Immune evasion, infectivity, and fusogenicity of SARS-CoV-2 BA.2.86 and FLip variants.

Evolution of SARS-CoV-2 requires the reassessment of current vaccine measures. Here, we characterized BA.2.86 and XBB-derived variant FLip by investigating their neutralization alongside D614G, BA.1, BA.2, BA.4/5, XBB.1.5, and EG.5.1 by sera from 3-dose-vaccinated and bivalent-vaccinated healthcare workers, XBB.1.5-wave-infected first responders, and monoclonal antibody (mAb) S309. We assessed the biology of the variant spikes by measuring viral infectivity and membrane fusogenicity. BA.2.86 is less immune evasive compared to FLip and other XBB variants, consistent with antigenic distances. Importantly, distinct from XBB variants, mAb S309 was unable to neutralize BA.2.86, likely due to a D339H mutation based on modeling. BA.2.86 had relatively high fusogenicity and infectivity in CaLu-3 cells but low fusion and infectivity in 293T-ACE2 cells compared to some XBB variants, suggesting a potentially different conformational stability of BA.2.86 spike. Overall, our study underscores the importance of SARS-CoV-2 variant surveillance and the need for updated COVID-19 vaccines.

Influenza, SARS-CoV-2, and Their Impact on Chronic Lung Diseases and Fibrosis: Exploring Therapeutic Options.

Respiratory tract infections represent a significant global public health concern, disproportionately affecting vulnerable populations such as children, the elderly, and immunocompromised individuals. RNA viruses, particularly influenza viruses and coronaviruses, significantly contribute to respiratory illnesses, especially in immunosuppressed and elderly individuals. Influenza A viruses (IAVs) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to pose global health threats due to their capacity to cause annual epidemics, with profound implications for public health. In addition, the increase in global life expectancy is influencing the dynamics and outcomes of respiratory viral infections. Understanding the molecular mechanisms by which IAVs and SARS-CoV-2 contribute to lung disease progression is therefore crucial. The aim of this review is to comprehensively explore the impact of IAVs and SARS-CoV-2 on chronic lung diseases, with a specific focus on pulmonary fibrosis in the elderly. It also outlines potential preventive and therapeutic strategies and suggests directions for future research.

Synergy Between NK Cells and Monocytes in Potentiating Cardiovascular Disease Risk in Severe COVID-19.

BACKGROUND: Evidence suggests that COVID-19 predisposes to cardiovascular diseases (CVDs). While monocytes/macrophages play a central role in the immunopathogenesis of atherosclerosis, less is known about their immunopathogenic mechanisms that lead to CVDs during COVID-19. Natural killer (NK) cells, which play an intermediary role during pathologies like atherosclerosis, are dysregulated during COVID-19. Here, we sought to investigate altered immune cells and their associations with CVD risk during severe COVID-19. METHODS: We measured plasma biomarkers of CVDs and determined phenotypes of circulating immune subsets using spectral flow cytometry. We compared these between patients with severe COVID-19 (severe, n=31), those who recovered from severe COVID-19 (recovered, n=29), and SARS-CoV-2-uninfected controls (controls, n=17). In vivo observations were supported using in vitro assays to highlight possible mechanistic links between dysregulated immune subsets and biomarkers during and after COVID-19. We performed multidimensional analyses of published single-cell transcriptome data of monocytes and NK cells during severe COVID-19 to substantiate in vivo findings. RESULTS: During severe COVID-19, we observed alterations in cardiometabolic biomarkers including oxidized-low-density lipoprotein, which showed decreased levels in severe and recovered groups. Severe patients exhibited dysregulated monocyte subsets, including increased frequencies of proinflammatory intermediate monocytes (also observed in the recovered) and decreased nonclassical monocytes. All identified NK-cell subsets in the severe COVID-19 group displayed increased expression of activation and tissue-resident markers, such as CD69 (cluster of differentiation 69). We observed significant correlations between altered immune subsets and plasma oxidized-low-density lipoprotein levels. In vitro assays revealed increased uptake of oxidized-low-density lipoprotein into monocyte-derived macrophages in the presence of NK cells activated by plasma of patients with severe COVID-19. Transcriptome analyses confirmed enriched proinflammatory responses and lipid dysregulation associated with epigenetic modifications in monocytes and NK cells during severe COVID-19. CONCLUSIONS: Our study provides new insights into the involvement of monocytes and NK cells in the increased CVD risk observed during and after COVID-19.

Regulation of Mesenchymal Cell Fate by Transfer of Active Gasdermin-D via Monocyte-Derived Extracellular Vesicles.

Fibrosis is characterized by inappropriately persistent myofibroblast accumulation and excessive extracellular matrix deposition with the disruption of tissue architecture and organ dysfunction. Regulated death of reparative mesenchymal cells is critical for normal wound repair, but profibrotic signaling promotes myofibroblast resistance to apoptotic stimuli. A complex interplay between immune cells and structural cells underlies lung fibrogenesis. However, there is a paucity of knowledge on how these cell populations interact to orchestrate physiologic and pathologic repair of the injured lung. In this context, gasdermin-D (GsdmD) is a cytoplasmic protein that is activated following cleavage by inflammatory caspases and induces regulated cell death by forming pores in cell membranes. This study was undertaken to evaluate the impact of human (Thp-1) monocyte-derived extracellular vesicles and GsdmD on human lung fibroblast death. Our data show that active GsdmD delivered by monocyte-derived extracellular vesicles induces caspase-independent fibroblast and myofibroblast death. This cell death was partly mediated by GsdmD-independent induction of cellular inhibitor of apoptosis 2 (cIAP-2) in the recipient fibroblast population. Our findings, to our knowledge, define a novel paradigm by which inflammatory monocytes may orchestrate the death of mesenchymal cells in physiologic wound healing, illustrating the potential to leverage this mechanism to eliminate mesenchymal cells and facilitate the resolution of fibrotic repair.

The aged extracellular matrix and the profibrotic role of senescence-associated secretory phenotype.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal lung disease that is primarily found in the elderly population, and several studies have demonstrated that aging is the major risk factor for IPF. IPF is characterized by the presence of apoptosis-resistant, senescent fibroblasts that generate an excessively stiff extracellular matrix (ECM). The ECM profoundly affects cellular functions and tissue homeostasis, and an aberrant ECM is closely associated with the development of lung fibrosis. Aging progressively alters ECM components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction through the expression of factors linked to a senescence-associated secretary phenotype (SASP). There is growing evidence that SASP factors affect various cell behaviors and influence ECM turnover in lung tissue through autocrine and/or paracrine signaling mechanisms. Since life expectancy is increasing worldwide, it is important to elucidate how aging affects ECM dynamics and turnover via SASP and thereby promotes lung fibrosis. In this review, we will focus on the molecular properties of SASP and its regulatory mechanisms. Furthermore, the pathophysiological process of ECM remodeling by SASP factors and the influence of an altered ECM from aged lungs on the development of lung fibrosis will be highlighted. Finally, recent attempts to target ECM alteration and senescent cells to modulate fibrosis will be introduced.NEW & NOTEWORTHY Aging is the most prominent nonmodifiable risk factor for various human diseases including Idiopathic pulmonary fibrosis. Aging progressively alters extracellular matrix components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction. In this review, we will discuss the pathological impact of aging and senescence on lung fibrosis via senescence-associated secretary phenotype factors and potential therapeutic approaches to limit the progression of lung fibrosis.

Both moderate- and high-intensity exercise training increase intramyocellular lipid droplet abundance and modify myocellular distribution in adults with obesity.

Exercise training modifies lipid metabolism in skeletal muscle, but the effect of exercise training on intramyocellular lipid droplet (LD) abundance, size, and intracellular distribution in adults with obesity remains elusive. This study compared high-intensity interval training (HIIT) with more conventional moderate-intensity continuous training (MICT) on intramyocellular lipid content, as well as LD characteristics (size and number) and abundance within the intramyofibrillar (IMF) and subsarcolemmal (SS) regions of type I and type II skeletal muscle fibers in adults with obesity. Thirty-six adults with obesity [body mass index (BMI) = 33 ± 3 kg/m2] completed 12 wk (4 days/wk) of either HIIT (10 × 1 min, 90% HRmax + 1-min active recovery; n = 19) or MICT (45-min steady-state exercise, 70% HRmax; n = 17), while on a weight-maintaining diet throughout training. Skeletal muscle biopsies were collected from the vastus lateralis before and after training, and intramyocellular lipid content and intracellular LD distribution were measured by immunofluorescence microscopy. Both MICT and HIIT increased total intramyocellular lipid content by more than 50% (P < 0.01), which was attributed to a greater LD number per µm2 in the IMF region of both type I and type II muscle fibers (P < 0.01). Our findings also suggest that LD lipophagy (autophagy-mediated LD degradation) may be transiently upregulated the day after the last exercise training session (P < 0.02 for both MICT and HIIT). In summary, exercise programs for adults with obesity involving either MICT or HIIT increased skeletal muscle LD abundance via a greater number of LDs in the IMF region of the myocyte, thereby providing more lipid in close proximity to the site of energy production during exercise.NEW & NOTEWORTHY In this study, 12 wk of either moderate-intensity continuous training (MICT) or high-intensity interval training (HIIT) enhanced skeletal muscle lipid abundance by increasing lipid droplet number within the intramyofibrillar (IMF) region of muscle. Because the IMF associates with high energy production during muscle contraction, this adaptation may enhance lipid oxidation during exercise. Despite differences in training intensity and energy expenditure between MICT and HIIT, their effects on muscle lipid abundance and metabolism were remarkably similar.

Biomechanical Force and Cellular Stiffness in Lung Fibrosis.

Lung fibrosis is characterized by the continuous accumulation of extracellular matrix (ECM) proteins produced by apoptosis-resistant (myo)fibroblasts. Lung epithelial injury promotes the recruitment and activation of fibroblasts, which are necessary for tissue repair and restoration of homeostasis. However, under pathologic conditions, a vicious cycle generated by profibrotic growth factors/cytokines, multicellular interactions, and matrix-associated signaling propagates the wound repair response and promotes lung fibrosis characterized not only by increased quantities of ECM proteins but also by changes in the biomechanical properties of the matrix. Importantly, changes in the biochemical and biomechanical properties of the matrix itself can serve to perpetuate fibroblast activity and propagate fibrosis, even in the absence of the initial stimulus of injury. The development of novel experimental models and methods increasingly facilitates our ability to interrogate fibrotic processes at the cellular and molecular levels. The goal of this review is to discuss the impact of ECM conditions in the development of lung fibrosis and to introduce new approaches to more accurately model the in vivo fibrotic microenvironment. This article highlights the pathologic roles of ECM in terms of mechanical force and the cellular interactions while reviewing in vitro and ex vivo models of lung fibrosis. The improved understanding of the fundamental mechanisms that contribute to lung fibrosis holds promise for identification of new therapeutic targets and improved outcomes.

Serum Ferritin as a Predictor of Outcomes in Hospitalized Patients with Covid-19 Pneumonia.

Purpose: Elevated ferritin levels are associated with poor outcomes in Covid-19 patients. Optimal timing of ferritin assessment and the merit of longitudinal values remains unclear. Methods: Patients admitted to Henry Ford Hospital with confirmed SARS-CoV-2 were studied. Regression models were used to determine the relation between ferritin and mortality, need for mechanical ventilation, ICU admission, and days on the ventilator. Results: 2265 patients were evaluated. Patients with an initial ferritin of > 490 ng/mL had an increased risk of death (OR 3.4, P < .001), admission to the ICU (OR 2.78, P < .001) and need for mechanical ventilation (OR 3.9, P < .001). There was no difference between admission and Day 1 ICU ferritin levels (611.5 ng/mL vs. 649 ng/mL respectively; P = .07). The decline in ferritin over ICU days 1-4 was similar between survivors and non-survivors. A change in ferritin levels from admission to ICU Day 1 (P = .330), or from ICU Day 1 to 2 (P = .788), did not predict days on the ventilator. Conclusions: Initial Ferritin levels were highly predictive of ICU admission, the need for mechanical ventilation and in-hospital mortality. However, longitudinal measures of ferritin throughout the hospital stay did not provide additional predictive value.

Update on the Features and Measurements of Experimental Acute Lung Injury in Animals: An Official American Thoracic Society Workshop Report.

Advancements in methods, technology, and our understanding of the pathobiology of lung injury have created the need to update the definition of experimental acute lung injury (ALI). We queried 50 participants with expertise in ALI and acute respiratory distress syndrome using a Delphi method composed of a series of electronic surveys and a virtual workshop. We propose that ALI presents as a "multidimensional entity" characterized by four "domains" that reflect the key pathophysiologic features and underlying biology of human acute respiratory distress syndrome. These domains are 1) histological evidence of tissue injury, 2) alteration of the alveolar-capillary barrier, 3) presence of an inflammatory response, and 4) physiologic dysfunction. For each domain, we present "relevant measurements," defined as those proposed by at least 30% of respondents. We propose that experimental ALI encompasses a continuum of models ranging from those focusing on gaining specific mechanistic insights to those primarily concerned with preclinical testing of novel therapeutics or interventions. We suggest that mechanistic studies may justifiably focus on a single domain of lung injury, but models must document alterations of at least three of the four domains to qualify as "experimental ALI." Finally, we propose that a time criterion defining "acute" in ALI remains relevant, but the actual time may vary based on the specific model and the aspect of injury being modeled. The continuum concept of ALI increases the flexibility and applicability of the definition to multiple models while increasing the likelihood of translating preclinical findings to critically ill patients.

Exercise training remodels subcutaneous adipose tissue in adults with obesity even without weight loss.

Excessive adipose tissue mass underlies much of the metabolic health complications in obesity. Although exercise training is known to improve metabolic health in individuals with obesity, the effects of exercise training without weight loss on adipose tissue structure and metabolic function remain unclear. Thirty-six adults with obesity (body mass index = 33 ± 3 kg · m-2 ) were assigned to 12 weeks (4 days week-1 ) of either moderate-intensity continuous training (MICT; 70% maximal heart rate, 45 min; n = 17) or high-intensity interval training (HIIT; 90% maximal heart rate, 10 × 1 min; n = 19), maintaining their body weight throughout. Abdominal subcutaneous adipose tissue (aSAT) biopsy samples were collected once before and twice after training (1 day after last exercise and again 4 days later). Exercise training modified aSAT morphology (i.e. reduced fat cell size, increased collagen type 5a3, both P ≤ 0.05, increased capillary density, P = 0.05) and altered protein abundance of factors that regulate aSAT remodelling (i.e. reduced matrix metallopeptidase 9; P = 0.02; increased angiopoietin-2; P < 0.01). Exercise training also increased protein abundance of factors that regulate lipid metabolism (e.g. hormone sensitive lipase and fatty acid translocase; P ≤ 0.03) and key proteins involved in the mitogen-activated protein kinase pathway when measured the day after the last exercise session. However, most of these exercise-mediated changes were no longer significant 4 days after exercise. Importantly, MICT and HIIT induced remarkably similar adaptations in aSAT. Collectively, even in the absence of weight loss, 12 weeks of exercise training induced changes in aSAT structure, as well as factors that regulate metabolism and the inflammatory signal pathway in adults with obesity. KEY POINTS: Exercise training is well-known to improve metabolic health in obesity, although how exercise modifies the structure and metabolic function of adipose tissue, in the absence of weight loss, remains unclear. We report that both 12 weeks of moderate-intensity continuous training (MICT) and 12 weeks of high-intensity interval training (HIIT) induced modifications in adipose tissue structure and factors that regulate adipose tissue remodelling, metabolism and the inflammatory signal pathway in adults with obesity, even without weight loss (with no meaningful differences between MICT and HIIT). The modest modifications in adipose tissue structure in response to 12 weeks of MICT or HIIT did not lead to changes in the rate of fatty acid release from adipose tissue. These results expand our understanding about the effects of two commonly used exercise training prescriptions (MICT and HIIT) on adipose tissue remodelling that may lead to advanced strategies for improving metabolic health outcomes in adults with obesity.

Pathogenesis of pneumonia and acute lung injury.

Pneumonia and its sequelae, acute lung injury, present unique challenges for pulmonary and critical care healthcare professionals, and these challenges have recently garnered global attention due to the ongoing Sars-CoV-2 pandemic. One limitation to translational investigation of acute lung injury, including its most severe manifestation (acute respiratory distress syndrome, ARDS) has been heterogeneity resulting from the clinical and physiologic diagnosis that represents a wide variety of etiologies. Recent efforts have improved our understanding and approach to heterogeneity by defining sub-phenotypes of ARDS although significant gaps in knowledge remain. Improving our mechanistic understanding of acute lung injury and its most common cause, infectious pneumonia, can advance our approach to precision targeted clinical interventions. Here, we review the pathogenesis of pneumonia and acute lung injury, including how respiratory infections and lung injury disrupt lung homoeostasis, and provide an overview of respiratory microbial pathogenesis, the lung microbiome, and interventions that have been demonstrated to improve outcomes-or not-in human clinical trials.

Exercise training decreases whole-body and tissue iron storage in adults with obesity.

NEW FINDINGS: What is the central question of this study? Does exercise training modify tissue iron storage in adults with obesity? What is the main finding and its importance? Twelve weeks of moderate-intensity exercise or high-intensity interval training lowered whole-body iron stores, decreased the abundance of the key iron storage protein in skeletal muscle (ferritin) and tended to lower hepatic iron content. These findings show that exercise training can reduce tissue iron storage in adults with obesity and might have important implications for obese individuals with dysregulated iron homeostasis. ABSTRACT: The regulation of iron storage is crucial to human health, because both excess and deficient iron storage have adverse consequences. Recent studies suggest altered iron storage in adults with obesity, with increased iron accumulation in their liver and skeletal muscle. Exercise training increases iron use for processes such as red blood cell production and can lower whole-body iron stores in humans. However, the effects of exercise training on liver and muscle iron stores in adults with obesity have not been assessed. The aim of this study was to determine the effects of 12 weeks of exercise training on whole-body iron stores, liver iron content and the abundance of ferritin (the key iron storage protein) in skeletal muscle in adults with obesity. Twenty-two inactive adults (11 women and 11 men; age, 31 ± 6 years; body mass index, 33 ± 3 kg/m2 ) completed 12 weeks (four sessions/week) of either moderate-intensity continuous training (MICT; 45 min at 70% of maximal heart rate; n = 11) or high-intensity interval training (HIIT; 10 × 1 min at 90% of maximal heart rate, interspersed with 1 min active recovery; n = 11). Whole-body iron stores were lower after training, as indicated by decreased plasma concentrations of ferritin (P = 3 × 10-5 ) and hepcidin (P = 0.02), without any change in C-reactive protein. Hepatic R2*, an index of liver iron content, was 6% lower after training (P = 0.06). Training reduced the skeletal muscle abundance of ferritin by 10% (P = 0.03), suggesting lower muscle iron storage. Interestingly, these adaptations were similar in MICT and HIIT groups. Our findings indicate that exercise training decreased iron storage in adults with obesity, which might have important implications for obese individuals with dysregulated iron homeostasis.

Printable Organic Electronic Materials for Precisely Positioned Cell Attachment.

Over the past 3 decades, there has been a vast expansion of research in both tissue engineering and organic electronics. Although the two fields have interacted little, the materials and fabrication technologies which have accompanied the rise of organic electronics offer the potential for innovation and translation if appropriately adapted to pattern biological materials for tissue engineering. In this work, we use two organic electronic materials as adhesion points on a biocompatible poly(p-xylylene) surface. The organic electronic materials are precisely deposited via vacuum thermal evaporation and organic vapor jet printing, the proven, scalable processes used in the manufacture of organic electronic devices. The small molecular-weight organics prevent the subsequent growth of antifouling polyethylene glycol methacrylate polymer brushes that grow within the interstices between the molecular patches, rendering these background areas both protein and cell resistant. Last, fibronectin attaches to the molecular patches, allowing for the selective adhesion of fibroblasts. The process is simple, reproducible, and promotes a high yield of cell attachment to the targeted sites, demonstrating that biocompatible organic small-molecule materials can pattern cells at the microscale, utilizing techniques widely used in electronic device fabrication.

Mechanisms for the Resolution of Organ Fibrosis.

Fibrosis is a dynamic process with the potential for reversibility and restoration of near-normal tissue architecture and organ function. Herein, we review mechanisms for resolution of organ fibrosis, in particular that involving the lung, with an emphasis on the critical roles of myofibroblast apoptosis and clearance of deposited matrix.

Skeletal muscle ferritin abundance is tightly related to plasma ferritin concentration in adults with obesity.

NEW FINDINGS: What is the central question of this study? Obesity is associated with complex perturbations to iron homeostasis: is plasma ferritin concentration (a biomarker of whole-body iron stores) related to the abundance of ferritin (the key tissue iron storage protein) in skeletal muscle in adults with obesity? What is the main finding and its importance? Plasma ferritin concentration was tightly correlated with the abundance of ferritin in skeletal muscle, and this relationship persisted when accounting for sex, age, body mass index and plasma C-reactive protein concentration. Our findings suggest that skeletal muscle may be an important iron store. ABSTRACT: Obesity is associated with complex perturbations to whole-body and tissue iron homeostasis. Recent evidence suggests a potentially important influence of iron storage in skeletal muscle on whole-body iron homeostasis, but this association is not clearly resolved. The primary aim of this study was to assess the relationship between whole-body and skeletal muscle iron stores by measuring the abundance of the key iron storage (ferritin) and import (transferrin receptor) proteins in skeletal muscle, as well as markers of whole-body iron homeostasis in men (n = 19) and women (n = 43) with obesity. Plasma ferritin concentration (a marker of whole-body iron stores) was highly correlated with muscle ferritin abundance (r = 0.77, P = 2 × 10-13 ) and negatively associated with muscle transferrin receptor abundance (r = -0.76, P = 1 × 10-12 ). These relationships persisted when accounting for sex, age, BMI and plasma C-reactive protein concentration. In parallel with higher whole-body iron stores in our male versus female participants, men had 2.2-fold higher muscle ferritin abundance (P = 1 × 10-4 ) compared with women. In accordance with lower muscle iron storage, women had 2.7-fold higher transferrin receptor abundance (P = 7 × 10-10 ) compared with men. We conclude that muscle iron storage and import proteins are tightly and independently related to plasma ferritin concentration in adults with obesity, suggesting that skeletal muscle may be an underappreciated iron store.

Glutaminolysis Epigenetically Regulates Antiapoptotic Gene Expression in Idiopathic Pulmonary Fibrosis Fibroblasts.

Fibrotic responses involve multiple cellular processes, including epigenetic changes. Epigenetic changes are sensitive to alterations in the tissue microenvironment such as the flux of tricarboxylic acid (TCA) cycle metabolites. TCA metabolites directly regulate epigenetic states, in part by regulating histone modification-related enzymes. Glutaminolysis is a critical metabolic process by which glutamine is converted to glutamate by glutaminase and then to α-ketoglutarate (α-KG), a TCA cycle metabolite. Idiopathic pulmonary fibrosis (IPF) is a disease characterized by aberrant metabolism, including enhanced glutaminolysis. IPF fibroblasts are apoptosis resistant. In this study, we explored the relationship between glutaminolysis and the resistance to apoptosis of IPF fibroblasts. Inhibition of glutaminolysis decreased expression of XIAP and survivin, members of the inhibitor of apoptosis protein (IAP) family. α-KG is a cofactor for JMJD3 histone demethylase, which targets H3K27me3. In the absence of glutamine, JMJD3 activity in fibroblasts is significantly decreased, whereas H3K27me3 levels are increased. Chromatin immunoprecipitation assays confirmed that JMJD3 directly interacts with XIAP and survivin promoter regions in a glutamine-dependent manner. Exogenous α-KG partially restores JMJD3 function and its interaction with the XIAP and survivin promoter regions under glutamine-deficient conditions. Interestingly, α-KG upregulates XIAP, but not survivin, suggesting differential α-KG-dependent and -independent mechanisms by which glutamine regulates these IAPs. Our data demonstrate a novel mechanism of metabolic regulation in which glutaminolysis promotes apoptosis resistance of IPF fibroblasts through epigenetic regulation of XIAP and survivin.

Discoidin Domain Receptor 2 Signaling Regulates Fibroblast Apoptosis through PDK1/Akt.

Progressive fibrosis is a complication of many chronic diseases, and collectively, organ fibrosis is the leading cause of death in the United States. Fibrosis is characterized by accumulation of activated fibroblasts and excessive deposition of extracellular matrix proteins, especially type I collagen. Extensive research has supported a role for matrix signaling in propagating fibrosis, but type I collagen itself is often considered an end product of fibrosis rather than an important regulator of continued collagen deposition. Type I collagen can activate several cell surface receptors, including α2ß1 integrin and discoidin domain receptor 2 (DDR2). We have previously shown that mice deficient in type I collagen have reduced activation of DDR2 and reduced accumulation of activated myofibroblasts. In the present study, we found that DDR2-null mice are protected from fibrosis. Surprisingly, DDR2-null fibroblasts have a normal and possibly exaggerated activation response to transforming growth factor-ß and do not have diminished proliferation compared with wild-type fibroblasts. DDR2-null fibroblasts are significantly more prone to apoptosis, in vitro and in vivo, than wild-type fibroblasts, supporting a paradigm in which fibroblast resistance to apoptosis is critical for progression of fibrosis. We have identified a novel molecular mechanism by which DDR2 can promote the activation of a PDK1 (3-phosphoinositide dependent protein kinase-1)/Akt survival pathway, and we have found that inhibition of PDK1 can augment fibroblast apoptosis. Furthermore, our studies demonstrate that DDR2 expression is heavily skewed to mesenchymal cells compared with epithelial cells and that idiopathic pulmonary fibrosis cells and tissue demonstrate increased activation of DDR2 and PDK1. Collectively, these findings identify a promising target for fibrosis therapy.

Regulation of fibroblast Fas expression by soluble and mechanical pro-fibrotic stimuli.

BACKGROUND: Fibroblast apoptosis is a critical component of normal repair and the acquisition of an apoptosis-resistant phenotype contributes to the pathogenesis of fibrotic repair. Fibroblasts from fibrotic lungs of humans and mice demonstrate resistance to apoptosis induced by Fas-ligand and prior studies have shown that susceptibility to apoptosis is enhanced when Fas (CD95) expression is increased in these cells. Moreover, prior work shows that Fas expression in fibrotic lung fibroblasts is reduced by epigenetic silencing of the Fas promoter. However, the mechanisms by which microenvironmental stimuli such as TGF-ß1 and substrate stiffness affect fibroblast Fas expression are not well understood. METHODS: Primary normal human lung fibroblasts (IMR-90) were cultured on tissue culture plastic or on polyacrylamide hydrogels with Young's moduli to recapitulate the compliance of normal (400 Pa) or fibrotic (6400 Pa) lung tissue and treated with or without TGF-ß1 (10 ng/mL) in the presence or absence of protein kinase inhibitors and/or inflammatory cytokines. Expression of Fas was assessed by quantitative real time RT-PCR, ELISA and Western blotting. Soluble Fas (sFas) was measured in conditioned media by ELISA. Apoptosis was assessed using the Cell Death Detection Kit and by Western blotting for cleaved PARP. RESULTS: Fas expression and susceptibility to apoptosis was diminished in fibroblasts cultured on 6400 Pa substrates compared to 400 Pa substrates. TGF-ß1 reduced Fas mRNA and protein in a time- and dose-dependent manner dependent on focal adhesion kinase (FAK). Surprisingly, TGF-ß1 did not significantly alter cell-surface Fas expression, but did stimulate secretion of sFas. Finally, enhanced Fas expression and increased susceptibility to apoptosis was induced by combined treatment with TNF-α/IFN-γ and was not inhibited by TGF-ß1. CONCLUSIONS: Soluble and matrix-mediated pro-fibrotic stimuli promote fibroblast resistance to apoptosis by decreasing Fas transcription while stimulating soluble Fas secretion. These findings suggest that distinct mechanisms regulating Fas expression in fibroblasts may serve different functions in the complex temporal and spatial evolution of normal and fibrotic wound-repair responses.

Plasma ferritin concentration is positively associated with in vivo fatty acid mobilization and insulin resistance in obese women.

NEW FINDINGS: What is the central question of this study? Do obese women with relatively high whole-body iron stores exhibit elevated in vivo rates of fatty acid (FA) release from adipose tissue compared with a well-matched cohort of obese women with relatively low iron stores? What is the main finding and its importance? Obese women with high plasma [ferritin] (a marker of whole-body iron stores) had greater FA mobilization, lipolytic activation in adipose tissue and insulin resistance (IR) compared with obese women with lower plasma [ferritin]. Given that elevated FA mobilization is intimately linked with the development of IR, these findings suggest that elevated iron stores might contribute to IR in obesity by increasing systemic FA availability. ABSTRACT: High rates of fatty acid (FA) mobilization from adipose tissue are associated with insulin resistance (IR) in obesity. In vitro evidence suggests that iron stimulates lipolysis in adipocytes, but whether iron is related to in vivo FA mobilization is unknown. We hypothesized that plasma ferritin concentration ([ferritin]), a marker of body iron stores, would be positively associated with FA mobilization. We measured [ferritin], the rate of appearance of FA in the systemic circulation (FA Ra; stable isotope dilution), key adipose tissue lipolytic proteins and IR (hyperinsulinaemic-euglycaemic clamp) in 20 obese, premenopausal women. [Ferritin] was correlated with FA Ra (r = 0.65; P = 0.002) and IR (r = 0.57; P = 0.008); these relationships remained significant after controlling for body mass index and plasma [C-reactive protein] (a marker of systemic inflammation) in multiple regression analyses. We then stratified subjects into tertiles based on [ferritin] to compare subjects with 'High-ferritin' versus 'Low-ferritin'. Plasma [hepcidin] was more than fivefold greater (P < 0.05) in the High-ferritin versus Low-ferritin group, but there was no difference in plasma [C-reactive protein] between groups, indicating that the large difference in plasma [ferritin] reflects a difference in iron stores, not systemic inflammation. We found that FA Ra, adipose protein abundance of hormone-sensitive lipase and adipose triglyceride lipase, and IR were significantly greater in subjects with High-ferritin versus Low-ferritin (all P < 0.05). These data provide the first evidence linking iron and in vivo FA mobilization and suggest that elevated iron stores might contribute to IR in obesity by increasing systemic FA availability.