Laminin-5 in Lung Develooment and Disease

基底膜（BM）是肺发育和肺损伤后正常肺泡结构恢复所必需的。然而，在牙槽骨损伤和修复的背景下，对牙槽骨的了解甚少。层粘连蛋白是整合的BM糖蛋白。层粘连蛋白-5（Ln-5）由层粘连蛋白链α 3、β 3和γ 2组成，通常影响上皮细胞结构和功能，并且存在于肺泡BM中，但其在肺发育和肺泡损伤中的具体作用尚不清楚。在Aim I中，我们将 使用由于靶向层粘连蛋白γ 2基因而导致Ln-5缺陷的小鼠研究Ln-5在肺发育和肺泡损伤中的作用。然而，层粘连蛋白γ 2基因完全敲除的小鼠在出生后5天内死亡，伴有粘膜和皮肤脱落。正因为如此，我们将产生肺特异性的，可诱导的层粘连蛋白γ 2缺陷小鼠。这些小鼠将用于检查Ln-5缺乏对肺发育后期的影响以及对各种肺泡损伤模型的反应，包括铜绿假单胞菌诱导的肺泡炎，博来霉素诱导的肺纤维化和香烟烟雾诱导的肺气肿。他们的肺将被评估的结构，细胞外基质基因的表达和炎症细胞的募集。目的二：探讨Ln-5对肺泡型上皮细胞的作用 (AEC)迁移这些研究将涉及从野生型小鼠和具有肺特异性、可诱导的层粘连蛋白72敲除的小鼠分离的AEC。在目标III中，使用如目标I和II中所述的体内和体外模型，我们将研究基质金属蛋白酶（MMPs）-2或-14缺陷的小鼠，以确定这些MMPs对肺泡损伤和AEC细胞迁移反应的作用。MMP-2和MMP-14是特别令人感兴趣的，因为已知它们通过对Ln-5的作用影响上皮细胞迁移，但它们在AEC功能中的作用尚未确定。由于中性粒细胞往往是突出的肺泡损伤，我们也将探讨中性粒细胞蛋白酶对Ln-5和AEC细胞功能的影响。本资助申请中提出的研究将提供有关肺泡BM的新信息， 在严重的、常见的肺部疾病中的重要结构，包括急性呼吸窘迫综合征、肺纤维化和肺气肿。

Basement membranes (BMs) are necessary for lung development and restoration of normal alveolar architecture when the lung has sustained damage. However, little is known about alveolar BMs in the context of alveolar damage and repair. Laminins are integral BM glycoproteins. Laminin-5 (Ln-5), comprised of laminin chains alpha3, beta3, and gamma2 affects epithelial cell structure and function generally, and is present in alveolar BMs, but its roles specifically in lung development and alveolar damage are unknown. In Aim I we will study the role of Ln-5 in lung development and alveolar damage using mice deficient in Ln-5 as a result of targeting the laminin gamma2 gene. However, mice with a global knockout of the laminin gamma2 gene die <5 days after birth with mucosal and cutaneous sloughing. Because of this we will generate lung-specific, inducible laminin gamma 2-deficient mice. These mice will be used to examine the effect of Ln-5 deficiency on the later stages of lung development and the response to various models of alveolar damage, including Pseudomonas aeruginosa-induced alveolitis, bleomycin-induced pulmonary fibrosis, and cigarette smoke-induced emphysema. Their lungs will be evaluated for structure, expression of extracellular matrix genes and inflammatory cell recruitment. In Aim II we will assess the effects of Ln-5 on alveolar type epithelial cell (AEC) migration. These studies will involve AECs isolated from wild type mice and mice with the lung- specific, inducible knockout of laminin 72. In Aim III, using in vivo and in vitro models like those described in Aims I and II, we will study mice with deficiencies of matrix metalloproteinases (MMPs) -2 or -14 to determine the roles of these MMPs on responses to alveolar injury and AEC cell migration. MMPs -2 and -14 are of particular interest because they are known to affect epithelial cell migration via effects upon Ln-5, but their role in AEC functions has not been determined. Because neutrophils are often prominent in alveolar injury we will also explore the effects of neutrophil proteinases upon Ln-5 and its effects on AEC cell functions. The studies proposed in this grant application will contribute new information about alveolar BMs, an important structure in serious, common lung disorders, including the acute respiratory distress syndrome, pulmonary fibrosis, and emphysema.