ONO-1301 enhanced SDF-1 secretion and BMC migration via SDF-1/CXCR4 signaling after MI.

<p>A–C) The SDF-1, HGF, and VEGF expression at the border zone of the infarcted area was measured by quantitative RT-PCR. The expression levels of these cytokines were higher in the ONO-1301-treated (O) group compared to the vehicle (V) group. (O group, n = 7; V group, n = 7–8; *<i>P</i><0.05 vs. V group). The expression relative to GAPDH is shown. D) BMC migration to ONO-1301-treated infarcted myocardium was evaluated using IVIS. Representative picture of IVIS at day 3. Left: 100 mg/Kg, Center: 0 mg/Kg, Right: 100 mg/Kg+AMD3100 (AMD). E) The number of accumulated BMCs was greater in the 100 mg/kg ONO-1301-treated infarcted heart compared to the 0 and 10 mg/kg ONO-1301-treated infarcted heart. When BMCs treated with AMD were injected, the BMC accumulation decreased in the 100 mg/Kg ONO-1301-treated infarcted heart compared with the untreated-BMC-injected heart (0 mg/Kg, n = 4; 10 mg/Kg, n = 8; 100 mg/Kg, n = 5; 100 mg/Kg+AMD3100, n = 4; *<i>P</i><0.05 vs. 0 mg/Kg, †<i>P</i><0.05 vs. 10 mg/Kg, ‡<i>P</i><0.05 vs. 100 mg/Kg).</p

Preservation of pulmonary arterial structure in fetuses with congenital diaphragmatic hernia (CDH) following prenatal ONO-1301SR treatment.

<p>Representative van Gieson-stained lung sections from control (<b>A</b>), CDH (<b>B</b>), and CDH + ONO-1301SR-treated (<b>C</b>) fetuses at 400× magnification. The medial wall thickness (MWT, %) of control (white bar; n = 6), CDH (black bar; n = 6), and CDH + ONO-1301SR-treated (gray bar; n = 6) groups (<b>D</b>). Representative α-smooth muscle actin (α-SMA) and Ki-67 immunostaining in fetal lung sections from control (<b>E</b>), CDH (<b>F</b>), and CDH + ONO-1301SR-treated (<b>G</b>) pups at 400× magnification. The percentage of Ki-67-positive cells relative to the total number of cells within the medial layers of the pulmonary arteries in the lungs from control (white bar; n = 5), CDH (black bar; n = 6), and CDH + ONO-1301SR-treated (gray bar; n = 5) pups (<b>H</b>). The values are expressed as the mean ± SEM. *** <i>P</i> < 0.001 versus control fetuses; ### <i>P</i> < 0.001 versus nitrofen-CDH fetuses.</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

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

Abolished BMC transplantation-induced cardiac function recovery by HMGB1-inhibition.

<p>Cardiac parameters were measured by echocardiography (<b>A–D</b>) and catheterization (<b>E–H</b>) on day 28 after each treatment. Cardiac function was improved by BMC transplantation (BMC group) compared to the PBS injection control (CON group), while this effect was eliminated by antibody neutralization of HMGB1 (AB group), but not by control IgG administration (IgG group). LVFAC, left ventricular fractional area change; ECA, endocardial area. *:<i>p</i><0.05 <i>versus</i> the CON group, ^†:<i>p</i><0.05 <i>versus</i> the BMC group. ^‡:<i>p</i><0.05 <i>versus</i> the IgG group, mean±SEM for n = 8∼10 in each group.</p

Enhanced alveolar development in fetuses with congenital diaphragmatic hernia (CDH) following prenatal ONO-1301SR treatment.

Representative Ttf-1 immunostaining in fetal lung sections from control, CDH, and CDH + ONO-1301SR-treated pups at 400× magnification (A). The percentage of Ttf-1 positive cells determined 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) are shown. The values are expressed as the mean ± SEM. *** P P < 0.001 versus nitrofen-CDH fetuses.</p

Preservation of lung structure in fetuses with congenital diaphragmatic hernia (CDH) following prenatal ONO-1301SR treatment.

<p>Representative hematoxylin-eosin stained lung sections from control (<b>A</b>), CDH (<b>B</b>), and CDH + ONO-1301SR-treated (<b>C</b>) fetuses at 100× magnification. The mean linear intercept (Lm, in μm) in the lungs from the control (white bar; n = 6), CDH (black bar; n = 6), and CDH + ONO-1301SR-treated (gray bar; n = 6) groups are shown (<b>D</b>). The values are expressed as the mean ± SEM. ** 0.001 < <i>P</i> <0.01, *** <i>P</i> < 0.001 versus control fetuses; # 0.01 < <i>P</i> < 0.05 versus nitrofen-CDH fetuses.</p

Effects of prenatal ONO-1301SR treatment on the macroscopic development of fetal hypoplastic lungs.

<p>The incidence of congenital diaphragmatic hernia (CDH) (<b>A</b>), fetal body weight (<b>B</b>), lung-to-body weight ratio (<b>C</b>), total DNA (<b>D</b>), and total protein content (<b>E</b>) in the fetal lung were evaluated in each pup of the control (white bars), nitrofen-induced CDH (black bars), and nitrofen-induced CDH treated with ONO-1301SR (CDH+ONO; gray bars) groups. The values are expressed as the mean ± SEM. * 0.01 < <i>P</i> < 0.05, *** <i>P</i> <0.001 versus control fetuses; # 0.01 < <i>P</i> < 0.05, ## 0.001< <i>P</i> < 0.01 versus nitrofen-CDH fetuses.</p

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

Upregulation of therapeutic factors in lungs with congenital diaphragmatic hernia (CDH) following prenatal ONO-1301SR treatment.

<p>Relative gene expression of <i>Vegf</i> (<b>A</b>), <i>Hgf</i> (<b>B</b>), and <i>Sdf1</i> (<b>C</b>) in the lungs from control (white bars; n = 5), CDH (black bars; n = 5), and CDH + ONO-1301SR-treated (gray bars; n = 5) pups. The average copy number of gene transcripts was normalized to that of the <i>Gapdh</i> gene. The values are expressed as the mean ± SEM. * 0.01 < <i>P</i><0.05, *** <i>P</i> < 0.001 versus control fetuses; # 0.01 < <i>P</i> < 0.05 versus nitrofen-CDH fetuses. Representative immunoblots of VEGF protein levels in fetal lungs from the control, CDH, and CDH + ONO-130-treated pups (<b>D</b>).</p