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

Abstract

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.

Figure 1

Combined administration of LM221 and ONO1301 induced chemotactic cytokine and angiogenic cytokine gene expression. The expression of SDF-1, PlGF, HGF, Ang1, FGF2, TIE2, and PDGF-B in the infarcted area was measured by quantitative reverse-transcription polymerase chain reaction. Data were normalised to GAPDH expression. *p < 0.05.

Figure 2

Combined administration of LM221 and ONO1301 induced chemotactic cytokine and angiogenic cytokine production. The protein expression of (a) SDF-1, (b) HGF, (c) PIGF, and (d) Ang1 in the implanted site was measured by immunohistochemistry. Representative microscopic images show cytokines (green) and nuclei (blue). Scale bar = 50 μm. The graph indicates the number of cytokine-positive cells at the implanted site. *p < 0.05.

Figure 3

Combined administration of LM221 and ONO1301 decreased cell apoptosis. The expression of p53, Bcl2 in the infarcted area was measured by quantitative reverse-transcription polymerase chain reaction. Data were normalised to GAPDH expression. *p < 0.05.

Figure 4

Combined administration of LM221 and ONO1301 increased ILB4-positive cells. The number of ILB4-positive cells in the implanted site increased following combined administration of LM221 and ONO1301, as observed by immunohistochemical analysis. Representative microscopic images show ILB4 (green) and nuclei (blue). Scale bar = 50 μm. The graph indicates the number of ILB4-positive cells at the implanted site. *p < 0.05.

Figure 5

Combined administration of LM221 and ONO1301 increased mature capillary density. Some ILB4-positive cells in the implanted site were coated with SMA-positive cells, as observed by immunohistochemical analysis. ILB4 and SMA double-positive cells increased following the combined administration of LM221 and ONO1301. Representative microscopic images show ILB4 (red), SMA (green), and nuclei (blue). Scale bar = 50 μm. The graph indicates the number of ILB4 and SMA double-positive cells at the implanted site. *p < 0.05.

Figure 6

Combined administration of LM221 and ONO1301 increased PDGFRα and CD90 double-positive cell accumulation. PDGFRα and CD90 double-positive cells increased after the combined administration of LM221 and ONO1301, as observed by immunohistochemical analysis. Representative microscopic images show PDGFRα (red), CD90 (green), and nuclei (blue). Scale bar = 50 μm. The graph indicates the number of PDGFRα and CD90 double-positive cells at the implanted site. *p < 0.05.

Figure 7

The combined administration of LM221 and ONO1301 reduced fibrosis. Fibrosis was observed by Picro-Sirius Red staining. (a) Representative microscopic images of the infarct size. Scale bars = 1000 μm. (b) The graph indicates the size of fibrosis at the implanted site 4 weeks after surgery. *p < 0.05.

Figure 8

The combined administration of LM221 and ONO1301 improved heart performance. (a,b) Line graphs depicting the left ventricle ejection fraction (EF, a) and fractional shortening (FS, b) 2 days and 2 and 4 weeks after treatment. (c,d) The statistical analysis of the echocardiographic results of the diastolic (LVIDd, c) and systolic (LVIDs, d) left ventricular internal diameter 4 weeks after treatment. *p < 0.05.

Figure 9

Schematic figure of proposed mechanisms underlying LM221 and ONO1301-administrated treatment of myocardial infarction heart. ONO1301 is linearly released and infiltrated into the cardiac tissue. IP receptor expressing cardiac cells, such as vascular smooth muscle cells, fibroblast 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.