Jansing Nicole L, McClendon Jazalle, Henson Peter M, Tuder Rubin M, Hyde Dallas M, Zemans Rachel L
1 Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, and.
2 Program in Cell Biology, Department of Pediatrics, National Jewish Health, Denver, Colorado.
Am J Respir Cell Mol Biol. 2017 Nov;57(5):519-526. doi: 10.1165/rcmb.2017-0037MA.
The alveolar epithelium consists of squamous alveolar type (AT) I and cuboidal ATII cells. ATI cells cover 95-98% of the alveolar surface, thereby playing a critical role in barrier integrity, and are extremely thin, thus permitting efficient gas exchange. During lung injury, ATI cells die, resulting in increased epithelial permeability. ATII cells re-epithelialize the alveolar surface via proliferation and transdifferentiation into ATI cells. Transdifferentiation is characterized by down-regulation of ATII cell markers, up-regulation of ATI cell markers, and cell spreading, resulting in a change in morphology from cuboidal to squamous, thus restoring normal alveolar architecture and function. The mechanisms underlying ATII to ATI cell transdifferentiation have not been well studied in vivo. A prerequisite for mechanistic investigation is a rigorous, unbiased method to quantitate this process. Here, we used SPCCreERT2;mTmG mice, in which ATII cells and their progeny express green fluorescent protein (GFP), and applied stereologic techniques to measure transdifferentiation during repair after injury induced by LPS. Transdifferentiation was quantitated as the percent of alveolar surface area covered by ATII-derived (GFP) cells expressing ATI, but not ATII, cell markers. Using this methodology, the time course and magnitude of transdifferentiation during repair was determined. We found that ATI cell loss and epithelial permeability occurred by Day 4, and ATII to ATI cell transdifferentiation began by Day 7 and continued until Day 16. Notably, transdifferentiation and barrier restoration are temporally correlated. This methodology can be applied to investigate the molecular mechanisms underlying transdifferentiation, ultimately revealing novel therapeutic targets to accelerate repair after lung injury.
肺泡上皮由扁平的肺泡I型（AT）细胞和立方状的ATII型细胞组成。AT I型细胞覆盖95%-98%的肺泡表面，因此在屏障完整性方面发挥着关键作用，且极其薄，从而允许高效的气体交换。在肺损伤期间，AT I型细胞死亡，导致上皮通透性增加。AT II型细胞通过增殖和转分化为AT I型细胞使肺泡表面重新上皮化。转分化的特征是AT II型细胞标志物下调、AT I型细胞标志物上调以及细胞铺展，导致形态从立方状变为扁平状，从而恢复正常的肺泡结构和功能。在体内，AT II型细胞向AT I型细胞转分化的机制尚未得到充分研究。进行机制研究的一个先决条件是要有一种严谨、无偏的方法来量化这个过程。在这里，我们使用了SPCCreERT2;mTmG小鼠，其中AT II型细胞及其后代表达绿色荧光蛋白（GFP），并应用体视学技术来测量脂多糖诱导损伤后修复过程中的转分化。转分化被量化为表达AT I型而非AT II型细胞标志物的AT II型来源（GFP）细胞覆盖的肺泡表面积百分比。使用这种方法，确定了修复过程中转分化的时间进程和程度。我们发现，到第4天出现AT I型细胞丢失和上皮通透性增加，到第7天开始出现AT II型细胞向AT I型细胞的转分化，并持续到第16天。值得注意的是，转分化和屏障恢复在时间上是相关的。这种方法可用于研究转分化的分子机制，最终揭示加速肺损伤后修复的新治疗靶点。
