ENDOTHELIAL CELL REACTIVITY IN SICKLE CELL VASOOCCLUSION

疼痛性血管闭塞是镰状细胞病的主要特征之一， 疾病它比任何其他并发症都更容易导致患者寻求 医疗护理。几种过程参与血管闭塞， 包括镰状红细胞对血管内皮细胞的粘附增加。 特异性细胞粘附受体、配体和异源细胞机制 已经并将继续被发现。我们 提出内皮细胞受体及其表达的变化 是血管阻塞的重要变量。 内皮细胞功能的显著变化受已知的 拮抗剂包括粘附性的改变， 显示在细胞表面，受体活性的变化，细胞 收缩和内皮细胞间隙形成。各种各样的 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粘附和内皮细胞粘附的激动剂， 介导增强粘附的细胞受体。我们将要研究的激动剂 包括凝血酶、组胺、肿瘤坏死因子-α、白细胞介素- 1 β和缺血/再灌注。用于评估的封闭剂 培养的内皮细胞上的受体参与基线和 诱导的粘附包括抗GP Ib的阻断性单克隆抗体， 含有RGD的寡肽，和阻断抗 整合素和整合素亚单位。此外，我们还将研究 尚未鉴定的粘附蛋白质。我们将 兔抗活化内皮细胞单克隆抗体的制备 作为替代，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。 单克隆抗体或重组抗体的亲和力 表达对表面抗原特异的抗体片段的噬菌体 将用于识别分子， 并评估其在诱导的 坚持。含有克隆人的噬菌体文库 免疫球蛋白可变区cDNA表达7 × 10 9抗体 特殊性我们还将研究细胞机制， 内皮细胞上受体的活性增加--定量 表达增加，细胞表面分布变化，细胞 收缩暴露先前占据的受体，并增加 受体的激活。这项工作补充了萨姆博士提出的工作 Test和Frans Kuypers，我们建议与他们合作研究。在 这项工作的后期阶段，我们将使用新西兰白色兔生产 克隆抗体我们的实验有望提高我们的 了解镰状红细胞的分子机制- 内皮细胞粘附，提高我们的病理生理学的把握， 镰状细胞血管闭塞，扩大我们对内皮细胞 生物学

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.