Simvastatin attenuated rat thoracic aorta remodeling by decreasing ROCK2‑mediated CyPA secretion and CD147‑ERK1/2‑cyclin pathway

Abstract

Reactive oxygen species‑induced cyclophilin A (CyPA) release from vascular smooth muscle cells (VSMCs) may be inhibited by simvastatin in vitro. The present study aimed to further examine the effect of simvastatin on serum CyPA levels and the basigin (CD147)‑extracellular signal‑regulated kinase (ERK) 1/2‑cyclin pathway during thoracic aorta remodeling. The mechanisms through which simvastatin may inhibit CyPA secretion from VSMCs were further investigated. Serum CyPA levels and the expression kinetics of CyPA‑associated signaling pathways were examined following simvastatin treatment in rat thoracic aortas during hypertension. Cell lysates were prepared from middle layer of thoracic aortas at 1, 4, 8 and 12 weeks subsequent to surgery. ELISA analysis revealed that serum CyPA levels were gradually increased with the progression of thoracic aorta remodeling. Western blotting demonstrated that the expression of CD147, phosphorylated‑ERK1/2, cyclin D1, cyclin A, and cyclin E were increased with the progression of thoracic aorta remodeling. Simvastatin administration for 4, 8 and 12 weeks diminished all these changes, as observed in the hypertensive group. VSMCs from simvastatin‑treated rats secreted a decreased amount of CyPA compared with VSMCs from hypertensive rats. In addition, pretreatment with geranylgeraniol partly reversed the inhibitory effect of simvastatin on LY83583‑induced CyPA secretion in cultured VSMCs, whereas GGTI‑298 and KD025 [a selective Rho‑associated protein kinase 2 (ROCK2) inhibitor] mimicked the inhibitory effect of simvastatin. The present study demonstrated that simvastatin alleviated thoracic aorta remodeling by reducing CyPA secretion and expression of the CD147‑ERK1/2‑cyclin signaling pathway. In addition, the results of the present study demonstrated that the Rho‑ROCK2 pathway mediated CyPA secretion from VSMCs.

Figure 1.

Simvastatin improves the remodeling of the rat thoracic aorta. (A) Representative images of cross sections of hematoxylin and eosin stained rat thoracic aortas. Scale bar, 500 µm (whole aorta ring; original magnification, ×40) and 50 µm (aorta section; original magnification, ×400). The bar graphs indicate the results of the analysis of the following parameters: (B) Media thickness; (C) media CSA; (D) systolic BP. Data are expressed as the mean ± standard deviation from 8 rats/group. *P<0.05, **P<0.01 vs. sham; ^#P<0.05, ^##P<0.01 vs. Htn. Htn, hypertension; Sim, simvastatin; CSA, cross-sectional area; BP, blood pressure.

Figure 2.

Rat serum CyPA and CyPA secretion in primary VSMCs from thoracic aortas is markedly decreased by treatment with simvastatin. (A) Serum CyPA levels gradually increased during the development of thoracic aorta remodeling, as assayed using a rat CyPA ELISA kit. Treatment with simvastatin for 4, 8 and 12 weeks reduced the serum CyPA concentration. (B) LY83583-induced CyPA secretion from primary VSMCs of simvastatin-treated rats was decreased compared with that from VSMCs of Htn rats at 12 weeks. Primary VSMCs were isolated from sham, Htn and simvastatin-treated rats at 12 weeks and cultured in vitro. Primary VSMCs were exposed to 1 µmol/l LY83583 for 2 h. The CyPA level in the CM and TCL were detected by western blotting. Data are expressed as mean ± standard deviation from 3 independent experiments. *P<0.05, **P<0.01 vs. sham; ^#P<0.05, ^##P<0.01 vs. Htn. Htn, hypertension; Sim, simvastatin; CM, conditioned medium; TCL, total cell lysate; CyPA, cyclophilin A.

Figure 3.

Upregulation of the CD147-ERK1/2-cyclin pathway with the extent of thoracic aorta remodeling is attenuated by treatment with simvastatin. Representative bands are from at least three independent experiments. CD147, basigin; ERK, extracellular signal-regulated kinase; p-, phosphorylated; Htn, hypertension; Sim, simvastatin.

Figure 4.

The Rho-ROCK2 pathway mediates simvastatin-inhibited CyPA secretion. (A) Growth-arrested VSMCs were pretreated with GGOL for 30 min, and simvastatin was added for 30 min prior to exposure to 1 µmol/l LY83583 for 2 h. (B) Growth-arrested VSMCs were pretreated with KD025 and GGTI-298 for 30 min prior to exposure to 1 µmol/l LY83583 for 2 h. CyPA in the CM and TCL were detected by western blotting. All the experiments were repeated at least three times. GGOL, geranylgeraniol; Sim, simvastatin; CM, conditioned medium; TCL, total cell lysate; CyPA, cyclophilin A; GGTI, geranylgeranyltransferase inhibitor; ROCK2, Rho-associated protein kinase 2; VSMCs, vascular smooth muscle cells.