Hemostasis System in Acute Inflammation-Sepsis

急性炎症败血症的止血系统

• 批准号: 6853266
• 负责人: FRANCIS J CASTELLINO
• 金额: $ 47.25万
• 依托单位: UNIVERSITY OF NOTRE DAME
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6853266
• 关键词: animal morbidity; anticoagulants; blood coagulation; cell adhesion molecules; cellular pathology; clotting factor; disease /disorder model; endotoxins; fibrinolysis; gene expression; gene mutation; gene targeting; genetically modified animals; hemostasis; inflammation; injury; laboratory mouse; lipopolysaccharides; molecular pathology; organ; pathologic process; septic shock; septicemia; tissue /cell culture

There is a strong relationship between inflammation and hemostasis that is based on the understanding that a proinflammatory environment is also procoagulant. Events that lead to the generation of the Factor (F) Vlla/Tissue Factor (TF) complex in an acute inflammatory state, such as gram-negative sepsis, begin with endotoxin-mediated activation of ieukocytes and endothelial cells, and cytokine and chemokine upregulation from these altered cells, along with expression of TF. The consequent generation of the procoagulants, thrombin and FXa, initiates signaling pathways via interactions of these proteases with protease activated cellular receptors, and thus mediate further cellular (e.g., platelet, endothelial cell, leukocyte) responses that are involved in blood coagulation, as well as inflammation and vessel formation. The activation of these cells also results in the expression of adhesion molecules on their surfaces, thereby facilitating leukocyte binding to the endothelium. This interaction is the first step in ultimate extravasation of neutrophils and macrophages into organs, thereby causing severe organ damage in the advanced septic state. Thus, it is our basic hypothesis that attenuation of inflammation may result from inhibition of coagulation and vice versa, and sepsis models using appropriate gene-targeted mice will allow an understanding of these relationships in vivo at the level of the gene. The overall goal of this proposal is to dissect the in vivo relationships between individual genes of hemostasis and inflammation that occur in an acute model of the serious inflammatory disease, gram-negative sepsis, with its progression to severe sepsis and septic shock. Mice with single and combined genetic alterations in the pathways of hemostasis will be employed with endotoxin (LPS)-mediated models of sepsis in order to monitor the relationships between hemostasis and inflammation in the progression of the disease. Specifically, 5 highly interconnected specific aims are proposed: (1) to employ mice (and isolated endothelial cells and adherent macrophages) with genetic alterations of specific hemostasis-related genes in examining the progression of induced sepsis to severe sepsis and septic shock, and ultimate survival; (2) to assess temporal responses, after injection of LPS, of the systemic coagulation, anticoagulation, and fibrinolytic systems in these genotypically-distinct mice; (3) to measure in these same mice, the temporal plasma and organ responses of specific cytokines, chemokines, soluble adhesion proteins, and other inflammatory mediators; (4) to determine the temporal nature of organ and cell damage in tissue slices in these mice after LPS administration; and (5) to employ additional mice with combined deficiencies of hemostasis- and inflammation-related genes to further understand the in vivo mechanisms involved in their sepsis-related effects. It is expected that the results of this study will allow an in vivo evaluation of the roles of specific hemostasis- and inflammation-related genes in the development and course of this model of acute inflammatory disease, and will provide groundwork for therapeutic interventions to attenuate the morbidity and mortality associated with its downward progression.

炎症和止血之间存在强烈的关系，这是基于促炎环境也是促凝血剂的理解。导致在急性炎症状态（例如革兰氏阴性脓毒症）中产生因子（F）VIIa/组织因子（TF）复合物的事件开始于内毒素介导的白细胞和内皮细胞的活化，以及来自这些改变的细胞的细胞因子和趋化因子上调，沿着TF的表达。随后产生的促凝血剂凝血酶和FXa通过这些蛋白酶与蛋白酶活化的细胞受体的相互作用启动信号传导途径，并因此介导进一步的细胞（例如，血小板、内皮细胞、白细胞）反应，其参与血液凝固以及炎症和血管形成。这些细胞的活化还导致粘附分子在其表面上的表达，从而 促进白细胞与内皮结合。这种相互作用是中性粒细胞和巨噬细胞最终外渗到器官中的第一步，从而在晚期脓毒症状态下引起严重的器官损伤。因此，这是我们的基本假设，即炎症的衰减可能是由于凝血的抑制，反之亦然，和败血症模型使用适当的基因靶向小鼠将允许在体内的基因水平上的这些关系的理解。本提案的总体目标是剖析在严重炎症性疾病（革兰氏阴性脓毒症）的急性模型中发生的止血和炎症的个体基因之间的体内关系，其进展为严重脓毒症和脓毒性休克。将使用在止血途径中具有单一和组合遗传改变的小鼠与内毒素（LPS）介导的脓毒症模型，以监测疾病进展中止血与炎症之间的关系。具体而言，提出了5个高度相互关联的具体目标：（1）使用具有特定止血相关基因遗传改变的小鼠（以及分离的内皮细胞和粘附的巨噬细胞）， 检查诱导的脓毒症向严重脓毒症和脓毒性休克的进展，以及最终的存活率;（2）评估在注射LPS后，这些基因型不同的小鼠中全身凝血、抗凝和纤维蛋白溶解系统的时间响应;（3）测量这些相同小鼠中特定细胞因子、趋化因子、可溶性粘附蛋白和其它炎症介质的时间血浆和器官响应;（4）确定LPS给药后这些小鼠组织切片中器官和细胞损伤的时间性质;和（5）采用另外的具有止血和炎症相关基因的组合缺陷的小鼠，以进一步了解涉及其脓毒症相关作用的体内机制。预期本研究的结果将允许在体内评价特异性止血和炎症相关基因在该模型的发展和过程中的作用。 急性炎症性疾病，并将提供基础的治疗干预，以减少发病率和死亡率与其向下发展。