Smooth muscle cell sheet transplantation preserve cardiac function and minimize cardiac remodeling in a rat myocardial infarction model

Abstract

Background: We examined whether a vascular smooth muscle cell (SMC) sheet is effective in the treatment of a rat myocardial infarction (MI) model.

Methods: We examined the effect of SMC sheet on the cardiac function and cardiac remodeling in a rat MI model in comparison with their effect of dermal fibroblast (DFB) sheet in vivo. Furthermore, we estimated the apoptosis and secretion of angiogenic factor of SMC under hypoxic condition in comparison with DFB. Seven days after MI, monolayer cell sheets were transplanted on the infarcted area (SMC transplantation group, SMC-Tx; DFB transplantation group, DFB-Tx; no cell sheet transplantation group, Untreated; neither MI nor cell sheet transplantation group, Sham). We evaluated cardiac function by echocardiogram, degree of cardiac remodeling by histological examination, and secretion of angiogenic growth factor by enzyme immunoassay.

Results: Twenty-eight days after transplantation, SMC-Tx showed the following characteristics compared with the other groups: 1) significantly greater fractional area shortening (SMC-Tx, 32.3 ± 2.1 %; DFB-Tx, 23.3 ± 2.1 %; untreated, 25.1 ± 2.6 %), 2) suppressed left ventricular dilation, smaller scar expansion, and preserved wall thickness of the area at risk and the posterior wall, 3) decreased fibrosis, preserved myocardium in the scar area, and greater number of arterioles in border-zone, 4) tight attachment of SMC sheets on the scarred myocardium, and less apoptotic cell death. In in vitro experiments, SMCs secreted higher amounts of basic fibroblast growth factor (SMC, 157.7 ± 6.4 pg/ml; DFB, 3.1 ± 1.0 pg/ml), and showed less apoptotic cell death under hypoxia.

Conclusions: Our results illustrate that transplantation of SMC sheets inhibited the progression of cardiac remodeling and improve cardiac function. These beneficial effects may be due to superior SMC survival.

Keywords: Cell sheet; Cell survival; Myocardial infarction; Smooth muscle cells.

Fig. 1

Representative pictures of cell sheet harvesting and transplantation. a; Cell sheet which was completely removed from temperature-responsive culture dish (35-mm diameter). Cell sheet shrinks into about 15-mm diameter. Scale bar = 10 mm. b; Cell sheet was put on a plastic sheet before transplantation. c; The cell sheet was applied face down onto the surface of the anterior scar. The plastic sheet was then carefully removed, leaving the monolayered cell sheet on the surface of the heart

Fig. 2

Fractional area shortening (Panel a) and ejection fraction (Panel b) assessed by echocardiography in experimental groups before cell sheet transplantation (baseline) and twenty-eight days after cell sheet transplantation (after treatment). *p < 0.05 vs. SMC-Tx. SMC-Tx, smooth muscle cell sheet transplantation group; DFB-Tx, dermal fibroblast sheet transplantation group; Untreated, untreated group. Each groups n = 10

Fig. 3

Representative pictures of myocardium (a, shown by #) in the scar area of SMC-Tx, DFB-Tx and untreated. b; % myocardium in the risk area was significantly higher in SMC-Tx. c; Representative pictures of α-SMA⁺ arterioles in border-zone between myocardium and scar area. Upper panels are less magnified pictures of border-zone, and lower panels are higher magnified pictures of border-zone. Scale bar = 100 μm. D; α-SMA⁺ arterioles in border-zone were significantly increased in SMC-Tx. *p < 0.05 vs. SMC-Tx; †p < 0.05 vs. other groups. SMC-Tx, smooth muscle cell sheet transplantation group; DFB-Tx, dermal fibroblast sheet transplantation group; Untreated, untreated group. Each groups n = 5

Fig. 4

Confirmation of the attachment of transplanted cell sheet at forty-eight hours (a–c) and twenty-eight days (d–f) after transplantation. Left panels are SMC-Tx (a, d), middle panels are DFB-Tx (b, e), and right panels are Untreated (c, f). Red signal, PKH stained cell sheet; blue signal, DAPI for nuclear staining. Microphotographs of transplanted cell sheet forty-eight hours after transplantation that has been stained with TUNEL assay in SMC-Tx (g) and DFB-Tx (h) groups. Numbers of TUNEL-positive grafted cell sheets (i). *p < 0.01 vs. SMC-Tx. Each groups n = 5. Scale bar = 500 μm (a–f), 50 μm (g, h). SMC-Tx, smooth muscle cell sheet transplantation group; DFB-Tx, dermal fibroblast sheet transplantation group; Untreated, untreated group

Fig. 5

Angiogenic factor secretion under normal and hypoxic condition in vitro. a; bFGF. b; VEGF. c; Apoptosis assay of cells cultured in vitro in hypoxic conditions. *p < 0.01 vs. SMC. Each groups n = 5

Fig. 6

Representative schema of custom-made apparatus to measure cell sheet strength (a). Cell sheets were stretched by weights and photographed. “Sinking Distance” was then measured (blue arrow). Representative pictures of stretched cell sheet by weight loading to measure cell sheet strength (b–d). SMC sheet at 5 mg (b), DFB sheet at 5 mg weight (c) and SMC sheet at 200 mg (d). “Sinking Distance” was measured as a distance within red bars and red arrows (yellow bicephalic arrows). Scale bar (white bar) = 5 mm. Sinking distance at 5 mg (e) and maximum weight tolerance (f) was measured. *p < 0.05 vs. DFB sheet. SMC, smooth muscle cell; DFB, dermal fibroblast. Each groups n = 3