Anti-CD3 Antibody Treatment Reduces Scar Formation in a Rat Model of Myocardial Infarction

Figures

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  Figure 1. Cell cultures of human peripheral blood mononuclear cell (PBMC)were supplemented with different doses of Anti-thymocyte globulin (ATG) or various anti-T-cell antibodies, such as treatment with anti- (1) IgG2a, (2) IgG1, (3) CD4, (4) CD8, (5) CD11a, (6) CD3, (7) CD28, (8) CD2, (9) HLA-DR. After 24 h incubation, supernatants were collected and the concentration of IL-8 and MCP-1 was assessed by enzyme-linked immunosorbent assays (ELISA). Only the addition of anti-CD3 antibodies led to a pronounced (a) IL-8 and (b) MCP-1 expression compared with other epitopes in PBMCs. (p-values were expressed as follows: ** p < 0.01, *** p < 0.001).

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  Figure 2. Rats were subjected to permanent left coronary artery (LCA) ligation. Rats were sacrificed 6 weeks after experimental infarction. Hearts were explanted and then sliced at three layers at the level of the largest extension of infarcted area and infarct sizes compared after staining with hematoxylin-eosin (H&E) and van Gieson (VG). (a) In animals treated with anti-CD-3 antibody (n = 13) left ventricular scar size was significantly reduced [12.6% (IQR 8.3–27.2) vs. 27.8% (IQR 16.2–34.9) p < 0.01] compared to vehicle treatment (n = 12). Representative (b) pictures are shown. (p-values were expressed as follows: ** p < 0.01).

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  Figure 3. human umbilical vein endothelial cell (HUVECs) were exposed to anti-CD3 treated PBMCs. Cell migration and angiogenesis were assessed by (a) tube assay and (b) tube formation, respectively. Compared to isotype control (100%), exposure to supernatant of anti-CD3 treated PBMC did not induce (a) endothelial cell tube formation (94 ± 2%; p = n.s. versus isotype control) (b) nor chemotaxis (90 ± 1%; p = n.s.) in HUVECs.

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  Figure 4. The effect of anti-CD3 treatment on intracellular pathways was investigated by Western Immunoblotting. In human cardiomyocytes, co-incubation with supernatant of PBMCs treated with anti-CD3 antibody did not alter phosphorylation of Akt (0.05 ± 0.01 versus 0.08 ± 0.02 protein expression/alpha-actinin; p = n.s.) and ERK1/2(0.30 ± 0.17 versus 0.46 ± 0.13 protein expression/alpha-actinin; p = n.s) compared to isotype control. Representative pictures of the isotype (1), ATG (2), anti-CD3 (3) and medium plus FCS (4) conditions are shown.

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  Figure 5. Exosome markers were assessed by (a) exoELISA and (b) FACS. The expression of the exosome marker (a) CD81 was not different (20.94 ± 4.84 × 106 exosomes/µL versus 39.36 ± 3.14 × 106 exosomes/µL; p = n.s.) compared to isotype control. The expression (b) of exosomes positive for CD9 (74 ± 4% versus 75 ± 2%; p = n.s), HSP70 (22 ± 8% versus 23 ± 8%; p = n.s) or CD81 (11 ± 3% versus 12 ± 4%; p = n.s.) and CD63+ exosomes (14 ± 1% versus 17 ± 2; p = n.s.) was unchanged upon anti-CD3 antibody treatment compared to isotype control.

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  Figure 6. miRNA contents were analyzed by next generation sequencing. In total, 322 miRNAs were detected across all samples at a sufficient level (RPM > 5) and considered for statistical analysis. Anti-CD3 stimulation led to alterations in 32 miRNAs (10%) compared to supernatant of non-stimulated PBMC, including miRNAs miR-19a-3p and miR-19b-3p, miR-26b-5p, miR-30e-5p, miR125a-5p, miR-181d-5p, miR-186-5p, miR-301a-3p and miR-335-5p.