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

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

Electropotential mapping was performed epicardially with a 16-needle probe at 2.0-ms intervals.

<p>In the premedication maps, one cardiac R-R-cycle is shown (images from 18 ms to 218 ms) at a normal rat heart rate (300 beats per minute, bpm). An area of reentry can be seen as red coloration in the middle of the border area measured. Sixty seconds after a bolus injection of isoproterenol, the reentry area was observed, while 120 s after the isoproterenol bolus, a full ventricular tachycardia has developed, with a heart rate of approximately 6000 bpm.</p

Panel A: Epicardial transplantation of myoblast sheets (Sheet group, n = 18) significantly improved the left ventricular ejection fraction (EF).

<p>In sham-treated animals (Control group, n = 19), EF decreased markedly during the 2-week follow-up. This decrease in EF was attenuated by myoblast intramyocardial injection therapy (Injection group, n = 17). Panel B: Significant improvement in fractional shortening (FS) was observed in the Sheet group. FS significantly deteriorated in the Control group, while in the Injection group this decrease was attenuated. Panel C: As a measure of left ventricular remodeling, the left ventricular end-diastolic diameter (LVEDD) decreased in the Sheet group and increased in the Control group. In the Injection group, the LVEDD remained similar to the baseline value. Panel D: Representative hematoxylin-eosin-stained paraffin-embedded sections of the heart on the midventricular short axis from each group. The myocardium in the Sheet group demonstrated less remodeling of the left ventricle than the Control group. A similar, but less pronounced, effect on remodeling was evident in the Injection group. * p < 0.05, ** p < 0.01 Sheet group vs. Control group; † p < 0.05, †† p < 0.01 Sheet group vs. Injection group.</p

Myocardial gene expressions of inflammatory genes in the sham-treated (Control group, n = 6) and in the groups receiving myoblast cell therapy as intramyocardial injections (Injection group, n = 6) or epicardial cell sheets (Sheet group, n = 6).

<p>Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01 compared with the Control group. † p < 0.05 compared with the Injection group.</p

Study protocol timeline.

<p>Myoblast cell therapy as intramyocardial injections or epicardial sheets was administered 2 weeks after induction of heart failure and ischemia by ligation of the left anterior coronary artery (Myocardial infarction). Electrocardiography (Holter) was monitored constantly, using implanted Holter monitors, and specific recordings were made on days 1, 7, and 14 after cell therapy administration. Echocardiography (Echo), as measured first before and then 2 weeks after myoblast transplantation, was used to evaluate therapy efficacy. Epicardial electropotential mapping (EEPM) was performed 14 days after therapy administration.</p

Ventricular premature contractions (VPCs) after epicardial myoblast sheet transplantation (Sheet group, n = 18) or intramyocardial myoblast injections (Injection group, n = 17).

<p>The Control group (n = 19) received, instead of myoblast therapy, a sham operation 2 weeks after myocardial infarction. Electrocardiography was monitored continuously. The number of VPCs was significantly higher in the Injection group on days 1 (p < 0.05) and 14 (p < 0.01) after myoblast transplantation than in the Control group (*) and significantly higher on days 7 (p < 0.05) and 14 (p < 0.01) than in the Sheet group (†). There was no ventricular tachycardia recorded in any of the animals.</p

Panels A-C: Representative immunofluorescence images of CD11b-expression, with DAPI counterstaining of the nuclei of the myocardium 2 weeks after myoblast cell therapy (4 weeks after myocardial infarction).

<p>Infiltration of CD11b-positive cells by epicardial transplantation (panel A) was similar to that of the Control group (panel C). Panel D: Clusters of CD11b-positive inflammatory cells can be seen in the myocardium of the Injection group at a higher magnification. Panel E shows densitometry quantitation of CD11b-expression in the various groups (n = 6). In the group receiving intramyocardial injections, an increased leukocyte infiltrate was evident in comparison to the Control or Sheet groups (*** p < 0.001 vs. Injection group). Panels F-I show CD68-staining similar to that of CD11b. Quantitation of the CD68-positive area showed increased CD68-positive cell infiltration in the Injection group in comparison to the Sheet and Control groups (panel J, * p < 0.05 for both comparisons).</p