ABSTRACT
The Acute Respiratory Distress Syndrome (ARDS), often preceded by acute lung injury (ALI), is characterized by the accumulation of inflammatory fluid in the lung alveoli, leaky alveolar epithelium and endothelium, and overexpression of pro-inflammatory cytokines. This progression from ALI to ARDS is a major contributor to the high mortality observed in COVID-19 patients. The Spike protein of SARS-CoV-2 binds to lung ACE2 and, in addition to facilitating viral cell entry, it plays an important role in the development of ALI and ARDS, especially in the later phases of COVID-19 as well as long COVID. Protein tyrosine phosphatase (PTP) 4A3 is a key mediator of ARDS pathology. This study tested the hypothesis that targeting PTP4A3 would prevent COVID-19 associated ALI. Intratracheal administration of SARS-CoV-2 Spike protein Subunit 1 to K18-hACE2 transgenic mice expressing human ACE2 elicited pulmonary and systemic inflammation, leaky alveoli, overexpression of cytokines, structural lung injury and lung dysfunction; all these symptoms were ameliorated by the selective, allosteric inhibitor of PTP4A3, KVX-053. These findings provide the first evidence supporting a role for PTP4A3 in the development of SARS-CoV-2- mediated ALI. Significance Statement This study tested the hypothesis that targeting PTP4A3 would prevent COVID-19 associated ALI/ARDS. Intratracheal administration of SARS-CoV-2 Spike protein Subunit 1 to K18-hACE2 transgenic mice expressing human ACE2 elicited pulmonary and systemic inflammation, leaky alveoli, overexpression of cytokines and chemokines, structural lung injury and lung dysfunction; all these symptoms were ameliorated by the selective, allosteric inhibitor of PTP4A3, KVX-053. These findings suggest that this novel PTP4A3 inhibitor may be useful against COVID-19 and potentially other viral-induced ARDS.
ABSTRACT
Hydrochloric acid is one of the most prevalent and dangerous chemicals. Accidental spills occur in industrial plants or during transportation. Exposure to HCl can induce severe health impairment, including acute and chronic pulmonary diseases. We have previously described the molecular, structural, and functional aspects of the development of chronic lung injury and pulmonary fibrosis caused by intratracheal instillation of HCl in mice. Although mouse models of human disease have many advantages, rodents are evolutionary far from human and exhibit significant anatomical and physiological differences. Genetic and anatomic similarities between rabbits and humans are significantly higher. Rabbit models of HCl-induced lung injury have been used sparsely to evaluate acute lung injury. In this study, for the first time, we utilized rabbits as a model of HCl-induced pulmonary fibrosis and chronic lung injury. We present molecular, histological, and functional evidence that demonstrate the utility of using this model for studying new pharmaceutics against pulmonary fibrosis.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents , Benzamides , Lung Injury , Pulmonary Fibrosis , Pyrazoles , Mice , Humans , Animals , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/metabolism , Lung Injury/chemically induced , Lung Injury/drug therapy , Hydrochloric Acid/toxicity , HSP70 Heat-Shock Proteins/metabolism , Endothelial Cells/metabolism , Antineoplastic Agents/adverse effects , Gefitinib/adverse effects , HSP90 Heat-Shock Proteins/metabolismABSTRACT
Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on the metabolic activities of cells and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for > 2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, > 20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-fivefold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.
Subject(s)
Pancreas , Pancreatic Neoplasms , Humans , Pancreatic Neoplasms/therapy , Cell Membrane , Apoptosis , Cell DeathABSTRACT
Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) in vitro were evaluated on cell viability and transcellular electrical resistance (TER). It was observed that treatment with ns-APPJ for >2 min disrupts Pan02 cell stability and resulted in over 30% cell death. Similarly, applying nsPEF alone, >20 pulses resulted in over 15% cell death. While the inactivation activity from the individual treatment is moderate, combined treatments resulted in 80% cell death, approximately 3-to-5-fold increase compared to the individual treatment. In addition, reactive oxygen species such as OH and O were identified at the plasma-liquid interface. The gas temperature of the plasma and the temperature of the cell solution during treatments were determined to be near room temperature.
ABSTRACT
The genesis of most older medicinal agents has generally been empirical. During the past one and a half centuries, at least in the Western countries, discovering and developing drugs has been primarily the domain of pharmaceutical companies largely built upon concepts emerging from organic chemistry. Public sector funding for the discovery of new therapeutics has more recently stimulated local, national, and international groups to band together and focus on new human disease targets and novel treatment approaches. This Perspective describes one contemporary example of a newly formed collaboration that was simulated by a regional drug discovery consortium. University of Virginia, Old Dominion University, and a university spinout company, KeViRx, Inc., partnered under a NIH Small Business Innovation Research grant, to produce potential therapeutics for acute respiratory distress syndrome resulting from the ongoing COVID-19 pandemic.
Subject(s)
COVID-19 , Respiratory Distress Syndrome , Humans , SARS-CoV-2 , Pandemics , Virginia , Drug Development , Drug Discovery , Respiratory Distress Syndrome/drug therapyABSTRACT
BACKGROUND: Bacterial pneumonia is a major risk factor for acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Pseudomonas aeruginosa (PA), an opportunistic pathogen with an increasing resistance acquired against multiple drugs, is one of the main causative agents of ALI and ARDS in diverse clinical settings. Given the anti-inflammatory role of the cannabinoid-2 receptor (CB2R), the effect of CB2R activation in the regulation of PA-induced ALI and inflammation was tested in a mouse model as an alternative to conventional antibiotic therapy. METHODS: In order to activate CB2R, a selective synthetic agonist, JWH133, was administered intraperitoneally (i.p.) to C57BL/6J mice. Furthermore, SR144528 (a selective CB2R antagonist) was administered in combination with JWH133 to test the specificity of the CB2R-mediated effect. PA was administered intratracheally (i.t.) for induction of pneumonia in mice. At 24 h after PA exposure, lung mechanics were measured using the FlexiVent system. The total cell number, protein content, and neutrophil population in the bronchoalveolar lavage fluid (BALF) were determined. The bacterial load in the whole lung was also measured. Lung injury was evaluated by histological examination and PA-induced inflammation was assessed by measuring the levels of BALF cytokines and chemokines. Neutrophil activation (examined by immunofluorescence and immunoblot) and PA-induced inflammatory signaling (analyzed by immunoblot) were also studied. RESULTS: CB2R activation by JWH133 was found to significantly reduce PA-induced ALI and the bacterial burden. CB2R activation also suppressed the PA-induced increase in immune cell infiltration, neutrophil population, and inflammatory cytokines. These effects were abrogated by a CB2R antagonist, SR144528, further confirming the specificity of the CB2R-mediated effects. CB2R-knock out (CB2RKO) mice had a significantly higher level of PA-induced inflammation as compared to that in WT mice. CB2R activation diminished the excess activation of neutrophils, whereas mice lacking CB2R had elevated neutrophil activation. Pharmacological activation of CB2R significantly reduced the PA-induced NF-κB and NLRP3 inflammasome activation, whereas CB2KO mice had elevated NLRP3 inflammasome. CONCLUSION: Our findings indicate that CB2R activation ameliorates PA-induced lung injury and inflammation, thus paving the path for new therapeutic avenues against PA pneumonia.
Subject(s)
Acute Lung Injury , Cannabinoids , Inflammation , Pseudomonas Infections , Receptor, Cannabinoid, CB2 , Animals , Mice , Acute Lung Injury/chemically induced , Acute Lung Injury/genetics , Acute Lung Injury/immunology , Acute Lung Injury/prevention & control , Cannabinoids/pharmacology , Cannabinoids/therapeutic use , Cytokines , Inflammasomes/genetics , Inflammasomes/immunology , Inflammation/genetics , Inflammation/immunology , Inflammation/prevention & control , Mice, Inbred C57BL , NLR Family, Pyrin Domain-Containing 3 Protein , Pseudomonas aeruginosa , Receptors, Cannabinoid , Respiratory Distress Syndrome , Receptor, Cannabinoid, CB2/genetics , Receptor, Cannabinoid, CB2/immunology , Pseudomonas Infections/genetics , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas Infections/prevention & control , Disease Models, AnimalABSTRACT
Exposure to high concentrations of hydrochloric acid (HCl) can lead to severe acute and chronic lung injury. In the aftermath of accidental spills, victims may be treated for the acute symptoms, but the chronic injury is often overlooked. We have developed a mouse model of acute and chronic lung injury, in which the peak of acute lung injury occurs on the day 4 after HCl exposure. We have also demonstrated that HSP90 inhibitors are effective antidotes when administered starting 24 h after HCl. In this study we examined the hypothesis that the novel oral HSP90 inhibitor TAS-116 can effectively ameliorate HCl-induced lung injury even when treatment starts at the peak of the acute injury, as late as 96 h after HCl. C57BI/6J mice were intratracheally instilled with 0.1N HCl. After 24 or 96 h, TAS-116 treatment began (3.5, 7 or 14 mg/kg, 5 times per week, p. o.) for either 2,3 or 4 or weeks. TAS-116 moderated the HCl-induced alveolar inflammation, as reflected in the reduction of white blood cells and total protein content in bronchoalveolar lavage fluid (BALF), overexpression of NLRP3 inflammasome, and inhibited the activation of pro-fibrotic pathways. Furthermore, TAS-116 normalized lung mechanics and decreased the deposition of extracellular matrix proteins in the lungs of mice exposed to HCl. Delayed and shortened treatment with TAS-116, successfully blocked the adverse chronic effects associated with acute exposure to HCl.
ABSTRACT
Cardiovascular disease (CVD) is the most prominent cause of death of adults in the United States with coronary artery disease being the most common type of CVD. Following a myocardial event, the coronary endothelium plays an important role in the recovery of the ischemic myocardium. Specifically, endothelial cells (EC) must be able to elicit a robust angiogenic response necessary for tissue revascularization and repair. However, local or distant cues may prevent effective revascularization. Extracellular vesicles (EV) are produced by all cells and endothelium is a rich source of EVs that have access to the main circulation thereby potentially impacting local and distant tissue function. Systemic inflammation associated with conditions such as obesity as well as the acute inflammatory response elicited by a cardiac event can significantly increase the EV release by endothelium and alter their miRNA, protein or lipid cargo. Our laboratory has previously shown that EVs released by adipose tissue endothelial cells exposed to chronic inflammation have angiostatic effects on naïve adipose tissue EC in vitro. Whether the observed effect is specific to EVs from adipose tissue endothelium or is a more general feature of the endothelial EVs exposed to pro-inflammatory cues is currently unclear. The objective of this study was to investigate the angiostatic effects of EVs produced by EC from the coronary artery and adipose microvasculature exposed to pro-inflammatory cytokines (PIC) on naïve coronary artery EC. We have found that EVs from both EC sources have angiostatic effects on the coronary endothelium. EVs produced by cells in a pro-inflammatory environment reduced proliferation and barrier function of EC without impacting cellular senescence. Some of these functional effects could be attributed to the miRNA cargo of EVs. Several miRNAs such as miR-451, let-7, or miR-23a impact on multiple pathways responsible for proliferation, cellular permeability and angiogenesis. Collectively, our data suggests that EVs may compete with pro-angiogenic cues in the ischemic myocardium therefore slowing down the repair response. Acute treatments with inhibitors that prevent endogenous EV release immediately after an ischemic event may contribute to better efficacy of therapeutic approaches using functionalized exogenous EVs or other pro-angiogenic approaches.
ABSTRACT
Exercise-induced increases in pulmonary blood flow normally increase pulmonary arterial pressure only minimally, largely due to a reserve of pulmonary capillaries that are available for recruitment to carry the flow. In pulmonary arterial hypertension, due to precapillary arteriolar obstruction, such recruitment is greatly reduced. In exercising pulmonary arterial hypertension patients, pulmonary arterial pressure remains high and may even increase further. Current pulmonary arterial hypertension therapies, acting principally as vasodilators, decrease calculated pulmonary vascular resistance by increasing pulmonary blood flow but have a minimal effect in lowering pulmonary arterial pressure and do not restore significant capillary recruitment. Novel pulmonary arterial hypertension therapies that have mainly antiproliferative properties are being developed to try and diminish proliferative cellular obstruction in precapillary arterioles. If effective, those agents should restore capillary recruitment and, during exercise testing, pulmonary arterial pressure should remain low despite increasing pulmonary blood flow. The effectiveness of every novel therapy for pulmonary arterial hypertension should be evaluated not only at rest, but with measurement of exercise pulmonary hemodynamics during clinical trials.
ABSTRACT
During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, alcohol consumption increased markedly. Nearly one in four adults reported drinking more alcohol to cope with stress. Chronic alcohol abuse is now recognized as a factor complicating the course of acute respiratory distress syndrome and increasing mortality. To investigate the mechanisms behind this interaction, a combined acute respiratory distress syndrome and chronic alcohol abuse mouse model was developed by intratracheally instilling the subunit 1 (S1) of SARS-CoV-2 spike protein (S1SP) in K18-human angiotensin-converting enzyme 2 (ACE2) transgenic mice that express the human ACE2 receptor for SARS-CoV-2 and were kept on an ethanol diet. Seventy-two hours after S1SP instillation, mice on an ethanol diet showed a strong decrease in body weight, a dramatic increase in white blood cell content of bronchoalveolar lavage fluid, and an augmented cytokine storm, compared with S1SP-treated mice on a control diet. Histologic examination of lung tissue showed abnormal recruitment of immune cells in the alveolar space, abnormal parenchymal architecture, and worsening Ashcroft score in S1SP- and alcohol-treated animals. Along with the activation of proinflammatory biomarkers [NF-κB, STAT3, NLR family pyrin domain-containing protein 3 (NLRP3) inflammasome], lung tissue homogenates from mice on an alcohol diet showed overexpression of ACE2 compared with mice on a control diet. This model could be useful for the development of therapeutic approaches against alcohol-exacerbated coronavirus disease 2019.
Subject(s)
Acute Lung Injury , Alcoholism , Angiotensin-Converting Enzyme 2 , COVID-19 , Respiratory Distress Syndrome , Acute Lung Injury/pathology , Acute Lung Injury/virology , Animals , COVID-19/pathology , Ethanol/adverse effects , Humans , Lung/pathology , Mice , Mice, Transgenic , Peptidyl-Dipeptidase A/metabolism , Respiratory Distress Syndrome/virology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/geneticsABSTRACT
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused more than 5 million deaths worldwide. Multiple reports indicate that the endothelium is involved during SARS-Cov-2-related disease (COVID-19). Indeed, COVID-19 patients display increased thrombophilia with arterial and venous embolism and lung microcapillary thrombotic disease as major determinants of deaths. The pathophysiology of endothelial dysfunction in COVID-19 is not completely understood. We have investigated the role of subunit 1 of the SARS-CoV-2 spike protein (S1SP) in eliciting endothelial barrier dysfunction, characterized dose and time relationships, and tested the hypothesis that heat shock protein 90 (HSP90) inhibitors would prevent and repair such injury. S1SP activated (phosphorylated) IKBα, STAT3, and AKT and reduced the expression of intercellular junctional proteins, occludin, and VE-cadherin. HSP90 inhibitors (AT13387 and AUY-922) prevented endothelial barrier dysfunction and hyperpermeability and reduced IKBα and AKT activation. These two inhibitors also blocked S1SP-mediated barrier dysfunction and loss of VE-cadherin. These data suggest that spike protein subunit 1 can elicit, by itself, direct injury to the endothelium and suggest a role of HSP90 inhibitors in preserving endothelial functionality.
ABSTRACT
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.
Subject(s)
Antineoplastic Agents , Lung Injury , Pulmonary Fibrosis , Animals , Antineoplastic Agents/pharmacology , Benzamides , Collagen/metabolism , Endothelial Cells/metabolism , HSP90 Heat-Shock Proteins/metabolism , Hydrochloric Acid/adverse effects , Inflammation/pathology , Isoindoles , Lung Injury/chemically induced , Lung Injury/drug therapy , Lung Injury/prevention & control , Mice , Mice, Inbred C57BL , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/drug therapy , Pulmonary Fibrosis/prevention & controlABSTRACT
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.
Subject(s)
Idiopathic Pulmonary Fibrosis , Inflammasomes , Animals , Carrier Proteins/metabolism , Humans , Idiopathic Pulmonary Fibrosis/drug therapy , Inflammasomes/metabolism , Interleukin-1beta/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , Pyrin DomainABSTRACT
Pulmonary arterial pressure rises minimally during exercise. The pulmonary microcirculation accommodates increasing blood flow via recruitment of pulmonary capillaries and, at higher flows, by distention of already perfused capillaries. The flow transition range between recruitment and distention has not been studied or compared across mammalian species, including humans. We hypothesised that the range would be similar. Functional pulmonary capillary surface area (FCSA) can be estimated using validated metabolic techniques. We reviewed data from previous studies in three mammalian species (perfused rabbit lungs and dog lung lobes, and exercising humans) and generated blood flow-FCSA curves over a range of flows. We noted where the curves diverged from the theoretical line of pure recruitment (Recruitment) and determined the flow where the curve slope equalled 50% that of Recruitment, or equalled that of a theoretical curve representing full capillary distention (Distention). The three mammalian species have similar flow ranges for the transition from predominantly recruitment to predominantly distention, with dogs having the highest transition point. Within the physiological range of most daily activity, the species are similar and accommodate increasing blood flow mainly via recruitment, with progressive distention at higher flows. This is highly relevant to pulmonary physiology during exercise.
Subject(s)
Capillaries , Pulmonary Circulation , Animals , Blood Pressure/physiology , Dogs , Hemodynamics/physiology , Humans , Lung/blood supply , Pulmonary Circulation/physiology , RabbitsABSTRACT
We previously reported that female mice exhibit protection against chemically induced pulmonary fibrosis and suggested a potential role of estrogen. Phytoestrogens act, at least in part, via stimulation of estrogen receptors; furthermore, compared to residents of Western countries, residents of East Asian countries consume higher amounts of phytoestrogens and exhibit lower rates of pulmonary fibrosis. Therefore, we tested the hypothesis that dietary phytoestrogens ameliorate the severity of experimentally induced pulmonary fibrosis. Male mice placed on either regular soybean diet or phytoestrogen-free diet were instilled with 0.1 N HCl to provoke pulmonary fibrosis. Thirty days later, lung mechanics were measured as indices of lung function and bronchoalveolar lavage fluid (BALF) and lung tissue were analyzed for biomarkers of fibrosis. Mice on phytoestrogen-free diet demonstrated increased mortality and stronger signs of chronic lung injury and pulmonary fibrosis, as reflected in the expression of collagen, extracellular matrix deposition, histology, and lung mechanics, compared to mice on regular diet. We conclude that dietary phytoestrogens play an important role in the pathogenesis of pulmonary fibrosis and suggest that phytoestrogens (e.g., genistein) may be useful as part of a therapeutic regimen against hydrochloric acid-induced lung fibrosis and chronic lung dysfunction.
Subject(s)
Diet , Lung Injury/chemically induced , Lung Injury/drug therapy , Phytoestrogens/therapeutic use , Pulmonary Fibrosis/drug therapy , Animals , Chronic Disease , Extracellular Matrix Proteins/metabolism , Hydrochloric Acid , Inflammation/pathology , Leukocyte Count , Lung/physiopathology , Lung Injury/complications , Lung Injury/mortality , Male , Mice, Inbred C57BL , Models, Biological , Phytoestrogens/pharmacology , Pulmonary Fibrosis/complications , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
Exposure to hydrochloric acid (HCl) represents a threat to public health. Children may inhale higher doses and develop greater injury because of their smaller airways and faster respiratory rate. We have developed a mouse model of pediatric exposure to HCl by intratracheally instilling p24 mice (mice 24 days old; 8-10 g) with 2 µL/g 0.1 N HCl, and compared the profile of lung injury to that in HCl-instilled adults (10 weeks old; 25-30 g) and their age-matched saline controls. After 30 days, alveolar inflammation was observed with increased proteinosis and mononuclear cells in the bronchoalveolar lavage fluid (BALF) in both HCl-instilled groups. Young p24 animals-but not adults-exhibited higher NLR family pyrin domain containing 3 (NLRP3) inflammasome levels. Increased amounts of Transforming Growth Factor-ß (TGF-ß) mRNA and its intracellular canonical and non-canonical pathways (p-Smad2 and p-ERK) were found in the lungs of both young and adult HCl-instilled mice. Constitutive age-related differences were observed in the levels of heat shock protein family (HSP70 and HSP90). HCl equally provoked the deposition of collagen and fibronectin; however, significant age-dependent differences were observed in the increase in elastin and tenascin C mRNA. HCl induced pulmonary fibrosis with an increased Ashcroft score, which was higher in adults, and a reduction in alveolar Mean Alveolar Linear Intercept (MALI). Young mice developed increased Newtonian resistance (Rn) and lower PV loops, while adults showed a higher respiratory system resistance and elastance. This data indicate that young p24 mice can suffer long-term complications from a single exposure to HCl, and can develop chronic lung injury characterized by a stronger persistent inflammation and lesser fibrotic pattern, mostly in the airways, differently from adults. Further data are required to characterize HCl time- and dose-dependent injury in young animals and to identify new key-molecular targets.
Subject(s)
Acute Lung Injury/pathology , Aging , Bronchoalveolar Lavage Fluid/chemistry , Hydrochloric Acid/toxicity , Inflammation/pathology , Pulmonary Fibrosis/pathology , Acute Lung Injury/chemically induced , Animals , Inflammation/chemically induced , Male , Mice , Mice, Inbred C57BL , Pulmonary Fibrosis/chemically inducedABSTRACT
Exposure to hydrochloric acid (HCl) leads acutely to asthma-like symptoms, acute respiratory distress syndrome (ARDS), including compromised alveolo-capillary barrier, and respiratory failure. To better understand the direct effects of HCl on pulmonary endothelial function, we studied the characteristics of HCl-induced endothelial barrier dysfunction in primary cultures of human lung microvascular endothelial cells (HLMVEC), defined the involved molecular pathways, and tested the potentially beneficial effects of Heat Shock Protein 90 (HSP90) inhibitors. HCl impaired barrier function in a time- and concentration-dependent manner and was associated with activation of Protein Kinase B (AKT), Ras homolog family member A (RhoA) and myosin light chain 2 (MLC2), as well as loss of plasmalemmal VE-cadherin, rearrangement of cortical actin, and appearance of inter-endothelial gaps. Pre-treatment or post-treatment of HLMVEC with AUY-922, a third-generation HSP90 inhibitor, prevented and restored HCl-induced endothelial barrier dysfunction. AUY-922 increased the expression of HSP70 and inhibited the activation (phosphorylation) of extracellular-signal regulated kinase (ERK) and AKT. AUY-922 also prevented the HCl-induced activation of RhoA and MLC2 and the internalization of plasmalemmal VE-cadherin. We conclude that, by increasing the expression of cytoprotective proteins, interfering with actomyosin contractility, and enhancing the expression of junction proteins, inhibition of HSP90 may represent a useful approach for the management of HCl-induced endothelial dysfunction and acute lung injury.
Subject(s)
Endothelial Cells/metabolism , Hydrochloric Acid/toxicity , Lung/metabolism , MAP Kinase Signaling System/drug effects , Microvessels/metabolism , Cardiac Myosins/metabolism , Endothelial Cells/pathology , HSP70 Heat-Shock Proteins/antagonists & inhibitors , HSP70 Heat-Shock Proteins/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/metabolism , Humans , Isoxazoles/pharmacology , Lung/blood supply , Lung/pathology , Microvessels/pathology , Myosin Light Chains/metabolism , Resorcinols/pharmacology , Respiratory Distress Syndrome/chemically induced , Respiratory Distress Syndrome/metabolism , Respiratory Distress Syndrome/pathology , rhoA GTP-Binding Protein/metabolismABSTRACT
We developed two models of chemically induced chronic lung injury and pulmonary fibrosis in mice (intratracheally administered hydrochloric acid (HCl) and intratracheally administered nitrogen mustard (NM)) and investigated male-female differences. Female mice exhibited higher 30-day survival and less weight loss than male mice. Thirty days after the instillation of either HCl or NM, bronchoalveolar lavage fluid displayed a persistent, mild inflammatory response, but with higher white blood cell numbers and total protein content in males vs. females. Furthermore, females exhibited less collagen deposition, milder pulmonary fibrosis, and lower Ashcroft scores. After instillation of either HCl or NM, all animals displayed increased values of phosphorylated (activated) Heat Shock Protein 90, which plays a crucial role in the alveolar wound-healing processes; however, females presented lower activation of both transforming growth factor-ß (TGF-ß) signaling pathways: ERK and SMAD. We propose that female mice are protected from chronic complications of a single exposure to either HCl or NM through a lesser activation of TGF-ß and downstream signaling. The understanding of the molecular mechanisms that confer a protective effect in females could help develop new, gender-specific therapeutics for IPF.
Subject(s)
Collagen/genetics , HSP90 Heat-Shock Proteins/genetics , Idiopathic Pulmonary Fibrosis/genetics , Transforming Growth Factor beta/genetics , Animals , Female , Gene Expression Regulation/genetics , Humans , Hydrochloric Acid/toxicity , Idiopathic Pulmonary Fibrosis/chemically induced , Idiopathic Pulmonary Fibrosis/pathology , Lung/pathology , MAP Kinase Signaling System/genetics , Male , Mechlorethamine/toxicity , Mice , Smad Proteins/geneticsABSTRACT
Acute lung injury (ALI) leading to acute respiratory distress syndrome is the major cause of COVID-19 lethality. Cell entry of SARS-CoV-2 occurs via the interaction between its surface spike protein (SP) and angiotensin-converting enzyme-2 (ACE2). It is unknown if the viral spike protein alone is capable of altering lung vascular permeability in the lungs or producing lung injury in vivo. To that end, we intratracheally instilled the S1 subunit of SARS-CoV-2 spike protein (S1SP) in K18-hACE2 transgenic mice that overexpress human ACE2 and examined signs of COVID-19-associated lung injury 72 h later. Controls included K18-hACE2 mice that received saline or the intact SP and wild-type (WT) mice that received S1SP. K18-hACE2 mice instilled with S1SP exhibited a decline in body weight, dramatically increased white blood cells and protein concentrations in bronchoalveolar lavage fluid (BALF), upregulation of multiple inflammatory cytokines in BALF and serum, histological evidence of lung injury, and activation of signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways in the lung. K18-hACE2 mice that received either saline or SP exhibited little or no evidence of lung injury. WT mice that received S1SP exhibited a milder form of COVID-19 symptoms, compared with the K18-hACE2 mice. Furthermore, S1SP, but not SP, decreased cultured human pulmonary microvascular transendothelial resistance (TER) and barrier function. This is the first demonstration of a COVID-19-like response by an essential virus-encoded protein by SARS-CoV-2 in vivo. This model of COVID-19-induced ALI may assist in the investigation of new therapeutic approaches for the management of COVID-19 and other coronaviruses.