Müller K-C, Welker L, Paasch K, Feindt B, Erpenbeck V J, Hohlfeld J M, Krug N, Nakashima M, Branscheid D, Magnussen H, Jörres R A, Holz O
Hospital Grosshansdorf, Center for Pneumology and Thoracic Surgery, D-22927 Grosshansdorf, Germany.
Respir Res. 2006 Feb 21;7(1):32. doi: 10.1186/1465-9921-7-32.
The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD.
Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant.
The median (quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor > or = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema.
These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.
肺泡壁的丧失是肺气肿的一个标志。由于成纤维细胞在维持肺泡结构中起重要作用，成纤维细胞表型的改变可能参与了该疾病的发病机制。在先前的一项研究中，我们发现肺气肿患者肺实质成纤维细胞的体外增殖率和群体倍增数降低，并且我们推测这些发现可能与这些细胞的过早细胞衰老有关。因此，在本研究中，我们比较了肺气肿患者和无慢性阻塞性肺疾病（COPD）的对照患者的肺成纤维细胞之间的细胞衰老标志物和相应基因的表达。
从13例中度至重度肺气肿患者（E组）和15例接受肺肿瘤切除或减容手术的对照患者（C组，n = 2）中获取原代肺成纤维细胞。对成纤维细胞（8例E组/9例C组）进行衰老相关β-半乳糖苷酶（SA-β-Gal）染色。在独立培养中，评估肺成纤维细胞（7例E组/8例C组）的DNA的平均端粒长度。使用两个探索性的12k cDNA微阵列评估混合成纤维细胞（3例E组/3例C组）中的基因表达。随后，通过定量PCR（qPCR）评估个体患者成纤维细胞（10例E组/病例C组）中选定基因的表达，并分析细胞培养上清液中的蛋白质浓度。
SA-β-Gal阳性的成纤维细胞的中位数（四分位数）百分比在对照组中为4.4（3.2；4.7）%，在肺气肿组中为16.0（10.0；24.8）%（p = 0.001），而端粒长度无差异。在阵列中差异表达基因的候选基因中（因子>或= 3），15个在肺气肿中上调而121个下调。qPCR证实胰岛素样生长因子结合蛋白（IGFBP）-3和IGFBP-rP1上调（p = 0.029，p = 0.0002），而IGFBP-5、-rP2（结缔组织生长因子）、-rP4（Cyr61）、FOSL1、LOXL2、OAZ1和CDK4的表达在两组之间无差异。与基因表达一致，我们发现在肺气肿中细胞培养上清液中IGFBP-3的浓度增加（p = 0.006）。
这些数据支持以下假设，即肺气肿中肺成纤维细胞通过不依赖端粒的机制发生过早衰老。肺气肿中衰老相关的IGFBP-3和-rP1的上调表明胰岛素和胰岛素样生长因子作用的抑制可能参与了体外增殖率的降低。
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