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. 2024 Nov 19;25(22):12406. doi: 10.3390/ijms252212406

Figure 6.

Figure 6

Extracellular matrix (ECM)-deposited EVs released by VSMCs under mineralization stress contain higher calcium (Ca) and decreased GRP levels and induce macrophage inflammation. (a) Calcium (Ca) content and (b) GRP quantification by ELISA, both normalized to protein levels, determined in VSMC-isolated EV populations as described in Figure 4 legend. SD was calculated from 3 independent experiments (n = 3), and unpaired t tests were used. Statistical significance was defined as p ≤ 0.05 (*), p ≤ 0.01 (**); ns, non-significant. (c,d) THP-1 differentiated macrophages (TPH-1 Mac) were treated with 5 ug/mL of VSMC EVs quantified according to total protein for each population, and the positive inflammatory control lipopolysaccharides (LPSs) (100 ng/mL), for 24 h. Levels of the proinflammatory cytokines TNFα (c) and interleukin-8 (IL-8) (d) released to the cell media were determined by ELISA. SD was calculated from 3 independent experiments (n = 3), and ANOVA, with comparison between groups by the Dunnett test, was performed relative to THP-1 Mac cells cultured in control conditions. Statistical significance was defined as p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****).