Gut microbiota modulates weight gain in mice after discontinued smoke exposure.

Cigarette smoking constitutes a leading global cause of morbidity and preventable death1, and most active smokers report a desire or recent attempt to quit2. Smoking-cessation-induced weight gain (SCWG; 4.5 kg reported to be gained on average per 6-12 months, >10 kg year-1 in 13% of those who stopped smoking3) constitutes a major obstacle to smoking abstinence4, even under stable5,6 or restricted7 caloric intake. Here we use a mouse model to demonstrate that smoking and cessation induce a dysbiotic state that is driven by an intestinal influx of cigarette-smoke-related metabolites. Microbiome depletion induced by treatment with antibiotics prevents SCWG. Conversely, fecal microbiome transplantation from mice previously exposed to cigarette smoke into germ-free mice naive to smoke exposure induces excessive weight gain across diets and mouse strains. Metabolically, microbiome-induced SCWG involves a concerted host and microbiome shunting of dietary choline to dimethylglycine driving increased gut energy harvest, coupled with the depletion of a cross-regulated weight-lowering metabolite, N-acetylglycine, and possibly by the effects of other differentially abundant cigarette-smoke-related metabolites. Dimethylglycine and N-acetylglycine may also modulate weight and associated adipose-tissue immunity under non-smoking conditions. Preliminary observations in a small cross-sectional human cohort support these findings, which calls for larger human trials to establish the relevance of this mechanism in active smokers. Collectively, we uncover a microbiome-dependent orchestration of SCWG that may be exploitable to improve smoking-cessation success and to correct metabolic perturbations even in non-smoking settings.

Serial Sampling of the Small Airway Epithelium to Identify Persistent Smoking-dysregulated Genes.

Rationale: The small airway epithelium (beyond the sixth generation), the initiation site of smoking-induced airway disorders, is highly sensitive to the stress of smoking. Because of variations over time in smoking habits, the small airway epithelium transcriptome is dynamic, fluctuating not only among smokers but also within each smoker. Objectives: To perform accurate assessment of the smoking-related dysregulation of the human small airway epithelium despite the variation of smoking within the same individual and of the effects of smoking cessation on the dysregulated transcriptome. Methods: We conducted serial sampling of the same smokers and nonsmoker control subjects over time to identify persistent smoking dysregulation of the biology of the small airway epithelium over 1 year. We conducted serial sampling of smokers who quit smoking, before and after smoking cessation, to assess the effect of smoking cessation on the smoking-dysregulated genes. Measurements and Main Results: Repeated measures ANOVA of the small airway epithelium transcriptome sampled four times in the same individuals over 1 year enabled the identification of 475 persistent smoking-dysregulated genes. Most genes were normalized after 12 months of smoking cessation; however, 53 (11%) genes, including CYP1B1, PIR, ME1, and TRIM16, remained persistently abnormally expressed. Dysregulated pathways enriched with the nonreversible genes included xenobiotic metabolism signaling, bupropion degradation, and nicotine degradation. Conclusions: Analysis of repetitive sampling of the same individuals identified persistent smoking-induced dysregulation of the small airway epithelium transcriptome and the effect of smoking cessation. These results help identify targets for the development of therapies that can be applicable to smoking-related airway diseases.

Dual-Function Platform Based on Postsynthetic Functionalization of a Water-Stable Hydrogen-Bonded Organic Framework: Ratiometric Sensing of Nicotine and Cotinine and Dynamic Anticounterfeiting for Information Encryption.

Nicotine and its major metabolite cotinine are widely used as markers of tobacco smoke abstinence as well as indicators of active smoking levels and the assessment of passive inhalation of tobacco smoke in nonsmokers. Therefore, using an easy-to-prepare sensing platform that can provide a rapid, highly sensitive response for the simultaneous detection of salivary nicotine levels and urinary cotinine levels is especially crucial for helping heavy cigarette smokers quit smoking and protecting public health. Hydrogen-bonded organic frameworks, as a novel class of porous crystalline materials, show immense potential for functional modification and optical sensing. Herein, a new HOF was prepared by a simple solvent evaporation method, and a dual-emitting material Eu(bpy)@HOF-215(1) was obtained by the postsynthetic modification of HOF by lanthanide luminescent complexes, which maintains favorable structural stability and introduces the characteristic emitting of Eu, allowing use as a ratiometric fluorescent sensor for salivary nicotine and urinary cotinine, with a limit of detection of nicotine of 0.045 µM in saliva and a limit of detection of cotinine of 0.591 µM in urine. Furthermore, luminescent inks based on HOF-215 have been fabricated based on the photoresponse variations of 1 to NIC and COT, which enables the multilevel encryption and decryption of information, in a dynamic and recyclable process. This work not only synthesizes a novel blue HOF but also provides a representative successful case of a dual-function platform for simultaneous application to ratiometric sensing and dynamic anticounterfeiting.

Mechanisms Contributing to the Comorbidity of COPD and Lung Cancer.

Lung cancer and chronic obstructive pulmonary disease (COPD) often co-occur, and individuals with COPD are at a higher risk of developing lung cancer. While the underlying mechanism for this risk is not well understood, its major contributing factors have been proposed to include genomic, immune, and microenvironment dysregulation. Here, we review the evidence and significant studies that explore the mechanisms underlying the heightened lung cancer risk in people with COPD. Genetic and epigenetic changes, as well as the aberrant expression of non-coding RNAs, predispose the lung epithelium to carcinogenesis by altering the expression of cancer- and immune-related genes. Oxidative stress generated by tobacco smoking plays a role in reducing genomic integrity, promoting epithelial-mesenchymal-transition, and generating a chronic inflammatory environment. This leads to abnormal immune responses that promote cancer development, though not all smokers develop lung cancer. Sex differences in the metabolism of tobacco smoke predispose females to developing COPD and accumulating damage from oxidative stress that poses a risk for the development of lung cancer. Dysregulation of the lung microenvironment and microbiome contributes to chronic inflammation, which is observed in COPD and known to facilitate cancer initiation in various tumor types. Further, there is a need to better characterize and identify the proportion of individuals with COPD who are at a high risk for developing lung cancer. We evaluate possible novel and individualized screening strategies, including biomarkers identified in genetic studies and exhaled breath condensate analysis. We also discuss the use of corticosteroids and statins as chemopreventive agents to prevent lung cancer. It is crucial that we optimize the current methods for the early detection and management of lung cancer and COPD in order to improve the health outcomes for a large affected population.

Biochemistry of nicotine metabolism and its relevance to lung cancer.

Nicotine is the key addictive constituent of tobacco. It is not a carcinogen, but it drives smoking and the continued exposure to the many carcinogens present in tobacco. The investigation into nicotine biotransformation has been ongoing for more than 60 years. The dominant pathway of nicotine metabolism in humans is the formation of cotinine, which occurs in two steps. The first step is cytochrome P450 (P450, CYP) 2A6-catalyzed 5'-oxidation to an iminium ion, and the second step is oxidation of the iminium ion to cotinine. The half-life of nicotine is longer in individuals with low P450 2A6 activity, and smokers with low activity often decrease either the intensity of their smoking or the number of cigarettes they use compared with those with "normal" activity. The effect of P450 2A6 activity on smoking may influence one's tobacco-related disease risk. This review provides an overview of nicotine metabolism and a summary of the use of nicotine metabolite biomarkers to define smoking dose. Some more recent findings, for example, the identification of uridine 5'-diphosphoglucuronosyltransferase 2B10 as the catalyst of nicotine N-glucuronidation, are discussed. We also describe epidemiology studies that establish the contribution of nicotine metabolism and CYP2A6 genotype to lung cancer risk, particularly with respect to specific racial/ethnic groups, such as those with Japanese, African, or European ancestry. We conclude that a model of nicotine metabolism and smoking dose could be combined with other lung cancer risk variables to more accurately identify former smokers at the highest risk of lung cancer and to intervene accordingly.

Predicting nicotine metabolism across ancestries using genotypes.

BACKGROUND: There is a need to match characteristics of tobacco users with cessation treatments and risks of tobacco attributable diseases such as lung cancer. The rate in which the body metabolizes nicotine has proven an important predictor of these outcomes. Nicotine metabolism is primarily catalyzed by the enzyme cytochrone P450 (CYP2A6) and CYP2A6 activity can be measured as the ratio of two nicotine metabolites: trans-3'-hydroxycotinine to cotinine (NMR). Measurements of these metabolites are only possible in current tobacco users and vary by biofluid source, timing of collection, and protocols; unfortunately, this has limited their use in clinical practice. The NMR depends highly on genetic variation near CYP2A6 on chromosome 19 as well as ancestry, environmental, and other genetic factors. Thus, we aimed to develop prediction models of nicotine metabolism using genotypes and basic individual characteristics (age, gender, height, and weight). RESULTS: We identified four multiethnic studies with nicotine metabolites and DNA samples. We constructed a 263 marker panel from filtering genome-wide association scans of the NMR in each study. We then applied seven machine learning techniques to train models of nicotine metabolism on the largest and most ancestrally diverse dataset (N=2239). The models were then validated using the other three studies (total N=1415). Using cross-validation, we found the correlations between the observed and predicted NMR ranged from 0.69 to 0.97 depending on the model. When predictions were averaged in an ensemble model, the correlation was 0.81. The ensemble model generalizes well in the validation studies across ancestries, despite differences in the measurements of NMR between studies, with correlations of: 0.52 for African ancestry, 0.61 for Asian ancestry, and 0.46 for European ancestry. The most influential predictors of NMR identified in more than two models were rs56113850, rs11878604, and 21 other genetic variants near CYP2A6 as well as age and ancestry. CONCLUSIONS: We have developed an ensemble of seven models for predicting the NMR across ancestries from genotypes and age, gender and BMI. These models were validated using three datasets and associate with nicotine dosages. The knowledge of how an individual metabolizes nicotine could be used to help select the optimal path to reducing or quitting tobacco use, as well as, evaluating risks of tobacco use.

Identification of 5-Hydroxycotinine in the Plasma of Nicotine-Treated Mice: Implications for Cotinine Metabolism and Disposition in Vivo.

Two oxidation products of cotinine, 5-hydroxycotinine (5-HC) and cotinine N-oxide (CNO), were identified for the first time in vivo in the plasma of C57BL/6 mice after injection of nicotine (1 mg/kg) or exposure to an e-cigarette (e-cig) containing 2.4% nicotine. Liquid chromatography-mass spectrometry was used to separate 3-hydroxycotinine (3-HC), 5-HC, and CNO and to quantify each by the sensitive direct detection of their parent ion with m/z of 193.097. In nicotine-injected mice, 5-HC was as abundant as 3-HC 15 minutes postinjection, and CNO was readily detectable. In e-cig-exposed mice with plasma nicotine levels resembling that of human smokers, plasma 5-HC and CNO, as well as 3-HC, were readily quantifiable at the end of the 4-hour exposure time. In nicotine-injected mice, the combined concentration of 3-HC plus 5-HC plus CNO, all formed from cotinine by CYP2A5, was higher (P < 0.01) in females than in males, although the male-female difference in cotinine plasma level did not reach statistical significance. The result highlights the importance of considering these three oxidation products of cotinine in examining cotinine metabolism and disposition. Coumarin 7-hydroxylase activity, a specific marker of CYP2A5, measured in the hepatic microsomes of untreated mice showed that females have higher activity (P < 0.001) than males (N = 8 per sex). The abundance of plasma 5-hydroxycotinine in nicotine-treated mice raises intriguing questions about the site of its origin (hepatic or possibly kidney CYP2A5) and the routes of its disposition because urinary excretion of 5-HC has not been detected by liquid chromatography with tandem mass spectrometry in mice and is controversial in human smokers. SIGNIFICANCE STATEMENT: Nicotine is the active ingredient of tobacco, but its elimination route through its biomarker cotinine is not fully understood. By liquid chromatography-mass spectrometry, this study has identified and quantified for the first time 5-hydroxycotinine and cotinine N-oxide, which are oxidation products of cotinine, in the plasma of mice treated with nicotine or exposed to e-cigarettes. The results raise intriguing questions about nicotine disposition in vivo in this well established preclinical model of human smokers.

Hepcidin Is Essential for Alveolar Macrophage Function and Is Disrupted by Smoke in a Murine Chronic Obstructive Pulmonary Disease Model.

Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease associated with cigarette smoking. Alterations in local lung and systemic iron regulation are associated with disease progression and pathogenesis. Hepcidin, an iron regulatory peptide hormone, is altered in subjects with COPD; however, the molecular role of hepcidin in COPD pathogenesis remains to be determined. In this study, using a murine model of smoke-induced COPD, we demonstrate that lung and circulating hepcidin levels are inhibited by cigarette smoke. We show that cigarette smoke exposure increases erythropoietin and bone marrow-derived erythroferrone and leads to expanded but inefficient erythropoiesis in murine bone marrow and an increase in ferroportin on alveolar macrophages (AMs). AMs from smokers and subjects with COPD display increased expression of ferroportin as well as hepcidin. Notably, murine AMs exposed to smoke fail to increase hepcidin in response to Gram-negative or Gram-positive infection. Loss of hepcidin in vivo results in blunted functional responses of AMs and exaggerated responses to Streptococcus pneumoniae infection.

Effect of smoking cessation on cardiac troponin I concentrations.

Chronic elevation of cardiac troponin I (cTnI) is associated with heart failure and cardiovascular death. Paradoxically, observational studies have indicated that current smokers have lower cTnI concentrations than non-smokers. We examined determinants of cTnI in smokers and the effect of smoking cessation on cTnI. Overweight or obese smokers received motivational support and varenicline to aid cessation and dietary advice to limit weight gain. Quitters were defined according to the Russell standard (≤5 cigarettes after the quit date) and validated with expired breath CO <10 ppm. Of the total 122 participants, 108 completed assessments at 12 weeks and 78 were classified as quitters versus 30 who continued smoking. cTnI was measured with a high-sensitivity assay with a limit of detection of 1.2 ng/L (Abbott Diagnostics), and concentrations (log-transformed) were compared between quitters and continuing smokers. cTnI concentrations were significantly higher in men than women and correlated with age, but not with number of cigarettes/day. Quitters had median baseline and 12-week levels of 1.4 ng/L (interquartile range [IQR] 1.2-2.5) and 1.4 ng/L (IQR 1.2-2.4), respectively, while nonquitters had baseline and 12-week levels of 1.5 ng/L (IQR 1.2-2.9) and 1.8 ng/L (IQR 1.3-3.4), respectively. The change in cTnI concentrations from baseline to 12 weeks did not differ between quitters and continuous smokers (p = .7). The results suggest that smoking cessation does not affect levels of cTnI, a marker of chronic subclinical myocardial injury, in contrast to prior observational data suggesting that tobacco smoking is associated with lower cTn concentrations.

Increased serum SP-D in identification of high-risk smokers at high risk of COPD.

Pulmonary surfactant protein D (SP-D) is an important component of the pulmonary innate immune system with the ability to dampen cigarette smoke-induced lung inflammation. However, cigarette smoking mediates translocation of SP-D from the lung to the blood, and serum SP-D (sSP-D) has therefore previously been suggested as marker for smoke-induced lung injury. In support of this notion, associations between high sSP-D and low lung function measurements have previously been demonstrated in smokers and in chronic obstructive lung disease (COPD). The present investigations employ a 12-yr longitudinal Danish twin study to test the hypothesis that baseline sSP-D variation has the capacity to identify smokers with normal baseline lung function who are at high risk of significant future smoke-induced lung function decline. We find that sSP-D is significantly increased in those with normal lung function at baseline who develop lung function decline during follow-up compared with those who stay lung healthy. Moreover, we demonstrate that it is the smoke-induced baseline sSP-D level, and not the constitutional level, which has capacity as biomarker, and which is linearly increased with the decline in lung function during follow-up. In conclusion, we here present first observation of increased sSP-D for identification of high-risk smokers.

Differential expression of sputum and serum autoantibodies in patients with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a complex and progressive respiratory disease. Autoimmune processes have been hypothesized to contribute to disease progression; however, the presence of autoantibodies in the serum has been variable. Given that COPD is a lung disease, we sought to investigate whether autoantibodies in sputum supernatant would better define pulmonary autoimmune processes. Matched sputum and serum samples were obtained from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study and at the Guangzhou Institute of Respiratory Health (GIRH). Samples were collected from patients with varying severity of COPD, asymptomatic smokers, and healthy control subjects. IgG and IgM autoantibodies were detected in sputum and serum of all subjects in both cohorts using a broad-spectrum autoantigen array. No differences were observed in sputum autoantibodies between COPD and asymptomatic smokers in either cohort. In contrast, 16% of detectable sputum IgG autoantibodies were decreased in subjects with COPD compared to healthy controls in the ADEPT cohort. Compared to asymptomatic smokers, approximately 13% of detectable serum IgG and 40% of detectable serum IgM autoantibodies were differentially expressed in GIRH COPD subjects. Of the differentially expressed specificities, anti-nuclear autoantibodies were predominately decreased. A weak correlation between increased serum IgM anti-tissue autoantibodies and a measure of airspace enlargement was observed. The differential expression of specificities varied between the cohorts. In closing, using a comprehensive autoantibody array, we demonstrate that autoantibodies are present in subjects with COPD, asymptomatic smokers, and healthy controls. Cohorts displayed high levels of heterogeneity, precluding the utilization of autoantibodies for diagnostic purposes.

FAM13A as potential therapeutic target in modulating TGF-ß-induced airway tissue remodeling in COPD.

Genome-wide association studies have shown that a gene variant in the Family with sequence similarity 13, member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with chronic obstructive pulmonary disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor-ß1 (TGF-ß1). To determine the role of FAM13A in TGF-ß1 signaling, FAM13A-/- airway epithelial cells were generated using CRISPR-Cas9, whereas overexpression of FAM13A was achieved using lipid nanoparticles. Wild-type (WT) and FAM13A-/- cells were treated with TGF-ß1, followed by gene and/or protein expression analyses. FAM13A-/- cells augmented TGF-ß1-induced increase in collagen type 1 (COL1A1), matrix metalloproteinase 2 (MMP2), expression compared with WT cells. This effect was mediated by an increase in ß-catenin (CTNNB1) expression in FAM13A-/- cells compared with WT cells after TGF-ß1 treatment. FAM13A overexpression was partially protective from TGF-ß1-induced COL1A1 expression. Finally, we showed that airway epithelial-specific FAM13A protein expression is significantly increased in patients with severe COPD compared with control nonsmokers, and negatively correlated with lung function. In contrast, ß-catenin (CTNNB1), which has previously been linked to be regulated by FAM13A, is decreased in the airway epithelium of smokers with COPD compared with non-COPD subjects. Together, our data showed that FAM13A may be protective from TGF-ß1-induced fibrotic response in the airway epithelium via sequestering CTNNB1 from its regulation on downstream targets. Therapeutic increase in FAM13A expression in the airway epithelium of smokers at risk for COPD, and those with mild COPD, may reduce the extent of airway tissue remodeling.

The effect of body mass index on smoking behaviour and nicotine metabolism: a Mendelian randomization study.

Given clear evidence that smoking lowers weight, it is possible that individuals with higher body mass index (BMI) smoke in order to lose or maintain their weight. We performed Mendelian randomization (MR) analyses of the effects of BMI on smoking behaviour in UK Biobank and the Tobacco and Genetics Consortium genome-wide association study (GWAS), on cotinine levels and nicotine metabolite ratio (NMR) in published GWAS and on DNA methylation in the Avon Longitudinal Study of Parents and Children. Our results indicate that higher BMI causally influences lifetime smoking, smoking initiation, smoking heaviness and also DNA methylation at the aryl-hydrocarbon receptor repressor (AHRR) locus, but we do not see evidence for an effect on smoking cessation. While there is no strong evidence that BMI causally influences cotinine levels, suggestive evidence for a negative causal influence on NMR may explain this. There is a causal effect of BMI on smoking, but the relationship is likely to be complex due to opposing effects on behaviour and metabolism.

Impact of chronic use of heat-not-burn cigarettes on oxidative stress, endothelial dysfunction and platelet activation: the SUR-VAPES Chronic Study.

Tobacco habit still represents the leading preventable cause of morbidity and mortality worldwide. Heat-not-burn cigarettes (HNBCs) are considered as an alternative to traditional combustion cigarettes (TCCs) due to the lack of combustion and the absence of combustion-related specific toxicants. The aim of this observational study was to assess the effect of HNBC on endothelial function, oxidative stress and platelet activation in chronic adult TCC smokers and HNBC users. The results showed that both HNBC and TCC display an adverse phenotype in terms of endothelial function, oxidative stress and platelet activation. Future randomised studies are strongly warranted to confirm these data.

Chr15q25 genetic variant (rs16969968) independently confers risk of lung cancer, COPD and smoking intensity in a prospective study of high-risk smokers.

IMPORTANCE: While cholinergic receptor nicotinic alpha 5 (CHRNA5) variants have been linked to lung cancer, chronic obstructive pulmonary disease (COPD) and smoking addiction in case-controls studies, their corelationship is not well understood and requires retesting in a cohort study. OBJECTIVE: To re-examine the association between the CHRNA5 variant (rs16969968 AA genotype) and the development of lung cancer, relative to its association with COPD and smoking. METHODS: In 9270 Non-Hispanic white subjects from the National Lung Screening Trial, a substudy of high-risk smokers were followed for an average of 6.4 years. We compared CHRNA5 genotype according to baseline smoking exposure, lung function and COPD status. We also compared the lung cancer incidence rate, and used multiple logistic regression and mediation analysis to examine the role of the AA genotype of the CHRNA5 variant in smoking exposure, COPD and lung cancer. RESULTS: As previously reported, we found the AA high-risk genotype was associated with lower lung function (p=0.005), greater smoking intensity (p<0.001), the presence of COPD (OR 1.28 (95% CI 1.10 to 1.49) p=0.0015) and the development of lung cancer (HR 1.41, (95% CI 1.03 to 1.93) p=0.03). In a mediation analyses, the AA genotype was independently associated with smoking intensity (OR 1.42 (95% CI 1.25 to 1.60, p<0.0001), COPD (OR 1.25, (95% CI 1.66 to 2.53), p=0.0015) and developing lung cancer (OR 1.37, (95% CI 1.03 to 1.82) p=0.03). CONCLUSION: In this large-prospective study, we found the CHRNA5 rs 16 969 968 AA genotype to be independently associated with smoking exposure, COPD and lung cancer (triple whammy effect).

Variation in the expression of cytochrome P450-related miRNAs and transcriptional factors in human livers: Correlation with cytochrome P450 gene phenotypes.

Cytochrome P450 (CYP) gene expression exhibits large interindividual variation attributable to diverse regulatory factors including microRNAs (miRNAs) and hepatic transcription factors (TFs). We used real-time qPCR with 106 human liver samples to measure the expression and interindividual variation of seven miRNAs and four TFs that have been reported to regulate the expression of CYPs; we also identified factors that influence their expression. The results show that expression of the seven miRNAs and the four TFs exhibits a non-normal distribution and the expression variability is high (89- to 618-fold for miRNA and 12- to 85-fold for TFs). Age contributed to the interindividual variation for miR-148a, miR-27b and miR-34a, whereas cigarette smoking and alcohol consumption significantly reduced HNF4α mRNA levels. Association analysis showed significant correlations among the seven miRNAs as well as the four TFs. Furthermore, we systematically evaluated the impact of the seven miRNAs and four TFs on protein content, mRNA levels, translation efficiency and activity of 10 CYPs. The results show that numerous associations (positive and negative) are present between the seven miRNAs or the four TFs and the 10 CYP phenotypes (as indicated by mRNA, protein and activity); specifically, miR-27b, miR-34a and all four TFs played key roles in the interindividual variation of CYPs. Our results extend previous findings and suggest that miR-27b and miR-34a may be potential direct or indirect master regulators of CYP expression and thereby contribute to the interindividual variations in CYP-mediated drug metabolism.

Association of lower vitamin D levels with inflammation and leucocytes parameters in patients with and without diabetes mellitus undergoing coronary angiography.

BACKGROUND: Diabetes mellitus has been associated with a chronic low-grade inflammation and a higher risk of cardiovascular and infectious disease, that could be prevented by the effects of vitamin D. We aimed at evaluating the impact of vitamin D levels on the biomarkers of acute-phase response, inflammation and glucose metabolism in a large cohort of diabetic patients with cardiovascular disease. MATERIALS AND METHODS: Consecutive patients undergoing coronary angiography were included. Diabetes mellitus was defined as previous diagnosis, specific treatment administration (oral drug or insulin), fasting glycaemia >6.99 mmol/L or HbA1c >48 mmol/L. Glucose parameters, white blood cells, Neutrophil-to-Lymphocyte Ratio (NLR), Monocyte-to-Lymphocyte Ratio (MLR), C-reactive protein (CRP) and vitamin D were measured at admission. Vitamin D levels were measured by chemiluminescence immunoassay kit LIAISON® Vitamin D assay (Diasorin Inc). RESULTS: We included 1472 diabetic patients and 2499 non-diabetic patients that were divided according to vitamin D tertiles. Among diabetic patients, lower levels of vitamin D were associated with female gender (P = .02), obesity (P = .004), active smoking and acute presentation (P < .001) and with a more atherogenic metabolic profile. The levels of white blood cells, leucocytes subfamilies, and inflammatory parameters significantly correlated with vitamin D levels in both patients with and without diabetes (diabetic: P = .012 for WBC, P = .004 for NLR and P < .001 for MLR and C-reactive protein, non-diabetic: P < .001 for WBC; NLR, MLR and C-reactive protein, respectively). Among diabetic patients, results were confirmed at multivariate analysis with no significant interaction according to glycaemic control. CONCLUSION: The present study demonstrates that, among patients with cardiovascular disease, vitamin D deficiency is associated with metabolic dysregulation and with an elevation of cellular and humoural inflammatory parameters, especially among diabetics, although not being dependent from glycaemic control.

Tunica-Specific Transcriptome of Abdominal Aortic Aneurysm and the Effect of Intraluminal Thrombus, Smoking, and Diameter Growth Rate.

OBJECTIVE: There is no medical treatment to prevent abdominal aortic aneurysm (AAA) growth and rupture, both of which are linked to smoking. Our objective was to map the tunica-specific pathophysiology of AAA with consideration of the intraluminal thrombus, age, and sex, and to subsequently identify which mechanisms were linked to smoking and diameter growth rate. Approach and Results: Microarray analyses were performed on 246 samples from 76 AAA patients and 13 controls. In media and adventitia, there were 5889 and 2701 differentially expressed genes, respectively. Gene sets related to adaptive and innate immunity were upregulated in both tunicas. Media-specific gene sets included increased matrix disassembly and angiogenesis, as well as decreased muscle cell development, contraction, and differentiation. Genes implicated in previous genome-wide association studies were dysregulated in media. The intraluminal thrombus had a pro-proteolytic and proinflammatory effect on the underlying media. Active smoking resulted in increased inflammation, oxidative stress, and angiogenesis in all tissues and enriched lipid metabolism in adventitia. Processes enriched with active smoking in control aortas overlapped to a high extent with those differentially expressed between AAAs and controls. The AAA diameter growth rate (n=24) correlated with T- and B-cell expression in media, as well as lipid-related processes in the adventitia. CONCLUSIONS: This tunica-specific analysis of gene expression in a large study enabled the detection of features not previously described in AAA disease. Smoking was associated with increased expression of aneurysm-related processes, of which adaptive immunity and lipid metabolism correlated with growth rate.

Body flexibility and incident hypertension: The Niigata wellness study.

A high level of physical fitness, especially cardiorespiratory fitness, is associated with lower incidence of hypertension. However, the relationship between flexibility, which is a component of physical fitness, and the incidence of hypertension is unknown. The purpose of this study was to investigate the relationship between flexibility and the incidence of hypertension in a cohort study. A total of 22,972 (14,805 men and 8167 women; median age 49 years) normotensive participants were included in this study. Between April 2001 and March 2002, flexibility (standing forward bending) was measured using a standing trunk flexion meter. The participants were divided into quartiles of flexibility by sex and age group. Hypertension was defined as systolic blood pressure ≥ 140 mm Hg, diastolic blood pressure ≥ 90 mm Hg, or a self-reported history of previously diagnosed hypertension or current medication for hypertension at a health examination between April 2002 and March 2008. Hazard ratios and 95% confidence intervals (95% CI) for the incidence of hypertension were estimated using Cox proportional hazards models after adjusting for age, sex, body mass index, exercise habits, smoking status, and drinking status. During 102,948 person years of follow-up (median 5.6 years), 4235 participants developed hypertension. Compared with the lowest flexibility (quartile 1), hazard ratios and 95% CI were 0.96 (0.88 - 1.04) for quartile 2, 0.94 (0.86 - 1.03) for quartile 3, and 0.83 (0.76 - 0.91) for quartile 4. A high level of flexibility was associated with lower incidence of hypertension, independent of other confounding factors.

Expression of the SARS-CoV-2 ACE2 Receptor in the Human Airway Epithelium.

Rationale: Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19), a predominantly respiratory illness. The first step in SARS-CoV-2 infection is binding of the virus to ACE2 (angiotensin-converting enzyme 2) on the airway epithelium.Objectives: The objective was to gain insight into the expression of ACE2 in the human airway epithelium.Methods: Airway epithelia sampled by fiberoptic bronchoscopy of trachea, large airway epithelia (LAE), and small airway epithelia (SAE) of nonsmokers and smokers were analyzed for expression of ACE2 and other coronavirus infection-related genes using microarray, RNA sequencing, and 10x single-cell transcriptome analysis, with associated examination of ACE2-related microRNA.Measurements and Main Results:1) ACE2 is expressed similarly in the trachea and LAE, with lower expression in the SAE; 2) in the SAE, ACE2 is expressed in basal, intermediate, club, mucus, and ciliated cells; 3) ACE2 is upregulated in the SAE by smoking, significantly in men; 4) levels of miR-1246 expression could play a role in ACE2 upregulation in the SAE of smokers; and 5) ACE2 is expressed in airway epithelium differentiated in vitro on air-liquid interface cultures from primary airway basal stem/progenitor cells; this can be replicated using LAE and SAE immortalized basal cell lines derived from healthy nonsmokers.Conclusions:ACE2, the gene encoding the receptor for SARS-CoV-2, is expressed in the human airway epithelium, with variations in expression relevant to the biology of initial steps in SARS-CoV-2 infection.