Molecular regulation of pulmonary tight junctions

描述(由申请人提供)：急性呼吸窘迫综合征(ARDS)的特征是终末肺泡上皮功能受损，导致空域泛滥、呼吸衰竭和死亡率增加。虽然在过去的40年里，人们对ARDS的病理生理学有了很多了解，但死亡率仍然高达~50%，令人无法接受。存活的ARDS与维持肺液清除的能力相关，而这反过来又需要肺内健康的气液屏障。这种屏障在肺泡上皮细胞的大部分紧密连接中维持，细胞-细胞接触部位的结构选择性地形成了 可渗透密封，用于调节水、离子和溶质的细胞旁扩散。我的实验室已经确定了被称为claudins的蛋白质在调节肺泡上皮紧密连接通透性中的特定作用。然而，在急性肺损伤(ALI)和ARDS中，克拉丁调节失调对下游的影响还没有完全阐明。此外，长期酗酒会加重ARDS的发病率和严重程度。我们发现，长期饮食酒精摄入改变了肺泡上皮细胞claudin的表达，这与屏障功能受损有关。在这里，我的实验室开发的分子工具，结合培养的细胞和损伤模型系统，将被用来了解claudin失调如何影响ARDS在ALI反应中的进展。该项目将确定肺泡上皮claudins的机制作用，包括与改善肺液清除相关的claudin-4和claudin-18，以及在酒精肺中上调并与肺泡屏障功能受损相关的claudin-5。已开发出一系列腺载体，可在体内通过培养的原代肺泡上皮细胞和肺上皮细胞操纵claudin的表达。这一方法将被用来测量增加claudin-4、claudin-5或claudin-18表达对肺泡上皮屏障功能的影响。增加claudin-4、claudin-5或claudin-18对体内肺液清除的影响将被评估，并与ALI期间紧密连接成分的变化相关联，这是由于两种不同的模型系统：1)气管内给药博莱霉素和2)盲肠结扎和穿孔诱导脓毒症。我们还将评估claudin-4和claudin-18逆转酒精肺有害影响的能力。 加重ALI病理后果的综合征。在不同的ALI模型系统中，测量肺泡克拉丁成分改变对紧密连接通透性和周转的影响将被用于评估治疗策略，以增强肺泡屏障功能和降低ARDS的易感性。

DESCRIPTION (provided by applicant): Acute Respiratory Distress Syndrome (ARDS) is characterized by compromised epithelial function in the terminal alveolus leading to airspace flooding, respiratory failure, and increased mortality. While much has been learned about the pathophysiology of ARDS in the past four decades, the mortality rate remains unacceptably high at ~50%. Surviving ARDS correlates with the ability to maintain lung fluid clearance which, in turn, requires a healthy air-liquid barrier in the lung. This barrier is maintained in large part b alveolar epithelial tight junctions, structures at cell-cell contact sites which form a selectively permeable seal to regulate paracellular diffusion of water, ions and solutes. My laboratory has identified specific roles for proteins known as claudins in regulating alveolar epithelial tight junction permeability. However, the downstream effects of claudin dysregulation in response to acute lung injury (ALI) and ARDS have not been fully elucidated. In addition, chronic alcohol abuse worsens the incidence and severity of ARDS. We have found that chronic dietary alcohol ingestion alters alveolar epithelial claudin expression which correlates with impaired barrier function. Here, molecular tools developed by my laboratory combined with cultured cell and model injury systems will be used to understand how claudin dysregulation affects the progression of ARDS in response to ALI. The project will define mechanistic roles for alveolar epithelial claudins, including claudin-4 and claudin-18 which correlate with improved lung fluid clearance and claudin-5 which is upregulated in the alcoholic lung and associates with impaired alveolar barrier function. A series of adenovectors were developed which allows manipulation of claudin expression by cultured primary alveolar epithelial cells and lung epithelia in vivo. This approach will be used to measure the effects of increasing claudin-4, claudin-5 or claudin-18 expression on alveolar epithelial barrier function. The effects of increasing claudin-4, claudin-5 or claudin-18 on lung fluid clearance in vivo will be assessed and correlated changes to tight junction composition during ALI due to two distinct model systems: 1) intratracheal administration of bleomycin and 2) cecal ligation and puncture to induce sepsis. We will also assess the ability of claudin-4 and claudin-18 to reverse the deleterious effects of alcoholic lung syndrome which exacerbate the pathologic consequences of ALI. Measuring the effects of altered alveolar claudin composition on tight junction permeability and turnover in diverse ALI model systems will be used to evaluate therapeutic strategies with the potential to strengthen alveolar barrier function and lessen susceptibility to ARDS.