Suzuki Jun, Jin Zheng-Gen, Meoli David F, Matoba Tetsuya, Berk Bradford C
Cardiovascular Research Institute, Department of Medicine, University of Rochester, NY 14642, USA.
Circ Res. 2006 Mar 31;98(6):811-7. doi: 10.1161/01.RES.0000216405.85080.a6. Epub 2006 Mar 9.
Reactive oxygen species (ROS) contribute to the pathogenesis of atherosclerosis in part by promoting vascular smooth muscle cell (VSMC) growth. Previously we demonstrated that cyclophilin A (CyPA) is a secreted oxidative stress-induced factor (SOXF) that promotes inflammation, VSMC growth, and endothelial cell apoptosis. However, the mechanisms that regulate CyPA secretion are unknown. In this study, we hypothesized that ROS-induced CyPA secretion from VSMC requires a highly regulated process of vesicle transport, docking, and fusion at the plasma membrane. Conditioned medium and plasma membrane sheets were prepared by exposing VSMC to 1 micromol/L LY83583, which generates intracellular superoxide. A vesicular transport mechanism was confirmed by colocalization at the plasma membrane with vesicle-associated membrane protein (VAMP). CyPA transport to the plasma membrane and secretion were significantly increased by LY83583. Reduction of VAMP-2 expression by small interfering RNA inhibited LY83583-induced CyPA secretion. Pretreatment with 3 micromol/L cytochalasin D, an actin depolymerizing agent, abrogated CyPA secretion. Infection with dominant-negative RhoA and Cdc42 adenovirus inhibited CyPA secretion by 72% and 63%, respectively, whereas dominant-negative Rac1 had a small effect (11%). Pretreatment with the Rho kinase inhibitor Y27632 (3 to 30 micromol/L) and myosin II inhibitor blebbistatin (1 to 10 micromol/L) inhibited CyPA secretion in a dose-dependent manner. Simvastatin (3 to 30 micromol/L) also dose-dependently inhibited LY83583-induced CyPA secretion likely via decreased isoprenylation of small GTPases. Our findings define a novel VSMC vesicular secretory pathway for CyPA that involves actin remodeling and myosin II activation via RhoA-, Cdc42-, and Rho kinase-dependent signaling events.
活性氧（ROS）部分通过促进血管平滑肌细胞（VSMC）生长而参与动脉粥样硬化的发病机制。此前我们证明亲环素A（CyPA）是一种分泌性氧化应激诱导因子（SOXF），可促进炎症、VSMC生长和内皮细胞凋亡。然而，调节CyPA分泌的机制尚不清楚。在本研究中，我们假设ROS诱导VSMC分泌CyPA需要在质膜上进行高度调控的囊泡运输、对接和融合过程。通过将VSMC暴露于1 μmol/L LY83583（可产生细胞内超氧化物）来制备条件培养基和质膜片。通过与囊泡相关膜蛋白（VAMP）在质膜上共定位证实了一种囊泡运输机制。LY83583显著增加了CyPA向质膜的运输和分泌。小干扰RNA降低VAMP - 2表达可抑制LY83583诱导的CyPA分泌。用3 μmol/L细胞松弛素D（一种肌动蛋白解聚剂）预处理可消除CyPA分泌。用显性负性RhoA和Cdc42腺病毒感染分别抑制CyPA分泌72%和63%，而显性负性Rac1的作用较小（11%）。用Rho激酶抑制剂Y27632（3至30 μmol/L）和肌球蛋白II抑制剂blebbistatin（1至10 μmol/L）预处理以剂量依赖方式抑制CyPA分泌。辛伐他汀（3至30 μmol/L）也可能通过降低小GTP酶的异戊二烯化而剂量依赖性地抑制LY83583诱导的CyPA分泌。我们的研究结果确定了一种新的CyPA的VSMC囊泡分泌途径，该途径涉及通过RhoA、Cdc42和Rho激酶依赖性信号事件进行的肌动蛋白重塑和肌球蛋白II激活。
