Mitochondrial Dysfunction in Lung Resident Mesenchymal Stem Cells from Idiopathic Pulmonary Fibrosis Patients.

Idiopathic pulmonary fibrosis (IPF) is characterized by an aberrant repair response with uncontrolled turnover of extracellular matrix involving mesenchymal cell phenotypes, where lung resident mesenchymal stem cells (LRMSC) have been supposed to have an important role. However, the contribution of LRMSC in lung fibrosis is not fully understood, and the role of LRMSC in IPF remains to be elucidated. Here, we performed transcriptomic and functional analyses on LRMSC isolated from IPF and control patients (CON). Both over-representation and gene set enrichment analyses indicated that oxidative phosphorylation is the major dysregulated pathway in IPF LRMSC. The most relevant differences in biological processes included complement activation, mesenchyme development, and aerobic electron transport chain. Compared to CON LRMSC, IPF cells displayed impaired mitochondrial respiration, lower expression of genes involved in mitochondrial dynamics, and dysmorphic mitochondria. These changes were linked to an impaired autophagic response and a lower mRNA expression of pro-apoptotic genes. In addition, IPF TGFß-exposed LRMSC presented different expression profiles of mitochondrial-related genes compared to CON TGFß-treated cells, suggesting that TGFß reinforces mitochondrial dysfunction. In conclusion, these results suggest that mitochondrial dysfunction is a major event in LRMSC and that their occurrence might limit LRMSC function, thereby contributing to IPF development.

Collagen 3D matrices as a model for the study of cell behavior in pulmonary fibrosis.

Purpose: Idiopathic pulmonary fibrosis (IPF) is a complex progressive chronic lung disease where epithelial to mesenchymal interaction, extracellular matrix (ECM) contact, and pro-fibrotic cytokines dynamics take part in the development of the disease. The study of IPF in the widespread in vitro two-dimensional (2 D) culture fails to explain the interaction of cells with the changing environment that occurs in fibrotic lung tissue. A three-dimensional (3 D) co-culture model might shed light on the pathogenesis of IPF by mimicking the fibrotic environment. Materials and Methods: Fibroblasts from nine IPF were isolated and embedded in collagen matrices with the alveolar epithelial human cell line (A549) on the top. Cells were also cultured in 2 D with and without TGF-ß1 as a conventional model to compare with. Both types of cells were isolated separately. Protein and gene expression of the main fibrotic markers were measured by qPCR, Western blot, and ELISA. Results: IPF fibroblasts to myofibroblasts differentiation was observed in the 3 D model and in cells stimulated with TGF-ß1. In addition, ECM-related genes were highly up-regulated in the 3 D collagen matrix. A549 co-cultured 3 D with IPF fibroblasts showed EMT activation, with down-regulation of E-cadherin (CDH1). However, other pro-fibrotic genes as VIM, TGFB1, and MMP7 were up-regulated in A549 co-cultured 3 D with fibroblasts. Conclusions: 3 D-collagen matrices might induce fibroblasts' fibrotic phenotype as in the classic TGF-ß1 model, by up-regulating genes associated with matrix production. In addition, IPF lung fibroblasts seem to exert a pro-fibrotic influence in A549 cells when they are co-cultured. These results suggest that an improved 3 D co-culture model might serve as an important tool to study the fibrotic process and its regulation.

Lung Transplant Improves Survival and Quality of Life Regardless of Telomere Dysfunction.

Introduction: Fibrotic interstitial lung diseases (ILDs) are the first indication for lung transplantation (LT). Telomere dysfunction has been associated with poor post-transplant outcomes. The aim of the study was to evaluate the morbi-mortality and quality of life in fibrotic ILDs after lung transplant depending on telomere biology. Methods: Fibrotic ILD patients that underwent lung transplant were allocated to two arms; with or without telomere dysfunction at diagnosis based on the telomere length and telomerase related gene mutations revealed by whole-exome sequencing. Post-transplant evaluation included: (1) short and long-term mortality and complications and (2) quality of life. Results: Fifty-five percent of patients that underwent LT carried rare coding mutations in telomerase-related genes. Patients with telomere shortening more frequently needed extracorporeal circulation and presented a higher rate of early post-transplant hematological complications, longer stay in the intensive care unit (ICU), and a higher number of long-term hospital admissions. However, post-transplant 1-year survival was higher than 80% regardless of telomere dysfunction, with improvement in the quality of life and oxygen therapy withdrawal. Conclusions: Post-transplant morbidity is higher in patients with telomere dysfunction and differs according to elapsed time from transplantation. However, lung transplant improves survival and quality of life and the associated complications are manageable.

Histamine signaling and metabolism identify potential biomarkers and therapies for lymphangioleiomyomatosis.

Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.

Membrane particles from mesenchymal stromal cells reduce the expression of fibrotic markers on pulmonary cells.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with limited treatment options in which the telomere shortening is a strong predictive factor of poor prognosis. Mesenchymal stromal cells (MSC) administration is probed in several experimental induced lung pathologies; however, MSC might stimulate fibrotic processes. A therapy that avoids MSC side effects of transformation would be an alternative to the use of living cells. Membranes particles (MP) are nanovesicles artificially generated from the membranes of MSC containing active enzymes involved in ECM regeneration. We aimed to investigate the anti-fibrotic role of MP derived from MSC in an in vitro model of pulmonary fibrosis. METHODS: Epithelial cells (A549) and lung fibroblasts, from IPF patients with different telomere length, were co-cultured with MP and TGF-ß for 48h and gene expression of major pro-fibrotic markers were analyzed. RESULTS: About 90% of both types of cells effectively took up MP without cytotoxic effects. MP decreased the expression of profibrotic proteins such as Col1A1, Fibronectin and PAI-1, in A549 cells. In fibroblasts culture, there was a different response in the inhibitory effect of MP on some pro-fibrotic markers when comparing fibroblast from normal telomere length patients (FN) versus short telomere length (FS), but both types showed an inhibition of Col1A1, Tenascin-c, PAI-1 and MMP-1 gene expression after MP treatment. CONCLUSIONS: MP conserve some of the properties attributed to the living MSC. This study shows that MP target lung cells, via which they may have a broad anti-fibrotic effect.

GSE4-loaded nanoparticles a potential therapy for lung fibrosis that enhances pneumocyte growth, reduces apoptosis and DNA damage.

Idiopathic pulmonary fibrosis is a lethal lung fibrotic disease, associated with aging with a mean survival of 2-5 years and no curative treatment. The GSE4 peptide is able to rescue cells from senescence, DNA and oxidative damage, inflammation, and induces telomerase activity. Here, we investigated the protective effect of GSE4 expression in vitro in rat alveolar epithelial cells (AECs), and in vivo in a bleomycin model of lung fibrosis. Bleomycin-injured rat AECs, expressing GSE4 or treated with GSE4-PLGA/PEI nanoparticles showed an increase of telomerase activity, decreased DNA damage, and decreased expression of IL6 and cleaved-caspase 3. In addition, these cells showed an inhibition in expression of fibrotic markers induced by TGF-ß such as collagen-I and III among others. Furthermore, treatment with GSE4-PLGA/PEI nanoparticles in a rat model of bleomycin-induced fibrosis, increased telomerase activity and decreased DNA damage in proSP-C cells. Both in preventive and therapeutic protocols GSE4-PLGA/PEI nanoparticles prevented and attenuated lung damage monitored by SPECT-CT and inhibited collagen deposition. Lungs of rats treated with bleomycin and GSE4-PLGA/PEI nanoparticles showed reduced expression of α-SMA and pro-inflammatory cytokines, increased number of pro-SPC-multicellular structures and increased DNA synthesis in proSP-C cells, indicating therapeutic efficacy of GSE4-nanoparticles in experimental lung fibrosis and a possible curative treatment for lung fibrotic patients.

Serum calprotectin as new biomarker for disease severity in idiopathic pulmonary fibrosis: a cross-sectional study in two independent cohorts.

BACKGROUND: Non-invasive biomarkers for the assessment of disease severity in idiopathic pulmonary fibrosis (IPF) are urgently needed. Calprotectin belongs to the S-100 proteins produced by neutrophils, which likely contribute to IPF pathogenesis. Calprotectin is a well-established biomarker in inflammatory bowel diseases. In this cross-sectional study, we aimed to establish the potential role of calprotectin as a biomarker in IPF. Specifically, we hypothesised that patients with IPF have higher serum calprotectin levels compared with healthy controls, and that calprotectin levels are associated with disease severity. METHODS: Blood samples were obtained from healthy volunteers (n=26) and from two independent IPF cohorts (derivation cohort n=26, validation cohort n=66). Serum calprotectin levels were measured with a commercial kit adapted for that purpose and compared between healthy controls and patients with IPF. Clinical parameters, including forced vital capacity, diffusing capacity for carbon monoxide (DLCO) and the Composite Physiologic Index (CPI), were correlated with calprotectin serum levels. RESULTS: The IPF derivation cohort showed increased serum calprotectin levels compared with healthy controls (2.47±1.67 vs 0.97±0.53 µg/mL, p<0.001). In addition, serum calprotectin levels correlated with DLCO% predicted (r=-0.53, p=0.007) and with CPI (r=0.66, p=0.007). These findings were confirmed in an independent IPF validation cohort. CONCLUSION: Serum calprotectin levels are significantly increased in patients with IPF compared with healthy controls and correlate with DLCO and CPI. Calprotectin might be a potential new biomarker for disease severity in IPF.

Predictive factors and prognostic effect of telomere shortening in pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: The abnormal shortening of telomeres is a mechanism linking ageing to idiopathic pulmonary fibrosis (IPF) that could be useful in the clinical setting. The objective of this study was to identify the IPF patients with higher risk for telomere shortening and to investigate the outcome implications. METHODS: Consecutive Spanish patients were included at diagnosis and followed up for 3 years. DNA blood samples from a Mexican cohort were used to validate the results found in Spanish sporadic IPF. Prior to treatment, telomere length was measured through quantitative polymerase chain reaction (qPCR) and Southern blot. Outcome was assessed according to mortality or need for lung transplantation. A multivariate regression logistic model was used for statistical analysis. RESULTS: Family aggregation, age of <60 years and the presence of non-specific immunological or haematological abnormalities were associated with a higher probability of telomere shortening. Overall, 66.6% of patients younger than 60 years with telomere shortening died or required lung transplantation, independent of functional impairment at diagnosis. By contrast, in patients older than 60 years with telomere shortening, the negative impact of telomere shortening in outcome was not significant. CONCLUSION: Our data indicate that young sporadic IPF patients (<60 years) with some non-specific immunological or haematological abnormalities had higher risk of telomere shortening, and furthermore, they presented a poorer prognosis.

Serum AGE/RAGEs as potential biomarker in idiopathic pulmonary fibrosis.

BACKGROUND: The soluble receptor for advanced glycation end-products (sRAGE) has been suggested that it acts as a decoy for capturing advanced glycation end-products (AGEs) and inhibits the activation of the oxidative stress and apoptotic pathways. Lung AGEs/sRAGE is increased in idiopathic pulmonary fibrosis (IPF). The objective of the study was to evaluate the AGEs and sRAGE levels in serum as a potential biomarker in IPF. METHODS: Serum samples were collected from adult patients: 62 IPF, 22 chronic hypersensitivity pneumonitis (cHP), 20 fibrotic non-specific interstitial pneumonia (fNSIP); and 12 healthy controls. In addition, 23 IPF patients were re-evaluated after 3-year follow-up period. Epidemiological and clinical features were recorded: age, sex, smoking habits, and lung function. AGEs and sRAGE were evaluated by ELISA, and the results were correlated with pulmonary functional test values. RESULTS: IPF and cHP groups presented a significant increase of AGE/sRAGE serum concentration compared with fNSIP patients. Moreover, an inverse correlation between AGEs and sRAGE levels were found in IPF, and serum sRAGE at diagnosis correlated with FVC and DLCO values. Additionally, changes in serum AGEs and sRAGE correlated with % change of FVC, DLCO and TLC during the follow-up. sRAGE levels below 428.25 pg/ml evolved poor survival rates. CONCLUSIONS: These findings demonstrate that the increase of AGE/sRAGE ratio is higher in IPF, although the levels were close to cHP. AGE/sRAGE increase correlates with respiratory functional progression. Furthermore, the concentration of sRAGE in blood stream at diagnosis and follow-up could be considered as a potential prognostic biomarker.

Stent-induced tracheal stenosis can be predicted by IL-8 expression in rabbits.

BACKGROUND: Bare metal stents may cause complications like fibrous encapsulation, granulation and tracheal stenosis. We investigated the behaviour of three commercially available stents in vivo (rabbits) and in vitro (coculture of those stents with epithelial and fibroblast cell lines). Also, we investigated whether development of tracheal stenosis could be predicted by any biological marker. MATERIALS AND METHODS: The tracheae of 30 rabbits were implanted with either nitinol stents, with or without paclitaxel elution, or a cobalt-based stent. An additional ten rabbits underwent mock implantation (controls). Serial peripheral venous blood samples were taken throughout the study, and several cytokines measured. Animals were euthanized on day 90, with immediate tracheal endoscopy and lavage performed, then necropsy. RESULTS: Rabbits with cobalt-based stent exhibited more inflammation and the highest stenosis incidence, with reduced survival. Both in vivo and in vitro, this stent induced higher IL-8 levels than nitinol stents. Most important, the presence of stent-induced tracheal stenosis was closely associated to increase in IL-8 expression in blood just 1 day after tracheal stent implantation: a 1·19-fold increase vs. baseline had 83% sensitivity, 83% specificity, 77% positive predictive value, 88% negative predictive value and 83% accuracy to predict development of stenosis. CONCLUSIONS: The cobalt-based stent had the highest incidence of tracheal inflammation and stenosis. On the other hand, the paclitaxel-eluting nitinol stent did not prevent those complications and provoked a marked reaction compared with the bare nitinol stent. Early increase in IL-8 expression in blood after stent implantation could predict development of tracheal stenosis in rabbits.

Increased AGE-RAGE ratio in idiopathic pulmonary fibrosis.

BACKGROUND: The abnormal epithelial-mesenchymal restorative capacity in idiopathic pulmonary fibrosis (IPF) has been recently associated with an accelerated aging process as a key point for the altered wound healing. The advanced glycation end-products (AGEs) are the consequence of non-enzymatic reactions between lipid and protein with several oxidants in the aging process. The receptor for AGEs (RAGEs) has been implicated in the lung fibrotic process and the alveolar homeostasis. However, this AGE-RAGE aging pathway has been under-explored in IPF. METHODS: Lung samples from 16 IPF and 9 control patients were obtained through surgical lung biopsy. Differences in AGEs and RAGE expression between both groups were evaluated by RT-PCR, Western blot and immunohistochemistry. The effect of AGEs on cell viability of primary lung fibrotic fibroblasts and alveolar epithelial cells was assessed. Cell transformation of fibrotic fibroblasts cultured into glycated matrices was evaluated in different experimental conditions. RESULTS: Our study demonstrates an increase of AGEs together with a decrease of RAGEs in IPF lungs, compared with control samples. Two specific AGEs involved in aging, pentosidine and Nε-Carboxymethyl lysine, were significantly increased in IPF samples. The immunohistochemistry identified higher staining of AGEs related to extracellular matrix (ECM) proteins and the apical surface of the alveolar epithelial cells (AECs) surrounding fibroblast foci in fibrotic lungs. On the other hand, RAGE location was present at the cell membrane of AECs in control lungs, while it was almost missing in pulmonary fibrotic tissue. In addition, in vitro cultures showed that the effect of AGEs on cell viability was different for AECs and fibrotic fibroblasts. AGEs decreased cell viability in AECs, even at low concentration, while fibroblast viability was less affected. Furthermore, fibroblast to myofibroblast transformation could be enhanced by ECM glycation. CONCLUSIONS: All of these findings suggest a possible role of the increased ratio AGEs-RAGEs in IPF, which could be a relevant accelerating aging tissue reaction in the abnormal wound healing of the lung fibrotic process.

Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices.

BACKGROUND: There is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars. METHODS: The present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann-Whitney U, Kruskal Wallis, Student's t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model. RESULTS: Our findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels. CONCLUSIONS: The use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.

Expression of aquaporins in bronchial tissue and lung parenchyma of patients with chronic obstructive pulmonary disease.

BACKGROUND: Aquaporins AQP1 and AQP5 are highly expressed in the lung. Recent studies have shown that the expression of these proteins may be mechanistically involved in the airway inflammation and in the pathogenesis of chronic obstructive pulmonary disease (COPD). The aim of this study was to investigate the expression of AQP1 and AQP5 in the bronchial tissue and the lung parenchyma of patients with COPD and COPD-resistant smokers. METHODS: Using a case-control design, we selected a group of 15 subjects with COPD and 15 resistant smokers (smokers without COPD) as a control, all of whom were undergoing lung resection surgery due to a lung neoplasm. We studied the expression of AQP1 and AQP5 in the bronchial tissue and the lung parenchyma by means of immunohistochemistry and reverse-transcription real-time polymerase chain reaction. Tissue expression of AQP1 and AQP5 was semi-quantitatively assessed in terms of intensity and expression by immunohistochemistry using a 4-point scale ranging from 0 (none) to 3 (maximum). RESULTS: There were no significant differences in gene expression between COPD patients and resistant smokers both in the bronchial tissue and in the lung parenchyma. However, AQP1 gene expression was 2.41-fold higher in the parenchyma of smokers with COPD compared to controls, whereas the AQP5 gene showed the opposite pattern, with a 7.75-fold higher expression in the bronchus of smokers with COPD compared with controls. AQP1 and AQP5 proteins were preferentially expressed in endothelial cells, showing a higher intensity for AQP1 (66.7% of cases with an intensity of 3, and 93.3% of subjects with an extension of 3 among patients with COPD). Subtle interstitial disease was associated with type II pneumocyte hyperplasia and an increased expression of AQP1. CONCLUSIONS: This study provides pilot observations on the differences in AQP1 and AQP5 expression between COPD patients and COPD-resistant smokers. Our findings suggest a potential role for AQP1 in the pathogenesis of COPD.

C-reactive protein and serum amyloid a overexpression in lung tissues of chronic obstructive pulmonary disease patients: a case-control study.

BACKGROUND: Although researchers have consistently demonstrated systemic inflammation in chronic obstructive pulmonary disease (COPD), its origin is yet unknown. We aimed to compare the lung bronchial and parenchymal tissues as potential sources of major acute-phase reactants in COPD patients and resistant smokers. METHODS: Consecutive patients undergoing elective surgery for suspected primary lung cancer were considered for the study. Patients were categorized as COPD or resistant smokers according to their spirometric results. Lung parenchyma and bronchus sections distant from the primary lesion were obtained. C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2 and SAA4) gene expressions were evaluated by RT-PCR. Protein levels were evaluated in paraffin embedded lung tissues by immunohistochemistry and in serum samples by nephelometry. RESULTS: Our study included 85 patients with COPD and 87 resistant smokers. In bronchial and parenchymal tissues, both CRP and SAA were overexpressed in COPD patients. In the bronchus, CRP, SAA1, SAA2, and SA4 gene expressions in COPD patients were 1.89-fold, 4.36-fold, 3.65-fold, and 3.9-fold the control values, respectively. In the parenchyma, CRP, SAA1, and SAA2 gene expressions were 2.41-, 1.97-, and 1.76-fold the control values, respectively. Immunohistochemistry showed an over-stained pattern of these markers on endovascular cells of COPD patients. There was no correlation with serum protein concentration. CONCLUSIONS: These results indicate an overexpression of CRP and SAA in both bronchial and parenchymal tissue in COPD, which differs between both locations, indicating tissue/cell type specificity. The endothelial cells might play a role in the production of theses markers.

Residual thrombosis after a first episode of proximal deep venous thrombosis.

The aim of this study was to analyse the normalization rate of compression ultrasonography after a first episode of proximal deep venous thrombosis (DVT). Patients underwent compressive ultrasound (C-US) examinations during the 3-6 months following the first proximal DVT episode. Normalization rate of compressive ultrasound (C-US) during the follow-up period was 26.4% [95% confidence interval, 16.3-37.3]. Allelic variation in factor XIII gene (Val34Leu) significantly affected the improvement of popliteal residual thrombi (P = 0.019). We also observed a significant negative correlation between D-dimer levels at 3 months and improvement of popliteal residual thrombi (P = 0.016). There was a significant positive correlation between baseline lumen diameter of the femoral thrombi and IL-8 cytokine (P = 0.015). A significant difference was also found between 1 month-tumor necrosis factor (TNF)-α levels and improvement of residual thrombi (P = 0.047). Our results show that normalization after a standard period of anticoagulation is not frequent and procoagulant and inflammatory biomarkers and also some genetic variations might be related to the resolution of thrombosis.