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. 2023 May 23;14:1104890. doi: 10.3389/fimmu.2023.1104890

Figure 3.

Figure 3

M1 macrophage-derived sEVs miR-503 induces HUVEC dysfunctions (A) The expression levels of miR-503 and miR-107 were examined in a medium containing sEVs extracted from PMA-treated THP-1 cells (M0-sEVs), medium containing sEVs extracted from M0 treated with 30 mM glucose ((HG+M0)-sEVs), or medium from M0 treated with 30 mM glucose and sEVs generation inhibitor GW4869 ((HG+M0)-sEVs-/-) using qRT-PCR. (B) sEVs extracted from M0 treated with 30 mM glucose ((HG+M0)-sEVs) were non-treated, treated with RNaseA, or treated with RNaseA plus TritonX-100 and examined for the expression level of miR-503 by qRT-PCR. (C) miR-503 overexpression or inhibition was achieved in macrophages by transfecting miR-503 mimics or inhibitor and confirmed using qRT-PCR. (D) M0 macrophages were transfected with miR-503 mimics or inhibitor, treated with HG, and examined for iNOS and Arg-1 levels by IF staining. (E) Then, M0 macrophages were transfected with miR-503 mimics or inhibitor, treated with HG, and extracted for sEVs. miR-503 expression in sEVs was determined by qRT-PCR. (F-I) HUVECs were cultured with sEVs from different groups and examined for cell viability by CCK-8 assay (F); tube formation capacity (G); cell migration by Wound healing (H); the protein levels of Ang1, Flk1, Vash1, TSP1I, ICAM-1, and VCAM-1 by Immunoblotting assay (I). n=3, ** p<0.01 vs.M0 (M0)-sEVs group or (HG+mimics NC)-sEVs group. ## p<0.01 vs. (HG+inhibitor NC)-sEVs group. * p<0.05.