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. 2015 Jan;8(1):167-74.
doi: 10.1161/CIRCHEARTFAILURE.114.001711. Epub 2014 Oct 23.

Stem cell factor gene transfer improves cardiac function after myocardial infarction in swine

Kiyotake Ishikawa  1 , Kenneth Fish  2 , Jaume Aguero  2 , Elisa Yaniz-Galende  2 , Dongtak Jeong  2 , Changwon Kho  2 , Lisa Tilemann  2 , Lauren Fish  2 , Lifan Liang  2 , Ahmed A Eltoukhy  2 , Daniel G Anderson  2 , Krisztina Zsebo  2 , Kevin D Costa  2 , Roger J Hajjar  2
Affiliations

Stem cell factor gene transfer improves cardiac function after myocardial infarction in swine

Kiyotake Ishikawa et al. Circ Heart Fail. 2015 Jan.

Abstract

Background: Stem cell factor (SCF), a ligand of the c-kit receptor, is a critical cytokine, which contributes to cell migration, proliferation, and survival. It has been shown that SCF expression increases after myocardial infarction (MI) and may be involved in cardiac repair. The aim of this study was to determine whether gene transfer of membrane-bound human SCF improves cardiac function in a large animal model of MI.

Methods and results: A transmural MI was created by implanting an embolic coil in the left anterior descending artery in Yorkshire pigs. One week after the MI, the pigs received direct intramyocardial injections of either a recombinant adenovirus encoding for SCF (Ad.SCF, n=9) or β-gal (Ad.β-gal, n=6) into the infarct border area. At 3 months post-MI, ejection fraction increased by 12% relative to baseline after Ad.SCF therapy, whereas it decreased by 4.2% (P=0.004) in pigs treated with Ad.β-gal. Preload-recruitable stroke work was significantly higher in pigs after SCF treatment (Ad.SCF, 55.5±11.6 mm Hg versus Ad.β-gal, 31.6±12.6 mm Hg, P=0.005), indicating enhanced cardiac function. Histological analyses confirmed the recruitment of c-kit(+) cells as well as a reduced degree of apoptosis 1 week after Ad.SCF injection. In addition, increased capillary density compared with pigs treated with Ad.β-gal was found at 3 months and suggests an angiogenic role of SCF.

Conclusions: Local overexpression of SCF post-MI induces the recruitment of c-kit(+) cells at the infarct border area acutely. In the chronic stages, SCF gene transfer was associated with improved cardiac function in a preclinical model of ischemic cardiomyopathy.

Keywords: angiogenesis; gene therapy; myocardial infarction; paracrine factor.

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Figures

Figure 1
Figure 1. Gene transfer efficacy and recruitment of c-kit+ cells 1 week after gene transfer
A. Infected area is clearly distinguishable by GFP expression. Blue; DAPI-stained nuclei, green; GFP. B. Confocal image represents c-kit membrane staining (Red). Clusters of c-kit+ cells were found at the injection sites after SCF gene transfer. Blue; DAPI-stained nuclei, red; c-kit, green; α-sarcomeric actin. C. SCF, PCNA, and P-H3 expression by Western blotting. Expression of SCF (P=0.001) as well as proliferation proteins (PCNA: P=0.08, P-H3: P=0.008) were increased in the SCF treated pigs. *; P<0.05, Abbreviations: GFP = Green fluorescent protein, SCF = stem cell factor, PCNA = proliferation cell nuclear antigen, P-H3 = phospho-histone h3.
Figure 2
Figure 2. Percent change of functional and volumetric parameters from 1 week (before gene transfer) to 6 weeks and to 3 months
Ad.SCF group showed significant improvement in EF (P=0.004) and trends towered improved dP/dt max (P=0.06) at 3 month. There were no statistically significant differences in EDVI (P=0.95), ESVI (P=0.35), and SVI changes (0.11) evaluated by left ventriculogram. *; P<0.05, †; P=0.06, Abbreviations: EF = left ventricular ejection fraction, EDVI = end-diastolic volume index, ESVI = end-systolic volume index, SVI = stroke volume index, dP/dt max = dP/dt maximum
Figure 3
Figure 3. Representative pressure-volume loops during inferior vena cava occlusion at 3 months post myocardial infarction
Left loop is from SCF treated pig and shows steeper end-systolic pressure-volume relationship with left ward shift relative to the right loop (Ad.β-gal).
Figure 4
Figure 4. Weight of the heart and the scar size
A. Ventricular weight/ body weight (g/Kg) was not different between the groups at 3 months (P=0.26). Left ventricular scar size was also similar after Ad.SCF (16.4 ± 5.5%) and Ad.β-gal injection (18.0 ± 4.4%, P = 0.61). B. Wall motion score index, an echocardiographic estimate of infarct size, was similar between the groups at 1 week which suggests similar scar size before the gene transfer (Ad.SCF, 2.01±0.16 vs Ad.β-gal, 2.01±0.56, P=0.98), whereas it was lower in the Ad.SCF group 3 months after the gene delivery without statistical significance (Ad.SCF, 1.68±0.40 vs Ad.β-gal, 1.99±0.36, P=0.18).
Figure 5
Figure 5. Angiogenesis and vasculogenesis at 3 months after myocardial infarction
A. Increased numbers of vasculatures co-stained with α-SMA and CD31 were found after Ad.SCF injection compared to the Ad.β-gal. Blue; DAPI-stained nuclei, red; α-SMA, green; CD31. B. Presumably functional vessel stained with α-SMA. Multiple layers of nuclei are found in the vessel wall suggesting that this is a functional arteriole. C. A significant increase in capillaries co-expressing isolectin IB4 (brown) was found at the infarct border after SCF gene transfer. D. Quantification of vessels (P=0.01) and capillaries (P=0.003) suggests angiogenic role of SCF. *; P<0.05 Abbreviations: α-SMA = α-smooth muscle actin, SCF = stem cell factor
Figure 6
Figure 6. Characterization of infarct border zone 1 week after the gene transfer
A. A few isolated c-kit+/CD45+ cells were found in clusters of c-kit+/CD45− cells. Blue; DAPI-stained nuclei, red; c-kit, green; CD45. B. Immunohistochemistry revealed increased PCNA expression at the injection site 1 week after gene transfer. Higher magnification image (right) shows that PCNA was expressed in both cardiomyocytes and non-myocytes. Blue; DAPI-stained nuclei, red; PCNA, green; α-sarcomeric actin. C. Representative examples of TUNEL staining and apoptosis index. Decreased apoptotic cells were found at the infarct border area after Ad.SCF injection (Apoptosis index: P<0.001). Blue; DAPI-stained nuclei, red; apoptotic nuclei. *; P<0.05
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