Overexpression of GREM1 Improves the Survival Capacity of Aged Cardiac Mesenchymal Progenitor Cells via Upregulation of the ERK/NRF2-Associated Antioxidant Signal Pathway

Figures

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  Figure 1. Decreased expression of GREM1 is associated with the aging/senescence of cardiac mesenchymal progenitor cells (hMPCs). (A) Gene expression of cell senescent markers (p16, p21, FOXO1a, and PSG5, as well as GREM1) in four lines of pediatric and aging hMPCs. (B) Western blot analysis for the protein expression of p16INK4A and GREM1 in four lines of pediatric and aging hMPCs. (C) Gene expression of p16INK4A and GREM1 in the pediatric hMPCs with 12 h doxorubicin-induced cell senescence. (D) Western blot analysis for the protein expression of p16INK4A and GREM1 in the pediatric hMPCs with 12 h doxorubicin-induced cell senescence. (E) Western blot analysis for the protein expression of p16INK4A and GREM1 in the pediatric hMPCs with different passage number following replicative senescence. Data are mean ± SEM. p value was calculated by unpaired Student’s t-test, n ≥ 3 independent experiments. * indicated the significant difference compared to the control group.

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  Figure 2. Overexpressing GREM1 rejuvenates the senescent phenotype of aged hMPCs. (A) Western blot analysis confirmed the overexpression of GREM1 after infecting hMPCs with lenti-GREM virus or control (Ctrl) lentivirus. n = 3 independent experiments. (B) qPCR for the gene expression of p16, p21, Foxo1a, and PSG-5 in aged hMPCs (AMC145) after overexpressing GREM1. n = 3 independent experiments. (C) Western blot analysis showed the protein level of p16 and p21 in aged hMPCs (AMC145) after overexpressing GREM1. n = 3 independent experiments. (D) Representative images and quantitative analysis of SA-β-gal staining in aged hMPCs (AMC145) after overexpressing GREM1. Data are mean ± SEM. p value was calculated by unpaired Student’s t-test, n ≥ 3 independent experiments. * indicated the significant difference compared to the control group.

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  Figure 3. Overexpressing GREM1 enhances the survival capacity of the hMPCs and increases the expression of anti-apoptotic genes and protein. (A) Overexpressing GREM1 in hMPCs significantly increased the cell survival capacity, as indicated by the decreased LDH release. (B) FACS analysis with annexin V/PI staining shows a significant increase in the number of live cells (Q3) and a decrease in the number of apoptotic cells (Q2 + Q4) and necrotic cells (Q1) after overexpressing GREM1 in hMPCs compared to the control cells. (C) Quantitative analysis for panel B. (D) Real-time quantitative PCR analysis with an anti-apoptotic primer library screening shows a significant increase of the indicated anti-apoptotic gene expression following 48 h of overexpressing GREM1 in hMPCs versus control cells. (E) Western blot analysis showed the protein level of BCL2L and BCL2 in hMPCs with overexpressing GREM1 versus control cells. Data are mean ± SEM. p value was calculated by unpaired Student’s t-test, n = 3 independent experiments. * indicated the significant difference compared to the control group.

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  Figure 4. Overexpressing GREM1 decrease the ROS generation in hMPCs and increase the expression of antioxidant proteins. (A) Representative merged histogram for mitochondrial ROS measurement for aged hMPCs overexpressing GREM1 vs. vector control by FACS analysis following TMRM live cell staining. (B) Quantitative analysis for panel A. (C) Representative images for DHE staining for aged hMPCs infected with GREM1 or vector lentivirus for 48 h. (D) Quantitative analysis for C. (E) Real-time quantitative PCR analysis with an antioxidant primer library screening shows the increased gene expression of CAT, SOD1, PRDX4, and PRDX6 in hMPCs with GREM1 overexpression. (F) Representative images and quantitative data of Western blot showing the expression level of antioxidant proteins (catalase and SOD1) after GREM1 was overexpressed. Full-length blots are presented in Supplemental Figure S2. Data are mean ± SEM. p value was calculated by unpaired Student’s t-test, n = 3 independent experiments. * indicated the significant difference compared to the control group.

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  Figure 5. Activation of NRF2/ERK signal pathway is associated with the cytoprotective effect of overexpressing GREM1. (A) Representative image of Western blots showing increased protein expression of total and phosphorylated ERK1/2 and NRF2 in hMPCs with GREM1 overexpression. (B) Quantitative analysis of panel A. (C) FACS analysis with annexin V/PI double staining shows that specific inhibition of ERK activity by U0126 diminishes the cytoprotective effect of overexpressing GREM1 in aged hMPCs, as indicated by the decreased number of live cells (Q3) and increased number of apoptotic cells (Q2 + Q4) compared with cells without U0126 treatment as control. (D) Quantitative analysis of the FACS assay shown in C. Data are mean ± SEM. p value was calculated by unpaired Students’ t-test, n = 3 independent experiments. * indicated the significant difference compared to the control group.