FOXO expressions in apoptotic EPCs.

<p>(A) Representative western blotting photos of expressions of FOXO3a and FOXO4 in EPCs and H₂O₂-treated-EPCs. EPCs were derived from atherosclerotic patients. (B) Pooled data of the FOXO3a/β-actin and FOXO4/β-actin expression ratios for the cells (*: p<0.05; n = 4, each). (C) A representative western blotting photo of expressions of FOXO4 in EPCs and H₂O₂-treated-EPCs. (D) Pooled data of the FOXO3a/β-actin and FOXO4/β-actin expression ratios of the cells. H-EPCs and P-EPCs indicate healthy volunteer-derived EPCs and atherosclerotic patient-derived EPCs, respectively (<b>*</b>: p<0.05; <b>^§</b>: p<0.01; n = 5, each).</p

Trends of hemodynamic changes in the absence or presence of C-B-SES to the lower limbs.

<p>The blood pressure was measured by the plethysmography. The heart rate, stroke volume, and cardiac output were measured by the percutaneous analyzer. The peripheral vascular resistance was calculated using the value of the blood pressure and stroke volume. Trends in 11 subjects are shown by a bar-graphic representation for each subject. Each value expressed by a gray or red bar graph was the value in testing whether each adopted synchro-time was suitable for ideal C-B-SES. The red bar graph indicates the value when we achieved an ideal C-B-SES whose plethysmography wave pattern was comparable with the pattern of IABP. The blue and yellow bar graphs indicate the values before and after the test, respectively. The ideal C-B-SES was not achieved in subjects #10 and #11. The values of blue and red bar graphs correspond to the values of ‘OFF’ and ‘ON’ in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187395#pone.0187395.t001" target="_blank">Table 1</a>, respectively.</p

Generation of FOXO4^KD-EPCs.

<p>(A) A representative western blotting photo of expressions of FOXO4 in EPCs, NT-EPCs, and FOXO4^KD-EPCs. (B) Pooled data of the FOXO4/β-actin expression ratios of the cells (*: p<0.001; n = 5, each).</p

Concentrations of EPC-secreted angiogenic proteins in the culture medium.

<p>VEGF: vascular endothelial growth factor; b-FGF: basic-fibroblast growth factor;</p><p>IGF-1: insulin-like growth factor-1; SDF-1α: stromal cell derived factor-1α.</p

Synchro-time of C-B-SES on electrocardiogram (ECG) and phonocardiogram (PCG).

<p>P, R, and T indicate the P wave, R wave, and T wave of the ECG, respectively. I and II indicate the sound of the closing of the mitral and aortic valves, respectively. RR-1 and RR-2 indicate the interval time between the tops of the two R waves. RT-1 and RT-2 indicate the trigger signal generation time between the top of the R wave (red dashed line) and the synchro-time (blue dashed line under the black diamond). The black diamond indicates the time phase of C-B-SES on the ECG and PCG charts.</p

Neovascularization capacity of FOXO4^KD-EPCs <i>in vivo</i>.

<p>(A) Representative laser Doppler blood flow images of both limbs of athymic nude rats (left panels) and fluorescence microscopic photos of CD31-positive endothelial capillaries in the ischemic limb (right panels) 14 days after intramuscular injection of PBS, EPCs, NT-EPCs, or FOXO4^KD-EPCs to the right ischemic limb. The hindlimbs enclosed by white squares indicate the right ischemic limbs. Red to white color and dark blue color on the image indicate high and low perfusion signals, respectively. CD31-positive endothelial capillaries were stained green. Scale bar: 100 μm. Pooled data of the ischemic/non-ischemic hindlimb blood flow ratio (B) and the capillary density (C) 14 days after the injection (*: p<0.05; **: p<0.01; ***: p<0.005;^§: p<0.0005; n = 5–6, each).</p

Baseline clinical characteristics of healthy volunteer- and atherosclerotic patient-derived EPCs.

<p>RASi = renin-angiotensin system inhibitor. Data are presented as the mean ± SEM.</p

Expressions of pro-apoptotic proteins of FOXO4^KD-EPCs.

<p>(A) A representative western blotting photo of expressions of FOXO4, BimEL, and cleaved caspase-3 in EPCs. (B) Pooled data of the FOXO4/β-actin, BimEL/β-actin, and cleaved caspase-3/β-actin expression ratios for EPCs. The expression ratios in non-transfected and H₂O₂-treated EPCs (purple bars) were control. (*: p<0.05; ^§: p<0.0005;^§§: p<0.0001; n = 5–7, each).</p

ROS production in athymic nude rat ischemic limbs.

<p>(A) Representative fluorescence microscopic images of DHE-stained tissues of the non-ischemic and ischemic limbs of athymic nude rats. DHE was stained red. Scale bar: 100 μm. (B) Pooled data of DHE fluorescence intensity of the rat non-ischemic and ischemic limbs (*: p<0.05; n = 12, each). (C) Representative fluorescence microscopic images of DHE-stained tissues of the ischemic limbs 24 h after intramuscular injection of PBS, EPCs, or FOXO4^KD-EPCs. Scale bar: 100 μm. (D) Pooled data of DHE fluorescence intensity of the ischemic limbs 24 h after intramuscular injection of PBS, EPCs, or FOXO4^KD-EPCs (n = 7, each).</p

Kinetics of EPCs for neovascularization <i>in vivo</i>.

<p>(A) Representative fluorescence microscopic images of EPCs incorporated in endothelial cells of the ischemic limbs of athymic nude rats 60 h after intramuscular injection of EPCs, NT-EPCs, or FOXO4^KD-EPCs. Rat specific CD31-positive endothelial cells were stained red. Human specific PCNA-positive cells, which were defined as human-derived EPCs, were stained green. Scale bar: 50 μm. (B) Pooled data of the number of PCNA-positive EPCs incorporated in endothelial cells of the rat ischemic limbs 60 h after the injection of EPCs, NT-EPCs, or FOXO4^KD-EPCs (*: p<0.01; **: p<0.005; n = 5, each). (C) A representative western blotting photo of expressions of human specific VEGF, b-FGF, SDF-1, and IGF-1 in the rat ischemic limb 60 h after intramuscular injection of EPCs, NT-EPCs, or FOXO4^KD-EPCs (n = 4, each).</p