Immunohistochemical Study of Airways Fibrous Remodeling in Smoking Mice.

The fibrotic remodeling in chronic obstructive pulmonary disease (COPD) is held responsible for narrowing of small airways and thus for disease progression. Oxidant damage and cell senescence factors are recently involved in airways fibrotic remodeling. Unfortunately, we have no indications on their sequential expression at anatomical sites in which fibrotic remodeling develops in smoking subjects. Using immunohistochemical techniques, we investigated in two strains of mice after cigarette smoke (CS) exposure what happens at various times in airway areas where fibrotic remodeling occurs, and if there also exists correspondence among DNA damage induced by oxidants, cellular senescence, the presence of senescence-secreted factors involved in processes that affect transcription, metabolism as well as apoptosis, and the onset of fibrous remodeling that appears at later times in mice exposed to CS. A clear positivity for fibrogenic cytokines TGF-ß, PDGF-B, and CTGF, and for proliferation marker PCNA around airways that will be remodeled is observed in both strains. Increased expression of p16ink4A senescence marker and MyoD is also seen in the same areas. p16ink4A and MyoD can promote cell cycle arrest, terminal differentiation of myofibroblasts, and can oppose their dedifferentiation. Of interest, an early progressive attenuation of SIRT-1 is observed after CS exposure. This intracellular regulatory protein can reduce premature cell senescence. These findings suggest that novel agents, which promote myofibroblast dedifferentiation and/or the apoptosis of senescent cells, may dampen progression of airway changes in smoking COPD subjects.

Smoking Cessation in Mice Does Not Switch off Persistent Lung Inflammation and Does Not Restore the Expression of HDAC2 and SIRT1.

Once COPD is established, pulmonary lesions can only progress and smoking cessation by itself is not sufficient to switch off persistent lung inflammation. Similarly, in former-smoker mice, neutrophil inflammation persists and lung lesions undergo progressive deterioration. The molecular mechanisms underlying disease progression and the inefficiency of smoking cessation in quenching neutrophilic inflammation were studied in male C57 Bl/6 mice after 6 months of rest from smoking cessation. As compared with the mice that continued to smoke, the former-smoker mice showed reduced expression of histone deacetylases HDAC2 and SIRT1 and marked expression of p-p38 MAPK and p-Ser10. All these factors are involved in corticosteroid insensitivity and in perpetuating inflammation. Former-smoker mice do show persistent lung neutrophilic influx and a high number of macrophages which account for the intense staining in the alveolar structures of neutrophil elastase and MMP-9 (capable of destroying lung scaffolding) and 8-OHdG (marker of oxidative stress). "Alarmins" released from necrotic cells together with these factors can sustain and perpetuate inflammation after smoking cessation. Several factors and mechanisms all together are involved in sustaining and perpetuating inflammation in former-smoker mice. This study suggests that a better control of COPD in humans may be achieved by precise targeting of the various molecular mechanisms associated with different phenotypes of disease by using a cocktail of drug active toward specific molecules.

Sarcopenia in Chronic Kidney Disease: Focus on Advanced Glycation End Products as Mediators and Markers of Oxidative Stress.

Sarcopenia is common in chronic kidney disease (CKD), and it is independently associated with morbidity and mortality. Advanced glycation end products (AGE) are mainly known as aging products. In CKD, AGE accumulate due to increased production and reduced kidney excretion. The imbalance between oxidant/antioxidant capacities in CKD patients is one of the main factors leading to AGE synthesis. AGE can, in turn, promote CKD progression and CKD-related complications by increasing reactive oxygen species generation, inducing inflammation, and promoting fibrosis. All these derangements can further increase AGE and uremic toxin accumulation and promote loss of muscle mass and function. Since the link between AGE and sarcopenia in CKD is far from being fully understood, we revised hereby the data supporting the potential contribution of AGE as mediators of oxidative stress in the pathogenesis of sarcopenia. Understanding how AGE and oxidative stress impact the onset of sarcopenia in CKD may help to identify new potential markers of disease progression and/or therapeutic targets.

In Vivo Electroporation-Mediated, Intrahepatic Alpha1 Antitrypsin Gene Transfer Reduces Pulmonary Emphysema in Pallid Mice.

RATIONALE: Mutation in the alpha1 antitrypsin (AAT) gene leads to low circulating levels of AAT, which is associated with several disease processes including pulmonary emphysema. The standard of care relies on substitution with plasma-purified AAT. We studied a novel approach to obtain sustained therapeutic levels of circulating AAT using nonviral in vivo electroporation-mediated gene transfer to the liver. METHODS: In vivo intrahepatic electroporation-mediated human AAT gene transfer was performed in C57 Bl/6J mice carrying a genetic deficiency of murine AAT (pallid mice) and suffering from pulmonary emphysema. The animals were evaluated for lung function using flexiVent and detailed stereological assessments. Lung neutrophilic burden was assessed. RESULTS: Pallid mice showed morphologically detectable pulmonary emphysema. Thirty days after in vivo electroporation-mediated gene transfer directly aimed at the liver, circulating human AAT was elevated and lung function was significantly improved compared to non-treated pallid mice. Stereological analysis revealed a reduction in pulmonary emphysema. CONCLUSION: Our data indicate that in vivo intrahepatic electroporation-mediated gene transfer of AAT is a safe and efficient procedure resulting in reduction of pulmonary emphysema in pallid mice.

SARS-CoV-2 COVID-19 susceptibility and lung inflammatory storm by smoking and vaping.

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.

Alveolar Macrophage Phenotype and Compartmentalization Drive Different Pulmonary Changes in Mouse Strains Exposed to Cigarette Smoke.

COPD can manifest itself with different clinical phenotypes characterized by different disease progression and response to therapy. Although a remarkable number of studies have been carried out, little is known about the mechanisms underlying phenotypes that could guide the development of viable future therapies. Several murine strains mirror some human phenotypes after smoke exposure. It was of interest to investigate in these strains whether different pattern of activation of macrophages, and their distribution in lungs, is associated to changes characterizing different phenotypes. We chose C57Bl/6, and Lck deficient mice, which show significant emphysema, DBA/2 mice that develop changes similar to those of "pulmonary fibrosis/emphysema syndrome", p66Shc ko mice that develop bronchiolitis with fibrosis but not emphysema, and finally ICR mice that do not develop changes at 7 months after smoke exposure. Unlike other strains, ICR mice show very few activated macrophages (Mac-3 positive) mostly negative to M1 or M2 markers. On the other hand, a large population of M1 macrophages predominates in the lung periphery of DBA/2, C57Bl/6 and in Lck deficient mice, where emphysema is more evident. M2 macrophages are mainly observed in subpleural and intraparenchymal areas of DBA/2 mice and around bronchioles of p66Shc ko mice where fibrotic changes are present. We observed slight but significant differences in mRNA expression of iNOS, ECF-L, arginase 1, IL-4, IL-13 and TGF-ß between air- and smoke-exposed mice. These differences together with the different compartmentalization of macrophages may offer an explanation for the diversity of lesions and their distribution that we observed among the strains.

Innate Immunity and Cell Surface Receptors in the Pathogenesis of COPD: Insights from Mouse Smoking Models.

Chronic obstructive pulmonary disease (COPD) is mainly associated with smoking habit. Inflammation is the major initiating process whereby neutrophils and monocytes are attracted into the lung microenvironment by external stimuli present in tobacco leaves and in cigarette smoke, which promote chemotaxis, adhesion, phagocytosis, release of superoxide anions and enzyme granule contents. A minority of smokers develops COPD and different molecular factors, which contribute to the onset of the disease, have been put forward. After many years of research, the pathogenesis of COPD is still an object of debate. In vivo models of cigarette smoke-induced COPD may help to unravel cellular and molecular mechanisms underlying the pathogenesis of COPD. The mouse represents the most favored animal choice with regard to the study of immune mechanisms due to its genetic and physiological similarities to humans, the availability of a large variability of inbred strains, the presence in the species of several genetic disorders analogous to those in man, and finally on the possibility to create models "made-to-measure" by genetic manipulation. The review outlines the different response of mouse strains to cigarette smoke used in COPD studies while retaining a strong focus on their relatability to human patients. These studies reveal the importance of innate immunity and cell surface receptors in the pathogenesis of pulmonary injury induced by cigarette smoking. They further advance the way in which we use wild type or genetically manipulated strains to improve our overall understanding of a multifaceted disease such as COPD. The structural and functional features, which have been found in the different strains of mice after chronic exposure to cigarette smoke, can be used in preclinical studies to develop effective new therapeutic agents for the different phenotypes in human COPD.

Functional contribution of sphingosine-1-phosphate to airway pathology in cigarette smoke-exposed mice.

BACKGROUND AND PURPOSE: A critical role for sphingosine kinase/sphingosine-1-phosphate (S1P) pathway in the control of airway function has been demonstrated in respiratory diseases. Here, we address S1P contribution in a mouse model of mild chronic obstructive pulmonary disease (COPD). EXPERIMENTAL APPROACH: C57BL/6J mice have been exposed to room air or cigarette smoke up to 11 months and killed at different time points. Functional and molecular studies have been performed. KEY RESULTS: Cigarette smoke caused emphysematous changes throughout the lung parenchyma coupled to a progressive collagen deposition in both peribronchiolar and peribronchial areas. The high and low airways showed an increased reactivity to cholinergic stimulation and α-smooth muscle actin overexpression. Similarly, an increase in airway reactivity and lung resistances following S1P challenge occurred in smoking mice. A high expression of S1P, Sph-K2 , and S1P receptors (S1P2 and S1P3 ) has been detected in the lung of smoking mice. Sphingosine kinases inhibition reversed the increased cholinergic response in airways of smoking mice. CONCLUSIONS AND IMPLICATIONS: S1P signalling up-regulation follows the disease progression in smoking mice and is involved in the development of airway hyperresponsiveness. Our study defines a therapeutic potential for S1P inhibitors in management of airways hyperresponsiveness associated to emphysema in smokers with both asthma and COPD.

Ongoing Lung Inflammation and Disease Progression in Mice after Smoking Cessation: Beneficial Effects of Formyl-Peptide Receptor Blockade.

The most important risk factor for chronic obstructive pulmonary disease (COPD) is cigarette smoking. Until now, smoking cessation (SC) is the only treatment effective in slowing down the progression of the disease. However, in many cases SC may only relieve the airflow obstruction and inflammatory response. Consequently, a persistent lung inflammation in ex-smokers is associated with progressive deterioration of respiratory functions. This is an increasingly important clinical problem whose mechanistic basis remains poorly understood. Available therapies do not adequately suppress inflammation and are not able to stop the vicious cycle that is at the basis of persistent inflammation. In addition, in mice after SC an ongoing inflammation and progressive lung deterioration is observed. After 4 months of smoke exposure mice show mild emphysematous changes. Lung inflammation is still present after SC, and emphysema progresses during the next 6-month period of observation. Destruction of alveolar walls is associated with airways remodeling (goblet cell metaplasia and peribronchiolar fibrosis). Modulation of formyl-peptide receptor signaling with antagonists mitigates inflammation and prevents deterioration of lung structures. This study suggests an important role for N-formylated peptides in the progression and exacerbation of COPD. Modulating formyl-peptide receptor signal should be explored as a potential new therapy for COPD.

Middermal Elastolysis: Dermal Fibroblasts Cooperate with Inflammatory Cells to the Elastolytic Disorder.

Little is known about the cause and pathophysiology of middermal elastolysis (MDE). In this condition, variable inflammatory infiltrate may be present or not together with loss of elastic fibres in the middermis that spares both papillary and lower reticular dermis. MDE may be a consequence of abnormal extracellular matrix degradation related to an imbalance between elastolytic enzymes released from inflammatory and resident cells and their naturally occurring inhibitors. However, the cause of this imbalance is still an object of investigation. In order to shed light on the role of fibroblasts in MDE, we used fibroblast cultures from MDE and control subjects to evaluate matrix metalloproteinases (MMPs) and their major inhibitor TIMP-1, which in combination with neutrophil or macrophage proteases released in inflamed areas may influence the elastolytic burden. We demonstrate that fibroblasts derived from MDE produce in vitro low levels of TIMP-1, the major inhibitor of MMPs. Elevated levels of MMP-2, MMP-14, and TIMP-2 capable to activate in a cooperative manner pro-MMP-2 are present in MDE tissue samples. Additionally, significant reaction for MMP-1 is present in the same MDE areas. These data all together suggest that ECM changes in MDE are due to cooperation of different cell populations (i.e., inflammatory cells and fibroblasts).

P2Y6 Receptor Activation Promotes Inflammation and Tissue Remodeling in Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease with a poor prognosis and very few available treatment options. The involvement of the purinergic receptor subtypes P2Y2 and P2X7 in fibrotic lung disease has been demonstrated recently. In this study, we investigated the role of P2Y6 receptors in the pathogenesis of IPF in humans and in the animal model of bleomycin-induced lung injury. P2Y6R expression was upregulated in lung structural cells but not in bronchoalveolar lavage (BAL) cells derived from IPF patients as well as in animals following bleomycin administration. Furthermore, BAL fluid levels of the P2Y6R agonist uridine-5'-diphosphate were elevated in animals with bleomycin-induced pulmonary fibrosis. Inflammation and fibrosis following bleomycin administration were reduced in P2Y6R-deficient compared to wild-type animals confirming the pathophysiological relevance of P2Y6R subtypes for fibrotic lung diseases. Experiments with bone marrow chimeras revealed the importance of P2Y6R expression on lung structural cells for pulmonary inflammation and fibrosis. Similar effects were obtained when animals were treated with the P2Y6R antagonist MRS2578. In vitro studies demonstrated that proliferation and secretion of the pro-inflammatory/pro-fibrotic cytokine IL-6 by lung fibroblasts are P2Y6R-mediated processes. In summary, our results clearly demonstrate the involvement of P2Y6R subtypes in the pathogenesis of pulmonary fibrosis. Thus, blocking pulmonary P2Y6 receptors might be a new target for the treatment of IPF.

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases.The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms.Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes.Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF.

Proteinase activated receptor-2 counterbalances the vascular effects of endothelin-1 in fibrotic tight-skin mice.

BACKGROUND AND PURPOSE: The majority of the severe vascular complications in fibrosis are a consequence of a deregulated activity of mediators controlling vasomotor tone. One of the most important of these mediators is endothelin-1 (ET-1). Here, we have investigated the role of proteinase-activated receptor 2 (PAR2) in the vascular dysfunction in a model of fibrosis, using tight-skin (Tsk) mice. EXPERIMENTAL APPROACH: Aortas were collected from Tsk, transgenic over-expressing PAR2 (TgPAR2), PAR2 deficient (PAR2-/- ) or the corresponding WT mice. Histological and immunohistochemistry analysis for α-smooth muscle actin, PAR2 and ET-1 receptors were performed on aorta sections. Vascular responses to phenylephrine, ET-1 and PAR2 activating peptide (PAR2-AP) were assessed on aortic rings. KEY RESULTS: In aortas from Tsk mice, responses to phenylephrine were reduced, contractions to ET-1 were increased and vasorelaxation to PAR2-AP was enhanced. These alterations matched changes observed in whole vessel architecture such as vascular fibre re-organization, increased collagen deposition and enhanced α-smooth muscle actin expression. Expression of both ETA receptors and PAR2 was enhanced in Tsk mice. Antagonism of PAR2 potentiated vascular effects of ET-1, whereas antagonism of ETA receptors increased vasorelaxation induced by PAR2-AP. In TgPAR2 mice, responses to ET-1 and ET-1 plasma levels were reduced. Conversely, PAR2-/- mice showed enhanced ET-1 induced contraction in aortic rings and higher circulating ET-1 levels. CONCLUSIONS AND IMPLICATIONS: Our data show that PAR2 counterbalanced enhanced contractions to ET-1 in aortas from Tsk mice. PAR2 could represent a possible target for novel drugs in the treatment of vascular complications in fibrosis. LINKED ARTICLES: This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

Ajulemic acid exerts potent anti-fibrotic effect during the fibrogenic phase of bleomycin lung.

BACKGROUND: Ajulemic acid (AjA) is a synthetic analogue of tetrahydrocannabinol that can prevent and limit progression of skin fibrosis in experimental systemic sclerosis. In this study we investigated whether AjA also prevents and modulates lung fibrosis induced by bleomycin (BLM) when administered in mice during the inflammatory or the fibrogenic phase of the model. METHODS: The anti-inflammatory and antifibrotic efficacy of AjA was evaluated in DBA/2 mice treated orally once a day starting either at day 0 (preventive treatment) or at day 8 (therapeutic treatment) after a single intratracheal instillation of BLM. AjA was given at a dose of 1 mg/kg or 5 mg/kg. Mice were sacrificed at day 8, 14 and 21 after BLM and lungs were processed for histology and morphometry, and examined for HO-proline content and for the expression of transforming growth factor beta 1 (TGF-ß1), phosphorylated Smad2/3 (pSMAD2/3), connective tissue growth factor (CTGF), alpha-smooth muscle actin (α-SMA) and peroxisome proliferator-activated receptor-gamma (PPAR-γ). RESULTS: In the 1st week after BLM challenge, an acute inflammation characterized by neutrophil and macrophage accumulation was the main change present in lung parenchyma. The "switch" between inflammation and fibrosis occurs between day 8 and 14 after BLM instillation and involves the bronchi and vasculature. In the subsequent week (at day 21 after BLM instillation) bronchiolocentric fibrosis with significant increase of tissue collagen develops. The fibrotic response evaluated by morphometry and quantified as HO-proline in lung tissue at day 21 after BLM treatment was significantly reduced in mice receiving either AjA in the inflammatory or in early fibrogenic phase. AjA induces marked change in the expression pattern of products implicated in fibrogenesis, such as TGF-ß1, pSMAD2/3, CTGF and α-SMA. In addition, AjA increases significantly the number of PPAR-γ positive cells and its nuclear localization. CONCLUSIONS: AjA treatment, starting either at day 0 or at day 8 after BLM challenge, counteracts the progression of pulmonary fibrosis. The anti-fibrotic effectiveness of AjA is irrespective of timing of compound administration. Further clinical studies are necessary to establish whether AjA may represent a new therapeutic option for treating fibrotic lung diseases.

Severe Reduction in Number and Function of Peripheral T Cells Does Not Afford Protection toward Emphysema and Bronchial Remodeling Induced in Mice by Cigarette Smoke.

The protein Lck (p56(Lck)) is a Src family tyrosine kinase expressed at all stages of thymocyte development and is required for maturation of T cells. The targeted disruption of Lck gene in mice results in severe block in thymocyte maturation with substantial reduction in the development of CD4(+)CD8(+) thymocytes, severe reduction of peripheral T cells, and disruption of T-cell receptor signaling with defective function of T-cell responses. To investigate the role of T lymphocyte in the development of cigarette smoke-induced pulmonary changes, Lck(-/-) mice and corresponding congenic wild-type mice were chronically exposed to cigarette smoke, and their lungs were analyzed by biochemical, immunologic, and morphometric methods. Smoking mice from both genotypes showed disseminated foci of emphysema and large areas of goblet cell metaplasia in bronchial and bronchiolar epithelium. Morphometric evaluation of lung changes and lung elastin determination confirmed that mice from both genotypes showed the same degree of emphysematous lesions. Thus, cigarette smoke exposure in the presence of severe reduction in number and function of peripheral T cells does not influence the development of pulmonary changes induced by cigarette smoke. The data obtained suggest that innate immunity is a leading actor in the early development of pulmonary changes in smoking mice and that the adaptive immune response may play a role at later stages.

Histopathological data of iron and calcium in the mouse lung after asbestos exposure.

This data article contains data related to the research article entitled, "Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice" [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation.

NTPDase1/CD39 and aberrant purinergic signalling in the pathogenesis of COPD.

Purinergic receptor activation via extracellular ATP is involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). Nucleoside triphosphate diphosphohydrolase-1/CD39 hydrolyses extracellular ATP and modulates P2 receptor signalling.We aimed to investigate the expression and function of CD39 in the pathogenesis of cigarette smoke-induced lung inflammation in patients and preclinical mouse models. CD39 expression and soluble ATPase activity were quantified in sputum and bronchoalveolar lavage fluid (BALF) cells in nonsmokers, smokers and COPD patients or mice with cigarette smoke-induced lung inflammation. In mice, pulmonary ATP and cytokine concentrations, inflammation and emphysema were analysed in the presence or absence of CD39.Following acute cigarette smoke exposure CD39 was upregulated in BALF cells in smokers with further increases in COPD patients. Acute cigarette smoke exposure induced CD39 upregulation in murine lungs and BALF cells, and ATP degradation was accelerated in airway fluids. CD39 inhibition and deficiency led to augmented lung inflammation; treatment with ATPase during cigarette smoke exposure prevented emphysema.Pulmonary CD39 expression and activity are increased in COPD. CD39 deficiency leads to enhanced emphysema in mice, while external administration of a functional CD39 analogue partially rescues the phenotype. The compensatory upregulation of pulmonary CD39 might serve as a protective mechanism in cigarette smoke-induced lung damage.