BMI1 Silencing Induces Mitochondrial Dysfunction in Lung Epithelial Cells Exposed to Hyperoxia

Acute Lung Injury (ALI), characterized by bilateral pulmonary infiltrates that restrict gas exchange, leads to respiratory failure. It is caused by an innate immune response with white blood cell infiltration of the lungs, release of cytokines, an increase in reactive oxygen species (ROS), oxidative stress, and changes in mitochondrial function. Mitochondrial alterations, changes in respiration, ATP production and the unbalancing fusion and fission processes are key events in ALI pathogenesis and increase mitophagy. Research indicates that BMI1 (B cell-specific Moloney murine leukemia virus integration site 1), a protein of the Polycomb repressive complex 1, is a cell cycle and survival regulator that plays a role in mitochondrial function. BMI1-silenced cultured lung epithelial cells were exposed to hyperoxia to determine the role of BMI1 in mitochondrial metabolism. Its expression significantly decreases in human lung epithelial cells (H441) following hyperoxic insult, as determined by western blot, Qrt-PCR, and functional analysis. This decrease correlates with an increase in mitophagy proteins, PINK1, Parkin, and DJ1; an increase in the expression of tumor suppressor PTEN; changes in the expression of mitochondrial biomarkers; and decreases in the oxygen consumption rate (OCR) and tricarboxylic acid enzyme activity. Our bioinformatics analysis suggested that the BMI1 multifunctionality is determined by its high level of intrinsic disorder that defines the ability of this protein to bind to numerous cellular partners. These results demonstrate a close relationship between BMI1 expression and mitochondrial health in hyperoxia-induced acute lung injury (HALI) and indicate that BMI1 is a potential therapeutic target to treat ALI and Acute Respiratory Distress Syndrome

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

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

Alda-1 Attenuates Hyperoxia-Induced Acute Lung Injury in Mice.

Acute lung injury (ALI), a milder form of acute respiratory distress syndrome (ARDS), is a leading cause of mortality in older adults with an increasing prevalence. Oxygen therapy, is a common treatment for ALI, involving exposure to a high concentration of oxygen. Unfortunately, hyperoxia induces the formation of reactive oxygen species which can cause an increase in 4-HNE (4-hydroxy 2 nonenal), a toxic byproduct of lipid peroxidation. Mitochondrial aldehyde dehydrogenase 2 (ALDH2) serves as an endogenous shield against oxidative stress-mediated damage by clearing 4-HNE. Alda-1 [(N-(1, 3 benzodioxol-5-ylmethyl)-2, 6- dichloro-benzamide)], a small molecular activator of ALDH2, protects against reactive oxygen species-mediated oxidative stress by promoting ALDH2 activity. As a result, Alda-1 shields against ischemic reperfusion injury, heart failure, stroke, and myocardial infarction. However, the mechanisms of Alda-1 in hyperoxia-induced ALI remains unclear. C57BL/6 mice implanted with Alzet pumps received Alda-1 in a sustained fashion while being exposed to hyperoxia for 48 h. The mice displayed suppressed immune cell infiltration, decreased protein leakage and alveolar permeability compared to controls. Mechanistic analysis shows that mice pretreated with Alda-1 also experience decreased oxidative stress and enhanced levels of p-Akt and mTOR pathway associated proteins. These results show that continuous delivery of Alda-1 protects against hyperoxia-induced lung injury in mice

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

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

Image2_A-Kinase Anchor Protein 1 deficiency causes mitochondrial dysfunction in mouse model of hyperoxia induced acute lung injury.TIFF

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.</p