Astrocyte swelling/Brain Edema in Acute Liver Failure: Role of Endothelial Cells

急性肝衰竭中的星形胶质细胞肿胀/脑水肿：内皮细胞的作用

• 批准号: 8443315
• 负责人: MICHAEL David NORENBERG
• 金额: --
• 依托单位: MIAMI VA HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8443315
• 关键词: Acute Liver Failure; Affect; Ammonia; Astrocytes; Blood; Brain Edema; Cells; Cerebrum; Complication; Conditioned Culture Media; Data; Development; Edema; Endothelial Cells; Event; Free Radicals; Generations; Goals; Individual; Inflammatory; Inflammatory Response; Intracranial Hypertension; Investigation; Ions; Knowledge; Life; Lipopolysaccharides; Liver diseases; Mediating; Mediator of activation protein; Mus; Neurons; Nitric Oxide; Outcome; Pathogenesis; Play; Positioning Attribute; Process; Production; Rattus; Reaction; Reducing Agents; Regulation; Research; Role; Secondary to; Sepsis; Series; Signal Pathway; Signal Transduction; Sodium-Potassium-Chloride Symporters; Swelling; Therapeutic; Toxin; Transgenic Mice; Up-Regulation; abstracting; aquaporin 4; brain cell; cytokine; cytotoxic; effective therapy; hepatic necrosis; in vivo; in vivo Model; new therapeutic target; protein expression; public health relevance; symporter; toll-like receptor 4; water channel

DESCRIPTION (provided by applicant): Project Summary/Abstract Brain edema and associated intracranial hypertension are major and potentially lethal complications of acute liver failure (ALF) that have limited therapeutic options It is generally believed that ammonia is largely responsible for the edema in ALF, and that the edema is principally "cytotoxic", i.e., due to astrocyte swelling. In addition to ammonia, emerging evidence also suggests other ALF-related toxins cytokines (CKs) and lipopolysaccharide (LPS), that are produced secondary to sepsis or liver necrosis, can generate an inflammatory response that may exacerbate the effect of ammonia on the astrocyte swelling and brain edema in ALF. Thus, ammonia and ALF-related toxins may directly impact astrocytes, or may influence other neural cells that indirectly contribute to astrocyte swelling. One likely candidate cell is the endothelial cell (EC), as this is the first cell to be affected by blood-borne ALF-related toxins (i.e., ammonia, LPS, CKs). Moreover, the close proximity of ECs to astrocytes places ECs in a strategic position whereby they can readily influence astrocytes under normal and pathological conditions. A major factor through which LPS and CKs influence ECs is the Toll-like receptor-4 (TLR4). However, the means by which ammonia impacts ECs is not known. Our overarching hypothesis is that (a) ammonia upregulates/activates TLR4 in ECs and such upregulation/ activation results in the generation of cell swelling mediators that contribute to the astrocyte swelling/brain edema in ALF, and (b) that LPS and CKs exacerbate the astrocyte swelling/brain edema in ALF through mechanisms similar to those of ammonia. In support of our hypothesis, we have generated preliminary data showing that the addition of conditioned media (CM) from ECs treated with ammonia results in astrocyte swelling; such swelling was markedly potentiated when CM from ECs treated with a combination of ALF- related toxins (ammonia, CKs, LPS) were added to astrocytes. Ammonia upregulated TLR4, whereas CM from ECs in which TLR4 was silenced and then added to astrocytes resulted a reduction in cell swelling, suggesting a critical role of endothelial TLR4 in the astrocyte swelling caused by CM from ECs. Likewise, transgenic mice (Tg) deficient in TLR4 were partially protected from the development of brain edema in ALF. Additionally, CM from ECs exposed to ammonia resulted in the upregulation of astrocytic Na+,K+, Cl- co-transporter (NKCC) and aquaporin-4 (AQP4), factors that represent penultimate events in the mechanism of astrocyte swelling/brain edema in ALF. The overall goal of this proposal is to investigate mechanisms by which ECs contribute to astrocyte swelling/brain edema in ALF and the critical role of TLR4 in that process. Aim #1 will examine the effect of ammonia in the upregulation/activation of TLR4; mechanisms by which ammonia upregulates TLR4, as well as potential additive/synergistic effects by other ALF-related toxins (LPS and CKs) on TLR4 upregulation. Aim #2 will examine the means by which TLR4 contributes to astrocyte swelling; the role of TLR4 in the activation of endothelial NF-6B; and the involvement of TLR4 in the formation and release of cell swelling mediators in ECs. Aim #3 will investigate mechanisms by which CM from ammonia-treated ECs cause astrocyte swelling by examining the effect of CM on activation of astrocytic NF-6B and regulation of NKCC and AQP4, the latter two representing penultimate factors in the mechanism of ammonia-induced astrocyte swelling. Aim #4 will establish which of the mechanisms identified in Specific Aims 1-3 that are involved in astrocyte swelling in culture, also contribute to the brain edema in vivo. Additionally, we will employ TLR4-KO mice to establish the role of endothelial TLR4 in the brain edema of ALF. We anticipate that a successful outcome of this proposal will not only advance our knowledge on the pathogenesis of the brain edema in ALF, but will also result in the identification of novel therapeutic targets aimed at alleviating this potentially lethal condition.

描述（由申请人提供）： 脑水肿和相关的颅内高压是急性肝功能衰竭（ALF）的主要和潜在致命的并发症，其具有有限的治疗选择。通常认为，氨是ALF中水肿的主要原因，并且水肿主要是“细胞毒性的”，即，因为星形胶质细胞肿胀除了氨， 证据还提示继发于脓毒症或肝坏死的其它ALF相关毒素细胞因子（CK）和脂多糖（LPS）可产生炎性反应，其可加剧氨对ALF中星形胶质细胞肿胀和脑水肿的影响。因此，氨和ALF相关毒素可能直接影响星形胶质细胞，或可能影响间接导致星形胶质细胞肿胀的其他神经细胞。一种可能的候选细胞是内皮细胞（EC），因为这是第一个受到血液传播的ALF相关毒素影响的细胞（即，氨、LPS、CK）。此外，EC与星形胶质细胞的紧密接近使EC处于战略地位，从而它们可以在正常和病理条件下容易地影响星形胶质细胞。Toll样受体4（TLR 4）是LPS和CK影响内皮细胞的主要因素。然而，氨影响EC的方式尚不清楚。我们的首要假设是：（a）氨上调/激活EC中的TLR 4，这种上调/激活导致细胞肿胀介质的产生，这些介质有助于ALF中的星形胶质细胞肿胀/脑水肿，以及（B）LPS和CK通过与氨类似的机制加剧ALF中的星形胶质细胞肿胀/脑水肿。为了支持我们的假设，我们已经产生了初步数据，显示添加来自用氨处理的EC的条件培养基（CM）导致星形胶质细胞肿胀;当将来自用ALF相关毒素（氨、CK、LPS）的组合处理的EC的CM添加到星形胶质细胞中时，这种肿胀显著增强。氨上调TLR 4，而CM从EC中TLR 4沉默，然后添加到星形胶质细胞导致细胞肿胀减少，这表明内皮细胞TLR 4在星形胶质细胞肿胀由CM从EC引起的关键作用。同样地，TLR 4缺陷的转基因小鼠（Tg）在ALF中部分免受脑水肿的发展。此外，来自暴露于氨的EC的CM导致星形胶质细胞Na+、K+、Cl-共转运蛋白（NKCC）和水通道蛋白-4（AQP 4）的上调，这些因子代表ALF中星形胶质细胞肿胀/脑水肿机制中的倒数第二个事件。本提案的总体目标是研究ECs促进ALF中星形胶质细胞肿胀/脑水肿的机制以及TLR 4在该过程中的关键作用。目的#1将检查氨在TLR 4的上调/活化中的作用;氨上调TLR 4的机制，以及其他ALF相关毒素（LPS和CK）对TLR 4上调的潜在累加/协同作用。目的#2将检查TLR 4促进星形胶质细胞肿胀的方式; TLR 4在内皮NF-6 B活化中的作用;以及TLR 4在星形胶质细胞中的作用。 TLR 4参与EC中细胞肿胀介质的形成和释放。目的#3将通过检查CM对星形胶质细胞NF-6 B活化以及NKCC和AQP 4调节的影响来研究来自氨处理的EC的CM引起星形胶质细胞肿胀的机制，后两个代表氨诱导的星形胶质细胞肿胀机制中的倒数第二个因素。目的#4将确定特定目的1-3中确定的参与培养物中星形胶质细胞肿胀的机制中的哪些机制也有助于体内脑水肿。此外，我们将使用TLR 4-KO小鼠来建立内皮TLR 4在ALF脑水肿中的作用。我们预计，这一建议的成功结果不仅将推进我们对ALF脑水肿发病机制的认识，而且还将导致识别旨在减轻这种潜在致命疾病的新治疗靶点。