Publications by authors named "Robert B Hamanaka"

40 Publications

The role of metabolic reprogramming and de novo amino acid synthesis in collagen protein production by myofibroblasts: implications for organ fibrosis and cancer.

Amino Acids 2021 May 8. Epub 2021 May 8.

Department of Medicine, Section of Pulmonary and Critical Care Medicine, The University of Chicago, 5841 S. Maryland Avenue, MC6026, Chicago, IL, 60637, USA.

Fibrosis is a pathologic condition resulting from aberrant wound healing responses that lead to excessive accumulation of extracellular matrix components, distortion of organ architecture, and loss of organ function. Fibrotic disease can affect every organ system; moreover, fibrosis is an important microenvironmental component of many cancers, including pancreatic, cervical, and hepatocellular cancers. Fibrosis is also an independent risk factor for cancer. Taken together, organ fibrosis contributes to up to 45% of all deaths worldwide. There are no approved therapies that halt or reverse fibrotic disease, highlighting the great need for novel therapeutic targets. At the heart of almost all fibrotic disease is the TGF-β-mediated differentiation of fibroblasts into myofibroblasts, the primary cell type responsible for the production of collagen and other matrix proteins and distortion of tissue architecture. Recent advances, particularly in the field of lung fibrosis, have highlighted the role that metabolic reprogramming plays in the pathogenic phenotype of myofibroblasts, particularly the induction of de novo amino acid synthesis pathways that are required to support collagen matrix production by these cells. In this review, we will discuss the metabolic changes associated with myofibroblast differentiation, focusing on the de novo production of glycine and proline, two amino acids which compose over half of the primary structure of collagen protein. We will also discuss the important role that synthesis of these amino acids plays in regulating cellular redox balance and epigenetic state.
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http://dx.doi.org/10.1007/s00726-021-02996-8DOI Listing
May 2021

Metabolic requirements of pulmonary fibrosis: role of fibroblast metabolism.

FEBS J 2021 Jan 3. Epub 2021 Jan 3.

Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, IL, USA.

Fibrosis is a pathologic condition characterized by excessive deposition of extracellular matrix and chronic scaring that can affect every organ system. Organ fibrosis is associated with significant morbidity and mortality, contributing to as many as 45% of all deaths in the developed world. In the lung, many chronic lung diseases may lead to fibrosis, the most devastating being idiopathic pulmonary fibrosis (IPF), which affects approximately 3 million people worldwide and has a median survival of 3.8 years. Currently approved therapies for IPF do not significantly extend lifespan, and thus, there is pressing need for novel therapeutic strategies to treat IPF and other fibrotic diseases. At the heart of pulmonary fibrosis are myofibroblasts, contractile cells with characteristics of both fibroblasts and smooth muscle cells, which are the primary cell type responsible for matrix deposition in fibrotic diseases. Much work has centered around targeting the extracellular growth factors and intracellular signaling regulators of myofibroblast differentiation. Recently, metabolic changes associated with myofibroblast differentiation have come to the fore as targetable mechanisms required for myofibroblast function. In this review, we will discuss the metabolic changes associated with myofibroblast differentiation, as well as the mechanisms by which these changes promote myofibroblast function. We will then discuss the potential for this new knowledge to lead to the development of novel therapies for IPF and other fibrotic diseases.
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http://dx.doi.org/10.1111/febs.15693DOI Listing
January 2021

TGF-β Promotes Metabolic Reprogramming in Lung Fibroblasts via mTORC1-dependent ATF4 Activation.

Am J Respir Cell Mol Biol 2020 11;63(5):601-612

Section of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Chicago, Chicago, Illinois.

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by the TGF-β (transforming growth factor-β)-dependent differentiation of lung fibroblasts into myofibroblasts, which leads to excessive deposition of collagen proteins and progressive scarring. We have previously shown that synthesis of collagen by myofibroblasts requires synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-β upregulates the expression of the enzymes of the serine-glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here, we demonstrate that TGF-β promotes accumulation of ATF4 (activating transcription factor 4), which is required for increased expression of the serine-glycine synthesis pathway enzymes in response to TGF-β. We found that induction of the integrated stress response (ISR) contributes to TGF-β-induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-β is activation of mTORC1 (mTOR Complex 1). TGF-β activates the PI3K-Akt-mTOR pathway, and inhibition of PI3K prevents activation of downstream signaling and induction of ATF4. Using a panel of mTOR inhibitors, we found that ATF4 activation is dependent on mTORC1, independent of mTORC2. Rapamycin, which incompletely and allosterically inhibits mTORC1, had no effect on TGF-β-mediated induction of ATF4; however, Rapalink-1, which specifically targets the kinase domain of mTORC1, completely inhibited ATF4 induction and metabolic reprogramming downstream of TGF-β. Our results provide insight into the mechanisms of metabolic reprogramming in myofibroblasts and clarify contradictory published findings on the role of mTOR inhibition in myofibroblast differentiation.
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http://dx.doi.org/10.1165/rcmb.2020-0143OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7605163PMC
November 2020

The Airway Epithelial Response to Air Pollution: It's Not Just Inflammation.

Am J Respir Cell Mol Biol 2020 08;63(2):139-140

Department of MedicineThe University of ChicagoChicago, Illinois.

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http://dx.doi.org/10.1165/rcmb.2020-0116EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397764PMC
August 2020

Endogenous itaconate is not required for particulate matter-induced NRF2 expression or inflammatory response.

Elife 2020 04 7;9. Epub 2020 Apr 7.

Department of Medicine, Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, United States.

Particulate matter (PM) air pollution causes cardiopulmonary mortality via macrophage-driven lung inflammation; however, the mechanisms are incompletely understood. RNA-sequencing demonstrated () as one of the top genes induced by PM in macrophages. encodes a mitochondrial enzyme that produces itaconate, which has been shown to exert anti-inflammatory effects via NRF2 after LPS. Here, we demonstrate that PM induces Acod1 and itaconate, which reduced mitochondrial respiration via complex II inhibition. Using mice, we found that Acod1/endogenous itaconate does not affect PM-induced inflammation or NRF2 activation in macrophages in vitro or in vivo. In contrast, exogenous cell permeable itaconate, 4-octyl itaconate (OI) attenuated PM-induced inflammation in macrophages. OI was sufficient to activate NRF2 in macrophages; however, NRF2 was not required for the anti-inflammatory effects of OI. We conclude that the effects of itaconate production on inflammation are stimulus-dependent, and that there are important differences between endogenous and exogenously-applied itaconate.
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http://dx.doi.org/10.7554/eLife.54877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185992PMC
April 2020

Suppression of Superoxide-Hydrogen Peroxide Production at Site IQ of Mitochondrial Complex I Attenuates Myocardial Stunning and Improves Postcardiac Arrest Outcomes.

Crit Care Med 2020 02;48(2):e133-e140

Section of Emergency Medicine, Department of Medicine, University of Chicago, Chicago, IL.

Objectives: Cardiogenic shock following cardiopulmonary resuscitation for sudden cardiac arrest is common, occurring even in the absence of acute coronary artery occlusion, and contributes to high rates of postcardiopulmonary resuscitation mortality. The pathophysiology of this shock is unclear, and effective therapies for improving clinical outcomes are lacking.

Design: Laboratory investigation.

Setting: University laboratory.

Subjects: C57BL/6 adult female mice.

Interventions: Anesthetized and ventilated adult female C57BL/6 wild-type mice underwent a 4, 8, 12, or 16-minute potassium chloride-induced cardiac arrest followed by 90 seconds of cardiopulmonary resuscitation. Mice were then blindly randomized to a single IV injection of vehicle (phosphate-buffered saline) or suppressor of site IQ electron leak, an inhibitor of superoxide production by complex I of the mitochondrial electron transport chain. Suppressor of site IQ electron leak and vehicle were administered during cardiopulmonary resuscitation.

Measurements And Main Results: Using a murine model of asystolic cardiac arrest, we discovered that duration of cardiac arrest prior to cardiopulmonary resuscitation determined postresuscitation success rates, degree of neurologic injury, and severity of myocardial dysfunction. Post-cardiopulmonary resuscitation cardiac dysfunction was not associated with myocardial necrosis, apoptosis, inflammation, or mitochondrial permeability transition pore opening. Furthermore, left ventricular function recovered within 72 hours of cardiopulmonary resuscitation, indicative of myocardial stunning. Postcardiopulmonary resuscitation, the myocardium exhibited increased reactive oxygen species and evidence of mitochondrial injury, specifically reperfusion-induced reactive oxygen species generation at electron transport chain complex I. Suppressor of site IQ electron leak, which inhibits complex I-dependent reactive oxygen species generation by suppression of site IQ electron leak, decreased myocardial reactive oxygen species generation and improved postcardiopulmonary resuscitation myocardial function, neurologic outcomes, and survival.

Conclusions: The severity of cardiogenic shock following asystolic cardiac arrest is dependent on the length of cardiac arrest prior to cardiopulmonary resuscitation and is mediated by myocardial stunning resulting from mitochondrial electron transport chain complex I dysfunction. A novel pharmacologic agent targeting this mechanism, suppressor of site IQ electron leak, represents a potential, practical therapy for improving sudden cardiac arrest resuscitation outcomes.
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http://dx.doi.org/10.1097/CCM.0000000000004095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6964871PMC
February 2020

Tissue-Resident Alveolar Macrophages Do Not Rely on Glycolysis for LPS-induced Inflammation.

Am J Respir Cell Mol Biol 2020 02;62(2):243-255

Department of Medicine, Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, Illinois.

Macrophage effector function is dynamic in nature and largely dependent on not only the type of immunological challenge but also the tissue-specific environment and developmental origin of a given macrophage population. Recent research has highlighted the importance of glycolytic metabolism in the regulation of effector function as a common feature associated with macrophage activation. Yet, most research has used macrophage cell lines and bone marrow-derived macrophages, which do not account for the diversity of macrophage populations and the role of tissue specificity in macrophage immunometabolism. Tissue-resident alveolar macrophages (TR-AMs) reside in an environment characterized by remarkably low glucose concentrations, making glycolysis-linked immunometabolism an inefficient and unlikely means of immune activation. In this study, we show that TR-AMs rely on oxidative phosphorylation to meet their energy demands and maintain extremely low levels of glycolysis under steady-state conditions. Unlike bone marrow-derived macrophages, TR-AMs did not experience enhanced glycolysis in response to LPS, and glycolytic inhibition had no effect on their proinflammatory cytokine production. Hypoxia-inducible factor 1α stabilization promoted glycolysis in TR-AMs and shifted energy production away from oxidative metabolism at baseline, but it was not sufficient for TR-AMs to mount further increases in glycolysis or enhance immune function in response to LPS. Importantly, we confirmed these findings in an influenza model in which infiltrating macrophages had significantly higher glycolytic and proinflammatory gene expression than TR-AMs. These findings demonstrate that glycolysis is dispensable for macrophage effector function in TR-AM and highlight the importance of macrophage tissue origin (tissue resident vs. recruited) in immunometabolism.
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http://dx.doi.org/10.1165/rcmb.2019-0244OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993551PMC
February 2020

Glutamine Metabolism Is Required for Collagen Protein Synthesis in Lung Fibroblasts.

Am J Respir Cell Mol Biol 2019 11;61(5):597-606

Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, Illinois; and.

Idiopathic pulmonary fibrosis (IPF) is characterized by the transforming growth factor (TGF)-β-dependent differentiation of lung fibroblasts into myofibroblasts, leading to excessive deposition of extracellular matrix proteins, which distort lung architecture and function. Metabolic reprogramming in myofibroblasts is emerging as an important mechanism in the pathogenesis of IPF, and recent evidence suggests that glutamine metabolism is required in myofibroblasts, although the exact role of glutamine in myofibroblasts is unclear. In the present study, we demonstrate that glutamine and its conversion to glutamate by glutaminase are required for TGF-β-induced collagen protein production in lung fibroblasts. We found that metabolism of glutamate to α-ketoglutarate by glutamate dehydrogenase or the glutamate-pyruvate or glutamate-oxaloacetate transaminases is not required for collagen protein production. Instead, we discovered that the glutamate-consuming enzymes phosphoserine aminotransferase 1 (PSAT1) and aldehyde dehydrogenase 18A1 (ALDH18A1)/Δ-pyrroline-5-carboxylate synthetase (P5CS) are required for collagen protein production by lung fibroblasts. PSAT1 is required for glycine production, whereas ALDH18A1/P5CS is required for proline production. Consistent with this, we found that TGF-β treatment increased cellular concentrations of glycine and proline in lung fibroblasts. Our results suggest that glutamine metabolism is required to promote amino acid biosynthesis and not to provide intermediates such as α-ketoglutarate for oxidation in mitochondria. In support of this, we found that inhibition of glutaminolysis has no effect on cellular oxygen consumption and that knockdown of oxoglutarate dehydrogenase has no effect on the ability of fibroblasts to produce collagen protein. Our results suggest that amino acid biosynthesis pathways may represent novel therapeutic targets for treatment of fibrotic diseases, including IPF.
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http://dx.doi.org/10.1165/rcmb.2019-0008OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6827066PMC
November 2019

Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis.

Am J Respir Crit Care Med 2019 06;199(12):1517-1536

4 Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, Illinois; and.

The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.
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http://dx.doi.org/10.1164/rccm.201712-2410OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580683PMC
June 2019

Particulate Matter Air Pollution: Effects on the Cardiovascular System.

Front Endocrinol (Lausanne) 2018 16;9:680. Epub 2018 Nov 16.

Section of Pulmonary and Critical Care Medicine, Department of Medicine, The University of Chicago, Chicago, IL, United States.

Air pollution is a complex mixture of gaseous and particulate components, each of which has detrimental effects on human health. While the composition of air pollution varies greatly depending on the source, studies from across the world have consistently shown that air pollution is an important modifiable risk factor for significantly increased morbidity and mortality. Moreover, clinical studies have generally shown a greater impact of particulate matter (PM) air pollution on health than the gaseous components. PM has wide-ranging deleterious effects on human health, particularly on the cardiovascular system. Both acute and chronic exposure to PM air pollution is associated with increased risk of death from cardiovascular diseases including ischemic heart disease, heart failure, and ischemic/thrombotic stroke. Particulate matter has also been shown to be an important endocrine disrupter, contributing to the development of metabolic diseases such as obesity and diabetes mellitus, which themselves are risk factors for cardiovascular disease. While the epidemiological evidence for the deleterious effects of PM air pollution on health is increasingly accepted, newer studies are shedding light on the mechanisms by which PM exerts its toxic effects. A greater understanding of how PM exerts toxic effects on human health is required in order to prevent and minimize the deleterious health effects of this ubiquitous environmental hazard. Air pollution is a growing public health problem and mortality due to air pollution is expected to double by 2050. Here, we review the epidemiological evidence for the cardiovascular effects of PM exposure and discuss current understanding about the biological mechanisms, by which PM exerts toxic effects on cardiovascular system to induce cardiovascular disease.
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http://dx.doi.org/10.3389/fendo.2018.00680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6250783PMC
November 2018

Metformin Targets Mitochondrial Electron Transport to Reduce Air-Pollution-Induced Thrombosis.

Cell Metab 2019 02 11;29(2):335-347.e5. Epub 2018 Oct 11.

Department of Medicine and Pulmonary and Critical Care Medicine, Northwestern University, 240 E Huron Street, M300, Chicago, IL 60611, USA.

Urban particulate matter air pollution induces the release of pro-inflammatory cytokines including interleukin-6 (IL-6) from alveolar macrophages, resulting in an increase in thrombosis. Here, we report that metformin provides protection in this murine model. Treatment of mice with metformin or exposure of murine or human alveolar macrophages to metformin prevented the particulate matter-induced generation of complex III mitochondrial reactive oxygen species, which were necessary for the opening of calcium release-activated channels (CRAC) and release of IL-6. Targeted genetic deletion of electron transport or CRAC channels in alveolar macrophages in mice prevented particulate matter-induced acceleration of arterial thrombosis. These findings suggest metformin as a potential therapy to prevent some of the premature deaths attributable to air pollution exposure worldwide.
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http://dx.doi.org/10.1016/j.cmet.2018.09.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365216PMC
February 2019

Alveolar Epithelial Cells Burn Fat to Survive Acute Lung Injury.

Am J Respir Cell Mol Biol 2019 02;60(2):135-136

1 Section of Pulmonary and Critical Care Medicine University of Chicago Chicago, Illinois.

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http://dx.doi.org/10.1165/rcmb.2018-0300EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6835041PMC
February 2019

Inhalational exposure to particulate matter air pollution alters the composition of the gut microbiome.

Environ Pollut 2018 Sep 18;240:817-830. Epub 2018 May 18.

Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, IL, 60637, USA. Electronic address:

Recent studies suggest an association between particulate matter (PM) air pollution and gastrointestinal (GI) disease. In addition to direct deposition, PM can be indirectly deposited in oropharynx via mucociliary clearance and upon swallowing of saliva and mucus. Within the GI tract, PM may alter the GI epithelium and gut microbiome. Our goal was to determine the effect of PM on gut microbiota in a murine model of PM exposure via inhalation. C57BL/6 mice were exposed via inhalation to either concentrated ambient particles or filtered air for 8-h per day, 5-days a week, for a total of 3-weeks. At exposure's end, GI tract tissues and feces were harvested, and gut microbiota was analyzed. Alpha-diversity was modestly altered with increased richness in PM-exposed mice compared to air-exposed mice in some parts of the GI tract. Most importantly, PM-induced alterations in the microbiota were very apparent in beta-diversity comparisons throughout the GI tract and appeared to increase from the proximal to distal parts. Changes in some genera suggest that distinct bacteria may have the capacity to bloom with PM exposure. Exposure to PM alters the microbiota throughout the GI tract which maybe a potential mechanism that explains PM induced inflammation in the GI tract.
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http://dx.doi.org/10.1016/j.envpol.2018.04.130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400491PMC
September 2018

Letter by Wu et al Regarding Article, "Mechanical Activation of Hypoxia-Inducible Factor 1α Drives Endothelial Dysfunction at Atheroprone Sites".

Arterioscler Thromb Vasc Biol 2017 12;37(12):e197-e198

Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, IL.

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http://dx.doi.org/10.1161/ATVBAHA.117.310335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5777320PMC
December 2017

Inhibition of Phosphoglycerate Dehydrogenase Attenuates Bleomycin-induced Pulmonary Fibrosis.

Am J Respir Cell Mol Biol 2018 05;58(5):585-593

1 Department of Medicine, Section of Pulmonary and Critical Care Medicine, the University of Chicago, Chicago, Illinois; and.

Organ fibrosis, including idiopathic pulmonary fibrosis, is associated with significant morbidity and mortality. Because currently available therapies have limited effect, there is a need to better understand the mechanisms by which organ fibrosis occurs. We have recently reported that transforming growth factor (TGF)-β, a key cytokine that promotes fibrogenesis, induces the expression of the enzymes of the de novo serine and glycine synthesis pathway in human lung fibroblasts, and that phosphoglycerate dehydrogenase (PHGDH; the first and rate-limiting enzyme of the pathway) is required to promote collagen protein synthesis downstream of TGF-β. In this study, we investigated whether inhibition of de novo serine and glycine synthesis attenuates lung fibrosis in vivo. We found that TGF-β induces mRNA and protein expression of PHGDH in murine fibroblasts. Similarly, intratracheal administration of bleomycin resulted in increased expression of PHGDH in mouse lungs, localized to fibrotic regions. Using a newly developed small molecule inhibitor of PHGDH (NCT-503), we tested whether pharmacologic inhibition of PHGDH could inhibit fibrogenesis both in vitro and in vivo. Treatment of murine and human lung fibroblasts with NCT-503 decreased TGF-β-induced collagen protein synthesis. Mice treated with the PHGDH inhibitor beginning 7 days after intratracheal instillation of bleomycin had attenuation of lung fibrosis. These results indicate that the de novo serine and glycine synthesis pathway is necessary for TGF-β-induced collagen synthesis and bleomycin-induced pulmonary fibrosis. PHGDH and other enzymes in the de novo serine and glycine synthesis pathway may be a therapeutic target for treatment of fibrotic diseases, including idiopathic pulmonary fibrosis.
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http://dx.doi.org/10.1165/rcmb.2017-0186OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946329PMC
May 2018

is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium.

Elife 2017 05 30;6. Epub 2017 May 30.

Department of Medicine, Section of Pulmonary and Critical Care Medicine, The University of Chicago, Chicago, United States.

Hemodynamic forces regulate vascular functions. Disturbed flow (DF) occurs in arterial bifurcations and curvatures, activates endothelial cells (ECs), and results in vascular inflammation and ultimately atherosclerosis. However, how DF alters EC metabolism, and whether resulting metabolic changes induce EC activation, is unknown. Using transcriptomics and bioenergetic analysis, we discovered that DF induces glycolysis and reduces mitochondrial respiratory capacity in human aortic ECs. DF-induced metabolic reprogramming required hypoxia inducible factor-1α (), downstream of NAD(P)H oxidase-4 ()-derived reactive oxygen species (ROS). increased glycolytic enzymes and pyruvate dehydrogenase kinase-1 (), which reduces mitochondrial respiratory capacity. Swine aortic arch endothelia exhibited elevated ROS, , , and glycolytic enzyme and expression, suggesting that DF leads to metabolic reprogramming in vivo. Inhibition of glycolysis reduced inflammation suggesting a causal relationship between flow-induced metabolic changes and EC activation. These findings highlight a previously uncharacterized role for flow-induced metabolic reprogramming and inflammation in ECs.
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http://dx.doi.org/10.7554/eLife.25217DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495571PMC
May 2017

PFKFB3, a Direct Target of p63, Is Required for Proliferation and Inhibits Differentiation in Epidermal Keratinocytes.

J Invest Dermatol 2017 06 17;137(6):1267-1276. Epub 2017 Jan 17.

Section of Pulmonary and Critical Care Medicine, Department of Medicine, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois, USA.

p63 is a transcription factor essential for epidermal development and homeostasis. p63 is a member of the p53 family of transcription factors, which are increasingly understood to be regulators of cellular metabolism. How p63 regulates metabolism in epidermal keratinocytes is incompletely understood, and it is unknown whether glycolytic regulation is essential to maintain the balance between proliferation and differentiation within the epidermis. We found that p63 promotes glycolytic metabolism in epidermal keratinocytes. p63 bound to consensus sites within the PFKFB3 gene and was required for PFKFB3 mRNA and protein expression. PFKFB3 overexpression inhibited differentiation of keratinocytes, whereas knockdown inhibited proliferation and increased the rate of differentiation. Furthermore, we found that PFKFB3 was highly expressed in psoriatic epidermis. Our results show that PFKFB3 is a key regulator of epidermal homeostasis and may represent a therapeutic target for epidermal diseases associated with hyperproliferation and impaired differentiation.
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http://dx.doi.org/10.1016/j.jid.2016.12.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5441935PMC
June 2017

Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3-Rac1 Signaling.

Am J Respir Crit Care Med 2017 03;195(5):639-651

1 Section of Pulmonary/Critical Care, Department of Medicine, The University of Chicago, Chicago, Illinois.

Rationale: Acute respiratory distress syndrome (ARDS) is caused by widespread endothelial barrier disruption and uncontrolled cytokine storm. Genome-wide association studies (GWAS) have linked multiple genes to ARDS. Although mechanosensitive transcription factor Krüppel-like factor 2 (KLF2) is a major regulator of endothelial function, its role in regulating pulmonary vascular integrity in lung injury and ARDS-associated GWAS genes remains poorly understood.

Objectives: To examine KLF2 expression in multiple animal models of acute lung injury and further elucidate the KLF2-mediated pathways involved in endothelial barrier disruption and cytokine storm in experimental lung injury.

Methods: Animal and in vitro models of acute lung injury were used to characterize KLF2 expression and its downstream effects responding to influenza A virus (A/WSN/33 [H1N1]), tumor necrosis factor-α, LPS, mechanical stretch/ventilation, or microvascular flow. KLF2 manipulation, permeability measurements, small GTPase activity, luciferase assays, chromatin immunoprecipitation assays, and network analyses were used to determine the mechanistic roles of KLF2 in regulating endothelial monolayer integrity, ARDS-associated GWAS genes, and lung pathophysiology.

Measurements And Main Results: KLF2 is significantly reduced in several animal models of acute lung injury. Microvascular endothelial KLF2 is significantly induced by capillary flow but reduced by pathologic cyclic stretch and inflammatory stimuli. KLF2 is a novel activator of small GTPase Ras-related C3 botulinum toxin substrate 1 by transcriptionally controlling Rap guanine nucleotide exchange factor 3/exchange factor directly activated by cyclic adenosine monophosphate, which maintains vascular integrity. KLF2 regulates multiple ARDS GWAS genes related to cytokine storm, oxidation, and coagulation in lung microvascular endothelium. KLF2 overexpression ameliorates LPS-induced lung injury in mice.

Conclusions: Disruption of endothelial KLF2 results in dysregulation of lung microvascular homeostasis and contributes to lung pathology in ARDS.
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http://dx.doi.org/10.1164/rccm.201604-0668OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5363977PMC
March 2017

Transforming Growth Factor (TGF)-β Promotes de Novo Serine Synthesis for Collagen Production.

J Biol Chem 2016 12 11;291(53):27239-27251. Epub 2016 Nov 11.

From the Departments of Medicine, Section of Pulmonary and Critical Care Medicine, and

TGF-β promotes excessive collagen deposition in fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). The amino acid composition of collagen is unique due to its high (33%) glycine content. Here, we report that TGF-β induces expression of glycolytic genes and increases glycolytic flux. TGF-β also induces the expression of the enzymes of the de novo serine synthesis pathway (phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH)) and de novo glycine synthesis (serine hydroxymethyltransferase 2 (SHMT2)). Studies in fibroblasts with genetic attenuation of PHGDH or SHMT2 and pharmacologic inhibition of PHGDH showed that these enzymes are required for collagen synthesis. Furthermore, metabolic labeling experiments demonstrated carbon from glucose incorporated into collagen. Lungs from humans with IPF demonstrated increased expression of PHGDH and SHMT2. These results indicate that the de novo serine synthesis pathway is necessary for TGF-β-induced collagen production and suggest that this pathway may be a therapeutic target for treatment of fibrotic diseases including IPF.
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http://dx.doi.org/10.1074/jbc.M116.756247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207151PMC
December 2016

The Mitochondrial Respiratory Chain Is Required for Organismal Adaptation to Hypoxia.

Cell Rep 2016 Apr 7;15(3):451-459. Epub 2016 Apr 7.

Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Electronic address:

Hypoxia-inducible factors (HIFs) are crucial for cellular and organismal adaptation to hypoxia. The mitochondrial respiratory chain is the largest consumer of oxygen in most mammalian cells; however, it is unknown whether the respiratory chain is necessary for in vivo activation of HIFs and organismal adaptation to hypoxia. HIF-1 activation in the epidermis has been shown to be a key regulator of the organismal response to hypoxic conditions, including renal production of erythropoietin (Epo). Therefore, we conditionally deleted expression of TFAM in mouse epidermal keratinocytes. TFAM is required for maintenance of the mitochondrial genome, and TFAM-null cells are respiratory deficient. TFAM loss in epidermal keratinocytes reduced epidermal levels of HIF-1α protein and diminished the hypoxic induction of HIF-dependent transcription in epidermis. Furthermore, epidermal TFAM deficiency impaired hypoxic induction of renal Epo expression. Our results demonstrate that the mitochondrial respiratory chain is essential for in vivo HIF activation and organismal adaptation to hypoxia.
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http://dx.doi.org/10.1016/j.celrep.2016.03.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4838509PMC
April 2016

Regulation of myofibroblast differentiation by cardiac glycosides.

Am J Physiol Lung Cell Mol Physiol 2016 05 5;310(9):L815-23. Epub 2016 Feb 5.

Section of Pulmonary and Critical Care Medicine, Department of Medicine, the University of Chicago, Chicago, Illinois;

Myofibroblast differentiation is a key process in pathogenesis of fibrotic diseases. Cardiac glycosides (ouabain, digoxin) inhibit Na(+)-K(+)-ATPase, resulting in increased intracellular [Na(+)]-to-[K(+)] ratio in cells. Microarray analysis suggested that increased intracellular [Na(+)]/[K(+)] ratio may promote the expression of cyclooxygenase-2 (COX-2), a critical enzyme in the synthesis of prostaglandins. Given antifibrotic effects of prostaglandins through activation of protein kinase A (PKA), we examined if cardiac glycosides stimulate COX-2 expression in human lung fibroblasts and how they affect myofibroblast differentiation. Ouabain stimulated a profound COX-2 expression and a sustained PKA activation, which was blocked by COX-2 inhibitor or by COX-2 knockdown. Ouabain-induced COX-2 expression and PKA activation were abolished by the inhibitor of the Na(+)/Ca(2+) exchanger, KB-R4943. Ouabain inhibited transforming growth factor-β (TGF-β)-induced Rho activation, stress fiber formation, serum response factor activation, and the expression of smooth muscle α-actin, collagen-1, and fibronectin. These effects were recapitulated by an increase in intracellular [Na(+)]/[K(+)] ratio through the treatment of cells with K(+)-free medium or with digoxin. Although inhibition of COX-2 or of the Na(+)/Ca(2+) exchanger blocked ouabain-induced PKA activation, this failed to reverse the inhibition of TGF-β-induced Rho activation or myofibroblast differentiation by ouabain. Together, these data demonstrate that ouabain, through the increase in intracellular [Na(+)]/[K(+)] ratio, drives the induction of COX-2 expression and PKA activation, which is accompanied by a decreased Rho activation and myofibroblast differentiation in response to TGF-β. However, COX-2 expression and PKA activation are not sufficient for inhibition of the fibrotic effects of TGF-β by ouabain, suggesting that additional mechanisms must exist.
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http://dx.doi.org/10.1152/ajplung.00322.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867347PMC
May 2016

Prolonged Exposures to Intermittent Hypoxia Promote Visceral White Adipose Tissue Inflammation in a Murine Model of Severe Sleep Apnea: Effect of Normoxic Recovery.

Sleep 2017 03;40(3)

Sections of Pediatric Sleep Medicine and Pulmonology, Department of Pediatrics, Pritzker School of Medicine, Biological Sciences Division, The University of Chicago, Chicago, IL.

Study Objective: Increased visceral white adipose tissue (vWAT) mass results in infiltration of inflammatory macrophages that drive inflammation and insulin resistance. Patients with obstructive sleep apnea (OSA) suffer from increased prevalence of obesity, insulin resistance, and metabolic syndrome. Murine models of intermittent hypoxia (IH) mimicking moderate-severe OSA manifest insulin resistance following short-term IH. We examined in mice the effect of long-term IH on the inflammatory cellular changes within vWAT and the potential effect of normoxic recovery (IH-R).

Methods: Male C57BL/6J mice were subjected to IH for 20 weeks, and a subset was allowed to recover in room air (RA) for 6 or 12 weeks (IH-R). Stromal vascular fraction was isolated from epididymal vWAT and mesenteric vWAT depots, and single-cell suspensions were prepared for flow cytometry analyses, reactive oxygen species (ROS), and metabolic assays.

Results: IH reduced body weight and vWAT mass and IH-R resulted in catch-up weight and vWAT mass. IH-exposed vWAT exhibited increased macrophage counts (ATMs) that were only partially improved in IH-R. IH also caused a proinflammatory shift in ATMs (increased Ly6c(hi)(+) and CD36(+) ATMs). These changes were accompanied by increased vWAT insulin resistance with only partial improvements in IH-R. In addition, ATMs exhibited increased ROS production, altered metabolism, and changes in electron transport chain, which were only partially improved in IH-R.

Conclusion: Prolonged exposures to IH during the sleep period induce pronounced vWAT inflammation and insulin resistance despite concomitant vWAT mass reductions. These changes are only partially reversible after 3 months of normoxic recovery. Thus, long-lasting OSA may preclude complete reversibility of metabolic changes.
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http://dx.doi.org/10.1093/sleep/zsw074DOI Listing
March 2017

Impaired clearance of influenza A virus in obese, leptin receptor deficient mice is independent of leptin signaling in the lung epithelium and macrophages.

PLoS One 2014 18;9(9):e108138. Epub 2014 Sep 18.

Department of Medicine, Division of Pulmonary and Critical Care Medicine, Northwestern University Feinberg School of Medicine, Chicago, Illinois, United States of America.

Rationale: During the recent H1N1 outbreak, obese patients had worsened lung injury and increased mortality. We used a murine model of influenza A pneumonia to test the hypothesis that leptin receptor deficiency might explain the enhanced mortality in obese patients.

Methods: We infected wild-type, obese mice globally deficient in the leptin receptor (db/db) and non-obese mice with tissue specific deletion of the leptin receptor in the lung epithelium (SPC-Cre/LepR fl/fl) or macrophages and alveolar type II cells (LysM-Cre/Lepr fl/fl) with influenza A virus (A/WSN/33 [H1N1]) (500 and 1500 pfu/mouse) and measured mortality, viral clearance and several markers of lung injury severity.

Results: The clearance of influenza A virus from the lungs of mice was impaired in obese mice globally deficient in the leptin receptor (db/db) compared to normal weight wild-type mice. In contrast, non-obese, SP-C-Cre+/+/LepR fl/fl and LysM-Cre+/+/LepR fl/fl had improved viral clearance after influenza A infection. In obese mice, mortality was increased compared with wild-type mice, while the SP-C-Cre+/+/LepR fl/fl and LysM-Cre+/+/LepR fl/fl mice exhibited improved survival.

Conclusions: Global loss of the leptin receptor results in reduced viral clearance and worse outcomes following influenza A infection. These findings are not the result of the loss of leptin signaling in lung epithelial cells or macrophages. Our results suggest that factors associated with obesity or with leptin signaling in non-myeloid populations such as natural killer and T cells may be associated with worsened outcomes following influenza A infection.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108138PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4169489PMC
April 2016

Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis.

Elife 2014 May 13;3:e02242. Epub 2014 May 13.

Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, United States

Recent epidemiological and laboratory-based studies suggest that the anti-diabetic drug metformin prevents cancer progression. How metformin diminishes tumor growth is not fully understood. In this study, we report that in human cancer cells, metformin inhibits mitochondrial complex I (NADH dehydrogenase) activity and cellular respiration. Metformin inhibited cellular proliferation in the presence of glucose, but induced cell death upon glucose deprivation, indicating that cancer cells rely exclusively on glycolysis for survival in the presence of metformin. Metformin also reduced hypoxic activation of hypoxia-inducible factor 1 (HIF-1). All of these effects of metformin were reversed when the metformin-resistant Saccharomyces cerevisiae NADH dehydrogenase NDI1 was overexpressed. In vivo, the administration of metformin to mice inhibited the growth of control human cancer cells but not those expressing NDI1. Thus, we have demonstrated that metformin's inhibitory effects on cancer progression are cancer cell autonomous and depend on its ability to inhibit mitochondrial complex I.DOI: http://dx.doi.org/10.7554/eLife.02242.001.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017650PMC
http://dx.doi.org/10.7554/eLife.02242DOI Listing
May 2014

Mitochondrial metabolism as a regulator of keratinocyte differentiation.

Cell Logist 2013 Jan 24;3(1):e25456. Epub 2013 Jun 24.

Department of Medicine, Division of Pulmonary and Critical Care Medicine and Department of Cell and Molecular Biology; Northwestern University Medical School; Chicago, IL USA.

Mitochondrial metabolism has traditionally been thought of as a source of cellular energy in the form of ATP. The recent renaissance in the study of cellular metabolism, particularly in the cancer field, has highlighted the fact that mitochondria are also critical biosynthetic and signaling hubs, making these organelles key governors of cellular outcomes. Using the epidermis as a model system, our recent study looked into the role that mitochondrial metabolism and ROS production play in cellular differentiation in vivo. We showed that conditional deletion of the mitochondrial transcription factor, TFAM within the basal cells of the epidermis results in loss of mitochondrial ROS production and impairs epidermal differentiation and hair growth. We demonstrated that mitochondrial ROS generation is required for the propagation of Notch and β-catenin signals which promote epidermal differentiation and hair follicle development respectively. This study bolsters accumulating evidence that oxidative mitochondrial metabolism plays a causal role in cellular differentiation programs. It also provides insights into the role that mitochondrial oxidative signaling plays in a cell type-dependent manner.
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http://dx.doi.org/10.4161/cl.25456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3891634PMC
January 2013

Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development.

Sci Signal 2013 Feb 5;6(261):ra8. Epub 2013 Feb 5.

Department of Medicine, The Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.
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http://dx.doi.org/10.1126/scisignal.2003638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4017376PMC
February 2013

microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.

Proc Natl Acad Sci U S A 2012 Aug 13;109(35):14030-4. Epub 2012 Aug 13.

Department of Dermatology, The Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

Notch plays a critical role in the transition from proliferation to differentiation in the epidermis and corneal epithelium. Furthermore, aberrant Notch signaling is a feature of diseases like psoriasis, eczema, nonmelanoma skin cancer, and melanoma where differentiation and proliferation are impaired. Whereas much is known about the downstream events following Notch signaling, factors responsible for negatively regulating Notch receptor signaling after ligand activation are incompletely understood. Notch can undergo hydroxylation by factor-inhibiting hypoxia-inducible factor 1 (FIH-1); however, the biological significance of this phenomenon is unclear. Here we show that FIH-1 expression is up-regulated in diseased epidermis and corneal epithelium. Elevating FIH-1 levels in primary human epidermal keratinocytes (HEKs) and human corneal epithelial keratinocytes (HCEKs) impairs differentiation in submerged cultures and in a "three-dimensional" organotypic raft model of human epidermis, in part, via a coordinate decrease in Notch signaling. Knockdown of FIH-1 enhances keratinocyte differentiation. Loss of FIH-1 in vivo increased Notch activity in the limbal epithelium, resulting in a more differentiated phenotype. microRNA-31 (miR-31) is an endogenous negative regulator of FIH-1 expression that results in keratinocyte differentiation, mediated by Notch activation. Ectopically expressing miR-31 in an undifferentiated corneal epithelial cell line promotes differentiation and recapitulates a corneal epithelium in a three-dimensional raft culture model. Our results define a previously unknown mechanism for keratinocyte fate decisions where Notch signaling potential is, in part, controlled through a miR-31/FIH-1 nexus.
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http://dx.doi.org/10.1073/pnas.1111292109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3435188PMC
August 2012

MicroRNA-31 targets FIH-1 to positively regulate corneal epithelial glycogen metabolism.

FASEB J 2012 Aug 24;26(8):3140-7. Epub 2012 Apr 24.

Department of Dermatology, The Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.

Corneal epithelium relies on abundant glycogen stores as its primary energy source. MicroRNA-31 (miR-31), a corneal epithelial-preferred miRNA, negatively regulates factor inhibiting hypoxia-inducible factor-1 (FIH-1). Since HIF-1α is involved in anaerobic energy production, we investigated the role that miR-31 and FIH-1 play in regulating corneal epithelial glycogen. We used antagomirs (antago) to reduce the level of miR-31 in primary human corneal epithelial keratinocytes (HCEKs), and a miR-31-resistant FIH-1 to increase FIH-1 levels. Antago-31 raised FIH-1 levels and significantly reduced glycogen stores in HCEKs compared to irrelevant-antago treatment. Similarly, HCEKs retrovirally transduced with a miR-31-resistant FIH-1 had markedly reduced glycogen levels compared with empty vector controls. In addition, we observed no change in a HIF-1α reporter or known genes downstream of HIF-1α indicating that the action of FIH-1 and miR-31 on glycogen is HIF-1α-independent. An enzyme-dead FIH-1 mutation failed to restore glycogen stores, indicating that FIH-1 negatively regulates glycogen in a hydroxylase-independent manner. FIH-1 overexpression in HCEKs decreased AKT signaling, activated GSK-3β, and inactivated glycogen synthase. Treatment of FIH-1-transduced HCEKs with either a myristolated Akt or a GSK-3β inhibitor restored glycogen stores, confirming the direct involvement of Akt/GSK-3β signaling. Silencing FIH-1 in HCEKs reversed the observed changes in Akt-signaling. Glycogen regulation in a HIF-1α-independent manner is a novel function for FIH-1 and provides new insight into how the corneal epithelium regulates its energy requirements.
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http://dx.doi.org/10.1096/fj.11-198515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405266PMC
August 2012