Results
Cardiac Allograft Vasculopathy
Mice treated with fasudil were well tolerated and showed no side effects, such as weight loss, hair loss, or diarrhea. A total of 2369 coronary arteries were evaluated by computer-assisted analysis in terms of the severity of CAV. Four weeks after the cardiac transplantation from AKR to C3H/He mice, neointima formation (evaluated by intima/vascular area ratio) and perivascular fibrosis of coronary arteries were markedly enhanced in the control allograft group compared with the isograft group (Figures 1 and 2⇓). By contrast, coronary veins were resistant to those changes (Figure 1E). In the isograft group, perivascular fibrosis was also developed probably due to a reperfusion injury alone (Figure 2B). Both neointima formation and perivascular fibrosis of the allografts were dose-dependently attenuated in the fasudil groups (Figures 1 and 2⇓). The high dose of fasudil inhibited the perivascular fibrosis to the level seen in the native hearts (Figure 2B). The medial area of the coronary artery was reduced only in the high-dose fasudil group compared with the control group (Figure 2C). The medial area in the high-dose fasudil group (0.31±0.01, n=9) was equal to that seen in the native hearts (0.36±0.01, n=10). At 4 weeks after the treatment with fasudil, plasma concentrations of hydroxyfasudil (ng/mL) increased from 0 (control animals) to 5.53±2.08 and 37.24±19.12 in the low-dose (n=6) and the high-dose (n=5) fasudil groups, respectively, specific therapeutic ranges of the Rho-kinase inhibitor.12 By contrast, fasudil was not detected in any groups.
Figure 1. Representative photomicrographs of a mouse coronary artery from an isograft (A) and from an allograft in the control (B), the low-dose (C) and the high-dose fasudil group (D) at 4 weeks after the transplantation (Masson’s trichrome staining, ×200). In the control allograft group, intimal thickening and perivascular fibrosis were noted, both of which were dose-dependently suppressed by fasudil treatment. Coronary vein, indicated by an asterisk, in the allograft is shown for comparison with coronary arteries (E). Bars=100 μm.
Figure 2. Long-term treatment with hydroxyfasudil inhibits the development of CAV in mice. In the control allograft group, intimal thickening (A, as expressed by intima/vascular area ratio) and perivascular fibrosis (B) developed at 4 weeks after the transplantation, both of which were dose-dependently suppressed by the fasudil treatment. By contrast, medial thickness was reduced only in the high-dose fasudil group (C).
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