-Increasing evidence shows that hyperoxia is a serious complication of oxygen therapy in acutely ill patients that causes excessive production of free radicals leading to hyperoxia-induced acute lung injury (HALI). Our previous studies have shown that P2X7 receptor activation is required for inflammasome activation during HALI. However, the role of P2X7 in HALI is unclear. The main aim of this study was to determine the effect of P2X7 receptor gene deletion on HALI. Wild-type (WT) and P2X7 knockout (P2X7 KO) mice were exposed to 100% O 2 for 72 h. P2X7 KO mice treated with hyperoxia had enhanced survival in 100% O 2 compared with the WT mice. Hyperoxia-induced recruitment of inflammatory cells and elevation of IL-1␤, TNF-␣, monocyte chemoattractant protein-1, and IL-6 levels were attenuated in P2X7 KO mice. P2X7 deletion decreased lung edema and alveolar protein content, which are associated with enhanced alveolar fluid clearance. In addition, activation of the inflammasome was suppressed in P2X7-deficient alveolar macrophages and was associated with suppression of IL-1␤ release. Furthermore, P2X7-deficient alveolar macrophage in type II alveolar epithelial cells (AECs) coculture model abolished protein permeability across mouse type II AEC monolayers. Deletion of P2X7 does not lead to a decrease in epithelial sodium channel expression in cocultures of alveolar macrophages and type II AECs. Taken together, these findings show that deletion of P2X7 is a protective factor and therapeutic target for the amelioration of hyperoxia-induced lung injury. acute lung injury; P2X7; NLRP3; inflammasome; hyperoxia ACUTE LUNG INJURY (ALI) IS a major clinical problem in the United States, accounting for one of the primary causes of in-hospital hypoxemic respiratory failure leading to morbidity and mortality (48). Prolonged exposure to hyperoxia has been conclusively demonstrated to cause ALI (22,58,62). Furthermore, numerous cardiovascular and pulmonary diseases require oxygen therapy (22, 28 -30, 58). Therefore, it is pertinent to understand the mechanism of damage in ALI, wherein the pathology manifests as alveolar damage from immune cell infiltration and pulmonary edema (31,63).Prolonged exposure to oxygen concentrations of FI O 2 Ͼ 0.8 has previously been shown to cause mortality in small animal and higher-order primate models (26). Hyperoxia-induced lung injury contributes to increasing the production of reactive oxygen species (ROS), inflammatory cytokines, as well as exudative pulmonary edema, collagen deposition, and damage to the alveolar epithelium (26, 32). Thus, using hyperoxia as a form of oxygen toxicity is clinically relevant to study the pathophysiology of ALI. In ALI, the disruption of the fine balance between proinflammatory and anti-inflammatory agents is the basic pathology with a concomitant activation of apoptotic signals (33, 59, 62). IL-1␤ was discovered to be one of the early inflammatory cytokines to appear in ALI patients and cause the release of additional cytokines (18). The caspase-1-mediated acti...
Prolonged exposure to oxidative stress causes Acute Lung Injury (ALI) and significantly impairs pulmonary function. Previously we have demonstrated that mitochondrial dysfunction is a key pathological factor in hyperoxic ALI. While it is known that hyperoxia induces the production of stable, but toxic 4-hydroxynonenal (4-HNE) molecule, it is unknown how the reactive aldehyde disrupts mitochondrial function. Our previous in vivo study indicated that exposure to hyperoxia significantly increases 4-HNE-Protein adducts, as well as levels of MDA in total lung homogenates. Based on the in vivo studies, we explored the effects of 4-HNE in human small airway epithelial cells (SAECs). Human SAECs treated with 25 μM of 4-HNE showed a significant decrease in cellular viability and increased caspase-3 activity. Moreover, 4-HNE treated SAECs showed impaired mitochondrial function and energy production indicated by reduced ATP levels, mitochondrial membrane potential, and aconitase activity. This was followed by a significant decrease in mitochondrial oxygen consumption and depletion of the reserve capacity. The direct effect of 4-HNE on the mitochondrial respiratory chain was confirmed using Rotenone. Furthermore, SAECs treated with 25 μM 4-HNE showed a time-dependent depletion of total Thioredoxin (Trx) proteins and Trx activity. Taken together, our results indicate that 4-HNE induces cellular and mitochondrial dysfunction in human SAECs, leading to an impaired endogenous antioxidant response.
Critically ill patients are commonly treated with high levels of oxygen, hyperoxia, for prolonged periods of time. Unfortunately, extended exposure to hyperoxia can exacerbate respiratory failure and lead to a high mortality rate. Mitochondrial A-kinase anchoring protein (Akap) has been shown to regulate mitochondrial function. It has been reported that, under hypoxic conditions, Akap121 undergoes proteolytic degradation and promotes cardiac injury. However, the role of Akap1 in hyperoxia-induced acute lung injury (ALI) is largely unknown. To address this gap in our understanding of Akap1, we exposed wild-type ( wt) and Akap1 mice to 100% oxygen for 48 h, a time point associated with lung damage in the murine model of ALI. We found that under hyperoxia, Akap1 mice display increased levels of proinflammatory cytokines, immune cell infiltration, and protein leakage in lungs, as well as increased alveolar capillary permeability compared with wt controls. Further analysis revealed that Akap1 deletion enhances lung NF-κB p65 activity as assessed by immunoblotting and DNA-binding assay and mitochondrial autophagy-related markers, PINK1 and Parkin. Ultrastructural analysis using electron microscopy revealed that Akap1 deletion was associated with remarkably aberrant mitochondria and lamellar bodies in type II alveolar epithelial cells. Taken together, these results demonstrate that Akap1 genetic deletion increases the severity of hyperoxia-induced acute lung injury in mice.
Idiopathic pulmonary fibrosis (IPF) is an age-related multifactorial disease featuring non-uniform lung fibrosis. The decisive cellular events at early stages of IPF are poorly understood. While the involvement of club cells in IPF pathogenesis is unclear, their migration has been associated with lung fibrosis. In this study, we labeled club cells immunohistochemically in IPF lungs using a club cell marker Claudin-10 (Cldn10), a unique protein based on the recent report which demonstrated that the appearance of Cldn10 in developing and repairing lungs precedes other club cell markers including club cell secretory protein (CCSP). Cldn10-positive cells in IPF lungs displayed marked pleomorphism and were found in varied arrangements, suggesting their phenoconversion. These results were corroborated by immunogold labeling for Cldn10. Further, immunohistochemical double-labeling for Cldn10 and α-smooth muscle actin (α-SMA) demonstrated that aberrant α-SMA signals are frequently encountered near disorganized Cldn10-positive cells in hyperplastic bronchiolar epithelium and thickened interstitium of IPF lungs. Collectively, these data indicate that club cells actively participate in the initiation and progression of IPF through phenoconversion involving the acquisition of proliferative and migratory abilities. Thus, our new findings open the possibility for club cell-targeted therapy to become a strategic option for the treatment of IPF.
The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 -/- ;Ybx3 -/- double mutants using a previously reported Ybx2 -/- model and a newly generated global Ybx3 -/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.
Mutations in the membrane frizzled-related protein (MFRP/Mfrp) gene, specifically expressed in the retinal pigment epithelium (RPE) and ciliary body, cause nanophthalmia or posterior microphthalmia with retinitis pigmentosa in humans, and photoreceptor degeneration in mice. To better understand MFRP function, microarray analysis was performed on eyes of homozygous Mfrprd6 and C57BL/6J mice at postnatal days (P) 0 and P14, prior to photoreceptor loss. Data analysis revealed no changes at P0 but significant differences in RPE and retina-specific transcripts at P14, suggesting a postnatal influence of the Mfrprd6 allele. A subset of these transcripts was validated by quantitative real-time PCR (qRT-PCR). In Mfrprd6 eyes, a significant 1.5- to 2.0-fold decrease was observed among transcripts of genes linked to retinal degeneration, including those involved in visual cycle (Rpe65, Lrat, Rgr), phototransduction (Pde6a, Guca1b, Rgs9), and photoreceptor disc morphogenesis (Rpgrip1 and Fscn2). Levels of RPE65 were significantly decreased by 2.0-fold. Transcripts of Prss56, a gene associated with angle-closure glaucoma, posterior microphthalmia and myopia, were increased in Mfrprd6 eyes by 17-fold. Validation by qRT-PCR indicated a 3.5-, 14- and 70-fold accumulation of Prss56 transcripts relative to controls at P7, P14 and P21, respectively. This trend was not observed in other RPE or photoreceptor mutant mouse models with similar disease progression, suggesting that Prss56 upregulation is a specific attribute of the disruption of Mfrp. Prss56 and Glul in situ hybridization directly identified Müller glia in the inner nuclear layer as the cell type expressing Prss56. In summary, the Mfrprd6 allele causes significant postnatal changes in transcript and protein levels in the retina and RPE. The link between Mfrp deficiency and Prss56 up-regulation, together with the genetic association of human MFRP or PRSS56 variants and ocular size, raises the possibility that these genes are part of a regulatory network influencing postnatal posterior eye development.
Atp8b1 (ATPase, aminophospholipid transporter, class I, type 8B, member 1) is a cardiolipin transporter in the apical membrane of lung epithelial cells. While the role of Atp8b1 in pneumonia-induced acute lung injury (ALI) has been well studied, its potential role in oxidative stress-induced ALI is poorly understood. We herein show that Atp8b1G308V/G308V mice under hyperoxic conditions display exacerbated cell apoptosis at alveolar epithelium and aberrant proliferation of club cells at bronchiolar epithelium. This hyperoxia-induced ambivalent response in Atp8b1G308V/G308V lungs was followed by patchy distribution of non-uniform interstitial fibrosis at late recovery phase under normoxia. Since this club cell abnormality is commonly observed between Atp8b1G308V/G308V lungs under hyperoxic conditions and IPF lungs, we characterized this mouse fibrosis model focusing on club cells. Intriguingly, subcellular morphological analysis of IPF lungs, using transmission electron microscopy (TEM), revealed that metaplastic bronchiolar epithelial cells in fibrotic lesions and deformed type II alveolar epithelial cells (AECs) in alveoli with mild fibrosis, have common morphological features including cytoplasmic vacuolation and dysmorphic lamellar bodies. In conclusion, the combination of Atp8b1 mutation and hyperoxic insult serves as a novel platform to study unfocused role of club cells in IPF.
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