Assessment of cardiac performance in the regional ischemia/reperfusion injury model.

<p>We evaluated cardiac function by ultrasound cardiography at 1 and 2 weeks after the procedure in controls and the activated protein C (APC) group (n = 10/group). The parameters were percent ejection fraction (EF), percent fractional shortening (FS), left ventricular end diastolic area (LVEDA) and left ventricular end systolic area (LVESA). Significant differences were observed between the two groups at 2 weeks after the procedure. Open circle indicate APC group and closed circle indicate controls. (*P<0.05; APC group versus controls, **P<0.01; APC group versus controls).</p

Assessment of myocardial infarct size after regional ischemia/reperfusion injury.

<p>We stained slices of left ventricular (LV) tissue with hematoxylin and eosin and Sirius red 2 weeks after inducing transient ischemia. The size of the infarct area was assessed by calculating the percentage of total LV area (% infarction) using Image J. The bar graph shows that the percent infarction in the activated protein C (APC) group was significantly smaller than the controls (* p<0.05; APC versus controls).</p

Assessment of endothelial function in the global injury model.

<p>Percent coronary flow (CF) increased in the activated protein C (APC) group compared with controls (p<0.01). Different vasodilator drugs were administered to investigate the response of enhanced endothelial function. One of them, acetylcholine (Ach), increased %CF only in the APC group (p<0.01, vs. controls), whereas nitroglycerine (NTG) increased %CF in both groups (p = 0.74).</p

Representative Western blots for AKT1 and p-AKT1.

<p>The photograph shows Western blots of myocardium for β-actin, whole AKT1, and p-AKT1in the global ischemia/reperfusion injury model during the time course. AKT1 was strongly detectable 30 min after reperfusion in the activated protein C (APC) group (p = 0.08, vs. control). The bar graph shows that p-AKT1 in the APC group was strongly and significantly expressed 30, 60, and 120 min after reperfusion, (* p<0.05; APC group versus controls).</p

Sustained-Release Delivery of Prostacyclin Analogue Enhances Bone Marrow-Cell Recruitment and Yields Functional Benefits for Acute Myocardial Infarction in Mice

<div><p>Background</p><p>A prostacyclin analogue, ONO-1301, is reported to upregulate beneficial proteins, including stromal cell derived factor-1 (SDF-1). We hypothesized that the sustained-release delivery of ONO-1301 would enhance SDF-1 expression in the acute myocardial infarction (MI) heart and induce bone marrow cells (BMCs) to home to the myocardium, leading to improved cardiac function in mice.</p><p>Methods and Results</p><p>ONO-1301 significantly upregulated SDF-1 secretion by fibroblasts. BMC migration was greater to ONO-1301-stimulated than unstimulated conditioned medium. This increase was diminished by treating the BMCs with a CXCR4-neutralizing antibody or CXCR4 antagonist (AMD3100). Atelocollagen sheets containing a sustained-release form of ONO-1301 (n = 33) or ONO-1301-free vehicle (n = 48) were implanted on the left ventricular (LV) anterior wall immediately after permanent left-anterior descending artery occlusion in C57BL6/N mice (male, 8-weeks-old). The SDF-1 expression in the infarct border zone was significantly elevated for 1 month in the ONO-1301-treated group. BMC accumulation in the infarcted hearts, detected by in vivo imaging after intravenous injection of labeled BMCs, was enhanced in the ONO-1301-treated hearts. This increase was inhibited by AMD3100. The accumulated BMCs differentiated into capillary structures. The survival rates and cardiac function were significantly improved in the ONO-1301-treated group (fractional area change 23±1%; n = 22) compared to the vehicle group (19±1%; n = 20; P = 0.004). LV anterior wall thinning, expansion of infarction, and fibrosis were lower in the ONO-1301-treated group.</p><p>Conclusions</p><p>Sustained-release delivery of ONO-1301 promoted BMC recruitment to the acute MI heart via SDF-1/CXCR4 signaling and restored cardiac performance, suggesting a novel mechanism for ONO-1301-mediated acute-MI heart repair.</p></div

BMCs differentiated into capillary structures in the infarcted area after MI and ONO-1301 treatment.

<p>Representative macro image of H and E staining seven days after MI and ONO-1301 treatment. The transplanted sheet is enclosed by a dashed line. A) Serial section of A. The BMCs displayed GFP. B) High-magnification image of the boxed region in A. C) Serial section of C. Arrowheads indicate vWF-expressing BMCs. Red indicates vWF; green, BMCs; and blue, nuclei. D) Representative images of isolectin-stained BMCs seven days after MI and ONO-1301 treatment. E) BMC accumulation and percentages of isolectin-positive BMCs. The number of BMCs that accumulated in the infarcted myocardium was greater in the ONO-1301-treated (O) group than in the vehicle (V) group. The percentage of isolectin-positive BMCs was also greater in the O group than in the V group. *<i>P</i><0.05 vs. V group. F) Small vessel density. Small vessels were detected by CD31 immunostaining. The density of small vessels in the O group was greater than in the V group. *P<0.05 vs. V group.</p

ONO-1301 enhanced SDF-1 secretion and BMC migration via SDF-1/CXCR4 signaling <i>in vitro</i>.

<p>NHDFs were stimulated with ONO-1301 for 72 hours, then the SDF-1 concentration in the culture medium was determined by ELISA (n = 3 each, *<i>P</i><0.05 vs. 0 nM). A) Number of BMCs that migrated toward the conditioned medium from ONO-1301-stimulated-NHDFs (0, 10, 100, or 1000 nM ONO-1301, n = 6; 1000 nM+nAB or 1000 nM+AMD, n = 3). *<i>P</i><0.05 vs. 0 nM, †<i>P</i><0.05 vs. 10 nM, ‡<i>P</i><0.05 vs. 1000 nM, §<i>P</i><0.05 vs. SDF-1. nAB, CXCR4-neutralizing antibody; AMD, CXCR4 antagonist AMD3100. B) Representative pictures of BMCs that had migrated to the medium from ONO-1301-stimulated BMCs. Green, BMCs.</p

Correlation of the maternal and fetal plasma ONO-1301 concentrations, and the location of prostacyclin receptor (IPR) expression in control fetal lungs and those with congenital diaphragmatic hernia (CDH).

<p>One-seventh of the maternal blood concentration of ONO-1301 was transferred to the fetal blood at E21.5, demonstrating the efficient placental permeability of this molecule (<b>A</b>). Immunohistochemistry images showing coexpression of IPR in α-smooth muscle actin (α-SMA)-positive cells in both control and CDH fetal lungs (<b>B</b>).</p

ONO-1301 treatment improved the cardiac performance and survival rate after MI.

<p>Survival rates after treatment. The ONO-1301-treated (O) group (n = 33) showed significantly better survival than the vehicle (V) group (n = 48). <i>*P</i><0.05 vs. V group. A) Evaluation of cardiac performance 4 weeks after treatment. In the O group, the LVESA was smaller, and the FAC was significantly higher compared to the V group (O group, n = 22; V group, n = 20; *<i>P</i><0.05 vs. V group). B) Representative macro images from each group. C) Quantification of anterior wall thickness. Anterior wall thickness was significantly thicker in the O group (n = 6) compared to the V group (n = 4). <i>*P</i><0.05 vs. V group. D) Quantification of percent infarction. Infarction was significantly smaller in the O group (n = 6) compared to the V group (n = 4). <i>*P</i><0.05 vs. V group. E) Representative Masson trichrome staining images at the border zone. F) Quantification of fibrosis. Fibrosis at the border zone was significantly smaller in the O group (n = 6) compared to the V group (n = 4). <i>*P</i><0.05 vs. V group.</p

Enhanced alveolar epithelial cell proliferation in fetus with congenital diaphragmatic hernia (CDH) following prenatal ONO-1301SR treatment.

<p>Representative Ttf-1 and Ki-67 immunostaining images of fetal lung sections from control (<b>A</b>), CDH (<b>B</b>), and CDH + ONO-1301SR-treated (<b>C</b>) pups at 400× magnification. The percentage of Ttf-1 and Ki-67 double-positive cells (white arrows) relative to the total number of cells in the lungs from control (white bar; n = 6), CDH (black bar; n = 6), and CDH + ONO-1301SR-treated (gray bar; n = 6) pups (<b>D</b>) are shown. The values are expressed as the mean ± SEM. ## 0.001 < <i>P</i> <0.01, versus nitrofen-CDH fetuses.</p