Representative data of graft survival in the full allomismatch combination. Mice treated with clarithromycin (CAM) (open circles) showed prolonged cardiac allograft survival in comparison with the nontreated mice (open squares). Nontreated allografts were acutely rejected (7.1 ± 0.2 days, n = 8). However, CAM administration statistically prolonged allograft survival (9.7 ± 0.2 days, n = 8). *p < 0.05 versus nontreated group.

Representative light micrographs of allografts from the nontreated group and the clarithromycin (CAM)-treated group. (A) Representative pathological findings with hematoxylin and eosin (HE) (a, b, e, and f) and Mallory (c, d, g, and h) stain in full allomismatch allografts. Although myocardial cell infiltration and fibrosis were observed in nontreatment (a, c, e, and g) on day 7, the CAM treatment (b, d, f, and h) markedly attenuated them. Scale bars = 2 mm (a to d) and 50 µm (e to h). (B) Representative pathological findings with HE (a and b), Mallory (c and d), and Elastica van Gieson (EvG) (e and f) stain in class II mismatch allografts. Although myocardial cell infiltration, fibrosis, and graft arterial disease (GAD) were observed in the nontreated allografts (a, c, and e) on day 60, CAM treatment (b, d, and f) markedly attenuated them. Scale bars = 2 mm (a to d) and 25 µm (e and f). (C) Quantitative data of cell infiltration (a) and fibrosis (b) in the full allomismatch combination. Quantitative data of cell infiltration (d), fibrosis (e) area, and percentage of neointimal thickening (c) in the class II mismatch combination. *p < 0.05 versus nontreated group.

Representative immunohistochemistry of allograft from the nontreated group and the clarithromycin (CAM)-treated group. The sections were incubated with CD4 (a and b), CD8 (c and d), CD11b (e and f), intercellular adhesion molecule (ICAM)-1 (g and h), and nuclear factor-B (NF-B) p65 (i and j). Although CD4⁺ (a), CD8⁺ (c), and CD11b⁺ (e) cells were observed in nontreatment on day 7, CAM treatment markedly attenuated them (b, d, and f). The expression of ICAM-1 (g) and NF-B p65 (i) were observed in nontreatment myocardial infiltrating area; however, CAM treatment markedly attenuated them (h and j). Scale bars = 50 µm (a to j).

(A) Representative gels in ribonuclease protection assay (RPA). Messenger ribonucleic acid (mRNA) was isolated from cardiac allografts on day 7. (B) The quantitative results. Increase of interleukin (IL)-10, IL-15, IL-6, and interferon (INF)-gamma mRNA levels were observed. However, clarithromycin (CAM) treatment markedly attenuated them. *p < 0.05 versus nontreated group. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

(A) Representative results of gelatinase's activity. In the matrix metalloproteinases (MMPs) inhibitor (–) group, gelatinase's activity was generally observed in the myocardial infiltrating area in full allografts (a). However, clarithromycin (CAM) treatment markedly attenuated gelatinase's activity (b). The MMPs inhibitor (+) films show the non–gelatinase-specific activity (c and d). Scale bars = 4 mm (a to d). (B) Representative results of immunohistochemistry. Although expression of MMP-9 was observed in the infiltrating cells in nontreatment allografts (a), CAM treatment suppressed the expression (b). Scale bars = 50 µm (a and b).

(A) Representative Western blot data detecting the expression of matrix metalloproteinases (MMP)-9 and -2 proteins in macrophages (a). The control group (Co) markedly enhanced the expression of MMP-9 compared with native (Na). The 0.2- and 20-µmol/l clarithromycin (CAM) treatment (0.2 CA and 20 CA) altered the MMP-9 protein levels compared with the control group. However, MMP-2 expression was not altered by CAM treatment. (b) Representative gels showed the expression of MMP-9, MMP-2, and tissue inhibitor metalloproteinase (TIMP)-1 messenger ribonucleic acid (mRNA) levels by reverse transcriptase-polymerase chain reaction (RT-PCR). The CAM treatment altered the expression of MMP-9 but not MMP-2 and TIMP-1. The treatment of 20 µmol/l CAM inhibited expression of MMP-9 more effectively than that of 0.2 µmol/l. (B) Representative RT-PCR data detecting the expression of MMP-9, MMP-2, and TIMP-1 mRNA in the smooth muscle cells (SMCs). The control group markedly expressed MMP-9, and CAM treatment inhibited the expression of MMP-9 mRNA levels. The CAM treatment did not alter the MMP-2 and TIMP-1 expression. GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

(A) Quantitative data of proliferation assay. Markedly increase proliferation of the smooth muscle cells (SMCs) was observed by stimulation of PDGF-BB (Biosource International, Camarillo, California) compared with those of the native group. The 0.2-µmol/l clarithromycin (CAM) treatment did not significantly inhibit the cell proliferation, whereas 2.0- and 20-µmol/l CAM treatment significantly inhibited the proliferation compared with the control group. *p < 0.05 versus control group. (B) Representative light micrographs of migration assay. Although the SMCs markedly migrated to a lower well by the stimulation of PDGF-BB (b) compared with those in the native group (a), CAM (20 µmol/l) treatment (c) significantly suppressed the SMC migration. (C) The quantitative data of migration assay. Counts of SMCs in a lower well significantly decreased in CAM-treatment group compared with the control group. *p < 0.05 versus control group.