Absence of HDAC3 by Matrix Stiffness Promotes Chromatin Remodeling and Fibroblast Activation in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic and fatal disease characterized by progressive and irreversible lung scarring associated with persistent activation of fibroblasts. Epigenetics could integrate diverse microenvironmental signals, such as stiffness, to direct persistent fibroblast activation. Histone modifications by deacetylases (HDAC) may play an essential role in the gene expression changes involved in the pathological remodeling of the lung. Particularly, HDAC3 is crucial for maintaining chromatin and regulating gene expression, but little is known about its role in IPF. In the study, control and IPF-derived fibroblasts were used to determine the influence of HDAC3 on chromatin remodeling and gene expression associated with IPF signature. Additionally, the cells were grown on hydrogels to mimic the stiffness of a fibrotic lung. Our results showed a decreased HDAC3 in the nucleus of IPF fibroblasts, which correlates with changes in nucleus size and heterochromatin loss. The inhibition of HDAC3 with a pharmacological inhibitor causes hyperacetylation of H3K9 and provokes an increased expression of Col1A1, ACTA2, and p21. Comparable results were found in hydrogels, where matrix stiffness promotes the loss of nuclear HDAC3 and increases the profibrotic signature. Finally, latrunculin b was used to confirm that changes by stiffness depend on the mechanotransduction signals. Together, these results suggest that HDAC3 could be a link between epigenetic mechanisms and the fibrotic microenvironment.

Functional Repercussions of Hypoxia-Inducible Factor-2α in Idiopathic Pulmonary Fibrosis.

Hypoxia and hypoxia-inducible factors (HIFs) are essential in regulating several cellular processes, such as survival, differentiation, and the cell cycle; this adaptation is orchestrated in a complex way. In this review, we focused on the impact of hypoxia in the physiopathology of idiopathic pulmonary fibrosis (IPF) related to lung development, regeneration, and repair. There is robust evidence that the responses of HIF-1α and -2α differ; HIF-1α participates mainly in the acute phase of the response to hypoxia, and HIF-2α in the chronic phase. The analysis of their structure and of different studies showed a high specificity according to the tissue and the process involved. We propose that hypoxia-inducible transcription factor 2a (HIF-2α) is part of the persistent aberrant regeneration associated with developing IPF.

Effect of Hypoxia in the Transcriptomic Profile of Lung Fibroblasts from Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated disease characterized by exacerbated extracellular matrix deposition that disrupts oxygen exchange. Hypoxia and its transcription factors (HIF-1α and 2α) influence numerous circuits that could perpetuate fibrosis by increasing myofibroblasts differentiation and by promoting extracellular matrix accumulation. Therefore, this work aimed to elucidate the signature of hypoxia in the transcriptomic circuitry of IPF-derived fibroblasts. To determine this transcriptomic signature, a gene expression analysis with six lines of lung fibroblasts under normoxia or hypoxia was performed: three cell lines were derived from patients with IPF, and three were from healthy donors, a total of 36 replicates. We used the Clariom D platform, which allows us to evaluate a huge number of transcripts, to analyze the response to hypoxia in both controls and IPF. The control's response is greater by the number of genes and complexity. In the search for specific genes responsible for the IPF fibroblast phenotype, nineteen dysregulated genes were found in lung fibroblasts from IPF patients in hypoxia (nine upregulated and ten downregulated). In this sense, the signaling pathways revealed to be affected in the pulmonary fibroblasts of patients with IPF may represent an adaptation to chronic hypoxia.

ADAR1 Isoforms Regulate Let-7d Processing in Idiopathic Pulmonary Fibrosis.

Double-stranded RNA adenosine deaminase 1 (ADAR1) is significantly down-regulated in fibroblasts derived from Idiopathic Pulmonary Fibrosis (IPF) patients, and its overexpression restored levels of miRNA-21, PELI1, and SPRY2. There are two ADAR1 isoforms in humans, ADAR1-p110 and ADAR1-p150, generated by an alternative promoter. Let-7d is considered an essential microRNA in Pulmonary Fibrosis (PF). In silico analysis revealed COL3A1 and SMAD2, proteins involved in the development of IPF, as Let-7d targets. We analyzed the role of ADAR1-p110 and ADAR1-p150 isoforms in the regulation of Let-7d maturation and the effect of this regulation on the expression of COL3A1 and SMAD2 in IPF fibroblast. We demonstrated that differential expression and subcellular distribution of ADAR1 isoforms in fibroblasts contribute to the up-regulation of pri-miR-Let-7d and down-regulation of mature Let-7d. Induction of overexpression of ADAR1 reestablishes the expression of pri-miR-Let-7d and Let-7d in lung fibroblasts. The reduction of mature Let-7d upregulates the expression of COL3A1 and SMAD2. Thus, ADAR1 isoforms and Let-7d could have a synergistic role in IPF, which is a promising explanation in the mechanisms of fibrosis development, and the regulation of both molecules could be used as a therapeutic approach in IPF.

Mitochondrial Dysfunction and Alterations in Mitochondrial Permeability Transition Pore (mPTP) Contribute to Apoptosis Resistance in Idiopathic Pulmonary Fibrosis Fibroblasts.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by increased activation of fibroblasts/myofibroblasts. Previous reports have shown that IPF fibroblasts are resistant to apoptosis, but the mechanisms remain unclear. Since inhibition of the mitochondrial permeability transition pore (mPTP) has been implicated in the resistance to apoptosis, in this study, we analyzed the role of mitochondrial function and the mPTP on the apoptosis resistance of IPF fibroblasts under basal conditions and after mitomycin C-induced apoptosis. We measured the release of cytochrome c, mPTP opening, mitochondrial calcium release, oxygen consumption, mitochondrial membrane potential, ADP/ATP ratio, ATP concentration, and mitochondrial morphology. We found that IPF fibroblasts were resistant to mitomycin C-induced apoptosis and that calcium, a well-established activator of mPTP, is decreased as well as the release of pro-apoptotic proteins such as cytochrome c. Likewise, IPF fibroblasts showed decreased mitochondrial function, while mPTP was less sensitive to ionomycin-induced opening. Although IPF fibroblasts did not present changes in the mitochondrial membrane potential, we found a fragmented mitochondrial network with scarce, thinned, and disordered mitochondria with reduced ATP levels. Our findings demonstrate that IPF fibroblasts are resistant to mitomycin C-induced apoptosis and that altered mPTP opening contributes to this resistance. In addition, IPF fibroblasts show mitochondrial dysfunction evidenced by a decrease in respiratory parameters.

Loss of MT1-MMP in Alveolar Epithelial Cells Exacerbates Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal age-related lung disease whose pathogenesis involves an aberrant response of alveolar epithelial cells (AEC). Activated epithelial cells secrete mediators that participate in the activation of fibroblasts and the excessive deposition of extracellular matrix proteins. Previous studies indicate that matrix metalloproteinase 14 (MMP14) is increased in the lung epithelium in patients with IPF, however, the role of this membrane-type matrix metalloproteinase has not been elucidated. In this study, the role of Mmp14 was explored in experimental lung fibrosis induced with bleomycin in a conditional mouse model of lung epithelial MMP14-specific genetic deletion. Our results show that epithelial Mmp14 deficiency in mice increases the severity and extension of fibrotic injury and affects the resolution of the lesions. Gain-and loss-of-function experiments with human epithelial cell line A549 demonstrated that cells with a deficiency of MMP14 exhibited increased senescence-associated markers. Moreover, conditioned medium from these cells increased fibroblast expression of fibrotic molecules. These findings suggest a new anti-fibrotic mechanism of MMP14 associated with anti-senescent activity, and consequently, its absence results in impaired lung repair. Increased MMP14 in IPF may represent an anti-fibrotic mechanism that is overwhelmed by the strong profibrotic microenvironment that characterizes this disease.

Dendrometric evaluation of a clonal population of Tectona grandis in forest-livestock system / Levantamento dendrométrico de um povoamento clonal de Tectona grandis, em sistema silvipastoril

ABSTRACT: Production of Tectona grandis (teak) in integrated systems with livestock or agriculture demonstrates high potential of financial return. However, studies on the development of teak are still scarce, especially in the northern region state of Mato Grosso. In this study we sought to evaluate dendometric variables of a clonal population of teak in a forest-livestock integration system (LFIS), during a period of 53 months in the city of Alta Floresta, Mato Grosso, Brazil. For this purpose, three installments were samples, a total of 360 individuals, and for each the diameter was measured at 1.3 meters from the base so as to calculate the total volume, the current annual increment (CAI) and mean annual increment (MAI), and five adjusted regression models. The Hoerl model provided the highest adjusted coefficient of determination (R2aj), lowest standard error of estimate (Syx), coefficient of variation (CV %), and from this the growth curves were developed. Clonal stands of teakin the forest-livestock system presented increases in DBH, height and volume were superior in relation to other scientific studies with teak, indicating their viability in integrated systems with pastures in the region.

RESUMO: A produção de madeira de Tectona grandis (teca) em sistemas integrados com pecuária ou agricultura demonstra alta perspectiva de retorno financeiro. Entretanto, estudos sobre o desenvolvimento da teca ainda são escassos, principalmente na região norte de Mato Grosso. O objetivo do trabalho foi avaliar o crescimento das variáveis dendrométricos de um povoamento clonal de teca, em um sistema silvipastoril (iPF), ao longo de 53 meses, no município de Alta Floresta-MT, Brasil. Para isso, foram amostradas três parcelas, totalizando 360 indivíduos, sendo que em cada indivíduo foi mensurado o diâmetro a 1,3 m do solo e altura total. Em seguida realizou-se o cálculo do volume. Para descrever o crescimento da variável diâmetro, altura total e volume foi realizado o ajuste através de cinco modelos matemáticos que expressam o crescimento ao longo do tempo. Posteriormente, foram calculados o incremento corrente anual (ICA) e incremento médio anual (IMA). O modelo de Hoerl apresentou melhores resultados de R²aj, obteve menores valores para o erro padrão de estimativa (Syx) e para o CV%, com isso, foi selecionado para a elaboração das curvas de crescimento. O povoamento clonal de teca no iPF apresentou incremento em DAP, Altura e volume foram superiores em relação a outros estudos científicos com teca, indicando sua viabilidade em sistemas integrados com pastagens na região.

Dendrometric evaluation of a clonal population of Tectona grandis in forest-livestock system / Levantamento dendrométrico de um povoamento clonal de Tectona grandis, em sistema silvipastoril

Production of Tectona grandis (teak) in integrated systems with livestock or agriculture demonstrates high potential of financial return. However, studies on the development of teak are still scarce, especially in the northern region state of Mato Grosso. In this study we sought to evaluate dendometric variables of a clonal population of teak in a forest-livestock integration system (LFIS), during a period of 53 months in the city of Alta Floresta, Mato Grosso, Brazil. For this purpose, three installments were samples, a total of 360 individuals, and for each the diameter was measured at 1.3 meters from the base so as to calculate the total volume, the current annual increment (CAI) and mean annual increment (MAI), and five adjusted regression models. The Hoerl model provided the highest adjusted coefficient of determination (R2aj), lowest standard error of estimate (Syx), coefficient of variation (CV %), and from this the growth curves were developed. Clonal stands of teakin the forest-livestock system presented increases in DBH, height and volume were superior in relation to other scientific studies with teak, indicating their viability in integrated systems with pastures in the region.(AU)

A produção de madeira de Tectona grandis (teca) em sistemas integrados com pecuária ou agricultura demonstra alta perspectiva de retorno financeiro. Entretanto, estudos sobre o desenvolvimento da teca ainda são escassos, principalmente na região norte de Mato Grosso. O objetivo do trabalho foi avaliar o crescimento das variáveis dendrométricos de um povoamento clonal de teca, em um sistema silvipastoril (iPF), ao longo de 53 meses, no município de Alta Floresta-MT, Brasil. Para isso, foram amostradas três parcelas, totalizando 360 indivíduos, sendo que em cada indivíduo foi mensurado o diâmetro a 1,3 m do solo e altura total. Em seguida realizou-se o cálculo do volume. Para descrever o crescimento da variável diâmetro, altura total e volume foi realizado o ajuste através de cinco modelos matemáticos que expressam o crescimento ao longo do tempo. Posteriormente, foram calculados o incremento corrente anual (ICA) e incremento médio anual (IMA). O modelo de Hoerl apresentou melhores resultados de R²aj, obteve menores valores para o erro padrão de estimativa (Syx) e para o CV%, com isso, foi selecionado para a elaboração das curvas de crescimento. O povoamento clonal de teca no iPF apresentou incremento em DAP, Altura e volume foram superiores em relação a outros estudos científicos com teca, indicando sua viabilidade em sistemas integrados com pastagens na região.(AU)

The Role of ADAR1 and ADAR2 in the Regulation of miRNA-21 in Idiopathic Pulmonary Fibrosis.

INTRODUCTION: microRNAs (miRNAs) are small non-coding 1RNAs that post-transcriptionally regulate gene expression. Recent evidence shows that adenosine deaminases that act on RNA (ADAR) can edit miRNAs. miRNAs are involved in the development of different diseases, such as idiopathic pulmonary fibrosis (IPF). In IPF, about 40% of the miRNAs are differentially expressed with respect to controls. Among these miRNAs, miRNA-21 has been found over-expressed in IPF and its targets are anti-fibrosing molecules such as PELI1 and SPRY2. The objective of this study is to determine the role of ADAR1 and 2 on the expression of miRNA-21 in human lung fibroblasts trough quantification of gene expression, protein levels, and overexpression of ADAR1 and 2. METHODS: Six control and six fibrotic primary fibroblast cell cultures were used for RNA extraction, ADAR1, ADAR2, PELI1, SPRY2, miRNA-21, and pri-miRNA-21 expression was measured. Subsequently, two fibrotic fibroblast cultures were used for overexpression of ADAR1 and ADAR2, and they were stimulated with TGFß1. Real-time PCR and Western blot were performed. RESULTS: ADAR1 is significantly downregulated in IPF fibroblasts; the overexpression of ADAR1 and ADAR2 reestablishes the expression levels of miRNA-21, PELI1, and SPRY2 in fibroblasts of patients with IPF. CONCLUSION: These changes in the processing of miRNAs have great value in pathology diagnosis, including lung diseases, and play an important role in the understanding of molecular mechanisms involved in the development of different pathologies, as well as representing new therapeutic targets.

Experience with the Compassionate Use Program of nintedanib for the treatment of Idiopathic Pulmonary Fibrosis in Argentina

Introduction: Idiopathic Pulmonary Fibrosis (IPF) is a diffuse lung disease (DLD) of unknown etiology that is chronic and progressive. It occurs in older adults; it is restricted to the lungs and it is associated with the anatomopathological and/or tomographic pattern of Usual Interstitial Pneumonia (UIP). The evolution of the disease is progressive and it is associated with a mean 5-year survival rate of 20%. Objectives: to identify the clinical and pulmonary function characteristics in the group of patients with IPF included in the Compassionate Use Program (NPU, Named Patient Use); to identify the safety profile reported with nintedanib. Materials and methods: a retrospective, descriptive and cross-sectional study including 54 patients enrolled in the NPU program from September 1st, 2015 to August 10th, 2016. Data were collected from the NPU program records. Results: fifty-four patients with IPF were included in the NPU program, of whom 47 received nintedanib; the data from the latter were analyzed. Thirty-seven (78.72%) were males, with a mean age at the beginning of treatment of 67.47 ± 7.85 years, and in 9 cases (19.14%) the diagnosis was confirmed by lung biopsy. The mean forced vital capacity (FVC) at the beginning of treatment was 65.87±19.23 and it is presented as the percentage of the predictive value; the mean carbon dioxide diffusing capacity (DLCO) presented as the percentage of the predictive value was 38.74 ± 3.09. The time of progression from the diagnosis of IPF to the beginning of the treatment with nintedanib was 27.17± 27.9 months (median 17). Average drug exposure to cut-off point was 9.92 weeks ± 2.15 (median: 10 weeks). In 7 cases (31.91%) the FVC was over 80%, in 22 (46.80%) cases it was between 50 and 79% and in 10 cases (21.27%) it was below 49%. In total, 7 patients (14.89%) exhibited adverse events: Five (10.6%) patients exhibited weight loss, 4 (8.51%) diarrhea, 2 patients had nausea, 1 (2.12%) an increase of the liver enzymes and 1 (2.12%) pruritus. In most cases, the adverse events appeared during the first 2 weeks after beginning the treatment with nintedanib. In 3 (6.38%) cases it was imperative to suspend nintedanib permanently due to the adverse effects and in 4 (8.51%) cases the dose had to be titrated to 100 mg every 12 hours. Out of the total of patients, 6 (12.76%) passed away due to the progression of their underlying disease. Conclusions: such as it was reported by other groups, nintedanib has a manageable and tolerable safety profile. In our series of 47 patients with IPF who received at least one dose of nintedanib, 14.89% had an adverse event that led to the permanent discontinuation of the drug in only 3 patients (6.38%).

Essential role for the ATG4B protease and autophagy in bleomycin-induced pulmonary fibrosis.

Autophagy is a critical cellular homeostatic process that controls the turnover of damaged organelles and proteins. Impaired autophagic activity is involved in a number of diseases, including idiopathic pulmonary fibrosis suggesting that altered autophagy may contribute to fibrogenesis. However, the specific role of autophagy in lung fibrosis is still undefined. In this study, we show for the first time, how autophagy disruption contributes to bleomycin-induced lung fibrosis in vivo using an Atg4b-deficient mouse as a model. Atg4b-deficient mice displayed a significantly higher inflammatory response at 7 d after bleomycin treatment associated with increased neutrophilic infiltration and significant alterations in proinflammatory cytokines. Likewise, we found that Atg4b disruption resulted in augmented apoptosis affecting predominantly alveolar and bronchiolar epithelial cells. At 28 d post-bleomycin instillation Atg4b-deficient mice exhibited more extensive and severe fibrosis with increased collagen accumulation and deregulated extracellular matrix-related gene expression. Together, our findings indicate that the ATG4B protease and autophagy play a crucial role protecting epithelial cells against bleomycin-induced stress and apoptosis, and in the regulation of the inflammatory and fibrotic responses.

Parental perception of functional status following tracheostomy in infancy: a single center study.

OBJECTIVE: To examine the functional outcomes of children who underwent a tracheostomy in the initial hospitalization after birth and to determine their correlates. STUDY DESIGN: We administered the validated 43-item Functional Status-II (FS-II) questionnaire by Stein and Jessop over the telephone to caregivers of surviving children. The FS-II items generated a total score, age-specific: (1) total; (2) general health (GH); and (3) responsiveness, activity, or interpersonal functioning (IPF) scores in specific age group categories. RESULTS: FS-II was administered to 51/62 (82.2%) survivors at a median (range) age of 5 (1-10) years; 27% children were on the ventilator and 43% required devices. About 40% of children had a median of 1 (1-4) hospitalization in the previous 6 months. Scores were >2 SD below means in 55%, 24%, and 55% cases for age-specific T, GH, and R/A/IPF scores respectively. The T and R/A/IPF scales were significantly higher in those with private, rather than public, maternal insurance, as were T and R/A/IPF scores for children ≥ 4 years, compared with younger children. On regression analysis, FS-II T, GH, and R/A/IPF scores were independently associated with maternal private insurance (P = .02). R/A/IPF scores were also significantly associated with corrected age at FS-II administration. CONCLUSIONS: One-third of surviving children who underwent tracheostomy during their initial hospitalization remained technology-dependent. The parental FS-II questionnaires revealed low R/A/IPF scores, especially at younger ages and in those with maternal public insurance. Further research on family-level interventions to improve functional outcomes in this population is warranted.