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Articles in PresS. Am J Physiol Lung Cell Mol Physiol (April 18, 2014). doi:10.1152/ajplung.00073.2014
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L-00073-2014 Revised (clean)
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Mitochondria in Lung Biology and Pathology:
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More than just a powerhouse
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Paul T. Schumacker1, Mark N. Gillespie2†,
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Kiichi Nakahira3, Augustine M.K. Choi3, Elliott D. Crouser4
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Claude A. Piantadosi5, and Jahar Bhattacharya6
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Northwestern University Feinberg School of Medicine, Department of
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Pediatrics, Chicago, IL, 2University of South Alabama College of Medicine,
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Department of Pharmacology, Mobile, AL, 3Weill Cornell Medical College,
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Department of Medicine, New York, NY, 4The Ohio State University College
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of Medicine, Department of Internal Medicine, Columbus, OH,
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University School of Medicine, Department of Medicine, Durham, NC, and
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Cellular Biophysics, New York, NY
Duke
Columbia University Medical Center, Department of Physiology and
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†
Correspondence:
e-mail:
telephone:
Mark N. Gillespie, Ph.D
Department of Pharmacology
College of Medicine
University of South Alabama
Mobile, AL 36688
[email protected]
(251) 460-6497
Copyright © 2014 by the American Physiological Society.
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ABSTRACT
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An explosion of new information about mitochondria reveals that their importance
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extends well beyond their time honored function as the “powerhouse of the cell.” In this
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Perspective, we summarize new evidence showing that mitochondria are at the center
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of a reactive oxygen species (ROS)-dependent pathway governing the response to
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hypoxia and to mitochondrial quality control. The potential role of the mitochondrial
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genome as a sentinel molecule governing cytotoxic responses of lung cells to ROS
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stress also is highlighted. Additional attention is devoted to the fate of damaged mtDNA
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relative to its involvement as a Damage Associated Molecular Pattern driving adverse
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lung and systemic cell responses in severe illness or trauma.
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strategies for replenishing normal populations of mitochondria after damage, either
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through promotion of mitochondrial biogenesis or via mitochondrial transfer, are
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discussed.
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Finally, emerging
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INTRODUCTION
“The powerhouse of the cell.”
All of the contributors to the 2013 ATS
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symposium forming the basis of this Perspective Article, and perhaps many of the
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attendees, too, recalled this as their first introduction to mitochondria and often their first
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memorable phrase in biology. Though still true, it now seems almost quaint given the
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explosion of information about mitochondrial functions unrelated to energy metabolism
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that has occurred over the past two decades or so. Our symposium was motivated
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partly by this “explosion” and partly by the equally rapid accumulation of evidence that
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mitochondria likely play crucial roles in lung disease with significant potential to serve as
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targets in pulmonary and critical care medicine.
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This Perspective article does not seek to comprehensively review all
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developments in the mitochondrial field that bear on lung disease. Rather, it highlights
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evolving concepts of how mitochondria contribute to normal lung health, and how
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mitochondria could be targeted for specific intervention in lung disease. Having
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registered this, we present concise summaries pertaining to mitochondria, reactive
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oxygen species (ROS) and oxygen sensing in the lung, the role of the mitochondrial
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genome as a sentinel molecule governing lung cell survival in response to oxidant
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stress, the nature of mtDNA Damage Associated Molecular Patterns (DAMPs)
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originating from the lung and their potential use and importance as biomarkers and
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proinflammatory alarm signals, respectively. Finally, we explore the intriguing notion
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that therapeutic strategies aimed at reconstituting normal mitochondrial population and
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function may comprise therapeutic approaches in acute lung injury.
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Mitochondrial oxygen sensing
Mitochondria consume O2 at cytochrome c oxidase, the terminal complex in the
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electron transport chain (ETC).
Electron flux along the ETC is linked to proton
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translocation across the inner mitochondrial membrane, resulting in the generation of an
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electrochemical gradient that is used by the ATP synthase to generate ATP during
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oxidative phosphorylation. As mitochondria convert O2 to water the intracellular O2
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tension decreases, thereby generating a gradient for O2 diffusion into the cell from the
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extracellular space. Mitochondria are the principal site of oxygen consumption in the
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cell, so their oxygen tension is lower than any other organelle. This characteristic has
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led investigators to ask whether mitochondria function as oxygen sensors in the cell
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(64). A conceptually simple idea is that decreases in cellular O2 levels (tissue hypoxia)
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would limit mitochondrial oxygen consumption, leading to redox changes as the ETC
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complexes became saturated with electrons due to the loss of cytochrome c oxidase
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activity (56).
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systems that feed electrons into the ETC, such as the Krebs cycle.
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conceptual problem with this idea arose from experimental studies showing that
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mitochondria do not become limited by oxygen availability until the cell is nearly anoxic
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(12, 95). Specifically, how could the mitochondria respond to physiological levels of
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hypoxia if their ability to signal via redox alterations was not activated until all of the
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oxygen was gone? For many years this paradox represented a conceptual barrier to
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the idea that mitochondria might be involved in oxygen sensing. Some investigators
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suggested that specialized oxygen sensors such as the glomus cells of the carotid body
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might circumvent this problem by expressing special isoforms of cytochrome c oxidase
These redox changes would then be reflected in the redox status of
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However, a
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with low apparent affinity for O2 (59). In those cells, electron transport and oxygen
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consumption would become O2 supply-limited at milder levels of hypoxia, allowing the
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mitochondria to detect subtle decreases in cellular oxygenation (58).
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experimental evidence for the existence of such a low-affinity oxidase has been
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reported. Moreover, mammalian cells with high-affinity forms of cytochrome c oxidase
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are still capable of detecting and responding to hypoxia.
However, no
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A second hypothesized model of oxygen sensing was promoted by Michelakis,
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Archer and Weir, who sought to explain how pulmonary artery smooth muscle cells
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detect hypoxia and trigger acute hypoxic pulmonary vasoconstriction (4). Their model
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proposed the existence of an intracellular oxidase that would generate more ROS at
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high O2 levels and less under hypoxic conditions. According to that model, ROS would
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then activate voltage-dependent potassium (Kv) channels in the plasma membrane.
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Conversely, the lesser oxidation of these channels during hypoxia would promote
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channel closure, causing membrane depolarization that would lead to the entry of
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extracellular Ca2+ through L-type calcium channels. However, when they tested the
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effect of gp91-(phox) deletion on this response, using mice with genetic deletion of
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NOX2, they found that the cellular response to hypoxia was still present (3). They
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subsequently considered that mitochondria were the source of these ROS signals,
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based on studies using various mitochondrial inhibitors (57). However, a conceptual
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problem with that model has been the consistent failure of diverse chemical antioxidants
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or the expression of enzymatic antioxidant systems to activate hypoxic responses in a
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normoxic cell.
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A third model of mitochondrial oxygen sensing (FIGURE 1) arose from initial
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studies showing that intracellular ROS levels actually increase during hypoxia (13, 26).
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Parallel studies using chemical mitochondrial inhibitors implicated complex III as the
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likely source of ROS generation.
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because it seemed counter-intuitive, and was based on the use of non-specific
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fluorescent probes to detect intracellular ROS levels and pharmacological inhibitors to
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block the ETC (34, 90).
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inhibitors at various concentrations reported conflicting responses. To address this,
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Waypa et al. utilized a novel genetically encoded fluorescent protein sensor to detect
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intracellular ROS levels. The initial sensor, HSP-FRET, exhibited ratiometric behavior
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that obviated an important limitation of the fluorescent chemical probes (91).
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Specifically, ratiometric sensors provide a signal that is independent of the light intensity
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used to excite the probe.
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pulmonary artery smooth muscle cells confirmed the previous observation that ROS
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levels increase in the cytosol during hypoxia (91).
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during hypoxia was shown to be required for the increase in cytosolic Ca2+ activation in
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pulmonary artery smooth muscle cells. Further advances in this hypoxia-induced ROS
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signaling model came with the introduction of roGFP, a redox-sensitive mutant of green
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fluorescent protein (24, 35). This ratiometric sensor provides a specific measure of
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protein thiol oxidation/reduction status in the cell. Two important features contributed to
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the importance of this advance. First, its oxidation is reversible in the cell allowing it to
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be calibrated, analogous to the behavior of ratiometric Ca2+ sensors.
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expression can be targeted to specific intracellular compartments, allowing assessment
This paradoxical response was initially criticized
Importantly, different laboratory groups using the same
Importantly, studies using HSP-FRET expressed in
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Moreover, the increase in ROS
Second, its
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of redox at the subcellular level.
Using roGFP expressed in cytosol, mitochondrial
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intermembrane space and in the mitochondrial matrix, Waypa et al. found that
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significant differences in basal oxidant status exist across these compartments in
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PASMC, with the cytosol existing in a more reduced state, the mitochondrial matrix in a
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highly oxidized state, and the intermembrane space at an intermediate level (92).
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During acute hypoxia, oxidation status in the intermembrane space and the cytosol
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increased, whereas oxidation in the mitochondrial matrix decreased. These findings
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were consistent with a model in which the release of superoxide from complex III to the
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intermembrane space increases during hypoxia, allowing the ROS to potentially exit to
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the cytosol and initiate redox signaling involved in triggering the release of intracellular
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calcium stores (94). By contrast, hypoxia caused an attenuation of oxidant stress in
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mitochondrial matrix.
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compartments could reflect a distinct operation of two independent mechanisms
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controlling ROS generation during hypoxia in the two domains.
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possibility that ROS alters the direction of ROS secretion from the membrane – toward
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the matrix during normoxia and toward the intermembrane space during hypoxia – has
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not been ruled out.
The opposing redox responses to hypoxia in these two
Alternatively, the
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In either case, studies were needed to more critically test the hypothesis that the
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mitochondrial electron transport chain is the source of ROS signaling during hypoxia.
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Accordingly, Waypa et al. developed a genetic model by generating a mouse to permit
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conditional deletion of the Rieske iron-sulfur protein (RISP) (93). This nuclear encoded
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protein is critical for the function of complex III, where it accepts an electron from
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ubiquinol, the carrier that moves electron into complex III from complexes I and II (34).
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ROS generation from complex III can occur after RISP accepts an electron from
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ubiquinol, generating the intermediate free radical, ubisemiquinone.
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electron on ubisemiquinone is normally transferred to the b-cytochromes in the
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complex, but can potentially be transferred instead to O2, generating superoxide. If
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RISP is deleted, ubisemiquinone, and thus superoxide, cannot be generated at complex
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III (33, 54). Accordingly, that group studied PASMC isolated from the conditional RISP
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mice and used Cre recombinase expression to delete the RISP gene. After loss of
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RISP was confirmed, subsequent roGFP studies demonstrated that the hypoxia-
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induced changes in redox status in the cytosol, intermembrane space and the
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mitochondrial matrix were abolished.
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functional complex III was required for the changes in redox observed in different
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subcellular compartments during hypoxia (93). In parallel studies, it was found that the
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transient increase in cytosolic Ca2+ in PASMC during acute hypoxia was abrogated in
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cells where RISP had been deleted (93).
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The remaining
These studies therefore demonstrated that a
A technical limitation of studies using cultured PASMC is the possibility that the
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cells will undergo a phenotypic shift in culture.
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cannot provide an assessment of the role of those responses in the intact lung.
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Accordingly, that group conducted in vivo studies in which RISP was deleted from
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smooth muscle cells by breeding the conditional RISP mice with a transgenic line
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expressing a smooth muscle-specific, tamoxifen-inducible Cre (96). The resulting mice
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were administered tamoxifen to activate nuclear localization of the Cre in smooth
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muscle cells, and subsequent deletion of RISP from smooth muscle was confirmed.
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The acute pulmonary vasoconstriction response was then compared in the knockout
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Moreover, studies in cultured cells
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and control mice, by measuring the right ventricular systolic pressure (RVSP) response
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to acute alveolar hypoxia. Control mice exhibited a significant increase in RVSP during
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5% O2 ventilation, which was significantly attenuated in the RISP-deficient mice (93).
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These findings indicate that a functional complex III is required for the acute
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vasopressor response to hypoxia in the lung.
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Does the same mitochondrial ROS signal trigger other functional responses to
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hypoxia? Mungai et al. tested the hypothesis that hypoxia activates the cellular energy
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sensor, AMP-dependent kinase (AMPK), through an ROS-dependent mechanism. In
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PASMC, they found that ROS generation during moderate hypoxia led to AMPK
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activation, even in the absence of any increase in AMP or ADP concentrations (60).
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Those findings suggest that mitochondrial ROS signals activate AMPK as an early
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response to hypoxia, allowing it to activate protective mechanisms before the cell
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progresses into a bioenergetic crisis where AMP and ADP concentrations are
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increased.
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Do specific antioxidants block these responses? To test this, Sabharwal et al.
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reasoned that the expression of an enzymatic scavenger of H2O2 exclusively in the
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mitochondrial intermembrane space (IMS) should abolish functional responses to
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hypoxia by preventing ROS released from complex III to reach the cytosol (79).
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Peroxiredoxin-5 (PRDX5) is an atypical 2-cysteine-containing peroxidase that is
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normally expressed in multiple subcellular compartments including the mitochondrial
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matrix (45). This enzyme functions as a monomer, whereas other H2O2 scavengers
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only function as multimers. They targeted its expression to the intermembrane space
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using the N-terminal sequence that directs the SMAC/Diablo protein to that
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compartment (66), and confirmed correct localization using immunogold electron
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microscopy (79).
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PASMC, it attenuated the increase in ROS in the IMS and the cytosol during hypoxia.
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Importantly, the PRDX5-expressing cells also showed a significant attenuation of the
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acute hypoxia-induced activation of cytosolic Ca2+.
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independent demonstration that ROS release from the mitochondrial inner membrane to
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the IMS and subsequently to the cytosol is critical for triggering acute responses to
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hypoxia in PASMC.
When PRDX5 was expressed in the intermembrane space of
These findings provide an
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Previous studies by this group had shown that the stabilization of Hypoxia-
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Inducible Factor-1alpha (HIF-1) is triggered by hypoxia-induced release of ROS from
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mitochondria. In PASMC, hypoxia also triggers stabilization of HIF-
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prolyl hydroxylase, which targets the protein for degradation by hydroxylating conserved
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proline residues during normoxia (38, 41).
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PRDX5 in the IMS, hypoxia-induced HIF-1 stabilization was inhibited in a dose-
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dependent manner (79).
In PASMC expressing graded levels of
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Collectively, these findings indicate that mitochondria function as more than
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simple factories supplying ATP to the cell. Through the regulated release of ROS to the
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cytosol, these organelles have the capacity to orchestrate a diverse group of adaptive
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responses to hypoxia. In that regard, the moniker of “cellular oxygen sensors” clearly
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applies to these organelles.
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Sentinel functions of mitochondrial DNA (mtDNA) in pulmonary
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vascular cells
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The nuclear and mitochondrial genomes differ in certain fundamental respects.
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For example, in comparison to the nuclear genome, the mitochondrial genome is tiny.
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Even if all the mtDNA molecules in cells with high mitochondrial densities are totaled, it
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is still a small fraction of the size of the nuclear genome. Following from this, mtDNA
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encodes for only a handful of proteins compared to the many thousands encoded by
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nuclear DNA. Second, the structure and regulation of the mitochondrial genome is
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comparatively simple. Unlike nuclear DNA, in which the state of compaction, its
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interactions with multiple transcription factors and co-activators, and epigenetic
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determinants are all exquisitely regulated processes culminating in precise control of
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gene expression, the mitochondrial genome is expressed as a single unit and its
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transcription is controlled by a handful of mtDNA binding proteins, particularly
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Mitochondrial Transcription Factor A (TFAM, (10, 44). Instead of compaction within a
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nucleosome context, a single molecule of mtDNA is associated with about 30 core
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proteins in a structure known as the nucleoid. Most if not all of the enzymes needed for
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mtDNA replication and mtRNA expression appear to be components of the nucleoid (9).
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Another difference between mitochondrial and nuclear DNA particularly relevant
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to the present discussion is that mtDNA is about 50-fold more sensitive to oxidative
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damage (7, 16, 32, 97). Reasons for this heightened sensitivity are not entirely known,
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but have been attributed to a comparatively simple process of mtDNA repair
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(mitochondria seem to contain only base excision repair while the nucleus has multiple,
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overlapping pathways (8), and the prospect that the open structure of mtDNA is more
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accessible to DNA-damaging ROS. In light of the forgoing comments that mitochondrial
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ROS generation plays a central role in hypoxic signal transduction, the question can be
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raised as to whether mtDNA is damaged by oxidation in hypoxic environments. While
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DNA integrity across the entire mitochondrial genome in hypoxic cells has not been
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examined, a large expanse of the mtDNA coding region - including the “common
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deletion sequence” known to be sensitive to oxidant stress (25) - fails to display
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oxidative base modifications in hypoxia (31). Perhaps this is because in hypoxia, the
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matrix, where mtDNA resides, becomes more reduced in hypoxia as ROS are vectored
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into the intermembrane space and cytosol (92).
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In marked contrast to the lack of effect of hypoxia-induced mitochondrial ROS
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generation on mtDNA integrity, when cells or intact lung tissue are challenged with
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exogenous ROS the nuclear genome is largely spared of lesions while mtDNA is
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extensively damaged (7, 16, 32, 97). One question that emerges from this interesting
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observation pertains to the biological value in having a mitochondrial genome sensitive
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to damage by exogenous oxidants while the nuclear genome is relatively resistant
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potential answer, discussed subsequently, is that mtDNA functions as a “fuse” or a
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“sentinel molecule” such that before an externally applied oxidant stress rises to a level
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that threatens the nuclear genome with mutation, oxidative mtDNA damage triggers
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both death of the affected cell and promotes the dissemination signals to “alarm”
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neighboring and itinerant cells.
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Multiple lines of evidence support the idea that mtDNA serves as a molecular
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sentinel dictating cell survival in response to oxidant stress. As just noted, in virtually all
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cell types studied thus far mtDNA is highly sensitive to ROS-induced damage (7, 32,
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97).
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mediated cell death, with the propensity for cytotoxicity inversely related to the efficiency
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of mtDNA repair (32, 37). Genetic modulation of the first and rate-limiting step in mtDNA
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repair, mediated by Ogg1 (19), a DNA glycosylase that detects and excises oxidatively
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damaged purines – coordinately regulates ROS- and reactive nitrogen species-induced
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mtDNA damage and cell death in all cultured cell populations so far examined (14, 21,
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47, 67, 72, 73). Particularly relevant here are studies using mitochondrially-targeted
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Ogg1 constructs, which eliminate the possibility that the biological consequences of
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Ogg1 over-expression are related to effects on nuclear DNA repair.
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pulmonary artery endothelial cells show that over-expression of mitochondrially-targeted
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Ogg1 reduces sensitivities to both oxidative mtDNA damage and cytotoxicity evoked by
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exogenous ROS stressors (21, 77).
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siRNA, while not sensitizing nuclear DNA to oxidative damage, leads to persistence of
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ROS-induced damage to the mitochondrial genome, increased apoptosis and
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cytotoxicity (78).
There is also a conspicuous association between mtDNA damage and ROS-
Studies in
Conversely, reduction in total cell Ogg1 using
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While these observations suggest that mtDNA integrity is a determinant of ROS-
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induced cytotoxicity, many questions remain about the specific pathway(s) by which
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oxidative mtDNA damage dictates lung cell survival and function. The link between
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ROS-induced mtDNA damage and cell death traditionally has been ascribed to the idea
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that persistent damage leads to diminished mtDNA transcription, defective ETC
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function, and resulting formation of a regenerative cycle of excessive pro-apoptotic ROS
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production and increasing mtDNA damage that ultimately culminates in mitochondrially-
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driven cell death (11, 39, 75). However, this traditional model has never been
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thoroughly explored, and more importantly, emerging evidence suggests that other
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pathways are equally plausible. Protective actions of mtDNA repair enzymes against
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ROS-induced lung cell death and dysfunction might not be linked to mtDNA repair, but
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rather, to their interaction with oxidatively damaged mtDNA or mtDNA damage-related
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proteins.
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defective Ogg1 mutant protected against asbestos-induced apoptosis in A549 cells (67).
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Because the repair-defective enzyme, like the wild-type enzyme, bound aconitase, the
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possibility was advanced that Ogg1 influences ROS-mediated signaling events
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downstream of mtDNA damage (80). In a related context, it has recently been shown
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that Ogg1 in complex with free 8-oxoguanine serves as a guanine exchange factor to
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activate Ras-mediated signaling (29), but whether this occurs in intact cells with ROS-
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induced mtDNA damage has not been established.
In this regard, over-expression of a mitochondrially-targeted DNA repair-
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As a means of exploring involvement of oxidative mtDNA damage in intact lung
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and animal models of pulmonary disease, Wilson and colleagues developed novel
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fusion-protein constructs targeting DNA repair glycosylases, either Ogg1 or the bacterial
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enzyme Endo III, to mitochondria (48). Early work showed that these fusion protein
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constructs prevented both hydrogen peroxide-induced mtDNA damage and increases in
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the vascular filtration coefficient in isolated perfused rat lungs (16). The Ogg1 construct
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also attenuated ventilator-induced lung injury and mortality in mice (36) and hyperoxia-
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induced dysmorphogenesis in fetal rat lung explants (28).
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suggest that mitochondrially-targeted Ogg1 inhibits mortality and acute lung injury
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evoked by intratracheal P. aeruginosa in rats (15).
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Preliminary studies also
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These observations on the effectiveness of increasing mtDNA repair in isolated
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lungs and animal models are a step towards determining if mtDNA integrity is a
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legitimate pharmacologic target in lung disease. However, there are many unresolved
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issues. As noted above, some pertain to the specific mechanism whereby increased
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mtDNA repair protects against cytotoxicity. As an extension of this, whereas protection
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of mtDNA integrity in cultured cells inhibits ROS-induced cytotoxicity, the involvement of
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cytotoxicity per se in intact animal models of acute lung injury is complex. Taking this
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analysis one step further, it is easy to envision how oxidative mtDNA damage could, by
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different mechanisms, mediate different types of cellular responses in lung disease. For
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example, ROS-induced cytotoxicity, driven by persistent mtDNA damage, defective
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mtDNA transcription and attendant bioenergetic insufficiency, could be lethal to some
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populations of lung cells, while oxidant-induced alterations in mtDNA-protein stability
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might acutely influence behavior of other lung cell populations secondary to alterations
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in cytoskeletal integrity (74).
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mechanism(s) of action and pharmacologic utility of protecting mtDNA integrity in the
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many pulmonary disorders where acute oxidant stress plays a pathogenic role.
Additional studies will be required to determine the
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It has been appreciated for some time that ROS contribute to the evolution of
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ALI/ARDS and other lung diseases, possibly through an adverse effect on mitochondrial
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function (11). Indeed, about 40 clinical trials have examined the effect of anti-oxidant
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strategies in acute lung injury and multiple organ system failure, but the therapeutic
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effects of anti-oxidant strategies have been unimpressive (88). These disappointing
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outcomes may be attributed in part to the heterogeneous nature and onset of ALI/ARDS
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that tend to confound design and interpretation of clinical trials. There also remains
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considerable scientific uncertainty about molecular targets of anti-oxidant drug action.
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For example, non-selective antioxidants may disrupt signaling required for physiologic
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adaption, such as hypoxia-mediated ventilation-perfusion matching in the injured lung,
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lung cell survival and recovery. Strategies currently available also may not target the
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key sentinel molecule(s) triggering adverse cellular effects of ROS.
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suggested by the foregoing discussion, mtDNA could be such a sentinel molecule.
Perhaps, as
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Mitochondrial DNA DAMP Release and its Cellular Targets in Lung
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Disease
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An intriguing question to emerge from the prospect that mtDNA damaged by
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oxidation in the setting of oxidant lung injury pertains to the fate of the molecule. As has
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been suggested from studies in other experimental systems (20), if mtDNA damage is
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severe and not promptly repaired, does the molecule fragment after which it is exported
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from the organelle, possibly as part of mitophagy? And if oxidatively-damaged mtDNA
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fragments are exported from mitochondria, what are the next destinations of the
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fragments and do they play a role in disease pathogenesis? These questions, depicted
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in FIGURE 2, are discussed below.
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If unrepaired, damaged mtDNA is rapidly degraded (81). Interestingly, mtDNA so
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damaged displays a higher abundance of sugar-phosphate backbone lesions and
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abasic sites relative to oxidized base products, which would be expected to fragment
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the DNA rather then lead to persistent mutagenic lesions. There is scant evidence
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concerning the fate of mtDNA fragments formed as a consequence of oxidative
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damage. In a study using isolated mitochondria subjected to an exogenous oxidant
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stress, Garcia and Chavez reported that mtDNA fragments less than 1000bp were lost
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from the organelle via the permeability transition pore (27). Studies in macrophages
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also indicate that the permeability transition pore is required for cytosolic accumulation
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of mtDNA after immunologic stimulation (61), but whether there is some specificity for
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fragment size or other attributes is unknown. In cardiomyocytes subjected to
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hemodymamic stress-induced mitochondrial damage, mtDNA fragments accumulate in
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autolysosomes where, on the one hand, Dnase II is capable of degrading the fragments
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to biological insignificance (65).
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degradation appear capable of autonomously activating endosomal TLR9 (65). In
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immunologically-stimulated macrophages, mtDNA fragments accumulating in the
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cytoplasm function as a co-activator in the NALP3-dependent activation of caspase 1
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(61). Importantly, both TLR9 and NALP3 activation can initiate downstream pro-
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inflammatory signaling.
On the other hand, mtDNA fragments escaping
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In severely injured (100) and critically ill human subjects (62) mtDNA
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accumulates in the circulation where it functions as a DAMP and causes TLR9-
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dependent activation of the innate immune system. Here, too, the mechanism by which
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mtDNA fragments exit cells has received little study.
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interferon-primed
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accumulation of mtDNA fragments in the extracellular space. The release process –
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described as “catapult-like” – occurred rapidly and was not dependent on death of the
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cells (98). Whether a similar process accounts for release of mtDNA from other types of
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oxidatively- or mechanically-stressed cells is entirely unknown. Indeed, whether mtDNA
eosinophils,
lipopolysaccharide
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In interleukin-5- or gamma
causes
the
ROS-dependent
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DAMP release is inextricably linked to autophagy, mitophagy or cell death remains to be
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established.
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As noted previously, mtDNA is normally localized in a multi-protein complex
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within the mitochondrial matrix, referred to as a nucleoid, which contains the essential
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transcription factor and structural protein TFAM (9, 30, 44). TFAM has a high binding
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affinity for mtDNA and remains associated when DNA is immunoprecipitated from lysed
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cells (43). TFAM is a functional and structural homologue of the nuclear DNA binding
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protein HMGB1, and both TFAM and HMGB1 are potent ligands for RAGE.
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context promoting inflammation, RAGE ligands are shown to amplify the immune
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responses to pattern-recognizing Toll-like receptors (43). In contrast to nuclear DNA
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which is non-immunogenic, mtDNA, like viral and bacterial DNA, is enriched with
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hypomethylated CpG motifs, which are natural ligands for TLR9, a highly specific
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intracellular (endosomal) pattern recognizing receptor (50).
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TFAM and mtDNA, it is not surprising that TFAM remains associated with mtDNA
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following cell necrosis, and this complex is particularly potent in terms of promoting
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TLR9-responsive immune cell activation (43).
In the
Given these properties of
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Whereas many cell types are capable of responding to exogenous and
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endogenous danger signals, including PMNs, monocytes, and even epithelial cells (50),
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dendritic cells are specialized for sensing and processing immunogenic antigens to
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initiate and coordinate complex and sustained immune responses.
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present in low abundance (<1%) in blood and other tissues, immature dendritic cells,
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particularly plasmacytoid DCs (pDCs) and immature myeloid DCs (iDCs), are uniquely
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equipped to initiate and ultimately sustain sterile immune responses to immunogenic
18
Despite being
425
DNA, including viral and bacterial DNA. Immature DCs are unique in their capacity to
426
simultaneously promote pro- and anti-inflammatory immune responses.
427
distinguished from other immune cell lines by the lack of expression of common T-cell,
428
B-cell or myeloid cell surface markers, and are further discriminated by their ability to
429
produce large quantities of type I interferons in response to immunogenic (e.g., viral or
430
bacterial) RNA and DNA, recognized by TLR7/8 and TLR9, respectively.
431
iDCs and pDCs, through the production of TNFa, further promote “bystander” activation
432
of regional macrophages, are induced to mature, and are targeted to various tissues via
433
expression of specific chemokine receptors [e.g., CCR2 targets DCs to lung (53), where
434
they are capable of presenting antigen via MHC class II molecules to promote potent T
435
cell-mediated adaptive and counter-regulatory immune responses (FIGURE 3).
They are
Activated
436
In the context of acute cell damage, immature DCs respond specifically to
437
mitochondrial danger signals; indeed, the TFAM/mtDNA complex has been shown to be
438
sufficient to promote the simultaneous release of TNFa and IFNa from purified pDCs
439
(42, 43) (FIGURE 3A).
440
approximating the complex interaction of immune cells in vivo, TFAM was found to
441
augment the immune response to CpGA DNA (a readily available surrogate for mtDNA).
442
Furthermore, the depletion of pDCs, representing a small fraction of the total splenocyte
443
culture population, dramatically suppressed the innate immune response to TFAM/CpG
444
complex, demonstrating the capacity of pDCs to amplify both proinflammatory and
445
counter-regulatory immune pathways (FIGURE 3B). Together, these data indicate that
446
TFAM remains associated with mtDNA following acute cell damage, that TFAM serves
447
to augment the immune response to mtDNA, and immature DCs are important sensors
Moreover, using a splenocyte culture model closely
19
448
for TFAM/mtDNA complex and important regulators of the sterile immune response to
449
these mitochondrial danger signals.
450
New evidence indicates that DCs, particularly pDC-like macrophages, are of
451
critical importance during the pathogenesis of acute lung injury in the context of sterile
452
inflammation, and mitochondrial danger signals are incriminated as proximal mediators.
453
Immunogenic DNA, such as is present in bacteria and mitochondria, is sufficient to
454
promote ALI in animal models (46, 87), and mtDNA has been specifically linked to the
455
pathogenesis of ALI in setting of sterile inflammation induced experimentally by acid
456
aspiration(17), surgical trauma (100), and acute liver necrosis (55).
457
through activation of TLR9, appear to play a major role in initiating ALI under these
458
conditions. By contrast, recruitment of pDC-like macrophages to the lung is shown to
459
attenuate ALI induced by LPS, particularly in the acute phase of lung injury (49). These
460
pDC-like macrophages express CCR2 and are recruited to the lung by CC chemokine
461
ligand-2 (CCL2), whereupon they facilitate the clearance of damaged cells (52). In
462
addition to enhanced clearance of damaged cells, pDC activation in the lungs results in
463
the release of Type 1 interferons which are capable of suppressing ALI by altering the
464
balance between pro-inflammatory (e.g. IL-1b, IL-6) and anti-inflammatory (e.g., IL-10)
465
cytokines (5). Finally, lung DCs present antigen to and thereby activate regulatory T
466
cells (Tregs), which, in turn, contribute to the resolution of inflammation during the sub-
467
acute phase of ALI(1) (FIGURE 3A).
468
acute damage inflicted by activated PMNs is counterbalanced by pDCs in the context of
469
ALI induced by mitochondrial danger signals.
Neutrophils,
Thus, the current evidence suggests that the
470
20
471
Circulating Mitochondria DNA: A Biomarker of Sepsis and ARDS in
472
the ICU
473
As noted above, accumulating evidence suggests that mitochondrial damage
474
may engender the export mtDNA DAMPs that activate innate immune responses and
475
possibly propagate injury to distant targets. In this regard, while biomarkers can be
476
useful for identifying patients with critically illness or assessing the responses to the
477
therapies for the patients in the intensive care unit (ICU), a small number of biomarkers
478
reflecting the disease severity are commonly measured in clinical practice (e.g. lactate,
479
(71). The question thus arises as to whether mtDNA DAMP may be a useful biomarker.
480
Inflammasomes are multi-protein complexes that activate caspase-1 and
481
downstream immune responses, including the maturation and secretion of pro-
482
inflammatory cytokines (i.e. interleukin (IL)-1β and IL-18) (18). Recent studies show
483
inflammasomes are involved in the pathogenesis of various diseases such as infection,
484
cancer and metabolic diseases (18). Inflammasomes are activated by a wide range of
485
signals of pathogenic and non-pathogenic origins (host cell derived molecules including
486
extracellular ATP, hyaluronan, amyloid-β fibrils and uric acid crystals) (18).
487
To examine the involvement of inflammasome in critical illnesses, Choi and
488
colleagues first performed gene expression profiling analysis of lungs from mice
489
subjected to mechanical ventilation (MV), an experimental animal model of acute lung
490
injury (ALI). Specific changes in expression were identified for caspase-activator
491
domain-10, interleukin-18 receptor-1 (Il18r1), and Il1r2 and interleukin-1 (Il1b) as
492
genes following MV (22, 23).
Since genes representing inflammasome complex
21
493
molecules and the downstream cytokines were regulated in animal models of ALI, it was
494
then determined whether inflammasome family genes are also regulated in human
495
critical illnesses such as acute respiratory distress syndrome (ARDS), a lethal disease
496
in ICU, and sepsis, a major cause of ARDS and a leading cause of death in ICU. Total
497
blood RNA was extracted from patients admitted in medical ICU to determine the global
498
gene expression profile of ICU controls, patients with systemic inflammatory response
499
syndrome (SIRS), sepsis, and sepsis/ARDS. The gene expression profiling analysis
500
revealed that inflammasome-associated genes (e.g. asc and Il1b) were up-regulated in
501
patients with sepsis/ARDS when compared to SIRS (23). In addition, plasma levels of
502
IL-18, a representative inflammasome-mediated pro-inflammatory cytokine, at the time
503
of medical ICU admission were higher with increasing disease severity categories
504
(control, SIRS, sepsis and sepsis/ARDS) and the highest in sepsis/ARDS patients (23).
505
Importantly, patients with elevated IL-18 levels at the time of medical ICU hospitalization
506
had increased mortality (23). In mice, MV enhanced IL-18 levels in the serum and
507
bronchoalveolar lavage fluid (23). Furthermore, IL-18 neutralizing antibody treatment, or
508
genetic deletion of IL-18 or caspase-1, reduced lung injury in response to MV (23).
509
These data suggest critical roles of inflammasome-mediated immune responses in ICU
510
patients.
511
Recent studies suggest mtDNA is a potent inflammasome activator (61) and is
512
released from mitochondria to the cytosol or the extracellular space as a mitochondrial
513
DAMP. In addition, exogenous administration of disrupted mitochondria containing
514
mtDNA induces acute inflammatory tissue injuries (100).
515
whether the levels of circulating mtDNA are associated with disease severity or mortality
22
Therefore, we examined
516
in ICU (62). Circulating cell-free mtDNA level was higher in ICU patients who died within
517
28 days of medical ICU admission as well as in patients with sepsis or ARDS in the
518
ICU. Medical ICU patients with an elevated mtDNA level had an increase in their odds
519
of dying within 28 days of ICU admission, while no evidence for association was noted
520
in non-medical ICU patients (e.g. surgical ICU patients). There was no evidence that the
521
association between mtDNA level and medical ICU mortality was attenuated in models
522
adjusted for clinical covariates, including Acute Physiology and Chronic Health
523
Evaluation (APACHE) II score, sepsis, or ARDS. The inclusion of an elevated mtDNA to
524
a clinical model (including age, gender, race/ethnicity, APACHE II score, and sepsis)
525
improved the net-reclassification of 28-day mortality among medical ICU patients. The
526
improvement in net reclassification remained significant even after additionally adjusting
527
for measures of procalcitonin or lactate, commonly used biomarkers in ICU. Similar
528
relationships between circulating mtDNA and outcome have been noted in patients with
529
severe trauma (82) or patients with sepsis in the emergency room (51).
530
circulating mtDNA could serve as a viable plasma biomarker in medical ICU patients
531
and, may represent an important pathogenic determinant that contributes to a
532
exaggerated systemic inflammatory response observed in patients with sepsis or
533
ARDS.
Thus,
534
535
Mitochondrial biogenesis to the rescue
536
Decades of laboratory and clinical studies have revealed that acute systemic
537
inflammatory syndromes, including sepsis, generate substantial macromolecular
23
538
damage to mitochondria, particularly in the cells of highly metabolically active tissues
539
like the liver, heart, and kidneys (83, 85). Such mitochondrial damage may be
540
indiscernible in vivo because it does not necessarily activate cell death pathways, and
541
some of the same factors that damage mitochondria – e.g., ROS - can also activate the
542
process of mitochondrial biogenesis (86). Recently mitochondrial biogenesis has even
543
been detected even in distal lung cells, including alveolar type II epithelial cells(6).
544
Indeed, all cells that rely on aerobic respiration and oxidative phosphorylation for energy
545
conservation express a robust genetic program of mitochondrial quality control that is
546
designed to maintain a fully functional complement of mitochondria through the
547
conjoined
548
(mitophagy) (89). These closely regulated processes serve a pro-survival capacity by
549
allowing cells to quickly replace metabolically dysfunctional mitochondria with fresh
550
organelles from a pool of undamaged organelles before energy failure (FIGURE 4).
activities
of
mitochondrial
biogenesis
and
mitochondrial
autophagy
551
During inflammation mitochondrial damage is caused by the over-production of
552
reactive oxygen and nitrogen species (ROS/RNS) at multiple sites, leading to the
553
inactivation of critical mitochondrial macromolecules through biochemical oxidation,
554
nitration, and/or S-nitrosation. The critical mitochondrial targets are diverse, including
555
many proteins, lipids, and mtDNA that embody integral mitochondrial processes
556
including protein importation, transcription, protein synthesis, TCA cycle function,
557
electron transport, heme synthesis, and control of the mitochondrial permeability
558
transition pore (69). The upshot of damage from elevated ROS production is the
559
compromised efficiency of oxidative phosphorylation, which may destabilize energy
560
homeostasis, and lead to further inflammation that may perpetuate the mitochondrial
24
561
damage (101). However, the production of mitochondrial ROS also provides signals to
562
activate redox-sensitive transcriptional pathways that regulate mitochondrial quality
563
control and trigger the synthesis of counter-inflammatory mediators, such as IL-10 (70).
564
Moreover, the enhanced induction of enzymatic NO and CO production also promotes
565
the expression of a large group of genes that is essential for mitochondrial biogenesis
566
(63, 84).
567
A number of the transcriptional pathways activated by the NO synthase 2 (NOS2)
568
and heme oxygenase-1 (HO-1) enzymes for the induction of mitochondrial biogenesis
569
during inflammation have been worked out over the last several years. For both
570
enzymes, the endogenous gaseous product (NO or CO) is directly involved in the
571
induction mechanisms through the activation of redox-sensitive pathways. For NOS2,
572
as for the other NOS isoforms, a key step involves the cyclic GMP-dependent
573
phosphorylation of cyclic AMP-responsive element-binding protein 1 (CREB1), which
574
undergoes translocation to the nucleus to activate the gene for PGC-1a, a critical co-
575
activator of mitochondrial biogenesis and oxidative metabolism. Activated PGC-1a
576
protein is a partner for nuclear respiratory factor-1 (NRF-1) and NRF-2 (GABPA); these
577
are both essential transcription factors for mitochondrial biogenesis in most cells. For
578
HO-1, heme catabolism releases CO, which binds to the reduced terminal oxidase of
579
the respiratory chain and increases the mitochondrial H2O2 leak rate. The increased
580
H2O2 production activates several pro-survival kinases, such as Akt, as well as the
581
transcription factor Nfe2l2 (Nrf2)(68). Nrf2, in its gene transactivation function, binds to
582
anti-oxidant response elements (ARE) in the NRF-1 gene promoter, while Akt
583
phosphorylates GSK-3b, which relieves a restriction on the entry of Nrf2 into the
25
584
nucleus.
Akt phosphorylates the NRF-1 protein as well, allowing both transcription
585
factors to gain entrance to the nucleus to induce an assemblage of genes required for
586
mitochondrial biogenesis.
587
At counterpoise to mitochondrial biogenesis is the removal of senescent or
588
damaged mitochondria that no longer perform oxidative phosphorylation or are
589
triggering oxidative damage to nearby cellular structures. Such damaged mitochondria
590
also propagate certain danger signals, for example, inflammasome activation (101). The
591
deconstruction of obsolescent mitochondria by mitophagy, a form of selective
592
macroautophagy, entails the envelopment of the mitochondrial cargo within double-
593
membrane vesicles or autophagosomes, which fuse with lysosomes to degrade the
594
cargo. The rate of mitophagy is governed by the extent of mitochondrial damage, but
595
also by the cell’s energy requirement and its capacity to meet this mitochondrial
596
disposal function (99). Thus, from the cellular perspective, optimal stress-stabilizing
597
strategies match energy requirements with the rates of mitophagy and mitochondrial
598
biogenesis in order to ensure energy sufficiency and mitochondrial quality control.
599
In the mammalian lung, mitochondrial energy provision is an important function of
600
many of the roughly 40 cell types present there. In the parenchyma for instance,
601
alveolar type II cells are enriched in mitochondria, which provide ATP to support
602
surfactant synthesis, secretion, and recycling. These cells also initiate mitochondrial
603
biogenesis during acute lung injury, pneumonia, hyperoxia, and other stresses through
604
induction of the HO-1/CO and NOS2 systems. These alveolar type II cell mitochondria
605
also proliferate to support not only surfactant metabolism, but multiple other energy-
606
dependent functions in the transition to type I epithelium, such as those involved in
26
607
maintaining alveolar barrier integrity. In fact, mitochondrial proliferation precedes the
608
proliferation of type II cells as well as their differentiation into type I epithelium. Under
609
many of these same conditions, as discussed elsewhere in this Perspective, autophagy
610
programs, including mitophagy, are activated in order to eliminate damaged proteins
611
and other damaged organelles from the cell.
612
In the future, our understanding of the basic intracellular mechanisms of
613
mitochondrial quality control in the lung will be brought into line with newly emerging
614
roles for mitochondrial depletion, immune cell scavenging of exported mitochondrial
615
components, and adoptive mitochondrial rescue-by-transfer during acute and chronic
616
lung injury. The importance of these intricate extra- and intercellular processes will
617
hinge on the relative effectiveness of mitochondrial biogenesis and mitophagy under
618
pathogenic conditions of varying type, intensity, and severity that are currently being
619
investigated in different experimental settings.
620
621
Mitochondrial transfer to the rescue
622
The foregoing discussion raises the prospect that mitochondrial oxygen sensing,
623
maintenance of mtDNA integrity to prevent mtDNA fragmentation and release in the
624
form of mtDNA DAMPs, and mitochondrial biogenesis may be important determinants of
625
outcome in sepsis.
626
severe illness is to prevent mitochondrial dysfunction (11).
627
decreased ATP production could causes failure of various aspects of cell function that in
628
combination could lead to multi-organ failure in ALI/ARDS. The cause of mitochondrial
A related and desirable goal of mitochondrial-directed therapy in
27
Clearly, the attendant
629
failure is not well understood, but one possibility is that NFκB activation, occurring
630
secondary to TLR4 ligation by bacterial endotoxin, causes transcriptional up regulation
631
of inducible nitric oxide synthase. The increased nitric oxide thus produced reacts with
632
mitochondrial superoxide to cause peroxynitration, hence dysfunction of mitochondrial
633
electron transport proteins (69). Although other mechanisms may also apply, the end
634
result is failure of cellular bioenergetics as well-documented by the reduction of tissue
635
ATP levels. The therapeutic objective therefore, would be to replenish dysfunctional
636
mitochondria of host cells with fresh mitochondria.
637
The question is how does one replenish mitochondria? One possibility is to give
638
exogenous mitochondria with the expectation that cells will take up the mitochondria by
639
endocytosis and thereby repair the ATP deficit. However the caveat here is that free
640
mitochondria in the extracellular space might act as DAMPs (damage-associated
641
molecular patterns) that activate pattern recognition receptors that exacerbate tissue
642
inflammation and injury (100).
643
The other option is to enable intercellular mitochondrial transfer, a process that
644
has been reported in cultured cells (76). Islam et al. now show that the process also
645
occurs in vivo and that mitochondrial transfer from exogenously administered BMSCs
646
(bone-marrow-derived stromal cells) to the alveolar epithelium protects against
647
lipopolysaccharide (LPS)-induced ALI (40). A more recent report by Ahmad et al. also
648
affirms that mitochondrial transfer from BMSCs to the airway epithelium occurs in vivo
649
under inflammatory conditions (2).
650
mitochondrial transfer abrogated airway inflammation and protected against bronchial
651
injury caused by mitochondrial dysfunction. These findings attest to the feasibility of
In this paper, the authors show that the
28
652
mitochondrial transfer between specific cell types in vivo, indicating that it might be
653
applicable as a therapeutic tool in that mitochondrial transfer might correct the deficit of
654
mitochondrial function in affected cells through donation of fresh mitochondria from
655
BMSCs.
656
Islam et al. noted two important features regarding mitochondrial transfer. First,
657
the transfer did not occur when BMSCs were instilled in control lungs not exposed to
658
LPS, indicating that pathologic stress to the alveolar epithelium was essential. Second,
659
stabilization of BMSCs on the alveolar epithelium was also essential. The stabilization
660
occurred through formation of gap junctional channels (GJCs) between the BMSCs and
661
the alveolar epithelium. Knockdown of connexin 43, a GJC protein, blocked the BMSC
662
stabilization and also diminished the survival benefit accruing from BMSC instillation.
663
These findings indicate that for mitochondrial transfer to occur from a donor to a
664
recipient cell, a pre-conditioning of the interacting cell surfaces is critical.
665
In the study by Islam et al. the mechanism of mitochondrial transfer in alveoli
666
occurred through a sequence in which BMSCs released mitochondria-containing
667
nanotubes and microvesicles, and then the alveolar epithelium engulfed the
668
microvesicles, internalizing the mitochondria (FIGURE 5).
669
Miro1, a mitochondrial Rho-GTPase, is critical for this process since BMSCs lacking
670
Miro1 fail to transfer mitochondria to the airway epithelium. The mechanisms that
671
regulate microvesicle engulfment and release of the internalized mitochondria in the
672
alveolar cytosol are not clear.
673
internalized mitochondria were functional, since progressive increases of ATP levels
674
were detectable in the mitochondria-recipient epithelium. Islam et al showed that
Ahmad et al. show that
However, in Islam and coworkers found that the
29
675
endotoxin-induced mouse mortality decreased markedly following instillation of BMSCs
676
carrying normal mitochondria, but not when the BMSC mitochondria were made
677
dysfunctional by knockdown of the electron-transporting Riske iron-sulfur protein that
678
determines mitochondrial ATP production. Although mitochondrial transfer has been
679
shown in vitro and in feral dogs (62), the reports by Islam et al. and Ahmad et al indicate
680
that the process occurs in vivo to promote tissue repair in organ systems undergoing
681
pathologic stress. The applicability of mitochondrial transfer as a viable therapeutic
682
modality in inflammatory lung disease requires further consideration.
683
684
685
Acknowledgements
686
The authors’ research described in this Perspective was supported by grants from the National
687
Institutes of Health (R01 HL35440 and R01 HL122062 to P.T.S., R01 HL058234 and R0 1
688
HL113614 to M.N.G., R01 HL055330 and P01 HL114501 to A.M.K.C., R01 AI62740 and R01
689
AI083912 to E.D.C. P01 HL108801 and R01 AI095424 C.A.P., and RO1 HL36024
and RO1
690
HL57556 to J.B.)
691
30
692
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38
1053
Figure Legends
1054
FIGURE 1:
1055
regulated generation of reactive oxygen species (ROS) that are released to the
1056
cytosol through the intermembrane space. IMS-PRDX5: Peroxiredoxin-5 targeted to
1057
the intermembrane space. PHD: HIF Prolyl Hydroxylase. III: Mitochondrial complex III.
1058
FIGURE 2: Fate and biological actions of the oxidatively damaged mitochondrial
1059
genome that warrant characterization of mtDNA as a “sentinel molecule”. See
1060
text for details. Abbreviations: DAMPs, Damage Associated Molecular Patterns; IMS,
1061
intermembrane space; PTP, permeability transition pore
1062
FIGURE 3: Plasmacytoid dendritic cells (pDCs) play a central role in orchestrating
1063
both pro- and anti-inflammatory responses.
1064
mitochondrial DNA
1065
contaminating bacteria is sensed by pDCs to promote simultaneous release of TNFα
1066
and IFNα, which promotes the simultaneous activation of proinflammatory and
1067
regulatory immune cells. Thus, pDCs are poised to orchestrate a localized, and self-
1068
limited inflammtory response. Presumably, excessive pDCs activation (e.g., massive
1069
cell injury) could promote systemic pro- and anti-inflammatory immune responses.
1070
Panel B: In splenocyte cultures representing complex immune cell interactions typically
1071
occurring in vivo release of IFNα and TNFα in response to immunogenic (CpGA) DNA is
1072
enhanced by TFAM, and depletion of pDCs [by magnetic MicroBead selection (Miltenyi
1073
Biotec, Auburn, CA)], representing <3% of the total cell populationfrom the splenocyte
1074
cultures, is shown to greatly suppress the immune response. Thus, pDCs are important
1075
“amplifiers” of the immune response to TFAM and DNA. (*p < 0.01, compared to
Conceptual model of oxygen sensing by mitochondria through the
(mtDNA) and TFAM
39
Panel A: During acute tissue injury
released from
damaged cells and
1076
corresponding CpGA DNA treatment alone, †p < 0.01; relative to matching treatment in
1077
the High pDC group, and ‡p < 0.01, compared to corresponding CpGA DNA treatment
1078
alone and the matching treatment in the High pDC group.) PMN = neutrophil; MФ =
1079
macrophage; T-eff = effector CD4+ T cell; T-reg = regulatory T cell
1080
FIGURE 4:
1081
maintenance of mitochondrial homeostasis. The diagram illustrates the simplified
1082
relationship between mitochondrial biogenesis and selective mitochondrial autophagy or
1083
mitophagy. The cycle is accelerated by oxidative stress and is monitored and regulated
1084
by redox sensors including the physiological gases.
1085
FIGURE 5: Sequence of events leading to mitochondrial transfer from alveolus-
1086
attached BMSCs to the alveolar epithelium. The sketch of the distal airway (LEFT)
1087
shows the arrival of airway instilled BMSCs (MSC) in alveoli composed of alveolar type
1088
1 and type 2 cells (AT2 and AT2, respectively) and alveolar macrophages (AM).
1089
Subsequent processes in the alveolus (RIGHT) are: (1) BMSCs adhere to the alveolar
1090
epithelium through Cx43 interactions; (2) Gap junctions form between BMSCs and the
1091
epithelium; (3) Calcium levels increase in the BMSCs; and (4) BMSCs release
1092
mitochondria-containing microvesicles which enter the epithelium and release their
1093
mitochondria in the cytosol.
The integrated process of mitochondrial quality control in the
1094
1095
1096
1097
40
1098
1099
41
FIGURE 1
FIGURE 2
ROS
D-loop
Bioenergetic
defect, cell death
and dysfunction
mtDNA
Damageinduced
mtDNA
Fragmentation
into DAMPs
Mitochondrial
Matrix
IMS
PTP
Cytoplasm
Cellautogenous
TLR9 activation
NALP3
Inflammasome
Activation
Endosome
Plasma Membrane
?
Extracellular Space
Local Inflammation,
TLR9-dependent Immune System Activation,
and Injury Propagation
FIGURE 3
A.
TFAM
RAGE
mtDNA
IFNα
T
reg
T-reg
TLR9
Cell Damage
+/Contamination
Tissue Repair
Antimicrobial
Defense
pDC
TNFα
MФ
T-eff
Local
Inflammation
Negative Feedback
*
6000
5000
No Treatment
CpGA DNA - IFNα
TFAM + CpGA - IFNα
CpGA DNA - TNFα
TFAM + CpGA - TNFα
*
4000
‡
3000
†
2000
†
1000
‡
pD
w
Lo
pD
Cs
Cs
0
Hi
gh
(p g /m l)
B.
S u p e rn a ta n t C yto k in e C o n c e n tra tio n
PMN
FIGURE 4
FIGURE 5
(1)
(2)
AM
MSC
(3)
Cx43
AT2
AT1
(4)