Abstract:Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with limited therapeutic options and poor prognosis. IPF has been associated with aberrant vascular remodelling, however the role of vascular remodelling in pulmonary fibrosis is poorly understood. Here, we used a novel segmental challenge model of bleomycin-induced pulmonary fibrosis in sheep to evaluate the remodelling of the pulmonary vasculature, and to investigate the changes to this remodelling after the administration of the KCa3.… Show more
“…This study is the first to report that the bleomycin treatment increases ER stress and apoptosis in type II AECs and lung macrophages, and this effect is significantly ameliorated by in vivo blockade of the KCa3.1 ion channel with the drug Senicapoc. Our previous studies using the sheep pulmonary fibrosis model reported that there were significant reductions in lung fibrosis and microvascular remodeling in sheep treated with Senicapoc [23,24]. The attenuated lung pathology in senicapoc-treated sheep coincided with improved lung function in these animals [24].…”
Section: Discussionmentioning
confidence: 99%
“…Importantly, stimulation of the KCa3.1 ion channel activates cells such as fibroblasts, macrophages and epithelial cells that are involved in the disease processes of IPF [18]. We have recently reported that blockade of the KCa3.1 ion channel using senicapoc (ICA-17043) attenuates interstitial lung fibrosis, improves lung compliance and attenuates microvascular remodeling in a sheep model for pulmonary fibrosis [23,24]. In the current study we utilized the sheep model to analyze the regulatory function(s) of the KCa3.1 ion channel in type II AECs and macrophages in fibrotic disease.…”
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.
“…This study is the first to report that the bleomycin treatment increases ER stress and apoptosis in type II AECs and lung macrophages, and this effect is significantly ameliorated by in vivo blockade of the KCa3.1 ion channel with the drug Senicapoc. Our previous studies using the sheep pulmonary fibrosis model reported that there were significant reductions in lung fibrosis and microvascular remodeling in sheep treated with Senicapoc [23,24]. The attenuated lung pathology in senicapoc-treated sheep coincided with improved lung function in these animals [24].…”
Section: Discussionmentioning
confidence: 99%
“…Importantly, stimulation of the KCa3.1 ion channel activates cells such as fibroblasts, macrophages and epithelial cells that are involved in the disease processes of IPF [18]. We have recently reported that blockade of the KCa3.1 ion channel using senicapoc (ICA-17043) attenuates interstitial lung fibrosis, improves lung compliance and attenuates microvascular remodeling in a sheep model for pulmonary fibrosis [23,24]. In the current study we utilized the sheep model to analyze the regulatory function(s) of the KCa3.1 ion channel in type II AECs and macrophages in fibrotic disease.…”
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.
“…Importantly, stimulation of the KCa3.1 ion channel activates cells such as fibroblasts, macrophages, and epithelial cells that are involved in the disease processes of IPF [ 18 ]. We have recently reported that blockade of the KCa3.1 ion channel using senicapoc (ICA-17043) attenuates interstitial lung fibrosis, improves lung compliance, and attenuates microvascular remodeling in a sheep model for pulmonary fibrosis [ 23 , 24 ]. In the current study, we utilized the sheep model to analyze the regulatory function(s) of the KCa3.1 ion channel in type II AECs and macrophages in fibrotic disease.…”
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.
“…However, senicapoc was demonstrated to be safe and well tolerated in humans, thereby allowing easier repurposing of the drug in other preclinical and clinical studies. Examples include neuroinflammation [ [5] , [6] , [7] , [8] ], cardiovascular and respiratory disorders (fibrosis or lung oedema) [ [9] , [10] , [11] , [12] ], other diseases with red cell dehydration [ 13 , 14 ] and, latest, a clinical trial using senicapoc to treat COVID-19 patients in intensive care (COVIPOC, EudraCT 2020-001420-34). Fig.…”
The clinically tested KCa3.1 channel blocker, senicapoc, has been proven to have excellent pharmacological properties and prior clinical trials found it to be safe for use in patients with sickle cell anaemia. Currently, several preclinical projects are aiming to repurpose senicapoc for other indications, but well-described analytical methods in the literature are lacking. Our aim was to develop a sensitive, rapid and accurate ultra-high-performance liquid chromatography-tandem mass spectrometry method using pneumatically assisted electrospray ionisation (UHPLC-ESI-MS/MS) suitable for the determination of senicapoc in plasma samples. Unfortunately, direct analysis of senicapoc in crude acetonitrile extracts of human plasma samples by UHPLC-ESI-MS/MS was subjected to significant and variable ion suppression from coeluting phospholipids (PLs). The interferences were mainly caused by the presence of phosphatidylcholine and phosphatidylethanolamine classes of PLs, including their lyso-products. However, the PLs were easily removed from crude extracts by filtration through a sorbent with Lewis acid properties which decreased the total ion suppression effect to approximately 5%. Based on this technique, a simple high-throughput UHPLC-MS/MS method was developed and validated for the determination of senicapoc in 100-μL plasma samples. The lower limit of quantification was 0.1 ng/mL. The mean true extraction recovery was close to 100%. The relative intra-laboratory reproducibility standard deviations of the measured concentrations were 8% and 4% at concentrations of 0.1 ng/mL and 250 ng/mL, respectively. The trueness expressed as the relative bias of the test results was within ± 2% at concentrations of 1 ng/mL or higher.
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