Combined administration of laminin-221 and prostacyclin agonist enhances endogenous cardiac repair in an acute infarct rat heart

Although endogenous cardiac repair by recruitment of stem cells may serve as a therapeutic approach to healing a damaged heart, how to effectively enhance the migration of stem cells to the damaged heart is unclear. Here, we examined whether the combined administration of prostacyclin agonist (ONO1301), a multiple-cytokine inducer, and stem cell niche laminin-221 (LM221), enhances regeneration through endogenous cardiac repair. We administered ONO1301- and LM221-immersed sheets, LM221-immersed sheets, ONO1301-immersed sheets, and PBS-immersed sheets (control) to an acute infarction rat model. Four weeks later, cardiac function, histology, and cytokine expression were analysed. The combined administration of LM221 and ONO1301 upregulated angiogenic and chemotactic factors in the myocardium after 4 weeks and enhanced the accumulation of ILB4 positive cells, SMA positive cells, and platelet-derived growth factor receptor alpha (PDGFRα) and CD90 double-positive cells, leading to the generation of mature microvascular networks. Interstitial fibrosis reduced and functional recovery was prominent in LM221- and ONO1301-administrated hearts as compared with those in ONO1301-administrated or control hearts. LM221 and ONO1301 combination enhanced recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute myocardial infarction hearts, highlighting a new promising acellular approach for the failed heart.

The protein expression of these cytokines in LM221-and ONO1301-administrated hearts was also significantly higher than that in other hearts (Fig. 2). Four weeks after treatment, the number of SDF-1-positive cells in LM221-and ONO1301-administrated hearts were 4689.3 ± 662.1 cells/mm 2 , which was significantly higher than that in control hearts (1453.3 ± 134.3 cells/mm 2 , p < 0.05) (Fig. 2a), and, that of PlGF-positive cells in LM221and ONO1301-administrated hearts was significantly higher than that in control hearts (160.5 ± 18.6 cells/mm 2 , p < 0.05) (Fig. 2c). Similarly, the number of HGF-positive cells in LM221-and ONO1301-administrated hearts was 447.7 ± 27.7 cells/mm 2 , which was significantly higher than that in control (91.0 ± 8.2 cells/mm 2 , p < 0.05), ONO1301-(169.1 ± 31.6 cells/mm 2 , p < 0.05), and LM221-administrated hearts (228.9 ± 22.5 cells/mm 2 , p < 0.05). On the other hand, the number of Ang1-positive cells in LM221-and ONO1301-administrated hearts was not a significant elevation compared to other hearts (Fig. 2d). effects of administration on cell death, we analysed apoptotic factors. The relative expression levels of p53 gene in ONO1301-administrated, and LM221-and ONO1301-administrated hearts were 0.67 ± 0.06, and 0.71 ± 0.11, respectively, which were significantly lower than that in control hearts (p < 0.05). On the other hand, the relative expression levels of Bcl2 gene in LM221-and ONO1301-administrated hearts was which was significantly higher than that in control hearts (p < 0.05) (Fig. 3).

Combined administration of LM221 and ONO1301 ameliorated the cardiac function.
To assess the pathological effect, we evaluated the cardiac function in each group. As shown in Fig. 8a,b, control hearts showed a time-dependent decrease in the percentage of ejection fraction (EF) and fractional shortening (FS). ONO1301-administrated hearts and LM221-administrated hearts didn't show much fluctuation in the percentage of EF and FS. However, the combined treatment with LM221 and ONO1301 displayed a time-dependent increase in the percentage of EF and FS. The relative EFs and FSs for LM221-and ONO1301-administrated hearts were significantly higher than that for control hearts 4 weeks after treatment (p < 0.05). However, there were no significant differences among LM221-and ONO1301-administrated hearts, LM221-administrated hearts, and ONO1301-administrated hearts (Fig. 8a,b). Although the left ventricle internal dimensions at diastole (LVIDd) in LM221-and ONO1301-administrated hearts was not a significant improvement, the left ventricle internal dimensions at systole (LVIDs) in the LM221-and ONO1301-administrated hearts was significantly improved compared to control hearts 4 weeks after treatment (Fig. 8c,d).

Discussion
Although cell-based therapy is a promising strategy for heart failure treatment, it is limited owing to the need for cell culture process, unsuitability with emergency use, and low versatility. To overcome these issues, we suggested a new acellular strategy in this study. Direct epicardial placement of LM221 and ONO1301 over the cardiac surface upregulated the expression of angiogenic and chemotactic factors in the myocardium at 4 weeks after transplantation. In addition, the combined administration of LM221 and ONO1301 suppressed cell death, and enhanced the accumulation of PDGFRα and CD90 double-positive cells or endothelial cells, leading to the generation of microvascular networks accompanied by SMA-positive cells. Lower fibrosis and effective functional recovery were shown in LM22-1 and ONO1301-administrated hearts than in ONO1301-administrated hearts, LM221 treated hearts and control hearts.
The vascular system is stabilized by the action of pericytes/vSMCs, a process termed as maturation. Vascular maturation has been reported to be dependent on the members of the vascular endothelial growth factor (VEGF) family, FGF2, PDGF-B, and Ang1. The VEGF family or FGF2 play a critical role in the expression and recruitment of endothelial cells 15 . PDGF-B secreted by endothelial cells controls mural cell recruitment to the endothelial cells 16 . Ang1 produced by mural cells activates endothelial cells and maximizes their interaction with mural cells 16 . These cytokine-related angiogenic processes produce a mature vascular system that avoids serious leakage and hemorrhage 15,16 . ONO1301 activates endothelial cells, vascular smooth muscle cells and fibroblasts to release multiple cytokines 3 . On the other hand, LM221 acts as a scaffold to store cells, growth factors and cytokines 7 . In the present study, we confirmed the significant upregulation in the expression of chemotactic factors and angiogenic factors (SDF-1, HGF, FGF2, PIGF, PDGF-B, and Ang1) after treatment with the combination of LM221 and ONO1301, suggesting that this combined strategy enhances vascular maturation mediated by cytokine cocktails. Although the expression of VEGF was not significantly different among the groups (data  16 . We speculate that vascular maturation in our model may be mediated by the mechanism described above.
The newly formed blood vessels through the action of a single cytokine may not have sufficient interaction with the surrounding vascular network and are not covered with mural cells. Vessels without mural cells demonstrate immature characteristics, leading to vascular leakage, hemorrhage, and early regression 15 . In this study, the combined administration of LM221 and ONO1301 significantly increased the number of structurally mature vessels, as evident from ILB4-positive endothelial cells accompanied with SMA-positive cells as compared to the The administration of LM221 alone was insufficient to accumulate PDGFRα and CD90 double-positive cells as compared with the combined treatment because these cells cannot be retained at the treatment site owing to the low expression of α7 integrin. These data suggest that SDF-1 induced by ONO1301 enhanced PDGFRα and CD90 double-positive cell recruitment into the transplanted site. In addition, the production of cytokines and the number of accumulated cells were higher in LM221-and ONO1301-administrated hearts than in the other hearts, suggesting the possibility that the cytokine expression was derived from endothelial cells activated by recruited cells.
Tissue homeostasis and wound repair are ensured by stem cells located within specialised microenvironments, referred to as niches. The stem cell niche comprises stem cells, neighboring cells, ECM, and secreted factors that are essential for cell survival and proliferation as well as to maintain the stability of undifferentiated stem cells 18 . The ECM is a scaffold for cells containing adhesion molecules such as integrins and is critical for the maintenance of the cytoskeleton as well as cell survival, growth, and fate 19 . LM221 is most abundantly expressed in the adult cardiac muscle and specifically binds to α7 integrin expressed in the myocardium 9,20 . LM221 is a scaffold for myocardiocytes and can influence cell maturation and survival through outside-in signaling 21 . These data suggest that the combined administration of LM221 and ONO1301 in vivo promotes stem cell differentiation, proliferation, and consequently improves cardiac function.
We proposed the following mechanisms in this paper. ONO1301 is linearly released and infiltrated into the cardiac tissue. IP receptor expressing cardiac cells, such as vascular smooth muscle cells, fibroblasts and endothelial cells, are activated by binding of ONO1301, leading to paracrine released cytokines, such as SDF-1, PIGF, or HGF. Cells, such as PDGFRα and CD90 double-positive cell, endothelial cell, or smooth muscle cell, are locally induced by SDF-1, and cells accumulate in the place using LM221 as a scaffold, leading to promotion of angiogenesis and recovering the cardiac function (Fig. 9). Totally, although the administration of ONO1301 alone or LM221 alone was insufficient in improvement in AMI model hearts, the combined administration with ONO1301 and LM221 was significantly more effective at suppression of infarct size and improvement of cardiac function. In conclusion, direct epicardial placement of LM221 and ONO301 promoted recruitment of PDGFRα and CD90 double-positive cells, maturation of vessels, and functional recovery in rat acute MI hearts, suggesting the promising role of this acellular approach for failed hearts.

Materials and methods
Animal care. Animal care was performed as previously described 11,22 . The animal care procedures used in this study was reviewed and proved by the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication no. 85-23, revised 1996 no. 85-23, revised 1996). The experimental protocols were  Ligation of the left coronary artery and epicardial placement of collagen sheet in rats. LM221 was resuspended in phosphate-buffered saline (PBS) at 100 nM concentration and applied to atelocollagen sheets (KOKEN, Tokyo, Japan) through immersion for 2 h at 37 °C. ONO1301 (Ono Pharmaceutical Co. Ltd., Osaka, Japan) was resuspended in saline and applied to atelocollagen sheets. Male athymic nude rats aged 6-8 weeks (F344 ⋅ NJcl-rnu/rnu) were obtained from CLEA Japan (Tokyo, Japan). Left thoracotomy was performed under general anesthesia via the fourth intercostal space, and the branch of the left coronary artery was permanently ligated at the bottom of the left atrial appendage. Five minutes after ligation, LM221-and ONO1301-immersed atelocollagen sheets (KOKEN, Tokyo, Japan) (n = 8), LM221-immersed atelocollagen sheets (n = 10), ONO1301immersed atelocollagen sheets (n = 10), or atelocollagen sheets (n = 11) were directly placed over the anterolateral surface of the left ventricular (LV) wall. After the approximation of the surgical wound, the rats were maintained in a temperature-controlled individual cage and then euthanized 4 weeks after the surgical procedure.
Statistical analysis. Data are expressed as mean ± standard error of the mean. Data distributions were checked for normality. Multiple comparisons were assessed using one-way analysis of variance (ANOVA) with Tukey-Kramer post-hoc testing. All p values were two-sided, and values of p < 0.05 were considered statistically significant.