Rapamycin Combined with α-Cyanoacrylate Contributes to Inhibiting Intimal Hyperplasia in Rat Models

Abstract

Background: Vein graft restenosis has an adverse impact on bridge vessel circulation and patient prognosis after coronary artery bypass grafting.

Objectives: We used the extravascular supporter α-cyanoacrylate (α-CA), the local application rapamycin/sirolimus (RPM), and a combination of the two (α-CA-RPM) in rat models of autogenous vein graft to stimulate vein graft change. The aim of our study was to observe the effect of α-CA, RPM, and α-CA-RPM on vein hyperplasia.

Methods: Fifty healthy Sprague Dawley (SD) rats were randomized into the following 5 groups: sham, control, α-CA, RPM, and α-CA-RPM. Operating procedure as subsequently described was used to build models of grafted rat jugular vein on carotid artery on one side. The level of endothelin-1 (ET-1) was determined by enzyme-linked immunosorbent assay (ELISA). Grafted veins were observed via naked eye 4 weeks later; fresh veins were observed via microscope and image-processing software in hematoxylin-eosin (HE) staining and immunohistochemistry after having been fixed and stored" (i.e. First they were fixed and stored, and second they were observed); α-Smooth Muscle Actin (αSMA) and von Willebrand factor (vWF) were measured with reverse transcription-polymerase chain reaction (RT-PCR). Comparisons were made with single-factor analysis of variance and Fisher's least significant difference test, with p < 0.05 considered significant.

Results: We found that intimal thickness of the α-CA, RPM, and α-CA-RPM groups was lower than that of the control group (p < 0.01), and the thickness of the α-CA-RPM group was notably lower than that of the α-CA and RPM groups (p < 0.05).

Conclusion: RPM combined with α-CA contributes to inhibiting intimal hyperplasia in rat models and is more effective for vascular patency than individual use of either α-CA or RPM.

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.

Figure 2

α-CA-RPM reduced intimal thickening of the vein graft. The vessel tissue was harvested 4 weeks after the operation, fixed in formalin, sliced to 4 µm tissue sections and stained with HE. Images (×100 objective lens) were collected and analyzed by Olympus micro-imaging system. The rats were divided into 5 group: Sham group (A), control group (B), α-cyanoacrylate group (C), Rapamycin group (D) and α-CA-RPM group (E). (F) Represented the statistical graph of each group’s intima thickness. The intima of control group was dramatically thicker than that of α-CA, RPM and α-CA-RPM groups; 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); the intima of the RPM group was thicker than that of α-CA group (133.6 ± 8.0 vs. 91.3 ± 3.9 µm, p < 0.05); the intima of α-CA group and RPM group was thicker than that of α-CA-RPM group (50.6 ± 5.4 vs. 91.3 ± 3.9 µm, 133.6 ± 8.0 µm, p < 0.05). * The control group had obvious difference with other groups, p < 0.05. ^# The α-cyanoacrylate group had obvious difference with other groups, p < 0.05. ^△ The rapamycin group had obvious difference with other groups, p < 0.05. ^☆ The α-CA-RPM group had obvious difference with other groups, p < 0.05.

Figure 3

α-CA-RPM decreased the proliferating index of vein graft. The vessel tissue was harvested 4 weeks after the operation, fixed with formalin, sliced to 4 µm tissue sections and stained with the primary antibody anti-PCNA. Images (×200 objective lens) were collected and analyzed by Olympus micro-imaging system. Likewise, the rats were also divided into 5 group: Sham group (A), control group (B), α-cyanoacrylate group (C), Rapamycin group (D) and α-CA-RPM group (E). (F) Represented the statistical graph of each group’s PCNA proliferation index. * The control group had obvious difference with other groups, p < 0.01. ^# The α-cyanoacrylate group had obvious difference with other groups, p < 0.01. ^△ The rapamycin group had obvious difference with other groups, p < 0.01. ^☆ The α-CA-RPM group had obvious difference with other groups, p < 0.01.

Figure 4

α-CA-RPM diminished the expression of αSMA and vWF and inflammatory responses. Four weeks after the surgery, RT-PCR was used to detect αSMA and vWF in grafted veins. (A) Value of αSMA in α-CA, RPM, and α-CA-RPM groups was much lower than in the control group, as detected by RT-PCR. Value of αSMA in α-CA-RPM group was lower than that of α-CA group and RPM group (p < 0.01). (B) Similar results were found in the value of vWF in α-CA-RPM group (p < 0.01). (C) The serum levels of ET-1 are shown for each group at different times. The level of ET-1 in the control group was significantly higher than that in the α-CA, RPM and α-CA-RPM groups 36 hours and 4 weeks after operation (96.1 ± 7.9 ng/l vs. 84.0 ± 10.9 ng/l, 79.5 ± 5.7 ng/l and 72.7 ± 9.9 ng/l; 99.7 ± 7.7 ng/l vs. 87.1 ± 13.3 ng/l, 65.4 ± 23.4 ng/l and 43.7 ± 20.1 ng/l; p < 0.05, respectively). Additionally, at 4 weeks after surgery, the level of ET-1 of the α-CA-RPM group was significantly lower than that of the α-CA, RPM, and control groups, (43.7 ± 20.1 ng/l vs. 87.1 ± 13.3 ng/l, 65.4 ± 23.4 ng/l and 99.5 ± 7.7 ng/l; p < 0.05, respectively). * The control group had obvious difference with other groups, p < 0.01. ^# The α-cyanoacrylate group had obvious difference with sham group, control group and α-CA-RPM group, p < 0.01. ^△ The rapamycin group had obvious difference with sham group, control group and α-CA-RPM groups, p < 0.01. ^☆ The α-CA-RPM group had obvious difference with other groups, p < 0.01.