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.

摘要 皮质下白色物质（WM）区域小脑血管卒中占所有卒中的25%。它导致血管性认知障碍和痴呆（VCID），是痴呆症的第二大原因。尽管有这样的临床重要性，缺血性WM损伤（WM）和VCID的病理生理学仍然知之甚少。此外，还没有批准的用于预防和/或治疗WM中风和VCID的疗法。在这里，我们建议Zlokovic和Griffin实验室之间的合作研究激活蛋白C（APC）通路在WM，并评估基于APC的治疗缺血性脑血管病的治疗潜力，使用模型的小脑血管收缩在WM。我们以前的研究使用大动脉梗死，脑外伤和神经退行性变的模型，导致发现血管保护，血脑屏障（BBB）稳定，神经保护和抗炎活性的APC及其细胞保护选择性突变体。2019年，这些发现已转化为成功完成的3 K3 A-APC缺血性卒中的2期试验，3 K3 A-APC是第二代细胞保护选择性APC类似物，具有>90%的抗凝活性损失。然而，在缺血性脑卒中期间，WM中APC通路的激活是否有益，仍然未知。我们的目标包括：1）为APC在WM中的保护活性的假设机制提供概念证明;和2）表征新的蛋白酶激活受体1（PAR 1）相关的P1-47和PAR 3相关的P3-42 APC模拟肽，和3）测试用于治疗和预防缺血性脑卒中和WM中风的改进的第三代APC R-46选择性生物制剂。我们的初步数据支持我们的假设：i）APC将通过PAR 1在Arg 46处切割以保护WM纤维束、少突胶质细胞和BBB免受缺血性脑损伤而有益于缺血性脑损伤（AIM 1）; ii）衍生自PAR 1和PAR 3序列的APC模拟肽（例如，P1-47和P3-42，（即，由APC裂解产生的系留PAR激动剂）表现出协同偏性激动作用，并将在WM卒中（AIM 2）后在体外和体内引起脑内皮和少突胶质细胞中的β-arrestin 2依赖性细胞保护信号（AIM 2）;和iii）E56 K-APC和D180 E-APC新工程改造的APC突变体具有增强的在Arg 46处切割PAR 1的能力，并将为WM中风治疗提供改进的APC生物制剂（AIM 3）.为了解决我们的假设，我们将使用i）中风的WM模型; ii）携带R41 Q-PAR 1和R46 Q-PAR 1点突变的新小鼠系，以及β-抑制蛋白2-/-和GST 12-/-小鼠; iii）具有各自的PAR 1和PAR 3拴系配体氨基酸序列的新APC模拟PAR 1和PAR 3相关肽; iv）新APC R46切割位点选择性生物制剂; v）WM病变体积、BBB完整性、血流、结构和连接性变化的体内多参数纵向MRI，以及用于回路水平分析的基于束追踪的连接组学; vi）行为测试; vii）免疫组织学、神经病理学;以及viii）少突胶质细胞培养物和体外BBB模型。如果成功，从该项目产生的新知识可以转化为临床WM卒中和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