Insufficient S-adenosylhomocysteine hydrolase compromises the beneficial effect of diabetic BMSCs on diabetic cardiomyopathy

Figures

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  Fig. 1. The distinctive effect on diabetic cardiomyopathy of BMSCs harvested from healthy and diabetic rats. BMSCs were infused 4 times, and cardiac function was measured using echocardiography (a) in control and DCM rats. The LVEF (b) and E′/A′ ratio (c) were used to assess the cardiac systolic and diastolic function, respectively (n = 7–8). Heart samples were harvested and fixed with paraformaldehyde (n = 6). The HE staining (upper) and Masson’s trichrome staining (lower) were conducted to assess myocardial morphology and cardiac fibrosis (d). The relative area of myocardial fibrosis (e) was estimated (scale bar = 20 μm). qPCR analysis showed the increased expression of cardiac stress-related genes, including Nppa, Nppb, and Myh7 (f) in rats with DCM (n = 4), suggesting adverse ventricular remodeling. The MDA levels (g, nmol/mg) and DHE staining (h, i) of cardiac tissue were measured to reflect the increased oxidative stress in rats with DCM (scale bar = 40 μm, n = 7). *P < 0.05 versus normal group, #P < 0.05 versus DCM group, and P < 0.05 versus DM-BSMC group

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  Fig. 2. The comparison of cellular properties between normal and diabetic BMSCs. The CCK-8 assay (a) showed that the cellular viability of DM-derived BMSCs was significantly worse than that of non-DM rats (n = 6). The levels of VEGF (b) and IGF-1 (c) in the medium were measured to reflect the paracrine function of BMSCs (n = 5). According to the DHE staining (d, e) and JC-1 staining (f, g), diabetic BMSCs had higher ROS level and mitochondrial dysfunction than nondiabetic BMSCs (n = 5–6). *P < 0.05 versus nondiabetic BMSCs group.

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  Fig. 3. SAH metabolism in normal and diabetic BMSCs. Diabetes BMSCs had a decreased expression of SAHH protein (a, b) compared with the nondiabetic BMSCs (n = 4). The increased metabolites SAH (c, nmol/mg) and Hcy (d, μmol/mg), especially SAH, in diabetes BMSCs also suggested an insufficient SAHH activity (n = 5). *P < 0.05 versus nondiabetic BMSCs group.

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  Fig. 4. The effect of SAHH inhibition in the normal BMSCs on endothelial cells. Western blots showed that plasmid containing SAHH shRNA and ADA significantly reduced the expression of SAHH in normal BMSCs (a, b, n = 5). According to the CCK-8 assay (c), SAHH inhibition reduced the cellular variability of BMSCs (n = 5). *P < 0.05 versus normal group. After SAHH inhibition, nondiabetic BMSCs could not promote endothelial proliferation (d, e) and tube formation (f, g, n = 5). *P < 0.05 versus normal group, #P < 0.05 versus BMSCs group. ROS generation (h, i) in endothelial cells under high glucose and fatty acids was measured using DHE staining after treatment with or without medium derived from normal BMSCs (n = 5). *P < 0.05 versus normal group, #P < 0.05 versus HG/PA group, and P < 0.05 versus HG/PA +BMSCs group.

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  Fig. 5. SAHH overexpression improved cellular activity of diabetic BMSCs . The plasmid containing the SAHH gene was transfected into diabetic BMSCs. 48 h later, the protein expression of SAHH in diabetic BMSCs was assessed by western blots (a, b, n = 4). The levels of VEGF (c) and IGF-1 (d) in the medium were measured to reflect the paracrine function of BMSCs (n = 5). *P < 0.05 versus control group, #P < 0.05 versus scramble (con) transfection group.

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  Fig. 6. SAHH overexpression improves cardioprotection of diabetic BMSCs. We further evaluated whether the overexpression of SAHH in BMSCs derived from diabetic rats can improve the therapeutic value of stem cells to DCM. Diabetic BMSCs with SAHH overexpression were infused 4 times. The LVEF (a) and E’/A’ ratio (b) were used to assess the cardiac function using echocardiography (n = 8). qPCR analysis showed the increased expression of cardiac stress-related genes, including Nppa, Nppb, and Myh7 (c) in rats with DCM (n = 4). Heart samples were harvested and fixed with paraformaldehyde. The immunofluorescence staining of vWF (d, e) and αSMA (f, g) was conducted to assess the pro-angiogenesis induced by BMSCs transplantation (n = 5). Sirius Red staining (h, i, n = 6) was conducted to assess to reflect myocardial fibrosis (scale bar = 100 μm). *P < 0.05 versus DCM group, #P < 0.05 versus DCM receiving diabetic BMSCs group.

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  Fig. 7. SAHH improves the biological activity of BMSCs partly by activating the Nrf2-mediated antioxidant signal . To further explore the underlying mechanism, we further explored whether SAHH activates the Nrf2-mediated antioxidant pathway to improve the activity and function of diabetic BMSCs. in vitro. The protein expression of Nrf2 and Keap1 was assessed in diabetic BMSCs after SAHH overexpression (a–c). After further treatment with ML385 (a Nrf2 inhibitor), the downstream molecules including anti-oxidative HO-1 (d, e), SOD1 (f), SOD2 (g), and pro-oxidative p67phox (h) were measured by western blots (n = 4). *P < 0.05 versus DM-BMSCs group, #P < 0.05 versus DM-BMSCs +SAHH group.