Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts

Abstract

Therapeutically delivered mesenchymal stem cells (MSCs) improve ventricular remodeling. However, the mechanism underlying MSC cardiac remodeling has not been clearly determined. Congestive heart failure (CHF) was induced in rats by cauterization of the left ventricular free wall. MSCs were cultured from autologous bone marrow and injected into the border zone and the remote myocardium 5 d after injury. Ten weeks later, when compared with sham operation, CHF significantly increased nucleus mitotic index, capillary density, and expression of insulin-like growth factor 1, hepatocyte growth factor and vascular endothelial growth factor in the border zone (P<0.01) and decreased each of them in the remote myocardium (P<0.05 or P<0.01). MSC implantation in CHF dramatically elevated expression of these growth factors in the remote myocardium and further elevated their expression in the border zone when compared with CHF without MSC addition (P<0.05 or P<0.01). This was paralleled by a higher nucleus mitotic index and a significantly increased capillary density both in the remote myocardium and in the border zone, and by a lower percentage of area of collagen and a higher percentage of area of myocardium in the border zone (P<0.05 or P<0.01), and cardiac remodeling markedly improved. Autologous MSC implantation promoted expression of growth factors in rat failing myocardium, which might enhance cardiomyogenesis and angiogenesis, and improved cardiac remodeling.

Fig. 1

Characterization of isolated MSCs Upper panel indicated cells cultured from rat bone marrow after plating at 38 h (a, ×100), 5 d (b, ×100) and 13 d (passage 1) (c, ×100). Lower panel showed FACS analysis with antibodies directed against CD44 (d), CD90 (e), and CD45 (f). The areas under black curves indicated isotype controls, and those under color ones demonstrated MSCs

Fig. 1

Characterization of isolated MSCs Upper panel indicated cells cultured from rat bone marrow after plating at 38 h (a, ×100), 5 d (b, ×100) and 13 d (passage 1) (c, ×100). Lower panel showed FACS analysis with antibodies directed against CD44 (d), CD90 (e), and CD45 (f). The areas under black curves indicated isotype controls, and those under color ones demonstrated MSCs

Fig. 1

Characterization of isolated MSCs Upper panel indicated cells cultured from rat bone marrow after plating at 38 h (a, ×100), 5 d (b, ×100) and 13 d (passage 1) (c, ×100). Lower panel showed FACS analysis with antibodies directed against CD44 (d), CD90 (e), and CD45 (f). The areas under black curves indicated isotype controls, and those under color ones demonstrated MSCs

Fig. 1

Characterization of isolated MSCs Upper panel indicated cells cultured from rat bone marrow after plating at 38 h (a, ×100), 5 d (b, ×100) and 13 d (passage 1) (c, ×100). Lower panel showed FACS analysis with antibodies directed against CD44 (d), CD90 (e), and CD45 (f). The areas under black curves indicated isotype controls, and those under color ones demonstrated MSCs

Fig. 1

Characterization of isolated MSCs Upper panel indicated cells cultured from rat bone marrow after plating at 38 h (a, ×100), 5 d (b, ×100) and 13 d (passage 1) (c, ×100). Lower panel showed FACS analysis with antibodies directed against CD44 (d), CD90 (e), and CD45 (f). The areas under black curves indicated isotype controls, and those under color ones demonstrated MSCs

Fig. 1

Characterization of isolated MSCs Upper panel indicated cells cultured from rat bone marrow after plating at 38 h (a, ×100), 5 d (b, ×100) and 13 d (passage 1) (c, ×100). Lower panel showed FACS analysis with antibodies directed against CD44 (d), CD90 (e), and CD45 (f). The areas under black curves indicated isotype controls, and those under color ones demonstrated MSCs

Fig. 2

Scar sizes and echocardiographic images (a–c) Examples from CHF rats with and without MSCs showed the scar areas on the global (a), epicardial (b) and endocardial (c) aspects of LV. (d) 2D echocardiographic images of LV geometric changes in systole and in diastole and (e) examples of pulse-wave of Doppler spectra of mitral inflow and aorta outflow are shown for representative animals. ET: ejection time; ICT: isovolumic contraction time; IRT: isovolumic relaxation time; LVEDd: LV end diastolic dimension

Fig. 2

Scar sizes and echocardiographic images (a–c) Examples from CHF rats with and without MSCs showed the scar areas on the global (a), epicardial (b) and endocardial (c) aspects of LV. (d) 2D echocardiographic images of LV geometric changes in systole and in diastole and (e) examples of pulse-wave of Doppler spectra of mitral inflow and aorta outflow are shown for representative animals. ET: ejection time; ICT: isovolumic contraction time; IRT: isovolumic relaxation time; LVEDd: LV end diastolic dimension

Fig. 2

Scar sizes and echocardiographic images (a–c) Examples from CHF rats with and without MSCs showed the scar areas on the global (a), epicardial (b) and endocardial (c) aspects of LV. (d) 2D echocardiographic images of LV geometric changes in systole and in diastole and (e) examples of pulse-wave of Doppler spectra of mitral inflow and aorta outflow are shown for representative animals. ET: ejection time; ICT: isovolumic contraction time; IRT: isovolumic relaxation time; LVEDd: LV end diastolic dimension

Fig. 2

Scar sizes and echocardiographic images (a–c) Examples from CHF rats with and without MSCs showed the scar areas on the global (a), epicardial (b) and endocardial (c) aspects of LV. (d) 2D echocardiographic images of LV geometric changes in systole and in diastole and (e) examples of pulse-wave of Doppler spectra of mitral inflow and aorta outflow are shown for representative animals. ET: ejection time; ICT: isovolumic contraction time; IRT: isovolumic relaxation time; LVEDd: LV end diastolic dimension

Fig. 2

Scar sizes and echocardiographic images (a–c) Examples from CHF rats with and without MSCs showed the scar areas on the global (a), epicardial (b) and endocardial (c) aspects of LV. (d) 2D echocardiographic images of LV geometric changes in systole and in diastole and (e) examples of pulse-wave of Doppler spectra of mitral inflow and aorta outflow are shown for representative animals. ET: ejection time; ICT: isovolumic contraction time; IRT: isovolumic relaxation time; LVEDd: LV end diastolic dimension

Fig. 3

Effects of MSC implantation on the mRNA expression of growth factors GAPDH: glyceroldehydes-3-phosphate-dehydrogenase; HGF: hepatocyte growth factor; IGF-1: insulin-like growth factor 1; VEGF-A: vascular endothelial growth factor A. ^a P<0.05 and ^b P<0.01 vs. sham: ^c P<0.05 and ^d P<0.01 vs. CHF. Values are mean±SEM

Fig. 4

Effects of MSC implantation on the protein expression of growth factors HGF: hepatocyte growth factor; IGF-1: insulin-like growth factor 1; VEGF-A: vascular endothelial growth factor A. ^a P<0.05 and ^b P<0.01 vs. sham; ^c P<0.05 and ^d P<0.01 vs. CHF. Values are mean±SEM

Fig. 5

Detection of implanted MSCs (a) DAPI-positive nucleus (arrow, ×400) and (b) endothelium with DAPI-positive nuclei in a blood vessel (arrowheads, ×800)

Fig. 5

Detection of implanted MSCs (a) DAPI-positive nucleus (arrow, ×400) and (b) endothelium with DAPI-positive nuclei in a blood vessel (arrowheads, ×800)

Fig. 6

Ki-67 labeling of mitotic nuclei in myocardium (a) Ki-67-labeled nucleus (green), (b) DAPI-labeled nuclei (blue), (c) α-sarcomeric actin antibody staining (red) and (d) merged image to illustrate a cycling cell nucleus that is in telophase (the arrowhead indicates the nucleus in caryokinesis). (e) The micrograph demonstrates in the LV myocardium of a sham-operated rat the combined labeling of myocyte cytoplasm by a-sarcomeric actin (red), nuclear staining by DAPI (blue) and cycling nuclei staining with Ki-67 (green; the arrow indicates nucleus in DNA synthesis). (f) The same staining is illustrated in the border zone of an injured heart (the arrowheads show nuclei in caryokinesis). Bars=10 μm

Fig. 6

Ki-67 labeling of mitotic nuclei in myocardium (a) Ki-67-labeled nucleus (green), (b) DAPI-labeled nuclei (blue), (c) α-sarcomeric actin antibody staining (red) and (d) merged image to illustrate a cycling cell nucleus that is in telophase (the arrowhead indicates the nucleus in caryokinesis). (e) The micrograph demonstrates in the LV myocardium of a sham-operated rat the combined labeling of myocyte cytoplasm by a-sarcomeric actin (red), nuclear staining by DAPI (blue) and cycling nuclei staining with Ki-67 (green; the arrow indicates nucleus in DNA synthesis). (f) The same staining is illustrated in the border zone of an injured heart (the arrowheads show nuclei in caryokinesis). Bars=10 μm

Fig. 6

Ki-67 labeling of mitotic nuclei in myocardium (a) Ki-67-labeled nucleus (green), (b) DAPI-labeled nuclei (blue), (c) α-sarcomeric actin antibody staining (red) and (d) merged image to illustrate a cycling cell nucleus that is in telophase (the arrowhead indicates the nucleus in caryokinesis). (e) The micrograph demonstrates in the LV myocardium of a sham-operated rat the combined labeling of myocyte cytoplasm by a-sarcomeric actin (red), nuclear staining by DAPI (blue) and cycling nuclei staining with Ki-67 (green; the arrow indicates nucleus in DNA synthesis). (f) The same staining is illustrated in the border zone of an injured heart (the arrowheads show nuclei in caryokinesis). Bars=10 μm

Fig. 6

Ki-67 labeling of mitotic nuclei in myocardium (a) Ki-67-labeled nucleus (green), (b) DAPI-labeled nuclei (blue), (c) α-sarcomeric actin antibody staining (red) and (d) merged image to illustrate a cycling cell nucleus that is in telophase (the arrowhead indicates the nucleus in caryokinesis). (e) The micrograph demonstrates in the LV myocardium of a sham-operated rat the combined labeling of myocyte cytoplasm by a-sarcomeric actin (red), nuclear staining by DAPI (blue) and cycling nuclei staining with Ki-67 (green; the arrow indicates nucleus in DNA synthesis). (f) The same staining is illustrated in the border zone of an injured heart (the arrowheads show nuclei in caryokinesis). Bars=10 μm

Fig. 6

Ki-67 labeling of mitotic nuclei in myocardium (a) Ki-67-labeled nucleus (green), (b) DAPI-labeled nuclei (blue), (c) α-sarcomeric actin antibody staining (red) and (d) merged image to illustrate a cycling cell nucleus that is in telophase (the arrowhead indicates the nucleus in caryokinesis). (e) The micrograph demonstrates in the LV myocardium of a sham-operated rat the combined labeling of myocyte cytoplasm by a-sarcomeric actin (red), nuclear staining by DAPI (blue) and cycling nuclei staining with Ki-67 (green; the arrow indicates nucleus in DNA synthesis). (f) The same staining is illustrated in the border zone of an injured heart (the arrowheads show nuclei in caryokinesis). Bars=10 μm

Fig. 6

Ki-67 labeling of mitotic nuclei in myocardium (a) Ki-67-labeled nucleus (green), (b) DAPI-labeled nuclei (blue), (c) α-sarcomeric actin antibody staining (red) and (d) merged image to illustrate a cycling cell nucleus that is in telophase (the arrowhead indicates the nucleus in caryokinesis). (e) The micrograph demonstrates in the LV myocardium of a sham-operated rat the combined labeling of myocyte cytoplasm by a-sarcomeric actin (red), nuclear staining by DAPI (blue) and cycling nuclei staining with Ki-67 (green; the arrow indicates nucleus in DNA synthesis). (f) The same staining is illustrated in the border zone of an injured heart (the arrowheads show nuclei in caryokinesis). Bars=10 μm

Fig. 7

Micrographs of the example sections from the remote myocardium and the border zone of injured hearts in CHF rats with and without MSCs, stained by a combination of vWF (red) and α-sarcomeric actin (green). Bar=20 μm

Fig. 8

Upper panel indicates the example mid longitudinal cross-sections from each group; lower panel shows the area of collagen and myocardium in the border zone (stained with Masson’s trichrome, ×200)