Transplantation of Fibroblast Sheets with Blood Mononuclear Cell Culture Exerts Cardioprotective Effects by Enhancing Anti-Inflammation and Vasculogenic Potential in Rat Experimental Autoimmune Myocarditis Model

Figures

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  Figure 1. Experimental protocol. (A) Schematic flowchart of the in vitro protocol; Differentiation and purification of QQMNCs from rat peripheral blood and three harvested QQMNCs and fibroblast sheets (F-sheets). (B) Schematic flowchart of the in vivo protocol; Immunization and transplant of QQMNCs and F-sheets. (C) Representative images of the entire heart and heart tissue section stained with H&E at 15 days after immunization with procaine cardiac myosin (a) Healthy control (×4), (b) EAM (×4), Scale bar = 200 μm. PB: peripheral blood, PBMNCs: peripheral blood mononuclear cells, QQMNCs: quality and quantity control culture of mononuclear cells, H&E: Hematoxylin and eosin stain, M1: M1 macrophage, M2: M2 macrophage, Treg: regulatory T cell, EPC: endothelial progenitor cell.

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  Figure 2. QQ culture in healthy control and rats with myocarditis. (A) The graph shows the total cell number of PBMNCs and QQMNCs isolated from 100 mL of peripheral blood. Cell counts showed significant differences between PBMNCs and QQMNCs derived from healthy controls and rats with myocarditis. Cell counts showed no significant difference in QQ culture between healthy controls and rats with myocarditis. Data are shown as the mean ± SE. N = 7 to 8. ** p < 0.01, *** p < 0.001, NS: not significant. (B) Representative picture of pEPC-CFU and dEPC-CFU in rats with myocarditis. Scale bar = 500 μm. (C) The graph shows EPC-CFU counts generated from PBMNCs and QQMNCs per 10 mL of peripheral blood in healthy controls and rats with myocarditis. Comparative analysis showing an increased number of EPC-CFU in QQMNCs of rats with healthy controls and myocarditis. (D) The graph shows pEPC-CFU and dEPC-CFU counts as a percentage of total EPC-CFU count per dish. Data are shown as mean ± SE. N = 6. ** p < 0.01, *** p < 0.001.

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  Figure 3. Flow cytometry analysis of QQMNCs derived from rats with EAM. (A) The upper bar graph in healthy control shows the ratio of each (%) cell positivity in QQMNCs relative to that in PBMNCs. The lower bar graph in EAM shows the ratio of each (%) cell positivity in QQMNCs relative to that in PBMNCs. The investigated cell surface markers were as follows: hematopoietic stem cell (CD34, CD133), endothelial cell (VEGFR-2+/CD34, CD31), T cell (CD3, CD4, CD8), dendric cell (CD11b/c), monocyte (CD68), M2 macrophage (CD163, CD206/CD86), and regulatory T cell (CD25/CD3+CD4). (B) FCM was performed on gated PBMNCs and QQMNCs (gated CD68, CD206, VEGFR-2, and CD34). Data shown as mean ± SE. N = 6, * p < 0.05, ** p < 0.01. VEGFR: vascular endothelial growth factor receptor.

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  Figure 4. qRT-PCR analysis of PBMNCs and QQMNCs derived from EAM rats. Data are shown as mean ± SE. N = 5 or 6, * p < 0.05, ** p < 0.01 vs. QQMNCs. Enhanced gene expression for vascular regeneration, anti-inflammatory, and cardioprotective effects in a rat model of myocarditis. (A) Proangiogenic growth and cardiogenic factors. (B) MMPs. (C) Inflammatory cytokines. (D) Anti-inflammatory cytokines and immune tolerance. Data are shown as mean ± SE. N = 5 or 6 per group. VEGF: vascular endothelial growth factor, HGF: hepatocyte growth factor, IL: interleukin, MRC1: mannose receptor (CD206), Igf-1: insulin-like growth factor 1, Mmp: matrix metalloproteinase, Tnf: tumor necrosis factor.

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  Figure 5. Analysis of gene expression profile in various F sheet with PBMNCs or QQMNCs derived from the EAM rats. QQMNCs were obtained from peripheral blood of the rat EAM model. Expression of genes encoding vascular regeneration, anti-inflammation, and cardioprotection factors (Vegf-b, Hgf, Il-10, Cd206/Mrc1) were upregulated in the QQ+F sheet. Inflammatory gene expression (Il-17) was downregulated in the QQ+F sheet. Inflammatory gene expression (Il1-β) was upregulated in the QQ+F sheet. Data are shown as mean ± SE. N = 5 per group. * p < 0.05, ** p < 0.01. QQ+F sheet group. qRT-PCR: quantitative real-time polymerase chain reaction, M + F sheet: co-culture sheets of fibroblasts and PBMNCs, QQ+F sheet: co-culture sheets of fibroblasts and QQMNCs.

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  Figure 6. Cardiac function on 15 days after transplantation of QQ+F sheets derived from EAM rats. Cardiac functions were recorded using a conductance catheter and the pressure–volume (P-V) loop was measured as described in Methods. (A,B) Representative P-V loops on 14 days after cell sheet transplantation. (A) The oblique lines indicate end-systolic pressure–volume relationship (ESPVR). (B) The oblique lines indicate end-diastolic pressure–volume relationship (EDPVR). (C) An schematic diagram showing hypothetical EDPVR. EDPVR describes LV compliance. A decrease in LV diastolic function owing to a decrease in cardiac compliance is indicated by an upward shift in EDPVR. (D) The ESPVR on volume load showed significant differences between sham and healthy control groups. N = 6 rats per group. (E) The EDPVR on volume load were significantly higher in the sham and F-sheet groups than in the healthy control group. There was no significant differences between the QQ+F sheet and healthy control groups. N = 6 rats per group. Data are shown as mean ± SE. NS, not significant, * p < 0.05, ** p < 0.01, vs. healthy control group.

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  Figure 7. The levels of Bnp gene expression in the heart at 14 days after transplantation of QQ+F sheets derived from EAM rats. Bnp mRNA levels, as measured by qRT-PCR, were significantly higher in the sham and F-sheet groups than in the healthy control group. There was no significant differences between the QQ+F sheet and healthy control groups. N = 5 rats per group. Data are shown as mean ± SE. * p < 0.05, vs. healthy control group. NS, not significant. Bnp, brain natriuretic peptide.

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  Figure 8. The effect of QQ+F sheets transplantation on left ventricular (LV) remodeling in the hearts of EAM rats. Heart tissues were harvested 14 days after cell sheet transplantation and markers for LV remodeling were detected using histological analysis, as described in Methods. The QQ+F sheet transplant group showed significantly reduced heart fibrosis. (A) Representative heart sections stained with picrosirius red (×2 and ×100) at 14 days after sheet transplantation. To evaluate fibrotic area, the heart tissue sections were treated with picrosirius red stain. The percentage of fibrotic area was determined using ImageJ software, and the threshold color plugin set to red fibrotic tissue was selected and converted to a binary image. Scale bar = 100 μm. (B) Representative heart sections were stained with immunofluorescence RECA-1(×20; Scale bar = 50 μm, ×40; Scale bar = 20 μm) at 14 days after sheet transplantation. RECA (red): anti-endothelial cell antibody (RECA-1), TnT (green): anti-cardiac troponin T antibody, DAPI (blue). (C) The average collagen deposition of three heart sections (cardiac base, papillary muscle, and apex) showed a significant decrease in the QQ+F sheet group…