Sidibé Adama, Mannic Tiphaine, Arboleas Mélanie, Subileau Mariela, Gulino-Debrac Danielle, Bouillet Laurence, Jan Mary, Vandhuick Thibault, Le Loët Xavier, Vittecoq Olivier, Vilgrain Isabelle
INSERM Unité 1036, Joseph Fourier University-Grenoble 1, and CEA Grenoble, Grenoble, France.
Arthritis Rheum. 2012 Jan;64(1):77-87. doi: 10.1002/art.33336.
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that principally attacks synovial joints. However, accelerated atherosclerosis and increased cardiovascular morbidity and mortality are major clinical consequences of endothelial dysfunction in RA patients. Tumor necrosis factor α (TNFα) is the major mediator of inflammation in RA, related to vascular injury by targeting VE-cadherin, an endothelium-specific adhesion molecule of vital importance for endothelium integrity and angiogenesis. We undertook this study to examine the mechanisms regulating VE-cadherin processing by TNFα and their occurrence in RA.
Human umbilical vein endothelial cells were used in primary culture and treated with recombinant TNFα to study VE-cadherin cleavage. Cell lysates and conditioned media were analyzed by Western blotting for VE-cadherin cytoplasmic domain and extracellular domain (VE-90) generation, respectively. VE-90 was analyzed at baseline and at the 1-year followup in sera from 63 RA patients (from the Very Early Rheumatoid Arthritis cohort) with disease duration of <6 months.
TNFα induced a time-dependent shedding of VE-90 in cell media. This effect was prevented by tyrosine kinase inhibitors (genistein and PP2) or by knocking down Src kinase. In contrast, tyrosine phosphatase blockade enhanced VE-cadherin cleavage, confirming the requirement of tyrosine phosphorylation processes. In addition, using the matrix metalloproteinase (MMP) activator APMA and the MMP inhibitor GM6001, we demonstrated that MMPs are involved in TNFα-induced VE-cadherin cleavage. Of major importance, VE-90 was detected in sera from the 63 RA patients and was positively correlated with the Disease Activity Score at baseline and after 1-year followup.
These findings provide the first evidence of VE-cadherin proteolysis upon TNFα stimulation and suggest potential clinical relevance of soluble VE-cadherin in management of RA.
类风湿关节炎（RA）是一种主要侵袭滑膜关节的慢性全身性炎症性疾病。然而，动脉粥样硬化加速以及心血管发病率和死亡率增加是RA患者内皮功能障碍的主要临床后果。肿瘤坏死因子α（TNFα）是RA炎症的主要介质，通过靶向血管内皮钙黏蛋白（VE-cadherin）导致血管损伤，VE-cadherin是一种对内皮完整性和血管生成至关重要的内皮特异性黏附分子。我们开展这项研究以探讨TNFα调节VE-cadherin加工的机制及其在RA中的发生情况。
采用人脐静脉内皮细胞进行原代培养，并用重组TNFα处理以研究VE-cadherin的裂解。分别通过蛋白质印迹法分析细胞裂解物和条件培养基中VE-cadherin胞质结构域和细胞外结构域（VE-90）的生成情况。对63例疾病病程<6个月的RA患者（来自极早期类风湿关节炎队列）血清中的VE-90在基线和1年随访时进行分析。
TNFα诱导细胞培养基中VE-90呈时间依赖性脱落。酪氨酸激酶抑制剂（染料木黄酮和PP2）或敲低Src激酶可阻止这种效应。相反，酪氨酸磷酸酶阻断增强了VE-cadherin的裂解，证实了酪氨酸磷酸化过程的必要性。此外，使用基质金属蛋白酶（MMP）激活剂APMA和MMP抑制剂GM6001，我们证明MMPs参与TNFα诱导的VE-cadherin裂解。最重要的是，在63例RA患者的血清中检测到VE-90，并且在基线和1年随访后与疾病活动评分呈正相关。
这些发现首次证明了TNFα刺激后VE-cadherin的蛋白水解，并提示可溶性VE-cadherin在RA管理中的潜在临床相关性。
