The administration of high-mobility group box 1 fragment prevents deterioration of cardiac performance by enhancement of bone marrow mesenchymal stem cell homing in the delta-sarcoglycan-deficient hamster

Abstract

Objectives: We hypothesized that systemic administration of high-mobility group box 1 fragment attenuates the progression of myocardial fibrosis and cardiac dysfunction in a hamster model of dilated cardiomyopathy by recruiting bone marrow mesenchymal stem cells thus causing enhancement of a self-regeneration system.

Methods: Twenty-week-old J2N-k hamsters, which are δ-sarcoglycan-deficient, were treated with systemic injection of high-mobility group box 1 fragment (HMGB1, n = 15) or phosphate buffered saline (control, n = 11). Echocardiography for left ventricular function, cardiac histology, and molecular biology were analyzed. The life-prolonging effect was assessed separately using the HMGB1 and control groups, in addition to a monthly HMGB1 group which received monthly systemic injections of high-mobility group box 1 fragment, 3 times (HMGB1, n = 11, control, n = 9, monthly HMGB1, n = 9).

Results: The HMGB1 group showed improved left ventricular ejection fraction, reduced myocardial fibrosis, and increased capillary density. The number of platelet-derived growth factor receptor-alpha and CD106 positive mesenchymal stem cells detected in the myocardium was significantly increased, and intra-myocardial expression of tumor necrosis factor α stimulating gene 6, hepatic growth factor, and vascular endothelial growth factor were significantly upregulated after high-mobility group box 1 fragment administration. Improved survival was observed in the monthly HMGB1 group compared with the control group.

Conclusions: Systemic high-mobility group box 1 fragment administration attenuates the progression of left ventricular remodeling in a hamster model of dilated cardiomyopathy by enhanced homing of bone marrow mesenchymal stem cells into damaged myocardium, suggesting that high-mobility group box 1 fragment could be a new treatment for dilated cardiomyopathy.

Fig 1

Changes in LVEF (a), LVDd (b), and LVDs (c) over time after the treatment. Diastolic and systolic dimensions of the left ventricle, and LVEF were measured before treatment, and reassessed at 4 and 6 weeks after treatment. The LVEF was significantly preserved until 6 weeks after the treatment in the HMGB1 group compared with the control group. LVEF, left ventricular ejection fraction; LVDd, left ventricular diastolic dimension; LVDs, left ventricular systolic dimension; HMGB1, high-mobility group box 1.

Fig 2. Suppression of myocardial fibrotic change in J2N-k hamsters by HMGB1 fragment.

(a), Representative photomicrographs (×20, scale bar = 1000μm) of picrosirius red staining. (b), Tissue sections were stained by picrosirius red and the fibrous area was quantified by image analysis. Percentage of myocardial fibrosis was significantly less in the HMGB1 group than in the control group. HMGB1, high-mobility group box 1.

Fig 3. Increased myocardial capillary density in J2N-k hamsters with HMGB1 fragment.

(a), Representative photomicrographs (×200, scale bar = 50μm) of anti-CD31 staining. (b), Tissue sections were stained for CD31 and the capillary density was measured. The HMGB1 group showed significantly higher capillary vascular density than the control group. HMGB1, high-mobility group box 1.

Fig 4. The increased accumulation of PDGFRα⁺ and CD106⁺ cells in the heart tissue with HMGB1 fragment.

(a), Representative photomicrographs (×1000, scale bar = 50 μm) of PDGFRα (green), CD106 (red) staining. (b), Tissue sections were stained for PDGFRα and CD106. The number of PDGFRα⁺ and CD106⁺ cells were measured. The HMGB1 group showed significantly increased numbers of PDGFRα⁺ and CD106⁺ cells than the control group. PDGFRα, platelet-derived growth factor receptor-alpha; HMGB1, high-mobility group box 1.

Fig 5. Increased CXCR4⁺ cells in SDF-1 positive area in the heart tissue by HMGB1 fragment.

(a), Representative photomicrographs (×600, scale bar = 50μm) of SDF-1 (green) and CXCR4 (red) staining. (b), Tissue sections were stained for CXCR4 and SDF-1. The number of CXCR4⁺ cells was measured and compared to the SDF-1 positive area. The HMGB1 group showed significantly higher CXCR4⁺ cells to SDF-1 positive area (mm²) in heart tissue than the control group. HMGB1, high-mobility group box 1; SDF-1, stromal derived factor-1.

Fig 6. Immunostaining for α-sarcoglycan and α-dystroglycan in cardiomyocytes.

(a), Representative photomicrographs (×600, scale bar = 50μm) of immunostaining of α-sarcoglycan and α-dystroglycan in cardiomyocytes. (b), Quantitative analysis of immunostaining showed significantly increased staining of both α-sarcoglycan and α-dystroglycan in the HMGB1 group than the control group. HMGB1, high-mobility group box 1.

Fig 7. Mitochondrial ultramicrostructure was detected by TEM.

Representative image (×12000, scale bar = 800nm) of mitochondrial morphology and cristae of myocardium in the HMGB1 group and the control group. The HMGB1 group showed a regular arrangement of mitochondrial cristae compared with the control group. TEM, Transmission electron microscopy; HMGB1, high-mobility group box 1.

Fig 8. Decreased oxidative stress in the heart tissue with HMGB1 fragment.

Representative photomicrographs of dihydroethidium staining (×400, scale bar = 50μm) (a) and 4-hydroxynonenal staining (×100, scale bar = 50μm) (b). Tissue sections were stained with dihydroethidium to estimate superoxide production, and 4-hydroxynonenal to estimate lipid peroxidation. The HMGB1 group showed significantly reduced production of superoxide (c) and a trend towards reduced lipid peroxidation (d) compared with the control group. HMGB1, high-mobility group box 1.

Fig 9. Decreased inflammatory response in the heart tissue with HMGB1 fragment.

Representative photomicrographs (×600, scale bar = 50μm) of CD68 staining (green). The HMGB1 group showed significantly reduced number of CD68⁺ cells compared with the control group. HMGB1, high-mobility group box 1.

Fig 10. Decreased cardiomyocyte apoptosis in the heart tissue with HMGB1 fragment.

Representative photomicrographs (×600, scale bar = 50μm) of annexin V staining (green). The HMGB1 group showed significantly reduced number of annexin V⁺ cells compared with the control group. HMGB1, high-mobility group box 1.

Fig 11. Expression of TSG-6, VEGF, CXCR4, and HGF in the heart tissue assessed with real-time PCR.

Intramyocardial mRNA levels of TSG-6, VEGF, and HGF were significantly higher in HMGB1 group compared with the control group. PCR, polymerase chain reaction, TSG-6, tumor necrosis factor-α stimulating gene 6, VEGF, vascular endothelial growth factor, HGF, hepatic growth factor, HMGB1, high-mobility group box 1.

Fig 12. Survival after each treatment assessed by the Kaplan–Meier method.

There was no significant difference between the single HMGB1 treatment (n = 11) group and the control group (n = 9), whereas the monthly HMGB1 group (n = 9) showed a significantly greater survival rate than control (p = 0.01). HMGB1, high-mobility group box 1.