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

描述(由申请人提供)： 急性肝功能衰竭(ALF)的主要和潜在的致死性并发症，治疗方法有限。一般认为，氨水是ALF中的水肿的主要原因，这种水肿主要是“细胞毒性”的，即星形胶质细胞肿胀。除了氨之外，还出现了 证据还表明，其他ALF相关毒素细胞因子(CKS)和脂多糖(LPS)，继发于脓毒症或肝坏死，可产生炎症反应，可能加剧氨对ALF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，也可能影响间接导致星形胶质细胞肿胀的其他神经细胞。一个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(即氨、脂多糖、Cks)影响的细胞。此外，内皮细胞与星形胶质细胞的接近使其处于战略地位，在正常和病理条件下，它们可以很容易地影响星形胶质细胞。Toll样受体-4(TLR4)是内毒素和CKs影响内皮细胞的一个主要因素。然而，氨影响内皮细胞的方式尚不清楚。我们的主要假设是：(A)氨上调/激活内皮细胞中的TLR4，这种上调/激活导致细胞肿胀介质的产生，从而导致ALF星形胶质细胞肿胀/脑水肿；(B)内毒素和CKS通过与氨相似的机制加重ALF星形胶质细胞肿胀/脑水肿。为了支持我们的假设，我们产生的初步数据显示，加入氨处理的内皮细胞的条件培养液(CM)会导致星形胶质细胞肿胀；当将经ALF相关毒素(氨、Cks、内毒素)组合处理的ECs的CM加入星形胶质细胞时，这种肿胀明显增强。氨水可上调TLR4的表达，而抑制TLR4的内皮细胞的CM可减少星形胶质细胞的肿胀，提示内皮TLR4在内皮细胞CM引起的星形胶质细胞肿胀中起关键作用。同样，TLR4基因缺陷的转基因小鼠(TG)可以部分保护ALF中脑水肿的发生。此外，氨暴露于内皮细胞的CM可导致ALF星形胶质细胞Na+，K+，Cl-共转运体(NKCC)和水通道蛋白4(AQP4)表达上调，这是ALF星形胶质细胞肿胀/脑水肿机制中的倒数第二个事件。这项建议的总体目标是研究内皮细胞在ALF中促进星形胶质细胞肿胀/脑水肿的机制以及TLR4在这一过程中的关键作用。目的#1研究氨在TLR4上调/激活中的作用；氨上调TLR4的机制，以及其他ALF相关毒素(LPS和Cks)对TLR4上调的潜在相加/协同作用。目的#2将研究TLR4促进星形胶质细胞肿胀的途径；TLR4在激活内皮细胞NF-6B中的作用；以及 TLR4参与内皮细胞肿胀介质的形成和释放。目的#3从氨水诱导的星形胶质细胞肿胀机制中的倒数第二个因素--NKCC和AQP4调节星形胶质细胞核因子-6B的活性，探讨氨水处理内皮细胞的CM引起星形胶质细胞肿胀的机制。目的#4将确定在特定的目标1-3中确定的哪些机制与培养中的星形胶质细胞肿胀有关，也与体内的脑水肿有关。此外，我们将使用TLR4-KO小鼠来确定内皮细胞TLR4在ALF脑水肿中的作用。我们预计，这一提议的成功结果不仅将促进我们对ALF脑水肿发病机制的了解，还将导致旨在缓解这一潜在致命疾病的新的治疗靶点的确定。

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.