Journal: Frontiers in Pharmacology

Article Title: Identification of an anti-inflammatory action of exosome release in P2Y 4 loss-mediated cardioprotection

doi: 10.3389/fphar.2025.1664015

Figure Lengend Snippet: Increase in plasma PD-L1 concentration and PD-L1 + exosome number in the plasma of ischemic P2Y 4 KO compared to WT mice. (A) Quantification of exosomal protein concentration by BCA assay on Exo-spin preparations from the plasma of WT and P2Y 4 KO mice, without MI (basal) or 24 h after MI (MI 24 h) (n = 20). The sex ratio was 40% male and 60% female in each group (n = 30 for basal conditions, n = 20 for MI conditions). Data were analyzed using a two-way ANOVA followed by a Bonferroni post hoc test. (B) Quantification of PD-L1 plasma concentrations by ELISA in WT and P2Y 4 KO mice, without MI (basal), 24 h post-MI (MI 24 h) or 24 h post-MI injected intraperitoneally with exosome inhibitor GW4869 (2.5 μg/g), 1 h before LAD ligation (MI 24 h + GW4869). The sex ratio was 50% male and 50% female in each group (n = 8–20), except for the P2Y 4 KO basal, WT MI 24h, and P2Y 4 KO MI 24 h + GW4869 groups (n = 15), which included 40% males and 60% females. Data were analyzed using a three-way ANOVA followed by a Bonferroni post hoc test. (C) Detection of adiponectin and CD63 exosome marker by Western blotting using exosomes from plasma of WT and P2Y 4 KO mice, without MI or 24 h post-MI (left graph). Quantification of adiponectin band density, normalized to CD63 band density. The sex ratio was 50% male and 50% female in each group (n = 4) (right graph). Data were analyzed using a two-way ANOVA followed by a Bonferroni post hoc test. (D) Quantification of different sized beads (200 nm–2000 nm) by flow cytometry. (E) Quantification of PD-L1 + particles (expressed as percentage of CD63 + exosomes) by flow cytometry, isolated using the Exo-spin Exosome Purification kit on the plasma of WT and P2Y 4 KO mice, 24 h after MI (MI 24 h). The sex ratio was 50% male and 50% female in both the WT MI 24 h and P2Y 4 KO MI 24 h groups (n = 14). Data were analyzed using an unpaired, two-tailed Student’s t-test (left graph). Representative flow cytometry dotplots for PD-L1 + particles in the plasma of WT and P2Y 4 KO mice, 24 h after MI (MI 24 h) (right graph). (F) Quantification of CD41 + and CD31 + particles (expressed as percentage of CD63 + exosomes) by flow cytometry, isolated using the Exo-spin Exosome Purification kit on the plasma of WT and P2Y 4 KO mice, 24 h after MI (MI 24 h). The sex ratio was 50% male and 50% female in each group (n = 4–6), except for the CD41/CD63 staining in the P2Y 4 KO MI 24 h group (n = 5), which included 40% males and 60% females. (G) Quantification of CD41 + and CD31 + particles (expressed as percentage of PD-L1 + exosomes) by flow cytometry, isolated using the Exo-spin Exosome Purification kit on the plasma of WT and P2Y 4 KO mice, 24 h after MI (MI 24 h) (n = 4–6) The sex ratio was 50% male and 50% female in each group (n = 4–6), except for the CD41/PD-L1 staining in the P2Y 4 KO MI 24 h group (n = 5), which included 40% males and 60% females (left graph). Representative flow cytometry dotplots for CD41/PD-L1 staining of particles in the plasma of WT and P2Y 4 KO mice, 24 h after MI (MI 24 h) (right graph). Data represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Plasma exosomes (45 μL) isolated using the Exo-spin Exosome Purification Kit were incubated with antibodies against CD63 (clone NVG-2, 2/500), PD-L1 (clone 10G.9G2, 2/500), CD31 (clone MEC 13.3, 2/250) and CD41 (clone MWReg30, 2/500).

Techniques: Clinical Proteomics, Concentration Assay, Protein Concentration, BIA-KA, Enzyme-linked Immunosorbent Assay, Injection, Ligation, Marker, Western Blot, Flow Cytometry, Isolation, Purification, Two Tailed Test, Staining