Small molecule inhibition of IRE1α kinase/RNase has anti-fibrotic effects in the lung

AbstractEndoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor that promotes apoptosis if ER stress remains high (i.e., a “terminal” UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the importance of IRE1α activation in bleomycin-induced pulmonary fibrosis. One such KIRA—KIRA7—provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.

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Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00082.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. L529-L536 ◽

Cited By ~ 58

Author(s):

Amiq Gazdhar ◽

Patrick Fachinger ◽

Coretta van Leer ◽

Jaroslaw Pierog ◽

Mathias Gugger ◽

...

Keyword(s):

Growth Factor ◽

Gene Transfer ◽

Pulmonary Fibrosis ◽

Wound Repair ◽

Alveolar Epithelial Cells ◽

Epithelial Repair ◽

Alveolar Epithelial ◽

Hepatocyte Growth

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-β1 (TGF-β1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-β1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.

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Protective Effect of Remdesivir Against Pulmonary Fibrosis in Mice

Frontiers in Pharmacology ◽

10.3389/fphar.2021.692346 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiaohe Li ◽

Rui Liu ◽

Yunyao Cui ◽

Jingjing Liang ◽

Zhun Bi ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Antiviral Drug ◽

Alveolar Epithelial Cells ◽

Lung Damage ◽

Lung Fibroblasts ◽

Mesenchymal Transition ◽

Alveolar Epithelial ◽

Tgf Β1

Pulmonary fibrosis is a known sequela of severe or persistent lung damage. Existing clinical, imaging and autopsy studies have shown that the lungs exhibit a pathological pulmonary fibrosis phenotype after infection with coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Pulmonary fibrosis may be one of the most serious sequelae associated with coronavirus disease 2019 (COVID-19). In this study, we aimed to examine the preventative effects of the antiviral drug remdesivir on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of remdesivir on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of remdesivir in lung fibroblasts and alveolar epithelial cells in vitro. The preventive remdesivir treatment was started on the day of bleomycin installation, and the results showed that remdesivir significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro experiments showed that remdesivir dose-dependently suppressed TGF-β1-induced lung fibroblast activation and improved TGF-β1-induced alveolar epithelial to mesenchymal transition. Our results indicate that remdesivir can preventatively alleviate the severity of pulmonary fibrosis and provide some reference for the prevention of pulmonary fibrosis in patients with COVID-19.

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In vivo blockade of KCa3.1 ion channel alleviates ER stress in type II alveolar epithelial cells and macrophages in pulmonary fibrosis

10.1183/13993003.congress-2019.pa5385 ◽

2019 ◽

Author(s):

K. Udari Eshani Perera ◽

Sasika Nimanthi Vithana Dewage ◽

Habtamu B. Derseh ◽

Paul John Benham ◽

Andrew Stent ◽

...

Keyword(s):

Epithelial Cells ◽

Pulmonary Fibrosis ◽

Ion Channel ◽

Er Stress ◽

Alveolar Epithelial Cells ◽

Type Ii ◽

Alveolar Epithelial

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Irradiation Activates MZF1 to Inhibit miR-541-5p Expression and Promote Epithelial-Mesenchymal Transition (EMT) in Radiation-Induced Pulmonary Fibrosis (RIPF) by Upregulating Slug

International Journal of Molecular Sciences ◽

10.3390/ijms222111309 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11309

Author(s):

Xinxin Liang ◽

Ziyan Yan ◽

Ping Wang ◽

Yuhao Liu ◽

Xingkun Ao ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Epithelial Mesenchymal Transition ◽

Therapeutic Targets ◽

Alveolar Epithelial Cells ◽

Mesenchymal Transition ◽

Alveolar Epithelial ◽

Radiation Induced ◽

Myeloid Zinc Finger

Understanding miRNAs regulatory roles in epithelial-mesenchymal transition (EMT) would help establish new avenues for further uncovering the mechanisms underlying radiation-induced pulmonary fibrosis (RIPF) and identifying preventative and therapeutic targets. Here, we demonstrated that miR-541-5p repression by Myeloid Zinc Finger 1 (MZF1) promotes radiation-induced EMT and RIPF. Irradiation could decrease miR-541-5p expression in vitro and in vivo and inversely correlated to RIPF development. Ectopic miR-541-5p expression suppressed radiation-induced-EMT in vitro and in vivo. Knockdown of Slug, the functional target of miR-541-5p, inhibited EMT induction by irradiation. The upregulation of transcription factor MZF1 upon irradiation inhibited the expression of endogenous miR-541-5p and its primary precursor (pri-miR-541-5p), which regulated the effect of the Slug on the EMT process. Our finding showed that ectopic miR-541-5p expression mitigated RIPF in mice by targeting Slug. Thus, irradiation activates MZF1 to downregulate miR-541-5p in alveolar epithelial cells, promoting EMT and contributing to RIPF by targeting Slug. Our observation provides further understanding of the development of RIPF and determines potential preventative and therapeutic targets.

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ER Stress is Involved in Epithelial-To-Mesenchymal Transition of Alveolar Epithelial Cells Exposed to a Hypoxic Microenvironment

International Journal of Molecular Sciences ◽

10.3390/ijms20061299 ◽

2019 ◽

Vol 20 (6) ◽

pp. 1299 ◽

Cited By ~ 4

Author(s):

Eva Delbrel ◽

Yurdagül Uzunhan ◽

Abdoulaye Soumare ◽

Thomas Gille ◽

Dominique Marchant ◽

...

Keyword(s):

Epithelial Cells ◽

Er Stress ◽

Epithelial To Mesenchymal Transition ◽

Alveolar Epithelial Cells ◽

Mesenchymal Transition ◽

Alveolar Epithelial ◽

Rat Lungs ◽

Fibrotic Process

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and fatal interstitial lung disease of unknown origin. Alveolar epithelial cells (AECs) play an important role in the fibrotic process as they undergo sustained endoplasmic reticulum (ER) stress, and may acquire a mesenchymal phenotype through epithelial-to-mesenchymal transition (EMT), two phenomena that could be induced by localized alveolar hypoxia. Here we investigated the potential links between hypoxia, ER stress and EMT in AECs. Methods: ER stress and EMT markers were assessed by immunohistochemistry, western blot and qPCR analysis, both in vivo in rat lungs exposed to normoxia or hypoxia (equivalent to 8% O2) for 48 h, and in vitro in primary rat AECs exposed to normoxia or hypoxia (1.5% O2) for 2–6 days. Results: Hypoxia induced expression of mesenchymal markers, pro-EMT transcription factors, and the activation of ER stress markers both in vivo in rat lungs, and in vitro in AECs. In vitro, pharmacological inhibition of ER stress by 4-PBA limited hypoxia-induced EMT. Calcium chelation or hypoxia-inducible factor (HIF) inhibition also prevented EMT induction under hypoxic condition. Conclusions: Hypoxia and intracellular calcium are both involved in EMT induction of AECs, mainly through the activation of ER stress and HIF signaling pathways.

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Increased Levels of ER Stress and Apoptosis in A Sheep Model for Pulmonary Fibrosis Are Alleviated by in Vivo Blockade of the KCa3.1 Ion Channel

10.21203/rs.3.rs-97842/v1 ◽

2020 ◽

Author(s):

Udari Eshani Perera ◽

Louise Organ ◽

Sasika N.V. Dewage ◽

Habtamu B Derseh ◽

Andrew Stent ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Ion Channel ◽

Er Stress ◽

Nuclear Dna ◽

Disease Process ◽

Alveolar Epithelial Cells ◽

Type Ii ◽

Sheep Model ◽

Alveolar Epithelial

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease, characterized by progressive damage to the lung tissues. Apoptosis and endoplasmic reticulum stress (ER stress) in type II alveolar epithelial cells (AECs) and lung macrophages has been linked with the development of IPF. Therefore, apoptosis- and ER stress-targeted therapies have drawn attention as potential avenues for treatment of IPF. The calcium-activated potassium ion channel KCa3.1 has been proposed as a potential therapeutic target for fibrotic diseases including IPF. While KCa3.1 is expressed in AECs and macrophages, its influence on ER stress and apoptosis during the disease process is unclear. Methods We utilized a novel sheep model of pulmonary fibrosis to demonstrate that apoptosis and ER stress occurs in type II AECs and macrophages in sheep with bleomycin-induced lung fibrosis. Apoptosis in type II AEC and macrophages was identified using the TUNEL method of tagging fragmented nuclear DNA, while ER stress was characterized by increased expression of GRP-78 ER chaperone proteins. Results We demonstrated that apoptosis and ER stress in type II AECs and macrophages increased significantly 2 weeks after the final bleomycin infusion and remained high for up to 7 weeks post-bleomycin injury. Senicapoc treatment significantly reduced the rates of ER stress in type II AECs and macrophages that were resident in bleomycin-infused lung segments. There were also significant reductions in the rates of apoptosis of type II AECs and macrophages in the lung segments of senicapoc-treated sheep. Conclusion In-vivo blockade of the KCa3.1 ion channel alleviates the ER stress and apoptosis in type II AECs and macrophages, and this effect potentially contributes to the antifibrotic effects of senicapoc.

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VEGF/Src signaling mediated pleural barrier damage and increased permeability contributes to sub-pleural pulmonary fibrosis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00436.2020 ◽

2021 ◽

Author(s):

Yu-Zhi Lu ◽

Li-Mei Liang ◽

Pei-Pei Cheng ◽

Li Xiong ◽

Meng Wang ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Alveolar Epithelial Cell ◽

Mesothelial Cell ◽

Fluorescein Isothiocyanate ◽

Alveolar Epithelial Cells ◽

Alveolar Epithelial ◽

Src Signaling ◽

Vitro Model

The distribution of fibrosis in idiopathic pulmonary fibrosis (IPF) is sub-pleural with basal predominance. Alveolar epithelial cell was considered as the key cell in the initial phase of IPF. However, the idea of activation and damage of alveolar epithelial cells is very difficult to explain why fibrosis distributes in the sub-pleural area. In this study, human pleural mesothelial cell (PMC) line and primary rat PMC was used as in vitro model. Intra-peritoneal injection of bleomycin was used for making a pulmonary fibrosis model. The integrity of cultured monolayer PMCs was determined by transepithelial electric resistance (TEER). Pleural permeability was estimated by measuring paracellular transport of fluorescein isothiocyanate (FITC)-conjugated dextran. Changes in lung tissue of IPF patients were analyzed by Masson's and immunofluorescence staining. We found bleomycin induced PMCs damage and increased PMCs permeability, increased PMCs permeability aggravated bleomycin-induced sub-pleural inflammation and pulmonary fibrosis. Moreover, bleomycin was found to activate VEGF/Src signaling which increased PMCs permeability. In vivo, inhibition of VEGF/Src signaling prevented bleomycin-induced sub-pleural pulmonary fibrosis. At last, activation of VEGF/Src signaling was confirmed in sub-pleural area in IPF patients. Taken together, our findings indicate that VEGF/Src signaling mediated pleural barrier damage and increased permeability which contributes to sub-pleural pulmonary fibrosis.

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Increased Levels of ER Stress and Apoptosis in a Sheep Model for Pulmonary Fibrosis Are Alleviated by In Vivo Blockade of the KCa3.1 Ion Channel

Canadian Respiratory Journal ◽

10.1155/2021/6683195 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Udari E. Perera ◽

Louise Organ ◽

Sasika N. V. Dewage ◽

Habtamu B. Derseh ◽

Andrew Stent ◽

...

Keyword(s):

Pulmonary Fibrosis ◽

Ion Channel ◽

Er Stress ◽

Nuclear Dna ◽

Disease Process ◽

Alveolar Epithelial Cells ◽

Type Ii ◽

Sheep Model ◽

Alveolar Epithelial

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease, characterized by progressive damage to the lung tissues. Apoptosis and endoplasmic reticulum stress (ER stress) in type II alveolar epithelial cells (AECs) and lung macrophages have been linked with the development of IPF. Therefore, apoptosis- and ER stress-targeted therapies have drawn attention as potential avenues for treatment of IPF. The calcium-activated potassium ion channel KCa3.1 has been proposed as a potential therapeutic target for fibrotic diseases including IPF. While KCa3.1 is expressed in AECs and macrophages, its influence on ER stress and apoptosis during the disease process is unclear. We utilized a novel sheep model of pulmonary fibrosis to demonstrate that apoptosis and ER stress occur in type II AECs and macrophages in sheep with bleomycin-induced lung fibrosis. Apoptosis in type II AEC and macrophages was identified using the TUNEL method of tagging fragmented nuclear DNA, while ER stress was characterized by increased expression of GRP-78 ER chaperone proteins. We demonstrated that apoptosis and ER stress in type II AECs and macrophages increased significantly 2 weeks after the final bleomycin infusion and remained high for up to 7 weeks post-bleomycin injury. Senicapoc treatment significantly reduced the rates of ER stress in type II AECs and macrophages that were resident in bleomycin-infused lung segments. There were also significant reductions in the rates of apoptosis of type II AECs and macrophages in the lung segments of senicapoc-treated sheep. In vivo blockade of the KCa3.1 ion channel alleviates the ER stress and apoptosis in type II AECs and macrophages, and this effect potentially contributes to the anti-fibrotic effects of senicapoc.

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iPLA2β Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury

Cells ◽

10.3390/cells10061446 ◽

2021 ◽

Vol 10 (6) ◽

pp. 1446

Author(s):

Tingting Jin ◽

Jun Lin ◽

Yingchao Gong ◽

Xukun Bi ◽

Shasha Hu ◽

...

Keyword(s):

Cell Death ◽

Heart Disease ◽

Myocardial Ischemia ◽

Ischemic Heart Disease ◽

Er Stress ◽

Ischemia Reperfusion ◽

Myocardial Ischemia Reperfusion ◽

Induced Apoptosis

Both calcium-independent phospholipase A2 beta (iPLA2β) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2β is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2β in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2β knockout mice and siRNA mediated iPLA2β knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2β. Our data demonstrate the increase of iPLA2β augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2β ameliorates ER stress and decreases cell death. Mechanistically, iPLA2β promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2β contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

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TRIM72 is required for effective repair of alveolar epithelial cell wounding

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00172.2014 ◽

2014 ◽

Vol 307 (6) ◽

pp. L449-L459 ◽

Cited By ~ 15

Author(s):

Seong Chul Kim ◽

Thomas Kellett ◽

Shaohua Wang ◽

Miyuki Nishi ◽

Nagaraja Nagre ◽

...

Keyword(s):

Epithelial Cells ◽

Molecular Mechanisms ◽

Striated Muscle ◽

Alveolar Epithelial Cell ◽

Alveolar Epithelial Cells ◽

Lung Cells ◽

Alveolar Epithelial ◽

High Tidal Volume Ventilation

The molecular mechanisms for lung cell repair are largely unknown. Previous studies identified tripartite motif protein 72 (TRIM72) from striated muscle and linked its function to tissue repair. In this study, we characterized TRIM72 expression in lung tissues and investigated the role of TRIM72 in repair of alveolar epithelial cells. In vivo injury of lung cells was introduced by high tidal volume ventilation, and repair-defective cells were labeled with postinjury administration of propidium iodide. Primary alveolar epithelial cells were isolated and membrane wounding and repair were labeled separately. Our results show that absence of TRIM72 increases susceptibility to deformation-induced lung injury whereas TRIM72 overexpression is protective. In vitro cell wounding assay revealed that TRIM72 protects alveolar epithelial cells through promoting repair rather than increasing resistance to injury. The repair function of TRIM72 in lung cells is further linked to caveolin 1. These data suggest an essential role for TRIM72 in repair of alveolar epithelial cells under plasma membrane stress failure.

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