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. 2014 Sep 25;5(5):111.
doi: 10.1186/scrt499.

Preconditioning of bone marrow mesenchymal stem cells by prolyl hydroxylase inhibition enhances cell survival and angiogenesis in vitro and after transplantation into the ischemic heart of rats

Xian-Bao Liu, Jian-An Wang, Xiao-Ya Ji, Shan Ping Yu, Ling Wei

Preconditioning of bone marrow mesenchymal stem cells by prolyl hydroxylase inhibition enhances cell survival and angiogenesis in vitro and after transplantation into the ischemic heart of rats

Xian-Bao Liu et al. Stem Cell Res Ther. .

Abstract

Introduction: Poor cell survival and limited functional benefits have restricted the efficacy of bone marrow mesenchymal stem cells (BMSCs) in the treatment of myocardial infarction. We showed recently that hypoxia preconditioning of BMSCs and neural progenitor cells before transplantation can enhance the survival and therapeutic properties of these cells in the ischemic brain and heart. The present investigation explores a novel strategy of preconditioning BMSCs using the Hypoxia-inducible factor 1α (HIF-α) prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) to enhance their survival and therapeutic efficacy after transplantation into infarcted myocardium.

Methods: BMSCs from green fluorescent protein transgenic rats were cultured with or without 1 mM DMOG for 24 hours in complete culture medium before transplantation. Survival and angiogenic factors were evaluated in vitro by trypan blue staining, Western blotting, and tube formation test. In an ischemic heart model of rats, BMSCs with and without DMOG preconditioning were intramyocardially transplanted into the peri-infarct region 30 minutes after permanent myocardial ischemia. Cell death was measured 24 hours after engraftment. Heart function, angiogenesis and infarct size were measured 4 weeks later.

Results: In DMOG preconditioned BMSCs (DMOG-BMSCs), the expression of survival and angiogenic factors including HIF-1α, vascular endothelial growth factor, glucose transporter 1 and phospho-Akt were significantly increased. In comparison with control cells, DMOG-BMSCs showed higher viability and enhanced angiogenesis in both in vitro and in vivo assays. Transplantation of DMOG-BMSCs reduced heart infarct size and promoted functional benefits of the cell therapy.

Conclusions: We suggest that DMOG preconditioning enhances the survival capability of BMSCs and paracrine effects with increased differentiation potential. Prolyl hydroxylase inhibition is an effective and feasible strategy to enhance therapeutic efficacy and efficiency of BMSC transplantation therapy after heart ischemia.

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Figures

Figure 1
Figure 1
Effect of dedimethyloxalylglycine preconditioning on the expression of prosurvival and angiogenic factors. (A) Rat bone marrow mesenchymal stem cells (BMSCs) were cultured with or without dedimethyloxalylglycine (DMOG, 1 mM) for 24 hours. Hypoxia-inducible factor (HIF)-1ɑ, glucose transporter 1 (Glut-1), vascular endothelial growth factor (VEGF) and phospho-Akt were then detected by western blotting. Beta-actin was used as the loading control. (B) Densitometric analysis was applied for comparison of the relative expression levels of different proteins in DMOG-BMSCs with respect to vehicle control BMSCs (C-BMSCs) that is arbitrarily presented as 1. n =3 independent assays. *P <0.05 compared with C-BMSC group. (C) Ratio of phospho-Akt against total Akt expression, quantified from the western blot gels. A large increase of 10-fold Akt phosphorylation was seen in DMOG-BMSCs compared with control BMSCs. n =3 independent assays. *P <0.05 vs. control cells.
Figure 2
Figure 2
Effect of dedimethyloxalylglycine preconditioning on cell death in vitro and after transplantation. (A) Hydrogen peroxide (H2O2)-induced cell death was measured in bone marrow mesenchymal stem cell (BMSC) cultures with and without dedimethyloxalylglycine (DMOG) preconditioning. After different durations of DMOG (1 mM) treatment, cell death was induced by H2O2 (100 μM) in a serum-free medium for 90 minutes. The membrane-permeable dye Trypan Blue was added 5 to 10 minutes before cell death measurement. Damaged BMSCs were identified as cells unable to exclude the blue color dye from the cytosol. n =3 independent tests in each time group. *P <0.05 compared with C-BMSC group. # P <0.05 compared with basal control group. (B) to (E) Green fluorescent protein (GFP)-positive BMSCs (green) were labeled with Hoechst 33342 (blue) before transplantation to further facilitate tracking transplanted cells. Cell death was evaluated 24 hours after transplantation using terminal deoxynucleotidyl transferase biotin-dUPT nick end labeling (TUNEL) staining (red) in heart sections. Co-labeling of GFP/Hoechst/TUNEL fluorescence was designated as transplanted dead BMSCs. (F) Summarized cell count data of GFP/Hoechst/TUNEL-positive cells against total exogenous (GFP/Hoechst-positive) cells. n =4 independent experiments in each group. *P <0.05 compared with C-BMSC group.
Figure 3
Figure 3
Effect of dedimethyloxalylglycine preconditioning on angiogenesis in vitro and after transplantation. (A), (B) Tube formation test stimulated by Matrigel was performed to identify the angiogenic activity of control bone marrow mesenchymal stem cells (C-BMSCs) and dedimethyloxalylglycine-preconditioned BMSCs (DMOG-BMSCs) in vitro. (C) to (H) Angiogenesis was inspected using von Willebrand factor staining (red) in heart sections from the myocardial infarction (MI), C-BMSC and DMOG-BMSC groups 4 weeks after MI. Hoechst staining (blue) shows the total cells. (I) Summary of total tube length measured in (A) and (B). The total tube length in the C-BMSC group was arbitrarily presented as 1. n =3 independent measurements. (J) Summary of total vessel density in different groups of in vivo experiments. n =8 animals in each group. *P <0.05 compared with C-BMSC group; # P <0.05 compared with MI control group.
Figure 4
Figure 4
Effect of bone marrow mesenchymal stem cell transplantation on ischemia-induced infarct formation. Heart infarct area and scar formation were determined using Masson’s Trichrome staining 4 weeks after myocardial infarction (MI). (A) to (C) Images of representative infarcted hearts from a MI control rat, a MI rat receiving control bone marrow mesenchymal stem cells (C-BMSCs), and a MI rat receiving dedimethyloxalylglycine-preconditioned BMSCs (DMOG-BMSCs). (D) Transplantation of BMSCs reduced heart infarction formation; the protective effects were significantly greater with transplantation of DMOG-BMSCs. n =5 rats in each group. *P <0.05 compared with MI group; # P <0.05 compared with C-BMSC group.
Figure 5
Figure 5
Effect of bone marrow mesenchymal stem cell transplantation on functional recovery after heart ischemia. Heart function was measured 4 weeks after myocardial infarction (MI) and cell transplantation treatment. MI rats showed functional deficits in all measurements of (A) left ventricular systolic pressure (LVSP), (B) left ventricular end-diastolic pressure (LVEDP), (C) maximum change rate of left ventricular pressure rise and fall (±dp/dt), (D) minimum change rate of left ventricular pressure rise and fall (–dp/dt), (E) time constant of the isovolumic pressure decline (Tau) and (F) left ventricular ejection fraction (LVEF). BMSC transplantation improved functional parameters of MI rats. Rats received dedimethyloxalylglycine-preconditioned BMSCs (DMOG-BMSCs) showed the enhanced functional recovery in all four functional parameters. n =8 rats in each group. *P <0.05 compared with MI-only group. # P <0.05 compared with the control BMSC (C-BMSC) group.
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