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: 6109522
• 负责人: Stephen H. Embury
• 金额: $ 23.29万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109522
• 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.

疼痛的血管阻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多，导致患者寻求 医疗护理。几个过程参与了血管牙合， 包括镰状RBC对血管内皮细胞的依从性增加。 特定的细胞粘附受体，配体和杂细胞机制 参与依从性已经并且正在继续被发现。我们 提出内皮细胞受体及其表达的变化 细胞激活发生的是血管牙合中的重要变量。 由已知的内皮细胞功能的急剧变化 拮抗剂包括粘附性的改变，曲目的变化 显示在细胞表面上，受体活性的变化，细胞 收缩和间皮细胞间隙形成。各种各样的 改变内皮细胞的激动剂是病理生理学 镰状细胞血管牙合。我们建议使用培养的内皮细胞 研究引起镰刀rbc依从性和内皮的激动剂 介导增强依从性的细胞受体。我们将学习的激动剂 包括凝血酶，组胺，肿瘤坏死因子-Alpha，白介素 - 1Beta和缺血/再灌注。用于评估的阻塞剂 培养的内皮细胞的受体与基线和 诱导的依从性包括对GP IB的封闭单克隆抗体， 含RGD的寡肽，并阻止单克隆抗体针对 整合素和整合素亚基。此外，我们将调查角色 使用免疫学方法尚未确定的粘合蛋白。我们将 准备针对活化的内皮细胞的兔单克隆抗体 并且，作为替代方案，请使用噬菌体显示技术来确定 激活导致内皮细胞的分子变化。 单克隆抗体的亲和力或重组 表达对表面抗原的抗体片段的噬菌体 在活化的内皮细胞上，将用于鉴定分子 已经被激动剂诱导，并评估其在诱导中的作用 坚持。包含克隆人的噬菌体库 免疫球蛋白可变区域cDNA表达7 x 10 9抗体 特殊性。我们还将研究负责的细胞机制 受体在内皮细胞上的活性增加 - 有疑问 表达增加，细胞表面分布的变化，细胞 收缩暴露先前占领的受体，并增加 受体的激活。这项工作补充了博士提出的。山姆 测试和Frans Kuypers，我们建议与之合作研究。在 这项工作的后期阶段，我们将使用新西兰白兔子生产 单克隆抗体。预计我们的实验可以增强我们的 了解负责镰刀RBC-的分子机制 内皮细胞依从性，改善我们对病理生理的掌握 镰状细胞血管咬合，并扩大我们对内皮细胞的了解 生物学。