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. 2009 Nov;297(5):R1503-15.
doi: 10.1152/ajpregu.00227.2009. Epub 2009 Sep 16.

Intramuscular VEGF repairs the failing heart: role of host-derived growth factors and mobilization of progenitor cells

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Intramuscular VEGF repairs the failing heart: role of host-derived growth factors and mobilization of progenitor cells

David Zisa et al. Am J Physiol Regul Integr Comp Physiol. 2009 Nov.

Abstract

Skeletal muscle produces a myriad of mitogenic factors possessing cardiovascular regulatory effects that can be explored for cardiac repair. Given the reported findings that VEGF may modulate muscle regeneration, we investigated the therapeutic effects of chronic injections of low doses of human recombinant VEGF-A(165) (0.1-1 microg/kg) into the dystrophic hamstring muscle in a hereditary hamster model of heart failure and muscular dystrophy. In vitro, VEGF stimulated proliferation, migration, and growth factor production of cultured C2C12 skeletal myocytes. VEGF also induced production of HGF, IGF2, and VEGF by skeletal muscle. Analysis of skeletal muscle revealed an increase in myocyte nuclear [531 +/- 12 VEGF 1 microg/kg vs. 364 +/- 19 for saline (number/mm(2)) saline] and capillary [591 +/- 80 VEGF 1 microg/kg vs. 342 +/- 21 for saline (number/mm(2))] densities. Skeletal muscle analysis revealed an increase in Ki67(+) nuclei in the VEGF 1 microg/kg group compared with saline. In addition, VEGF mobilized c-kit(+), CD31(+), and CXCR4(+) progenitor cells. Mobilization of progenitor cells was consistent with higher SDF-1 concentrations found in hamstring, plasma, and heart in the VEGF group. Echocardiogram analysis demonstrated improvement in left ventricular ejection fraction (0.60 +/- 0.02 VEGF 1 microg/kg vs. 0.45 +/- 0.01 mm for saline) and an attenuation in ventricular dilation [5.59 +/- 0.12 VEGF 1 microg/kg vs. 6.03 +/- 0.09 for saline (mm)] 5 wk after initiating therapy. Hearts exhibited higher cardiomyocyte nuclear [845 +/- 22 VEGF 1 microg/kg vs. 519 +/- 40 for saline (number/mm(2))] and capillary [2,159 +/- 119 VEGF 1 microg/kg vs. 1,590 +/- 66 for saline (number/mm(2))] densities. Myocardial analysis revealed approximately 2.5 fold increase in Ki67+ cells and approximately 2.8-fold increase in c-kit(+) cells in the VEGF group, which provides evidence for cardiomyocyte regeneration and progenitor cell expansion. This study provides novel evidence of a salutary effect of VEGF in the cardiomyopathic hamster via induction of myogenic growth factor production by skeletal muscle and mobilization of progenitor cells, which resulted in attenuation of cardiomyopathy and repair of the heart.

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Figures

Fig. 1.
Fig. 1.
Vascular endothelial growth factor (VEGF) induces growth factors in cultured C2C12 skeletal myocytes. Cells were plated at 5×105 cells per 35-mm dish, and after 24 h, cells were switched to DMEM/F12 containing 0.5% horse serum. After 4 days of differentiation, cells were washed with HBSS three times and then stimulated with 100 ng/ml human VEGF-A165 in the absence of serum. Total RNA was isolated, and cultured media were collected after 3 days. A: qRT-PCR analysis of growth factor gene expression. *P < 0.05 vs. saline. B: ELISA analysis of culture media for mouse hepatocyte growth factor (HGF), IGF2, NGF, and VEGF contents after 3 days. *P < 0.01 vs. saline. Results presented are representative of two independent assays (n = 5 for A and n = 3 for B).
Fig. 2.
Fig. 2.
VEGF stimulates skeletal myocyte proliferation and migration in vitro. A: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell proliferation assays were performed at day 0 prior to VEGF addition and both 2 and 4 days after VEGF addition (10 and 100 ng/ml VEGF; n = 3). B: C2C12 cell migration in response to VEGF after 24 h. Migration index is the ratio of migrated cells in response to VEGF to the number of cells migrated without VEGF. Results are representative of two independent experiments. *P < 0.01 vs. saline. #P < 0.05 vs. 10 ng/ml VEGF (n = 3).
Fig. 3.
Fig. 3.
Intramuscular VEGF injections induce skeletal muscle growth factor production. A: qRT-PCR analysis of injected hamstring 3 days and 5 wk after initiation of 1 μg/kg VEGF injections. Fold change of 1 indicates no effect of VEGF. #P < 0.05 and *P < 0.05 vs. saline 3 days and 5 wk, respectively. †P < 0.05 5 wk vs. 3 days (n = 3). B: ELISA assays of HGF, IGF2, NGF, and VEGF using injected hamstring tissue homogenates from 3 days and 1 mo. #P < 0.05 and *P < 0.05 vs. saline 3 days and 5 wk, respectively. †P < 0.05 5 wk vs. 3 days (n = 3 per group).
Fig. 4.
Fig. 4.
Increased myofiber nuclear and capillary densities in VEGF-injected hamstrings. VEGF denotes 1 μg/kg dosage. A: representative images of saline and VEGF-injected muscle with myofiber and capillary staining using Troponin I antibody (red) and FITC-labeled GSL-IB4 lectin (green). Nuclei were stained by DAPI (blue), ×200. B: representative image of Ki67+ cells (pink nuclei) in VEGF-injected hamstring. Myofibers were stained by a myosin heavy chain antibody (green), and nuclei were stained by DAPI (blue), ×200. C: hamstring myofiber nuclear densities expressed as numbers of myofiber nuclei per mm2. D: hamstring capillary densities are expressed as GSL-IB4 lectin-stained capillaries per mm2. E: percentage of Ki67+ nuclei in hamstring muscle. F and G: qRT-PCR analysis of cell cycle and stem cell markers (F) and endothelial markers (G). *P < 0.05 vs. saline (n = 3 for each panel).
Fig. 5.
Fig. 5.
VEGF injection mobilizes bone marrow progenitor cells and modulates SDF-1 levels in tissue and plasma. VEGF denotes 1 μg/kg dosage. A: Circulating c-kit+, CD31+, or CXCR4+ cells were quantified by flow cytometry 5 wk after VEGF injection. Cell numbers per million peripheral blood mononuclear cells were presented (n = 6 per group). **P < 0.001 vs. saline. *P < 0.05 vs. saline. †P < 0.05 vs. VEGF 0.1 μg/kg. B: ELISA assay of SDF-1 using injected hamstring tissue homogenates from 3 days and 5 wk. *P < 0.05 vs. saline. C: ELISA assay of SDF-1 levels in plasma from 3 days and 5 wk. *P < 0.005 (n = 4 per group). D: ELISA assay of SDF-1 using heart tissue homogenates from 3 days and 5 wk. **P < 0.001 vs. saline. †P < 0.001 vs. 3 days (n = 3–6 per group).
Fig. 6.
Fig. 6.
Intramuscular VEGF injections improve cardiac function. A: left ventricular ejection fraction (LVEF) was measured preinjection, 2 wk, and 5 wk after initiation of injections. Normal age-matched F1B hamsters maintained a stable LVEF of ∼70%. B: left ventricular diastolic dimension (LVDd) in mm. Normal age-matched F1B hamsters maintained a LVDd of 4.5–5 mm. *P < 0.05 vs. preinjection. #P < 0.05 vs. saline (n = 6 per group).
Fig. 7.
Fig. 7.
Intramuscular VEGF injections attenuate myocardial tissue injury and fibrosis. A: plasma levels of cardiac troponin-I 5 wk after VEGF treatment (1 μg/kg). Cardiac troponin-I was not detected (ND) in F1B plasma. B: quantification of cardiomyocyte and noncardiomyocyte apoptosis using the ApopTag kit. Apoptotic cells were not detected (ND) in F1B hearts. C: representative images of trichrome-stained heart sections showing fibrosis (blue), ×200. D: computer-assisted quantification of fibrotic areas using Trichrome-stained sections. *P < 0.05 vs. TO2 saline control. **P < 0.001 vs. TO2 saline control. #P < 0.05 vs. F1B (n = 3 for each panel).
Fig. 8.
Fig. 8.
Intramuscular VEGF injections promote myocardial regeneration. A: cardiomyocyte nuclear densities expressed as numbers of nuclei per mm2. B: myocardial capillary densities expressed as GSL-IB4 lectin-stained capillaries per mm2. C: representative image showing Ki-67+ (pink nuclei) cardiomyocyte and noncardiomyocyte. Cardiomyocytes were stained green with a cardiac troponin T antibody, and nuclei were stained blue with DAPI, ×630. D: total Ki67+ nuclei and Ki67+ cardiomyocytes. E: representative image showing c-kit staining of interstitial cells (pink cytoplasmic) in the heart. Cardiomyocytes were stained green with a cardiac troponin T antibody, and nuclei were stained blue with DAPI, ×630. F: quantification of % c-kit+ cells in the heart. G: image showing c-kit (pink) and Ki-67 (green) double staining of interstitial cells in the heart. Nuclei were stained blue with DAPI, ×630. *P < 0.01 vs. TO2 saline control. **P < 0.05 vs. TO2 saline control. #P < 0.01 vs. F1B. ##P < 0.05 vs. F1B. †P < 0.01 vs. TO2 0.1 μg/kg VEGF (n = 3 for each panel).
Fig. 9.
Fig. 9.
Myocardial regeneration is associated with progenitor cell recruitment and new myocyte formation. A: qRT-PCR analysis of cell cycle and stem cell markers in the heart 5 wk after initiation of treatment. *P < 0.05 vs. saline. B: average cardiomyocyte cross-section diameter. *P < 0.01 vs. saline. C: frequency histogram of cardiomyocyte cross-section diameters showing an increased number of smaller cardiomyocytes after VEGF injections in both groups. VEGF denotes 1 μg/kg dosage. *P < 0.01 vs. saline (n = 3).

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