Platelet-Mediated Neutrophil Extracellular Traps Regulate Ischemic Stroke Injury

尽管取得了进步，但美国每年仍有约70万人患有缺血性中风。 目前对急性缺血性中风的治疗主要局限于溶栓或血栓切除术，对于这些 许多中风患者没有资格。因此，开发治疗中风的新疗法是一个重要的公众 健康需要。了解中风的细胞机制对新的中风的发展至关重要 中风疗法。中性粒细胞和血小板是缺血性卒中损伤的重要调节因子。在人类和 小鼠缺血性卒中后血小板-中性粒细胞聚集性增加，中性粒细胞胞外陷阱增加 (Nets)，中性粒细胞活化的标志。Net在炎症和感染过程中起关键作用，并被释放 由中性粒细胞捕获病原体。虽然网络有助于对抗感染，但过度的网络形成可能会 通过促进血栓形成对宿主有害。然而，净释放的病理作用并不是 对缺血性卒中损伤进行了研究。此外，在过程中触发网络形成的分子调节器 中风仍不清楚。最后，如果在缺血性卒中损伤期间靶向净释放可以改善预后 完全未知。在这里，我们将检验这一创新假设，即血小板是 缺血性卒中时的净释放和以新型内源性网络为靶点的网络形成- 抑制因子(NNIF)，将改善中风预后。我们将采用补充临床，体外， 而在体内的方法，以及最先进的技术和模型来严格测试这一假设。 具体目标1将确定是否在缺血性中风后释放Net，以及它们是否存在于大脑中 对人类中风患者和实验性中风后的小鼠的影响。此外，我们将检查中性粒细胞是否 为缺血性中风患者释放Net做好准备。特定目标2将确定血小板是否过高 血小板活化后释放的一种危险相关分子模式--迁移率组盒1(HMGB1)， 调节缺血性卒中损伤期间的网络形成。特定目标3将确定是否具有治疗性nNIF 给药阻断实验性中风期间的网络形成并改善急性和长期中风 结果，包括运动和神经功能。这些目标的成功完成将决定1)是否 Net存在于缺血性中风损伤中，包括血管内和血管外；(2)建立 中性粒细胞和血小板是否已准备好参与缺血性卒中的网络形成，(3) 确定血小板HMGB1是否是缺血性卒中时血小板介导的NETase的关键调节因子； 以及(4)确定净抑制是否改善卒中结果和与以下相关的治疗窗口 病理性网状结构。这份建议书中产生的数据将极大地提高我们对 血小板如何参与缺血性卒中及其相关神经系统的病理性网络形成 受伤。

Despite advancements, about 700,000 people in the U.S. still experience an ischemic stroke annually. Current treatments for acute ischemic stroke are largely restricted to thrombolysis or thrombectomy, for which many stroke patients are ineligible for. Therefore, developing novel therapies for stroke is a significant public health need. Understanding the cellular mechanisms underlying stroke is critical for the development of new stroke therapies. Neutrophils and platelets are critical regulators of ischemic stroke injury. In humans and mice, platelet-neutrophil aggregates increase after ischemic stroke as well do neutrophil extracellular traps (NETs), a marker of neutrophil activation. NETs are critical during inflammation and infection and are released by neutrophils to trap pathogens. While NETs help fight infection, excessive NET formation can be detrimental to the host by promoting thrombosis. However, the pathological role of NET release has not been studied in ischemic stroke injury. Furthermore, the molecular regulators that trigger NET formation during stroke remain unclear. Finally, if targeting NET release during ischemic stroke injury improves outcomes is completely unknown. Here, we will test the innovative hypothesis that platelets are a primary driver of NET release during ischemic stroke and targeting NET formation with a novel, endogenous NET- inhibitory factor (nNIF), will improve stroke outcomes. We will employ complementary clinical, in vitro, and in vivo approaches, along with state-of-the-art techniques and models to rigorously test this hypothesis. Specific Aim 1 will determine if NETs are released after ischemic stroke and if they are present in the brains of human stroke patients and mice after experimental stroke. Furthermore, we will examine if neutrophils are primed to release NETs in ischemic stroke patients. Specific Aim 2 will establish whether platelet high mobility group box 1 (HMGB1), a danger associated molecular pattern released by platelets after activation, regulates NET formation during ischemic stroke injury. Specific Aim 3 will determine if therapeutic nNIF administration blocks NET formation during experimental stroke and improves acute and long-term stroke outcomes, including motor and neurological function. Successful completion of these aims will 1) determine if NETs are present in ischemic stroke injury including intravascular and extravascular locations; (2) establish whether neutrophils and platelets are primed to participate in NET formation during ischemic stroke, (3) determine whether platelet HMGB1 is a critical regulator of platelet-mediated NETosis during ischemic stroke; and (4) determine if NET inhibition improves stroke outcomes and the therapeutic window associated with pathological NET formation. Data generated in this proposal will significantly increase our understanding of how platelets contribute to pathological NET formation during ischemic stroke and associated neurological injury.