Role of the PHD2-HIF-VEGF/Epo axis in neurons for neuroplasticity and functional neurological recovery long-term after stroke

虽然中风是世界范围内第二大最常见的死亡原因和长期残疾的主要原因，但组织型纤溶酶原激活剂静脉溶栓和血管内机械取栓是目前唯一被批准用于治疗急性缺血性中风的临床疗法。此外，除了物理治疗和认知训练外，目前还没有临床证实有效的治疗方法来促进脑卒中患者的脑组织再生和神经功能的长期康复。临床前研究已经证明，脑缺血期间氧分压的下降启动中枢神经系统（CNS）的适应性过程，促进急性神经元存活和长期神经再生。后者包括血管生成、神经生成和可塑性相关过程。其中，常驻脑细胞的血管内皮生长因子（VEGF）和红细胞生成素（Epo）的表达和释放在缺血损伤时增加。这两种因子不仅通过直接的细胞保护作用保护神经元免受急性缺血损伤，而且还控制梗死组织的新生血管形成、神经发生和神经可塑性。VEGF和Epo都是缺氧诱导转录因子（hif）的靶基因，hif的活性受脯氨酸-4-羟化酶结构域（PHD）蛋白家族以氧依赖的方式严格调控。因此，我们假设PHD-HIF轴对脑缺血的适应具有重要意义。因此，我们使用小鼠缺血性中风模型的初步工作证明了以下几点：(1) HIF抑制因子PHD2的神经元特异性基因失活促进HIF依赖的基因组反应，并减轻（亚）急性卒中后的脑损伤和功能损害；(2)神经元HIF- α缺乏加重了卒中亚急性早期的组织损伤和功能损害；(3)PHD2缺陷小鼠脑组织保存和感觉运动功能的改善主要是由于HIF依赖机制。(4)用药理学PHD抑制剂进行全身治疗可改善急性脑缺血引起的脑组织损伤。基于我们的初步工作，本项目旨在阐明PHD/HIF氧传感机制是否也控制中风后中枢神经系统的长期再生过程。沿着这条路线，将解决以下主要问题：(i)细胞特异性基因消融神经元中PHD2和hif - α对脑卒中后神经可塑性和功能恢复的影响，（ii） hif依赖性激活VEGF和Epo对脑卒中后神经可塑性和功能恢复的重要性，以及（iii）延迟治疗PHD抑制剂对脑卒中后结构和功能长期再生的治疗效果。

Although stroke is the second most common cause of death and major cause of long-term disability worldwide, intravenous thrombolysis with tissue-type plasminogen activator and endovascular mechanical thrombectomy are the only clinical therapies currently approved for treatment of acute ischemic stroke. Moreover, apart from physical therapy and cognitive training, there is no therapeutic approach with proven clinical efficacy available that aims to promote brain tissue regeneration and long-term convalescence of neurological functions in stroke patients. Pre-clinical studies have already demonstrated that decline of the oxygen partial pressure during cerebral ischemia initiates adaptive processes in the central nervous system (CNS) that promote both acute neuronal survival and long-term neuroregeneration. The latter comprises angiogenic, neurogenic, and plasticity-related processes. Among others the expression and release of the vascular endothelial growth factor (VEGF) and erythropoietin (Epo) by resident brain cells is increased upon ischemic insult. Both factors not only protect neurons from acute ischemic injury through direct cytoprotective effects, but also control neovascularization of the infarcted tissue, neurogenesis and neuroplasticity. VEGF and Epo are both target genes of the hypoxia-inducible transcription factors (HIFs) whose activity is strictly regulated through the family of prolyl-4-hydroxylase domain (PHD) proteins in an oxygen-dependent manner. Thus, we hypothesize that the PHD-HIF axis is of great importance for the adaption to cerebral ischemia. Accordingly, our preliminary work using murine models of ischemic stroke have demonstrate the following: (1) neuron-specific gene inactivation of the HIF suppressor PHD2 promotes the HIF-dependent genomic response, and reduces brain injury and functional impairment after (sub)acute stroke, (2) neuronal HIF-alpha deficiency worsens tissue injury and functional impairment in the early subacute stage upon stroke, (3) improved brain tissue preservation and sensorimotor function in PHD2 deficient mice is predominantly due to HIF-dependent mechanisms, and (4) systemic treatment with pharmacological PHD inhibitors ameliorates brain tissue damage resulting from acute cerebral ischemia. Based on our preliminary work, the present project aims to clarify whether the PHD/HIF oxygen sensing machinery controls long-term regenerative processes in the CNS post-stroke as well. Along this line, following main issues will be addressed: (i) influence of cell-specific genetic ablation of PHD2 and HIF-alpha in neurons for neuroplasticity and functional recovery after stroke, (ii) importance of HIF-dependent activation of VEGF and Epo for post-stroke neuroplasticity and functional recovery, and (iii) therapeutic efficacy of delayed treatment with PHD inhibitors on structural and functional long-term regeneration after stroke.