Role of TRP channels in secondary injury after brain trauma

描述（由申请人提供）：创伤性脑损伤（TBI）不可避免地伴有微血管功能障碍和/或衰竭，导致水肿的形成，甚至更严重的是“进行性继发性出血”（PSH）。这些异常导致缺血、血液和炎症细胞外渗、中枢神经系统组织的自我破坏和神经预后的恶化。我们最近发现NCCa-ATP通道在PSH中起关键作用，有强有力的证据表明通道的调节亚基磺酰脲受体1 （SUR1）起关键作用。新的初步数据强烈表明，瞬时受体电位（TRP）家族的一个不寻常的成员TRPM4作为通道的成孔亚基，并且该通道在微血管内皮细胞中通过NF-?的机械激活而转录上调。B，在体内组织和体外培养的内皮细胞的力传递后几分钟内发生。我们的首要假设是，创伤相关的机械力激活了机械敏感转录因子NF-？B，导致微血管内皮中TRPM4通道的转录上调，这些通道在TBI后进行性继发性出血（PSH）的病理生理中起关键作用。这一假设的许多分支将使用分子和细胞生物学以及膜片钳电生理学进行测试。在Specific Aim (SA) 1中，我们将使用TRPM4基因敲除小鼠来确定TRPM4通道在多大程度上促进PSH的整体病理生理。在同一目标中，我们还将评估两种具有强大翻译潜力的治疗方法：使用IV反义寡脱氧核苷酸（AS-ODN）进行短暂（24小时）TRPM4基因抑制，以及利鲁唑（一种fda批准的多效药物，是TRPM4通道的有效阻滞剂）。在SA2中，我们将在tbi小鼠新鲜分离的CNS毛细血管和暴露于“创伤样”机械刺激（拉伸）的培养内皮细胞中检查通道的生理调节，并确定通道在毛细血管内皮细胞功能障碍和死亡中的具体作用。在SA3中，我们将研究机械激活转录因子NF-？B，在通道的转录调节中，我们将评估在TBI中靶向该转录因子是否因干扰TRPM4通道的表达而有益。创伤性脑损伤仍然是导致死亡和残疾的主要原因，目前还没有减少继发性损伤的治疗方法。我们希望发现TRPM4在TBI后毛细血管破坏导致PSH的最终刽子手中起着关键作用，并且通过基因抑制，在转录水平或药理学上阻断TRPM4将显著减少PSH和出血引起的继发性损伤的多重级联。这些发现将在TBI中建立一个新的和强大的治疗靶点，导致进一步的as - odn和/或利鲁唑的临床前和临床试验，我们预测这将对人类急性TBI治疗产生重大影响。

DESCRIPTION (provided by applicant): Traumatic brain injury (TBI) is inevitably complicated by microvascular dysfunction and/or failure, resulting in formation of edema and, even worse, "progressive secondary hemorrhage" (PSH). These abnormalities lead to ischemia, extravasation of blood and inflammatory cells, autodestruction of CNS tissues, and worsening of neurological outcome. We recently discovered that the NCCa-ATP channel is critically involved in PSH, with strong evidence showing critical involvement of sulfonylurea receptor 1 (SUR1), the regulatory subunit of channel. New preliminary data strongly suggest that an unusual member of the transient receptor potential (TRP) family, TRPM4, acts as the pore-forming subunit of the channel, and that the channel is transcriptionally upregulated in microvascular endothelial cells via mechano-activation of NF-?B, which occurs within minutes of force delivery to tissues in vivo and to cultured endothelial cells in vitro. Our overarching hypothesis is that trauma-associated mechanical forces activate the mechano-sensitive transcription factor, NF-?B, resulting in transcriptional upregulation of TRPM4 channels in microvascular endothelium, and that these channels play a critical role in the pathophysiology of progressive secondary hemorrhage (PSH) following TBI. Numerous ramifications of this hypothesis will be tested using molecular and cell biology, and patch clamp electrophysiology. In Specific Aim (SA) 1, we will use TRPM4 gene knock-out mice to determine the extent to which TRPM4 channels contribute to the overall pathophysiology of PSH. In the same Aim, we will also evaluate 2 treatments with strong translational potential: brief (24-hr) TRPM4 gene suppression using IV antisense oligodeoxynucleotide (AS-ODN), and riluzole, a pleiotropic FDA-approved drug that is a potent blocker of TRPM4 channels. In SA2, we will examine physiological modulation of the channel in freshly isolated CNS capillaries from TBI-mice, and in cultured endothelial cells exposed to a "trauma-like" mechanical stimulus (stretch), and we will determine the specific role of the channel in dysfunction and death of capillary endothelial cells. In SA3, we will examine the role of the mechano-activated transcription factor, NF-?B, in transcriptional regulation of the channel, and we will assess whether targeting this transcription factor in TBI is beneficial due to interference with expression of TRPM4 channels. TBI continues to be a leading cause of death and disability, with no treatments yet available to reduce secondary injury. We expect to find that TRPM4 plays a critical role as end-executioner in capillary destruction leading to PSH following TBI, and that block of TRPM4 by gene suppression, at the transcriptional level, or pharmacologically will yield significant reductions in the multiple cascades of secondary injury attributable to PSH and hemorrhage. These findings would establish a novel and powerful therapeutic target in TBI, leading to further pre-clinical as well as clinical trials with AS-ODN and/or riluzole, which we predict will have a major impact on acute treatment of TBI in humans. PUBLIC HEALTH RELEVANCE: TBI continues to be a leading cause of death and disability, with no treatments yet available to reduce secondary injury. We expect to find that TRPM4 plays a critical role as end-executioner in capillary destruction leading to PSH following TBI, and that block of TRPM4 by gene suppression, at the transcriptional level, or pharmacologically will yield significant reductions in the multiple cascades of secondary injury attributable to PSH and hemorrhage. These findings would establish a novel and powerful therapeutic target in TBI, leading to further pre-clinical as well as clinical trials with AS-ODN and/or riluzole, which we predict will have a major impact on acute treatment of TBI in humans.