Nanog overexpression enhances the therapeutic efficacy of ADMSCs in AMI rats via the upregulation of JAK/STAT3 signaling and cyclin-mitochondrial expression

Abstract

Background: This study investigated whether Nanog-overexpressing adipose-derived mesenchymal stem cells (Nanog^OE-ADMSCs) are superior to unmodified ADMSCs in improving the left ventricular ejection fraction (LEVF) in acute myocardial infarction (AMI) patients. Methods: We utilized silencing and overexpression of Nanog gene in ADMSCs and performed a wound healing assay/transwell migration assay/MTT cell viability assay/left coronary artery ligation for AMI induction. Additionally, we categorized the cells into three classes [i.e., (ADMSCs and Nanog^OE-ADMSCs); A₁ (ADMSCs)/A₂ (ADMSCs + CoCl₂)/A₃ (Nanog^OE-ADMSCs + CoCl₂)/A₄ (siRNA-Nanog-ADMSCs) + CoCl₂); B₁ (ADMSCs)/B₂ (ADMSCs + H₂O₂)/B₃ (Nanog^OE-ADMSCs + H₂O₂)/B₄ (siRNA-Nanog gene in ADMSCs + H₂O₂)], and the rats (n=50) were evenly divided into Groups 1 (sham-operated control)/2 (AMI)/3 (AMI+ADMSCs)/4 (AMI+Nanog^OE-ADMSCs)/5 (AMI+siRNA-Nanog-ADMSCs). The hearts were harvested on Day 35. Results: In vitro experiments revealed significantly higher ATP, relative mitochondrial DNA/Nonog gene expression, mitochondrial cytochrome C+ cell, angiogenesis and exosome-specific marker (Alix/CD81/CD63/CD9) levels in Nanog^OE-ADMSCs than in ADMSCs. The cell viability, wound healing, and migration were highest in A1, lowest in A4, and significantly greater in A3 than in A2, whereas early/late apoptosis and intracellular and mitochondrial ROS displayed the opposite pattern of cell viability among the groups (all P<0.001). Additionally, the proteins expressions of phosphorylation (p) of the PI3K/Akt/mTOR, p-JAK2/p-STAT3, and Ras/Raf/MEK_1/2/ERK_1/2 signaling pathways were highest in A3, lowest in A4 and significantly greater in A1 than in A2 (all P<0.001). The levels of cell cycle proteins and mitochondrial electron transport train (ETC) complex I/II/III/IV components exhibited identical patterns as PI3K/Akt/mTOR among the groups B1 to B4 (all P<0.001). On Day 35, the LVEF was highest in Group 1, lowest in Group 2, significantly greater in Group 4 than in Groups 3 and 5, and significantly greater in Group 3 than in Group 5, with the opposite pattern for the LV remodeling index, infarct and fibrosis areas, and LV chamber size (all P < 0.0001). The p-AK/p-STAT3, p-PI3K/p-Akt/p-mTOR, and Ras/Raf/MEK_1/2/ERK_1/2 protein levels displayed the same pattern as the LVEF among the groups (all P < 0.001). Conclusion: Nanog^OE-ADMSCs rescued LVEF by upregulating JAK/STAT3-mediated cell proliferation/cell stress pathways and accelerating the cell cycle.

Keywords: Nanog gene overexpression in adipose-derived mesenchymal stem cells; acute myocardial infarction; cell stress signaling.

Figure 1

Nanog^OE-ADMSCs ensured the AMDMSCs survival in hypoxic environment. A) MTT assay for identification of cell viability at 24 h, * vs. other groups with different symbols (†, ‡, §), P<0.0001. B) MTT assay for identification of cell viability at 48 h, * vs. other groups with different symbols (†, ‡, §), P<0.0001. C) MTT assay for identification of cell viability at 48 h, * vs. other groups with different symbols (†, ‡, §), P<0.0001. D1 to D4) Illustrating the microscopic finding (100x) for identification of cell migratory ability (pink color). Scale bar in right lower corner represents 50µm. D5) Analytical result of cell migratory ability, * vs. other groups with different symbols (†, ‡, §), P<0.0001. E1 to E4) Illustrating the wound healing process at baseline. Analytical result, P>0.5. F1 to F4) Illustrating the wound healing process at 24 h. F5) Analytical result of wound healing ability, * vs. other groups with different symbols (†, ‡, §), P<0.0001. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=3-6) for each group). Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Cell grouping: A₁ = ADMSCs, A₂ = ADMSCs + CoCl₂, A₃ = Nanog^OE-ADMSCs + CoCl₂, A4 = siRNA-Nanog in ADMSCs + CoCl₂. ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene. CoCl₂ serviced as a hypoxia condition.

Figure 2

Impact of Nanog^OE in ADMSCs ensured cell survival in hypoxia environments. A1 to A4) Illustrating the flow cytometric analysis for identification of early (V+/PI-) and late (V+/PI+) apoptosis. A5) Analytical result of early apoptosis, * vs. other groups with different symbols (†, ‡, §), P<0.0001. A6) Analytical result of late apoptosis, * vs. other groups with different symbols (†, ‡, §), P<0.0001. B1 to B4) Illustrating the flow cytometric analysis for identification of total intracellualr reactive oxygen species (ROS) (i.e., by H₂DCFDA dye stain). B5) Analytical result of the total intracellular ROS, * vs. other groups with different symbols (†, ‡), P<0.001. C1 to C4) Showing the flow cytometric analysis for identification of mitochondrial ROS (by mitoSOX dye stain). C5) Analytical result of mitochondrial ROS by 72h, * vs. other groups with different symbols (†, ‡), P<0.001. Note: * indicated the control group. D) Protein level of Nanog, * vs. other groups with different symbols (†, ‡, §), P<0.001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6) for each group. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Cell grouping: A₁ = ADMSCs, A₂ = ADMSCs + CoCl₂, A₃ = Nanog^OE-ADMSCs + CoCl₂, A4 = siRNA-Nanog in ADMSCs + CoCl₂. ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 3

ATP concentration, relative mitochondria DNA expression, mitochondrial cytochrome-C, and angiogenesis were upregulated by Nanog gene overexpression. A) Relative gene expression of Nanog in ADMSCs with and without Nanog overexpression, * vs. †, P<0.001. B) Protein expression of Nanog in ADMSCs with and without Nanog overexpression, * vs. †, P<0.001. C) ATP concentration in ADMSCs with and without Nanog overexpression, * vs. †, P<0.001. D) Relative mitochondrial DNA expression, * vs. †, P<0.0001. E1 and E2) Illustrating the immunofluorescent (IF) microscopic finding (400x) for identification of mitochondrial cytochrome C (yellow-green color of a merge picture of double stains including red-color Hsp60 stain and green color cytochrome C stain). The scale bars in right lower corner represent 20 µm. E3) Analytical result of number of mitochondrial cytochrome C in ADMSCs with and without Nanog overexpression, * vs. †, P < 0.0001. F) Illustrating the interpretation of Matrigel assay for identification of angiogenesis, including tubular formation (blue length), cluster formation (red circle) network formation (green triangle). G1 and G2) Illustrating one sample of Matrigel assay for identification of rat circulatory derived endothelial progenitor cells (EPCs)-formed angiogenesis in ADMSCs-derived medium treated EPCs (G1) and Nanog^OE-ADMSCs-derived medium treated EPCs (G2). H1) Number of tubular formation, * vs. †, P < 0.001. H2) Total tubular length, * vs. †, P<0.001. H3) Mean tubular length, * vs. †, P<0.001. H4) Cluster formation, * vs. †, P<0.001. H5) Network formation. I) Protein expression of Alix, * vs. †, P<0.001. J) Protein expression of CD81, * vs. †, P < 0.001. K) Protein expression of CD63, * vs. †, P<0.001. L) Protein expression of CD9, * vs. †, P<0.001. Note the typical specific biomarkers, including ALIX, CD81, CD63 and C9 were identified from those of ADMSCs- or Nanog^OE-ADMSCs-derived exosomes. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 3-6) for each group. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 4

Nanog gene overexpression in ADMSCs suppressed oxidative stress and cellular apoptosis and upregulated cell stress signaling. A) Protein expression of NOX-1, * vs. other groups with different symbols (†, ‡), P < 0.001. B) Protein expression of NOX-2, Analytical result of wound healing ability, * vs. other groups with different symbols (†, ‡), P < 0.001. C) Protein expression of cleaved caspase 3 (c-Casp3), * vs. other groups with different symbols (†, ‡), P < 0.001. D) Protein expression of c-Casp9, * vs. other groups with different symbols (†, ‡), P<0.0001. E) Protein expression of phosphorylated (p)-JAK2, * vs. other groups with different symbols (†, ‡, §), P<0.001. F) Protein expression of p-STAT, * vs. other groups with different symbols (†, ‡, §), P<0.001. G) Protein expression of p-m-TOR, * vs. other groups with different symbols (†, ‡, §), P < 0.001. H) Protein expression of p-PI3K, * vs. other groups with different symbols (†, ‡, §), P < 0.0001. I) Protein expression of p-Akt, * vs. other groups with different symbols (†, ‡, §), P < 0.001. J) Protein expression of Ras, * vs. other groups with different symbols (†, ‡, §), P<0.001. K) Protein expression of Raf, * vs. other groups with different symbols (†, ‡, §), P<0.0001. L) Protein expression of p-MEK1/2, * vs. other groups with different symbols (†, ‡, §), P<0.001. M) Protein expression of p-ERK1/2, * vs. other groups with different symbols (†, ‡, §), P < 0.001. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 3) for each group. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Cell grouping: A₁ = ADMSCs, A₂ = ADMSCs + CoCl₂, A₃ = Nanog^OE-ADMSCs + CoCl₂, A4 = siRNA-Nanog in ADMSCs + CoCl₂. ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 5

Nanog gene expression activated cell cycle and upregulated mitochondrial electron transport chain (ETC) in oxidative stress condition. A) Protein expression of Cyclin D, * vs. other groups with different symbols (†, ‡, §), P < 0.0001. B) Protein expression of Cyclin E, * vs. other groups with different symbols (†, ‡, §), P < 0.001. C) Protein expression of CKD2, * vs. other groups with different symbols (†, ‡, §), P < 0.0001. D) Protein expression of CKD4, * vs. other groups with different symbols (†, ‡, §), P<0.0001. E) Protein expression of complex I, * vs. other groups with different symbols (†, ‡, §), P < 0.001. F) Protein expression of complex II, * vs. other groups with different symbols (†, ‡, §), P < 0.001. G) Protein expression of complex III, * vs. other groups with different symbols (†, ‡, §), P < 0.001. H) Protein expression of complex IV, * vs. other groups with different symbols (†, ‡, §), P < 0.0001. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6) for each group. Symbols (*, †, ‡, §) indicate significance (at 0.05 level). Cell grouping: B₁ = ADMSCs, B₂ = ADMSCs + H₂O₂, B₃ = Nanog^OE-ADMSCs + H₂O₂, B₄ = siRNA-Nanog gene in ADMSCs + H₂O₂. ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 6

Time courses of LVEF, LVRI and LVEDd, and LV chamber area by day 35. A1 to A5) Illustrating the M-mode findings of transthoracic echocardiographic study at the time point of day 35 after AMI induction in each group of the animal. The left ventricular end diastolic dimension (LVEDd) was notably increased in groups 2 and 5 than in other groups by day 35 after AMI induction. B1) left ventricular ejection fraction (LVEF) at baseline, P > 0.5. B2) LVRI at baseline, P > 0.5. B3) LVEDd at baseline, P > 0.5. C1) LVEF at day 14 after AMI induction, * vs. other groups with different symbols (†, ‡, §), P < 0.0001. C2) LVRI at day 14 after AMI induction, * vs. other groups with different symbols (†, ‡), P < 0.0001. C3) LVEDd at day 14 after AMI induction, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. D1) LVEF at day 35 after AMI induction, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. D2) LVRI at day 35 after AMI induction, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. D3) LVEDd at day 35 after AMI induction, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. E1 to E5) Illustrating the vertical microscopic (cross sections) finding (12.5x) of H.E., stain for identification of LV chamber area. All scale bars in right lower corner represents 12.5 mm. E6) Analytical result of LV chamber area, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 8-10) for each group. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). Group 1 = sham-operated control (SC), group 2 = AMI, group 3 = AMI + ADMSCs, group 4 = AMI + Nanog^OE in ADMSCs, 5 = AMI + siRNA-Nanog in ADMSCs. LV = left ventricular; AMI = acute myocardial infarction; ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 7

Protein levels of cell proliferation and cell-stress signalings in LV myocardium by day 35 after AMI induction. A) Protein expression of phosphorylated (p)-JAK, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. B) Protein expression of p-STAT3, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. C) Protein expression of p-PI3K, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. D) Protein expression of p-Akt, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. E) Protein expression of p-mTOR, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. F) Protein expression of RAS, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. G) Protein expression of RAF, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. H) Protein expression of p-MEK1/2, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. I) Protein expression of ERK1/2, * vs. other groups with different symbols (†, ‡, §, ¶), P<0.0001. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n=6) for each group. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). LV = left ventricular; SC = sham-operated control; AMI = acute myocardial infarction; ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 8

Protein expressions of cell cycle and mitochondrial ETC complexes in LV myocardium by day 35 after AMI induction. A) Protein expression of cyclin D, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. B) Protein expression of cyclin E, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. C) Protein expression of CDK2, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. D) Protein expression of CDK4, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. E) Protein expression of complex I, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. F) Protein expression of complex II, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. G) Protein expression of complex III, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. H) Protein expression of complex IV, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6) for each group. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). LV = left ventricular; SC = sham-operated control; AMI = acute myocardial infarction; ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 9

Protein expressions of oxidative stress and apoptosis, cardiomyocyte size and LV myocardial infarct area. A) Protein expression of NOX-1, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. B) Protein expression of NOX-2, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. C) Protein expression of cleaved caspase 3 (c-Casp3, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. D) Protein expression of c-Casp9, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. E1 to E5) Illustrating the microscopic finding (200x) of H.E., stain for identification of infarct area of LV myocardium (white color). All scale bar in right lower corner represents 50 µm. E6) Analytical result of infarct area, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. F1 to F5) Showing the microscopic finding (400x) of H.E., stain for identification of cardiomyocyte size (black dotted line) of LV myocardium. F6) Analytical result of cardiomyocyte size, * vs. other groups with different symbols (†, ‡, §, ¶), P < 0.0001. All scale bar in right lower corner represents 20.0 µm. Note: * indicated the control group. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6) for each group. Symbols (*, †, ‡, §, ¶) indicate significance (at 0.05 level). LV = left ventricular; SC = sham-operated control; AMI = acute myocardial infarction; ADMSCs = adipose-derived mesenchymal stem cells; Nanog^OE = overexpression of Nanog gene.

Figure 10

Underlying mechanism of Nanog gene on affecting the outcomes of AMI. Figure 10 illustrates the overexpression of Nanog gene on ADMSCs (i.e., Nanog^OE-ADMSCs) on upregulating the cell stress/cell proliferation signalings, resulting on improving the outcomes in setting of AMI. ADMSCs = adipose-derived mesenchymal stem cells; AMI = acute myocardial infarction; LVEF = left ventricular ejection fraction.