MACROPHAGE-DERIVED ANGIOGENIC ACTIVITY

我们最近证明了巨噬细胞衍生的血管生成 活动(MDAA)相同或密切相关 肿瘤坏死因子-α(TNF-α)的免疫学。 重组(R)肿瘤坏死因子-α在大鼠体内的血管生成作用 角膜和鸡绒毛膜尿囊膜，是一种化学诱导剂 对于体外培养的牛毛细血管内皮细胞(BCE)，以及 诱导在胶原胶上培养的BCE融合单层 侵入凝胶并形成毛细血管样小管结构。MDAA 巨噬细胞培养的条件培养液中和 兔抗鼠肿瘤坏死因子-α多克隆抗体。我们还有 研究表明，转化生长因子-β(TGF-β)是一种产品 正常(血小板和活化的淋巴细胞)以及 转化细胞，是一种对人单核细胞有效的化学诱导剂 在体外，最大的活动在股骨膜。在皮摩尔 浓度，转化生长因子-β诱导血管生成活性的表达 通过单核细胞。此应用程序中的项目旨在 研究：(A)与启动有关的事件 血管生成活性的单核/巨噬细胞表达(MDAA/TNF- Alpha)。转化生长因子-β和氧/乳酸浓度将 学习。将使用从Genentech，Inc.获得的C-DNA克隆 探讨肿瘤坏死因子-α、转化生长因子-α和转化生长因子-β的表达水平。 单核/巨噬细胞。表达的蛋白质水平也将是 下定决心。(B)与以下有关的肿瘤坏死因子-α结构特征 它的血管生成活性。五株抗肿瘤坏死因子-α的单抗， 两个中和细胞毒活性的，三个不起作用的，将成为 比较其抗血管生成活性。特定的蛋白水解物和 将进行肿瘤坏死因子-α的化学切割，并 经血管生成和细胞毒活性测试的多肽。合成的 活性序列的多肽类似物最终将在 与潜在的抑制物有关。(C)的行动机制 肿瘤坏死因子-α作为血管生成剂，通过研究其对血管生成的影响 培养中的毛细血管内皮细胞。内皮细胞受体 对于肿瘤坏死因子-α，将使用125I标记的肿瘤坏死因子-α进行研究。这个 肿瘤坏死因子-α对碱性成纤维细胞生长因子基因表达的影响 将对级别进行检查。金属蛋白酶的分泌，如 胶原酶和TIMP(金属蛋白酶组织抑制物)将 被研究。(D)肿瘤坏死因子-α在体内的表达 对伤口、炎症和肿瘤进行原位检查 杂交。要么是标记的肿瘤坏死因子寡核苷酸探针- 将使用Alpha或RNA探针(“RiboProbe”)。(E) 将使用以下方法研究肿瘤坏死因子-α对体内伤口修复的影响 植入的聚乙烯(Lvalon)海绵和植入的Gortex 微渗透管材模型。

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.