ANGIOGENIC FACTORS ASSOCIATED WITH UVEAL MELANOMA

葡萄膜黑色素瘤是成人中最常见的原发性眼部肿瘤。 葡萄膜黑色素瘤的年发病率相当于视网膜色素变性的新发病例数。 这种疾病的遗传改变尚不清楚，临床上几乎没有可以观察到的预后指标，这有助于将眼球摘除术作为主要的治疗方法。不幸的是，导致全身转移的事件可能在识别眼部症状时已经发生，并且通常会发生由于肝病和其他并发症导致的死亡。显然，需要改进的方法来早期发现和治疗这种疾病。本申请集中于血管生成生长因子和抑制剂对疾病进展的贡献。 我们推测，这种肿瘤的恶性和转移特性取决于多种血管生成因子，特别是Cyr 61，组织因子和VEGF的同时表达。 此外，这些生长因子和抑制剂的表达之间的平衡决定了可检测转移之前的静止期。具体目标包括：1。使用存档标本和肿瘤组织的新鲜活检标本验证Cyr 61、组织因子和VEGF在原发性眼部黑色素瘤中的表达。 检测方法包括免疫组织化学、北方印迹杂交、RT-PCR和原位杂交。 表达将与血管密度相关。 2.使用动物模型评估Cyr 61、组织因子和VEGF在原发性肿瘤生长及其转移中的重要性。 将具有这三种基因的不同表达水平的葡萄膜黑色素瘤的转染细胞系移植到裸鼠的前房中，并测量原发性眼部肿瘤的生长、肝损伤的程度和两个部位的血管密度。3.使用免疫细胞化学方法和细胞分级分离的组合确定Cyr 61、组织因子和VEGF的细胞定位。 4.通过RT_PCR、免疫印迹和北方印迹杂交鉴定与葡萄膜黑色素瘤相关的其他血管生成生长因子（angiopoietin-1）和抑制剂（angiostatin和endostatin）。

Uveal melanoma is the most prevalent primary ocular tumor found in adults. The annual incidence of uveal melanoma is equivalent to the number of new cases of retinitis pigmentosa. The genetic alterations underlying the disease are unknown, and there are few prognostic indicators that can be observed clinically, contributing to enucleation as a principal method of treatment. Unfortunately, events leading to systemic metastases may have occured by the time the ocular symptoms are recognized, and death due to hepatic disease and other complications usually ensues. Clearly, improved methods are needed for the early detection and treatment of the disease. The current application focuses on the contributions of angiogenic growth factors and inhibitors to the progression of the disease. We hypothesize that the malignant and metastatic properties of such tumors depend on the simultaneous expression of multiple angiogenic factors, specifically Cyr61, Tissue Factor, and VEGF. Further, the balance between the expression of these growth factors and inhibitors determines the quiescent period prior to detectable metastases. Specific Aims include: 1. Verify the expression of Cyr61, Tissue Factor and VEGF in primary ocular melanoma using both archival specimens and fresh biopsies of tumor tissue. Methods of detection will include immunohistochemistry, Northern blot hybridization, RT-PCR, and in situ hybridization. Expression will be correlated with vascular density. 2. Assess the importance of Cyr61, Tissue Factor and VEGF during growth of the primary tumor and its metastases using an animal model. Transfected cell lines of uveal melanoma with varying levels of expression of these three genes will be transplanted into the anterior chambers of nude mice, and the growth of the primary ocular tumors, the extent of hepatic lesions, and the vascular density at both sites will be measured. 3. Determine the cellular localization of Cyr61, Tissue Factor and VEGF using a combination of immunocytochemical methods and cellular fractionation. 4. Identify additional angiogenic growth factors (angiopoietin-1) and inhibitors (angiostatin and endostatin) associated with uveal melanoma by RT_PCR, immunoblotting and Northern blot hybridization.