A multimodal exercise countermeasure prevents the negative impact of head-down tilt bed rest on muscle volume and mitochondrial health in older adults.

Mitochondrial dysfunctions are thought to contribute to muscle atrophy and weakness that develop during ageing and mechanical unloading caused by immobilization, bed rest and microgravity. Older adults are at greater risk of developing muscle and mitochondrial dysfunctions in response to unloading. Although exercise is well known to promote muscle and mitochondrial health, its protective effect during mechanical unloading in older adults remains largely unexplored. Here, we investigated the impact of 14 days of head-down tilt bed rest (HDBR) with and without a multimodal exercise countermeasure in older men and women (55-65 years). Leg muscle volume was assessed using magnetic resonance imaging. Biopsies of the vastus lateralis were performed to assess markers of mitochondrial content, respiration, reactive oxygen species (ROS) production and calcium retention capacity (mCRC). Indices of mitochondrial quality control (MQC), including markers of fusion (MFN1 and 2), fission (Drp1), mitophagy (Parkin) and autophagy (p62 and LC3I and II) were measured using immunoblots. Muscle cross-sections were stained for neural cell adhesion molecule (NCAM, a marker of denervation). HDBR triggered muscle atrophy, decreased mitochondrial content and respiration and increased mitochondrial ROS production. HDBR had no impact on mCRC or MQC markers but increased markers of autophagy and denervation. Exercise prevented the deleterious effects of HDBR on leg muscle volume, mitochondrial ROS production and markers of autophagy and denervation. Exercise also increased mitochondrial content and respiration without altering mCRC and MQC markers. Collectively, our results indicate that an exercise countermeasure that can be performed in bed is effective in protecting muscle and mitochondrial health during HDBR in older adults. KEY POINTS: Conditions associated with muscle unloading, such as immobilization, bed rest or microgravity, result in muscle atrophy and weakness, particularly in older adults. Mitochondrial dysfunctions are thought to contribute to muscle atrophy caused by unloading and ageing. However, whether exercise can counteract the deleterious effects of unloading in older adults remains largely unexplored. Here, we report that older adults exposed to 14 days of head-down tilt bed rest (HDBR) displayed upper leg muscle atrophy, a decrease in mitochondrial content and respiration, an increase in H2O2 emission, and an increase in autophagy and denervation markers. No impact of HDBR on mitochondrial quality control was observed. A multimodal exercise countermeasure prevented the deleterious effects of HDBR on upper leg muscle volume, mitochondrial reactive oxygen species emission, and markers of autophagy and denervation and increased mitochondrial content and respiration. These findings highlight the effectiveness of exercise in promoting muscle and mitochondrial health in older adults undergoing bed rest.

Lack of compensatory mitophagy in skeletal muscles during sepsis.

Skeletal muscle dysfunction is a major problem in critically ill patients suffering from sepsis. This condition is associated with mitochondrial dysfunction and increased autophagy in skeletal muscles. Autophagy is a proteolytic mechanism involved in eliminating dysfunctional cellular components, including mitochondria. The latter process, referred to as mitophagy, is essential for maintaining mitochondrial quality and skeletal muscle health. Recently, a fluorescent reporter system called mito-QC (i.e. mitochondrial quality control) was developed to specifically quantify mitophagy levels. In the present study, we used mito-QC transgenic mice and confocal microscopy to morphologically monitor mitophagy levels during sepsis. To induce sepsis, Mito-QC mice received Escherichia coli lipopolysaccharide (10 mg kg-1 i.p.) or phosphate-buffered saline and skeletal muscles (hindlimb and diaphragm) were excised 48 h later. In control groups, there was a negative correlation between the basal mitophagy level and overall muscle mitochondrial content. Sepsis increased general autophagy in both limb muscles and diaphragm but had no effect on mitophagy levels. Sepsis was associated with a downregulation of certain mitophagy receptors (Fundc1, Bcl2L13, Fkbp8 and Phbb2). The present study suggests that general autophagy and mitophagy can be dissociated from one another, and that the characteristic accumulation of damaged mitochondria in skeletal muscles under the condition of sepsis may reflect a failure of adequate compensatory mitophagy. KEY POINTS: There was a negative correlation between the basal level of skeletal muscle mitophagy and the mitochondrial content of individual muscles. Mitophagy levels in limb muscles and the diaphragm were unaffected by lipopolysaccharide (LPS)-induced sepsis. With the exception of BNIP3 in sepsis, LPS administration induced either no change or a downregulation of mitophagy receptors in skeletal muscles.

MFN2 overexpression in skeletal muscles of young and old mice causes a mild hypertrophy without altering mitochondrial respiration and H2O2 emission.

AIM: Sarcopenia, the aging-related loss of muscle mass and function, is a debilitating process negatively impacting the quality of life of affected individuals. Although the mechanisms underlying sarcopenia are incompletely understood, impairments in mitochondrial dynamics, including mitochondrial fusion, have been proposed as a contributing factor. However, the potential of upregulating mitochondrial fusion proteins to alleviate the effects of aging on skeletal muscles remains unexplored. We therefore hypothesized that overexpressing Mitofusin 2 (MFN2) in skeletal muscle in vivo would mitigate the effects of aging on muscle mass and improve mitochondrial function. METHODS: MFN2 was overexpressed in young (7 mo) and old (24 mo) male mice for 4 months through intramuscular injections of an adeno-associated viruses. The impacts of MFN2 overexpression on muscle mass and fiber size (histology), mitochondrial respiration, and H2O2 emission (Oroboros fluororespirometry), and various signaling pathways (qPCR and western blotting) were investigated. RESULTS: MFN2 overexpression increased muscle mass and fiber size in both young and old mice. No sign of fibrosis, necrosis, or inflammation was found upon MFN2 overexpression, indicating that the hypertrophy triggered by MFN2 overexpression was not pathological. MFN2 overexpression even reduced the proportion of fibers with central nuclei in old muscles. Importantly, MFN2 overexpression had no impact on muscle mitochondrial respiration and H2O2 emission in both young and old mice. MFN2 overexpression attenuated the increase in markers of impaired autophagy in old muscles. CONCLUSION: MFN2 overexpression may be a viable approach to mitigate aging-related muscle atrophy and may have applications for other muscle disorders.

Autophagy ablation in skeletal muscles worsens sepsis-induced muscle wasting, impairs whole-body metabolism, and decreases survival.

Septic patients frequently develop skeletal muscle wasting and weakness, resulting in severe clinical consequences and adverse outcomes. Sepsis triggers sustained induction of autophagy, a key cellular degradative pathway, in skeletal muscles. However, the impact of enhanced autophagy on sepsis-induced muscle dysfunction remains unclear. Using an inducible and muscle-specific Atg7 knockout mouse model (Atg7iSkM-KO), we investigated the functional importance of skeletal muscle autophagy in sepsis using the cecal ligation and puncture model. Atg7iSkM-KO mice exhibited a more severe phenotype in response to sepsis, marked by severe muscle wasting, hypoglycemia, higher ketone levels, and a decreased in survival as compared to mice with intact Atg7. Sepsis and Atg7 deletion resulted in the accumulation of mitochondrial dysfunction, although sepsis did not further worsen mitochondrial dysfunction in Atg7iSkM-KO mice. Overall, our study demonstrates that autophagy inactivation in skeletal muscles triggers significant worsening of sepsis-induced muscle and metabolic dysfunctions and negatively impacts survival.

PHAF1/MYTHO is a novel autophagy regulator that controls muscle integrity.

Skeletal muscles play key roles in movement, posture, thermogenesis, and whole-body metabolism. Autophagy plays essential roles in the regulation of muscle mass, function and integrity. However, the molecular machinery that regulates autophagy is still incompletely understood. In our recent study, we identified and characterized a novel Forkhead Box O (FoxO)-dependent gene, PHAF1/MYTHO (phagophore assembly factor 1/macro-autophagy and youth optimizer), as a novel autophagy regulator that controls muscle integrity. MYTHO/PHAF1 is upregulated in multiple conditions leading to muscle atrophy, and downregulation of its expression spares muscle atrophy triggered by fasting, denervation, cachexia and sepsis. Overexpression of PHAF1/MYTHO is sufficient to induce muscle atrophy. Prolonged downregulation of PHAF1/MYTHO causes a severe myopathic phenotype, which is characterized by impaired autophagy, muscle weakness, myofiber degeneration, mammalian target of rapamycin complex 1 (mTORC1) hyperactivation and extensive ultrastructural defects, such as accumulation of proteinaceous and membranous structures and tubular aggregates. This myopathic phenotype is attenuated upon administration of the mTORC1 inhibitor rapamycin. These findings position PHAF1/MYTHO as a novel regulator of skeletal muscle autophagy and tissue integrity.

MYTHO is a novel regulator of skeletal muscle autophagy and integrity.

Autophagy is a critical process in the regulation of muscle mass, function and integrity. The molecular mechanisms regulating autophagy are complex and still partly understood. Here, we identify and characterize a novel FoxO-dependent gene, d230025d16rik which we named Mytho (Macroautophagy and YouTH Optimizer), as a regulator of autophagy and skeletal muscle integrity in vivo. Mytho is significantly up-regulated in various mouse models of skeletal muscle atrophy. Short term depletion of MYTHO in mice attenuates muscle atrophy caused by fasting, denervation, cancer cachexia and sepsis. While MYTHO overexpression is sufficient to trigger muscle atrophy, MYTHO knockdown results in a progressive increase in muscle mass associated with a sustained activation of the mTORC1 signaling pathway. Prolonged MYTHO knockdown is associated with severe myopathic features, including impaired autophagy, muscle weakness, myofiber degeneration, and extensive ultrastructural defects, such as accumulation of autophagic vacuoles and tubular aggregates. Inhibition of the mTORC1 signaling pathway in mice using rapamycin treatment attenuates the myopathic phenotype triggered by MYTHO knockdown. Skeletal muscles from human patients diagnosed with myotonic dystrophy type 1 (DM1) display reduced Mytho expression, activation of the mTORC1 signaling pathway and impaired autophagy, raising the possibility that low Mytho expression might contribute to the progression of the disease. We conclude that MYTHO is a key regulator of muscle autophagy and integrity.

The impact of high-fat feeding and parkin overexpression on skeletal muscle mass, mitochondrial respiration, and H2O2 emission.

Obesity is a major risk factor for developing various health problems, including insulin resistance and type 2 diabetes. Although controversial, accumulation of mitochondrial dysfunction, and notably an increase in mitochondrial reactive oxygen species (ROS) production, was proposed as a key contributor leading to obesity-induced insulin resistance. Here, our goal was to investigate whether Parkin overexpression, a key regulator of the removal of dysfunctional mitochondria through mitophagy, could confer protection against obesity-induced mitochondrial dysfunction. To this end, intramuscular injections of adeno-associated viruses (AAVs) were performed to overexpress Parkin in limb muscle of 6-mo-old mice fed a control diet (CD) or a high-fat diet (HFD) for 12 wk. An AAV-expressing the green fluorescent protein (GFP) was used as control. HFD increased fat mass, altered glycemia, and resulted in insulin resistance. Parkin overexpression resulted in an increase in muscle mass in both CD and HFD mice. In CD mice, Parkin overexpression increased maximal mitochondrial respiration and lowered H2O2 emission. HFD increased mitochondrial respiration and, surprisingly, also lowered H2O2 emission. Parkin overexpression did not significantly impact mitochondrial function in HFD mice. Taken altogether, our results indicate that Parkin overexpression positively impacts muscle and mitochondrial health under basal conditions and challenges the notion that intrinsic mitochondrial dysfunction is involved in the development of insulin resistance caused by high-fat feeding.

Parkin: a potential target to promote healthy ageing.

Parkin is an E3 ubiquitin ligase mostly known for its role in regulating the removal of defective mitochondria via mitophagy. However, increasing experimental evidence that Parkin regulates several other aspects of mitochondrial biology in addition to its role in mitophagy has emerged over the past two decades. Indeed, Parkin has been shown to regulate mitochondrial biogenesis and dynamics and mitochondria-derived vesicle formation, suggesting that Parkin plays key roles in maintaining healthy mitochondria. While Parkin is commonly described as a cytosolic E3 ubiquitin ligase, it was also detected in other cellular compartments, including the nucleus, where it regulates transcription factors and acts as a transcription factor itself. New evidence also suggests that Parkin overexpression can be leveraged to delay ageing. In Drosophila, for example, Parkin overexpression extends lifespan. In mammals, Parkin overexpression delays hallmarks of ageing in several tissues and cell types. Parkin overexpression also confers protection in various models of cellular senescence and neurological disorders closely associated with ageing, such as Alzheimer's and Parkinson's diseases. Recently, Parkin overexpression has also been shown to suppress tumour growth. In this review, we discuss newly emerging biological roles of Parkin as a modulator of cellular homeostasis, survival and healthy ageing, and we explore potential mechanisms through which Parkin exerts its beneficial effects on cellular health.

Characterization of skeletal muscle wasting pathways in diaphragm and limb muscles of cystic fibrosis mice.

Patients with cystic fibrosis (CF) often suffer from skeletal muscle atrophy, most often attributed to physical inactivity and nutritional factors. CF is also characterized by abnormally elevated systemic inflammation. However, it is unknown whether the lack of a functional CF transmembrane conductance regulator (CFTR) gene predisposes to exaggerated inflammation-induced muscle proteolysis. CF mice (CFTR-/-) and their wild-type (WT = CFTR+/+) littermate controls were systemically injected with Pseudomonas-derived lipopolysaccharide (LPS). After 24 h, the diaphragm and limb muscles (fast-twitch tibialis anterior, and slow-twitch soleus) were assessed for induction of inflammatory cytokines (TNFα, IL1ß, and IL6), oxidative stress, canonical muscle proteolysis pathways (Calpain, Ubiquitin-Proteasome, Autophagy), muscle fiber histology, and diaphragm contractile function. At baseline, CF and WT muscles did not differ with respect to indices of inflammation, proteolysis, or contractile function. After LPS exposure, there was significantly greater induction of all proteolysis pathways (calpain activity; ubiquitin-proteasome: MuRF1 and Atrogin1; autophagy: LC3B, Gabarapl-1, and BNIP3) in CF mice for the diaphragm and tibialis anterior, but not the soleus. Proteolysis pathway upregulation and correlations with inflammatory cytokine induction were most prominent in the tibialis anterior. Diaphragm force normalized to muscle cross-sectional area was reduced by LPS to an equivalent degree in CF and WT mice. CF skeletal muscles containing a high proportion of fast-twitch fibers (diaphragm, tibialis anterior) exhibit abnormally exaggerated upregulation of multiple muscle wasting pathways after exposure to an acute inflammatory stimulus, but not under basal conditions.

Long Non-coding RNA Rhabdomyosarcoma 2-Associated Transcript Regulates Angiogenesis in Endothelial Cells.

Background: Long non-coding RNAs (lncRNAs) are non-coding RNAs that have more than 200 nucleotides. They have recently emerged as important regulators of angiogenesis. To identify novel lncRNAs that may be involved in the regulation of angiogenesis, we detected the mRNA of 84 lncRNAs in human umbilical vein endothelial cells (HUVECs) exposed to hypoxia for 24h. One of these, rhabdomyosarcoma 2-associated transcript (RMST), is significantly upregulated by hypoxia. Little is known about the presence and roles of RMST in EC function. Objective: The main objective of the study was to investigate the regulation of RMST in ECs and to determine its role in EC survival, proliferation, migration, and differentiation. Methods: Using qPCR, basal mRNA levels of 10 RMST isoforms in HUVECs were measured. Levels were then measured in response to 24h of hypoxia, 7days of differentiation in a co-culture assay, and exposure to four different angiogenesis factors. Functional roles of RMST in EC survival, migration, and differentiation were quantified by using a loss-of-function approach (transfection with single-stranded antisense LNA GapmeRs). EC survival was measured using cell counts and crystal violet assays. Cell migration and differentiation were measured using scratch wound healing and Matrigel® differentiation assays, respectively. Results: Five RMST isoforms (RMST-202, -203, -204, -206, and -207) were detected in HUVECs and human microvascular endothelial cells (HMEC-1s). Other types of vascular cells, including human aortic valve interstitial cells and human aortic smooth muscle cells, did not display this expression profile. RMST was significantly upregulated in response to 24h of hypoxia and in response to 7days of HUVEC co-culture with human lung fibroblasts. RMST was significantly downregulated by angiopoietin-2 (Ang-2), but not by VEGF, FGF-2, or angiopoietin-1 (Ang-1). Selective knockdown of RMST demonstrated that it promotes EC survival in response to serum deprivation. It is also required for VEGF- and Ang-1-induced EC survival and migration, but not for differentiation. Conclusion: We conclude that RMST is expressed in human ECs and that this expression is upregulated in response to hypoxia and during differentiation into capillary-like structures. We also conclude that RMST plays important roles in EC survival and migration.

ETS1, ELK1, and ETV4 Transcription Factors Regulate Angiopoietin-1 Signaling and the Angiogenic Response in Endothelial Cells.

BACKGROUND: Angiopoietin-1 (Ang-1) is the main ligand of Tie-2 receptors. It promotes endothelial cell (EC) survival, migration, and differentiation. Little is known about the transcription factors (TFs) in ECs that are downstream from Tie-2 receptors. OBJECTIVE: The main objective of this study is to identify the roles of the ETS family of TFs in Ang-1 signaling and the angiogenic response. METHODS: In silico enrichment analyses that were designed to predict TF binding sites of the promotors of eighty-six Ang-1-upregulated genes showed significant enrichment of ETS1, ELK1, and ETV4 binding sites in ECs. Human umbilical vein endothelial cells (HUVECs) were exposed for different time periods to recombinant Ang-1 protein and mRNA levels of ETS1, ELK1, and ETV4 were measured with qPCR and intracellular localization of these transcription factors was assessed with immunofluorescence. Electrophoretic mobility shift assays and reporter assays were used to assess activation of ETS1, ELK1, and ETV4 in response to Ang-1 exposure. The functional roles of these TFs in Ang-1-induced endothelial cell survival, migration, differentiation, and gene regulation were evaluated by using a loss-of-function approach (transfection with siRNA oligos). RESULTS: Ang-1 exposure increased ETS1 mRNA levels but had no effect on ELK1 or ETV4 levels. Immunostaining revealed that in control ECs, ETS1 has nuclear localization whereas ELK1 and ETV4 are localized to the nucleus and the cytosol. Ang-1 exposure increased nuclear intensity of ETS1 protein and enhanced nuclear mobilization of ELK1 and ETV4. Selective siRNA knockdown of ETS1, ELK1, and ETV4 showed that these TFs are required for Ang-1-induced EC survival and differentiation of cells, while ETS1 and ETV4 are required for Ang-1-induced EC migration. Moreover, ETS1, ELK1, and ETV4 knockdown inhibited Ang-1-induced upregulation of thirteen, eight, and nine pro-angiogenesis genes, respectively. CONCLUSION: We conclude that ETS1, ELK1, and ETV4 transcription factors play significant angiogenic roles in Ang-1 signaling in ECs.

Mitochondrial Dynamics and Mitophagy in Skeletal Muscle Health and Aging.

The maintenance of mitochondrial integrity is critical for muscle health. Mitochondria, indeed, play vital roles in a wide range of cellular processes, including energy supply, Ca2+ homeostasis, retrograde signaling, cell death, and many others. All mitochondria-containing cells, including skeletal muscle cells, dispose of several pathways to maintain mitochondrial health, including mitochondrial biogenesis, mitochondrial-derived vesicles, mitochondrial dynamics (fusion and fission process shaping mitochondrial morphology), and mitophagy-the process in charge of the removal of mitochondria though autophagy. The loss of skeletal muscle mass (atrophy) is a major health problem worldwide, especially in older people. Currently, there is no treatment to counteract the progressive decline in skeletal muscle mass and strength that occurs with aging, a process termed sarcopenia. There is increasing data, including our own, suggesting that accumulation of dysfunctional mitochondria contributes to the development of sarcopenia. Impairments in mitochondrial dynamics and mitophagy were recently proposed to contribute to sarcopenia. This review summarizes the current state of knowledge on the role played by mitochondrial dynamics and mitophagy in skeletal muscle health and in the development of sarcopenia. We also highlight recent studies showing that enhancing mitophagy in skeletal muscle is a promising therapeutic target to prevent or even treat skeletal muscle dysfunction in the elderly.

Regulation of muscle and mitochondrial health by the mitochondrial fission protein Drp1 in aged mice.

KEY POINTS: The maintenance of mitochondrial integrity is critical for skeletal muscle health. Mitochondrial dynamics play key roles in mitochondrial quality control; however, the exact role that mitochondrial fission plays in the muscle ageing process remains unclear. Here we report that both Drp1 knockdown and Drp1 overexpression late in life in mice is detrimental to skeletal muscle function and mitochondrial health. Drp1 knockdown in 18-month-old mice resulted in severe skeletal muscle atrophy, mitochondrial dysfunction, muscle degeneration/regeneration, oxidative stress and impaired autophagy. Overexpressing Drp1 in 18-month-old mice resulted in mild skeletal muscle atrophy and decreased mitochondrial quality. Our data indicate that silencing or overexpressing Drp1 late in life is detrimental to skeletal muscle integrity. We conclude that modulating Drp1 expression is unlikely to be a viable approach to counter the muscle ageing process. ABSTRACT: Sarcopenia, the ageing-related loss of skeletal muscle mass and function, is a debilitating process negatively impacting the quality of life of afflicted individuals. Although the mechanisms underlying sarcopenia are still only partly understood, impairments in mitochondrial dynamics, and specifically mitochondrial fission, have been proposed as an underlying mechanism. Importantly, conflicting data exist in the field and both excessive and insufficient mitochondrial fission were proposed to contribute to sarcopenia. In Drosophila melanogaster, enhancing mitochondrial fission in midlife through overexpression of dynamin-1-like protein (Drp1) extended lifespan and attenuated several key hallmarks of muscle ageing. Whether a similar outcome of Drp1 overexpression is observed in mammalian muscles remains unknown. In this study, we investigated the impact of knocking down and overexpressing Drp1 protein for 4 months in skeletal muscles of late middle-aged (18 months) mice using intra-muscular injections of adeno-associated viruses expressing shRNA targeting Drp1 or full Drp1 cDNA. We report that knocking down Drp1 expression late in life triggers severe muscle atrophy, mitochondrial dysfunctions, degeneration/regeneration, oxidative stress and impaired autophagy. Drp1 overexpression late in life triggered mild muscle atrophy and decreased mitochondrial quality. Taken altogether, our results indicate that both overexpression and silencing of Drp1 in late middle-aged mice negatively impact skeletal muscle mass and mitochondrial health. These data suggest that Drp1 content must remain within a narrow physiological range to preserve muscle and mitochondrial integrity during ageing. Altering Drp1 expression is therefore unlikely to be a viable target to counter sarcopenia.

Prostaglandin E2 amplifies IL-17 production by γδ T cells during barrier inflammation.

Interleukin-17 (IL-17)-producing γδ (γδ17) T cells are innate-like lymphocytes that contribute to protective anti-microbial responses but are also implicated in pathogenic inflammation at barrier sites. Understanding tissue-specific signals that regulate this subset is important to boost host defense mechanisms, but also to mitigate immunopathology. Here, we demonstrate that prostaglandin E2 (PGE2), a cyclooxygenase-dependent member of the eicosanoid family, directly enhances cytokine production by circulating and tissue-specific γδ17 T cells in vitro. Gain- and loss-of-function in vivo approaches further reveal that although provision of PGE2 amplifies psoriasiform inflammation, ablation of host mPGES1-dependent PGE2 synthesis is dispensable for cutaneous γδ17 T cell activation. By contrast, loss of endogenous PGE2 production or depletion of the gut microbiota compromises intestinal γδ17 T cell responses and increases disease severity during experimental colitis. Together, our results demonstrate how a lipid mediator can synergize with tissue-specific signals to enhance innate lymphocyte production of IL-17 during barrier inflammation.

Surrogate Humane Endpoints in Small Animal Models of Acute Lung Injury: A Modified Delphi Consensus Study of Researchers and Laboratory Animal Veterinarians.

OBJECTIVES: In many jurisdictions, ethical concerns require surrogate humane endpoints to replace death in small animal models of acute lung injury. Heterogenous selection and reporting of surrogate endpoints render interpretation and generalizability of findings between studies difficult. We aimed to establish expert-guided consensus among preclinical scientists and laboratory animal veterinarians on selection and reporting of surrogate endpoints, monitoring of these models, and the use of analgesia. DESIGN: A three-round consensus process, using modified Delphi methodology, with researchers who use small animal models of acute lung injury and laboratory animal veterinarians who provide care for these animals. Statements on the selection and reporting of surrogate endpoints, monitoring, and analgesia were generated through a systematic search of MEDLINE and Embase. Participants were asked to suggest any additional potential statements for evaluation. SETTING: A web-based survey of participants representing the two stakeholder groups (researchers, laboratory animal veterinarians). Statements were rated on level of evidence and strength of support by participants. A final face-to-face meeting was then held to discuss results. SUBJECTS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Forty-two statements were evaluated, and 29 were rated as important, with varying strength of evidence. The majority of evidence was based on rodent models of acute lung injury. Endpoints with strong support and evidence included temperature changes and body weight loss. Behavioral signs and respiratory distress also received support but were associated with lower levels of evidence. Participants strongly agreed that analgesia affects outcomes in these models and that none may be necessary following nonsurgical induction of acute lung injury. Finally, participants strongly supported transparent reporting of surrogate endpoints. A prototype composite score was also developed based on participant feedback. CONCLUSIONS: We provide a preliminary framework that researchers and animal welfare committees may adapt for their needs. We have identified knowledge gaps that future research should address.

Angiotensin-converting enzyme 2 expression in COPD and IPF fibroblasts: the forgotten cell in COVID-19.

The COVID-19 pandemic is associated with severe pneumonia and acute respiratory distress syndrome leading to death in susceptible individuals. For those who recover, post-COVID-19 complications may include development of pulmonary fibrosis. Factors contributing to disease severity or development of complications are not known. Using computational analysis with experimental data, we report that idiopathic pulmonary fibrosis (IPF)- and chronic obstructive pulmonary disease (COPD)-derived lung fibroblasts express higher levels of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 entry and part of the renin-angiotensin system that is antifibrotic and anti-inflammatory. In preclinical models, we found that chronic exposure to cigarette smoke, a risk factor for both COPD and IPF and potentially for SARS-CoV-2 infection, significantly increased pulmonary ACE2 protein expression. Further studies are needed to understand the functional implications of ACE2 on lung fibroblasts, a cell type that thus far has received relatively little attention in the context of COVID-19.