Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.

Sarcoidosis, alveolar β-actin and pulmonary fibrosis

BACKGROUND: Sarcoidosis is a multisystem granulomatous disease of unknown aetiology. Proteins present within the alveolar space early in sarcoidosis disease may provide an insight into novel mechanisms for the development of fibrotic disease and in particular pulmonary fibrosis.

METHODS: A modified two-dimensional difference gel electrophoresis protocol was applied to the human bronchoalveolar lavage fluid (hBALF) of four patients with non-persistent pulmonary interstitial disease at 4-year follow-up (defined as mild disease) and four patients who developed pulmonary interstitial disease at 4-year follow-up (defined as severe disease). The protein β-actin was identified by LC-MS/MS from a preparative gel and found to be significantly elevated in early lavages from the severe disease group. To look at the potential pro-fibrotic effects of this protein, primary human pulmonary fibroblasts (CCD-19Lu) were treated with recombinant β-actin following which qPCR and ELISA assays were used to measure any effects.

RESULTS: We found that β-actin levels were significantly elevated in early hBALF samples in patients who subsequently developed severe disease when compared to the mild group. Treating primary human pulmonary fibroblasts with recombinant β-actin led to enhanced gene expression of the pro-fibrotic markers alpha smooth muscle actin and collagen 1 as well as the increased secretion of interleukin-13 and metalloproteinases 3 and 9.

CONCLUSION: Free β-actin within the lungs of sarcoidosis patients potentially may contribute to disease pathogenesis particularly in the context of abnormal remodelling and the development of pulmonary fibrosis.

Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion: a new tumor entity.

We present clinicopathologic data on 10 pulmonary myxoid sarcomas, which are defined by distinctive histomorphologic features and characterized by a recurrent fusion gene, that appear to represent a distinct tumor entity at this site. The patients [7 female, 3 male; aged 27 to 67 y (mean, 45 y)] presented with local or systemic symptoms (n=5), symptoms from cerebral metastasis (1), or incidentally (2). Follow-up of 6 patients showed that 1 with brain metastasis died shortly after primary tumor resection, 1 developed a renal metastasis but is alive and well, and 4 are disease free after 1 to 15 years. All tumors involved pulmonary parenchyma, with a predominant endobronchial component in 8 and ranged from 1.5 to 4 cm. Microscopically, they were lobulated and composed of cords of polygonal, spindle, or stellate cells within myxoid stroma, morphologically reminiscent of extraskeletal myxoid chondrosarcoma. Four cases showed no or minimal atypia, 6 showed focal pleomorphism, and 5 had necrosis. Mitotic indices varied, with most tumors not exceeding 5/10 high-power fields. Tumors were immunoreactive for only vimentin and weakly focal for epithelial membrane antigen. Of 9 tumors, 7 were shown to harbor a specific EWSR1-CREB1 fusion by reverse transcription-polymerase chain reaction and direct sequencing, with 7 of 10 showing EWSR1 rearrangement by fluorescence in situ hybridization. This gene fusion has been described previously in 2 histologically and behaviorally different sarcomas: clear cell sarcoma-like tumors of the gastrointestinal tract and angiomatoid fibrous histiocytomas; however, this is a novel finding in tumors with the morphology we describe and that occur in the pulmonary region.

Is There More to This Case than Mere Pulmonary Alveolar Proteinosis? A Clinical Case Presentation

Introduction: Pulmonary alveolar proteinosis (PAP) is a rare disease, associated with excess accumulation of surfactant proteins and lipids in the alveoli. Clinical presentation: We report the case of a 46-year-old woman with a combined presentation of PAP, myelodysplasia and recurrent miscarriages. Conclusions: The concomitant presentation of the above might be compatible with a mutation of the haematopoietic transcription factor gene GATA2.

The Consequences of Not Looking at the Eosinophil Count: Pulmonary Infiltrates and Eosinophilia Syndrome (PIE) Probably Caused by Ciprofloxacin

Objectives: To describe a case of pulmonary infiltrates and eosinophilia (PIE syndrome) probably caused by ciprofloxacin. Materials and methods: A 64-year-old woman was admitted to our department with suspected hospital-acquired pneumonia and treated with antibiotics. She had no symptoms but had peripheral eosinophilia. She had recently been given ciprofloxacin for a urinary tract infection. Results: The patient spontaneously improved after exhaustive negative investigations. Conclusion: We concluded that this patient had PIE syndrome probably caused by ciprofloxacin.

Cardiac magnetic resonance imaging in the diagnosis and determination of outcome of pulmonary hypertension

Pulmonary hypertension (PH) is a rare but serious condition that causes progressive right ventricular (RV) failure and death. PH may be idiopathic, associated with underlying connective-tissue disease or hypoxic lung disease, and is also increasingly being observed in the setting of heart failure with preserved ejection fraction (HFpEF). The management of PH has been revolutionised by the recent development of new disease-targeted therapies which are beneficial in pulmonary arterial hypertension (PAH), but can be potentially harmful in PH due to left heart disease, so accurate diagnosis and classification of patients is essential. These PAH therapies improve exercise capacity and pulmonary haemodynamics, but their overall effect on the right ventricle remains unclear. Current practice in the UK is to assess treatment response with 6-minute walk test and NYHA functional class, neither of which truly reflects RV function. Cardiac magnetic resonance (CMR) imaging has been established as the gold standard for the evaluation of right ventricular structure and function, but it also allows a non-invasive and accurate study of the left heart. The aims of this thesis were to investigate the use of CMR in the diagnosis of PH, in the assessment of treatment response, and in predicting survival in idiopathic and connective-tissue disease associated PAH. In Chapter 3, a left atrial volume (LAV) threshold of 43 ml/m2 measured with CMR was able to distinguish idiopathic PAH from PH due to HFpEF (sensitivity 97%, specificity 100%). In Chapter 4, disease-targeted PAH therapy resulted in significant improvements in RV and left ventricular ejection fraction (p<0.001 and p=0.0007, respectively), RV stroke volume index (p<0.0001), and left ventricular end-diastolic volume index (p=0.0015). These corresponded to observed improvements in functional class and exercise capacity, although correlation coefficients between Δ 6MWD and Δ RVEF or Δ LVEDV were low. Finally, in Chapter 5, one-year and three-year survival was worse in CTD-PAH (75% and 53%) than in IPAH (83% and 74%), despite similar baseline clinical characteristics, lung function, pulmonary haemodynamics and treatment. Baseline right ventricular stroke volume index was an independent predictor of survival in both conditions. The presence of LV systolic dysfunction was of prognostic significance in CTD-PAH but not IPAH, and a higher LAV was observed in CTD-PAH suggesting a potential contribution from LV diastolic dysfunction in this group.

PULMONARY REHABILITATION PROGRAMS IN LUNG TRANSPLANT: A LITERATURE REVIEW

Objective: To analyze, using a literature review, Pulmonary Rehabilitation (RP) Programs in lung transplant. Methods: A literature review in July 2014 in Ebsco Host, Periódicos Capes, BVS and Science Direct data bases using descriptors in English (“lung transplantation”, “lung transplant” AND/OR “rehabilitation”) and Portuguese (“reabilitação” AND/OR “transplante pulmonar”). The eligibility criterions were interventional studies of PR before and/or after lung transplant; participants who were candidates to lung transplant or lung transplant recipients; studies that applied any kind of PR program (hospital-based, homebased or outpatient) and articles published in English, Spanish or Portuguese. Literature reviews, guidelines and case reports were excluded. The search process yielded 46 articles of which two were duplicated. After title and abstract screening 13 articles remained for full text reading. Six studies met the inclusion eligibility and were included in the review. Results: The studies involved patients with Chronic Obstructive Pulmonary Disease, Cystic Fibrosis, Pulmonary Hypertension, Interstitial Lung Disease and Pulmonary Fibrosis. Pulmonary function, exercise capacity, quality of life (QoL) and quadriceps force were evaluated. Most interventions were outpatient programs with three months duration, three times a week and session with at least one hour. Protocols included physical training, educational approach and just one included nutritional, psychiatric and social assistant follow-up. The studies presented significant change in the six-minute walking distance, QoL and quadriceps force after PR programs. Conclusion: This review showed the benefits of the PR in the QoL and exercise capacity contributing to the Health Promotion of the patients.

Plasma inflammatory biomarkers response to aerobic versus resisted exercise training for chronic obstructive pulmonary disease patients.

Background: Chronic obstructive pulmonary disease (COPD) is a main risk for morbidity, associated with alterations in systemic inflammation. Recent studies proved that morbidity and mortality of COPD is related to systemic inflammation as it contributes to the pathogenesis of atherosclerosis and cardiovascular disease. However, increase of inflammatory cytokines adversely affects quality of life, alteration in ventilatory and skeletal muscles functions. Moreover, exercise training has many beneficial effects in correction of the adverse effects of COPD. Objective: This study aimed to compare the response of inflammatory cytokines of COPD to aerobic versus resisted exercises. Materials and methods: One hundred COPD diseased patients participated in this study and were randomly included in two groups; the first group received aerobic exercise, whereas the second group received resisted exercise training for 12 weeks. Results: The mean values of TNF-α, Il-2, IL-4, IL-6 and CRP were significantly decreased in both groups. Also; there was a significant difference between both groups at the end of the study with more reduction in patients who received aerobic exercise training. Conclusion: Aerobic exercise is more appropriate than resisted exercise training in modulating inflammatory cytokines level in patients with chronic obstructive pulmonary disease.

Pulmonary vascular resistance: A meaningless variable?

Almost 20 years ago, Adriaan Versprille published an editorial in this journal to explain why, in his opinion, the calculation of pulmonary vascular resistance (PVR) is meaningless [1]. The uncertainties of PVR were underscored a year later by McGregor and Sniderman in the American Journal of Cardiology [2]. Obviously, both papers failed to convince. A Medline search from 1985 to the end of 2002 reveals no less than 7,158 papers with PVR calculations. What is it that could be wrong in all this literature?.

Edoxaban for preventing stroke and systemic embolism in people with non-valvular atrial fibrillation: A Single Technology Appraisal. Evidence Review Group Report to NICE

1.1 Edoxaban is recommended, within its marketing authorisation, as an option for preventing stroke and systemic embolism in adults with non-valvular atrial fibrillation with one or more risk factors, including: congestive heart failure hypertension diabetes prior stroke or transient ischaemic attack age 75 years or older

Association of Celiac Disease With Idiopathic Pulmonary Hemosiderosis; Lane Hamilton Syndrome

Introduction: Idiopathic Pulmonary Hemosiderosis (IPH) is a rare cause of alveolar hemorrhage, which is seen primarily in childhood. Celiac disease is defined as a chronic, immune-mediated enteropathy of the small intestine, caused by exposure to dietary gluten in genetically pre-disposed individuals. Association of IPH and celiac disease is known as Lane Hamilton syndrome. There are limited number of case reports of this syndrome in literature. Case Presentation: Although there were no growth and developmental delay and gastrointestinal symptoms like chronic diarrhea, chronic constipation, vomiting, abdominal bloating and pain in the two patients with IPH, they were diagnosed with Lane Hamilton Syndrome. After initiation of gluten-free diet, their IPH symptoms disappeared and hemoglobin levels were observed to return to normal. Conclusions: Even if there were no gastrointestinal symptoms in a patient with IPH, celiac disease should be investigated. These patients may benefit from gluten free diet and IPH symptoms may disappear.

Association of myocardial T1-mapping CMR with hemodynamics and RV performance in pulmonary hypertension

Early detection of right ventricular (RV) involvement in chronic pulmonary hypertension (PH) is essential due to prognostic implications. T1 mapping by cardiac magnetic resonance (CMR) has emerged as a noninvasive technique for extracellular volume fraction (ECV) quantification. We assessed the association of myocardial native T1 time and equilibrium contrast ECV (Eq-ECV) at the RV insertion points with pulmonary hemodynamics and RV performance in an experimental model of chronic PH. Right heart catheterization followed by immediate CMR was performed on 38 pigs with chronic PH (generated by surgical pulmonary vein banding) and 6 sham-operated controls. Native T1 and Eq-ECV values at the RV insertion points were both significantly higher in banded animals than in controls and showed significant correlation with pulmonary hemodynamics, RV arterial coupling, and RV performance. Eq-ECV values also increased before overt RV systolic dysfunction, offering potential for the early detection of myocardial involvement in chronic PH.