Tadalafil, a long acting phosphodiesterase inhibitor, promotes bone marrow stem cell survival and their homing into ischemic myocardium for cardiac repair

Figures

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  Figure 1. Effect of tadalafil treatment on in vitro MSCs: MSCs viability (CCK‐8 Assay, A) and cGMP activity (B) were assessed. The expression of p‐STAT3, p‐Erk1/2, p‐Akt, Bcl‐xl, p‐VASP, p‐GSK β, PKG1, and Fas (C) were assessed in MSCs extracts by western blots. Also, in MSCs extracts, SDF‐1α (D) and NO levels (E) (ELISA assay), and iNOS, CXCR4 and total‐Akt expressions (western blots assay) were assessed in ± NOS (L‐NAME) or CXCR4 (AMD3100) inhibitors (F). Values are mean ± SE; n = 6; P‐value <0.05 is significant.

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  Figure 2. Tadalafil induced cytoprotection of in vitro MSCs under oxidative stress through PKG‐MAPK signaling pathways: Cell viability (CCK‐8 assay) (A) and cell death (LDH assay) (B) of MSCs after exposure to 100 μmol/L H2O2 for 4 h (oxidative stress) were assessed in control and tadalafil. The level of NOS (ELISA assay, C) levels were assessed under oxidative stress in control and tadalafil in ± L‐NAME inhibitors. p‐VASP, PKG1, Fas, BcL‐xl, p‐ p‐STAT3 and Erk1/2 expressions (western blots, D) were assessed under oxidative stress in control and tadalafil in ± MAPK (U0126) and PKG1 (KT5823) inhibitors. Values are mean ± SE; n = 6; P‐value < 0.05 is significant.

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  Figure 3. Tadalafil induced cytoprotection of MSCs under oxidative stress through an anti‐apoptotic signaling pathway. Number of TUNEL + cells (A; ×200, Red = TUNEL + nuclei; blue=DAPI, B; TUNEL + cells numbers) and MSCs viability (CCK‐8 assay, C) were assessed in tadalafil‐treated versus DMSO‐treated MSCs in vitro after 4 h exposure to 100 μmol/L H2O2 in ± MAPK (U0126) and PKG1 (KT5823) inhibitors. Values are mean ± SE; n = 6; P‐value < 0.05 is significant.

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  Figure 4. Tadalafil increased miR‐21 expression of in vitro MSCs under oxidative stress. Tadalafil induced miR‐21 up‐regulation (microRNA‐array analysis, (A). miR‐21 expression was increased in MSCs‐treated by tadalafil versus control (B). RT‐PCR: miR‐21 expression bands; and quantitative analysis of miR‐21/U6 expression was assessed in ± MAPK (U0126) and PKG1 (KT5823) inhibitors (PCR‐gel bands and densitometry assay, (C). Values are mean ± SE; n = 6; P‐value <0.05 is significant.

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  Figure 5. Cytoprotective effects of tadalafil were abrogated by miR‐21 inhibition in in vitro MSCs under oxidative stress. MSCs were transfected with pEZX‐Luc vector containing Fas 3′‐UTR together with a plasmid encoding miR‐21; or MSCs were transfected with anti‐miR‐21 using siPORT™ NeoFx™ transfection agent. Number of TUNEL + cells (A‐B) and cell viability (CCK‐8 assay, C), luciferase activity % (D), STAT3 and Fas expressions (western blots, E) were assessed after treatment with ± miR‐21 inhibitors. Also, p‐VASP, PKG1, Fas and BcL‐xl expressions (western blots, F) were assessed under oxidative stress in control and tadalafil in ± FasL inhibitors. Values are mean ± SE; n = 6; P‐value < 0.05 is significant.

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  Figure 6. Tadalafil improved in vivo left ventricular (LV) function of infarcted hearts: LV‐ESD (A), LV‐EDD (B), LV‐ESV (C), LV‐EDV (D), LV‐EF (E), and LV‐FS (F) were assessed by echocardiography in control and MSCs transplanted ± tadalafil groups after 2 weeks and 1 month post‐treatment. At 7 days, both SDF‐1α (G) and NO (H) levels (ELISA assay), iNOS, p‐GSK β, Bcl‐xl, Bcl‐2, total‐Akt, and Fas expressions (western blots, I) were assessed in LV tissues extracts in control and MSCs transplanted ± tadalafil groups. Values are mean ± SE; n = 8; P‐value <0.05 is significant.

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  Figure 7. Tadalafil improved grafted cell survival in vivo and increased cell homing to the infarcted heart. LV‐ESD (A), LV‐EDD (B), LV‐ESV (C), LV‐EDV (D), LV‐EF% (E) and LV‐FS% (F) were assessed in control (Saline, IP) and IP tadalafil ± NOS (L‐NAME) or CXCR4 (AMD3100) inhibitors groups after 2 weeks and 1 month post‐treatment. In other groups, at 7 days, tadalafil effects on NO and CXCR4 signaling pathways were assessed after LAD ligation in 1 month post‐myeloablated rats with successful IV MSCs‐GFP +. In the LV tissue extracts, SDF‐1α (G) and NO (H) levels (ELISA assay); and PKG1, VEGF and SDF‐1α expressions (western blots, I) were assessed in ± L‐NAME and AMD3100. Values are mean ± SE; n = 8; P‐value < 0.05 is significant.

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  Figure 8. Tadalafil induced in vivo mobilization and homing of transplanted 111In‐oxine–labeled MSCs into infarcted heart. The SPECT/CT images of the chest and abdomin of mice were performed at 2 h, 24 h and 48 h to detect the uptake of 111In‐oxine–labeled MSCs‐GFP + in ischemia‐induced infarcted heart of mice injected intravenously (IV) 1 h after ± IP tadalafil (A). At day 7, the specific activity of 111In‐oxine was assessed in heart, lung, spleen, kidney and liver organs in tadalafil‐treated versus control mice (B). Ex‐vivo fluorescent imaging was assessed to detect the uptake of 111In‐oxine–labeled MSCs‐GFP + in hearts of mice treated with tadalafil versus controls (C). Values are mean ± SE; n = 8; P‐value < 0.05 is significant.