Role of RBC NO and ATP in Sickle Vasculopathy

描述（由申请人提供）：血管收缩、血细胞粘附和炎症都是镰状细胞病（SCD）的潜在破坏性事件。镰状红细胞（SS红细胞）表现出复杂的膜和生物学异常。由红细胞传递的一氧化氮（NO）在维持血管舒张和有效抗炎剂中都是至关重要的。ATP也由红细胞释放，并在血流中增加信号以满足氧气需求，通常通过增强NO合成。正常（AA）红细胞通过释放ATP和生物活性一氧化氮（NO）作为缺氧传感器，导致NO依赖性血管舒张，而SS红细胞则缺乏释放NO和ATP的含量和能力。SS红细胞粘附内皮并激活白细胞和其他细胞的能力，以及它们诱导肺血管舒张的失败，可能部分原因是它们缺乏膜结合生物活性s -亚硝基硫醇（SNO）和ATP。我们的初步数据显示，在体内加载NO/SNO可下调SS红细胞粘附、S红细胞刺激的白细胞粘附和血管闭塞，并调节血管收缩性肺表型。红细胞对ATP释放的抑制也会引起红细胞粘连和肺血管收缩。在SCD中，异常血管张力、细胞粘附、白细胞活化和炎症都被认为是血管闭塞的病理生理因素，这是疼痛危象和急慢性器官损伤的核心。因此，我们的中心假设是，SS红细胞中的NO和ATP缺陷直接导致SS红细胞粘附、S红细胞诱导的白细胞活化和肺血管收缩。我们进一步假设，SS或输注（储存）AA红细胞中NO和ATP含量的恢复也将改善肺中SS红细胞的一些可测量的不良反应。为了验证我们的假设并在改善SCD治疗方面取得进展，我们将研究人员的努力与SS红细胞生物学、输血医学和肺生理学的专业知识结合起来，以便：1)确定NO/SNO-和atp -充血对分离肺和完整小鼠肺血流动力学和气体交换的影响，这些小鼠单独输注SS红细胞或与调节红细胞粘附事件的策略相结合；2)在体内小鼠模型中检测SS红细胞或储存的AA红细胞是否能缓解血管闭塞；3)确定SS红细胞NO和ATP对体外红细胞粘附受体激活的影响、参与的信号通路以及红细胞对白细胞的激活。我们的长期目标是通过识别可修复的SS红细胞异常来改善SCD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的血管闭塞和组织损伤。

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.