ENDOTHELIAL CELL REACTIVITY IN SICKLE CELL VASOOCCLUSION

镰状细胞血管闭塞中的内皮细胞反应性

• 批准号: 6272592
• 负责人: Stephen H. Embury
• 金额: $ 23.56万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-24 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6272592
• 关键词: blood vessel occlusion; cell adhesion; cell adhesion molecules; genetic library; hemoglobin Ss; laboratory rabbit; medical complication; monoclonal antibody; receptor binding; receptor expression; sickle cell anemia; tissue /cell culture; vascular endothelium

Painful vascular occlusion is one of the cardinal features of sickle cell disease. It, more than any other complication, leads patients to seek medical attention. Several processes participate in vasoocclusion, including increased adherence of sickle RBC to vascular endothelial cells. Specific cytoadhesion receptors, ligands, and heterocellular mechanisms involved in adherence have been and are continuing to be discovered. We propose that endothelial cell receptors and changes in their expression that occur with cell activation are important variables in vasoocclusion. The dramatic changes in endothelial cell function effected by known antagonists include alterations in adhesivity, a shift in the repertoire displayed on cell surfaces, changes in receptor activity, cell contraction, and interendothelial cell gap formation. A wide variety of agonists that alter endothelial cells are germane to the pathophysiology of sickle cell vasoocclusion. We propose to use cultured endothelial cells to study agonists that induce sickle RBC adherence and the endothelial cell receptors that mediate enhanced adherence. The agonists we will study include thrombin, histamine, tumor necrosis factor-alpha, interleukin- 1beta, and ischemia/reperfusion. The blocking agents to be used to assess the receptors on cultured endothelial cells involved in baseline and induced adherence include a blocking monoclonal antibody against GP Ib, RGD-containing oligopeptides, and blocking monoclonal antibodies against integrin and integrin subunits. In addition we will investigate the role of yet unidentified adhesive proteins using immunologic methods. We will prepare rabbit monoclonal antibodies against activated endothelial cells and, as an alternative, use phage display technology to determine molecular changes in endothelial cells that result from activation. Affinity of monoclonal antibodies or, alternatively, recombinant bacteriophage expressing antibody fragments specific for surface antigens on activated endothelial cells will be used to identify molecules that have been induced by agonists and to assess their role in induced adherence. The bacteriophage library containing cloned human immunoglobulin variable region cDNA expresses 7 x 10 9 antibody specificities. We will also study the cell mechanisms responsible for increased activity of receptors on endothelial cells--quantitatively increased expression, changes in cell surface distribution, cell contraction exposing previously occupied receptors, and increased activation of receptors. This work complements that proposed by Drs. Sam Test and Frans Kuypers, with whom we propose collaborative studies. In the later phases of this work we will use New Zealand White rabbits to produce monoclonal antibodies. Our experiments are anticipated to enhance our knowledge of the molecular mechanisms responsible for sickle RBC- endothelial cell adherence, improve our grasp of the pathophysiology of sickle cell vasoocclusion, and expand our knowledge of endothelial cell biology.

痛苦的血管闭塞是镰状细胞的基本特征之一 疾病。它比任何其他并发症都更能引导患者寻求 医疗救治。几个过程参与了血管闭塞， 包括镰状红细胞与血管内皮细胞的粘附性增加。 特异性细胞黏附受体、配体和异源细胞机制 已经并正在继续被发现与坚持有关。我们 血管内皮细胞受体及其表达的变化 是血管闭塞的重要变量。 已知的影响内皮细胞功能的戏剧性变化 拮抗剂包括粘附性的改变，曲目的改变 显示在细胞表面，受体活性的变化，细胞 收缩，内皮细胞间隙形成。种类繁多的 改变内皮细胞的激动剂与病理生理学密切相关 镰状细胞血管闭塞。我们建议使用培养的内皮细胞 研究诱导镰状红细胞黏附和内皮细胞黏附的激动剂 介导增强粘附性的细胞受体。我们将研究的激动剂 包括凝血酶、组胺、肿瘤坏死因子-α、白介素2 1β，缺血/再灌流。用于评估的封闭剂 培养的内皮细胞上的受体参与了基线和 诱导的黏附包括封闭的抗GP Ib的单抗， 含RGD的寡肽和封闭的抗RGD的单抗 整合素和整合素亚基。此外，我们还将调查 使用免疫学方法对尚未鉴定的黏附蛋白进行鉴定。我们会 兔抗活化内皮细胞单抗的制备 作为替代方案，使用噬菌体展示技术来确定 由激活引起的内皮细胞的分子变化。 单抗或重组抗体的亲和力 表达表面抗原特异性抗体片段的噬菌体 在激活的内皮细胞上将被用来识别 都是由激动剂诱导的，并评估它们在诱导 坚持不懈。克隆人噬菌体文库的构建 免疫球蛋白可变区基因表达7×109抗体 具体细节。我们还将研究负责的细胞机制 内皮细胞上受体活性的增加--定量 表达增加，细胞表面分布改变，细胞 收缩暴露先前占据的受体，并增加 受体的激活。这项工作是对萨姆博士提出的工作的补充 Test和Frans Kuypers，我们建议与他们合作研究。在 这项工作的后续阶段我们将使用新西兰白兔来生产 单克隆抗体。我们的实验有望增强我们的 了解导致镰状红细胞的分子机制- 内皮细胞黏附，提高我们对血管内皮细胞黏附的病理生理的掌握 镰状细胞血管闭塞，扩大我们对内皮细胞的认识 生物学。