A-Kinase Anchor Protein 1 deficiency causes mitochondrial dysfunction in mouse model of hyperoxia induced acute lung injury.

Background: Critically ill patients on supplemental oxygen therapy eventually develop acute lung injury (ALI). Reactive oxygen species (ROS) produced during ALI perturbs the mitochondrial dynamics resulting in cellular damage. Genetic deletion of the mitochondrial A-kinase anchoring protein 1 (Akap1) in mice resulted in mitochondrial damage, Endoplasmic reticulum (ER) stress, increased expression of mitophagy proteins and pro-inflammatory cytokines, exacerbating hyperoxia-induced Acute Lung Injury (HALI). Objective: Despite a strong causal link between mitochondrial dysfunction and HALI, the mechanisms governing the disease progression at the transcriptome level is unknown. Methods: In this study, RNA sequencing (RNA-seq) analysis was carried out using the lungs of Akap1 knockout (Akap1 -/-) mice exposed to normoxia or 48 h of hyperoxia followed by quantitative real time PCR and Ingenuity pathway analysis (IPA). Western blot analysis assessed mitochondrial dysfunction, OXPHOS complex (I-V), apoptosis and antioxidant proteins. Mitochondrial enzymatic assays was used to measure the aconitase, fumarase, citrate synthase activities in isolated mitochondria from Akap1 -/- vs. Wt mice exposed to hyperoxia. Results: Transcriptome analysis of Akap1 -/- exposed to hyperoxia reveals increases in transcripts encoding electron transport chain (ETC) and tricarboxylic acid cycle (TCA) proteins. Ingenuity pathway analysis (IPA) shows enrichment of mitochondrial dysfunction and oxidative phosphorylation in Akap1 -/- mice. Loss of AKAP1, coupled with oxidant injury, significantly decreases the activities of TCA enzymes. Mechanistically, a significant loss of dynamin-related protein 1 (Drp1) phosphorylation at the protein kinase A (PKA) site Serine 637 (Ser637), decreases in Akt phosphorylation at Serine 437 (Ser47) and increase in the expression of pro-apoptotic protein Bax indicate mitochondrial dysfunction. Heme oxygenase-1 (HO-1) levels significantly increased in CD68 positive alveolar macrophages in Akap1 -/- lungs, suggesting a strong antioxidant response to hyperoxia. Conclusion: Overall these results suggest that AKAP1 overexpression and modulation of Drp1 phosphorylation at Ser637 is an important therapeutic strategy for acute lung injury.

Aberrant Expression of ACO1 in Vasculatures Parallels Progression of Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by mitochondrial dysfunction. However, details about the non-mitochondrial enzymes that sustain the proliferative nature of IPF are unclear. Aconitases are a family of enzymes that sustain metabolism inside and outside mitochondria. It is hypothesized that aconitase 1 (ACO1) plays an important role in the pathogenesis of IPF given that ACO1 represents an important metabolic hub in the cytoplasm. Objectives: To determine if ACO1 expression in IPF lungs shows specific patterns that may be important in the pathogenesis of IPF. To determine the similarities and differences in ACO1 expression in IPF, bleomycin-treated, and aging lungs. Methods: ACO1 expression in IPF lungs were characterized and compared to non-IPF controls by western blotting, immunostaining, and enzymatic activity assay. ACO1-expressing cell types were identified by multicolor immunostaining. Using similar methods, the expression profiles of ACO1 in IPF lungs versus bleomycin-treated and aged mice were investigated. Measurements and main results: Lower lobes of IPF lungs, unlike non-IPF controls, exhibit significantly high levels of ACO1. Most of the signals colocalize with von Willebrand factor (vWF), a lineage marker for vascular endothelial cells. Bleomycin-treated lungs also show high ACO1 expressions. However, most of the signals colocalize with E-cadherin and/or prosurfactant protein C, representative epithelial cell markers, in remodeled areas. Conclusions: A characteristic ACO1 expression profile observed in IPF vasculatures may be a promising diagnostic target. It also may give clues as to how de novo angiogenesis contributes to the irreversible nature of IPF.

Matrix Metalloproteinase 7 Expression and Apical Epithelial Defects in Atp8b1 Mutant Mouse Model of Pulmonary Fibrosis.

Abnormalities in airway epithelia and lung parenchyma are found in Atp8b1 mutant mice, which develop pulmonary fibrosis after hyperoxic insult. Microarray and ingenuity pathway analysis (IPA) show numerous transcripts involved in ciliogenesis are downregulated in 14-month (14 M) -old Atp8b1 mouse lung compared with wild-type C57BL/6. Lung epithelium of Atp8b1 mice demonstrate apical abnormalities of ciliated and club cells in the bronchial epithelium on transmission electron microscopy (TEM). Matrix metalloproteinase 7 (MMP7) regulates of ciliogenesis and is a biomarker for idiopathic pulmonary fibrosis (IPF) in humans. Mmp7 transcript and protein expression are significantly upregulated in 14 M Atp8b1 mutant mouse lung. MMP7 expression is also increased in bronchoalveolar lavage fluid (BAL). Immunohistochemistry is localized MMP7 to bronchial epithelial cells in the Atp8b1 mutant. In conclusion, MMP7 is upregulated in the aged Atp8b1 mouse model, which displays abnormal ciliated cell and club cell morphology. This mouse model can facilitate the exploration of the role of MMP7 in epithelial integrity and ciliogenesis in IPF. The Atp8b1 mutant mouse is proposed as a model for IPF.

Lung fibrosis is induced in ADAR2 overexpressing mice via HuR-induced CTGF signaling.

Adenosine deaminase acting on RNA 2 (ADAR2), an RNA editing enzyme is involved in a site-selective modification of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Its role in the lungs is unknown. The phenotypic characterization of Adarb1 mice that lacked ADAR2 auto-regulation due to the deletion of editing complementary sequence (ΔECS mice) determined the functional role of ADAR2 in the lungs. ADAR2 protein expression increased in the ΔECS mice. These mice display immune cell infiltration and alveolar disorganization. The lung wet by dry ratio indicates there is no lung edema in ΔECS mice. Bronchoalveolar lavage (BAL) analysis of ΔECS mice reveals a significant increase in neutrophils. Interestingly, ΔECS mice spontaneously develop lung fibrosis as indicated by Sirius red staining of collagen fibers in the lung sections and a significant increase in hydroxyproline level in their lungs. ADAR2 expression increased significantly in a bleomycin mouse model, implicating a role of ADAR2 in lung fibrosis. Furthermore, there is a likely possibility that the genetically modified ΔECS mice does not model the physiological or pathophysiological process of lung fibrosis. Nevertheless, this model is useful in interrogating the role of ADAR2 in the lungs. The Ctgf mRNA and connective tissue growth factor (CTGF) protein significantly increased in ΔECS lungs and occurs in bronchial epithelial cells. There is a significant increase in Human antigen R (ELAVL1; HuR) protein levels in ΔECS lungs and suggests a role in stabilizing Ctgf mRNA. Lung mechanics such as total respiratory resistance, Newtonian resistance and tissue damping were increased, whereas inspiratory capacity was decreased in the ΔECS mice. Taken together, these data indicate that overexpression of ADAR2 causes spontaneous lung fibrosis via HuR-mediated CTGF signaling and implicate a role for ADAR2 auto-regulation in lung homeostasis. The identification of ADAR2 target genes in ΔECS mice would facilitate a mechanistic understanding of the role of ADAR2 in the lungs and provide a therapeutic strategy for lung fibrosis.

Nicotine in E-cigarette smoke: cancer culprit?

Tobacco smoke's harmful effects are well-known; the harmful effects of tobacco smoke have been well-investigated. Nicotine in tobacco smoke contributes to the pathogenesis of various conditions, such as lung cancer, coronary artery disease and asthma. A decade ago, a seemingly safer alternative to tobacco cigarettes was introduced- the E-cigarette. However, studies have found that E-cigarette smoke (ECS) not only induces DNA damage but also reduces DNA repair activity via BER and NER pathways. Further research conducted with cells damaged by Ultra-Violet (UV) light or hydrogen peroxide (H2O2) indicates that ECS can function as a comutagen; nicotine can amplify mutagenic activity by merging with other mutagens. The downstream metabolites derived from nicotine found in ECS put E-cigarette smokers at a higher risk for developing lung or bladder cancers or heart disease than their non-smoking counterparts. Overall, these findings are instrumental in our understanding of the harmful effects of ECS.

Alda-1 attenuates hyperoxia-induced mitochondrial dysfunction in lung vascular endothelial cells.

Acute lung injury (ALI) is a major cause of morbidity and mortality worldwide, especially in aged populations. Mitochondrial damage is one of the key features of ALI. Hyperoxia-induced lung injury model in mice has been widely used for ALI study because it features many ALI phenotypes including, but not limited to, mitochondrial and vascular endothelial cell damage. Recently, accumulating evidence has shown that mitochondrial aldehyde dehydrogenase 2 (ALDH2) has a protective effect against oxidative stress mediated cell damage in epithelial cells. However, it is not known whether ALDH2 protects against oxidative stress in vascular endothelial cells. In this current study, we attempted to find the capacity of Alda-1 [(N-(1,3benzodioxol-5-ylmethyl)-2,6- dichloro-benzamide), an ALDH2 activator] to protect against oxidative stress in human microvascular endothelial cells (HMVEC). HMVEC pretreated with Alda-1 prior to hyperoxic exposure vs non-treated controls showed i) lower 4-hydroxynonenal (4-HNE) levels, ii) significantly decreased expressions of Bax and Cytochrome C, iii) partially restored activity and expression of ALDH2 and iv) significantly improved mitochondrial membrane potential. These results suggest that ALDH2 protein in lung vascular endothelial cells is a promising therapeutic target for the treatment of ALI and that Alda-1 is a potential treatment option.

Akap1 genetic deletion increases the severity of hyperoxia-induced acute lung injury in mice.

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.