Heart failure therapy mediated by the trophic activities of bone marrow mesenchymal stem cells: a noninvasive therapeutic regimen

Abstract

Heart failure carries a poor prognosis with few treatment options. While myocardial stem cell therapeutic trials have traditionally relied on intracoronary infusion or intramyocardial injection routes, these cell delivery methods are invasive and can introduce harmful scar tissue, arrhythmia, calcification, or microinfarction in the heart. Given that patients with heart failure are at an increased surgical risk, the development of a noninvasive stem cell therapeutic approach is logistically appealing. Taking advantage of the trophic effects of bone marrow mesenchymal stem cells (MSCs) and using a hamster heart failure model, the present study demonstrates a novel noninvasive therapeutic regimen via the direct delivery of MSCs into the skeletal muscle bed. Intramuscularly injected MSCs and MSC-conditioned medium each significantly improved ventricular function 1 mo after MSC administration. MSCs at 4 million cells/animal increased fractional shortening by approximately 40%, enhanced capillary and myocyte nuclear density by approximately 30% and approximately 80%, attenuated apoptosis by approximately 60%, and reduced fibrosis by approximately 50%. Myocyte regeneration was evidenced by an approximately twofold increase in the expression of cell cycle markers (Ki67 and phosphohistone H(3)) and an approximately 13% reduction in mean myocyte diameter. Increased circulating levels of hepatocyte growth factor (HGF), leukemia inhibitory factor, and macrophage colony-stimulating factor were associated with the mobilization of c-Kit-positive, CD31-positive, and CD133-positive progenitor cells and a subsequent increase in myocardial c-Kit-positive cells. Trophic effects of MSCs further activated the expression of HGF, IGF-II, and VEGF in the myocardium. The work highlights a cardiac repair mechanism mediated by trophic cross-talks among the injected MSCs, bone marrow, and heart that can be explored for noninvasive stem cell therapy.

Fig. 1.

Cardiac functional improvement by intramuscular injection of mesenchymal stem cells (MSCs) and MSC-conditioned medium. A and B: percent fractional shortening (%FS) and left ventricular diastolic diameter (LVDd) at preinjection and 1 mo postinjection (n = 5 animals/group) were obtained by blinded echocardiography. MSCs at the indicated cell dosages were injected into hamstring muscles of 4-mo-old TO2 hamsters. Control TO2 hamsters received HBSS injections. Normal F1B hamsters were not injected and are shown as a reference for A–D. C and D: %FS and LVDd before and 1 mo after the first intramuscular injection of MSC-conditioned medium versus control medium (n = 4 animals/group). The MSC-conditioned media used here were from the same batch. Note that the 0.25 million MSC dosage group exhibited a higher mean %FS at pretreatment than the 1 million MSC dosage group. MSC-mediated improvements in %FS between the two cell dosage groups were statistically similar after normalization to the pretreatment level. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; #P < 0.05 vs. pretreatment; ##P < 0.01 vs. pretreatment; ###P < 0.001 vs. pretreatment.

Fig. 2.

Increased capillary and myocyte nuclear densities after MSC administration. A: representative images of capillary and myocyte staining using FITC-labeled Griffonia simplicifolia isolectin B₄ (GSL-IB₄; green) and troponin I (TnI) antibody (red), respectively. 4′,6-Diamidino-2-phenylindole (DAPI; blue) was used for nuclear staining. B and C: computer analysis of capillary and myocyte nuclear densities (expressed as numbers/mm²). D: computer analysis of myocyte cross-sectional diameters. At least 15 fields of ×200 magnification and >350 myocytes were evaluated in each hamster. E: frequency histogram of diameters showing a greater number of smaller myocytes in the MSC-treated group (n = 4 animals/group). *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control.

Fig. 3.

MSC administration augments cell cycle activities in the myocardium. A: representative image of Ki67-positive cells (pink nuclei). Myocytes were stained by a troponin T (TnT) antibody (green). Nuclei were stained by DAPI (blue). B and C: percentages of Ki67-positive myocytes and total Ki67-positive nuclei (n = 4 animals/group). D: representative image of phospho-histone H₃ (p-HH₃)-positive cells (pink nuclei). E and F: percentages of p-HH₃-positive myocytes and total p-HH₃-positive nuclei (n = 4 animals/group). *P < 0.05 vs. control.

Fig. 4.

MSC administration reduces myocardial apoptosis and tissue damage. A and B: percentages of myocyte and nonmyocyte apoptosis. At least 25 fields of ×200 magnification and >8,000 cells were evaluated from each hamster. C: decreased circulating levels of cardiac TnI (cTnI) after MSC administration (n = 3 animals/group). *P < 0.05 vs. control; **P < 0.01 vs. control.

Fig. 5.

Attenuation of myocardial fibrosis by MSCs. A: hematoxylin-esosin-stained (top) and Masson trichrome-stained (bottom) heart sections. B: quantification of fibrotic areas (n = 5 animals/group). C: quantitative RT-PCR analysis of expression of genes involved in extracellular tissue remodeling. Col1a1, collagen type I-α₁; Col1a2, collagen type I-α₁; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase. Results are representative of 2 independent experiments. *P < 0.05 vs. control; **P < 0.01 vs. control; ***P < 0.001 vs. control; †P < 0.001 vs. F1B.

Fig. 6.

Increased circulating and myocardial trophic factors. A: circulating (plasma) levels of hepatocyte growth factor (HGF), leukemia inhibitory factor (LIF), and granulocyte/macrophage colony-stimulating factor (G/M-CSF) 1 mo after MSC administration (n = 3 animals/group). B: heart tissues were collected 1 mo after MSC administration and homogenized. Clarified lysates were assayed by HGF, IGF-II, and VEGF ELISA kits (n = 4 animals/group). *P < 0.05 vs. the HBSS control.

Fig. 7.

Mobilization of bone marrow progenitor cells. Circulating c-Kit-positive, CD133-positive, and CD31-positive cells were quantified by flow cytometry 3 days after MSC injections. Cell numbers per 1 million peripheral blood mononuclear cells are shown (n = 3 animals/group). *P < 0.05 vs. control.

Fig. 8.

Increased myocardial c-Kit-positive progenitor cells. A: representative images of interstitial c-Kit-positive cells (red). Myocytes were stained by a TnT antibody (green). Nuclei were stained by DAPI (blue). B: quantification of c-Kit-positive cells. C: quantitative RT-PCR confirmed the quantification data in B. Note that the expression of CD45 (leukocyte common antigen) was not affected by MSC administration. **P < 0.01 vs. the HBSS control.