Role of RBC NO and ATP in Sickle Vasculopathy

描述（由申请人提供）：血管收缩、血细胞粘附和炎症是镰状细胞病（SCD）的潜在破坏性事件。镰状红细胞（SS RBC）表现出复杂的膜和生物学异常。由RBC递送的一氧化氮（NO）在维持血管舒张和有效的抗炎剂中都是至关重要的。红细胞也释放ATP，并通过增加NO合成来增加血流以满足O2需求。虽然正常（AA）RBC通过释放ATP和生物活性一氧化氮（NO）两者而充当缺氧传感器，导致NO依赖性血管舒张，但SS RBC在释放NO和ATP两者的含量和能力方面都有缺陷。SS RBC粘附于内皮并活化白细胞以及其他细胞的能力，沿着它们不能诱导肺血管舒张，可能部分地由于它们缺乏膜结合的生物活性S-亚硝基硫醇（SNO）和ATP。我们的初步数据表明，加载SS红细胞与NO/SNO下调SS红细胞粘附，S红细胞刺激的白细胞粘附，血管闭塞在体内和调节血管收缩肺表型。抑制红细胞释放ATP也可诱导红细胞粘附和肺血管收缩。在SCD中，异常的血管张力、细胞粘附、白细胞活化和炎症都被认为有助于血管闭塞的病理生理学，这是疼痛危象和急性和慢性器官损伤的核心。因此，我们的中心假设是SS RBC中的NO和ATP缺陷直接导致SS RBC粘附、S RBC诱导的白细胞活化和肺血管收缩。我们进一步假设，SS或输血（储存）AA RBC中NO和ATP含量的恢复也将改善SS RBC在肺中的一些可测量的不良反应。为了测试我们的假设和实现SCD的改进疗法的进展，我们结合了具有SS RBC生物学、输血医学和肺生理学专业知识的研究者的努力，以1）确定NO/SNO-和ATP-补充对单独输注SS RBC或与调节RBC粘附事件的策略组合输注SS RBC的离体肺和完整小鼠的肺血流动力学和气体交换的影响; 2）测试SS RBC或储存的AA RBC的NO和ATP补充是否可以在体内缓解小鼠模型中的血管闭塞;和3）确定SS RBC NO和ATP对体外RBC粘附受体的活化、所涉及的信号传导途径和白细胞的RBC活化的影响。我们的长期目标是通过识别可治疗的SS RBC异常来改善SCD中的血管张力、细胞粘附和细胞活化，从而减少血管闭塞。这项工作将允许开发新的治疗方法来预防和控制SCD中的血管闭塞和组织损伤。 公共卫生相关性：相关性镰状细胞病（SCD）影响美国约10万人和全球数百万人。开发治疗或预防SCD中血管闭塞、炎症和肺功能异常的新疗法对于降低疾病的发病率和死亡率至关重要。我们建议测试的假设，支持我们的初步数据，即一氧化氮和ATP的镰状红细胞的缺陷，导致异常红细胞粘附，细胞间相互作用，炎症性白细胞活化，血管闭塞，并在各种模型系统镰状红细胞引起的肺血流动力学的不良变化。

DESCRIPTION (provided by applicant): Vasoconstriction, blood cell adhesion, and inflammation are each potentially devastating events in sickle cell disease (SCD). Sickle red cells (SS RBCs) demonstrate complex membrane and biologic abnormalities. Nitric oxide (NO) delivered by RBCs is both critical in the maintenance of vasodilation and a potent anti-inflammatory agent. ATP is also released by RBCs and signals increases in blood flow to meet O2 demand, typically by enhancing NO synthesis. While normal (AA) RBCs act as a hypoxia sensor by releasing both ATP and bioactive nitric oxide (NO), leading to NO-dependent vasodilation, SS RBCs are deficient in both content and ability to release both NO and ATP. The ability of SS RBCs to adhere to the endothelium and to activate leukocytes as well as other cells, along with their failure to induce pulmonary vasodilation, may result in part from their deficiencies in membrane-bound bioactive S-nitrosothiol (SNO) and ATP. Our preliminary data show that loading SS RBCs with NO/SNO down-regulates SS RBC adhesion, S RBC-stimulated leukocyte adhesion, and vaso-occlusion in vivo and modulates the vasoconstrictive pulmonary phenotype. Inhibition of ATP release by RBCs also induces RBC adhesion and pulmonary vasoconstriction. In SCD, abnormal vascular tone, cell adhesion, leukocyte activation, and inflammation are all believed to contribute to the pathophysiology of vaso-occlusion, which is central to both painful crises and acute and chronic organ damage. Thus, our central hypothesis is that NO and ATP deficits in SS RBCs directly contribute to both SS RBC adhesion, S RBC-induced activation of leukocytes, and pulmonary vasoconstriction. We further postulate that restoration of NO and ATP content in either SS or transfused (stored) AA RBCs will also improve some of the measurable adverse effects of SS RBCs in the lung. To test our hypothesis and progress toward achieving improved therapies for SCD, we have combined the efforts of investigators with expertise in SS RBC biology, transfusion medicine, and pulmonary physiology in order to 1) Determine the influence of NO/SNO- and ATP-repletion on pulmonary hemodynamics and gas exchange in isolated lungs and intact mice transfused with SS RBCs alone or in combination with strategies that modulate RBC adhesive events; 2) Test whether NO and ATP repletion of SS RBCs or stored AA RBCs can relieve vaso-occlusion in a mouse model in vivo; and 3) Determine the effect of SS RBC NO and ATP on the activation of RBC adhesion receptors in vitro, the signaling pathways involved, and RBC activation of leukocytes. Our long-term goal is to improve vascular tone, cell adhesion, and cell activation in SCD by identifying remediable SS RBC abnormalities and thereby reduce vaso-occlusion. This work will allow development of new therapeutic approaches to prevent and control vaso-occlusion and tissue damage in SCD. PUBLIC HEALTH RELEVANCE: Relevance Sickle cell disease (SCD) affects approximately 100,000 individuals in the USA and several million worldwide. Development of new therapies to treat or prevent vaso-occlusion, inflammation and abnormal lung function in SCD is critical to reducing the disease's morbidity and mortality. We propose to test the hypothesis, supported by our preliminary data, that the nitric oxide and ATP deficiencies of sickle red blood cells contribute to the abnormal red cell adhesion, cell-cell interactions, inflammatory leukocyte activation, vaso-occlusion, and adverse pulmonary hemodynamic changes caused by sickle red cells in a variety of model systems.