Research Paper Volume 13, Issue 11 pp 14687—14708

Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells

Autophagy is associated with EV secretion and/or cargo in Sca-1+ bone marrow cells. (A) Extracellular vesicle (EV) particle concentration isolated from Y-Sca-1+ CM±CLQ, ±GW4869, ±DMSO and O-Sca-1+ conditioned medium (CM). EV production was inhibited in Y-Sca-1+ bone marrow cells (BMCs) using GW4869 (GW). Autophagy was inhibited using chloroquine (CLQ). (B–D) EVs were purified from CM of the same treatment groups, and added to cultured old cardiac fibroblasts. Treatment groups are abbreviated as: Y-Sca-1+ EV, Y-Sca-1+ CLQ EV and O-Sca-1+ EV. (B) Representative images from scratch wound and proliferation assays with old fibroblasts treated with Y-Sca-1+ EV, Y-Sca-1+ CLQ EV and O-Sca-1+ EV for 48, or 24 hours, respectively. Dashed yellow line indicates the wound edge at 0 hours. (C) Percent wound closure (after completing the scratch wound assay) was measured using ImageJ. (D) Percentage of BrdU+ cells, normalized to total cell number. Scale bars represent 100 μm. Data analysis used one-way ANOVA. Data presented as mean ± SEM; n=4-8. *p≤0.05; **p≤0.01; ns: not statistically significant.

Figure 4. Autophagy is associated with EV secretion and/or cargo in Sca-1+ bone marrow cells. (A) Extracellular vesicle (EV) particle concentration isolated from Y-Sca-1+ CM±CLQ, ±GW4869, ±DMSO and O-Sca-1+ conditioned medium (CM). EV production was inhibited in Y-Sca-1+ bone marrow cells (BMCs) using GW4869 (GW). Autophagy was inhibited using chloroquine (CLQ). (BD) EVs were purified from CM of the same treatment groups, and added to cultured old cardiac fibroblasts. Treatment groups are abbreviated as: Y-Sca-1+ EV, Y-Sca-1+ CLQ EV and O-Sca-1+ EV. (B) Representative images from scratch wound and proliferation assays with old fibroblasts treated with Y-Sca-1+ EV, Y-Sca-1+ CLQ EV and O-Sca-1+ EV for 48, or 24 hours, respectively. Dashed yellow line indicates the wound edge at 0 hours. (C) Percent wound closure (after completing the scratch wound assay) was measured using ImageJ. (D) Percentage of BrdU+ cells, normalized to total cell number. Scale bars represent 100 μm. Data analysis used one-way ANOVA. Data presented as mean ± SEM; n=4-8. *p≤0.05; **p≤0.01; ns: not statistically significant.