Vascular responses as therapeutic targets after SCI

描述（由申请人提供）：内质网（ER）应激反应（ERSR）是防止细胞损伤的主要防御机制之一，但如果不加抑制，则会导致细胞凋亡。ERSR有三个臂，分别由PERK、IRE 1、ATF 6启动。初步数据显示SCI后内皮细胞（EC）中所有三种ERSR信号通路的急性激活。最重要的是，我们表明CHOP-/-（PERK的下游效应子）小鼠或静脉注射salubrinal（维持蛋白质合成抑制）后PERK信号转导的减弱导致WT小鼠SCI后功能恢复增强。我们发现SCI后有一个急性血管收缩期，血管扩张剂尼莫地平加血管保护剂格列本脲可增强WT小鼠的EC保护作用。具体而言，目标1将描绘ERSR介导的EC死亡的PERK信号的具体效应。我们将确定减少SCI后ECs中的PERK或ATF 4信号传导是否会增强SCI后的功能恢复。这将使用可用的转基因小鼠（Aim 1a）和siRNA方法（Aim 1b）完成。我们推测ERSR通路抑制发生的越早，血管保护和恢复的范围越广。目的2将表征当一种信号传导途径缺失时，FACS纯化的EC中ERSR的急性激活概况（目的2a）、它们对脊髓微血管系统的影响（目的2b）和功能后果（目的2c）。目的3将测试当与血管扩张剂尼莫地平或MgSO 4（CNS血管痉挛的主要治疗方法）组合时，ER应激抑制剂的EC拯救是否可以改善。目标3a将优化血管扩张方案。目标3b将优化salubrinal和两种影响ERSR信号传导的化学分子伴侣的治疗方案：TUDCA（用于ALS的临床试验）和PBA（FDA批准）。然后，我们将使用药理学和遗传学方法来测试优化的血管舒张是否会进一步提高这些药物的疗效。目标3c将定义确定治疗窗。总的来说，这3个目标中概述的实验描绘了一种策略，以最佳地抑制EC中的ER应激，以最大限度地恢复SCI后的功能，并确定这种方法是否具有临床意义。

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.