HSP70 Is a Critical Regulator of HSP90 Inhibitor's Effectiveness in Preventing HCl-Induced Chronic Lung Injury and Pulmonary Fibrosis.

Exposure to hydrochloric acid (HCl) can provoke acute and chronic lung injury. Because of its extensive production for industrial use, frequent accidental exposures occur, making HCl one of the top five chemicals causing inhalation injuries. There are no Food and Drug Administration (FDA)-approved treatments for HCl exposure. Heat shock protein 90 (HSP90) inhibitors modulate transforming growth factor-ß (TGF-ß) signaling and the development of chemical-induced pulmonary fibrosis. However, little is known on the role of Heat Shock Protein 70 (HSP70) during injury and treatment with HSP90 inhibitors. We hypothesized that administration of geranylgeranyl-acetone (GGA), an HSP70 inducer, or gefitinib (GFT), an HSP70 suppressant, alone or in combination with the HSP90 inhibitor, TAS-116, would improve or worsen, respectively, HCl-induced chronic lung injury in vivo and endothelial barrier dysfunction in vitro. GGA, alone, improved HCl-induced human lung microvascular endothelial cells (HLMVEC) barrier dysfunction and, in combination with TAS-116, improved the protective effect of TAS-116. In mice, GGA reduced HCl toxicity and while TAS-116 alone blocked HCl-induced chronic lung injury, co-administration with GGA, resulted in further improvement. Conversely, GFT potentiated HCl-induced barrier dysfunction and impaired the antidotal effects of TAS-116. We conclude that combined treatments with HSP90 inhibitors and HSP70 inducers may represent a novel therapeutic approach to manage HCl-induced chronic lung injury and pulmonary fibrosis.

The Inflammasome NLR Family Pyrin Domain-Containing Protein 3 (NLRP3) as a Novel Therapeutic Target for Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a dramatic disease without cure. The US Food and Drug Administration-approved drugs, pirfenidone and nintedanib, only slow disease progression. The clinical investigation of novel therapeutic approaches for IPF is an unmet clinical need. Nucleotide-binding oligomerization domain-like receptor or NOD-like receptors are pattern recognition receptors capable of binding a large variety of stress factors. NLR family pyrin domain-containing protein 3 (NLRP3), once activated, promotes IL-1ß, IL-18 production, and innate immune responses. Multiple reports indicate that the inflammasome NLRP3 is overactivated in IPF patients, leading to increased production of class I IL and collagens. Similarly, data from animal models of pulmonary fibrosis confirm the role of NLRP3 in the development of chronic lung injury and pulmonary fibrosis. This report provides a review of the evidence of NLRP3 activation in IPF and of NLRP3 inhibition in different animal models of fibrosis, and highlights the recent advances in direct and indirect NLRP3 inhibitors.

The Heat Shock Protein 90 Inhibitor, AT13387, Protects the Alveolo-Capillary Barrier and Prevents HCl-Induced Chronic Lung Injury and Pulmonary Fibrosis.

Hydrochloric acid (HCl) exposure causes asthma-like conditions, reactive airways dysfunction syndrome, and pulmonary fibrosis. Heat Shock Protein 90 (HSP90) is a molecular chaperone that regulates multiple cellular processes. HSP90 inhibitors are undergoing clinical trials for cancer and are also being studied in various pre-clinical settings for their anti-inflammatory and anti-fibrotic effects. Here we investigated the ability of the heat shock protein 90 (HSP90) inhibitor AT13387 to prevent chronic lung injury induced by exposure to HCl in vivo and its protective role in the endothelial barrier in vitro. We instilled C57Bl/6J mice with 0.1N HCl (2 µL/g body weight, intratracheally) and after 24 h began treatment with vehicle or AT13387 (10 or 15 mg/kg, SC), administered 3×/week; we analyzed histological, functional, and molecular markers 30 days after HCl. In addition, we monitored transendothelial electrical resistance (TER) and protein expression in a monolayer of human lung microvascular endothelial cells (HLMVEC) exposed to HCl (0.02 N) and treated with vehicle or AT13387 (2 µM). HCl provoked persistent alveolar inflammation; activation of profibrotic pathways (MAPK/ERK, HSP90); increased deposition of collagen, fibronectin and elastin; histological evidence of fibrosis; and a decline in lung function reflected in a downward shift in pressure-volume curves, increased respiratory system resistance (Rrs), elastance (Ers), tissue damping (G), and hyperresponsiveness to methacholine. Treatment with 15 mg/kg AT13387reduced alveolar inflammation, fibrosis, and NLRP3 staining; blocked activation of ERK and HSP90; and attenuated the deposition of collagen and the development of chronic lung injury and airway hyperreactivity. In vitro, AT13387 prevented HCl-induced loss of barrier function and AKT, ERK, and ROCK1 activation, and restored HSP70 and cofilin expression. The HSP90 inhibitor, AT13387, represents a promising drug candidate for chronic lung injury that can be administered subcutaneously in the field, and at low, non-toxic doses.

Post-treatment with a heat shock protein 90 inhibitor prevents chronic lung injury and pulmonary fibrosis, following acute exposure of mice to HCl.

Aim/Purpose: Exposure to high levels of hydrochloric acid (HCl) is associated with severe lung injury including both acute inflammation and chronic lung disease, which leads to the development of pulmonary fibrosis. Currently, there are no specific therapeutic agents for HCl-induced lung injury. Heat shock protein 90 (HSP90) has been implicated in the pathogenesis of pulmonary fibrosis. Thus, we have used a murine model of intra-tracheal acid instillation to investigate the antidotal effects of AUY-922, a small molecule HSP90 inhibitor, already in clinical trials for various types of cancer, against HCl-induced chronic lung injury and pulmonary fibrosis.Methods: HCl (0.1 N, 2 µl/g body weight) was instilled into male C57Bl/6J mice at day 0. After 24 h, mice began receiving 1 mg/kg AUY-922, 2x/week for 15 or 30 days.Results: AUY-922 suppressed the HCl-induced sustained inflammation, as reflected in the reduction of leukocyte and protein concentrations in bronchoalveolar lavage fluid, and inhibited the activation of pro-fibrotic biomarkers, ERK and HSP90. Furthermore, AUY-922 improved lung function, decreased the overexpression and accumulation of extracellular matrix proteins and dramatically reduced histologic evidence of fibrosis in the lungs of mice exposed to HCl.Conclusions: We conclude that AUY-922, and possibly other HSP90 inhibitors, successfully block the adverse effects associated with acute exposures to HCl and may represent an effective antidote against HCl-induced chronic lung injury and fibrosis.

Development of chronic lung injury and pulmonary fibrosis in mice following acute exposure to nitrogen mustard.

Objective: Sulfur mustards are toxic agents used as a chemical warfare in the twentieth century. Exposure to nitrogen mustards (NM), their more water-soluble analogs, is associated with respiratory, dermatological, neurological, and systemic symptoms whose severity depends on dose and length of contact. Long-term effects of acute inhalation have been related to the development of chronic lung injury and pulmonary fibrosis whose precise mechanisms and potential antidotes are yet to be discovered.Materials and methods: We have developed a model of NM-induced pulmonary fibrosis by intratracheally instilling mechlorethamine hydrochloride into C57Bl/6J male mice.Results and Discussion: Following mechlorethamine exposure, strong early and milder late inflammatory responses were observed. Initially, the number of white blood cells and levels of protein and pro-inflammatory cytokines in the bronchoalveolar lavage fluid (BALF) increased, followed by increases in the number of macrophages and the levels of transforming growth factor-ß (TGF-ß), a pro-fibrotic mediator. Analysis of lung homogenates revealed increased phosphorylation of pro-fibrotic biomarkers, serine/threonine-selective protein kinases (p-ERK), and heat shock protein 90 (P-HSP90) at 10 and 30 days after exposure. Total collagen expression and deposition of extracellular matrix proteins also increased. Lung function measurements demonstrated the presence of both obstructive and restrictive disease in agreement with evidence of increased lower airway peribronchial collagen deposition and parenchymal fibrosis.Conclusions: We conclude that the mouse represents a useful model of NM-induced acute lung injury and chronic pulmonary fibrosis, the latter driven by the overexpression of TGF-ß, p-ERK, and P-HSP90. This model may prove useful in the pre-clinical development of antidotes and other countermeasures.

Acute exposure of mice to hydrochloric acid leads to the development of chronic lung injury and pulmonary fibrosis.

Objective: Accidental exposure to hydrochloric acid (HCl) is associated with acute lung injury in humans, development of long-term chronic airway obstruction, and fibrosis. However, the mechanisms responsible for the progression to pulmonary fibrosis remain unclear. We utilized a mouse model of progressive lung injury from a single exposure to HCl to investigate the effects of HCl on the lower respiratory tract. Materials and methods: HCl (0.05-0.3 N) or saline was injected intratracheally into male C57Bl/6J mice. At 1, 4, 10 and 30 days post instillation, bronchoalveolar lavage fluid (BALF) and lung tissues were collected and examined for multiple outcomes. Results and discussion: We observed an early inflammatory response and a late mild inflammation present even at 30 d post HCl exposure. Mice treated with HCl exhibited higher total leukocyte and protein levels in the BALF compared to the vehicle group. This was characterized by increased number of neutrophils, monocytes, and lymphocytes as well as pro-inflammatory cytokines during the first 4 d of injury. The late inflammatory response exhibited a predominant presence of mononuclear cells, increased permeability to protein, and higher levels of the pro-fibrotic mediator TGFß. Pro-fibrotic protein biomarkers, phosphorylated ERK, and HSP90, were also overexpressed at 10 and 30 d following HCl exposure. In vivo lung function measurements demonstrated lung dysfunction and chronic lung injury associated with increased lung hydroxyproline content and increased expression of extracellular matrix (ECM) proteins. The acute inflammation and severity of fibrosis increased in HCl-concentration dependent manner. Conclusions: Our findings suggest that the initial inflammatory response and pro-fibrotic biomarker upregulation may be linked to the progression of pulmonary fibrosis and airway dysfunction and may represent valuable therapeutic targets.