PGE2 EP1 and EP3 receptors as therapeutic targets in intracerebral hemorrhage

描述（申请人提供）：PGE2 EP1和EP3受体作为脑出血的治疗靶点脑出血（ICH）是死亡率和发病率最高的中风亚型；不幸的是，与缺血性中风相比，这种疾病受到的研究关注度要低得多。炎症机制，包括由某些前列腺素 (PG) 介导的炎症机制，已被认为有助于 ICH 损伤的进展。特别是，PGE2 在大脑中占主导地位，在血肿周围区域产生并积聚。 PGE2 通过四种 G 蛋白偶联受体亚型（称为 EP1-EP4）发挥作用。在脑缺血动物模型中，这些受体中的每一种都具有独特的信号级联反应。 EP1 和 EP3 受体的基因缺失或选择性抑制已被证明可以减少体内和体外缺血性脑损伤。本次活动的总体目标 R01旨在研究PGE2 EP1和EP3受体的抑制是否可以用作ICH的神经保护疗法，如果可以，则生成临床前疗效数据并阐明潜在机制。我们一直在研究 PGE2 受体在 ICH 损伤中的具体作用，初步数据表明，删除或抑制 EP1 或 EP3 受体可减少 ICH 损伤并改善功能结果。我们的数据还表明 ICH 后 Src 激酶介导 EP1 毒性，Rho 激酶 (ROCK) 介导 EP3 毒性。因此，我们将检验以下假设：PGE2 EP1 受体阻断可通过 Src 激酶信号通路减少 ICH 损伤，而 EP3 受体阻断可通过 ROCK 信号通路减少 ICH 损伤。我们设计了三个具体目标，利用自体血液 ICH 模型和胶原酶诱导的 ICH 模型。考虑到 PGE2 产生的增加会导致与年龄相关的炎症反应功能障碍，并且衰老和性别差异会影响 ICH 结果，因此本研究将在年轻雄性和雌性以及老年雄性小鼠中进行，以增强临床相关性。目标 1 将确定抑制 EP1 或 EP3 受体是否可以改善 ICH 结果；目标 2 将确定 Src 激酶信号传导的抑制是否有助于 ICH 后 EP1 受体阻断所提供的神经保护，以及 EP1 受体抑制是否会减少凝血酶诱导的脑损伤；目标 3 将确定 ROCK 信号传导的抑制是否有助于 ICH 后 EP3 受体阻断所提供的神经保护，以及 EP3 受体抑制是否会减少凝血酶诱导的脑损伤。通过药理学、遗传学、影像学、组织学、分子和细胞生物学方法，这项工作将提供有关选择性 PGE2 EP1 和 EP3 受体抑制的功效的新信息，以防止 ICH 诱导的脑损伤以及发生这种保护的机制。需要这些概念验证信息来计划对 ICH 中的选择性 EP1 和 EP3 受体拮抗剂进行更详细的临床前评估。 公众健康相关性：脑出血 (ICH) 是一种毁灭性的临床事件，其中破裂的血管将血液释放到大脑中。我们建议在两种小鼠 ICH 模型中测试 PGE2 EP1 和 EP3 受体抑制的功效，并阐明这种抑制提供神经保护的潜在机制。如果 EP1 和 EP3 受体抑制在两种小鼠 ICH 模型中显示出一致的神经保护作用，则可能有助于设计针对 ICH 的新型治疗干预措施。

DESCRIPTION (provided by applicant): PGE2 EP1 and EP3 receptors as therapeutic targets in intracerebral hemorrhage Intracerebral hemorrhage (ICH) is the stroke subtype with the highest mortality and morbidity; unfortunately, this condition has received far less research attention than has ischemic stroke. Inflammatory mechanisms, including those mediated by certain prostaglandins (PGs), have been suggested to contribute to the progression of ICH injury. In particular, PGE2, which is predominant in the brain, is produced and accumulates in the perihematomal region. PGE2 acts through four G-protein-coupled receptor subtypes known as EP1- EP4. Each of these receptors has distinct signaling cascades in animal models of cerebral ischemia. Genetic deletion or selective inhibition of the EP1 and EP3 receptors has been shown to reduce ischemic brain injury both in vivo and in vitro. The overall objective of this R01 is to investigate whether inhibition of PGE2 EP1 and EP3 receptors can be used as neuroprotective therapy in ICH, and if so, to generate preclinical efficacy data and elucidate the underlying mechanisms. We have been investigating the specific roles of PGE2 receptors in ICH injury and have preliminary data showing that deletion or inhibition of the EP1 or EP3 receptor reduces ICH injury and improves functional outcomes. Our data also indicate that Src kinase mediates EP1 toxicity and Rho kinase (ROCK) mediates EP3 toxicity after ICH. Consequently, we will test the hypothesis that PGE2 EP1 receptor blockade reduces ICH injury through the Src kinase signaling pathway, whereas EP3 receptor blockade reduces ICH injury through the ROCK signaling pathway. We have designed three specific aims that utilize the autologous blood ICH model and collagenase-induced ICH model. Considering that an increase in PGE2 production contributes to age-related dysfunction of the inflammatory responses and that aging and sex differences affect ICH outcomes, this research will be carried out in young male and female and aged male mice to enhance the clinical relevance. Aim 1 will determine whether inhibition of EP1 or EP3 receptors improves ICH outcomes; Aim 2 will determine whether inhibition of Src kinase signaling contributes to the neuroprotection afforded by EP1 receptor blockade after ICH and whether EP1 receptor inhibition decreases thrombin-induced brain damage; and Aim 3 will determine whether inhibition of ROCK signaling contributes to the neuroprotection afforded by EP3 receptor blockade after ICH and whether EP3 receptor inhibition decreases thrombin-induced brain damage. Through pharmacologic, genetic, imaging, histologic, molecular, and cellular biologic approaches, this work will provide novel information about the efficacy of selective PGE2 EP1 and EP3 receptor inhibition to protect against ICH-induced brain injury and the mechanisms by which such protection occurs. This proof-of-concept information is required to plan more detailed preclinical assessment of selective EP1 and EP3 receptor antagonists in ICH. PUBLIC HEALTH RELEVANCE: Intracerebral hemorrhage (ICH) is a devastating clinical event in which a burst blood vessel releases blood into the brain. We propose to test the efficacy of PGE2 EP1 and EP3 receptor inhibition in two mouse ICH models and to elucidate the underlying mechanisms by which such inhibition provides neuroprotection. If EP1 and EP3 receptor inhibition shows consistent neuroprotection in the two mouse ICH models, it could contribute to the design of novel therapeutic interventions for ICH.