Vascular responses as therapeutic targets after SCI

描述(申请人提供)：内质网(ER)应激反应(ERSR)是保护细胞免受伤害的主要防御机制之一，但如果不加以控制会导致细胞凋亡。ERSR有三个臂，分别由PERK、IRE1和ATF6启动。初步数据显示，脊髓损伤后内皮细胞(ECs)中所有三条ERSR信号通路均被急性激活。最重要的是，我们发现在CHOP-/-(PERK的下游效应)小鼠体内或静脉注射后，PERK信号的衰减。Salubrine(维持蛋白质合成抑制)可促进WT小鼠脊髓损伤后的功能恢复。我们发现了脊髓损伤后的急性血管收缩阶段，血管扩张剂尼莫地平和血管保护剂格列本脲可以增强血管内皮细胞的保护作用。具体地说，目标1将描述ERSR通过PERK信号介导的EC死亡背后的特定效应器。我们将确定减少脊髓损伤后内皮细胞中的PERK或ATF4信号是否会促进脊髓损伤后的功能恢复。这将使用现有的转基因小鼠(目标1a)和siRNA方法(目标1b)来完成。我们假设，在ERSR通路中，抑制发生得越早，血管保护和恢复就越广泛。目的2将表征当一个信号通路缺失时，FACS纯化的内皮细胞中ERSR的急性激活情况(目标2a)，它们对脊髓微血管系统的影响(目标2b)以及功能后果(目标2c)。目的3将测试内质网应激抑制剂联合血管扩张剂、尼莫地平或硫酸镁等中枢神经系统血管痉挛的主要治疗方法是否可以改善EC的挽救。AIM 3a将优化血管扩张方案。目的3b将优化Salubrine和两种影响ERSR信号转导的化学伴侣的治疗方案：TUDCA(ALS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测试优化的血管扩张是否会进一步提高这些药物的疗效。Aim 3c将定义确定治疗窗口。总而言之，这3个实验旨在勾勒出一种策略，以最佳地抑制内皮细胞的内质网应激，以最大限度地促进脊髓损伤后的功能恢复，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.