Division of Vascular and Endovascular Surgery, Department of Surgery, Stanford University, Stanford, CA 94304, USA.
J Vasc Surg. 2011 Apr;53(4):1044-51. doi: 10.1016/j.jvs.2010.10.117. Epub 2011 Jan 31.
Emerging evidence showed that resistin induces vascular smooth muscle cell (VSMC) migration, a critical step in initiating vascular restenosis. Adhesion molecule expression and cytoskeletal rearrangement have been observed in this progress. Given that matrix metalloproteinases (MMPs) also regulate cell migration, we hypothesized that MMPs may mediate resistin-induced VSMC migration.
Human VSMCs were treated with recombinant human resistin at physiologic (10 ng/mL) and pathologic (40 ng/mL) concentrations for 24 hours. Cell migration was determined by the Boyden chamber assay. MMP and tissue inhibitor metalloproteinase (TIMP) mRNA and protein levels were measured with real-time PCR and ELISA. MMP enzymatic activity was measured by zymography. In another experiment, neutralizing antibodies against MMP-2 and MMP-9 were coincubated with resistin in cultured VSMCs. The regulation of MMP by protein kinase C (PKC) was determined by εV1-2, a selective PKCε inhibitor.
Resistin-induced smooth muscle cell (SMC) migration was confirmed by the Boyden chamber assay. Forty nanograms/milliliter resistin increased SMC migration by 3.7 fold. Additionally, resistin stimulated MMP-2 and -MMP9 mRNA and protein expressions. In contrast, the TIMP-1 and TIMP-2 mRNA levels were inhibited by resistin. Neutralizing antibodies against MMP-2 and MMP-9 effectively reversed VSMC migration. Furthermore, resistin activated PKCε, but selective PKCε inhibitor suppressed resistin-induced MMP expression, activity, and cell migration.
Our study confirmed that resistin increased vascular smooth muscle cell migration in vitro. In terms of mechanism, resistin-stimulated cell migration was associated with increased MMP expression, which was dependent on PKCε activation.
新出现的证据表明，抵抗素可诱导血管平滑肌细胞（VSMC）迁移，这是引发血管再狭窄的关键步骤。在这个过程中观察到黏附分子的表达和细胞骨架的重排。鉴于基质金属蛋白酶（MMPs）也可调节细胞迁移，我们假设 MMPs 可能介导抵抗素诱导的 VSMC 迁移。
用生理浓度（10ng/ml）和病理浓度（40ng/ml）的重组人抵抗素处理人 VSMC 24 小时。通过 Boyden 室测定细胞迁移。用实时 PCR 和 ELISA 测定 MMP 和组织抑制剂金属蛋白酶（TIMP）的 mRNA 和蛋白水平。用明胶酶谱法测定 MMP 酶活性。在另一个实验中，在培养的 VSMC 中用中和 MMP-2 和 MMP-9 的抗体与抵抗素共同孵育。用选择性 PKCε抑制剂 εV1-2 测定 PKC 对 MMP 的调节。
Boyden 室测定证实抵抗素诱导平滑肌细胞（SMC）迁移。40ng/ml 抵抗素使 SMC 迁移增加 3.7 倍。此外，抵抗素刺激 MMP-2 和 MMP-9 的 mRNA 和蛋白表达。相反，抵抗素抑制 TIMP-1 和 TIMP-2 的 mRNA 水平。中和 MMP-2 和 MMP-9 的抗体有效逆转 VSMC 迁移。此外，抵抗素激活 PKCε，但选择性 PKCε 抑制剂抑制抵抗素诱导的 MMP 表达、活性和细胞迁移。
我们的研究证实，抵抗素在体外增加血管平滑肌细胞迁移。就机制而言，抵抗素刺激的细胞迁移与 MMP 表达增加有关，而 MMP 表达增加依赖于 PKCε 的激活。
