Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration.尽管在多数哺乳动物中，衰老会导致肺切除术（pneumonectomy, PNX）后再生能力下降，但这一生物学现象尚未在小鼠的整个生命周期中得到系统性表征。本研究以雌性C57BL/6小鼠为对象，检测了不同年龄（3、9、24月龄）下肺切除术对小鼠生理状态、形态计量学指标、细胞增殖与凋亡、全基因表达谱，以及肺成纤维细胞表型与克隆形成能力的影响。研究数据显示，仅3月龄小鼠可在术后7天恢复肺容积，并于21天内完全恢复肺泡总表面积。9月龄小鼠的再生速率则有所减缓（术后21天仍未完成再生），而24月龄小鼠在肺切除术后21天完全无组织再生。再生速率的早期下降与II型肺泡上皮细胞（alveolar epithelial cell type II, AECII）的增殖或凋亡速率变化无关。但24月龄小鼠在肺切除术后，其总细胞与II型肺泡上皮细胞均出现显著凋亡，且细胞增殖受到明显抑制。对9月龄与3月龄小鼠肺切除术后1、3、7天的基因表达分析结果显示，9月龄小鼠呈现肌成纤维细胞特征（Tnc、Lox1、Col3A1、Eln及Tnfrsf12a表达上调），且术后α平滑肌肌动蛋白（alpha smooth muscle actin, αSMA）阳性的肌成纤维细胞数量多于3月龄小鼠。分离得到的肺成纤维细胞克隆形成能力随年龄增长显著下降。此外，从9月龄与17月龄小鼠体内分离的肺成纤维细胞，其αSMA、Col3A1、Fn1及S100A表达水平更高，而存活基因Mdk的表达水平更低，这与细胞发生终末分化的特征相符。上述数据表明，克隆形成能力的伴随性丧失与进行性肌成纤维细胞分化共同导致了肺再生速率随年龄增长而下降的现象。