Suppression of inflammation and acute lung injury by Miz1 via repression of C/EBP-δ.

Inflammation is essential for host defense but can cause tissue damage and organ failure if unchecked. How the inflammation is resolved remains elusive. Here we report that the transcription factor Miz1 was required for terminating lipopolysaccharide (LPS)-induced inflammation. Genetic disruption of the Miz1 POZ domain, which is essential for the transactivation or repression activity of Miz1, resulted in hyperinflammation, lung injury and greater mortality in LPS-treated mice but a lower bacterial load and mortality in mice with Pseudomonas aeruginosa pneumonia. Loss of the Miz1 POZ domain prolonged the expression of proinflammatory cytokines. After stimulation, Miz1 was phosphorylated at Ser178, which was required for recruitment of the histone deacetylase HDAC1 to repress transcription of the gene encoding C/EBP-δ, an amplifier of inflammation. Our data provide a long-sought mechanism underlying the resolution of LPS-induced inflammation.

Dynamin-related protein 1 is a critical regulator of mitochondrial calcium homeostasis during myocardial ischemia/reperfusion injury.

Dynamin-related protein 1 (Drp1) is a cytosolic GTPase protein that when activated translocates to the mitochondria, meditating mitochondrial fission and increasing reactive oxygen species (ROS) in cardiomyocytes. Drp1 has shown promise as a therapeutic target for reducing cardiac ischemia/reperfusion (IR) injury; however, the lack of specificity of some small molecule Drp1 inhibitors and the reliance on the use of Drp1 haploinsufficient hearts from older mice have left the role of Drp1 in IR in question. Here, we address these concerns using two approaches, using: (a) short-term (3 weeks), conditional, cardiomyocyte-specific, Drp1 knockout (KO) and (b) a novel, highly specific Drp1 GTPase inhibitor, Drpitor1a. Short-term Drp1 KO mice exhibited preserved exercise capacity and cardiac contractility, and their isolated cardiac mitochondria demonstrated increased mitochondrial complex 1 activity, respiratory coupling, and calcium retention capacity compared to controls. When exposed to IR injury in a Langendorff perfusion system, Drp1 KO hearts had preserved contractility, decreased reactive oxygen species (ROS), enhanced mitochondrial calcium capacity, and increased resistance to mitochondrial permeability transition pore (MPTP) opening. Pharmacological inhibition of Drp1 with Drpitor1a following ischemia, but before reperfusion, was as protective as Drp1 KO for cardiac function and mitochondrial calcium homeostasis. In contrast to the benefits of short-term Drp1 inhibition, prolonged Drp1 ablation (6 weeks) resulted in cardiomyopathy. Drp1 KO hearts were also associated with decreased ryanodine receptor 2 (RyR2) protein expression and pharmacological inhibition of the RyR2 receptor decreased ROS in post-IR hearts suggesting that changes in RyR2 may have a role in Drp1 KO mediated cardioprotection. We conclude that Drp1-mediated increases in myocardial ROS production and impairment of mitochondrial calcium handling are key mechanisms of IR injury. Short-term inhibition of Drp1 is a promising strategy to limit early myocardial IR injury which is relevant for the therapy of acute myocardial infarction, cardiac arrest, and heart transplantation.

Anoctamin-1 is induced by TGF-ß and contributes to lung myofibroblast differentiation.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-ß (TGF-ß) is one of the most established drivers of fibrotic processes. TGF-ß promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-ß robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle α-actin (SMA)-positive myofibroblasts. TGF-ß-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA-mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-ß signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-ß treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing "with-no-lysine (K)" kinase (WNK1). WNK1 siRNA significantly attenuates TGF-ß-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-ß-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-ß; and 2) ANO1 mediates TGF-ß-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity.NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-ß (TGF-ß) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation.

WITHDRAWN: Update in Critical Care 2021.

Ahead of Print article withdrawn by publisher.

Late onset infectious complications and safety of tocilizumab in the management of COVID-19.

BACKGROUND: Tocilizumab (TCZ) has been used in the management of COVID-19-related cytokine release syndrome (CRS). Concerns exist regarding the risk of infections and drug-related toxicities. We sought to evaluate the incidence of these TCZ complications among COVID-19 patients. METHODS: All adult inpatients with COVID-19 between 1 March and 25 April 2020 that received TCZ were included. We compared the rate of late-onset infections (>48 hours following admission) to a control group matched according to intensive care unit admission and mechanical ventilation requirement. Post-TCZ toxicities evaluated included: elevated liver function tests (LFTs), GI perforation, diverticulitis, neutropenia, hypertension, allergic reactions, and infusion-related reactions. RESULTS: Seventy-four patients were included in each group. Seventeen infections in the TCZ group (23%) and 6 (8%) infections in the control group occurred >48 hours after admission (P = .013). Most infections were bacterial with pneumonia being the most common manifestation. Among patients receiving TCZ, LFT elevations were observed in 51%, neutropenia in 1.4%, and hypertension in 8%. The mortality rate among those that received TCZ was greater than the control (39% versus 23%, P = .03). CONCLUSION: Late onset infections were significantly more common among those receiving TCZ. Combining infections and TCZ-related toxicities, 61% of patients had a possible post-TCZ complication. While awaiting clinical trial results to establish the efficacy of TCZ for COVID-19 related CRS, the potential for infections and TCZ related toxicities should be carefully weighed when considering use.

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

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-ß-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.

Influenza A Virus Infection Induces Muscle Wasting via IL-6 Regulation of the E3 Ubiquitin Ligase Atrogin-1.

Muscle dysfunction is common in patients with adult respiratory distress syndrome and is associated with morbidity that can persist for years after discharge. In a mouse model of severe influenza A pneumonia, we found the proinflammatory cytokine IL-6 was necessary for the development of muscle dysfunction. Treatment with a Food and Drug Administration-approved Ab antagonist to the IL-6R (tocilizumab) attenuated the severity of influenza A-induced muscle dysfunction. In cultured myotubes, IL-6 promoted muscle degradation via JAK/STAT, FOXO3a, and atrogin-1 upregulation. Consistent with these findings, atrogin-1+/- and atrogin-1-/- mice had attenuated muscle dysfunction following influenza infection. Our data suggest that inflammatory endocrine signals originating from the injured lung activate signaling pathways in the muscle that induce dysfunction. Inhibiting these pathways may limit morbidity in patients with influenza A pneumonia and adult respiratory distress syndrome.

FGFR2 Is Required for AEC2 Homeostasis and Survival after Bleomycin-induced Lung Injury.

Alveolar epithelial cell (AEC) injury is central to the pathogenesis of pulmonary fibrosis. Epithelial FGF (fibroblast growth factor) signaling is essential for recovery from hyperoxia- and influenza-induced lung injury, and treatment with FGFs is protective in experimental lung injury. The cell types involved in the protective effect of FGFs are not known. We hypothesized that FGF signaling in type II AECs (AEC2s) is critical in bleomycin-induced lung injury and fibrosis. To test this hypothesis, we generated mice with tamoxifen-inducible deletion of FGFR1-3 (fibroblast growth factor receptors 1, 2, and 3) in surfactant protein C-positive (SPC+) AEC2s (SPC triple conditional knockout [SPC-TCKO]). In the absence of injury, SPC-TCKO mice had fewer AEC2s, decreased Sftpc (surfactant protein C gene) expression, increased alveolar diameter, and increased collagen deposition. After intratracheal bleomycin administration, SPC-TCKO mice had increased mortality, lung edema, and BAL total protein, and flow cytometry and immunofluorescence revealed a loss of AEC2s. To reduce mortality of SPC-TCKO mice to less than 50%, a 25-fold dose reduction of bleomycin was required. Surviving bleomycin-injured SPC-TCKO mice had increased collagen deposition, fibrosis, and ACTA2 expression and decreased epithelial gene expression. Inducible inactivation of individual Fgfr2 or Fgfr3 revealed that Fgfr2, but not Fgfr3, was responsible for the increased mortality and lung injury after bleomycin administration. In conclusion, AEC2-specific FGFR2 is critical for survival in response to bleomycin-induced lung injury. These data also suggest that a population of SPC+ AEC2s require FGFR2 signaling for maintenance in the adult lung. Preventing epithelial FGFR inhibition and/or activating FGFRs in alveolar epithelium may therefore represent a novel approach to treating lung injury and reducing fibrosis.

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

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 in vivo 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.

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

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease characterized by the TGF-ß (transforming growth factor-ß)-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 de novo synthesis of glycine, the most abundant amino acid found in collagen protein. TGF-ß upregulates the expression of the enzymes of the de novo serine-glycine synthesis pathway in lung fibroblasts; however, the transcriptional and signaling regulators of this pathway remain incompletely understood. Here, we demonstrate that TGF-ß 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-ß. We found that induction of the integrated stress response (ISR) contributes to TGF-ß-induced ATF4 activity; however, the primary driver of ATF4 downstream of TGF-ß is activation of mTORC1 (mTOR Complex 1). TGF-ß 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-ß-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-ß. 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.

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

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.

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

Rationale: 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. Objectives: 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. Methods: 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 in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: 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. Conclusions: 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.

P311 Promotes Lung Fibrosis via Stimulation of Transforming Growth Factor-ß1, -ß2, and -ß3 Translation.

Interstitial lung fibrosis, a frequently idiopathic and fatal disease, has been linked to the increased expression of profibrotic transforming growth factor (TGF)-ßs. P311 is an RNA-binding protein that stimulates TGF-ß1, -ß2, and -ß3 translation in several cell types through its interaction with the eukaryotic translation initiation factor 3b. We report that P311 is switched on in the lungs of patients with idiopathic pulmonary fibrosis (IPF) and in the mouse model of bleomycin (BLM)-induced pulmonary fibrosis. To assess the in vivo role of P311 in lung fibrosis, BLM was instilled into the lungs of P311-knockout mice, in which fibrotic changes were significantly decreased in tandem with a reduction in TGF-ß1, -ß2, and -ß3 concentration/activity compared with BLM-treated wild-type mice. Complementing these findings, forced P311 expression increased TGF-ß concentration/activity in mouse and human lung fibroblasts, thereby leading to an activated phenotype with increased collagen production, as seen in IPF. Consistent with a specific effect of P311 on TGF-ß translation, TGF-ß1-, -ß2-, and -ß3-neutralizing antibodies downregulated P311-induced collagen production by lung fibroblasts. Furthermore, treatment of BLM-exposed P311 knockouts with recombinant TGF-ß1, -ß2, and -ß3 induced pulmonary fibrosis to a degree similar to that found in BLM-treated wild-type mice. These studies demonstrate the essential function of P311 in TGF-ß-mediated lung fibrosis. Targeting P311 could prove efficacious in ameliorating the severity of IPF while circumventing the development of autoimmune complications and toxicities associated with the use of global TGF-ß inhibitors.

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

Idiopathic pulmonary fibrosis (IPF) is characterized by the transforming growth factor (TGF)-ß-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-ß-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)/Δ1-pyrroline-5-carboxylate synthetase (P5CS) are required for collagen protein production by lung fibroblasts. PSAT1 is required for de novo glycine production, whereas ALDH18A1/P5CS is required for de novo proline production. Consistent with this, we found that TGF-ß 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.

Anti-fibrotic effects of tannic acid through regulation of a sustained TGF-beta receptor signaling.

BACKGROUND: Pulmonary fibrosis is a progressive disease characterized by structural distortion of the lungs. Transforming growth factor-beta (TGF-beta) is a key cytokine implicated in the pathogenesis of pulmonary fibrosis. TGF-beta-induced myofibroblast differentiation characterized by expression of smooth muscle alpha-actin and extracellular matrix proteins is a key process in pathogenesis of fibrotic disease. Tannic acid is a natural polyphenol with diverse applications. In this study, we investigated the effect of tannic acid on myofibroblast differentiation and pulmonary fibrosis in cultured cells and in bleomycin model of the disease. METHODS: Primary cultured human lung fibroblasts (HLF) were used. The relative levels of proteins were determined by Western blotting. HLF contraction was measured by traction microscopy. Bleomycin-induced pulmonary fibrosis in mice was used as the disease model. RESULTS: Tannic acid inhibited TGF-beta-induced expression of collagen-1 and smooth muscle alpha-actin (SMA) as well as force generation by HLF. Tannic acid did not affect initial phosphorylation of Smad2 in response to TGF-beta, but significantly inhibited sustained Smad2 phosphorylation, which we recently described to be critical for TGF-beta-induced myofibroblast differentiation. Accordingly, tannic acid inhibited Smad-dependent gene transcription in response to TGF-beta, as assessed using luciferase reporter for the activity of Smad-binding elements. Finally, in mouse model of bleomycin-induced pulmonary fibrosis, therapeutic application of tannic acid resulted in a significant reduction of lung fibrosis, decrease in collagen-1 content and of Smad2 phosphorylation in the lungs. CONCLUSIONS: This study demonstrates the anti-fibrotic effect of tannic acid in vitro and in vivo through a regulation of sustained Smad2 phosphorylation.

Inhibition of Phosphoglycerate Dehydrogenase Attenuates Bleomycin-induced Pulmonary Fibrosis.

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)-ß, 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-ß. In this study, we investigated whether inhibition of de novo serine and glycine synthesis attenuates lung fibrosis in vivo. We found that TGF-ß 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-ß-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-ß-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.

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

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.

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

TGF-ß 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-ß induces expression of glycolytic genes and increases glycolytic flux. TGF-ß 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-ß-induced collagen production and suggest that this pathway may be a therapeutic target for treatment of fibrotic diseases including IPF.

SIRT3 blocks myofibroblast differentiation and pulmonary fibrosis by preventing mitochondrial DNA damage.

Myofibroblast differentiation is a key process in the pathogenesis of fibrotic diseases. Transforming growth factor-ß1 (TGF-ß1) is a powerful inducer of myofibroblast differentiation and is implicated in pathogenesis of tissue fibrosis. This study was undertaken to determine the role of mitochondrial deacetylase SIRT3 in TGF-ß1-induced myofibroblast differentiation in vitro and lung fibrosis in vivo. Treatment of human lung fibroblasts with TGF-ß1 resulted in increased expression of fibrosis markers, smooth muscle α-actin (α-SMA), collagen-1, and fibronectin. TGF-ß1 treatment also caused depletion of endogenous SIRT3, which paralleled with increased production of reactive oxygen species (ROS), DNA damage, and subsequent reduction in levels of 8-oxoguanine DNA glycosylase (OGG1), an enzyme that hydrolyzes oxidized guanine (8-oxo-dG) and thus protects DNA from oxidative damage. Overexpression of SIRT3 by adenovirus-mediated transduction reversed the effects of TGF-ß1 on ROS production and mitochondrial DNA damage and inhibited TGF-ß1-induced myofibroblast differentiation. To determine the antifibrotic role of SIRT3 in vivo, we used the bleomycin-induced mouse model of pulmonary fibrosis. Compared with wild-type controls, Sirt3-knockout mice showed exacerbated fibrosis after intratracheal instillation of bleomycin. Increased lung fibrosis was associated with decreased levels of OGG1 and concomitant accumulation of 8-oxo-dG and increased mitochondrial DNA damage. In contrast, the transgenic mice with whole body Sirt3 overexpression were protected from bleomycin-induced mtDNA damage and development of lung fibrosis. These data demonstrate a critical role of SIRT3 in the control of myofibroblast differentiation and lung fibrosis.