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

Y-Sca-1+ BMCs enhance old cardiac fibroblast function via autophagy-dependent TGF-β1 secretion. (A) Y-Sca-1+ bone marrow cells (BMCs) were treated with pharmacological inhibitors of autophagy (chloroquine, CLQ) and extracellular vesicle (EV) secretion (via GW4869). Conditioned medium (CM) was then collected and added to cultured cardiac fibroblasts isolated from old mice. Treatments are abbreviated as: Y-Sca-1+ CM±CLQ, Y-Sca-1+ CM±GW4869 or O-Sca-1+ CM. Representative images from scratch wound and proliferation assays after old cardiac fibroblasts were treated with CM from Y-Sca-1+ CM±CLQ, ±GW4869 and O-Sca-1+ CM for 48, or 24 hours, respectively. (B) Percent wound closure (after completing the scratch wound assay) was measured using ImageJ. Dashed yellow line indicates the wound edge at 0 hours. (C) Percentage of BrdU+ cells, normalized to total cell number. (D) TGF-β1 levels were measured in CM using ELISA. (E) Representative images from scratch wound and proliferation assays after old fibroblasts were treated with CM in the presence of TGF-β1 blocking antibody (Ab) for 48 hours. Treatment groups are abbreviated as: Y-Sca-1+ CM± TGF-β1 Ab and O-Sca-1+ CM± TGF-β1 Ab. Dashed yellow line indicates the wound edge at 0 hours. (F) Percent wound closure (after completing the scratch wound assay) was measured using ImageJ. (G) Percentage of BrdU+ cells, normalized to total cell number. Scale bars represent 100 μm. One-way ANOVA used to analyze data. n=3-4 *p≤0.05; **p≤0.01; ns: not statistically significant.

Figure 5. Y-Sca-1+ BMCs enhance old cardiac fibroblast function via autophagy-dependent TGF-β1 secretion. (A) Y-Sca-1+ bone marrow cells (BMCs) were treated with pharmacological inhibitors of autophagy (chloroquine, CLQ) and extracellular vesicle (EV) secretion (via GW4869). Conditioned medium (CM) was then collected and added to cultured cardiac fibroblasts isolated from old mice. Treatments are abbreviated as: Y-Sca-1+ CM±CLQ, Y-Sca-1+ CM±GW4869 or O-Sca-1+ CM. Representative images from scratch wound and proliferation assays after old cardiac fibroblasts were treated with CM from Y-Sca-1+ CM±CLQ, ±GW4869 and O-Sca-1+ CM for 48, or 24 hours, respectively. (B) Percent wound closure (after completing the scratch wound assay) was measured using ImageJ. Dashed yellow line indicates the wound edge at 0 hours. (C) Percentage of BrdU+ cells, normalized to total cell number. (D) TGF-β1 levels were measured in CM using ELISA. (E) Representative images from scratch wound and proliferation assays after old fibroblasts were treated with CM in the presence of TGF-β1 blocking antibody (Ab) for 48 hours. Treatment groups are abbreviated as: Y-Sca-1+ CM± TGF-β1 Ab and O-Sca-1+ CM± TGF-β1 Ab. Dashed yellow line indicates the wound edge at 0 hours. (F) Percent wound closure (after completing the scratch wound assay) was measured using ImageJ. (G) Percentage of BrdU+ cells, normalized to total cell number. Scale bars represent 100 μm. One-way ANOVA used to analyze data. n=3-4 *p≤0.05; **p≤0.01; ns: not statistically significant.