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. 2012;7(7):e37948.
doi: 10.1371/journal.pone.0037948. Epub 2012 Jul 18.

Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis

Fengdi Yan  1 , Yuyu Yao, Lijuan Chen, Yefei Li, Zulong Sheng, Genshan Ma
Affiliations

Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis

Fengdi Yan et al. PLoS One. 2012.

Abstract

Background: Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis.

Methods and results: CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100.

Conclusions: Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Characterization of cultured CPCs.
(A) Typical phase-contrast images of the outgrowth from enzymatically digested cardiac explants after planted for 1 h (×100 magnification). (B) Cells (small, round, phase-bright) migrated from the cardiac explants, aggregated and proliferated over the fibroblast layer after culturing for ten days (×100 magnification). (C) Cells migrated from the cardiac explants after culturing for 14 days (×200 magnification). (D) Representative clone generated by CPCs (×200 magnification). (E) Phase-contrast image of CPCs after passage (×100 magnification). (F) Representative immunocytochemistry staining of CPCs for expression of cell surface marker c-kit. (×200 magnification) (G) Negative control immunocytochemistry staining of c-kit. (H) Representative flow cytometric analyses of CPCs for expression of the cell surface markers c-kit, Sca-1.
Figure 2
Figure 2. Effects of hypoxia preconditioning on CPCs in vitro.
CPCs were cultured under either normoxia (Nx) or hypoxia (Hx) for 3, 6,12, and 24 hours. (A) Flow cytometry analysis of apoptosis in CPCs cultured under either normoxia or hypoxia at different times. (B) Quantitative analysis of apoptotic cells identified by flow cytometry. Data were obtained from three independent experiments and are expressed as mean ± SD. *P<0.01 vs. normoxia (Nx) group. (C) Representative Western blots of HIF-1α, CXCR4, and β-actin protein expression in CPCs (top); quantification of HIF-1α and CXCR4 protein levers (normalized to β-actin protein levels ). (D) Representative Western blots of p-Akt, Bcl-2 and β-actin protein expression in CPCs (top); quantification of p-Akt and Bcl-2 protein levers (normalized to β-actin protein levels). (E) Supernatant VEGF and SDF-1α concentrations measured by ELISA in CPCs that underwent hypoxia preconditioning. Values are mean ± SD. n = 3. *P<0.01vs.normoxia.
Figure 3
Figure 3. CPC migration and survival influenced by hypoxia preconditioning.
CPCs were cultured under either normoxic (Nx) or hypoxic (Hx) conditions for six hours with or without AMD3100 (CXCR4-selective antagonist, 5 µg/mL). (A) Representative migrated CPCs (stained with crystal violet) are shown (×200 magnification). (C) Quantitative analysis of migrated cells. Data are presented as mean ± SD. n = 5. *P<0.01. (B) CPCs were exposed to hypoxia along with serum deprivation for 48 hours. Representative flow cytometry analysis of apoptotic cells after being labeled with annexin V and propidium iodide. (D) Quantitative analysis of apoptosis evaluated by annexin V staining. Data were obtained from five independent experiments and are expressed as mean ± SD. *P<0.01 vs. normoxia (Nx) group & normoxia (Nx) + AMD group & hypoxia (Hx) + AMD group.
Figure 4
Figure 4. Effect of hypoxia preconditioned CPCs in treating MI.
CPCs were cultured under either normoxia (Nx) or hypoxia (Hx) for six hours with or without AMD3100 (CXCR4-selective antagonist, 5 µg/mL) and then intramyocardially injected into mice after surgical MI. Mice in the control group were administered DMEM. (A) Echocardiographic measurements were performed seven days after CPC transplantation. Representative M-mode images at the level of papillary muscles were recorded. (B) Representative Masson’s trichrome staining of transverse heart sections seven days after coronary ligation and CPC administration. (C) Representative phase micrographs show TUNEL-positive apoptotic cells in bordering myocardium adjacent to the infarct zone seven days after coronary ligation (×200 magnification). (D) Quantitative analysis of infarct size. Values are expressed as mean ± SD. n = 5. *P<0.05 vs. control group, **P<0.01 vs. control group, # P<0.05 vs. normoxia (Nx) & hypoxia (Hx) + AMD group. (E) Quantitative analysis of apoptotic cells in bordering myocardium. Values are expressed as mean ± SD. n = 5. *P<0.01 vs. control group, # P<0.01 vs. normoxia (Nx) & hypoxia (Hx) + AMD group. (F) Representative TUNEL+ apoptotic engrafted CPCs in heart tissues which were collected 72 h after cell transplantation (×200 magnification). (G) The percentage of apoptotic engrafted CPCs. The number of apoptotic engrafted CPCs was assessed by double positive of GFP+/TUNEL+ (yellow). Values are expressed as mean ± SD. n = 5. *P<0.01 vs. normoxia (Nx) group & hypoxia (Hx) + AMD group.
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