Platelet Activating Factor Receptor and Intercellular Adhesion Molecule-1 Expression Increases in the Small Airway Epithelium and Parenchyma of Patients with Idiopathic Pulmonary Fibrosis: Implications for Microbial Pathogenesis.

Background: Idiopathic pulmonary fibrosis (IPF) is an irreversible lung fibrotic disorder of unknown cause. It has been reported that bacterial and viral co-infections exacerbate disease pathogenesis. These pathogens use adhesion molecules such as platelet activating factor receptor (PAFR) and intercellular adhesion molecule-1 (ICAM-1) to gain cellular entry, causing infections. Methods: Immunohistochemical staining was carried out for lung resections from IPF patients (n = 11) and normal controls (n = 12). The quantification of PAFR and ICAM-1 expression is presented as a percentage in the small airway epithelium. Also, type 2 pneumocytes and alveolar macrophages were counted as cells per mm2 of the parenchymal area and presented as a percentage. All image analysis was done using Image Pro Plus 7.0 software. Results: PAFR expression significantly increased in the small airway epithelium (p < 0.0001), type 2 pneumocytes (p < 0.0001) and alveolar macrophages (p < 0.0001) compared to normal controls. Similar trend was observed for ICAM-1 expression in the small airway epithelium (p < 0.0001), type 2 pneumocytes (p < 0.0001) and alveolar macrophages (p < 0.0001) compared to normal controls. Furthermore, the proportion of positively expressed type 2 pneumocytes and alveolar macrophages was higher in IPF than in normal control. Conclusions: This is the first study to show PAFR and ICAM-1 expression in small airway epithelium, type 2 pneumocytes and alveolar macrophages in IPF. These findings could help intervene microbial impact and facilitate management of disease pathogenesis.

Differential expression of mast cells in the small airways and alveolar septa of current smokers and patients with small airway disease and COPD.

Background: COPD patients suffer from dysregulated and suppressed immune functionality, determined by their loss of degranulating capacity. Here we provide crucial information on the presence of degranulated mast cells (MCs) in COPD airways and demonstrate their relationship to lung physiology and airway remodelling. Methods: Small airway lung resections from non-smoking controls (NC), normal lung function smokers (NLFS), small airway disease (SAD), and mild-to-moderate COPD current smokers (COPD-CS) and ex-smokers (COPD-ES) were dual immuno-stained with MC tryptase and degranulation marker lysosome-associated membrane protein (LAMP)-1. Total MCs, degranulating MCs and non-MCs were enumerated in small airway epithelium and subepithelium, and in alveolar septa. Results: In the small airway wall subepithelial areas, COPD-CS and COPD-ES patients had significantly lower MCs than the NC group (p<0.05), although the numbers were considerably higher in the small airway epithelium (p<0.01). Degranulating non-MCs were higher in SAD (p<0.05) than in COPD in the small airway subepithelium. In contrast, there were significant increases in total MCs (degranulated and non-degranulated) and degranulated non-MCs in the alveolar septum of COPD patients compared with the NC group (p<001). The lower numbers of MCs in the subepithelium correlated with lower forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC) and forced expiratory flow at 25-75% of FVC (FEF25-75%), higher smoking rates in COPD patients, and increased small airway wall thickness and extracellular matrix. The increase in MCs in the alveolar septum negatively correlated with FEF25-75%. Conclusions: This study is the first to assess the differential pattern of MC, degranulating MC and non-MC populations in the small airways and alveoli of COPD patients. The spatial positioning of the MCs within the airways showed variable correlations with lung function.

TGF-ß1, pSmad-2/3, Smad-7, and ß-Catenin Are Augmented in the Pulmonary Arteries from Patients with Idiopathic Pulmonary Fibrosis (IPF): Role in Driving Endothelial-to-Mesenchymal Transition (EndMT).

Background: We have previously reported that endothelial-to-mesenchymal transition (EndMT) is an active process in patients with idiopathic pulmonary fibrosis (IPF) contributing to arterial remodelling. Here, we aim to quantify drivers of EndMT in IPF patients compared to normal controls (NCs). Methods: Lung resections from thirteen IPF patients and eleven NCs were immunohistochemically stained for EndMT drivers, including TGF-ß1, pSmad-2/3, Smad-7, and ß-catenin. Intima, media, and adventitia were analysed for expression of each EndMT driver in pulmonary arteries. Computer- and microscope-assisted Image ProPlus7.0 image analysis software was used for quantifications. Results: Significant TGF-ß1, pSmad-2/3, Smad-7, and ß-catenin expression was apparent across all arterial sizes in IPF (p < 0.05). Intimal TGF-ß1, pSmad-2/3, Smad-7, and ß-catenin were augmented in the arterial range of 100-1000 µm (p < 0.001) compared to NC. Intimal TGF-ß1 and ß-catenin percentage expression showed a strong correlation with the percentage expression of intimal vimentin (r' = 0.54, p = 0.05 and r' = 0.61, p = 0.02, respectively) and intimal N-cadherin (r' = 0.62, p = 0.03 and r' = 0.70, p = 0.001, respectively). Intimal TGF-ß1 and ß-catenin expression were significantly correlated with increased intimal thickness as well (r' = 0.52, p = 0.04; r' = 0.052, p = 0.04, respectively). Moreover, intimal TGF-ß1 expression was also significantly associated with increased intimal elastin deposition (r' = 0.79, p = 0.002). Furthermore, total TGF-ß1 expression significantly impacted the percentage of DLCO (r' = -0.61, p = 0.03). Conclusions: This is the first study to illustrate the involvement of active TGF-ß/Smad-2/3-dependent and ß-catenin-dependent Wnt signalling pathways in driving EndMT and resultant pulmonary arterial remodelling in patients with IPF. EndMT is a potential therapeutic target for vascular remodelling and fibrosis in general in patients with IPF.

Endothelial to mesenchymal transition is an active process in smokers and patients with early COPD contributing to pulmonary arterial pathology.

Background: We have previously reported pulmonary arterial remodelling in smokers and patients with early COPD, which can be attributed to endothelial to mesenchymal transition (EndMT). In this study, we aimed to evaluate if EndMT is an active mechanism in smokers and COPD. Methods: Immunohistochemical staining for the EndMT biomarkers CD31, N-cadherin, vimentin and S100A4 was done on lung resection tissue from 49 subjects. These comprised 15 nonsmoker controls (NC), six normal lung function smokers (NLFS), nine patients with small airway disease (SAD), nine current smokers with mild-moderate COPD (COPD-CS) and 10 ex-smokers with COPD (COPD-ES). Pulmonary arteries were analysed using Image ProPlus software v7.0. Results: We noted reduced junctional CD31+ endothelial cells (p<0.05) in the intimal layer of all smoking groups compared to NC. We also observed increased abundance of the mesenchymal markers N-cadherin (p<0.05) and vimentin (p<0.001) in all smoking groups and across all arterial sizes versus NC, except for N-cadherin in large arteries in COPD-CS. The abundance of S100A4 correlated with arterial thickness (small: r=0.29, p=0.05; medium: r=0.33, p=0.03; large: r=0.35, p=0.02). Vimentin in the small arterial wall negatively correlated with forced expiratory volume in 1 s/forced vital capacity (r= -0.35, p=0.02) and forced expiratory flow rate at 25-75% of forced vital capacity (r= -0.34, p=0.03), while increased cytoplasmic CD31 abundance in the intimal layer of medium and large arteries negatively correlated with predicted diffusing capacity of the lung for carbon monoxide (medium: r= -0.35, p=0.04; large: r= -0.39, p=0.03). Conclusion: This is the first study showing the acquisition of mesenchymal traits by pulmonary endothelial cells from NLFS, SAD and mild-moderate COPD patients through EndMT. This informs on the potential early origins of pulmonary hypertension in smokers and patients with early COPD.

Airway inflammatory changes in the lungs of patients with asthma-COPD overlap (ACO): a bronchoscopy endobronchial biopsy study.

BACKGROUND: Although asthma and chronic obstructive pulmonary disease (COPD) are two distinct chronic airway inflammatory diseases, they often co-exist in a patient and the condition is referred to as asthma-COPD overlap (ACO). Lack of evidence regarding the inflammatory cells in ACO airways has led to their poor prognosis and treatment. The objective of this endobronchial biopsy (EBB) study was to enumerate inflammatory cellular changes in the airway wall of ACO compared with asthma, COPD current smokers (CS) and ex-smokers (ES), normal lung function smokers (NLFS), and non-smoker controls (HC). METHODS: EBB tissues from 74 patients were immunohistochemically stained for macrophages, mast cells, eosinophils, neutrophils, CD8+ T-cells and CD4+ T-cells. The microscopic images of stained tissues were evaluated in the epithelium, reticular basement membrane (RBM) cells/mm RBM length, and lamina propria (LP) cells/mm2 up to a depth of 120 µM using the image analysis software Image-Pro Plus 7.0. The observer was blinded to the images and disease diagnosis. Statistical analysis was performed using GraphPad Prism v9. RESULTS: The tissue macrophages in ACO were substantially higher in the epithelium and RBM than in HC (P < 0.001 for both), COPD-ES (P < 0.001 for both), and -CS (P < 0.05 and < 0.0001, respectively). The ACO LP macrophages were significantly higher in number than COPD-CS (P < 0.05). The mast cell numbers in ACO were lower than in NLFS (P < 0.05) in the epithelium, lower than COPD (P < 0.05) and NLFS (P < 0.001) in RBM; and lower than  HC (P < 0.05) in LP. We noted lower eosinophils in ACO LP than HC (P < 0.05) and the lowest neutrophils in both ACO and asthma. Furthermore, CD8+ T-cell numbers increased in the ACO RBM than HC (P < 0.05), COPD-ES (P < 0.05), and NLFS (P < 0.01); however, they were similar in number in epithelium and LP across groups. CD4+ T-cells remained lower in number across all regions and groups. CONCLUSION: These results suggest that the ACO airway tissue inflammatory cellular profile differed from the contributing diseases of asthma and COPD with a predominance of macrophages.

Endothelial-to-mesenchymal transition: a precursor to pulmonary arterial remodelling in patients with idiopathic pulmonary fibrosis.

Background: We have previously reported arterial remodelling in patients with idiopathic pulmonary fibrosis (IPF) and suggested that endothelial-to-mesenchymal transition (EndMT) might be central to these changes. This study aims to provide evidence for active EndMT in IPF patients. Methods: Lung resections from 13 patients with IPF and 15 normal controls (NCs) were immunostained for EndMT biomarkers: vascular endothelial cadherin (VE-cadherin), neural cadherin (N-cadherin), S100A4 and vimentin. Pulmonary arteries were analysed for EndMT markers by using computer- and microscope-assisted image analysis software Image ProPlus7.0. All the analysis was done with observer blinded to subject and diagnosis. Results: Increased expression of mesenchymal markers N-cadherin (p<0.0001), vimentin (p<0.0001) and S100A4 (p<0.05) was noted with downregulation of junctional endothelial VE-cadherin (p<0.01) in the intimal layer of the arteries from patients with IPF compared to NCs. Cadherin switch was observed in IPF patients, showing increase in endothelial N-cadherin and decrease in VE-cadherin (p<0.01). There was also VE-cadherin shift from junctions to cytoplasm (p<0.01), effecting endothelial cell integrity in patients with IPF. In IPF, individual mesenchymal markers vimentin and N-cadherin negatively correlated with diffusing capacity of the lungs for carbon monoxide (r'= -0.63, p=0.03 and r'= -0.66, p=0.01). Further, N-cadherin positively correlated with arterial thickness (r'=0.58, p=0.03). Conclusion: This is the first study to demonstrate active EndMT in size-based classified pulmonary arteries from IPF patients and potential role in driving remodelling changes. The mesenchymal markers had a negative impact on the diffusing capacity of the lungs for carbon monoxide. This work also informs early origins of pulmonary hypertension in patients with IPF.

Arterial remodelling in smokers and in patients with small airway disease and COPD: implications for lung physiology and early origins of pulmonary hypertension.

Introduction: Pulmonary vascular remodelling in chronic obstructive pulmonary disease (COPD) has detrimental consequences for lung physiology. The aim of our study was to provide a comprehensive size-based morphometric quantification of pulmonary arterial remodelling in smokers and in patients with small airway disease (SAD) or COPD. Method: Movat's pentachrome staining was performed on lung resections for 46 subjects: 12 never-smoker normal controls (NC), six normal lung function smokers (NLFS), nine patients with SAD, nine patients with mild-to-moderate COPD who were current smokers (COPD-CS) and 10 patients with mild-to-moderate COPD who were ex-smokers (COPD-ES). Following a size-based classification of pulmonary arteries, image analysis software was used to measure their number, total wall thickness, individual layer thickness and elastin percentage. Results: All pathological groups showed decreased numbers of pulmonary arteries compared with the NC group in all artery sizes. Arterial wall thickness was greater in NLFS and COPD-CS than in NC. Thickness in COPD-ES was decreased compared with COPD-CS. Intimal thickness was greater in all pathological groups in all arterial sizes than in the NC group. Medial thickness was also greater in small and medium arteries. Intimal thickness of larger arteries in COPD-CS correlated negatively to forced expiratory volume in 1 s/forced vital capacity (FVC) % and forced expiratory flow at 25-75% of FVC. Elastin deposition in small arteries was greatest in COPD-CS. Intimal elastin deposition had a more negative correlation with intimal thickness in NLFS and SAD than in COPD-CS. Conclusion: Smoking, SAD and mild-to-moderate COPD are associated with pruning and a decrease in the number of pulmonary arteries, increased wall thickness and variable elastin deposition. These changes were associated with worse airway obstruction.

Differential airway remodeling changes were observed in patients with asthma COPD overlap compared to patients with asthma and COPD alone.

Management of patients with asthma COPD overlap (ACO) is clinically challenging due to insufficient evidence of pathological changes in these patients. In this cross-sectional study, we evaluated airway remodeling in endobronchial biopsies from a total of 90 subjects, which included 12 ACO, 14 patients with asthma, 12 COPD exsmokers (ES), 11 current smokers (CS), 28 healthy controls (HC), and 13 normal lung function smokers (NLFS). Tissue was stained with Masson's trichrome. Epithelium, goblet cells, reticular basement membrane (RBM), cellularity, lamina propria (LP), and smooth muscle (SM) changes were measured using Image-Pro Plus v7 software. Differential airway remodeling pattern was seen in patients with ACO. A limited change was noted in the ACO epithelium compared with other pathological groups. RBM was substantially thicker in patients with ACO than in HC (P < 0.0002) and tended to be thicker than in patients with asthma and NLFS. The total RBM cells were higher in ACO than in the HC (P < 0.0001), COPD-CS (P = 0.0559), -ES (P = 0.0345), and NLFS (P < 0.0002), but did not differ from patients with asthma. Goblet cells were higher in the ACO than in the HC (P = 0.0028) and COPD-ES (P = 0.0081). The total LP cells in ACO appeared to be higher than in HC, COPD-CS, and NLFS but appeared to be lower than in patients with asthma. Finally, SM area was significantly lower in the ACO than in patients with asthma (P = 0.001), COPD-CS (=0.0290), and NLFS (P = 0.0011). This first comprehensive study suggests that patients with ACO had distinguishable tissue remodeling that appeared to be more severe than patients with asthma and COPD. This study will help in informed decision-making for better patient management in clinical practice.

Vascular remodelling in idiopathic pulmonary fibrosis patients and its detrimental effect on lung physiology: potential role of endothelial-to-mesenchymal transition.

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible fibrotic interstitial lung disease. We performed size-based quantitation of pulmonary arterial remodelling in IPF and examined the role of endothelial-to-mesenchymal transition (EndMT) and effects on lung physiology. Methods: Resected lung tissues from 11 normal controls (NCs), and 13 IPF patients were differentially stained using the Movat Pentachrome technique. Size-based classification for pulmonary arteries was conducted in NC and IPF tissues. For each pulmonary artery, arterial size, luminal diameter, thickness of the intima, media and adventitia, and elastin deposition were quantified using Image ProPlus7.0 software. In addition, immunohistochemical staining was performed for EndMT markers and collagen. Results: Large and medium-size arterial numbers were significantly reduced in IPF compared to NCs (p<0.0001). Intima thickness was highest in the arterial range of 200-399 µm and 600-1000 µm (p<0.0001), while medial and adventitial thickness was significant across 200-1000 µm (p<0.05) compared to NC. Medial thickness was found to significantly affect the diffusing capacity of the lungs for carbon monoxide (D LCO) (r=-0.8, p=0.01). Total arterial elastin in IPF was higher across all arterial ranges except 100-199 µm in IPF than in NC, with the greatest differences in 200-399 µm (p<0.001) and 600-1000 µm (p<0.001). Total elastin also negatively correlated with D LCO (r'=-0.63, p=0.04) in IPF. An increase in EndMT markers and collagen type I/ IV was observed. Conclusions: This is the first study demonstrating size-based differences in pulmonary arteries in IPF and its detrimental effect on lung physiology. The process of EndMT might be central to these vascular remodelling changes and could be a potential novel therapeutic target.

Clinical Application of Forced Oscillation Technique (FOT) in Early Detection of Airway Changes in Smokers.

The forced oscillation technique (FOT) is a non-invasive method to assess airway function by emitting oscillatory signals into the respiratory tract during tidal ventilation. This opinion piece discusses the current use, trialled modification and future directions in utilizing FOT as a novel diagnostic tool for early detection of small airway changes in smokers. The published evidence to date has shown that FOT parameters could be a sensitive diagnostic tool to detect early respiratory changes in smokers. Multiple frequencies and the frequency dependence of resistance and reactance can provide the most valuable and early information regarding smoking induced changes in airways. Considering its non-invasiveness, lower level of discomfort to patients than spirometry, feasibility, and cost effectiveness, it could be the first-choice diagnostic technique for detection of early respiratory changes in smokers. The finding of FOT could further be supported and correlated with inflammatory markers.

Effect of long term high altitude exposure on cardiovascular autonomic adjustment during rest and post-exercise recovery.

BACKGROUND: Despite the successful adaptation to high altitude, some differences do occur due to long term exposure to the hypoxic environment. The effect of long term high altitude exposure on cardiac autonomic adjustment during basal and post-exercise recovery is less known. Thus we aimed to study the differences in basal cardiac autonomic adjustment and its response to exercise in highlanders and to compare it with lowlanders. METHODS: The study was conducted on 29 healthy highlander males who were born and brought up at altitude of 3000 m and above from the sea level, their cardiac autonomic adjustment was compared with age, sex, physical activity and ethnicity-matched 29 healthy lowlanders using Heart Rate Variability (HRV) during rest and recovery from sub-maximal exercise (3 m step test). Intergroup comparison between the highlanders and lowlanders and intragroup comparison between the rest and the postexercise recovery conditions were done. RESULTS: Resting heart rate and HRV during rest was comparable between the groups. However, heart rate recovery after 3 min step test was faster in highlanders (p < 0.05) along with significantly higher LF power and total power during the recovery phase. Intragroup comparison of highlanders showed higher SDNN (p < 0.05) and lower LF/HF ratio (p < 0.05) during recovery phase compared to rest which was not significantly different in two phases in lowlanders. Further highlander showed complete recovery of RMSSD, NN50, pNN50 and HF power back to resting level within five minutes, whereas, these parameters failed to return back to resting level in lowlanders within the same time frame. CONCLUSION: Highlanders completely recovered back to their resting state within five minutes from cessation of step test with parasympathetic reactivation; however, recovery in lowlanders was delayed.

Pattern of thyroid dysfunction in patients with metabolic syndrome and its relationship with components of metabolic syndrome.

BACKGROUND: Thyroid dysfunction (TD) and metabolic syndrome (MetS) are known risk factors for atherosclerotic cardiovascular disease (ASCVD). TD is risk factor for ASCVD mediated by the effects of thyroid hormones on lipid metabolism and blood pressure hence the components of MetS. It is possible that coexistence of these two disease entities and unrecognized TD in patients with MetS might substantially increase ASCVD risk. Moreover, little is known about the relationship between TD and the components of MetS. Thus, the purpose of this study was to evaluate the pattern of TD in patients with MetS and its relationship with components of the MetS. METHODS: A total of 358 previously diagnosed patients with MetS were recruited in the study. The thyroid function test parameters were measured to classify TD at Dhulikhel Hospital-Kathmandu University Hospital, Dhulikhel, Nepal. Statistical analyses were performed using SPSS version 16.0 to evaluate pattern and relationship. RESULTS: The overall prevalence of TD in patients with MetS was 31.84% with high prevalence of subclinical hypothyroidism (29.32%). We found no evidence of a relationship between TD and components of MetS, although there was significant difference in waist circumference between four groups of TD. CONCLUSION: Patients with MetS had subclinical hypothyroidism greatly. Although there was no evidence of any relationship between thyroid status and all components of MetS, TD should be taken into account when evaluating and treating patients with MetS to reduce the impending risk.