Using VEGF expression in inflammatory arthritis to induce targeted apoptosis

描述(申请人提供)：类风湿性关节炎(RA)涉及慢性炎症和滑膜增殖，部分原因是致病效应细胞凋亡缺陷。不适当的低凋亡率可能是类风湿性关节炎滑膜细胞持续增殖的原因。在炎症性关节炎的动物模型中，Fas受体的激动剂激活可以改善疾病，然而，一个挑战是仅在致病细胞中特异性地诱导Fas介导的细胞凋亡。RA患者滑膜细胞产生高水平的血管内皮生长因子(VEGF)，而正常细胞则没有。我们的应用将利用炎症滑膜分泌的高局部浓度的血管内皮生长因子直接靶向Fas激动剂，诱导这些部位的细胞凋亡。我们开发了一种新型的融合蛋白，其Fas配体连接到一个血管内皮生长因子结合结构域(R1FasL)。我们发现，R1FasL可触发细胞凋亡，并且只有在通过血管内皮生长因子聚集的情况下才具有细胞毒性。我们的应用将检测血管内皮生长因子对炎症性关节炎炎症和细胞凋亡的影响，并将新型R1FasL融合蛋白用于治疗炎症性关节炎，以确定我们是否可以选择性地消除产生血管内皮生长因子的区域的致病细胞。我们的具体目标是：1)检测血管内皮生长因子及其受体在小鼠炎性关节炎模型和胶原性关节炎模型中的表达。2)检测有或无Fas配体刺激的炎性关节炎小鼠滑膜细胞、巨噬细胞和破骨细胞经血管内皮生长因子诱导的细胞因子。3)检测血管内皮生长因子对Fas介导的滑膜细胞、巨噬细胞和破骨细胞凋亡的影响。4)用炎性关节炎模型SKG和CIA检测R1FasL的治疗效果。 公共卫生相关性：类风湿性关节炎是一种慢性和衰弱的炎症性疾病，尽管在治疗方面取得了重大进展，但由于现有治疗方法缺乏反应或副作用，患者继续患有活动性疾病。我们的应用是研究血管内皮细胞生长因子在本病发病机制中的作用，并利用滑膜细胞异常产生的血管内皮生长因子选择性地清除这些致病细胞。我们的研究将进一步加深我们对炎症性关节炎发病机制的理解，并探索一种新型的治疗这种疾病的动物模型。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) involves chronic inflammation and proliferation of the synovium, which is in part due to defective apoptosis of pathogenic effector cells. Inappropriately low apoptosis likely contributes to persistent synovial cell proliferation in rheumatoid arthritis. In animal models of inflammatory arthritis, agonist activation of the Fas Receptor improves disease, however a challenge is to specifically induce Fas-mediated apoptosis only in pathogenic cells. RA patients have high levels of Vascular Endothelial Growth Factor (VEGF) produced by synoviocytes, not found with normal cells. Our application will exploit the high local concentrations of VEGF secreted by inflamed synovium to directly target a Fas agonist to induce apoptosis at those sites. We developed a novel fusion protein with Fas ligand linked to a VEGF binding domain (R1FasL). We show that R1FasL triggers apoptosis and is cytotoxic only with clustering by VEGF. Our application will examine VEGF effects on inflammation and apoptosis in inflammatory arthritis and utilize the novel R1FasL fusion protein as a treatment for inflammatory arthritis to determine if we can selectively eliminate pathogenic cells in areas where VEGF is produced. Our specific aims are: 1) Determine VEGF and VEGF receptor expression in the murine inflammatory arthritis models SKG and collagen induced arthritis (CIA). 2) Determine cytokines induced by VEGF with or without Fas ligand stimulation of synoviocytes, macrophages and osteoclasts from mice with inflammatory arthritis. 3) Determine the effect of VEGF on Fas-mediated apoptosis of synoviocytes, macrophages and osteoclasts. 4) Determine the effect of R1FasL treatment using the inflammatory arthritis models SKG and CIA. PUBLIC HEALTH RELEVANCE: Rheumatoid Arthritis is a chronic and debilitating inflammatory disease and despite significant advances in treatment, patients continue with active disease due to a lack of response or side effects with available therapies. Our application investigates the role of VEGF or vascular endothelial growth factor in pathogenesis of this disease and utilizes the abnormal production of VEGF by synovial cells to selectively remove these pathogenic cells. Our studies will further our understanding of disease pathogenesis in inflammatory arthritis and examine a novel type of therapeutic in animal models for this disease.