Bernau Ksenija, Ngam Caitlyn, Torr Elizabeth E, Acton Benjamin, Kach Jacob, Dulin Nickolai O, Sandbo Nathan
Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA.
Department of Medicine, University of Chicago, Chicago, IL, USA.
Respir Res. 2015 Mar 27;16(1):45. doi: 10.1186/s12931-015-0206-6.
Fibrosing disorders of the lung, such as idiopathic pulmonary fibrosis, are characterized by progressive extracellular matrix accumulation that is driven by myofibroblasts. The transcription factor megakaryoblastic leukemia-1 (MKL1) mediates myofibroblast differentiation in response to several profibrotic stimuli, but the role it plays in mediating pulmonary fibrosis has not been fully elucidated. In this study, we utilized mice that had a germline deletion of MKL1 (MKL1 (-,-)) to determine the role that MKL1 plays in the development of bleomycin-induced pulmonary fibrosis.
Bleomycin or normal saline were intratracheally delivered to 9 to 12 week old female MKL1 (+,+) and MKL1 (-,-) mice. Mice were assessed for weight loss and survival to 28 days. Inflammatory responses were assessed through bronchoalveolar lavage at days 3 and 7 post-treatment. The development of pulmonary fibrosis was characterized using hydroxyproline assay and histological staining. MKL1 (+,+) and MKL1 (-,-) mouse lung fibroblasts were isolated to compare morphologic, gene expression and functional differences.
MKL1 (-,-) mice demonstrated increased survival, attenuated weight loss, and decreased collagen accumulation compared to wild-type animals 28-days after intratracheal instillation of bleomycin. Histological analysis demonstrated decreased trichrome, smooth muscle α-actin, and fibronectin staining in MKL1(-,-) mice compared to MKL1 (+,+) controls. Differential cell counts from bronchoalveolar lavage demonstrated that there was attenuated neutrophilia 3 days after bleomycin administration, but no difference at day 7. Isolated mouse lung fibroblasts from MKL1 (-,-) mice had decreased contractility and deposited less fibronectin matrix compared to wild-type controls, suggesting a defect in key remodeling functions.
Altogether, these data demonstrate that MKL1 plays a significant role in mediating the fibrotic response to bleomycin injury. Loss of MKL1 attenuated early neutrophil influx, as well as myofibroblast-mediated remodeling. Targeting MKL1 activity may therefore be a useful strategy in treating pulmonary fibrosis.
肺部纤维化疾病，如特发性肺纤维化，其特征是由肌成纤维细胞驱动的细胞外基质进行性积累。转录因子巨核细胞白血病-1（MKL1）介导肌成纤维细胞对多种促纤维化刺激的分化，但它在介导肺纤维化中所起的作用尚未完全阐明。在本研究中，我们利用MKL1基因敲除小鼠（MKL1(-,-)）来确定MKL1在博来霉素诱导的肺纤维化发展过程中所起的作用。
将博来霉素或生理盐水经气管内注入9至12周龄的雌性MKL1(+/+)和MKL1(-,-)小鼠体内。评估小鼠体重减轻情况及28天的生存率。在治疗后第3天和第7天通过支气管肺泡灌洗评估炎症反应。使用羟脯氨酸测定法和组织学染色来表征肺纤维化的发展。分离MKL1(+/+)和MKL1(-,-)小鼠的肺成纤维细胞，比较形态学、基因表达和功能差异。
与野生型动物相比，在气管内注入博来霉素28天后，MKL1(-,-)小鼠显示出更高的生存率、减轻的体重减轻以及减少的胶原蛋白积累。组织学分析表明，与MKL1(+/+)对照组相比，MKL1(-,-)小鼠的三色染色、平滑肌α-肌动蛋白和纤连蛋白染色减少。支气管肺泡灌洗的差异细胞计数表明，博来霉素给药后3天中性粒细胞增多有所减轻，但在第7天无差异。与野生型对照相比，从MKL1(-,-)小鼠分离的肺成纤维细胞收缩性降低，纤连蛋白基质沉积减少，表明关键重塑功能存在缺陷。
总之，这些数据表明MKL1在介导对博来霉素损伤的纤维化反应中起重要作用。MKL1的缺失减弱了早期中性粒细胞流入以及肌成纤维细胞介导的重塑。因此，靶向MKL1活性可能是治疗肺纤维化的一种有用策略。
