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Abstract Plasminogen activator inhibitor-1 (PAI-1) is a vital regulator of the fibrinolytic mechanism and has been intricately involved in various physiological and clinical processes, including cancer, thrombosis, and wound healing. The PAI-1 signaling pathway is multifaceted, encompassing numerous signaling molecules and nodes. Recent studies have revealed a novel contribution of PAI-1 during cellular senescence. This review introduces a pathway resource detailing the signaling network events mediated by PAI-1. The literature curated on the PAI-1 system was manually compiled from various published studies, our analysis presents a signaling pathway network of PAI-1, which includes various events like enzyme catalysis, molecular association, gene regulation, protein expression, and protein translocation. This signaling network aims to provide a detailed analysis of the existing understanding of the PAI-1 signaling pathway in the context of cellular senescence across various research models. By developing this pathway, we aspire to deepen our understanding of aging and senescence research, ultimately contributing to the pursuit of effective therapeutic approaches for these complex chronic diseases.

Abstract The low resolution of spatial transcriptomics data necessitates additional information for optimal use. We developed scDOT, which combines spatial transcriptomics and single cell RNA sequencing to improve the ability to reconstruct single cell resolved spatial maps and identify senescent cells. scDOT integrates optimal transport and expression deconvolution to learn non-linear couplings between cells and spots and to infer cell placements. Application of scDOT to lung spatial transcriptomics data improves on prior methods and allows the identification of the spatial organization of senescent cells, their neighboring cells and novel genes involved in cell-cell interactions that may be driving senescence.

Abstract Transforming Growth Factor Beta1 (TGF-β1) signaling is upregulated in Chronic Obstructive Pulmonary disease (COPD), smokers, and people living with HIV. Cigarette smoking and HIV are also independent risk factors for COPD. Chronic inflammation is a hallmark of COPD. However, the underlying mechanisms remain unknown. Previous research has suggested that TGF-β1 alters the airway epithelial microRNAome and transcriptome, potentially contributing to lung inflammation. The Lactoperoxidase (LPO) system is an integral component of innate immunity within the airway. LPO plays a crucial role in host defense by catalyzing the oxidation of thiocyanate to hypothiocyanite in the presence of hydrogen peroxide (H2O2), generating a potent antibacterial and antiviral agent. Additionally, the LPO system potentially aids in maintaining cellular redox balance by reducing the levels of H2O2, thus mitigating oxidative stress within the airway epithelium. LPO dysfunction can impair immune responses and exacerbate inflammatory processes in respiratory diseases. In this study, primary bronchial epithelial cells and bronchial cell lines were treated with TGF-β1 and exposed to cigarette smoke to characterize the effect of these factors on LPO and their downstream effects. RT-qPCR and Western Blot were applied to quantify mRNA and proteins’ expression. The levels of H2O2 were detected using the Amplex Red Assay. Magnetofection and transfection were applied to probe the effect of miR-449b-5p. Staining procedures using the MitoTracker Green and C12FDG dyes were used to establish mitochondria mass and senescence. The levels of pro-inflammatory cytokines were measured via Luminex assays. We found that TGF-β1 and cigarette smoke suppressed airway LPO expression, increasing H2O2 levels. This increase in H2O2 had downstream effects on mitochondrial homeostasis, epithelial cellular senescence, and the pro-inflammatory cytokine response. We demonstrate for the first time that airway LPO is regulated by TGF-β1-induced miRNA-mediated post-transcriptional silencing through miR-449b-5p in the lungs. Further, we identify and validate miR-449-5p as the candidate miRNA upregulated by TGF-β1, which is involved in LPO suppression. This paper demonstrates a new mechanism by which TGF-β1 can lead to altered redox status in the airway.

Emerging tobacco products such as electronic nicotine delivery systems (ENDS) and heated tobacco products (HTPs) have a dynamic landscape and are becoming widely popular as they claim to offer a low-risk alternative to conventional smoking. Most pre-clinical laboratories currently exploit in vitro , ex vivo , and in vivo experimental models to assess toxicological outcomes as well as to develop risk-estimation models. While most laboratories have produced a wide range of cell culture and mouse model data utilizing current smoke/aerosol generators and standardized puffing profiles, much variation still exists between research studies, hindering the generation of usable data appropriate for the standardization of these tobacco products. In this review, we discuss current state-of-the-art in vitro and in vivo models and their challenges, as well as insights into risk estimation of novel products and recommendations for toxicological parameters for reporting, allowing comparability of the research studies between laboratories, resulting in usable data for regulation of these products before approval by regulatory authorities.

In this narrative review, we highlight the challenges of comparing emissions from different tobacco products under controlled laboratory settings (using smoking/ vaping machines). We focus on tobacco products that generate inhalable smoke or aerosol, such as cigarettes, cigars, hookah, electronic cigarettes, and heated tobacco products. We discuss challenges associated with sample generation including variability of smoking/vaping machines, lack of standardized adaptors that connect smoking/vaping machines to different tobacco products, puffing protocols that are not representative of actual use, and sample generation session length (minutes or number of puffs) that depends on product characteristics. We also discuss the challenges of physically characterizing and trapping emissions from products with different aerosol characteristics. Challenges to analytical method development are also covered, highlighting matrix effects, order of magnitude differences in analyte levels, and the necessity of tailored quality control/quality assurance measures. The review highlights two approaches in selecting emissions to monitor across products, one focusing on toxicants that were detected and quantified with optimized methods for combustible cigarettes, and the other looking for productspecific toxicants using non-targeted analysis. The challenges of data reporting and statistical analysis that allow meaningful comparison across products are also discussed. We end the review by highlighting that even if the technical challenges are overcome, emission comparison may obscure the absolute exposure from novel products if we only focus on relative exposure compared to combustible products.

Abstract Environmental tobacco smoke (ETS) is known to cause lung inflammatory and injurious responses. Smoke exposure is associated with the pathobiology related to lung fibrosis, whereas the mechanism that ETS exposure augments pulmonary fibrogenesis is unclear. We hypothesized that ETS exposure could exacerbate fibrotic responses via collagen dynamic dysregulation and complement activation. C57BL/6J and p16-3MR mice were exposed to ETS followed by bleomycin administration. ETS exposure exacerbated bleomycin-induced collagen and lysyl oxidase overexpression in the fibrotic lesion. ETS exposure also led to augmented bleomycin-induced upregulation of C3 and C3AR, which are pro-fibrotic markers. Moreover, overexpressed collagens and C3 levels were highly significant in males than females. The old mice (17 months old) were exposed to ETS and treated with bleomycin to induce fibrogenesis which is considered as an aging-associated disease. Fewer gene and protein dysregulations trends were identified between ETS exposure with the bleomycin group and the bleomycin alone group in old mice. Based on our findings, we suggested that ETS exposure increases the risk of developing severe lung fibrotic responses via collagen overexpression and lysyl oxidase-mediated collagen stabilization in the fibrotic lesion, and potentially affected the complement system activation induced by bleomycin. Further, male mice were more susceptible than females during fibrogenesis exacerbation. Thus ETS and bleomycin induced lung fibrotic changes via collagen-lysyl oxidase in an age-dependent mechanism.

Abstract Inhaling xenobiotics, such as tobacco smoke is a major risk factor for pulmonary diseases, e.g., COPD/emphysema, interstitial lung disease, and pre‐invasive diseases. Shelterin complex or telosome provides telomeric end protection during replication. Telomere protection protein 1 (TPP1) is one of the main six subunits of the shelterin complex supporting the telomere stability and genomic integrity. Dysfunctional telomeres and shelterin complex are associated as a disease mechanism of tobacco smoke‐induced pulmonary damage and disease processes. The airway epithelium is critical to maintaining respiratory homeostasis and is implicated in lung diseases. Club cells (also known as clara cells) play an essential role in the immune response, surfactant production, and metabolism. Disrupted shelterin complex may lead to dysregulated cellular function, DNA damage, and disease progression. However, it is unknown if the conditional removal of TPP1 from Club cells can induce lung disease pathogenesis caused by tobacco smoke exposure. In this study, conditional knockout of Club‐cell specific TPP1 demonstrated the instability of other shelterin protein subunits, such as TRF1, dysregulation of cell cycle checkpoint proteins, p53 and downstream targets, and dysregulation of telomeric genes. This was associated with age‐dependent senescence‐associated genes, increased DNA damage, and upregulated RANTES/IL13/IL33 mediated lung inflammation and injury network by cigarette smoke (CS). These phenomena are also associated with alterations in cytochrome P450 and glutathione transferases, upregulated molecular pathways promoting lung lesions, bronchial neoplasms, and adenocarcinomas. These findings suggest a pivotal role of TPP1 in maintaining lung homeostasis and injurious responses in response to CS. Thus, these data TPP1 may have therapeutic value in alleviating telomere‐related chronic lung diseases.

Abstract Background Electronic nicotine delivery systems (ENDS) or electronic cigarettes (e-cigarettes) aerosolize an e-liquid composed of propylene glycol (PG) and vegetable glycerin (VG) as humectants, flavoring chemicals, and nicotine. Nicotine naturally occurs in two isomers R- and S-nicotine, with tobacco-derived nicotine (TDN) composed of S-nicotine, and tobacco-free/synthetic nicotine (TFN) composed of a racemic mixture of R- and S-nicotine. Currently, there is limited knowledge of the potential differences in the toxicity of TFN versus TDN. We hypothesized that exposure of TFN and TDN salts to C57BL/6J mice would result in a differential response in lung inflammation and protease/ antiprotease imbalance. Methods Five-week-old male and female C57BL/6J mice were exposed to air, PG/VG, PG/VG with TFN salts (TFN), or PG/VG with TDN salts (TDN) by nose-only exposure. Lung inflammatory cell counts, cytokine/chemokine levels, and matrix metalloproteinase (MMP) protein abundance and activity levels were determined by flow cytometry, ELISA, immunoblotting, and gel zymography, respectively. Results Exposure to the humectants (PG/VG) alone increased cytokine levels- IL-6, KC, and MCP-1 in the BALF and KC levels in lung homogenate of exposed mice. While no change was observed in the cytokine levels in lung homogenate of TDN aerosol exposed mice, exposure to TFN aerosols resulted in an increase in KC levels in the lungs of these mice compared to air controls. Interestingly, exposure to TDN aerosols increased MMP-9 protein abundance in the lungs of female mice, while exposure to TFN aerosol showed no change. The metabolism of nicotine or the clearance of cotinine for TFN exposure may differ from that for TDN. Conclusion Exposure to humectants, PG/VG alone, induces an inflammatory response in C57BL/6J mice. TFN and TDN salts show distinct changes in inflammatory responses and lung proteases on acute exposures. These data suggest variable toxicological profiles of the two forms of nicotine in vivo. Future work is thus warranted to delineate the harmful effects of synthetic/natural nicotine with humectants to determine the potential toxicological risks for users.

Abstract Background Electronic cigarette (e-cig) vaping has increased in the past decade in the US, and e-cig use is misleadingly marketed as a safe cessation for quitting smoking. The main constituents in e-liquid are humectants, such as propylene glycol (PG) and vegetable glycerine (VG), but different flavoring chemicals are also used. However, the toxicology profile of flavored e-cigs in the pulmonary tract is lacking. We hypothesized that menthol and tobacco-flavored e-cig (nicotine-free) exposure results in inflammatory responses and dysregulated repair in lung fibroblast and epithelium. Method We exposed lung fibroblast (HFL-1) and epithelium (BEAS-2B) to Air, PG/VG, menthol flavored, or tobacco-flavored e-cig, and determined the cytotoxicity, inflammation, and wound healing ability in 2D cells and 3D microtissue chip models. Results After exposure, HFL-1 showed decreased cell number with increased IL-8 levels in the tobacco flavor group compared to air. BEAS-2B also showed increased IL-8 secretion after PG/VG and tobacco flavor exposure, while menthol flavor exposure showed no change. Both menthol and tobacco-flavored e-cig exposure showed decreased protein abundance of type 1 collagen α 1 (COL1A1), α-smooth-muscle actin (αSMA), and fibronectin as well as decreased gene expression level of αSMA (Acta2) in HFL-1. After tobacco flavor e-cig exposure, HFL-1 mediated wound healing and tissue contractility were inhibited. Furthermore, BEAS-2B exposed to menthol flavor showed significantly decreased tight junction gene expressions, such as CDH1, OCLN, and TJP1. Conclusion Overall, tobacco-flavored e-cig exposure induces inflammation in both epithelium and fibroblasts, and tobacco-flavored e-cig inhibits wound healing ability in fibroblasts.

BackgroundRecently, the US Food and Drug Administration implemented enforcement priorities against all flavored, cartridge-based e-cigarettes other than menthol and tobacco flavors. This ban undermined the products’ appeal to vapers, so e-cigarette manufacturers added flavorants of other attractive flavors into tobacco-flavored e-cigarettes and reestablished appeal. ObjectiveThis review aims to analyze the impact of the addition of other flavorants in tobacco-flavored e-cigarettes on both human and public health issues and to propose further research as well as potential interventions. MethodsSearches for relevant literature published between 2018 and 2023 were performed. Cited articles about the toxicity of e-cigarette chemicals included those published before 2018, and governmental websites and documents were also included for crucial information. ResultsBoth the sales of e-cigarettes and posts on social media suggested that the manufacturers’ strategy was successful. The reestablished appeal causes not only a public health issue but also threats to the health of individual vapers. Research has shown an increase in toxicity associated with the flavorants commonly used in flavored e-cigarettes, which are likely added to tobacco-flavored e-cigarettes based on tobacco-derived and synthetic tobacco-free nicotine, and these other flavors are associated with higher clinical symptoms not often induced solely by natural, traditional tobacco flavors. ConclusionsThe additional health risks posed by the flavorants are pronounced even without considering the toxicological interactions of the different tobacco flavorants, and more research should be done to understand the health risks thoroughly and to take proper actions accordingly for the regulation of these emerging products.

This viewpoint aims to provide a comprehensive understanding of vaping from various perspectives that contribute to the invention, development, spread, and consequences of e-cigarette products and vaping. Our analysis showed that the specific characteristics of e-cigarette products as well as marketing strategies, especially social media marketing, fostered the spread of vaping and the subsequent effects on human health and toxicity. We analyzed the components of e-cigarette devices and e-liquids, including the latest variants whose impacts were often overlooked. The different forms of nicotine, including salts and freebase nicotine, tobacco-derived nicotine, tobacco-free nicotine, and cooling agents (WS3 and WS23), have brought more choices for vapers along with more ways for e-cigarette manufacturers to advertise false understandings and present a greater threat to vapers’ health. Our work emphasized the products of brands that have gained significant influence recently, which are contributing to severe public health issues. On the other hand, we also discussed in detail the toxicity of e-liquid components and proposed a toxicity mechanism. We also noticed that nicotine and other chemicals in e-liquids promote each other’s negative effects through the oxidative stress and inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, a mechanism leading to pulmonary symptoms and addiction. The impact of government regulations on the products themselves, including flavor bans or regulations, has been limited. Therefore, we proposed further interventions or harm reduction strategies from a public health perspective.

The molecular clock REV-ERBα regulates lung injury during fibrosis, but the role of REV-ERBα in fibrogenesis remains unknown. Here, the authors show that REV-ERBα interacts with the lysyl oxidase-collagen axis during fibrogenesis and demonstrate the therapeutic potential of Rev-erbα agonist against lung fibrosis.

Macrophages treated with the flavoring chemicals found in flavored electronic cigarettes have been shown to induce an inflammatory response, however, limited data are available on the effect of aerosol exposure to these chemicals. We hypothesized that aerosol exposure to flavoring chemicals found in commercially available flavored e-liquids would result in an increase in pro-inflammatory cytokines in macrophages. Raw264.7 macrophage cell lines were exposed to a low and high dose of propylene glycol/vegetable glycerin (PG/VG) with almond flavoring benzaldehyde (280 μg/ml and 2.1 mg/ml), PG/VG with spicy/clove flavoring eugenol (3.5 mg/ml and 12 mg/ml), or PG/VG with apple flavoring hexyl acetate (500 μg/ml and 2.5 mg/ml). Exposure to PG/VG with 2.1 mg/ml benzaldehyde resulted in a significant increase in KC levels compared to air and PG/VG exposed cells. Exposure to PG/VG with both doses of hexyl acetate resulted in a significant increase in KC and IL-6 levels compared to air exposed cells. Exposure to PG/VG with both doses of eugenol resulted in a significant increase in KC and IL-6 levels compared to air and PG/VG exposed cells. These data indicate the ability of aerosol exposure to e-cigarette flavoring chemicals to significantly increase pro-inflammatory cytokine release in macrophages.

BackgroundSocializing is one of the main motivations for water pipe smoking. Restrictions on social gatherings during the COVID-19 pandemic might have influenced water pipe smokers’ behaviors. As one of the most popular social media platforms, Reddit has been used to study public opinions and user experiences. ObjectiveIn this study, we aimed to examine the influence of the COVID-19 pandemic on public perception and discussion of water pipe tobacco smoking using Reddit data. MethodsWe collected Reddit posts between December 1, 2018, and June 30, 2021, from a Reddit archive (PushShift) using keywords such as “waterpipe,” “hookah,” and “shisha.” We examined the temporal trend in Reddit posts mentioning water pipes and different locations (such as homes and lounges or bars). The temporal trend was further tested using interrupted time series analysis. Sentiment analysis was performed to study the change in sentiment of water pipe–related posts before and during the pandemic. Topic modeling using latent Dirichlet allocation (LDA) was used to examine major topics discussed in water pipe–related posts before and during the pandemic. ResultsA total of 45,765 nonpromotion water pipe–related Reddit posts were collected and used for data analysis. We found that the weekly number of Reddit posts mentioning water pipes significantly increased at the beginning of the COVID-19 pandemic (P

In the original publication [...]

Electronic cigarette (e-cig) aerosol exposures are strongly associated with pulmonary dysfunctions, and the airway epithelial cells (AECs) of respiratory passages play a pivotal role in understanding this association. However, not much is known about the effect of synthetic cooling agents such as WS-23 on AECs. WS-23 is a synthetic menthol-like cooling agent widely used to enhance the appeal of e-cigs and to suppress the harshness and bitterness of other e-cig constituents. Using primary human AECs, we compared the effects of aerosolized WS-23 with propylene glycol/vegetable glycerin (PG/VG) vehicle control and nicotine aerosol exposures. AECs treated with 3 % WS-23 aerosols showed a significant increase in viable cell numbers compared to PG/VG-vehicle aerosol exposed cells and cell growth was comparable following 2.5 % nicotine aerosol exposure. AEC inflammatory factors, IL-6 and ICAM-1 levels were significantly suppressed by WS-23 aerosols compared to PG/VG-controls. When differentiated AECs were challenged with WS-23 aerosols, there was a significant increase in secretory mucin MUC5AC expression with no discernible change in airway inflammatory SCGB1A1 expression. Compared to PG/VG-controls, WS-23 or nicotine aerosols presented with increased MUC5AC expression, but there was no synergistic effect of WS-23 + nicotine combination exposure. Thus, WS-23 and nicotine aerosols modulate the AEC responses and induce goblet cell hyperplasia, which could impact the airway physiology and susceptibility to respiratory diseases.

There has been a substantial rise in e-cigarette (e-cig) use or vaping in the past decade, prompting growing concerns about their adverse health effects. Recently, e-cig manufacturers have been using synthetic cooling agents, like WS-23 and WS-3, to provide a cooling sensation without the “menthol taste”. Studies have shown that aerosols/vapes generated by e-cigs can contain significant levels of reactive oxygen species (ROS). However, studies investigating the role of synthetic coolants in modulating ROS levels generated by e-cigs are lacking. This study seeks to understand how synthetic coolants, e-cig additives that have become increasingly prevalent in e-liquids sold in the United States (US), impact acellular ROS production from e-liquid aerosols as well as cellular ROS levels from pulmonary epithelial cells exposed to these e-liquids. To further explain, our study aims to understand whether the addition of WS-3 and WS-23 to e-liquid base and e-liquid base with nicotine significantly modifies generated acellular ROS levels within aerosolized e-liquids, as well as cellular ROS within BEAS-2B cells treated with these same e-liquids. Aerosols were generated from e-liquids with and without synthetic coolants through a single-puff aerosol generator; subsequently, acellular ROS was semi-quantified in H2O2 equivalents via fluorescence spectroscopy. Our acellular ROS data suggest that adding WS-3 to e-liquid base (PG:VG), regardless of nicotine content, has a minimal impact on modifying e-cig generated acellular ROS levels. Additionally, we also measured cellular ROS in lung epithelial cells using both e-liquids containing and not containing synthetic coolants via the CellROX Green fluorescent sensor. Similar comparable results were found in BEAS2B cells though ROS was increased by WS-3 and WS-23 treated in e-cig nicotine groups. Altogether, our data suggest that neither the addition of WS-23 nor WS-3 to e-liquid base solution, with and without nicotine, significantly modifies e-cig generated acellular ROS levels within aerosolized e-liquids and cellular ROS levels within treated BEAS-2B cells. Together, our data provide insight into whether synthetic coolants added to e-liquids could impact vaping-induced oxidative stress in the lungs.

Oral nicotine pouches are emerging as a new “modern oral” nicotine product. These prefilled pouches contain nicotine, flavorings, and filling agents that dissolve in the mouth. Nicotine can be derived from tobacco leaf or chemical synthesis. Traces of TSNAs and toxic chromium were detected in the pouch products. This raises the concern about general and periodontal health. This review aims to update the current oral nicotine products research relating to periodontal disease and its relevance in periodontal inflammation. Nicotine interacts with host cells and affects inflammatory responses to microbial challenges. It may directly or indirectly deteriorate periodontal tissues by activating nicotinic acetylcholine receptors, repressing PDL fibroblasts cells, increasing cellular ROS and cytokines/chemokines, growth factors, breaking microbiota balance, and dysregulating miRNAs expression. Studies show that appealing flavorings contained in nicotine pouches pose harm to periodontal innate immune responses and increase penetration of nitrosamines. In addition, flavored ONPs increase the risk of dual or poly-tobacco products among young adults, stacking up detrimental effects on the periodontium. Given the recent growth of users, further studies are needed to elucidate the impact of ONPs, even poly-tobacco use, on systemic and periodontal health. Moreover, policymakers should ensure to avoid generating a new wave of nicotine addiction among youths in the U.S.

Abstract Background Electronic-cigarette (e-cig) usage, particularly in the youth population, is a growing concern. It is known that e-cig causes endothelial dysfunction, which is a risk factor for the development of cardiovascular diseases; however, the mechanisms involved remain unclear. We hypothesized that long noncoding RNAs (lncRNAs) may play a role in e-cig-induced endothelial dysfunction. Methods Here, we identified lncRNAs that are dysregulated in human induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) following 24 h of e-cig aerosol extract treatment via microarray analysis. We performed Gene Ontology and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analyses of the dysregulated mRNAs following e-cig exposure and constructed co-expression networks of the top 5 upregulated lncRNAs and the top 5 downregulated lncRNAs and the mRNAs that are correlated with them. Furthermore, the functional effects of knocking down lncRNA lung cancer-associated transcript 1 (LUCAT1) on EC phenotypes were determined as it was one of the significantly upregulated lncRNAs following e-cig exposure based on our profiling. Results 183 lncRNAs and 132 mRNAs were found to be upregulated, whereas 297 lncRNAs and 413 mRNAs were found to be downregulated after e-cig exposure. We also observed that e-cig caused dysregulation of endothelial metabolism resulting in increased FAO activity, higher mitochondrial membrane potential, and decreased glucose uptake and glycolysis. These results suggest that e-cig alters EC metabolism by increasing FAO to compensate for energy deficiency in ECs. Finally, the knockdown of LUCAT1 prevented e-cig-induced EC dysfunction by maintaining vascular barrier, reducing reactive oxygen species level, and increasing migration capacity. Conclusion This study identifies an expression profile of differentially expressed lncRNAs and several potential regulators and pathways in ECs exposed to e-cig, which provide insights into the regulation of lncRNAs and mRNAs and the role of lncRNA and mRNA networks in ECs associated e-cig exposure.

Mitochondrial quality control is sustained by Miro1 (Rhot1), a calcium-binding membrane-anchored GTPase during mitophagy. The exact mechanism that operates the interaction of Miro1 with mitophagy machinery and their role in cigarette smoke (CS)-induced mitochondrial dysfunction that often results in lung inflammation is unclear. We hypothesized that Miro1 plays an important role in regulating mitophagy machinery and the resulting lung inflammation by CS exposure to mice. The lung epithelial Rhot1fl/fl (WT) and Rhot1CreCC10 mice were exposed to mainstream CS for 3 days (acute) and 4 months (chronic). Acute CS exposure showed a notable increase in the total inflammatory cells, macrophages, and neutrophils that are associated with inflammatory mediators. Chronic exposure showed increased infiltration of neutrophils versus air controls. The effects of acute and chronic CS exposure were augmented in the Rhot1CreCC10 group, indicating that epithelial Miro1 ablation led to the augmentation of inflammatory cell infiltration with alteration in the inflammatory mediators. Thus, Rhot1/Miro1 plays an important role in regulating CS-induced lung inflammatory responses with implications in mitochondrial quality control.

BackgroundOral nicotine pouches are a new form of tobacco-free nicotine products launched in recent years with a variety of flavors. ObjectiveThis study aims to examine the public perceptions and discussions of oral nicotine pouches on Reddit, a popular social media platform for sharing user experiences. MethodsBetween February 15, 2019, and February 12, 2021, a total of 2410 Reddit posts related to oral nicotine pouches were obtained over a 2-year period. After the removal of unrelated or commercial posts, 653 Reddit posts related to oral nicotine pouches remained. Topics and sentiments related to oral nicotine pouches on Reddit were hand coded. ResultsThe number of Reddit posts related to oral nicotine pouches increased during the study period. Content analysis showed that the most popular topic was “sharing product information and user experience” (366/653, 56%), in which sharing oral nicotine pouch products and user experiences were dominant. The next popular topic was “asking product-related questions” (product properties and product recommendations; 115/653, 17.6%), followed by “quitting nicotine products” such as vaping or smoking through use of oral nicotine pouches or quitting the oral nicotine pouches themselves (83/653, 12.7%) and “discussing oral nicotine pouch–related health” symptoms or concerns related to oral nicotine pouches (74/653, 11.3%). The least popular topic was “legality and permissions” related to oral nicotine pouches (15/653, 2.3%). In addition, a greater number of Reddit posts described positive attitudes compared to negative attitudes toward oral nicotine pouches (354/653, 54.2% vs 101/653, 15.5%; P

Research ImpactCigarette smoke (CS) exposure is strongly associated with chronic obstructive pulmonary disease (COPD). In respiratory airways, CS exposure disrupts airway barrier functions, mucous/phlegm production, and basic immune responses of airway epithelial cells. Based on our recent identification of a specific immunomodulatory long noncoding RNA (lncRNA), we investigated its role in CS-induced responses in bronchial airways of cynomolgus macaque model of CS-induced COPD and in former smokers with and without COPD. The lncRNA was significantly upregulated in CS-induced macaque airways and in COPD airways that exhibited higher mucus expression and goblet cell hyperplasia. Experimental models of cells derived from COPD subjects recapitulated the augmented inflammation and mucus expression following the smoke challenge. Blocking of lncRNA expression in cell culture setting suppressed the smoke-induced and COPD-associated dysregulated mucoinflammatory response suggesting that this airway specific immunomodulatory lncRNA may represent a novel target to mitigate the smoke-mediated inflammation and mucus hyperexpression.RationaleIn conducting airways, CS disrupts airway epithelial functions, mucociliary clearances, and innate immune responses that are primarily orchestrated by human bronchial epithelial cells (HBECs). Mucus hypersecretion and dysregulated immune response are the hallmarks of chronic bronchitis (CB) that is often exacerbated by CS. Notably, we recently identified a long noncoding RNA (lncRNA) antisense to ICAM-1 (LASI) that mediates airway epithelial responses.ObjectiveTo investigate the role of LASI lncRNA in CS-induced airway inflammation and mucin hyperexpression in an animal model of COPD, and in HBECs and lung tissues from former smokers with and without COPD. To interrogate LASI lncRNA role in CS-mediated airway mucoinflammatory responses by targeted gene editing.MethodsSmall airway tissue sections from cynomolgus macaques exposed to long-term mainstream CS, and those from former smokers with and without COPD were analyzed. The structured-illumination imaging, RNA fluorescence in-situ hybridization (FISH), and qRT-PCR were used to characterize lncRNA expression and the expression of inflammatory factors and airway mucins in a cell culture model of CS extract (CSE) exposure using HBECs from COPD (CHBEs) in comparison with cells from normal control (NHBEs) subjects. The protein levels of mucin MUC5AC, and inflammatory factors ICAM-1, and IL-6 were determined using specific ELISAs. RNA silencing was used to block LASI lncRNA expression and lentivirus encoding LASI lncRNA was used to achieve LASI overexpression (LASI-OE).ResultsCompared to controls, LASI lncRNA was upregulated in CS-exposed macaques and in COPD smoker airways, correlating with mucus hyperexpression and mucus cell hyperplasia in severe COPD airways. At baseline, the unstimulated CHBEs showed increased LASI lncRNA expression with higher expression of secretory mucin MUC5AC, and inflammatory factors, ICAM-1, and IL-6 compared to NHBEs. CSE exposure of CHBEs resulted in augmented inflammation and mucus expression compared to controls. While RNA silencing-mediated LASI knockdown suppressed the mucoinflammatory response, cells overexpressing LASI lncRNA showed elevated mRNA levels of inflammatory factors.ConclusionsAltogether, LASI lncRNA may represent a novel target to control the smoke-mediated dysregulation in airway responses and COPD exacerbations.

Background Electronic cigarette (e-cigarette) vaping, containing nicotine and/or Δ8, Δ9 or Δ10 or Δo tetrahydrocannabinol (Δn-THC), is associated with an outbreak of e-cigarette, or vaping, product use-associated lung injury (EVALI). Despite thousands being hospitalised with EVALI, much remains unknown about diagnosis, treatment and disease pathogenesis. Biomarkers of inflammation, oxidative stress and lipid mediators may help identify e-cigarette users with EVALI. Methods We collected plasma and urine along with demographic and vaping-related data of EVALI subjects (age 18–35 years) and non-users matched for sex and age in a pilot study. Biomarkers were assessed by ELISA/EIA and Luminex-based assays. Results Elevated levels of THC metabolite (11-nor-9-carboxy-Δ9-THC) were found in plasma from EVALI subjects compared to non-users. Levels of 8-hydroxy-2′-deoxyguanosine (8-OHdG), an oxidative DNA damage biomarker, and 8-isoprostane, an oxidative stress marker, were slightly increased in urine samples from EVALI subjects compared to non-users. Conversely, plasma levels of lipid mediators, including resolvin D1 (RvD1) and prostaglandin E2 (PGE2), were significantly lower in EVALI subjects compared to non-users. Both pro-inflammatory biomarkers, such as tumour necrosis factor-α, macrophage inflammatory protein-1β, RANTES (regulated on activation, normal T-cell expressed and secreted) and granulocyte–macrophage colony-stimulating factor, as well as anti-inflammatory biomarkers, such as interleukin-9 and CC10/16, were decreased in plasma from EVALI subjects compared to non-users, supportive of a possible dysregulated inflammatory response in EVALI subjects. Conclusions Significant elevations in urine and plasma biomarkers of oxidative stress, as well as reductions in lipid mediators, were shown in EVALI subjects. These noninvasive biomarkers (8-OHdG, 8-isoprostane, RvD1 and CC10/16), either individually or collectively, may serve as tools in diagnosing future EVALI subjects.

Introduction Flavors other than tobacco flavor have been identified as a major reason for electronic nicotine delivery system (ENDS) initiation in youth and are thought to contribute to the continued use of ENDS in users of all ages. Our previous research showed a significant association between overall ENDS use and COPD. This study aims to identify the association of ENDS flavor categories with self-reported COPD. Methods The data analysis included 4909 adults from Population Assessment of Tobacco and Health (PATH) Wave 4 data who were ever established ENDS users and responded to an item about diagnosis of COPD. Weighted multivariable logistic regression models were used to examine the association between different ENDS flavors and self-reported COPD considering complex sampling design. Results Among 4909 ever established ENDS users, 418 adults (weighted percentage 9.8%) had self-reported COPD. Self-reported COPD prevalence differed between different ENDS flavor categories, with the highest (weighted percentage 19.9%) occurring among tobacco flavor users. Compared to non-tobacco flavor categories, tobacco flavor category showed significantly higher association with self-reported COPD (AOR=2.05; 95% CI: 1.20–3.53), after adjusting for potential confounding variables. No significant associations with self-reported COPD were found for other examined ENDS flavor categories including menthol/mint, fruit, candy/ desserts/other-sweets, and other flavors, compared to their corresponding non-users. Conclusions Tobacco flavored ENDS use was significantly associated with self-reported COPD. Future studies are needed to confirm the biological and epidemiological association of flavored ENDS use with COPD.

Abstract Background Electronic cigarettes (e-cigs) vaping, cigarette smoke, and waterpipe tobacco smoking are associated with various cardiopulmonary diseases. microRNAs are present in higher concentration in exosomes that play an important role in various physiological and pathological functions. We hypothesized that the non-coding RNAs transcript may serve as susceptibility to disease biomarkers by smoking and vaping. Methods Plasma exosomes/EVs from cigarette smokers, waterpipe smokers and dual smokers (cigarette and waterpipe) were characterized for their size, morphology and TEM, Nanosight and immunoblot analysis. Exosomal RNA was used for small RNA library preparation and the library was quantified using the High Sensitivity DNA Analysis on the Agilent 2100 Bioanalyzer system and sequenced using the Illumina NextSeq 500 and were converted to fastq format for mapping genes. Results Enrichment of various non-coding RNAs that include microRNAs, tRNAs, piRNAs, snoRNAs, snRNAs, Mt-tRNAs, and other biotypes are shown in exosomes. A comprehensive differential expression analysis of miRNAs, tRNAs and piRNAs showed significant changes across different pairwise comparisons. The seven microRNAs that were common and differentially expressed of when all the smoking and vaping groups were compared with non-smokers (NS) are hsa-let-7a-5p, hsa-miR-21-5p, hsa-miR-29b-3p, hsa-let-7f-5p, hsa-miR-143-3p, hsa-miR-30a-5p and hsa-let-7i-5p. The e-cig vs. NS group has differentially expressed 5 microRNAs (hsa-miR-224-5p, hsa-miR-193b-3p, hsa-miR-30e-5p, hsa-miR-423-3p, hsa-miR-365a-3p, and hsa-miR-365b-3p), which are not expressed in other three groups. Gene set enrichment analysis of microRNAs showed significant changes in the top six enriched functions that consisted of biological pathway, biological process, molecular function, cellular component, site of expression and transcription factor in all the groups. Further, the pairwise comparison of tRNAs and piRNA in all these groups revealed significant changes in their expressions. Conclusions Plasma exosomes of cigarette smokers, waterpipe smokers, e-cig users and dual smokers have common differential expression of microRNAs which may serve to distinguish smoking and vaping subjects from NS. Among them has-let-7a-5p has high sensitivity and specificity to distinguish NS with the rest of the users, using ROC curve analysis. These findings will pave the way for the utilizing the potential of exosomes/miRNAs as a novel theranostic agents in lung injury and disease caused by tobacco smoking and vaping.

Abstract The current pandemic of COVID-19 has caused severe morbidity and mortality across the globe. People with a smoking history have severe disease outcomes by COVID-19 infection. Epidemiological studies show that old age and pre-existing disease conditions (hypertension and diabetes) result in severe disease outcome and mortality amongst COVID-19 patients. Evidences suggest that the S1 domain of the SARS-CoV-2 (causative agent of COVID-19) membrane spike has a high affinity towards the angiotensin-converting enzyme 2 (ACE2) receptor found on the host’s lung epithelium. Likewise, TMPRSS2 protease has been shown to be crucial for viral activation thus facilitating the viral engulfment. The viral entry has been shown to cause ‘cytokine storm’ involving excessive production of pro-inflammatory cytokines/chemokines including IL-6, TNF-α, IFN-γ, IL-2, IL-7, IP-10, MCP-3 or GM-CSF, which is augmented by smoking. Future research could target these inflammatory-immunological responses to develop effective therapy for COVID-19. This mini-review provides a consolidated account on the role of inflammation and immune responses, proteases, and epithelial permeability by smoking and vaping during SARS-CoV2 infection with future directions of research, and provides a list of the potential targets for therapies particularly controlling cytokine storms in the lung.

Abstract Electronic cigarette (e-cig) vaping is increasing rapidly in the United States, as e-cigs are considered less harmful than combustible cigarettes. However, limited research has been conducted to understand the possible mechanisms that mediate toxicity and pulmonary health effects of e-cigs. We hypothesized that sub-chronic e-cig exposure induces inflammatory response and dysregulated repair/extracellular matrix (ECM) remodeling, which occur through the α7 nicotinic acetylcholine receptor (nAChRα7). Adult wild-type (WT), nAChRα7 knockout (KO), and lung epithelial cell-specific KO (nAChRα7 CreCC10) mice were exposed to e-cig aerosol containing propylene glycol (PG) with or without nicotine. Bronchoalveolar lavage fluids (BALF) and lung tissues were collected to determine e-cig induced inflammatory response and ECM remodeling, respectively. Sub-chronic e-cig exposure with nicotine increased inflammatory cellular influx of macrophages and T-lymphocytes including increased pro-inflammatory cytokines in BALF and increased SARS-Cov-2 Covid-19 ACE2 receptor, whereas nAChRα7 KO mice show reduced inflammatory responses associated with decreased ACE2 receptor. Interestingly, matrix metalloproteinases (MMPs), such as MMP2, MMP8 and MMP9, were altered both at the protein and mRNA transcript levels in female and male KO mice, but WT mice exposed to PG alone showed a sex-dependent phenotype. Moreover, MMP12 was increased significantly in male mice exposed to PG with or without nicotine in a nAChRα7-dependent manner. Additionally, sub-chronic e-cig exposure with or without nicotine altered the abundance of ECM proteins, such as collagen and fibronectin, significantly in a sex-dependent manner, but without the direct role of nAChRα7 gene. Overall, sub-chronic e-cig exposure with or without nicotine affected lung inflammation and repair responses/ECM remodeling, which were mediated by nAChRα7 in a sex-dependent manner.

RNA-seq is a high-throughput sequencing technology widely used for gene transcript discovery and quantification under different biological or biomedical conditions. A fundamental research question in most RNA-seq experiments is the identification of differentially expressed genes among experimental conditions or sample groups. Numerous statistical methods for RNA-seq differential analysis have been proposed since the emergence of the RNA-seq assay. To evaluate popular differential analysis methods used in the open source R and Bioconductor packages, we conducted multiple simulation studies to compare the performance of eight RNA-seq differential analysis methods used in RNA-seq data analysis (edgeR, DESeq, DESeq2, baySeq, EBSeq, NOISeq, SAMSeq, Voom). The comparisons were across different scenarios with either equal or unequal library sizes, different distribution assumptions and sample sizes. We measured performance using false discovery rate (FDR) control, power, and stability. No significant differences were observed for FDR control, power, or stability across methods, whether with equal or unequal library sizes. For RNA-seq count data with negative binomial distribution, when sample size is 3 in each group, EBSeq performed better than the other methods as indicated by FDR control, power, and stability. When sample sizes increase to 6 or 12 in each group, DESeq2 performed slightly better than other methods. All methods have improved performance when sample size increases to 12 in each group except DESeq. For RNA-seq count data with log-normal distribution, both DESeq and DESeq2 methods performed better than other methods in terms of FDR control, power, and stability across all sample sizes. Real RNA-seq experimental data were also used to compare the total number of discoveries and stability of discoveries for each method. For RNA-seq data analysis, the EBSeq method is recommended for studies with sample size as small as 3 in each group, and the DESeq2 method is recommended for sample size of 6 or higher in each group when the data follow the negative binomial distribution. Both DESeq and DESeq2 methods are recommended when the data follow the log-normal distribution.

Background: Chronic lung diseases, such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are linked to several mitochondrial alterations. Cigarette smoke (CS) alters the structure and function of mitochondria. OPA1 is the main inner mitochondrial GTPase responsible for the fusion events. OPA1 undergoes proteolytic cleavage from long to short forms during acute stress and mitophagy. However, the exact role of OPA1 isoforms and related proteins during CS-induced mitophagy and COPD is not clear. Methods: Lung tissues from non-smokers, smokers, COPD and IPF were used to determine the relative expression of OPA1 and related proteins. Additionally, we used mouse lungs from chronic (6 months) CS exposure to evaluate the status of OPA1. Primary lung fibroblasts from normal and COPD patients and naked mole rat (NMR) lung fibroblasts, human fetal lung fibroblast (HFL1), mouse embryonic fibroblast from wild type (WT), OPA1−/−, MFN1 and MFN2−/− were used to determine the effect of CS on OPA1 isoforms. Various mitochondrial fusion promoters/activators (BGP-15, leflunomide, M1) and fission inhibitor (DRP1) were used to determine their effect on OPA1 status and cigarette smoke extract (CSE)-induced lung epithelial (BEAS2B) cell damage, respectively. Seahorse flux analyzer was used to determine the effect of these compounds in BEAS2B cells with and without CSE exposure. Findings: Short OPA1 isoforms were predominantly detected and significantly increased in COPD subjects. Acute CSE treatment in various cell lines except NMR was found to increase the conversion of long to short OPA1 isoforms. CSE treatment significantly increased mitochondrial stress-related protein SLP2 in all the cells used. OPA1 interacting partners like prohibitins (PHB1 and 2) were also altered depending on the CS exposure. Finally, BGP-15 and leflunomide treatment were able to preserve the long OPA1 isoform in cells treated with CSE. Interpretation/conclusion: The long OPA1 isoform along with SLP2 and prohibitins play a crucial role in CS-induced lung damage, causing mitophagy/mitochondrial dysfunction in COPD, which may be used as a novel therapeutic target in COPD.

Oral nicotine pouches (ONPs) are a modern form of smokeless tobacco products sold by several brands in the U.S., which comprise a significant portion of non-combustible nicotine-containing product (NCNP) sales to date. ONPs are available in various flavors and may contain either tobacco-derived nicotine (TDN) or tobacco-free nicotine (TFN). The growth in popularity of these products has raised concerns that flavored ONPs may cause adverse oral health effects and promote systemic toxic effects due to nicotine and other ONP by-products being absorbed into the circulatory system through oral mucosa. We hypothesized that flavored ONPs are unsafe and likely to cause oral and pulmonary inflammation in oral and respiratory epithelial cells. Before analyzing the effects of ONPs, we first classified ONPs sold in the U.S. based on their flavor and the flavor category to which they belonged using a wheel diagram. Human gingival epithelial cells (HGEP) were treated with flavored ONP extracts of tobacco (original, smooth), menthol (wintergreen and cool cider), and fruit flavor (americana and citrus), each from the TDN and TFN groups. The levels of ONP-induced inflammatory cytokine release (TNF-α, IL-6, and IL-8) by ELISA, cellular reactive oxygen species (ROS) production by CellRox Green, and cytotoxicity by lactate dehydrogenase (LDH) release assay in HGEP cells were assessed. Flavored ONP extracts elicited differential toxicities in a dose- and extract-dependent manner in HGEP cells 24 h post-treatment. Both fruit TDN and TFN extracts resulted in the greatest cytotoxicity. Tobacco- and fruit-flavored, but not menthol-flavored, ONPs resulted in increased ROS production 4 h post-treatment. Flavored ONPs led to differential cytokine release (TNF-α, IL-6, and IL-8) which varied by flavor (menthol, tobacco, or fruit) and nicotine (TDN vs. TFN) 24 h post-treatment. Menthol-flavored ONPs led to the most significant TNF-α release; fruit TFN resulted in the most significant IL-6 release; and fruit TDN and tobacco TFN led to the highest release of IL-8. Subsequently, human bronchial epithelial cells (16-HBE and BEAS-2B) were also treated with flavored ONP extracts, and similar assays were evaluated. Here, the lowest concentration treatments displayed increased cytotoxicity. The most striking response was observed among cells treated with spearmint and tobacco flavored ONPs. Our data suggest that flavored ONPs are unsafe and likely to cause systemic and local toxicological responses during chronic usage.

Cigarette smoke (CS) is the main etiological factor in the pathogenesis of emphysema/chronic obstructive pulmonary disease (COPD), which is associated with abnormal epithelial-mesenchymal transition (EMT). Previously, we have shown an association among circadian rhythms, CS-induced lung inflammation, and nuclear heme receptor α (REV-ERBα), acting as an antiinflammatory target in both pulmonary epithelial cells and fibroblasts. We hypothesized that molecular clock REV-ERBα plays an important role in CS-induced circadian dysfunction and EMT alteration. C57BL/6J WT and REV-ERBα heterozygous (Het) and –KO mice were exposed to CS for 30 days (subchronic) and 4 months (chronic), and WT mice were exposed to CS for 10 days with or without REV-ERBα agonist (SR9009) administration. Subchronic/chronic CS exposure caused circadian disruption and dysregulated EMT in the lungs of WT and REV-ERBα–KO mice; both circadian and EMT dysregulation were exaggerated in the REV-ERBα–KO condition. REV-ERBα agonist, SR9009 treatment reduced acute CS-induced inflammatory response and abnormal EMT in the lungs. Moreover, REV-ERBα agonist (GSK4112) inhibited TGF-β/CS–induced fibroblast differentiation in human fetal lung fibroblast 1 (HFL-1). Thus, CS-induced circadian gene alterations and EMT activation are mediated through a Rev-erbα–dependent mechanism, which suggests activation of REV-ERBα as a novel therapeutic approach for smoking-induced chronic inflammatory lung diseases.

Abstract Nicotine inhalation via electronic cigarettes (e‐cigs) is an emerging concern. However, little is known about the acute toxicity in the lungs following inhalation of nicotine‐containing e‐cig aerosols. We hypothesized that acute exposure to aerosolized nicotine causes lung toxicity by eliciting inflammatory and dysregulated repair responses. Adult C57BL/6J mice were exposed 2 hours daily for 3 days to e‐cig aerosols containing propylene glycol (PG) with or without nicotine. Acute exposure to nicotine‐containing e‐cig aerosols induced inflammatory cell influx (neutrophils and CD8a+ T lymphocytes), and release of pro‐inflammatory cytokines in bronchoalveolar lavage fluid in a sex‐dependent manner. Inhalation of e‐cig aerosol containing PG alone significantly augmented the lung levels of various homeostasis/repair mediators (PPARγ, ADRP, ACTA2, CTNNB1, LEF1, β‐catenin, E‐cadherin, and MMP2) in a sex‐dependent manner when compared to air controls. These findings were accompanied by an increase in protein abundance and altered gene expression of lipogenic markers (PPARγ, ADRP) and myogenic markers (fibronectin, α‐smooth muscle actin and β‐catenin), suggesting a dysregulated repair response in mouse lungs. Furthermore, exposure to nicotine‐containing e‐cig aerosols or PG alone differentially affected the release of pro‐inflammatory cytokines in healthy and COPD human 3D EpiAirway tissues. Overall, acute exposure to nicotine‐containing e‐cig aerosols was sufficient to elicit a pro‐inflammatory response and altered mRNA and protein levels of myogenic, lipogenic, and extracellular matrix markers in mouse lung in a sex‐dependent manner. Thus, acute exposure to inhaled nicotine via e‐cig leads to dysregulated repair and inflammatory responses, which may lead to airway remodeling in the lungs.

Flavoring chemicals in electronic nicotine delivery systems have been shown to cause cellular inflammation; meanwhile, the effects of fruit and tobacco flavors on lung inflammation by nose-only exposures to mice are relatively unknown. We hypothesized that exposure to flavored e-cigarettes would cause lung inflammation in C57BL/6 J mice. The mice were exposed to air, propylene glycol/vegetable glycerin, and flavored e-liquids: Apple, Cherry, Strawberry, Wintergreen, and Smooth & Mild Tobacco, one hour per day for three days. Quantification of flavoring chemicals by proton nuclear magnetic resonance spectroscopy (1H NMR), differential cell counts by flow cytometry, pro-inflammatory cytokines/chemokines by ELISA, and matrix metalloproteinase levels by western blot were performed. Exposure to PG/VG increased neutrophil cell count in lung bronchoalveolar lavage fluid (BALF). KC and IL6 levels were increased by PG/VG exposure and female mice exposed to Cherry flavored e-cigarettes, in lung homogenate. Mice exposed to PG/VG, Apple, Cherry, and Wintergreen increased MMP2 levels. Our results revealed flavor- and sex-based e-cigarette effects in female mice exposed to cherry-flavored e-liquids and male mice exposed to tobacco-flavored e-liquids, namely, increased lung inflammation.

Background: Tobacco smoking is known to be involved in the pathogenesis of several cardiopulmonary diseases. Additionally, smokers are highly susceptible to infectious agents due to weakened immunity. However, the progression of lung injury based on SARS-CoV-2-mediated COVID-19 pathogenesis amongst smokers and those with pre-existing pulmonary diseases is not known. We determined the systemic levels and activity of COVID-19 associated proteins, cytokine/chemokines, and lipid mediators (lipidomics) amongst COVID-19 patients with and without a history of smoking to understand the underlying susceptible factor in the pathogenesis of COVID-19.Methods: We obtained serum from healthy (CoV−), COVID-19 positive (CoV+), and COVID-19 recovered (CoV Rec) subjects with and without a history of smoking. We conducted a Luminex multiplex assay (cytokine levels), LC/MS (eicosanoids or oxylipin panel), and ACE2 enzymatic activity assays on the serum samples to determine the systemic changes in COVID-19 patients.Results: On comparing the levels of serum ACE2 amongst COVID-19 (positive and recovered) patients and healthy controls, we found a pronounced increase in serum ACE2 levels in patients with COVID-19 infection. Furthermore, ACE2 enzyme activity was significantly increased amongst COVID-19 patients with a smoking history. Also, we analyzed the levels of Angiotensin 1–7 (Ang1–7) peptide, the product of enzymatic action of ACE2, in the serum samples. We found significantly high levels of Ang1–7 in the serum of both CoV+ and CoV Rec patients. Our data further demonstrated a smoking-induced increase in serum furin and inflammatory cytokine [IFNγ(p = 0.0836), Eotaxin (p < 0.05), MCP-1 (p < 0.05), and IL-9 (p = 0.0991)] levels in COVID-19 patients as compared to non-smoking controls. Overall, our results show that smoking adversely affects the levels of systemic inflammatory markers and COVID-19 associated proteins, thus suggesting that COVID-19 infection may have severe outcomes amongst smokers.

Global usage of electronic nicotine delivery systems (ENDS) has been increasing in the last decade. ENDS are non-combustible tobacco products that heat and aerosolize a liquid containing humectants, with added flavorings and often nicotine. Though ENDS are promoted as a less harmful alternative to smoking, current evidence links their use to a wide range of deleterious health effects including acute and chronic lung damage. ENDS can elicit an inflammatory response and impair the innate immune response in the lungs. Exposure to ENDS flavorings results in abnormal activation of the lung epithelial cells and β-defensins, dysfunction of the macrophage phagocytic activity, increased levels of mucin (MUC5AC) and abnormal activation of the neutrophilic response (NETosis). ENDS menthol flavorings disrupt innate immunity and might be associated with allergies and asthma through activation of transient receptor potential ankyrin 1 (TRAP1). Recent studies have expanded our understanding of the relationship between the homeostasis of lung innate immunity and the immunomodulatory effect of the host-microbiota interaction. Alterations of the normal respiratory microbiota have been associated with chronic obstructive pulmonary disease (COPD), asthma, atopy and cystic fibrosis complications which are strongly associated with smoking and potentially with ENDS use. Little is known about the short-and long-term effects of ENDS on the respiratory microbiota, their impact on the innate immune response and their link to pulmonary health and disease. Here we review the interaction between the innate immune system and the respiratory microbiota in the pathogenesis of ENDS-induced pulmonary dysfunction and identify future areas of research.

Introduction Cigarette smoking and oxidative stress are common risk factors for the multi-morbidities associated with chronic obstructive pulmonary disease (COPD). Elevated levels of advanced glycation endproducts (AGE) increase the risk of cardiovascular disease (CVD) comorbidity and mortality. The enzyme fructosamine-3-kinase (FN3K) reduces this risk by lowering AGE levels.Methods The distribution and expression of FN3K protein in lung tissues from stable COPD and control subjects, as well as an animal model of COPD, was assessed by immunohistochemistry. Serum FN3K protein and AGE levels were assessed by ELISA in patients with COPD exacerbations receiving metformin. Genetic variants within the FN3K and FN3K-RP genes were evaluated for associations with cardiorespiratory function in the Subpopulations and Intermediate Outcome Measures in COPD Study cohort.Results This pilot study demonstrates that FN3K expression in the blood and human lung epithelium is distributed at either high or low levels irrespective of disease status. The percentage of lung epithelial cells expressing FN3K was higher in control smokers with normal lung function, but this induction was not observed in COPD patients nor in a smoking model of COPD. The top five nominal FN3K polymorphisms with possible association to decreased cardiorespiratory function (p

Electronic nicotine delivery systems (ENDS) containing synthetic nicotine have yet to be classified as tobacco products; consequently, there is ambiguity over whether Food and Drug Administration (FDA) regulatory authority can be extended to include tobacco-free nicotine (TFN) e-cigarettes. In recent years, a more significant number of e-cigarette companies have been manufacturing TFN-containing e-cigarettes and e-liquids to circumvent FDA regulations. While studies have shown that aerosols generated from tobacco-derived nicotine-containing e-cigarettes contain significant reactive oxygen species (ROS) levels, no comparison studies have been conducted using TFN e-cigarettes. This study uses a single puff aerosol generator to aerosolize TFN and tobacco-derived nicotine-containing vape products and subsequently involves semi-quantifying the ROS generated by these vape products in H2O2 equivalents. We found that the differences between ROS levels generated from TFN and tobacco-derived nicotine-containing vape products vary by flavor. TFN tobacco flavored and fruit flavored products are more toxic in terms of ROS generation than menthol/ice and drink/beverage flavored products using TFN. Our study provides further insight into understanding how flavoring agents used in vape products impact ROS generation from e-cigarettes differently in TFN e-cigarettes than e-cigarettes using tobacco-derived nicotine.

Electronic nicotine delivery systems/devices (ENDS) such as electronic cigarettes (e-cigarettes) have been made available globally, with the intent to reduce tobacco smoking. To make these products more appealing to young adults, many brands have added flavoring agents. However, these flavoring agents are shown to progressively result in lung toxicity when inhaled via e-cigarettes. While recent federal regulations have banned the sale of flavored e-cigarettes other than tobacco or menthol flavors, concerns have been raised about the health effects of even these flavors. In this review, we evaluate the current toxicological data with regard to effects upon exposure in animal models and in vitro cell culture for these popular flavorants. We have tabulated the current e-cigarette products containing these most common flavors (menthol, mint, and tobacco) in the market. We have also indicated the prevalence of tobacco and menthol-flavor use among e-cigarette users and highlighted the possible challenges and benefits that will result from new federal regulations.

BackgroundAging is one of the key contributing factors for chronic obstructive pulmonary diseases (COPD) and other chronic inflammatory lung diseases. Here, we determined how aging contributes to the altered gene expression related to mitochondrial function, cellular senescence, and telomeric length processes that play an important role in the progression of COPD and idiopathic pulmonary fibrosis (IPF).MethodsTotal RNA from the human lung tissues of non-smokers, smokers, and patients with COPD and IPF were processed and analyzed using a Nanostring platform based on their ages (younger: 55 years).ResultsSeveral genes were differentially expressed in younger and older smokers, and patients with COPD and IPF compared to non-smokers which were part of the mitochondrial biogenesis/function (HSPD1, FEN1, COX18, COX10, UCP2 & 3), cellular senescence (PCNA, PTEN, KLOTHO, CDKN1C, TNKS2, NFATC1 & 2, GADD45A), and telomere replication/maintenance (PARP1, SIRT6, NBN, TERT, RAD17, SLX4, HAT1) target genes. Interestingly, NOX4 and TNKS2 were increased in the young IPF as compared to the young COPD patients. Genes in the mitochondrial dynamics and quality control mechanisms like FIS1 and RHOT2 were decreased in young IPF compared to their age matched COPD subjects. ERCC1 and GADD45B were higher in young COPD as compared to IPF. Aging plays an important role in various infectious diseases including the SARS-CoV-2 infection. Lung immunoblot analysis of smokers, COPD and IPF subjects revealed increased abundance of proteases and receptor/spike protein like TMPRSS2, furin, and DPP4 in association with a slight increase in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor ACE2 levels.ConclusionsOverall, these findings suggest that altered transcription of target genes that regulate mitochondrial function, cellular senescence, and telomere attrition in the pathobiology of lung aging in COPD and IPF is associated with alterations in SARS-CoV-2 ACE2-TMPRSS2-Furin-DPP4 axis as pharmacological targets for COVID-19.

Electronic cigarette (e-cig) usage has risen dramatically worldwide over the past decade. While they are touted as a safe alternative to cigarettes, recent studies indicate that high levels of nicotine and flavoring chemicals present in e-cigs may still cause adverse health effects. We hypothesized that an e-liquid containing a mixture of tobacco, coconut, vanilla, and cookie flavors would induce senescence and disrupt wound healing processes in pulmonary fibroblasts. To test this hypothesis, we exposed pulmonary fibroblasts (HFL-1) to e-liquid at varying doses and assessed cytotoxicity, inflammation, senescence, and myofibroblast differentiation. We found that e-liquid exposure caused cytotoxicity, which was accompanied by an increase in IL-8 release in the conditioned media. E-liquid exposure resulted in elevated senescence-associated beta-galactosidase (SA-β-gal) activity. Transforming growth factor-β1 (TGF-β1) induced myofibroblast differentiation was inhibited by e-liquid exposure, resulting in decreased α-smooth muscle actin and fibronectin protein levels. Together, our data suggest that an e-liquid containing a mixture of flavors induces inflammation, senescence and dysregulated wound healing responses.

Background: Histological chorioamnionitis (HCA) is an infection/inflammation of fetal membranes and complicates 5.2–28.5% of all live births. Exposure to HCA can have long-term consequences including abnormal neurodevelopment and an increased risk for allergic disorders and asthma later in childhood. HCA may incite epigenetic changes, which have the potential to modulate both the immune and neurological systems as well as increase the risk of related disorders later in life. However, there is limited data on the impact of HCA on epigenetics, in particular DNA methylation, and changes to immune and neurological systems in full-term human neonates.Objective: To determine differential DNA methylation in cord blood mononuclear leukocytes from neonates exposed to HCA.Methods: Cord blood was collected from 10 term neonates (5 with HCA and 5 controls without HCA) and mononuclear leukocytes were isolated. Genome-wide DNA methylation screening was performed on Genomic DNA extracted from mononuclear leukocytes.Results: Mononuclear leukocytes from cord blood of HCA-exposed neonates showed differential DNA methylation of 68 probe sets compared to the control group (44 hypermethylated, 24 hypomethylated) with a p ≤ 0.0001. Several genes involved in immune modulation and nervous system development were found to be differentially methylated. Important canonical pathways as revealed by Ingenuity Pathway Analysis (IPA) were CREB Signaling in Neurons, FcγRIIB Signaling in B Lymphocytes, Cell Cycle: G1/S Checkpoint Regulation, Interleukin-1, 2, 3, 6, 8, 10, 17, and 17A signaling, p53 signaling, dopamine degradation, and serotonin degradation. The diseases and disorders picked up by IPA were nervous system development and function, neurological disease, respiratory disease, immune cell trafficking, inflammatory response, and immunological disease.Conclusions: HCA induces differential DNA methylation in cord blood mononuclear leukocytes. The differentially methylated genes may contribute to inflammatory, immunological and neurodevelopmental disorders in neonates exposed to HCA.

The abnormal inflammatory responses due to the lung tissue damage and ineffective repair/resolution in response to the inhaled toxicants result in the pathological changes associated with chronic respiratory diseases. Investigation of such pathophysiological mechanisms provides the opportunity to develop the molecular phenotype-specific diagnostic assays and could help in designing the personalized medicine-based therapeutic approaches against these prevalent diseases. As the central hubs of cell metabolism and energetics, mitochondria integrate cellular responses and interorganellar signaling pathways to maintain cellular and extracellular redox status and the cellular senescence that dictate the lung tissue responses. Specifically, as observed in chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, the mitochondria-endoplasmic reticulum (ER) crosstalk is disrupted by the inhaled toxicants such as the combustible and emerging electronic nicotine-delivery system (ENDS) tobacco products. Thus, the recent research efforts have focused on understanding how the mitochondria-ER dysfunctions and oxidative stress responses can be targeted to improve inflammatory and cellular dysfunctions associated with these pathologic illnesses that are exacerbated by viral infections. The present review assesses the importance of these redox signaling and cellular senescence pathways that describe the role of mitochondria and ER on the development and function of lung epithelial responses, highlighting the cause and effect associations that reflect the disease pathogenesis and possible intervention strategies.

IntroductionElectronic cigarette use (vaping) has become popular in recent years. The number of Americans with a variety of cognitive deficits has been increasing dramatically. This study aimed to examine the potential association of vaping with subjective cognitive complaints in US adults.MethodsA combined 2016 and 2017 Behavioral Risk Factor Surveillance System (BRFSS) national survey dataset yielded 886,603 adults who indicated their smoking and vaping status, as well as whether they had subjective cognitive complaints. With this dataset, the cross-sectional association of electronic cigarette use with subjective cognitive complaints was examined using multivariable weighted logistic regression models.ResultsBoth dual users (adjusted Odds Ratio [aOR] = 2.07; 95% Confidence Interval [CI] = 1.66 to 2.60) and current vapers who were either ex-smokers (aOR = 1.94; 95% CI = 1.40 to 2.71) or never smoked (aOR = 1.96; 95% CI = 1.16 to 3.30) showed a significantly higher association with subjective cognitive complaints than never users. Current smokers (aOR = 1.49; 95% CI = 1.32 to 1.69) and ex-smokers (aOR = 1.25; 95% CI = 1.11 to 1.41) had a significantly higher association with subjective cognitive complaints compared to never users. Compared to current smokers, the ex-smokers showed a lower association with subjective cognitive complaints (aOR = 0.84; 95% CI = 0.73 to 0.96). Finally, the association of vaping with subjective cognitive complaints was not statistically significant in individual age group.ConclusionSimilar to smoking, vaping is associated with subjective cognitive complaints in US adults. These results provide preliminary evidence for a cross-sectional association of vaping with potential cognitive health effects in adults.

Long non-coding RNAs (lncRNAs) are the varied set of transcripts that play a critical role in biological processes like gene regulation, transcription, post-transcriptional modification, and chromatin remodeling. Recent studies have reported the presence of lncRNAs in the exosomes that are involved in regulating cell-to-cell communication in lung pathologies including lung cancer, chronic obstructive pulmonary disease (COPD), asthma, and idiopathic pulmonary fibrosis (IPF). In this study, we compared the lncRNA profiles in the plasma-derived exosomes amongst non-smokers (NS), cigarette smokers (CS), E-cig users (E-cig), waterpipe smokers (WP) and dual smokers (CSWP) using GeneChip™ WT Pico kit for transcriptional profiling. We found alterations in a distinct set of lncRNAs among subjects exposed to E-cig vapor, cigarette smoke, waterpipe smoke and dual smoke with some overlaps. Gene enrichment analyses of the differentially expressed lncRNAs demonstrated enrichment in the lncRNAs involved in crucial biological processes including steroid metabolism, cell differentiation and proliferation. Thus, the characterized lncRNA profiles of the plasma-derived exosomes from smokers, vapers, waterpipe users, and dual smokers will help identify the biomarkers relevant to chronic lung diseases such as COPD, asthma or IPF.

New emerging tobacco products, especially electronic cigarettes (E-Cig) or electronic nicotine delivery systems (ENDS), have gained a huge popularity, particularly in younger populations. The lack of sufficient evidence-based health effect studies has promoted widespread use/abuse with the assumption that E-Cig or ENDS and/or vaping products are safer and less toxic than conventional tobacco smoking. However, the recent escalation in acute lung injuries and their associated fatalities among ENDS or vaping product users has now brought attention to this silent epidemic via investigation into the constituents of ENDS/vaping products and their toxic effects on pulmonary health. Accordingly, CDC has declared an “outbreak” of the e-cigarette or vaping product use associated lung injury (EVALI). EVALI is characterized by sterile exogenous pneumonitis like reaction with substantial involvement of innate immune mechanisms. Vitamin-E acetate (VEA) is found in counterfeit cartridges and bronchoalveolar lavage fluid of EVALI patients. Other reports implicated the presence of aromatic/volatile hydrocarbons and oils consisting of medium-chain triglycerides (MCT oil), including terpenes and mineral oil in tetrahydrocannabinol (THC) containing counterfeit vaping products. These compounds are involved in oxidative stress and inflammatory responses in the lung. Here, we provide the perspectives on the recent case reports on EVALI, etiology, and discuss pulmonary toxicity as well as the mechanisms underlying EVALI susceptibility and lung pathophysiology.