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

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

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

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

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

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

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