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Plants and other organisms, but not insects or vertebrates, express the auxiliary respiratory enzyme alternative oxidase (AOX) that bypasses mitochondrial respiratory complexes III and/or IV when impaired. Persistent expression of AOX from Ciona intestinalisin mammalian models has previously been shown to be effective in alleviating some metabolic stresses produced by respiratory chain inhibition while exacerbating others. This implies that chronic AOX expression may modify or disrupt metabolic signaling processes necessary to orchestrate adaptive remodeling, suggesting that its potential therapeutic use may be confined to acute pathologies, where a single course of treatment would suffice. One possible route for administering AOX transiently is AOX-encoding nucleic acid constructs. Here we demonstrate that AOX-encoding chemically-modified RNA (cmRNA), sequence-optimized for expression in mammalian cells, was able to support AOX expression in immortalized mouse embryonic fibroblasts (iMEFs), human lung carcinoma cells (A549) and primary mouse pulmonary arterial smooth muscle cells (PASMCs). AOX protein was detectable as early as 3 h after transfection, had a half-life of ~4 days and was catalytically active, thus supporting respiration and protecting against respiratory inhibition. Our data demonstrate that AOX-encoding cmRNA optimized for use in mammalian cells represents a viable route to investigate and possibly treat mitochondrial respiratory disorders.

The NLRP3 inflammasome is linked to sterile and pathogen-dependent inflammation, and its dysregulation underlies many chronic diseases. Mitochondria have been implicated as regulators of the NLRP3 inflammasome through several mechanisms including generation of mitochondrial reactive oxygen species (ROS). Here, we report that mitochondrial electron transport chain (ETC) complex I, II, III and V inhibitors all prevent NLRP3 inflammasome activation. Ectopic expression of Saccharomyces cerevisiaeNADH dehydrogenase (NDI1) or Ciona intestinalisalternative oxidase, which can complement the functional loss of mitochondrial complex I or III, respectively, without generation of ROS, rescued NLRP3 inflammasome activation in the absence of endogenous mitochondrial complex I or complex III function. Metabolomics revealed phosphocreatine (PCr), which can sustain ATP levels, as a common metabolite that is diminished by mitochondrial ETC inhibitors. PCr depletion decreased ATP levels and NLRP3 inflammasome activation. Thus, the mitochondrial ETC sustains NLRP3 inflammasome activation through PCr-dependent generation of ATP, but via a ROS-independent mechanism.

Plants and many lower organisms, but not mammals, express alternative oxidases (AOXs) that branch the mitochondrial respiratory chain, transferring electrons directly from ubiquinol to oxygen without proton pumping. Thus, they maintain electron flow under conditions when the classical respiratory chain is impaired, limiting excess production of oxygen radicals and supporting redox and metabolic homeostasis. AOX from Ciona intestinalis has been used to study and mitigate mitochondrial impairments in mammalian cell lines, Drosophila disease models and, most recently, in the mouse, where multiple lentivector-AOX transgenes conferred substantial expression in specific tissues. Here, we describe a genetically tractable mouse model in which Ciona AOX has been targeted to the Rosa26 locus for ubiquitous expression. The AOXRosa26 mouse exhibited only subtle phenotypic effects on respiratory complex formation, oxygen consumption or the global metabolome, and showed an essentially normal physiology. AOX conferred robust resistance to inhibitors of the respiratory chain in organello; moreover, animals exposed to a systemically applied LD50 dose of cyanide did not succumb. The AOXRosa26 mouse is a useful tool to investigate respiratory control mechanisms and to decipher mitochondrial disease aetiology in vivo.

Respiratory progenitors can be efficiently generated from pluripotent stem cells (PSCs). However, further targeted differentiation into bronchoalveolar sublineages is still in its infancy, and distinct specifying effects of key differentiation factors are not well explored. Focusing on airway epithelial Clara cell generation, we analyzed the effect of the glucocorticoid dexamethasone plus cAMP-elevating agents (DCI) on the differentiation of murine embryonic and induced pluripotent stem cells (iPSCs) into bronchoalveolar epithelial lineages, and whether keratinocyte growth factor (KGF) might further influence lineage decisions. We demonstrate that DCI strongly induce expression of the Clara cell marker Clara cell secretory protein (CCSP). While KGF synergistically supports the inducing effect of DCI on alveolar markers with increased expression of surfactant protein (SP)-C and SP-B, an inhibitory effect on CCSP expression was shown. In contrast, neither KGF nor DCI seem to have an inducing effect on ciliated cell markers. Furthermore, the use of iPSCs from transgenic mice with CCSP promoter-dependent lacZ expression or a knockin of a YFP reporter cassette in the CCSP locus enabled detection of derivatives with Clara cell typical features. Collectively, DCI was shown to support bronchoalveolar specification of mouse PSCs, in particular Clara-like cells, and KGF to inhibit bronchial epithelial differentiation. The targeted in vitrogeneration of Clara cells with their important function in airway protection and regeneration will enable the evaluation of innovative cellular therapies in animal models of lung diseases.

Dedifferentiation is a common phenomenon among plants but has only been found rarely in vertebrates where it is mostly associated with regenerative responses such as formation of blastemae in amphibians to initiate replacement of lost body parts. Relatively little attention has been paid to dedifferentiation processes in mammals although a decline of differentiated functions and acquisition of immature, "embryonic" properties is seen in various disease processes. Dedifferentiation of parenchymal cells in mammals might serve multiple purposes including (1) facilitation of tissue regeneration by generation of progenitor-like cells and (2) protection of cells from hypoxia by reduction of ATP consumption due to changes in energy metabolism and/or inactivation of energy-intensive "specialized" functions. We recently found that an inflammatory cytokine of the interleukin 6 family, oncostatin M (OSM), initiates dedifferentiation of cardiomyocytes both in vitro and in vivo. Interestingly, activation of the OSM signaling pathway protects the heart from acute myocardial ischemia but has a negative impact when continuously activated thereby promoting dilative cardiomyopathy. The strong presence of the OSM receptor on cardiomyocytes and the unique features of the OSM signaling circuit suggest a major role of OSM for cardiac protection and repair. We propose that continuous activation or malfunctions of the cellular dedifferentiation machinery might contribute to different disease conditions.

Endothelin-1 (ET-1) is considered to be involved in the development and progression of heart failure. Therefore, we analysed the expression of endothelin-converting enzyme-1 (ECE-1), endothelin receptors A (ETA) and B (ETB) mRNAs by standard-calibrated, competitive reverse transcriptase-PCR using an internal-deleted in vitro-transcribed cRNA standard. ET-1 peptide levels were measured using isoform-specific rabbit antibodies against synthetic ET-1. mRNA and protein expression was determined in the right atrial myocardium of New York Heart Association class I patients and class IV patients undergoing aorto-coronary bypass surgery. ECE-1 mRNA was upregulated in failing atrial myocardium. Furthermore, ET-1 peptide levels were increased in failing atrial myocardium. Atrial ETA mRNA expression was not changed, while ETB mRNA was downregulated in the failing atrial myocardium. Our results support an upregulation of ET-1 synthesis by induction of ECE-1 in failing atrial myocardium. Pharmacological inhibition of augmented ECE-1 expression might provide a new therapeutic perspective in the treatment of heart failure.

Redox reactions and electron flow through the respiratory chain are the hallmarks of mitochondria. By supporting oxidative phosphorylation and metabolite transport, mitochondrial redox reactions are of central importance for cellular energy conversion. In the present review, we will discuss two other aspects of the mitochondrial redox state: (i) its control of mitochondrial Ca2+ homeostasis, and (ii) the intramitochondrial formation of reactive oxygen or nitrogen species that strongly influence electron flow of the respiratory chain.

We here report that aging increases expression of endothelin-1 and NO synthases in the vasculature and kidney of normotensive rats in vivo. Expression of preproendothelin-1 mRNA was quantified by RT-PCR and in situ hybridization, and endothelin-1 protein was determined by radioimmunoassay/HPLC. Vascular mRNA expression of NO synthase isoforms II and III was analyzed by RT-PCR. In young animals, vascular endothelin-1 protein was differentially expressed (aorta < renal artery < carotid artery) and increased with aging in all vascular beds (P < 0.05). In the intact aorta of aged rats, mRNA expression of preproendothelin-1, “inducible” NO synthase II, and endothelial cell NO synthase III gene was up-regulated (P < 0.05). Moreover, preproendothelin-1 mRNA expression increased in glomeruli and tubulointerstitial cells (P < 0.05). To our knowledge this is the first study demonstrating local vascular up-regulation of the trophic factor endothelin under physiological conditions. Activation of vascular endothelin and NO synthases may be important, pressure-independent factors contributing to structural and functional abnormalities of age-dependent diseases, including atherosclerosis.

1In this study, the effect of shear stress on the expression of genes of the human endothelin‐1 system was examined. Primary cultures of human umbilical vein endothelial cells (HUVEC) were exposed to laminar shear stress of 1, 15 or 30 dyn cm−2(i.e. 0.1, 1.5 or 3 N m−2) (venous and two different arterial levels of shear stress) in a cone‐and‐plate viscometer.2Laminar shear stress transiently upregulates preproendothelin‐1 (ppET‐1) mRNA, reaching its maximum after 30 min (approx 1.7‐fold increase). In contrast, long‐term application of shear stress (24 h) causes downregulation of ppET‐1 mRNA in a dose‐dependent manner.3Arterial levels of shear stress result in downregulation of endothelin‐converting enzyme‐1 isoform ECE‐1a (predominating in HUVEC) to 36.2 ± 8.5%, and isoform ECE‐1b mRNA to 72.3 ± 1.9% of static control level.4The endothelin‐1 (ET‐1) release is downregulated by laminar shear stress in a dose‐dependent manner.5This downregulation of ppET‐1 mRNA and ET‐1 release is not affected by inhibition of protein kinase C (PKC), or tyrosine kinase. Inhibition of endothelial NO synthase (L‐NAME, 500 μm) prevents downregulation of ppET‐1 mRNA by shear stress.6In contrast, increasing degrees of long‐term shear stress upregulate endothelin receptor type B (ETB) mRNA by a NO‐ and PKC‐, but not tyrosine kinase‐dependent mechanism.7In conclusion, our data suggest the downregulation of human endothelin synthesis, and an upregulation of the ETBreceptor by long‐term arterial laminar shear stress. These effects might contribute to the vasoprotective and anti‐arteriosclerotic potential of arterial laminar shear stress.

Idiopathic pulmonary fibrosis (IPF) is a form of progressive interstitial lung disease with unknown etiology. Due to a lack of effective treatment, IPF is associated with a high mortality rate. The hallmark feature of this disease is the accumulation of activated myofibroblasts that excessively deposit extracellular matrix proteins, thus compromising lung architecture and function and hindering gas exchange. Here we investigated the origin of activated myofibroblasts and the molecular mechanisms governing fibrosis formation and resolution. Genetic engineering in mice enables the time-controlled labeling and monitoring of lipogenic or myogenic populations of lung fibroblasts during fibrosis formation and resolution. Our data demonstrate a lipogenic-to-myogenic switch in fibroblastic phenotype during fibrosis formation. Conversely, we observed a myogenic-to-lipogenic switch during fibrosis resolution. Analysis of human lung tissues and primary human lung fibroblasts indicates that this fate switching is involved in IPF pathogenesis, opening potential therapeutic avenues to treat patients.