Figure 1.
Effect of genistein on ECFC migration and proliferation.
(A) ECFCs were incubated for 12 h with various concentrations of genistein (10−10–10−5 M) and then stained with 5 µM Calcein AM. Fluorescence was quantified with a plate reader. (B) The in vitro ECFC wound-healing motility assay was performed in the absence and presence of genistein. Ten fields per plate were examined. (Scale bar: 100 µm). (C) ECFCs were incubated for 12 h with various concentrations of genistein, and CDK 2, cyclin E, CDK 4, and cyclin D1 were assessed by western blotting. (D) ECFCs were incubated for 12 h with various concentrations of genistein, and assessed by MTT. (E) ECFCs were treated with genistein and then washed with PBS, fixed, stained, and analyzed by flow cytometry. Gates were manually configured to determine the percentage of cells in S phase based on the DNA content (n = 5). *P<0.05 vs. CTRL (indicates control genistein untreated ECFCs).
Figure 2.
ILK, α-parvin and F-actin mediated genistein-induced ECFC migration.
(A) ECFCs were treated with genistein for 0–24 h, and ILK, α-parvin and F-actin was detected by western blotting. (B) ECFCs were transfected with ILK, α-parvin, and TRIOBP (F-actin) small interfering RNA (siRNA) (ILK, α-parvin, and TRIOBP-specific siRNA; 200 pmol) for 24 h before genistein treatment and staining with Calcein AM. Fluorescence in the analytical zone was quantified with a plate reader. *P<0.05 vs. CTRL (indicates control genistein untreated ECFCs), **P<0.05 vs. genistein. (C) ECFCs were transfected with ILK siRNA (ILK-specific siRNA; 200 pmol) for 24 h before genistein (10−10 M) treatment, and the cells were injected into the tail veins of mice 30 min after left anterior descending (LAD) artery ligation. Staining of ECs with isolectin B4 (green) showed human nuclei antibody (HNA) (red)-positive cell incorporation into the border zone of left ventricular (LV) infarct at 3 days after myocardial infarction (MI) (Scale bar: 20 µm). Inset in higher magnification of the yellow-boxed area. Arrows indicate of isolectin B4+HNA+DAPI+cells. (D) The bar graph shows quantitative analysis of the number of HNA+cells associated with isolectin B4+vasculature (n = 5). HPF indicates high-power field. *P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC).
Figure 3.
ERK1/2-mediated genistein-induced ECFC proliferation and survival in the border zone of the left ventricular (LV) infarct at 3 days after myocardial infarction (MI).
(A) ECFCs were pretreated with U0126 (ERK1/2 inhibitor, 10−6 M) for 30 min prior to 12 h of genistein treatment and then washed with PBS, fixed, stained, and analyzed by flow cytometry. Gates were manually configured to determine the percentage of cells in S phase based on DNA content (n = 5). *P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC). ECFCs were pretreated with U0126 for 30 min prior to a 12 h genistein (10−10 M) treatment, and the cells were transplanted into the ischemic region. (B) Proliferating cell nuclear antigen (PCNA) staining to detect ECFC proliferation. PCNA+ cell zone, yellow-boxed area. (Scale bar: 100 µm). (C) Quantification of PCNA-positive cells at 3 d after MI. (D) Co-immunofluorescent staining to detect proliferation (Ki67 [proliferation marker, red] and of hECFCs [human nuclear antigen (HNA)-positive cells, green] and DAPI [blue] for nuclear staining). Arrows indicate Ki67+ HNA+ DAPI+ cells. (E) Quantitative analysis of Ki67/HNA/DAPI triple-positive cells at 3 days after MI. (F) Coimmunofluorescent staining to detect apoptosis (caspase-3, apoptosis marker, green) and of hEPCs (HNA-positive cells, red) and DAPI (blue) by nuclear staining. Arrows indicate caspase-3+ HNA+ DAPI+ cells. (Scale bar: 20 µm). (G) Quantitative analysis of caspase-3/HNA/DAPI triple-positive cells at 3 days after MI. (n = 6) *P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC).
Figure 4.
Enhanced secretion of angiogenic growth factors in the ischemic border zone following transplantation of GS-ECFCs.
(A) ECFCs were cultured in serum-free medium for 24 h and then treated with genistein (10−10 M) for 12 h. SDF-1, HGF, and FGF-2 were determined by western blotting. (B) Western blot analyses of SDF-1, HGF, and FGF-2 at 3 day after genistein stimulate-ECFC (GS-ECFC) or CTRL (indicates control genistein untreated ECFC) were transplanted. The lower parts (A, B) depict the mean ± SE of 4 independent experiments for each condition, as determined from densitometry relative to β-actin. P<0.05 vs. CTRL (indicates control genistein untreated ECFC), **P<0.05 vs. genistein stimulate-ECFC (GS-ECFC). (C) IF staining for SDF-1, HGF, and FGF-2 in ischemic heart tissue at 3 days after ECFCs were grafted as genistein stimulate-ECFC (GS-ECFC) or CTRL (indicates control genistein untreated ECFC) (Cytokine expression zone, yellow-boxed area) (Scale bar: 50 µm) (n = 5).
Figure 5.
GS-ECFC-mediated neovascularization in border zone of LV infarct at 28 days post-MI.
(A) IF staining for CD 31 (green) in ischemic heart tissue at 28 days after ECFCs were grafted as genistein stimulate-ECFC (GS-ECFC) or CTRL (control untreated ECFC). (Scale bar: 50 µm). (B) The bar graph shows the quantification of CD31+ capillary density. (C) Engraftment of ECFC (HNA+, red fluorescence) into vascular structures (CD31 staining for capillaries green fluorescence) is seen as yellow structures. Insert is higher magnification of the yellow-boxed area. (D) The bar graph shows the quantification of HNA+ cells associated with CD31+ vasculature. (E) Engraftment of ECFC (HNA+, green fluorescence) into arteriole structures (α-SMA staining for arterioles red fluorescence) is seen as yellow structures. Insert is higher magnification of the yellow-boxed area. (F) The bar graph shows the quantification of HNA+ cells associated with α-SMA + arterioles. HPF indicates high-power field. (n = 6) *P<0.05 vs. CTRL (indicates control genistein untreated ECFC).
Figure 6.
Enhancement of functional recovery by transplantation of GS-ECFC.
M-mode echocardiographic tracing at 28 days after myocardial infarction (MI) in the CTRL (control untreated ECFC) and primed with genistein stimulate-ECFC (GS-ECFC) groups. Analysis of left ventricular (LV) diameter in (A) %EF and (B) %FS. %EF, percent of ejection fraction; and %FS, percent of functional shortening. (C) H&E and Trichrome-stained heart sections (28 days after MI). (D) The bar graph shows quantitative analysis of the fibrosis area at 28 days after MI (n = 9). *P<0.05 vs. CTRL (indicates control genistein untreated ECFC).