In vitro and in vivo effects of bone marrow stem cells on cardiac structure and function

Abstract

It is hypothesized that the protection of bone marrow stem cells (BMSCs) on ischemic myocardium might be related to the anti-apoptotic effect via paracrine mechanisms. In this study, a wide array of cytokines including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor-1 (SDF-1) and insulin growth factor-1 (IGF-1) were detected in the BMSCs cultured medium by ELISA. Myocyte apoptosis was assayed by DNA fragmentation and annexin-V staining. Myocardial infarction model was produced by ligation of mouse left anterior descending coronary artery (LAD). Before LAD ligation, mice were myoablated by irradiation and transplanted with bone marrow cells from transgenic mice expressing green fluorescent protein (GFP). After LAD ligation, animals were administered stem cell factor (SCF, 200 mug/day/kg, i.p.) or saline for 6 days. Animals were sacrificed at 4 weeks after SCF treatment. Apoptotic cardiomyocytes were analyzed by TUNEL. Myocardial function was analyzed by echocardiography and pressure-volume system. Bcl-2 protein was analyzed by Western blotting. Our results showed that cultured BMSCs released VEGF, bFGF, SDF-1 and IGF-1. Hypoxia-induced cell apoptosis was diminished in cardiomyocytes co-cultured with BMSCs. Smaller LV dimension and increased LV ejection fraction were seen in SCF-treated animals. SCF significantly reduced cardiomyocytes apoptosis within peri-infarct area and increased up-regulation expression of Bcl-2 in ischemic area. Moreover, conditioned medium from cultured BMSCs also induced up-regulation of Bcl-2 protein in cardiomyocytes. It is concluded that paracrine mediators secreted by BMSCs might be involved in early repair of ischemic heart by preventing cardiomyocytes apoptosis and improving cardiac function.

Figure 1

BMSCs secreted cytokines under in vitro conditions. A, A significant amount of VEGF, bFGF, SDF-1 and IGF-1 was released from BMSCs (n=8). MC = cardiomyocytes

Figure 2

BMSCs enhanced cardiomyocyte survival under hypoxia. Panel A and B: Representative photographs show typical of Annexin-V positive cells. Red fluorescence shows apoptotic cells stained with PE-labeled annexin-V. Nuclei were counterstained with DAPI (blue). A: cardiomyocytes; B: co-culture. Panel C and D: Representative FACS of count Annexin-V-PE positive cells in cardiomyocytes (C) and co-culture (D), respectively. Panel E: quantitation of annexin-V positive cells. Data are shown as mean ± SEM. * p < 0.05 vs cardiomyocytes culture alone. Panel F: DNA fragmentation in cardiomyocytes alone or co-cultured with BMSCs following hypoxia [lane 1: DNA marker; lane 2: cardiomyocyte (normoxia); lane 3: co-culture (normoxia); lane 4: cardiomyocytes (hypoxia); lane 5: co-culture (hypoxia); lane 6: positive control].

Figure 3

Representative western blotting of Bcl-2 protein in dual-set system cultured cardiomyocytes exposed to hypoxia for 24 hours (panel A). Panel B: Bcl-2 was quantified by western blots in cultured cardiomyocytes. Data are shown as mean ± SEM. * p < 0.01 vs cardiomyocytes culture alone.

Figure 4

Apoptosis of cardiomyocytes after 1 week of MI. A-B, Representative photomicrographs showing TUNEL staining in peri-infarct area. Arrow indicates TUNEL positive cardiomyocytes (A: saline control mice; B: SCF treated mice) (× 400). Asterisks show infarct area. C, Percentage of TUNEL positive cardiomyocytes in both groups (n = 4). * p < 0.05 vs saline control.

Figure 5

Representative western blotting of Bcl-2 protein within peri-infarct myocardium 1 week after MI (panel A). Panel B: Bcl-2 was quantified by western blots (n = 4) in peri-infarcted myocardium. Data are shown as mean ± SEM. * p < 0.01 vs saline control.

Figure 6

LV function assessed by echocardiography. Shown are representative LV M-mode echocardiographic recordings in both SCF treated and saline treated mice at 4 weeks after MI. IVS, interventricular septum; LVPW, left ventricular posterior wall; LVID, left ventricular internal dimensions.

Figure 7

Representative LV Pressure-Volume Relationships obtained 4 weeks after LAD occlusion. Slope of the regression line (end-systolic pressure volume relationship, red color) represents ventricular end-systolic elastance (Ees). Ees of SCF-treated mice (B) was 2-fold greater than control group (A). Besides, Ves and Ved of SCF treated mice were obviously less shifted rightward which supports the observation that LV contractility of SCF-treated group was significantly improved. In SCF treated mice, LV systolic (Pmax, E-max) and diastolic functions (Pmin, tau-W) were significantly better than the control group (C). Data are mean ± SEM, n = 6 in each group, * p < 0.05 vs. control. Pmax indicates maximum pressure; Pmin, minimum pressure; Emax, maximal elastance; Tau-W, Tau-Weiss method.

Figure 8

Incorporation of green BMSCs into cardiomyocytes and smooth muscle cells. Representative fluroscence micrographs of peri-infarct area at 4 weeks after MI. A-B, GFP-positive cells (arrow) is shown as a desmin-positive cardiomyocytes (red). Yellow in (B) indicates cardiomyocytes derived from GFP-positive cells (× 1000). C-D, GFP-positive cells expressed SMA (arrow). Yellow labeling indicates vascular smooth muscle cells derived from GFP-positive cells (× 400). E. Percentage of GFP-positive cardiomyocytes. Total 15 sections were examined in both groups (n = 6). Data are shown as mean ± SEM. * p < 0.05. Bar scale, 20 μm.