MACROPHAGE-DERIVED ANGIOGENIC ACTIVITY

我们最近已经证明，巨噬细胞源性血管生成 活动（MDAA）是相同的或密切相关的 肿瘤坏死因子-α（TNF-α）。 重组（r）TNF-α在大鼠体内具有强效血管生成作用 角膜和鸡绒毛尿囊膜，是一种化学引诱物 体外牛毛细血管内皮细胞（BCE），和 诱导在胶原凝胶上培养的BCE的汇合单层， 侵入凝胶并从毛细管状管状结构侵入。 MDAA 在巨噬细胞培养物的条件培养基中， 兔抗鼠TNF-α多克隆抗体。 我们还 显示转化生长因子-β（TGF-β），一种 正常（血小板和活化淋巴细胞）以及 转化的细胞，是一种有效的化学引诱人单核细胞 在体外，最大活性为飞摩尔。 皮摩尔 浓度，TGF-β诱导血管生成活性的表达 单核细胞。 此应用程序中的项目旨在 研究：（a）涉及激活的事件 单核细胞/巨噬细胞表达血管生成活性（MDAA/TNF-α） α）。 TGF-β和氧/乳酸盐浓度将在 研究了 C-DNA克隆获自Genentech，Inc.将使用 为了探测TNF-α、TGF-α和TGF-β mRNA水平， 单核细胞/巨噬细胞。 表达的蛋白质水平也将是 测定 (b)TNF-α的结构特征与 它的血管生成活性 五种TNF-α的单克隆抗体， 两种中和细胞毒活性，三种不中和，将B 比较抗血管生成活性。 特异性蛋白水解和 将进行TNF-α的化学裂解， 测试肽的血管生成和细胞毒性活性。 合成 活性序列的肽类似物最终将在 与潜在的抑制剂有关。 (c)的作用机制 TNF-α作为血管生成剂，通过研究其对 培养的毛细血管内皮细胞。 内皮细胞受体 将使用125 I标记的TNF-α进行研究。 的 TNF-α对碱性成纤维细胞生长因子mRNA表达的影响 水平将被检查。 分泌金属蛋白酶，如 胶原酶和TIMP（金属蛋白酶组织抑制剂）将 被研究。 (d)TNF-α在体内的表达将是 在伤口、炎症和肿瘤中进行原位检查， 杂交方法 用于TNF-α的标记寡核苷酸探针 α或RNA探针（“Riboprobe”）。 (e)的 TNF-α对体内伤口修复的影响将被研究，使用 植入的聚乙烯（Ivalon）海绵和植入的Gortex 微渗透管模型。

We have demonstrated recently that Macrophage-derived angiogenic activity (MDAA) is either identical or closely related immunologically to Tumor Necrosis Factor-alpha (TNF-alpha). Recombinant (r) TNF-alpha is potently angiogenic in vivo in the rat cornea and chick chorioallantoic membrane, is a chemoattractant for bovine capillary endothelial cells (BCE's) in vitro, and induces confluent monolayers of BCE's cultured on collagen gels to invade the gels and from capillary-like tubular structures. MDAA in conditioned media of macrophage cultures is neutralized by a rabbit anti-murine TNF-alpha polyclonal antibody. We have also shown that Transforming Growth Factor-beta (TGF-beta), a product of both normal (platelets and activated lymphocytes) as well as transformed cells, is a potent chemoattractant for human monocytes in vitro, with maximal activity in the femtomolar. At picomolar concentrations, TGF-beta induces expression of angiogenic activity by monocytes. The projects in this application are designed to study: (a) Events involved in the activation of monocyte/macrophage expression of angiogenic activity (MDAA/TNF- alpha). TGF-beta and oxygen/lactate concentrations will be studied. C-DNA clones obtained from Genentech, Inc. will be used to probe for TNF-alpha, TGF-alpha and TGF-beta mRNA levels in monocytes/macrophages. Levels of expressed protein will also be determined. (b) Structural features of TNF-alpha that relate to its angiogenic activity. Five monoclonal antibodies to TNF-alpha, two that neutralize cytotoxic activity, three that do not, will b compared for anti-angiogenic activity. Specific proteolytic and chemical cleavage of TNF-alpha will be carried out, and the peptides tested for angiogenic and cytotoxic activity. Synthetic peptide analogs of active sequences will ultimately be examined in relation to potential inhibitors. (c) The mechanism of action of TNF-alpha as an angiogenic agent, by studying its effects on capillary endothelial cells in culture. Endothelial cell receptors for TNF-alpha will be studied using 125I-labelled TNF-alpha. The effects of TNF-alpha on basic Fibroblast Growth Factor (bFGF) mRNA levels will be examined. Secretion of metalloproteinases, such as collagenase, and TIMP (Tissue Inhibitor of Metalloproteinases) will be studied. (d) The expression of TNF-alpha in vivo will be examined in wounds, inflammation and tumors, by in situ hybridization. Either a labelled oligonucleotide probe for TNF- alpha, or an RNA probe ("Riboprobe"), will be used. (e) The effects of TNF-alpha on wound repair in vivo will be studied, using the implanted polyvinyl (lvalon) sponge and the implanted Gortex micropermeable tubing models.