Autoregulation of insulin receptor signaling through MFGE8 and the αvß5 integrin.

The role of integrins, in particular αv integrins, in regulating insulin resistance is incompletely understood. We have previously shown that the αvß5 integrin ligand milk fat globule epidermal growth factor like 8 (MFGE8) regulates cellular uptake of fatty acids. In this work, we evaluated the impact of MFGE8 on glucose homeostasis. We show that acute blockade of the MFGE8/ß5 pathway enhances while acute augmentation dampens insulin-stimulated glucose uptake. Moreover, we find that insulin itself induces cell-surface enrichment of MFGE8 in skeletal muscle, which then promotes interaction between the αvß5 integrin and the insulin receptor leading to dampening of skeletal-muscle insulin receptor signaling. Blockade of the MFGE8/ß5 pathway also enhances hepatic insulin sensitivity. Our work identifies an autoregulatory mechanism by which insulin-stimulated signaling through its cognate receptor is terminated through up-regulation of MFGE8 and its consequent interaction with the αvß5 integrin, thereby establishing a pathway that can potentially be targeted to improve insulin sensitivity.

Reversible Airflow Obstruction Predicts Future Chronic Obstructive Pulmonary Disease Development in the SPIROMICS Cohort: An Observational Cohort Study.

Rationale: Chronic obstructive pulmonary disease (COPD) is defined by fixed spirometric ratio, FEV1/FVC < 0.70 after inhaled bronchodilators. However, the implications of variable obstruction (VO), in which the prebronchodilator FEV1/FVC ratio is less than 0.70 but increases to 0.70 or more after inhaled bronchodilators, have not been determined. Objectives: We explored differences in physiology, exacerbations, and health status in participants with VO compared with reference participants without obstruction. Methods: Data from the SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study) cohort were obtained. Participants with VO were compared with reference participants without obstruction. Measurements and Main Results: We assessed differences in baseline radiographic emphysema and small airway disease at study entry, baseline, and change in lung function by spirometry, functional capacity by 6-minute walk, health status using standard questionnaires, exacerbation rates, and progression to COPD between the two groups. All models were adjusted for participant characteristics, asthma history, and tobacco exposure. We assessed 175 participants with VO and 603 reference participants without obstruction. Participants with VO had 6.2 times the hazard of future development of COPD controlling for other factors (95% confidence interval, 4.6-8.3; P < 0.001). Compared with reference participants, the VO group had significantly lower baseline pre- and post-bronchodilator (BD) FEV1, and greater decline over time in post-BD FEV1, and pre- and post-BD FVC. There were no significant differences in exacerbations between groups. Conclusions: Significant risk for future COPD development exists for those with pre- but not post-BD airflow obstruction. These findings support consideration of expanding spirometric criteria defining COPD to include pre-BD obstruction. Clinical trial registered with www.clinicaltrials.gov (NCT01969344).

Longitudinal Follow-Up of Participants With Tobacco Exposure and Preserved Spirometry.

Importance: People who smoked cigarettes may experience respiratory symptoms without spirometric airflow obstruction. These individuals are typically excluded from chronic obstructive pulmonary disease (COPD) trials and lack evidence-based therapies. Objective: To define the natural history of persons with tobacco exposure and preserved spirometry (TEPS) and symptoms (symptomatic TEPS). Design, Setting, and Participants: SPIROMICS II was an extension of SPIROMICS I, a multicenter study of persons aged 40 to 80 years who smoked cigarettes (>20 pack-years) with or without COPD and controls without tobacco exposure or airflow obstruction. Participants were enrolled in SPIROMICS I and II from November 10, 2010, through July 31, 2015, and followed up through July 31, 2021. Exposures: Participants in SPIROMICS I underwent spirometry, 6-minute walk distance testing, assessment of respiratory symptoms, and computed tomography of the chest at yearly visits for 3 to 4 years. Participants in SPIROMICS II had 1 additional in-person visit 5 to 7 years after enrollment in SPIROMICS I. Respiratory symptoms were assessed with the COPD Assessment Test (range, 0 to 40; higher scores indicate more severe symptoms). Participants with symptomatic TEPS had normal spirometry (postbronchodilator ratio of forced expiratory volume in the first second [FEV1] to forced vital capacity >0.70) and COPD Assessment Test scores of 10 or greater. Participants with asymptomatic TEPS had normal spirometry and COPD Assessment Test scores of less than 10. Patient-reported respiratory symptoms and exacerbations were assessed every 4 months via phone calls. Main Outcomes and Measures: The primary outcome was assessment for accelerated decline in lung function (FEV1) in participants with symptomatic TEPS vs asymptomatic TEPS. Secondary outcomes included development of COPD defined by spirometry, respiratory symptoms, rates of respiratory exacerbations, and progression of computed tomographic-defined airway wall thickening or emphysema. Results: Of 1397 study participants, 226 had symptomatic TEPS (mean age, 60.1 [SD, 9.8] years; 134 were women [59%]) and 269 had asymptomatic TEPS (mean age, 63.1 [SD, 9.1] years; 134 were women [50%]). At a median follow-up of 5.76 years, the decline in FEV1 was -31.3 mL/y for participants with symptomatic TEPS vs -38.8 mL/y for those with asymptomatic TEPS (between-group difference, -7.5 mL/y [95% CI, -16.6 to 1.6 mL/y]). The cumulative incidence of COPD was 33.0% among participants with symptomatic TEPS vs 31.6% among those with asymptomatic TEPS (hazard ratio, 1.05 [95% CI, 0.76 to 1.46]). Participants with symptomatic TEPS had significantly more respiratory exacerbations than those with asymptomatic TEPS (0.23 vs 0.08 exacerbations per person-year, respectively; rate ratio, 2.38 [95% CI, 1.71 to 3.31], P < .001). Conclusions and Relevance: Participants with symptomatic TEPS did not have accelerated rates of decline in FEV1 or increased incidence of COPD vs those with asymptomatic TEPS, but participants with symptomatic TEPS did experience significantly more respiratory exacerbations over a median follow-up of 5.8 years.

Cell division cycle 7 kinase is a negative regulator of cell-mediated collagen degradation.

Although extensive work has delineated many of the mechanisms of extracellular matrix (ECM) production, far less is known about pathways that regulate ECM degradation. This is particularly true of cellular internalization and degradation of matrix, which play an underappreciated role in ECM metabolism and lung fibrosis. For example, genetic perturbation of this pathway leads to exacerbated fibrosis in experimental animal models. In this work, we present the results of an unbiased screen of Drosophila phagocytes that yielded multiple genes that, when silenced, led to increased collagen uptake. We further describe the function of cell division cycle 7 kinase (CDC7) as a specific suppressor of collagen uptake. We show that the genetic or pharmacological inhibition of CDC7 results in increased expression of the collagen endocytic receptor Endo180. Chromobox 5 (CBX5) is a putative target of CDC7, and genetic silencing of CBX5 also results in increased Endo180 and collagen uptake. Finally, CRISPR-mediated activation of Endo180 expression results in increased collagen uptake, suggesting that CDC7 regulates collagen internalization through increased Endo180 expression. Targeting the regulatory elements of the collagen degradative machinery may be a useful therapeutic approach in diseases of fibrosis or malignancy.

Mfge8 suppresses airway hyperresponsiveness in asthma by regulating smooth muscle contraction.

Airway obstruction is a hallmark of allergic asthma and is caused primarily by airway smooth muscle (ASM) hypercontractility. Airway inflammation leads to the release of cytokines that enhance ASM contraction by increasing ras homolog gene family, member A (RhoA) activity. The protective mechanisms that prevent or attenuate the increase in RhoA activity have not been well studied. Here, we report that mice lacking the gene that encodes the protein Milk Fat Globule-EGF factor 8 (Mfge8(-/-)) develop exaggerated airway hyperresponsiveness in experimental models of asthma. Mfge8(-/-) ASM had enhanced contraction after treatment with IL-13, IL-17A, or TNF-α. Recombinant Mfge8 reduced contraction in murine and human ASM treated with IL-13. Mfge8 inhibited IL-13-induced NF-κB activation and induction of RhoA. Mfge8 also inhibited rapid activation of RhoA, an effect that was eliminated by an inactivating point mutation in the RGD integrin-binding site in recombinant Mfge8. Human subjects with asthma had decreased Mfge8 expression in airway biopsies compared with healthy controls. These data indicate that Mfge8 binding to integrin receptors on ASM opposes the effect of allergic inflammation on RhoA activity and identify a pathway for specific inhibition of ASM hypercontractility in asthma.

Always cleave up your mess: targeting collagen degradation to treat tissue fibrosis.

Pulmonary fibrosis is a vexing clinical problem with no proven therapeutic options. In the normal lung there is continuous collagen synthesis and collagen degradation, and these two processes are precisely balanced to maintain normal tissue architecture. With lung injury there is an increase in the rate of both collagen production and collagen degradation. The increase in collagen degradation is critical in preventing the formation of permanent scar tissue each time the lung is exposed to injury. In pulmonary fibrosis, collagen degradation does not keep pace with collagen production, resulting in extracellular accumulation of fibrillar collagen. Collagen degradation occurs through both extracellular and intracellular pathways. The extracellular pathway involves cleavage of collagen fibrils by proteolytic enzyme including the metalloproteinases. The less-well-described intracellular pathway involves binding and uptake of collagen fragments by fibroblasts and macrophages for lysosomal degradation. The relationship between these two pathways and their relevance to the development of fibrosis is complex. Fibrosis in the lung, liver, and skin has been associated with an impaired degradative environment. Much of the current scientific effort in fibrosis is focused on understanding the pathways that regulate increased collagen production. However, recent reports suggest an important role for collagen turnover and degradation in regulating the severity of tissue fibrosis. The objective of this review is to evaluate the roles of the extracellular and intracellular collagen degradation pathways in the development of fibrosis and to examine whether pulmonary fibrosis can be viewed as a disease of impaired matrix degradation rather than a disease of increased matrix production.

Treating COVID-19: Evolving approaches to evidence in a pandemic.

The rapid pace of the COVID-19 pandemic precluded traditional approaches to evaluating clinical research and guidelines. We highlight notable successes and pitfalls of clinicians' new approaches to managing evidence amidst an unprecedented crisis. In "Era 1" (early 2020), clinicians relied on anecdote and social media, which democratized conversations on guidelines, but also led clinicians astray. "Era 2" (approximately late 2020 to early 2021) saw preprints that accelerated new interventions but suffered from a surfeit of poor-quality data. In the current era, clinicians consolidate the evidentiary gains of Era 2 with living, online clinical guidelines, but the public suffers from misinformation. The COVID-19 pandemic is a laboratory on how clinicians adapt to an absence of clinical guidance amidst an informational and healthcare crisis. Challenges remain as we integrate new approaches to innovations made in the traditional guideline process to confront both the long tail of COVID-19 and future pandemics.

Isoflurane inhibits growth but does not cause cell death in hippocampal neural precursor cells grown in culture.

BACKGROUND: Isoflurane causes long-term hippocampal-dependent learning deficits in rats despite limited isoflurane-induced hippocampal cell death, raising questions about the causality between isoflurane-induced cell death and isoflurane-induced cognitive function. Neurogenesis in the dentate gyrus is required for hippocampal-dependent learning and thus constitutes a potential alternative mechanism by which cognition can be altered after neonatal anesthesia. The authors tested the hypothesis that isoflurane alters proliferation and differentiation of hippocampal neural progenitor cells. METHODS: Multipotent neural progenitor cells were isolated from pooled rat hippocampi (postnatal day 2) and grown in culture. These cells were exposed to isoflurane and evaluated for cell death using lactate dehydrogenase release, caspase activity, and immunocytochemistry for nuclear localization of cleaved caspase 3. Growth was assessed by cell counting and BrdU incorporation. Expression of markers of stemness (Sox2) and cell division (Ki67) were determined by quantitative polymerase chain reaction. Cell fate selection was assessed using immunocytochemistry to stain for neuronal and glial markers. RESULTS: Isoflurane did not change lactate dehydrogenase release, activity of caspase 3/7, or the amount of nuclear cleaved caspase 3. Isoflurane decreased caspase 9 activity, inhibited proliferation, and decreased the proportion of cells in s-phase. messenger ribonucleic acid expression of Sox2 (stem cells) and Ki67 (proliferation) were decreased. Differentiating neural progenitor cells more often select a neuronal fate after isoflurane exposure. CONCLUSIONS: The authors conclude that isoflurane does not cause cell death, but it does act directly on neural progenitor cells independently of effects on the surrounding brain to decrease proliferation and increase neuronal fate selection. These changes could adversely affect cognition after isoflurane anesthesia.

A prospective study of primary surrogate decision makers' knowledge of intensive care.

OBJECTIVES: We sought to determine 1) primary surrogate decision makers' knowledge of their family members' intensive care and resuscitation status; 2) whether characteristics such as low education level and lack of English language fluency were associated with poor knowledge of intensive care; 3) surrogates' ratings of intensive care unit team communication; and 4) barriers to communication. DESIGN: Prospective study. SETTING: Medical intensive care units of a county hospital located in an urban area. SUBJECTS: Primary surrogate decision makers of all adults admitted >48 hrs from August 2005 to April 2006, enrolled sequentially. INTERVENTIONS: Using a structured instrument consisting of visual analog scales, Likert-type questions, and an objective assessment of knowledge of family member's current intensive care, we interviewed primary surrogate decision makers after they had at least one bedside patient visit. MEASUREMENTS AND MAIN RESULTS: Of eligible primary surrogate decision makers, 81 were enrolled; 96% had spoken to hospital staff. On a scale in which 0 indicated the worst possible communication and 10 indicated the best possible communication, primary surrogate decision makers' mean (SD) ratings were 8.6 (2.3) for nurses and 7.8 (2.8) for doctors. Forty-seven percent of primary surrogate decision makers met the predetermined criteria for good understanding with no significant difference between college-educated (44%) and non-college-educated (50%) primary surrogate decision makers; more non-English-speaking primary surrogate decision makers (63%), however, had poor understanding than English speakers (40%) (mean difference in proportions 23%, 95% confidence interval 1% to 46%). Seventy-three percent (95% confidence interval 61-82) of primary surrogate decision makers correctly identified their family members' resuscitation orders (do-not-resuscitate, limited resuscitation, or full code). The primary reasons cited for poor communication/understanding were as follows: not given enough time (21%), explanations too complicated (16%), and too emotionally upset (5%). CONCLUSIONS: Although most primary surrogate decision makers reported good understanding and excellent staff communication, almost half had poor understanding on objective testing; non-English speakers were more likely to have poor understanding.

500 Million Alveoli from 30,000 Feet: A Brief Primer on Lung Anatomy.

The lungs are a complex organ that fulfill multiple life-sustaining roles including transfer of oxygen and carbon dioxide between the ambient environment and the bloodstream, host defense, and immune homeostasis. As in any biological system, an understanding of the underlying anatomy is prerequisite for successful experimental design and appropriate interpretation of data, regardless of the precise experimental model or procedure in use. This chapter provides an overview of human lung anatomy focused on the airways, the ultrastructure or parenchyma of the lung, the pulmonary vasculature, the innervation of the lungs, and the pulmonary lymphatic system. We will also discuss notable anatomic differences between mouse and human lungs.

α8ß1 integrin regulates nutrient absorption through an Mfge8-PTEN dependent mechanism.

Coordinated gastrointestinal smooth muscle contraction is critical for proper nutrient absorption and is altered in a number of medical disorders. In this work, we demonstrate a critical role for the RGD-binding integrin α8ß1 in promoting nutrient absorption through regulation of gastrointestinal motility. Smooth muscle-specific deletion and antibody blockade of α8 in mice result in enhanced gastric antral smooth muscle contraction, more rapid gastric emptying, and more rapid transit of food through the small intestine leading to malabsorption of dietary fats and carbohydrates as well as protection from weight gain in a diet-induced model of obesity. Mechanistically, ligation of α8ß1 by the milk protein Mfge8 reduces antral smooth muscle contractile force by preventing RhoA activation through a PTEN-dependent mechanism. Collectively, our results identify a role for α8ß1 in regulating gastrointestinal motility and identify α8 as a potential target for disorders characterized by hypo- or hyper-motility.

Functional genomic screen identifies novel mediators of collagen uptake.

Tissue fibrosis occurs when matrix production outpaces matrix degradation. Degradation of collagen, the main component of fibrotic tissue, is mediated through an extracellular proteolytic pathway and intracellular pathway of cellular uptake and lysosomal digestion. Recent studies demonstrate that disruption of the intracellular pathways can exacerbate fibrosis. These pathways are poorly characterized. Here we identify novel mediators of the intracellular pathway of collagen turnover through a genome-wide RNA interference screen in Drosophila S2 cells. Screening of 7505 Drosophila genes conserved among metazoans identified 22 genes that were required for efficient internalization of type I collagen. These included proteins involved in vesicle transport, the actin cytoskeleton, and signal transduction. We show further that the flotillin genes have a conserved and central role in collagen uptake in Drosophila and human cells. Short hairpin RNA-mediated silencing of flotillins in human monocyte and fibroblasts impaired collagen uptake by promoting lysosomal degradation of the endocytic collagen receptors uPARAP/Endo180 and mannose receptor. These data provide an initial characterization of intracellular pathways of collagen turnover and identify the flotillin genes as critical regulators of this process. A better understanding of these pathways may lead to novel therapies that reduce fibrosis by increasing collagen turnover.

Mfge8 promotes obesity by mediating the uptake of dietary fats and serum fatty acids.

Fatty acids are integral mediators of energy storage, membrane formation and cell signaling. The pathways that orchestrate uptake of fatty acids remain incompletely understood. Expression of the integrin ligand Mfge8 is increased in human obesity and in mice on a high-fat diet, but its role in obesity is unknown. We show here that Mfge8 promotes the absorption of dietary triglycerides and the cellular uptake of fatty acid and that Mfge8-deficient (Mfge8(-/-)) mice are protected from diet-induced obesity, steatohepatitis and insulin resistance. Mechanistically, we found that Mfge8 coordinates fatty acid uptake through αvß3 integrin- and αvß5 integrin-dependent phosphorylation of Akt by phosphatidylinositide-3 kinase and mTOR complex 2, leading to translocation of Cd36 and Fatp1 from cytoplasmic vesicles to the cell surface. Collectively, our results imply a role for Mfge8 in regulating the absorption and storage of dietary fats, as well as in the development of obesity and its complications.

Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages.

Milk fat globule epidermal growth factor 8 (Mfge8) is a soluble glycoprotein known to regulate inflammation and immunity by mediating apoptotic cell clearance. Since fibrosis can occur as a result of exaggerated apoptosis and inflammation, we set out to investigate the hypothesis that Mfge8 might negatively regulate tissue fibrosis. We report here that Mfge8 does decrease the severity of tissue fibrosis in a mouse model of pulmonary fibrosis; however, it does so not through effects on inflammation and apoptotic cell clearance, but by binding and targeting collagen for cellular uptake through its discoidin domains. Initial analysis revealed that Mfge8-/- mice exhibited enhanced pulmonary fibrosis after bleomycin-induced lung injury. However, they did not have increased inflammation or impaired apoptotic cell clearance after lung injury compared with Mfge8+/+ mice; rather, they had a defect in collagen turnover. Further experiments indicated that Mfge8 directly bound collagen and that Mfge8-/- macrophages exhibited defective collagen uptake that could be rescued by recombinant Mfge8 containing at least one discoidin domain. These data demonstrate a critical role for Mfge8 in decreasing the severity of murine tissue fibrosis by facilitating the removal of accumulated collagen.