To investigate the effect and molecular mechanism of rapamycin (RAP) on inhibiting the proliferation of rat vascular smooth muscle cells (VSMCs) with abnormal proliferation activity.

Rat aortic VSMCs were treated with different concentrations of platelet-derived growth factor (PDGF) as an inducer to construct an in vitro model of vascular malformation. They were divided into Blank group, PDGF group, PDGF+RAP group and PDGF+VECs (vascular endothelial cells) group. The changes in the proliferation activity of VSMCs were observed and the effects of different treatments on the autophagy level of VSMCs were detected. The independent sample t test was used for comparison between two groups and the repeated measures analysis of variance was used for comparison among multiple groups. The mRNA and protein expression among multiple groups were compared via One-Way analysis of variance.

The results of CCK-8 and EdU experiments showed that the activity of VSMCs treated with PDGF significantly increased in a time-dependent manner. Compared with the PDGF group, at 48 and 72 hours, the cell proliferation activity of the PDGF+RAP group was reduced by 24.8% (0.129 ± 0.010 vs. 0.172 ± 0.012, t = 4.787, P = 0.009) and 45.1% (0.170 ± 0.012 vs. 0.292 ± 0.046, t = 4.431, P = 0.011) , respectively. The cell proliferation activity of PDGF+VECs group decreased by 10.0% (0.155 ± 0.013 vs. 0.172 ± 0.012, t = 2.357, P = 0.076) and 8.9% (0.266 ± 0.022 vs. 0.292 ± 0.046, t = 1.718, P = 0.161) , respectively. Real-time quantitative PCR and Western blot results showed that, compared with the Blank group, PDGF could inhibit the expression of Smoothelin-B (0.486 ± 0.057 vs. 1.005 ± 0.067, t = 10.192, P = 0.001) and promote the expression of cellular retinol binding protein-1 (CRBP-1) (3.185 ± 0.091 vs. 0.991 ± 0.056, t = 35.461, P<0.001) . In the PDGF+RAP group and PDGF+VECs group, the expression of Smoothelin-B increased (0.857 ± 0.091 vs. 0.486 ± 0.057, t = 5.943, P = 0.004; 0.563 ± 0.067 vs. 0.486 ± 0.057, t = 1.501, P = 0.208) and the expression of CRBP-1 decreased (1.579 ± 0.042 vs. 3.185 ± 0.091, t = 27.707, P<0.001; 2.951 ± 0.144 vs 3.185 ± 0.091, t = 2.382, P = 0.076) . Compared with the Blank group, the expression of LC3B (LC3-Ⅱ/Ⅰ) in PDGF group significantly decreased (0.175 ± 0.003 vs. 1.020 ± 0.020, t = 72.263, P<0.001) , and the expression of p62 significantly increased (2.632 ± 0.098 vs. 1.005 ± 0.007, t = 28.562, P = 0.001) . Compared with PDGF group, the expression of LC3B in PDGF+RAP group significantly increased (0.316 ± 0.037 vs. 0.175 ± 0.003, t = 6.529, P = 0.022) , and the relative expression of p62 significantly decreased (1.396 ± 0.070 vs. 2.632 ± 0.098, t = 17.771, P<0.001) . The expression of LC3B in the PDGF+VECs group slightly increased (0.206 ± 0.014 vs. 0.175 ± 0.003, t = 3.687, P = 0.021) , while the expression of p62 slightly reduced (2.400 ± 0.076 vs. 2.632 ± 0.098, t = 3.220, P = 0.032) . Transmission electron microscope observation showed that autophagosomes in PDGF+RAP group and PDGF+VECs group were higher than those in PDGF group.

High concentration of PDGF can increase the proliferation activity of VSMCs. RAP or VECs can reduce the cell activity of VSMCs by activating autophagy and inhibiting the PI3K/AKT signaling pathway. Moreover, RAP or VECs can also promote the transformation of VSMCs to contractile phenotype as well as reverse the effect of PDGF.