Hypoxic preconditioning enhances the benefit of cardiac progenitor cell therapy for treatment of myocardial infarction by inducing CXCR4 expression

Abstract

Myocardial infarction rapidly depletes the endogenous cardiac progenitor cell pool, and the inefficient recruitment of exogenously administered progenitor cells limits the effectiveness of cardiac cell therapy. Recent reports indicate that interactions between the CXC chemokine stromal cell-derived factor 1 and its receptor CXC chemokine receptor 4 (CXCR4) critically mediate the ischemia-induced recruitment of bone marrow-derived circulating stem/progenitor cells, but the expression of CXCR4 in cardiac progenitor cells is very low. Here, we studied the influence of hypoxia on CXCR4 expression in cardiac progenitor cells, on the recruitment of intravenously administered cells to ischemic heart tissue, and on the preservation of heart function in a murine myocardial infarction model. We found that hypoxic preconditioning increased CXCR4 expression in CLK (cardiosphere-derived, Lin(-)c-kit(+) progenitor) cells and markedly augmented CLK cell migration (in vitro) and recruitment (in vivo) to the ischemic myocardium. Four weeks after surgically induced myocardial infarction, infarct size and heart function were significantly better in mice administered hypoxia-preconditioned CLK cells than in mice treated with cells cultured under normoxic conditions. Furthermore, these effects were largely abolished by the addition of a CXCR4 inhibitor, indicating that the benefits of hypoxic preconditioning are mediated by the stromal cell-derived factor 1/CXCR4 axis, and that therapies targeting this axis may enhance cardiac-progenitor cell-based regenerative therapy.

Figure 1. Isolation, expansion, and phenotypic characterization of cardiosphere-derived, Lin⁻c-kit⁺ progenitor (CLK) cells

(A) Isolation and expansion of CLK cells. Step 1: cardiac explants were cultured for 2-3 weeks (panels 1 and 2); phase-contrast microscopy revealed cells migrating from the primary culture of mouse ventricular explants (panel 3, blue arrow); the migrated cells (round, phase-bright) aggregated and proliferated over the fibroblast coating (panel 4). Step 2: CLK cells were isolated from the phase-bright cells via lineage-depletion and c-kit⁺ enrichment (panels 1 and 2), then expanded in culture (panel 3). (B) Flow cytometric analyses of CLK cells for expression of the cell-surface markers c-kit, Sca-1, Flk-1, CD34, CD31, and CD45. (C) Immunofluorescent staining of CLK cells for expression of the cardiac transcription factors GATA4 (green) and Nkx2.5 (red); cell nuclei were counterstained with DAPI (blue). (D) GFP expression in CLK cells transfected with a lentiviral vector coding for GFP expression regulated by the human Nkx2.5 promoter.

Figure 2. CXCR4 expression in CLK cells

(A) CXCR4 mRNA expression in CLK cells and in freshly-isolated BMMNCs was evaluated with quantitative real-time RT-PCR. Values are presented as mean±SEM (n=3 per group, ***p<0.001). (B) Flow cytometry analyses of CXCR4 protein expression in CLK cells cultured under hypoxic or normoxic conditions for 4 hours. (C) Representative Western blots of HIF-1α, CXCR4, and GADPH protein expression in CLK cells cultured under normoxic or hypoxic conditions for 2, 4, 6, 8, 10, 16, and 24 hours (upper panel); quantification of HIF-1α and CXCR4 protein levels (normalized to GADPH protein levels) (lower panels). Representative data from three independent experiments are shown. (D) Representative Western blots of CXCR4, HIF-1α, and GADPH expression in CLK cells transfected with a CXCR4-expressing plasmid (pORF9-CXCR4), HIF-1α siRNA, non-targeting (NT) siRNA, or the control vehicle and cultured under hypoxic conditions for 8 hours (upper panel); quantification of CXCR4 and HIF-1α protein levels (normalized to GADPH protein levels) expressed as a ratio relative to the level observed in normoxia-cultured cells transfected with the empty vector (lower panels) (n=3 independent experiments; **, p<0.01 versus normoxia+empty vector group; §, p<0.05 versus hypoxia+HIF-1α siRNA group). (E) Immunofluorescent staining for HIF-1α (green) and CXCR4 (red) protein expression in CLK cells cultured under hypoxic or normoxic conditions for 8 hours; nuclei were counterstained with DAPI (blue).

Figure 3. CLK-cell migration, recruitment, and differentiation

(A) CLK cells were seeded into the upper chamber (4×10⁴ cells/well) of the ChemoTx cell-migration system, and the lower chamber was filled with 125 ng/mL recombinant human SDF-1, then the chambers were incubated at 37°C for 4 hours under hypoxic or normoxic conditions (n=8 per group); a subset of CLK cells were transfected with lentiviral CXCR4 shRNA before culture. The number of cells that had migrated to the lower chamber were quantified with a nucleic acid-binding fluorescent dye (***p < 0.001). (B-C) CLK cells were transfected with a vector coding for lacZ expression, cultured under hypoxic (Hypo) or normoxic (Norm) conditions for 6 hours, then intravenously injected into mice within 1 hour after surgically induced MI. (B) One day later, hearts were harvested, and recruitment was quantified by measuring β-galactosidase enzymatic activity in protein extracts from the ischemic heart tissue (**p<0.01). (C) One month after surgical MI and CLK-cell injection, hearts were harvested and sectioned, then sections were double-immunostained for co-expression of lacZ (green) and the lineage-specific proteins (red) cardiac troponin I (cTnI), von Willebrand factor (vWF), or smooth muscle actin (SMA); nuclei were counterstained with Draq5 (blue). The double-positive cells (yellow, identified with white arrows) in panels 1, 2, and 3 identified CLK cells that had differentiated into cardiomyocytes, endothelial cells, and smooth-muscle cells, respectively. Representative sections from 3 infarcted hearts in animals administered hypoxia-preconditioned CLK cells are displayed.

Figure 4. Therapeutic potential of systemically infused CLK cells

CLK cells were cultured under normoxic or hypoxic conditions for 6 hours with or without the CXCR4 antagonist AMD3100 (5 μg/mL), then intravenously injected into mice after surgical MI; control mice were administered CLK-cell medium. Echocardiographic measurements were performed 1, 2, and 4 weeks after CLK-cell administration, and mice were sacrificed for histological examinations at week 4. (A) Representative echocardiographic images at week 4. (B) Cardiac function during the 4-week period after surgical MI and CLK-cell administration. FS indicates left-ventricular fractional shortening; EF, left-ventricular ejection fraction. (**, p<0.005 versus any other treatment group). (C) Capillary density in peri-infarct myocardium 4 weeks after MI (**, p<0.01 versus medium group; ***, p<0.001 versus medium group; §, p<0.001 versus hypoxia+AMD3100 group). (D-E) Infarct wall thickness (D) and infarct size (E) 4 weeks after surgical MI and CLK-cell administration. (*, p<0.05 versus medium group; ***, p<0.001 versus medium group; §, p<0.001 versus hypoxia+AMD3100 group). (F) Heart weight (HW), body weight (BW), and heart-weight:body-weight ratio (HW/BW) 4 weeks after surgical MI and CLK-cell administration. (**, p<0.005 versus any other treatment group).