound closure.

(A) 4-mm punch biopsy of resealing colonic wounds (1 day after injury, red circle) and nonwounded mucosa (blue circle) were rinsed in sterile PBS to remove cellular debris and immediately placed into a 24-well culture plate (3 wounds from each mouse per well, n = 4). Photographic images of a wound (1- to 2-mm size) and confocal images of resealing mucosal wound showing F-actin (Alexa Fluor 555 phalloidin, pink) and nuclei (TO-PRO-3, blue) are shown (n = 4). Scale bar: 100 μm. (B) Immunoblot showing ANXA1 in the supernatant and lysate of the ex vivo culture. Densitometry analysis shows that ANXA1 protein in culture supernatants derived from wounded cultures is significantly increased versus nonwounded cells (1.979-fold increase, P = 0.0024 average of n = 3 immunoblots). In total lysate, the ratio of ANXA1 expression in wounded tissue versus nonwounded tissue is 0.762 (P = 0.0005 average of n = 3 immunoblots). (C) EVs (microparticles and exosomes) were stained with BODIPY-Texas Red and conjugated CD11b, A33, and ANXA1 fluorescent antibodies. Data are expressed as mean ± SEM; n = 4 per group. (D) EVs derived from Anxa1^+/+ and Anxa1^–/– mice were added to monolayers of cultured murine epithelial cells, and scratch wound resealing assay was performed. Increased wound closure was observed after incubation with ANXA1-containing EVs (**P = 0.0025) compared with EVs lacking ANXA1. Mean ± SEM; n = 15 mice per group. Statistical comparisons were performed by ANOVA with Tukey’s multiple comparison post-test.