Vascular responses as therapeutic targets after SCI

内质网（ER）应激反应（ERSR）是保护细胞免受损伤的主要防御机制之一，但如果不加以控制，则会导致细胞凋亡。ERSR有三条臂，分别由PERK、IRE1、ATF6发起。初步数据显示，损伤后内皮细胞（ECs）的所有三种ERSR信号通路均被急性激活。最重要的是，我们发现CHOP-/- （PERK的下游效应物）小鼠或静脉注射salubrinal（维持蛋白质合成抑制）后PERK信号的衰减导致WT小鼠脊髓损伤后功能恢复增强。我们发现脊髓损伤后出现急性血管收缩期，血管扩张剂尼莫地平加血管保护剂格列本脲可以增强WT小鼠的EC保护。具体来说，Aim 1将描述ersr通过PERK信号介导的EC死亡的特定效应物。我们将确定减少脊髓损伤后ECs中的PERK或ATF4信号是否会增强脊髓损伤后的功能恢复。这将使用现有的转基因小鼠（Aim 1a）和siRNA方法（Aim 1b）来完成。我们假设ERSR通路中抑制发生得越早，血管保护和恢复就越广泛。Aim 2将描述当一个信号通路被删除时，FACS纯化的ECs中ERSR的急性激活特征（Aim 2a），它们对脊髓微血管的影响（Aim 2b）和功能后果（Aim 2c）。目的3将测试内质网应激抑制剂与血管扩张剂、尼莫地平或MgSO4（主要用于治疗中枢神经系统血管痉挛）联合使用时，是否能改善EC的抢救效果。Aim 3a将优化血管舒张方案。Aim 3b将优化salubrinal和两种影响ERSR信号传导的化学伴侣的治疗方案：TUDCA（在ALS的临床试验中）和PBA （fda批准）。然后，我们将测试优化的血管舒张是否会使用药理学和遗传学方法进一步提高这些药物的疗效。Aim 3c将定义确定治疗窗口期。总的来说，这3个目的概述的实验描述了一种策略，以最佳地抑制内质网应激，最大限度地恢复脊髓损伤后的功能，并确定该方法是否具有临床相关性。

DESCRIPTION (provided by applicant): The endoplasmic reticulum (ER) stress response (ERSR) is one of the major defense mechanisms that protect against cellular insult but if unchecked leads to apoptotic cell death. The ERSR has three arms initiated by PERK, IRE1, ATF6, respectively. Preliminary data show the acute activation of all three ERSR signaling pathways in endothelial cells (ECs) after SCI. Most importantly, we show that attenuation of PERK signaling in CHOP-/- (the downstream effector of PERK) mice or after i.v. salubrinal (which sustains protein synthesis inhibition) leads to enhanced functional recovery after SCI in WT mice. We found an acute vasoconstrictive phase following SCI and can enhance EC protection by the vasodilator nimodipine plus the vasoprotector glibenclamide in WT mice. Specifically, Aim 1 will delineate the specific effectors that underlie ERSR-mediated EC death by PERK signaling. We will determine if reducing PERK or ATF4 signaling in ECs after SCI will enhance functional recovery after SCI. This will be done using available transgenic mice (Aim 1a) and siRNA methods (Aim 1b). We hypothesize that the earlier in the ERSR pathway that inhibition occurs, the more extensive the vasoprotection and recovery. Aim 2 will characterize the acute activation profile of the ERSR in FACS purified ECs when one signaling pathway is deleted (Aim 2a), their effects on spinal cord microvasculature (Aim 2b) and the functional consequences (Aim 2c). Aim 3 will test whether EC rescue by ER stress inhibitors can be improved when combined with the vasodilators, nimodipine or MgSO4, the mainstay treatments for CNS vasospasm. Aim 3a will optimize vasodilation protocols. Aim 3b will optimize treatment regimens for salubrinal and two chemical chaperones that influence ERSR signaling: TUDCA (in clinical trials for ALS) and PBA (FDA-approved). We will then test whether optimized vasodilation would further improve the efficacy of those drugs using both pharmacological and genetic approaches. Aim 3c will define determine the therapeutic window. Collectively, the experiments outlined in these 3 Aims delineate a strategy to optimally inhibit ER stress in ECs to maximize functional recovery after SCI and determine whether this approach is clinically relevant.