Calcineurin Inhibition: Lung Reperfusion Injury

描述(由申请人提供)： 大约25%的肺移植接受者和15%的 接受慢性肺栓塞症治疗的患者将发展为 手术肺缺血再灌注损伤(IRI)。治疗是保守的 而这一问题仍然是严重的发病率和死亡率的原因。 已确定了对几种促炎介质的要求。 然而，单一调解人的对抗很少能产生戏剧性的保护。 因此，使用定向抗体的临床治疗很可能是 昂贵、不切实际，而且略微有效。更有效的干预措施 将同时阻止多个调解人，可能是通过在 转录水平。钙调神经磷酸酶抑制核因子-kB的激活 可减少多种促炎介质的表达。自.以来 环孢素(CsA)和他克莫司(TAC)是钙调神经磷酸酶抑制剂， 已经在临床上可用的，最好是检查它们的效果 在实验性肺IRI中。CsA和TAC可减少其他组织中的IRI，并可以 在体外和体内持续降低核因子-kB的活性。在……里面 初步研究，我们已经可靠地建立了一种肺IRI的动物模型 结果表明，肺缺血再灌注后核因子-kB的激活是 与一些炎性细胞因子的出现和 趋化因子。因此，我们的假设是，钙调神经磷酸酶介导了核因子-kB。 肺IRI激活导致转录上调 关键的促炎介质与组织的最终发展 受伤。我们将从以下几个方面系统地阐述这一假设 体内干预性研究和机理分析 体内和体外。我们将追求三个相关目标：目标1：确定 钙调神经磷酸酶抑制是否对肺的发育有保护作用 IRI。我们将使用我们的动物模型来实现这一点。目标2：确定 钙调神经磷酸酶抑制是否导致转录下调 介质通过NFkB依赖机制参与肺IRI。我们会 衡量CSA/TAC提供的保护并评估相关更改 核蛋白中细胞因子的产生和NFkB。目标3：定义 肺IRI关键介质的细胞来源及其对细胞的依赖 钙调神经磷酸酶驱动转录和细胞间相互作用。我们会 通过培养肺泡巨噬细胞、II型肺泡细胞和 肺动脉内皮细胞(单独和组合)和 使它们处于低氧和复氧状态。

DESCRIPTION (provided by applicant): Approximately 25 percent of lung transplant recipients and 15 percent of patients treated for chronic pulmonary embolic disease will develop post- operative lung ischemia reperfusion injury (IRI). Treatment is conservative and this problem continues to account for significant morbidity and mortality. Requirements for several proinflammatory mediators have been characterized. However, antagonism of a single mediator rarely produces dramatic protection. Clinical treatment with directed antibodies is likely therefore to be expensive, impractical and marginally effective. More effective interventions would block several mediators at once, potentially by acting at the transcriptional level. Inhibition of calcineurin mediated activation of NF-kB can reduce expression of a variety of proinflammatory mediators. Since cyclosporin (CSA) and tacrolimus (TAC) are calcineurin inhibitors that are already available clinically, it would be desirable to examine their effects in experimental lung IRI. CSA and TAC reduce IRI in other tissues and can consistently reduce NF-kB activation both in vitro and in vivo. In preliminary studies, we have reliably established an animal model of lung IRI and shown that NF-kB activation follows lung IRI and that activation is associated with the appearance of a number of inflammatory cytokines and chemokines. Our hypothesis, therefore, is that calcineurin mediates NF-kB activation with lung IRI which results in transcriptional upregulation of critical pro-inflammatory mediators and the ultimate development of tissue injury. We will address this hypothesis systematically beginning with interventional studies in vivo and proceed to mechanistic analysis both in vivo and in vitro. We will pursue three related aims: Aim 1: To determine whether calcineurin inhibition is protective against the development of lung IRI. We will accomplish this using our animal model. Aim 2: To determine whether calcineurin inhibition results in transcriptional down-regulation of mediators involved in lung IRI through an NFkB-dependent mechanism. We will measure protection offered by CSA/TAC and assess associated changes in cytokine production and NFkB in nuclear protein. Aim 3: To define the cellular sources of critical mediators of lung IRI and their dependence on calcineurin driven transcription and cell-cell interactions. We will accomplish this by culturing alveolar macrophages , type II pneumocytes and pulmonary artery endothelial cells (individually and in combination) and subject them to hypoxia and reoxygenation.