Department of Internal Medicine, Justus-Liebig University (JLU) Giessen, Giessen, Hessen, Germany.
Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Centre for Lung Research (DZL), Giessen, Hessen, Germany.
Clin Transl Med. 2022 Jul;12(7):e935. doi: 10.1002/ctm2.935.
Exaggerated fibroblast proliferation is a well-known feature in idiopathic pulmonary fibrosis (IPF) which may be - in part - due to insufficient autophagy, a lysosome dependent cellular surveillance pathway. Bcl2-associated athanogene 3 (BAG3) is a pivotal co-chaperone of the autophagy pathway. Here, we studied whether therapeutic modulation of BAG3-mediated autophagy can rescue insufficient autophagy and impact IPF fibroblast proliferation.
Primary interstitial fibroblasts or precision cut lung slices (PCLS) of IPF lungs were treated with (1) the antifibrotic drug pirfenidone (Pirf), (2) the demethylating agent 5-azacytidine (Aza), (3) the BAG3 modulator cantharidin (Ctd). Autophagy flux was measured following pretreatment with the autophagy inhibitors or by GFP-RFP-LC3B transfection followed by drug treatments. Proliferation was measured by 5-bromo-2'-deoxyuridine assay. BAG3, filamin C (FLNC), proliferating-cell-nuclear-antigen (PCNA), collagen1A1 (COL1A1) and autophagy proteins were assessed by immunoblotting or immunofluorescence. Loss of function experiments were performed by siRNA mediated knockdown of BAG3.
In comparison with healthy donors, increased BAG3 protein was observed in IPF lung homogenates and IPF fibroblasts. In addition, the substrate of BAG3-mediated autophagy, FLNC, was increased in IPF fibroblasts, implying insufficient activation of BAG3-dependent autophagy. Therapeutic modulation of this pathway using Aza and Ctd alone or in combination with the IPF therapy drug Pirf rescued the insufficient BAG3-mediated autophagy and decreased fibroblast proliferation. Such effects were observed upon therapeutic modulation of BAG3 but not upon knock down of BAG3 per se in IPF fibroblasts. Similarly, PCLS of IPF patients showed a significant decrease in collagen deposition in response to these drugs, either alone or in a more potent form in combination with Pirf.
Our study reveals that repurposing drugs that modulate autophagy regulating proteins render therapeutic benefits in IPF. Fine tuning of this pathway may hence signify a promising therapeutic strategy to ameliorate antifibrotic properties and augment the efficacy of current IPF therapy.
过度的成纤维细胞增殖是特发性肺纤维化（IPF）的一个显著特征，这可能部分归因于自噬不足，这是一种依赖溶酶体的细胞监视途径。Bcl2 相关抗凋亡基因 3（BAG3）是自噬途径的关键伴侣蛋白。在这里，我们研究了 BAG3 介导的自噬的治疗调节是否可以挽救自噬不足并影响 IPF 成纤维细胞增殖。
用（1）抗纤维化药物吡非尼酮（Pirf）、（2）去甲基化剂 5-氮杂胞苷（Aza）、（3）BAG3 调节剂斑蝥素（Ctd）处理原代肺间质成纤维细胞或 IPF 肺的精密切割肺切片（PCLS）。在用自噬抑制剂预处理后或用 GFP-RFP-LC3B 转染后用药物处理，测量自噬通量。通过 5-溴-2'-脱氧尿苷测定法测量增殖。通过免疫印迹或免疫荧光法评估 BAG3、细丝蛋白 C（FLNC）、增殖细胞核抗原（PCNA）、胶原 1A1（COL1A1）和自噬蛋白。通过 BAG3 的 siRNA 介导的敲低进行功能丧失实验。
与健康供体相比，IPF 肺匀浆和 IPF 成纤维细胞中观察到 BAG3 蛋白增加。此外，IPF 成纤维细胞中 BAG3 介导的自噬的底物 FLNC 增加，暗示 BAG3 依赖性自噬的激活不足。使用 Aza 和 Ctd 单独或与 IPF 治疗药物 Pirf 联合治疗来调节这种途径可挽救不足的 BAG3 介导的自噬并减少成纤维细胞增殖。在 IPF 成纤维细胞中，这种作用是通过治疗性调节 BAG3 而不是通过 BAG3 本身的敲低观察到的。同样，IPF 患者的 PCLS 对这些药物的胶原沉积也表现出显著减少，无论是单独使用还是与 Pirf 联合使用时效果更显著。
我们的研究表明，重新利用调节自噬调节蛋白的药物可在 IPF 中提供治疗益处。对这种途径的微调可能是改善抗纤维化特性和增强当前 IPF 治疗效果的有前途的治疗策略。
