ABC | Volume 112, Nº1, January 2019

Original Article Arq Bras Cardiol. 2019; 112(1):3-10 Tianshu-Chu et al Rapamycin combined with α -cyanoacrylate Figure 1 – Rats survived well 4 weeks after operation. Operating procedure as previously described was used to build models of grafted rat jugular vein on carotid artery on one side. Post-operation, the transplanted veins are well filled and the blood vessels beat well, and the glue was spread evenly over the surface of the veins in the α-CA and α-CA-RPM groups (arrow). Four weeks after operation, veins in the sham group slightly expanded; the control group had new granulation tissue, thickening tubes, edema and light stiffness; the α-CA group had few fresh tissues which were easily separated, with no obvious expansion and clear boundary from the surrounding, and the glue was not fully degraded (arrow); the RPM group had clear boundaries from the surrounding tissue, and they were fresh and no obvious expansion. The general form of α-CA-RPM group was similar to α-CA group. Statistical analysis All data were analyzed using statistical analysis software SPSS 17.0. Data are presented as mean ± standard deviation. Because data showed a normal distribution, comparison among multiple groups was analyzed by single-factor analysis of variance (ANOVA) and comparison between two groups was conducted by Fisher's least significant difference (LSD) test. A value of p < 0.05 was considered statistically significant. Results Rats survived well 4 weeks after operation Operating procedure as previously described was used to build models of grafted rat jugular vein on carotid artery on one side. Post-operation, the transplanted veins were well filled and the blood vessels beat well; the glue was spread evenly over the surface of the veins in the α -CA and α -CA-RPM group. Rats’ vital status and incisions were checked every day. Subsequently, we found that one rat in the RPM group and one rat in the α -CA group had died of low temperature 2 weeks after operation and the other rats survived and recovered well with strong pulse in grafted veins. The rats were euthanized 4 weeks after surgery; notably, there were only 2 rats who presented venous occlusion, one in the α -CA group and one the RPM group. Correspondingly, blood flow in other grafted veins was patent. Veins in the sham group slightly expanded. What is more, veins in the control group had new granulation tissue, thickened tubes, edema, and light stiffness; however, veins in the α -CA, RPM, and α -CA-RPM groups had few fresh tissues which were easily separated, with no obvious expansion and clear boundary from the surrounding, and the glue was not fully degraded (Figure 1). α-CA-RPM reduced intimal thickening of the vein graft In order to observe what impacts each group’s intervention had on intimal hyperplasia, grafted veins were stained with HE 4 weeks after surgery. Afterwards, we used computer image analysis system to analyze intimal hyperplasia. This showed that the intima of the control group was strikingly thicker than that of α -CA group, RPM group, and α -CA-RPM group; the difference was statistically significant (91.3 ± 3.9, 133.6 ± 8.0, 50.6 ± 5.4 vs. 233.6 ± 29.1 μm, p < 0.01; Figure 2B, C, D, E and F); the intima of the RPM group was thicker than that of α -CA group; the difference was statistically significant (133.6 ± 8.0 vs. 91.3 ± 3.9 μm, p < 0.05; Figure 2C, D and F); the intima of α -CA group and RPM groups was thicker than that of α -CA-RPM group; the difference was statistically significant (50.6 ± 5.4 vs. 91.3 ± 3.9 μm, 133.6 ± 8.0 μm, p < 0.05; Figure 2 C, D, E and F). What is more, as shown in Figure 3, our results from immunohistochemical staining of PCNA demonstrate that the control, α -CA, RPM, and α ‑CA‑RPM groups had a significantly higher proliferating index than the sham group (p < 0.01; Figure 3A, B, C, D, E and F), and the percentage of PCNA-positive cells in the α -CA, RPM, and α -CA-RPM groups was significantly less than in the control group (p < 0.01; Figure 3B, C, D, E and F). Moreover, it is worth noting that the proliferating index in the α -CA-RPM group was markedly less than in the α -CA or RPM group (p < 0.01; Figure 3C, D, E and F). Taken together, our results strongly demonstrate that α -CA, RPM, and α -CA-RPM inhibit intimal hyperplasia in vein grafts, and the effect of α -CA-RPM is stronger than that of α -CA or RPM. α-CA-RPM diminished intimal hyperplasia and inflammatory responses In order to further study the mechanism through which the three intervening methods prevent intimal hyperplasia, we examined the value of α SMA and vWF in grafted veins 4 weeks after surgery. The α SMA values in the α -CA, RPM, and α ‑CA‑RPM groups were much lower than in the control group, as detected by RT-PCR (p < 0.01; Figure 4A). The α SMA values in the α -CA-RPM group were lower than in the α -CA and RPM groups (p < 0.01; Figure 4A). Similar results were found in the value of vWF in α -CA-RPM group (p < 0.01; Figure 4B). This result verified that α -CA, RPM, and α -CA-RPM inhibition might reduce intimal hyperplasia by blocking α SMA and vWF over-expression. 5