Okubo Masashi, Kimura Tokuhiro, Fujita Yoshinari, Mochizuki Satsuki, Niki Yasuo, Enomoto Hiroyuki, Suda Yasunori, Toyama Yoshiaki, Okada Yasunori
Department of Pathology, School of Medicine, Keio University, Tokyo, Japan.
Arthritis Rheum. 2011 Oct;63(10):3000-9. doi: 10.1002/art.30482.
Vascular endothelial growth factor 165 (VEGF165) and its receptors, including neuropilin 1 (NRP-1), are overexpressed in human osteoarthritic (OA) articular cartilage, although their functional roles in the cartilage are not fully understood. An axon-guidance molecule, semaphorin 3A (Sema3A), which binds to NRP-1, acts as an antagonist of VEGF signaling in endothelial cells. The aim of this study was to examine the expression of Sema3A and the functions of the VEGF165/Sema3A/NRP-1 axis in OA cartilage.
The expression of Sema3A in OA and normal cartilage samples was examined by real-time polymerase chain reaction and immunohistochemical analyses. Functional analyses of VEGF165 and Sema3A were carried out using OA chondrocytes in culture. The migration activity of chondrocytes was examined in a monolayer wound assay. The effects of Sema3A on VEGF165-induced up-regulation of matrix metalloproteinases (MMPs) and intracellular signaling were also studied in cultured chondrocytes.
Sema3A expression was significantly elevated in OA cartilage as compared to normal cartilage. Sema3A immunoreactivity directly correlated with the Mankin score and with chondrocyte cloning. VEGF165 promoted the migration of chondrocytes, and this activity was suppressed by VEGF receptor 2 tyrosine kinase inhibitors. Sema3A antagonized the chondrocyte migration promoted by VEGF165, and the activity was blocked by a selective inhibitor of, or small interfering RNA for, Sema3A. VEGF165-induced overexpression of MMPs and phosphorylation of ERK and focal adhesion kinase in chondrocytes were inhibited by Sema3A.
Our findings provide the first evidence that Sema3A is overexpressed, with a direct correlation with cloning, in OA cartilage and that it suppresses the VEGF165-promoted migration of chondrocytes. Our findings also suggest that Sema3A plays a role in chondrocyte cloning through inhibition of cell migration in OA cartilage.
血管内皮生长因子165（VEGF165）及其受体，包括神经纤毛蛋白1（NRP - 1），在人类骨关节炎（OA）关节软骨中过度表达，尽管它们在软骨中的功能作用尚未完全明确。一种轴突导向分子，即与NRP - 1结合的信号素3A（Sema3A），在内皮细胞中作为VEGF信号的拮抗剂发挥作用。本研究的目的是检测Sema3A在OA软骨中的表达以及VEGF165/Sema3A/NRP - 1轴的功能。
通过实时聚合酶链反应和免疫组织化学分析检测OA和正常软骨样本中Sema3A的表达。使用培养的OA软骨细胞对VEGF165和Sema3A进行功能分析。在单层伤口试验中检测软骨细胞的迁移活性。还在培养的软骨细胞中研究了Sema3A对VEGF165诱导的基质金属蛋白酶（MMPs）上调和细胞内信号传导的影响。
与正常软骨相比，OA软骨中Sema3A表达显著升高。Sema3A免疫反应性与Mankin评分及软骨细胞克隆直接相关。VEGF165促进软骨细胞迁移，且该活性被VEGF受体2酪氨酸激酶抑制剂抑制。Sema3A拮抗VEGF165促进的软骨细胞迁移，且该活性被Sema3A的选择性抑制剂或小干扰RNA阻断。Sema3A抑制了VEGF165诱导的软骨细胞中MMPs的过度表达以及ERK和粘着斑激酶的磷酸化。
我们的研究结果首次证明，Sema3A在OA软骨中过度表达，且与克隆直接相关，并且它抑制VEGF165促进的软骨细胞迁移。我们的研究结果还表明，Sema3A通过抑制OA软骨中的细胞迁移在软骨细胞克隆中发挥作用。
