Aging and ARDS: Novel Mechanistic Role of Nox4/D in Age-Dependent Barrier Dysfunction

急性呼吸窘迫综合征（ARDS）的死亡率仍然高得令人无法接受（35-40%）， 也没有FDA批准的药物治疗大量数据表明，ARDS患者 老年人的结果明显更差。然而，我们还不了解 与年龄相关的易感性的机制。NADPH氧化酶（Nox）是 活性氧（ROS）产生和信号传导的重要细胞来源。我们之前 表明Nox 4依赖的ROS在介导内皮细胞（EC）屏障中起关键作用 在肺损伤急性反应期间的功能。然而，越来越多的证据表明， 过量的Nox 4衍生的ROS和许多与年龄相关的疾病。我们已经证明，在 老年小鼠对肺损伤的反应表现出持续升高的Nox 4/ROS水平， 对严重临床前ARDS的易感性和未能解决损伤。我们发现了高度发散的 在“年轻”对照与衰老EC中Nox 4/ROS诱导;对照EC表现出快速和短暂的 Nox 4的诱导，而衰老的EC表现出持续升高的Nox 4/ROS ROS水平， 屏障功能受损。我们发现了一种新的机制，通过这种机制，Nox 4的表达可以迅速改变， 在内皮细胞中通过翻译后修饰。此外，衰老的内皮细胞表现出缺陷的泛素介导的 Nox 4降解，促进持续的Nox 4/ROS水平。最后，我们发现了一个小分子 Nox 4的重剪接变体（Nox 4D）仅在具有严重临床前病变的老年小鼠的肺中表达 在3个不同队列的ARDS患者的肺中。最后，靶向Nox 4/D依赖性ROS 在衰老/老化中提供了最显著的治疗益处（与年轻相比），并且可能是 改善老年ARDS患者预后的关键。拟议的研究将检验中心假设 年龄依赖性的持续Nox 4/D表达促进氧化还原失衡，导致衰老- 相关的屏障破坏性EC表型和更大的炎性损伤，最终导致 老年人对ARDS的易感性增加和缓解受损。目标1研究将确定 Nox 4介导年龄依赖性屏障破坏性EC表型和临床前ARDS的严重程度/缓解 目的2研究Nox 4D在ARDS患者中的表达及其在ALI中的作用。 目的3研究Nox 4/D在衰老细胞中的持续存在的翻译后机制， EC。这些研究将为深入了解Nox 4/D在调节EC屏障和炎症反应中的作用提供线索。 反应，以及Nox 4/D的年龄依赖性功能障碍如何有助于ARDS发病机制。这些研究 将提供一种新的机制联系，可能有助于解释ARDS发病率和死亡率的增加 与老年人相关，并将为治疗开发提供概念验证。

The mortality rate for acute respiratory distress syndrome (ARDS) remains unacceptably high (35-40%), and there are no FDA-approved drug treatments. Overwhelming data demonstrate that ARDS patient outcomes are significantly worse among the elderly population. However, we do not yet understand the mechanisms that account for this age-associated predisposition. The NADPH oxidase (Nox) enzymes are an important cellular source of reactive oxygen species (ROS) generation and signaling. We previously demonstrated that Nox4-dependent ROS plays a critical role in mediating endothelial cell (EC) barrier function during acute responses to lung injury. However, a growing body of evidence supports a link between excessive Nox4-derived ROS and numerous age-related diseases. We have demonstrated that in response to lung injury, aged mice exhibit persistently elevated Nox4/ROS levels that result in susceptibility to severe pre-clinical ARDS and failure to resolve injury. We discovered highly divergent induction of Nox4/ROS in “young” control vs. senescent ECs; control ECs exhibit a rapid and transient induction of Nox4, whereas senescent ECs exhibit persistently elevated levels of Nox4/ROS ROS leading to impaired barrier function. We identified a novel mechanism by which Nox4 expression can be rapidly altered in ECs via post-translational modification. Further, senescent ECs exhibit defective ubiquitin-mediated degradation of Nox4, which promotes sustained Nox4/ROS levels. Finally, we identified a small molecular weight splice variant of Nox4 (Nox4D) to be expressed only in the lungs of aged mice with severe pre-clinical ARDS and in the lungs of ARDS patients from 3 different cohorts. Finally, targeting of Nox4/D-dependent ROS in senescence/aging provided the most significant therapeutic benefit (as compared to young), and may be the key to improving outcomes for elderly ARDS patients. The proposed studies will test the central hypothesis that age-dependent persistent Nox4/D expression promotes redox imbalance leading to senescence- associated barrier disruptive EC phenotypes and greater inflammatory injury, ultimately resulting in the increased susceptibility and impaired resolution of ARDS in aging. Aim 1 studies will determine the role of Nox4 in mediating age-dependent barrier disruptive EC phenotypes and severity/resolution of pre-clinical ARDS. Aim 2 studies will investigate the association of Nox4D in ARDS patients and its functional role in ALI in vivo. Aim 3 studies will evaluate post-translational mechanisms contributing to the persistence of Nox4/D in senescent ECs. The proposed studies will provide insight into the roles of Nox4/D in regulating EC barrier and inflammatory responses, and how age-dependent dysfunction of Nox4/D contributes to ARDS pathogenesis. These studies will offer a novel mechanistic link that may help to explain the increased incidence and mortality of ARDS associated with the elderly and will provide proof-of-concept for therapeutic development.