Activated protein C mechanisms of brain white matter protection and new therapies for brain white matter ischemic injury

激活蛋白C脑白质保护机制及脑白质缺血性损伤新疗法

• 批准号: 10391557
• 负责人: William J Mack
• 金额: $ 84.37万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10391557
• 关键词: Address; Agonist; Amino Acid Sequence; Anti-Inflammatory Agents; Anticoagulants; Apoptotic; Arteries; Astrocytes; Axon; Biological Products; Blood - brain barrier anatomy; Blood Vessels; Blood flow; Brain; Cells; Clinic; Clinical; Clinical Trials; Collaborations; Data; Dementia; Endothelium; Engineering; Exhibits; Extravasation; Factor VIIIa; Factor Va; Fiber; Functional disorder; GTP-Binding Proteins; Gene Expression; Generations; Goals; In Vitro; Inflammatory; Injury; Ischemic Stroke; Knowledge; Lesion; Ligands; Magnetic Resonance Imaging; Microglia; Modeling; Mus; Mutation; N-terminal; Nerve Degeneration; Neurons; Oligodendroglia; PAR-1 Receptor; Pathway interactions; Peptides; Platelet Activation; Point Mutation; Prevention therapy; Proteolysis; Role; Signal Transduction; Site; Stroke; Testing; Therapeutic; Thrombin; Toxic effect; Translating; Traumatic Brain Injury; activated Protein C; activated protein C receptor; analog; arrestin 2; base; behavior test; cell type; disability; improved; in vivo; in vivo Model; insight; ischemic injury; mimetics; mortality; mutant; neuropathology; novel; novel therapeutics; peptidomimetics; phase II trial; prevent; protective effect; response; stroke model; stroke patient; stroke therapy; success; vascular cognitive impairment and dementia; vasoconstriction; white matter; white matter injury

ABSTRACT Stroke in small brain vessels in subcortical white matter (WM) regions account for 25% of all strokes. It leads to vascular cognitive impairment and dementia (VCID), and is the second leading cause of dementia overall. Despite such clinical importance, the pathophysiology of ischemic WM injury (WMI) and VCID is still poorly understood. Moreover, there is no yet an approved therapy for prevention and/or treatment of WM strokes and VCID. Here, we propose collaborative studies between the Zlokovic and Griffin labs on activated protein C (APC) pathways in the WM, and to evaluate therapeutic potential of APC-based therapies for ischemic WMI using a model of vasoconstriction of small brain vessels in the WM. Our previous studies using models of large artery infracts, brain trauma and neurodegeneration led to discovery of vasculoprotective, blood-brain barrier (BBB)-stabilizing, neuroprotective, and anti-inflammatory activities of APC and its cytoprotective-selective mutants. In 2019, these findings have been translated into successfully completed phase 2 trial for ischemic stroke of 3K3A-APC, a 2nd generation cytoprotective-selective APC analog with >90% loss of anticoagulant activity. However, whether activation of APC pathways in the WM is beneficial or not during ischemic WMI, remains unknown. Our goals include: 1) providing proof of concept for hypothesized mechanisms for protective activities of APC in the WM; and 2) characterizing novel protease activated receptor 1 (PAR1)-related P1-47 and PAR3-related P3-42 APC-mimetic peptides, and 3) testing improved 3rd generation APC R-46-selective biologics for treating and preventing ischemic WMI and WM stroke. Our pilot data support our hypotheses that: i) APC will be beneficial for ischemic WMI via PAR1 cleavage at Arg46 to protect WM fiber tracts, oligodendrocytes and BBB from ischemic WMI (AIM 1); ii) APC-mimetic peptides derived from PAR1 and PAR3 sequences (e.g,, P1-47 and P3-42, (i.e., the tethered PAR agonists created by APC cleavages) exhibit synergistic biased agonism, and will elicit -arrestin 2-dependent cytoprotective signaling in brain endothelium and oligodendrocytes in vitro and in vivo after WM stroke (AIM 2); and iii) E56K-APC and D180E-APC newly engineered APC mutants have enhanced ability to cleave PAR1 at Arg46 and will provide improved APC biologics for WM stroke therapy (AIM 3). To address our hypotheses, we will use i) WM model of stroke; ii) new mouse lines carrying R41Q-PAR1 and R46Q-PAR1 point mutations, and -arrestin 2-/- and G12-/- mice; iii) new APC-mimetic PAR1- and PAR3-related peptides with the respective PAR1 and PAR3 tethered-ligand amino acid sequences; iv) new APC R46-cleavage site selective biologics; v) in vivo mutiparametric longitudinal MRI of WM lesion volume, BBB integrity, blood flow, structural and connectivity changes, and tract-tracing based connectomics for circuit level analysis; vi) behavior tests; vii) immunohistology, neuropathology; and viii) oligodendrocyte cultures and in vitro BBB model. If successful, new knowledge generated from this project could translate to the clinic as new therapies for WM stroke and VCID.

抽象的 皮质下白质 (WM) 区域的小脑血管中风占所有中风的 25%，它会导致血管性认知障碍和痴呆 (VCID)，并且是导致痴呆的第二大原因，尽管具有如此的临床重要性，但其病理生理学特征却不容忽视。缺血性 WM 损伤 (WMI) 和 VCID 的关系仍知之甚少，此外，目前还没有批准的用于预防和/或治疗 WM 中风和 VCID 的疗法。 Zlokovic 和 Griffin 实验室研究 WM 中的活化蛋白 C (APC) 通路，并使用 WM 中小脑血管的血管收缩模型来评估基于 APC 的疗法对缺血性 WMI 的治疗潜力。梗塞、脑外伤和神经退行性疾病导致人们发现了 APC 及其化合物的血管保护、血脑屏障 (BBB) 稳定、神经保护和抗炎活性。 2019 年，这些发现已转化为成功完成的 3K3A-APC 治疗缺血性中风的 2 期试验，3K3A-APC 是第二代细胞保护选择性 APC 类似物，抗凝活性丧失 > 90%。 WM 中的通路在缺血性 WMI 期间是否有益仍然未知，我们的目标包括：1）为所利用的保护活动机制提供概念证明。 WM 中的 APC；2) 表征新型蛋白酶激活受体 1 (PAR1) 相关的 P1-47 和 PAR3 相关的 P3-42 APC 模拟肽，以及 3) 测试改进的第三代 APC R-46 选择性生物制剂用于治疗我们的试验数据支持我们的假设：i) APC 将通过 PAR1 对缺血性 WMI 有益。在 Arg46 处裂解，以保护 WM 纤维束、少突胶质细胞和 BBB 免受缺血性 WMI (AIM 1) 的影响；ii) 源自 PAR1 和 PAR3 序列的 APC 模拟肽（例如，P1-47 和 P3-42，（即束缚的 PAR） APC 裂解产生的激动剂）表现出协同偏向激动作用，并会引发WM 中风后脑内皮和少突胶质细胞体外和体内的 -arrestin 2 依赖性细胞保护信号传导 (AIM 2)；以及 iii) E56K-APC 和 D180E-APC 新设计的 APC 突变体增强了在 Arg46 处切割 PAR1 的能力，并且将为 WM 中风治疗提供改进的 APC 生物制剂 (AIM 3) 为了验证我们的假设，我们将使用 i)。中风的 WM 模型；ii) 携带 R41Q-PAR1 和 R46Q-PAR1 点突变的新小鼠系，以及 -arrestin 2-/- 和 G12-/- 小鼠；iii) 新的 APC 模拟 PAR1- 和 PAR3 相关小鼠；具有各自 PAR1 和 PAR3 系留配体氨基酸序列的肽 iv) 新的 APC R46 切割位点选择性；生物制剂；v) WM 病变体积、血脑屏障完整性、血流、结构和连接变化的体内多参数纵向 MRI，以及用于回路水平分析的纤维束追踪；vii) 免疫组织学、神经病理学；如果成功，该项目产生的新知识可以转化为临床，作为 WM 中风和 VCID 的新疗法。

项目成果

Protection of ischemic white matter and oligodendrocytes in mice by 3K3A-activated protein C.

• DOI: 10.1084/jem.20211372
• 发表时间: 2022-01-03
• 期刊: The Journal of experimental medicine
• 作者: Huuskonen MT;Wang Y;Nikolakopoulou AM;Montagne A;Dai Z;Lazic D;Sagare AP;Zhao Z;Fernandez JA;Griffin JH;Zlokovic BV
• 通讯作者: Zlokovic BV