Long Noncoding RNA FENDRR Inhibits Lung Fibroblast Proliferation via a Reduction of ß-Catenin.

Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive, and usually lethal lung disease and it has been widely accepted that fibroblast proliferation is one of the key characteristics of IPF. Long noncoding RNAs (lncRNAs) play vital roles in the pathogenesis of many diseases. In this study, we investigated the role of lncRNA FENDRR on fibroblast proliferation. Human lung fibroblasts stably overexpressing FENDRR showed a reduced cell proliferation compared to those expressing the control vector. On the other hand, FENDRR silencing increased fibroblast proliferation. FENDRR bound serine-arginine rich splicing factor 9 (SRSF9) and inhibited the phosphorylation of p70 ribosomal S6 kinase 1 (PS6K), a downstream protein of the mammalian target of rapamycin (mTOR) signaling. Silencing SRSF9 reduced fibroblast proliferation. FENDRR reduced ß-catenin protein, but not mRNA levels. The reduction of ß-catenin protein levels in lung fibroblasts by gene silencing or chemical inhibitor decreased fibroblast proliferation. Adenovirus-mediated FENDRR transfer to the lungs of mice reduced asbestos-induced fibrotic lesions and collagen deposition. RNA sequencing of lung tissues identified 7 cell proliferation-related genes that were up-regulated by asbestos but reversed by FENDRR. In conclusion, FENDRR inhibits fibroblast proliferation and functions as an anti-fibrotic lncRNA.

SIRT3 Overexpression Ameliorates Asbestos-Induced Pulmonary Fibrosis, mt-DNA Damage, and Lung Fibrogenic Monocyte Recruitment.

Alveolar epithelial cell (AEC) mitochondrial (mt) DNA damage and fibrotic monocyte-derived alveolar macrophages (Mo-AMs) are implicated in the pathobiology of pulmonary fibrosis. We showed that sirtuin 3 (SIRT3), a mitochondrial protein regulating cell fate and aging, is deficient in the AECs of idiopathic pulmonary fibrosis (IPF) patients and that asbestos- and bleomycin-induced lung fibrosis is augmented in Sirt3 knockout (Sirt3-/-) mice associated with AEC mtDNA damage and intrinsic apoptosis. We determined whether whole body transgenic SIRT3 overexpression (Sirt3Tg) protects mice from asbestos-induced pulmonary fibrosis by mitigating lung mtDNA damage and Mo-AM recruitment. Crocidolite asbestos (100 µg/50 µL) or control was instilled intratracheally in C57Bl6 (Wild-Type) mice or Sirt3Tg mice, and at 21 d lung fibrosis (histology, fibrosis score, Sircol assay) and lung Mo-AMs (flow cytometry) were assessed. Compared to controls, Sirt3Tg mice were protected from asbestos-induced pulmonary fibrosis and had diminished lung mtDNA damage and Mo-AM recruitment. Further, pharmacologic SIRT3 inducers (i.e., resveratrol, viniferin, and honokiol) each diminish oxidant-induced AEC mtDNA damage in vitro and, in the case of honokiol, protection occurs in a SIRT3-dependent manner. We reason that SIRT3 preservation of AEC mtDNA is a novel therapeutic focus for managing patients with IPF and other types of pulmonary fibrosis.

Magneto mitochondrial dysfunction mediated cancer cell death using intracellular magnetic nano-transducers.

Mitochondria are crucial regulators of the intrinsic pathway of cancer cell death. The high sensitivity of cancer cells to mitochondrial dysfunction offers opportunities for emerging targets in cancer therapy. Herein, magnetic nano-transducers, which convert external magnetic fields into physical stress, are designed to induce mitochondrial dysfunction to remotely kill cancer cells. Spindle-shaped iron oxide nanoparticles were synthesized to maximize cellular internalization and magnetic transduction. The magneto-mechanical transduction of nano-transducers in mitochondria enhances cancer cell apoptosis by promoting a mitochondrial quality control mechanism, referred to as mitophagy. In the liver cancer animal model, nano-transducers are infused into the local liver tumor via the hepatic artery. After treatment with a magnetic field, in vivo mitophagy-mediated cancer cell death was also confirmed by mitophagy markers, mitochondrial DNA damage assay, and TUNEL staining of tissues. This study is expected to contribute to the development of nanoparticle-mediated mitochondria-targeting cancer therapy and biological tools, such as magneto-genetics.

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment-both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (C57Bl6) mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7-21 following bleomycin and day 14-21 or day 30-60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis.

Lipid Mediators Regulate Pulmonary Fibrosis: Potential Mechanisms and Signaling Pathways.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease of unknown etiology characterized by distorted distal lung architecture, inflammation, and fibrosis. The molecular mechanisms involved in the pathophysiology of IPF are incompletely defined. Several lung cell types including alveolar epithelial cells, fibroblasts, monocyte-derived macrophages, and endothelial cells have been implicated in the development and progression of fibrosis. Regardless of the cell types involved, changes in gene expression, disrupted glycolysis, and mitochondrial oxidation, dysregulated protein folding, and altered phospholipid and sphingolipid metabolism result in activation of myofibroblast, deposition of extracellular matrix proteins, remodeling of lung architecture and fibrosis. Lipid mediators derived from phospholipids, sphingolipids, and polyunsaturated fatty acids play an important role in the pathogenesis of pulmonary fibrosis and have been described to exhibit pro- and anti-fibrotic effects in IPF and in preclinical animal models of lung fibrosis. This review describes the current understanding of the role and signaling pathways of prostanoids, lysophospholipids, and sphingolipids and their metabolizing enzymes in the development of lung fibrosis. Further, several of the lipid mediators and enzymes involved in their metabolism are therapeutic targets for drug development to treat IPF.

Mitochondrial 8-oxoguanine DNA glycosylase mitigates alveolar epithelial cell PINK1 deficiency, mitochondrial DNA damage, apoptosis, and lung fibrosis.

Alveolar epithelial cell (AEC) apoptosis, arising from mitochondrial dysfunction and mitophagy defects, is important in mediating idiopathic pulmonary fibrosis (IPF). Our group established a role for the mitochondrial (mt) DNA base excision repair enzyme, 8-oxoguanine-DNA glycosylase 1 (mtOGG1), in preventing oxidant-induced AEC mtDNA damage and apoptosis and showed that OGG1-deficient mice have increased lung fibrosis. Herein, we determined whether mice overexpressing the mtOGG1 transgene (mtOgg1tg) are protected against lung fibrosis and whether AEC mtOGG1 preservation of mtDNA integrity mitigates phosphatase and tensin homolog-induced putative kinase 1 (PINK1) deficiency and apoptosis. Compared with wild type (WT), mtOgg1tg mice have diminished asbestos- and bleomycin-induced pulmonary fibrosis that was accompanied by reduced lung and AEC mtDNA damage and apoptosis. Asbestos and H2O2 promote the MLE-12 cell PINK1 deficiency, as assessed by reductions in the expression of PINK1 mRNA and mitochondrial protein expression. Compared with WT, Pink1-knockout (Pink1-KO) mice are more susceptible to asbestos-induced lung fibrosis and have increased lung and alveolar type II (AT2) cell mtDNA damage and apoptosis. AT2 cells from Pink1-KO mice and PINK1-silenced (siRNA) MLE-12 cells have increased mtDNA damage that is augmented by oxidative stress. Interestingly, mtOGG1 overexpression attenuates oxidant-induced MLE-12 cell mtDNA damage and apoptosis despite PINK1 silencing. mtDNA damage is increased in the lungs of patients with IPF as compared with controls. Collectively, these findings suggest that mtOGG1 maintenance of AEC mtDNA is crucial for preventing PINK1 deficiency that promotes apoptosis and lung fibrosis. Given the key role of AEC apoptosis in pulmonary fibrosis, strategies aimed at preserving AT2 cell mtDNA integrity may be an innovative target.

Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts.

The sphingosine kinase 1 (SPHK1)/sphingosine-1-phosphate (S1P) signaling axis is emerging as a key player in the development of idiopathic pulmonary fibrosis (IPF) and bleomycin (BLM)-induced lung fibrosis in mice. Recent evidence implicates the involvement of the Hippo/Yes-associated protein (YAP) 1 pathway in lung diseases, including IPF, but its plausible link to the SPHK1/S1P signaling pathway is unclear. Herein, we demonstrate the increased co-localization of YAP1 with the fibroblast marker FSP1 in the lung fibroblasts of BLM-challenged mice, and the genetic deletion of Sphk1 in mouse lung fibroblasts (MLFs) reduced YAP1 localization in fibrotic foci. The PF543 inhibition of SPHK1 activity in mice attenuated YAP1 co-localization with FSP1 in lung fibroblasts. In vitro, TGF-ß stimulated YAP1 translocation to the nucleus in primary MLFs, and the deletion of Sphk1 or inhibition with PF543 attenuated TGF-ß-mediated YAP1 nuclear localization. Moreover, the PF543 inhibition of SPHK1, or the verteporfin inhibition of YAP1, decreased the TGF-ß- or BLM-induced mitochondrial reactive oxygen species (mtROS) in human lung fibroblasts (HLFs) and the expression of fibronectin (FN) and alpha-smooth muscle actin (α-SMA). Furthermore, scavenging mtROS with MitoTEMPO attenuated the TGF-ß-induced expression of FN and α-SMA. The addition of the S1P antibody to HLFs reduced TGF-ß- or S1P-mediated YAP1 activation, mtROS, and the expression of FN and α-SMA. These results suggest a role for SPHK1/S1P signaling in TGF-ß-induced YAP1 activation and mtROS generation, resulting in fibroblast activation, a critical driver of pulmonary fibrosis.

A spatially restricted fibrotic niche in pulmonary fibrosis is sustained by M-CSF/M-CSFR signalling in monocyte-derived alveolar macrophages.

Ontologically distinct populations of macrophages differentially contribute to organ fibrosis through unknown mechanisms.We applied lineage tracing, single-cell RNA sequencing and single-molecule fluorescence in situ hybridisation to a spatially restricted model of asbestos-induced pulmonary fibrosis.We demonstrate that tissue-resident alveolar macrophages, tissue-resident peribronchial and perivascular interstitial macrophages, and monocyte-derived alveolar macrophages are present in the fibrotic niche. Deletion of monocyte-derived alveolar macrophages but not tissue-resident alveolar macrophages ameliorated asbestos-induced lung fibrosis. Monocyte-derived alveolar macrophages were specifically localised to fibrotic regions in the proximity of fibroblasts where they expressed molecules known to drive fibroblast proliferation, including platelet-derived growth factor subunit A. Using single-cell RNA sequencing and spatial transcriptomics in both humans and mice, we identified macrophage colony-stimulating factor receptor (M-CSFR) signalling as one of the novel druggable targets controlling self-maintenance and persistence of these pathogenic monocyte-derived alveolar macrophages. Pharmacological blockade of M-CSFR signalling led to the disappearance of monocyte-derived alveolar macrophages and ameliorated fibrosis.Our findings suggest that inhibition of M-CSFR signalling during fibrosis disrupts an essential fibrotic niche that includes monocyte-derived alveolar macrophages and fibroblasts during asbestos-induced fibrosis.

Role of phospholipase D in bleomycin-induced mitochondrial reactive oxygen species generation, mitochondrial DNA damage, and pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a pernicious lung disease characterized by alveolar epithelial apoptosis, dysregulated repair of epithelial injury, scar formation, and respiratory failure. In this study, we identified phospholipase D (PLD)-generated phosphatidic acid (PA) signaling in the development of pulmonary fibrosis (PF). Of the PLD isoenzymes, the protein expression of PLD2, but not PLD1, was upregulated in lung tissues from IPF patients and bleomycin challenged mice. Both PLD1 (Pld1-/-)- and PLD2 (Pld2-/-)-deficient mice were protected against bleomycin-induced lung inflammation and fibrosis, thereby establishing the role of PLD in fibrogenesis. The role of PLD1 and PLD2 in bleomycin-induced lung epithelial injury was investigated by infecting bronchial airway epithelial cells (Beas2B) with catalytically inactive mutants of PLD (hPLD1-K898R or mPld2-K758R) or downregulation of expression of PLD1 or PLD2 with siRNA. Bleomycin stimulated mitochondrial (mt) superoxide production, mtDNA damage, and apoptosis in Beas2B cells, which was attenuated by the catalytically inactive mutants of PLD or PLD2 siRNA. These results show a role for PLD1 and PLD2 in bleomycin-induced generation of mt reactive oxygen species, mt DNA damage, and apoptosis of lung epithelial cells in mice. Thus, PLD may be a novel therapeutic target in ameliorating experimental PF in mice.

The mitochondria in lung fibrosis: friend or foe?

Idiopathic pulmonary fibrosis (IPF) and other forms of lung fibrosis are age-associated diseases with increased deposition of mesenchymal collagen that promotes respiratory malfunction and eventual death from respiratory failure. Our understanding of the pathobiology underlying pulmonary fibrosis is incomplete and current therapies available to slow or treat lung fibrosis are limited. Evidence reviewed herein demonstrates key involvement of mitochondrial dysfunction in diverse pulmonary cell populations, including alveolar epithelial cells (AEC), fibroblasts, and macrophages and/or immune cells that collectively advances the development of pulmonary fibrosis. The mitochondria have an important role in regulating whether fibrogenic stimuli results in the return of normal healthy function ("friend") or the development of pulmonary fibrosis ("foe"). In particular, we summarize the evidence suggesting that AEC mitochondrial dysfunction is important in mediating lung fibrosis signaling via mechanisms involving imbalances in the levels of reactive oxygen species, endoplasmic reticulum stress response, mitophagy, apoptosis and/or senescence, and inflammatory signaling. Further, we review the emerging evidence suggesting that dysfunctional mitochondria in AECs and other cell types play crucial roles in modulating nearly all aspects of the 9 hallmarks of aging in the context of pulmonary fibrosis as well as some novel molecular pathways that have recently been identified. Finally, we discuss the potential translational aspects of these studies as well as the key knowledge gaps necessary for better informing our understanding of the pathobiology of the mitochondria in mediating pulmonary fibrosis. We reason that targeting deficient mitochondria-derived pathways may provide innovative future treatment strategies that are urgently needed for lung fibrosis.

Klotho, an antiaging molecule, attenuates oxidant-induced alveolar epithelial cell mtDNA damage and apoptosis.

Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from "exaggerated" lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an antiaging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression, whereas Klotho overexpression is protective while Klotho silencing augments AEC mtDNA damage. Compared with wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in wild-type but not mitochondrial catalase overexpressing (MCAT) mice 3 wk following exposure to asbestos and that EUK-134, a MnSOD/catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent on AKT activation arising from upstream fibroblast growth factor receptor 1 activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.

miR-363-3p inhibits tumor growth by targeting PCNA in lung adenocarcinoma.

Increasing evidence suggests that microRNAs play key roles in lung cancer. Our previous study demonstrated that microRNA 363-3p (miR-363-3p) is downregulated in lung cancer tissues. In this study, we demonstrated that overexpression of miR-363-3p inhibits the proliferation and colony formation of A549 and H441 cells, while silencing of miR-363-3p has the converse effects. The anti-oncogenic function of miR-363-3p was verified in a mouse tumor xenograft model. Furthermore, cell cycle analysis showed miR-363-3p can induce S phase arrest by downregulating Cyclin-D1 and upregulating Cyclin-dependent kinase-2 in lung adenocarcinoma cells. Additionally, miR-363-3p enhances cell apoptosis, whereas miR-363-3p inhibitor prevents apoptosis and leads to downregulation of Bax and Bak expression. The anti-proliferative function of miR-363-3p toward lung cancer cells may be explained by its ability to inhibit the activation of the mTOR and ERK signaling pathways. Using target prediction software and luciferase reporter assays, we identified PCNA as a specific target of miR-363-3p. miR-363-3p can decreased the accumulation of endogenous PCNA in lung adenocarcinoma cells. Moreover, exogenous expression of PCNA relieve the inhibition of miR-363-3p on cell proliferation, colony formation and mTOR and ERK signaling pathways. Taken together, our data indicate that miR-363-3p suppresses tumor growth by targeting PCNA in lung adenocarcinoma.

SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis.

Alveolar epithelial cell (AEC) mitochondrial dysfunction and apoptosis are important in idiopathic pulmonary fibrosis and asbestosis. Sirtuin 3 (SIRT3) detoxifies mitochondrial reactive oxygen species, in part, by deacetylating manganese superoxide dismutase (MnSOD) and mitochondrial 8-oxoguanine DNA glycosylase. We reasoned that SIRT3 deficiency occurs in fibrotic lungs and thereby augments AEC mtDNA damage and apoptosis. Human lungs were assessed by using immunohistochemistry for SIRT3 activity via acetylated MnSODK68 Murine AEC SIRT3 and cleaved caspase-9 (CC-9) expression were assayed by immunoblotting with or without SIRT3 enforced expression or silencing. mtDNA damage was measured by using quantitative PCR and apoptosis via ELISA. Pulmonary fibrosis after asbestos or bleomycin exposure was evaluated in 129SJ/wild-type and SIRT3-knockout mice (Sirt3-/- ) by using fibrosis scoring and lung collagen levels. Idiopathic pulmonary fibrosis lung alveolar type II cells have increased MnSODK68 acetylation compared with controls. Asbestos and H2O2 diminished AEC SIRT3 protein expression and increased mitochondrial protein acetylation, including MnSODK68 SIRT3 enforced expression reduced oxidant-induced AEC OGG1K338/341 acetylation, mtDNA damage, and apoptosis, whereas SIRT3 silencing promoted these effects. Asbestos- or bleomycin-induced lung fibrosis, AEC mtDNA damage, and apoptosis in wild-type mice were amplified in Sirt3-/- animals. These data suggest a novel role for SIRT3 deficiency in mediating AEC mtDNA damage, apoptosis, and lung fibrosis.-Jablonski, R. P., Kim, S.-J., Cheresh, P., Williams, D. B., Morales-Nebreda, L., Cheng, Y., Yeldandi, A., Bhorade, S., Pardo, A., Selman, M., Ridge, K., Gius, D., Budinger, G. R. S., Kamp, D. W. SIRT3 deficiency promotes lung fibrosis by augmenting alveolar epithelial cell mitochondrial DNA damage and apoptosis.

Lrp5/ß-Catenin Signaling Controls Lung Macrophage Differentiation and Inhibits Resolution of Fibrosis.

Previous studies established that attenuating Wnt/ß-catenin signaling limits lung fibrosis in the bleomycin mouse model of this disease, but the contribution of this pathway to distinct lung cell phenotypes relevant to tissue repair and fibrosis remains incompletely understood. Using microarray analysis, we found that bleomycin-injured lungs from mice that lack the Wnt coreceptor low density lipoprotein receptor-related protein 5 (Lrp5) and exhibit reduced fibrosis showed enrichment for pathways related to extracellular matrix processing, immunity, and lymphocyte proliferation, suggesting the contribution of an immune-matrix remodeling axis relevant to fibrosis. Activation of ß-catenin signaling was seen in lung macrophages using the ß-catenin reporter mouse, Axin2+/LacZ. Analysis of lung immune cells by flow cytometry after bleomycin administration revealed that Lrp5-/- lungs contained significantly fewer Siglec Flow alveolar macrophages, a cell type previously implicated as positive effectors of fibrosis. Macrophage-specific deletion of ß-catenin in CD11ccre;ß-cateninflox mice did not prevent development of bleomycin-induced fibrosis but facilitated its resolution by 8 weeks. In a nonresolving model of fibrosis, intratracheal administration of asbestos in Lrp5-/- mice also did not prevent the development of fibrosis but hindered the progression of fibrosis in asbestos-treated Lrp5-/- lungs, phenocopying the findings in bleomycin-treated CD11ccre;ß-cateninflox mice. Activation of ß-catenin signaling using lithium chloride resulted in worsened fibrosis in wild-type mice, further supporting that the effects of loss of Lrp5 are directly mediated by Wnt/ß-catenin signaling. Together, these data suggest that lung myeloid cells are responsive to Lrp5/ß-catenin signaling, leading to differentiation of an alveolar macrophage subtype that antagonizes the resolution of lung fibrosis.

Mitochondrial catalase overexpressed transgenic mice are protected against lung fibrosis in part via preventing alveolar epithelial cell mitochondrial DNA damage.

RATIONALE: Alveolar epithelial cell (AEC) injury and mitochondrial dysfunction are important in the development of lung fibrosis. Our group has shown that in the asbestos exposed lung, the generation of mitochondrial reactive oxygen species (ROS) in AEC mediate mitochondrial DNA (mtDNA) damage and apoptosis which are necessary for lung fibrosis. These data suggest that mitochondrial-targeted antioxidants should ameliorate asbestos-induced lung. OBJECTIVE: To determine whether transgenic mice that express mitochondrial-targeted catalase (MCAT) have reduced lung fibrosis following exposure to asbestos or bleomycin and, if so, whether this occurs in association with reduced AEC mtDNA damage and apoptosis. METHODS: Crocidolite asbestos (100µg/50µL), TiO2 (negative control), bleomycin (0.025 units/50µL), or PBS was instilled intratracheally in 8-10 week-old wild-type (WT - C57Bl/6J) or MCAT mice. The lungs were harvested at 21d. Lung fibrosis was quantified by collagen levels (Sircol) and lung fibrosis scores. AEC apoptosis was assessed by cleaved caspase-3 (CC-3)/Surfactant protein C (SFTPC) immunohistochemistry (IHC) and semi-quantitative analysis. AEC (primary AT2 cells from WT and MCAT mice and MLE-12 cells) mtDNA damage was assessed by a quantitative PCR-based assay, apoptosis was assessed by DNA fragmentation, and ROS production was assessed by a Mito-Sox assay. RESULTS: Compared to WT, crocidolite-exposed MCAT mice exhibit reduced pulmonary fibrosis as measured by lung collagen levels and lung fibrosis score. The protective effects in MCAT mice were accompanied by reduced AEC mtDNA damage and apoptosis. Similar findings were noted following bleomycin exposure. Euk-134, a mitochondrial SOD/catalase mimetic, attenuated MLE-12 cell DNA damage and apoptosis. Finally, compared to WT, asbestos-induced MCAT AT2 cell ROS production was reduced. CONCLUSIONS: Our finding that MCAT mice have reduced pulmonary fibrosis, AEC mtDNA damage and apoptosis following exposure to asbestos or bleomycin suggests an important role for AEC mitochondrial H2O2-induced mtDNA damage in promoting lung fibrosis. We reason that strategies aimed at limiting AEC mtDNA damage arising from excess mitochondrial H2O2 production may be a novel therapeutic target for mitigating pulmonary fibrosis.

SIRT3 is attenuated in systemic sclerosis skin and lungs, and its pharmacologic activation mitigates organ fibrosis.

Constitutive fibroblast activation is responsible for organ fibrosis in fibrotic disorders including systemic sclerosis (SSc), but the underlying mechanisms are not fully understood, and effective therapies are lacking. We investigated the expression of the mitochondrial deacetylase sirtuin 3 (SIRT3) and its modulation by hexafluoro, a novel fluorinated synthetic honokiol analogue, in the context of fibrosis. We find that augmenting cellular SIRT3 by forced expression in normal lung and skin fibroblasts, or by hexafluoro treatment, blocked intracellular TGF-ß signaling and fibrotic responses, and mitigated the activated phenotype of SSc fibroblasts. Moreover, hexafluoro attenuated mitochondrial and cytosolic reactive oxygen species (ROS) accumulation in TGF-ß-treated fibroblasts. Remarkably, we found that the expression of SIRT3 was significantly reduced in SSc skin biopsies and explanted fibroblasts, and was suppressed by TGF-ß treatment in normal fibroblasts. Moreover, tissue levels of acetylated MnSOD, a sensitive marker of reduced SIRT3 activity, were dramatically enhanced in lesional skin and lung biopsies from SSc patients. Mice treated with hexafluoro showed substantial attenuation of bleomycin-induced fibrosis in the lung and skin. Our findings reveal a cell-autonomous function for SIRT3 in modulating fibrotic responses, and demonstrate the ability of a novel pharmacological SIRT3 agonist to attenuate fibrosis in vitro and in vivo. In light of the impaired expression and activity of SIRT3 associated with organ fibrosis in SSc, pharmacological approaches for augmenting SIRT3 might have therapeutic potential.

Pro-inflammatory response and oxidative stress induced by specific components in ambient particulate matter in human bronchial epithelial cells.

Previous studies have shown that biological effect of particulate matter (PM2.5) is involved in including chemical composition and mass concentration, but the precise components and biological action on human bronchial epithelial cell line (BEAS-2B) are still unclear. The aim of this study was to evaluate the in vitro toxicity of PM2.5 collected at six urban sites in China, and to investigate how particle composition affects cytotoxicity. We used human bronchial epithelial (BEAS-2B) cell lines as model in vitro to expose to PM2.5 from different source, and then reactive oxygen species (ROS), superoxide dismutase activity and total antioxidant capacity were analyzed. Furthermore, we estimated the polycyclic aromatic hydrocarbon (PAH) and transition metal and the endotoxin contents. The mRNA expression of IL-1ß and IL-10 following exposure to PM2.5 was measured by QRT-PCR. We also observed the mitochondrial membrane potential (MMP) using JC-1 staining, and apoptosis of BEAS-2B using flow cytometry. In addition, double-stranded DNA breaks (DSBs) were assessed using γ-H2AX immunofluorescence. Our results show that high concentrations of PAHs and elemental Ni were strongly associated with high apoptosis rates and high expression of IL-1ß, in addition, Fe element was associated with the ROS level, furthermore, Fe and Cr element were associated with DNA damage in BEAS-2B cells. The cytotoxic effects of urban PM2.5 derived from six different cities in China appear dependent on the specific components in each. Our results indicate that air quality standards based on PM2.5 components may be more relevant than concentration-response functions (CRF). © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 923-936, 2016.

The Role of Mitochondrial DNA in Mediating Alveolar Epithelial Cell Apoptosis and Pulmonary Fibrosis.

Convincing evidence has emerged demonstrating that impairment of mitochondrial function is critically important in regulating alveolar epithelial cell (AEC) programmed cell death (apoptosis) that may contribute to aging-related lung diseases, such as idiopathic pulmonary fibrosis (IPF) and asbestosis (pulmonary fibrosis following asbestos exposure). The mammalian mitochondrial DNA (mtDNA) encodes for 13 proteins, including several essential for oxidative phosphorylation. We review the evidence implicating that oxidative stress-induced mtDNA damage promotes AEC apoptosis and pulmonary fibrosis. We focus on the emerging role for AEC mtDNA damage repair by 8-oxoguanine DNA glycosylase (OGG1) and mitochondrial aconitase (ACO-2) in maintaining mtDNA integrity which is important in preventing AEC apoptosis and asbestos-induced pulmonary fibrosis in a murine model. We then review recent studies linking the sirtuin (SIRT) family members, especially SIRT3, to mitochondrial integrity and mtDNA damage repair and aging. We present a conceptual model of how SIRTs modulate reactive oxygen species (ROS)-driven mitochondrial metabolism that may be important for their tumor suppressor function. The emerging insights into the pathobiology underlying AEC mtDNA damage and apoptosis is suggesting novel therapeutic targets that may prove useful for the management of age-related diseases, including pulmonary fibrosis and lung cancer.

Circulating microRNA Signatures Associated with Childhood Asthma.

BACKGROUND: MicroRNAs (miRNAs) exist stably and reproducibly in plasma and may be used as biomarkers for various diseases. Little is known about circulating miRNAs in the peripheral blood of juvenile patients with asthma. METHODS: In this study, we used hybridization arrays to compare the miRNA expression profiles among 6 juvenile patients with or without asthma. Using quantitative PCR (qPCR), we verified the expression levels of these miRNAs in plasma from patients with asthma (n = 40) and healthy subjects (n = 14). RESULTS: Our results showed that the levels of plasma miR-Let7C, miR-486, and miR-1260a in childhood asthma patients were significantly higher than in healthy controls (p < 0.01). Additionally, miR-1260a is correlated with the treatment schedule of these patients and patients with long treatment times had higher expression of miR-1260a than short treatment times; miR494 was significantly associated with challenge, and miR-3162-3p was significantly associated with MEF25 in asthma patients suggesting a potential correlation of miRNA levels with clinical disease parameters. Receiver operator characteristic analysis confirmed that the levels of miR-3162-3p could be used to discriminate childhood asthma patients from healthy subjects (area under the curve of 0.821), suggesting it may be a potential diagnostic biomarker. CONCLUSIONS: These results indicate that circulating miR-3162-3p and miR-1260a should be further evaluated as potential non-invasive biomarkers in diagnosis and treatment for childhood asthma.