Preconditioning diabetic mesenchymal stem cells with myogenic medium increases their ability to repair diabetic heart

Abstract

Introduction: Mesenchymal stem cells (MSCs) have the potential for treatment of diabetic cardiomyopathy; however, the repair capability of MSCs declines with age and disease. MSCs from diabetic animals exhibit impaired survival, proliferation, and differentiation and therefore require a strategy to improve their function. The aim of the study was to develop a preconditioning strategy to augment the ability of MSCs from diabetes patients to repair the diabetic heart.

Methods: Diabetes was induced in C57BL/6 mice (6 to 8 weeks) with streptozotocin injections (55 mg/kg) for 5 consecutive days. MSCs isolated from diabetic animals were preconditioned with medium from cardiomyocytes exposed to oxidative stress and high glucose (HG/H-CCM).

Results: Gene expression of VEGF, ANG-1, GATA-4, NKx2.5 MEF2c, PCNA, and eNOS was upregulated after preconditioning with HG/H-CCM, as evidenced by reverse transcriptase/polymerase chain reaction (RT-PCR). Concurrently, increased AKT phosphorylation, proliferation, angiogenic ability, and reduced levels of apoptosis were observed in HG/H-CCM-preconditioned diabetic MSCs compared with nontreated controls. HG/H-CCM-preconditioned diabetic-mouse-derived MSCs (dmMSCs) were transplanted in diabetic animals and demonstrated increased homing concomitant with augmented heart function. Gene expression of angiogenic and cardiac markers was significantly upregulated in conjunction with paracrine factors (IGF-1, HGF, SDF-1, FGF-2) and, in addition, reduced fibrosis, apoptosis, and increased angiogenesis was observed in diabetic hearts 4 weeks after transplantation of preconditioned dmMSCs compared with hearts with nontreated diabetic MSCs.

Conclusions: Preconditioning with HG/H-CCM enhances survival, proliferation, and the angiogenic ability of dmMSCs, augmenting their ability to improve function in a diabetic heart.

Figure 1

Preconditioning strategy for the treatment of dmMSCs. (A) Blood glucose levels during streptozotocin administration until 60 days in C57BL/6 wild-type mice. (B) Gene-expression profiling of normal and diabetic MSCs with RT-PCR. (C) Gel band quantification by Image J. *P < 0.05; **P < 0.01; and ***P < 0.001 significance for normal MSCs versus diabetic MSCs. (D) VEGF release in cardiomyocytes subjected to 60 and 90 minutes of H₂O₂-induced oxidative stress combined with varying glucose concentrations, as measured with ELISA. (E, F) Gene-expression analysis of dmMSCs preconditioned with media from cardiomyocytes subjected to no treatment or H₂O₂ treatment in the presence of low or high glucose. (G, H) Gene expression of dmMSCs preconditioned with different concentrations of VEGF. (I) Gene-expression analysis of p53 between different CCM-treated groups compared with nontreatment (NT) along with corresponding quantification. NT versus HG/H, *P < 0.05; **P < 0.01; and ***P < 0.001). (J, K) Comparison of gene expression between dmMSCs preconditioned with HG/H-CCM or VEGF (0.065 ng/ml). (L) Analysis of gene expression for VEGF, NKx2.5, and GATA-4 by using real-time quantitative PCR. NT versus VEGF, *P < 0.05; **P < 0.01; and ***P < 0.001; NT versus HG/H-CCM, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; VEGF versus HG/H-CCM, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001.

Figure 2

Increased survival and proliferation of dmMSCs after preconditioning. (A, B) Increased AKT phosphorylation in dmMSCs treated with HG/H-CCM compared with VEGF (0.065 ng/ml). (C) Trypan blue viability assay. NT versus HG/H-CCM *P < 0.05; **P < 0.01; and ***P < 0.001; VEGF versus HG/H-CCM ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001. (D) LDH-release assay. (E)PCNA gene expression after preconditioning with HG/H-CCM and VEGF (0.065 ng/ml). (F) Quantification of PCNA gel bands was done by using Image J software. (G) Quantification of Annexin-V (%) in nontreated, HG/H-CCM and VEGF (0.065 ng/ml) preconditioned dmMSCs along with quantification. NT versus VEGF, *P < 0.05; **P < 0.01; and ***P < 0.001; NT versus HG/H-CCM, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; VEGF versus HG/H-CCM, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001. (H) XTT cell-proliferation assay confirms increased proliferation after HG/H-CCM treatment compared with VEGF (0.065 ng/ml) or nontreated control cells. NT versus HG/H-CCM, *P < 0.05; **P < 0.01; and ***P < 0.001; HG/H-CCM versus VEGF, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001.

Figure 3

Effect of preconditioning on the angiogenic ability of dmMSCs. (A-D) Matrigel assay of preconditioned and nontreated dmMSCs. Images show increased tube formation after HG/H-CCM group versus VEGF (0.065 ng/ml) versus nontreated dmMSCs. Scale bar about 20 μm. (E, F) Gene expression of CD31 and CD34 endothelial lineage markers. Quantification of gel bands by using Image J software. (G) NOS activity of dmMSCs preconditioned with HG/H-CCM or VEGF. NT versus VEGF, *P < 0.05; **P < 0.01; and ***P < 0.001; NT versus HG/H-CCM, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; VEGF versus HG/H-CCM, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001.

Figure 4

Increased homing and paracrine signaling of preconditioned dmMSCs. (A, B) Increased homing of preconditioned dmMSCs compared with nontreated MSCs along with corresponding quantification in (C); Scale bar about 20 μm. (D) Gene-expression analysis of paracrine factors in all animal groups by using quantitative real-time PCR. (E) Gene-expression analysis in hearts from all animal groups. (F) Gel quantification using image J. Group II versus group III, *P < 0.05; **P < 0.01, and ***P < 0.001; group II versus group IV, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; group III versus group IV, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001.

Figure 5

Cardiac function. Graphic representation of following parameters: (A) Ejection fraction (%) in four groups. (B) dp/dt. (C and D) represent the left ventricle end-diastolic pressure and left ventricle diastolic pressure, respectively. Group II versus group III, *P < 0.05; **P < 0.01; and ***P < 0.001; group II versus group IV, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; group III versus Group IV, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001.

Figure 6

Preconditioned dmMSCs enhance angiogenesis in diabetic heart. (A through D) Blood-vessel density of cells expressing smooth muscle actin along with corresponding quantification in (E); scale bar about 20 μm. (F through J) Quantification of eNOS-positive cells in all the groups. Group II versus group IV, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; group III versus group IV, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001. (K) Gene-expression analysis of eNOS and iNOS in all groups with RT-PCR. (L) Quantification of gel bands by using Image J software. Group II versus group III, *P < 0.05; **P < 0.01; and ***P < 0.001; Group II versus group IV, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; group III versus group IV, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001.

Figure 7

Decreased fibrosis and apoptosis in diabetic hearts transplanted with preconditioned dmMSCs. Analysis of fibrosis in (A) group I, (B) group II, (C) group III, and (D) group IV with Masson trichrome staining of diabetic heart sections. (E) Quantification of fibrosis in all animal groups. Cleaved casapse-3 staining of diabetic heart for the identification of apoptosis. Images represent (F) group I; (G) group II; (H) group III, (I) group IV (scale bar about 20 μm). (J) Cleaved caspase-3 analysis of apoptosis in all experimental groups. Quantification was done with image J software. Group II versus group III, *P < 0.05; **P < 0.01; and ***P < 0.001; group II versus group IV, ^#P < 0.05; ^##P < 0.01; and ^###P < 0.001; group III versus group IV, ^φP < 0.05; ^φφP < 0.01; and ^φφφP < 0.001.