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1.
Eur Respir J ; 60(5)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35798357

RESUMO

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , Doenças Pulmonares Intersticiais , Humanos , Camundongos , Animais , Bleomicina/farmacologia , Plasmócitos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/metabolismo , Doenças Pulmonares Intersticiais/induzido quimicamente
2.
Thorax ; 76(1): 73-82, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33214245

RESUMO

INTRODUCTION: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection. METHODS: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression. RESULTS: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor ß1 (TGF-ß1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-ß1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. CONCLUSION: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.


Assuntos
Regulação da Expressão Gênica , Fibrose Pulmonar Idiopática/genética , RNA/genética , Transcriptoma/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/metabolismo , Pulmão/patologia , Transdução de Sinais , Regulação para Cima
3.
Am J Respir Crit Care Med ; 201(5): 564-574, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31710517

RESUMO

Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease characterized by scarring of the lung that is believed to result from an atypical response to injury of the epithelium. Genome-wide association studies have reported signals of association implicating multiple pathways including host defense, telomere maintenance, signaling, and cell-cell adhesion.Objectives: To improve our understanding of factors that increase IPF susceptibility by identifying previously unreported genetic associations.Methods: We conducted genome-wide analyses across three independent studies and meta-analyzed these results to generate the largest genome-wide association study of IPF to date (2,668 IPF cases and 8,591 controls). We performed replication in two independent studies (1,456 IPF cases and 11,874 controls) and functional analyses (including statistical fine-mapping, investigations into gene expression, and testing for enrichment of IPF susceptibility signals in regulatory regions) to determine putatively causal genes. Polygenic risk scores were used to assess the collective effect of variants not reported as associated with IPF.Measurements and Main Results: We identified and replicated three new genome-wide significant (P < 5 × 10-8) signals of association with IPF susceptibility (associated with altered gene expression of KIF15, MAD1L1, and DEPTOR) and confirmed associations at 11 previously reported loci. Polygenic risk score analyses showed that the combined effect of many thousands of as yet unreported IPF susceptibility variants contribute to IPF susceptibility.Conclusions: The observation that decreased DEPTOR expression associates with increased susceptibility to IPF supports recent studies demonstrating the importance of mTOR signaling in lung fibrosis. New signals of association implicating KIF15 and MAD1L1 suggest a possible role of mitotic spindle-assembly genes in IPF susceptibility.


Assuntos
Fibrose Pulmonar Idiopática/genética , Idoso , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Feminino , Expressão Gênica , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/genética , Masculino , Pessoa de Meia-Idade , Medição de Risco , Transdução de Sinais , Fuso Acromático , Serina-Treonina Quinases TOR/metabolismo
4.
Biochem Biophys Res Commun ; 510(2): 198-204, 2019 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-30685089

RESUMO

Malignant mesothelioma is an aggressive fibrous tumor, predominantly of the pleura, with a very poor prognosis. Cell-matrix interactions are recognized important determinants of tumor growth and invasiveness but the role of the extracellular matrix in mesothelioma is unknown. Mesothelioma cells synthesize collagen as well as transforming growth factor-beta (TGF-ß), a key regulator of collagen production. This study examined the effect of inhibiting collagen production on mesothelioma cell proliferation in vitro and tumor growth in vivo. Collagen production by mesothelioma cells was inhibited by incubating cells in vitro with the proline analogue thiaproline (thiazolidine-4-carboxylic acid) or by oral administration of thiaproline in a murine tumor model. Cell cytotoxicity was measured using neutral red uptake and lactate dehydrogenase assays. Proliferation was measured by tritiated thymidine incorporation, and inflammatory cell influx, proliferation, apoptosis and angiogenesis in tumors examined by immunohistochemical labelling. Tumor size was determined by tumor weight and collagen production was measured by HPLC. Thiaproline at non-toxic doses significantly reduced basal and TGF-ß-induced collagen production by over 50% and cell proliferation by over 65%. In vivo thiaproline administration inhibited tumor growth at 10 days, decreasing the median tumor weight by 80%. The mean concentration of collagen was 50% lower in the thiaproline-treated tumors compared with the controls. There were no significant differences in vasculature or inflammatory cell infiltration but apoptosis was increased in thiaproline treated tumors at day 10. In conclusion, these observations strongly support a role for collagen in mesothelioma growth and establish the potential for inhibitors of collagen synthesis in mesothelioma treatment.


Assuntos
Colágeno/biossíntese , Neoplasias Pulmonares/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Colágeno/antagonistas & inibidores , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Humanos , Inflamação , Neoplasias Pulmonares/patologia , Mesotelioma/patologia , Mesotelioma Maligno , Camundongos , Camundongos Endogâmicos CBA , Neoplasias Pleurais/patologia , Tiazolidinas/farmacologia , Fator de Crescimento Transformador beta/metabolismo
5.
Thorax ; 73(9): 847-856, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29748250

RESUMO

INTRODUCTION: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. METHODS: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. RESULTS: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and ßENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. CONCLUSION: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.


Assuntos
Fibrose Cística/genética , Fibrose Cística/patologia , Canais Epiteliais de Sódio/genética , Inativação Gênica , RNA Interferente Pequeno , Transfecção/métodos , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Humanos , Camundongos , Nanopartículas
6.
Int J Mol Sci ; 19(8)2018 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-30103548

RESUMO

Prostacyclins are extensively used to treat pulmonary arterial hypertension (PAH), a life-threatening disease involving the progressive thickening of small pulmonary arteries. Although these agents are considered to act therapeutically via the prostanoid IP receptor, treprostinil is the only prostacyclin mimetic that potently binds to the prostanoid EP2 receptor, the role of which is unknown in PAH. We hypothesised that EP2 receptors contribute to the anti-proliferative effects of treprostinil in human pulmonary arterial smooth muscle cells (PASMCs), contrasting with selexipag, a non-prostanoid selective IP agonist. Human PASMCs from PAH patients were used to assess prostanoid receptor expression, cell proliferation, and cyclic adenosine monophosphate (cAMP) levels following the addition of agonists, antagonists or EP2 receptor small interfering RNAs (siRNAs). Immunohistochemical staining was performed in lung sections from control and PAH patients. We demonstrate using selective IP (RO1138452) and EP2 (PF-04418948) antagonists that the anti-proliferative actions of treprostinil depend largely on EP2 receptors rather than IP receptors, unlike MRE-269 (selexipag-active metabolite). Likewise, EP2 receptor knockdown selectively reduced the functional responses to treprostinil but not MRE-269. Furthermore, EP2 receptor levels were enhanced in human PASMCs and in lung sections from PAH patients compared to controls. Thus, EP2 receptors represent a novel therapeutic target for treprostinil, highlighting key pharmacological differences between prostacyclin mimetics used in PAH.


Assuntos
Proliferação de Células/efeitos dos fármacos , Epoprostenol/análogos & derivados , Hipertensão Pulmonar/tratamento farmacológico , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Receptores de Prostaglandina E Subtipo EP2/biossíntese , Regulação para Cima/efeitos dos fármacos , Adolescente , Adulto , Criança , Epoprostenol/farmacologia , Feminino , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Masculino , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Receptores de Prostaglandina E Subtipo EP2/antagonistas & inibidores , Sistemas do Segundo Mensageiro/efeitos dos fármacos
7.
Thorax ; 71(6): 565-7, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26911575

RESUMO

Caffeine is a commonly used food additive found naturally in many products. In addition to potently stimulating the central nervous system caffeine is able to affect various systems within the body including the cardiovascular and respiratory systems. Importantly, caffeine is used clinically to treat apnoea and bronchopulmonary dysplasia in premature babies. Recently, caffeine has been shown to exhibit antifibrotic effects in the liver in part through reducing collagen expression and deposition, and reducing expression of the profibrotic cytokine TGFß. The potential antifibrotic effects of caffeine in the lung have not previously been investigated. Using a combined in vitro and ex vivo approach we have demonstrated that caffeine can act as an antifibrotic agent in the lung by acting on two distinct cell types, namely epithelial cells and fibroblasts. Caffeine inhibited TGFß activation by lung epithelial cells in a concentration-dependent manner but had no effect on TGFß activation in fibroblasts. Importantly, however, caffeine abrogated profibrotic responses to TGFß in lung fibroblasts. It inhibited basal expression of the α-smooth muscle actin gene and reduced TGFß-induced increases in profibrotic genes. Finally, caffeine reduced established bleomycin-induced fibrosis after 5 days treatment in an ex vivo precision-cut lung slice model. Together, these findings suggest that there is merit in further investigating the potential use of caffeine, or its analogues, as antifibrotic agents in the lung.


Assuntos
Cafeína/farmacologia , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Biomarcadores/metabolismo , Células Cultivadas , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Fibrose Pulmonar/genética , Transdução de Sinais/efeitos dos fármacos
8.
Thorax ; 71(8): 701-11, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27103349

RESUMO

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is the most rapidly progressive and fatal of all fibrotic conditions with no curative therapies. Common pathomechanisms between IPF and cancer are increasingly recognised, including dysfunctional pan-PI3 kinase (PI3K) signalling as a driver of aberrant proliferative responses. GSK2126458 is a novel, potent, PI3K/mammalian target of rapamycin (mTOR) inhibitor which has recently completed phase I trials in the oncology setting. Our aim was to establish a scientific and dosing framework for PI3K inhibition with this agent in IPF at a clinically developable dose. METHODS: We explored evidence for pathway signalling in IPF lung tissue and examined the potency of GSK2126458 in fibroblast functional assays and precision-cut IPF lung tissue. We further explored the potential of IPF patient-derived bronchoalveolar lavage (BAL) cells to serve as pharmacodynamic biosensors to monitor GSK2126458 target engagement within the lung. RESULTS: We provide evidence for PI3K pathway activation in fibrotic foci, the cardinal lesions in IPF. GSK2126458 inhibited PI3K signalling and functional responses in IPF-derived lung fibroblasts, inhibiting Akt phosphorylation in IPF lung tissue and BAL derived cells with comparable potency. Integration of these data with GSK2126458 pharmacokinetic data from clinical trials in cancer enabled modelling of an optimal dosing regimen for patients with IPF. CONCLUSIONS: Our data define PI3K as a promising therapeutic target in IPF and provide a scientific and dosing framework for progressing GSK2126458 to clinical testing in this disease setting. A proof-of-mechanism trial of this agent is currently underway. TRIAL REGISTRATION NUMBER: NCT01725139, pre-clinical.


Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/uso terapêutico , Sulfonamidas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Proliferação de Células , Ensaios Clínicos como Assunto , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/patologia , Piridazinas , Transdução de Sinais , Resultado do Tratamento
9.
Clin Sci (Lond) ; 130(8): 575-86, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26744410

RESUMO

Fibroblasts derived from the lungs of patients with idiopathic pulmonary fibrosis (IPF) and systemic sclerosis (SSc) produce low levels of prostaglandin (PG) E2, due to a limited capacity to up-regulate cyclooxygenase-2 (COX-2). This deficiency contributes functionally to the fibroproliferative state, however the mechanisms responsible are incompletely understood. In the present study, we examined whether the reduced level of COX-2 mRNA expression observed in fibrotic lung fibroblasts is regulated epigenetically. The DNA methylation inhibitor, 5-aza-2'-deoxycytidine (5AZA) restored COX-2 mRNA expression by fibrotic lung fibroblasts dose dependently. Functionally, this resulted in normalization of fibroblast phenotype in terms of PGE2 production, collagen mRNA expression and sensitivity to apoptosis. COX-2 methylation assessed by bisulfite sequencing and methylation microarrays was not different in fibrotic fibroblasts compared with controls. However, further analysis of the methylation array data identified a transcriptional regulator, chromosome 8 open reading frame 4 (thyroid cancer protein 1, TC-1) (c8orf4), which is hypermethylated and down-regulated in fibrotic fibroblasts compared with controls. siRNA knockdown of c8orf4 in control fibroblasts down-regulated COX-2 and PGE2 production generating a phenotype similar to that observed in fibrotic lung fibroblasts. Chromatin immunoprecipitation demonstrated that c8orf4 regulates COX-2 expression in lung fibroblasts through binding of the proximal promoter. We conclude that the decreased capacity of fibrotic lung fibroblasts to up-regulate COX-2 expression and COX-2-derived PGE2 synthesis is due to an indirect epigenetic mechanism involving hypermethylation of the transcriptional regulator, c8orf4.


Assuntos
Ciclo-Oxigenase 2/genética , Metilação de DNA , Epigênese Genética , Fibroblastos/enzimologia , Pulmão/enzimologia , Proteínas de Neoplasias/genética , Fibrose Pulmonar/genética , Escleroderma Sistêmico/genética , Idoso , Sítios de Ligação , Estudos de Casos e Controles , Proliferação de Células , Células Cultivadas , Ciclo-Oxigenase 2/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Dinoprostona/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação Neoplásica da Expressão Gênica , Genótipo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/patologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Escleroderma Sistêmico/enzimologia , Escleroderma Sistêmico/patologia , Transcrição Gênica , Transfecção
10.
medRxiv ; 2024 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-38293162

RESUMO

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition that is more prevalent in males than females. The reasons for this are not fully understood, with differing environmental exposures due to historically sex-biased occupations, or diagnostic bias, being possible explanations. To date, over 20 independent genetic variants have been identified to be associated with IPF susceptibility, but these have been discovered when combining males and females. Our aim was to test for the presence of sex-specific associations with IPF susceptibility and assess whether there is a need to consider sex-specific effects when evaluating genetic risk in clinical prediction models for IPF. Methods: We performed genome-wide single nucleotide polymorphism (SNP)-by-sex interaction studies of IPF risk in six independent IPF case-control studies and combined them using inverse-variance weighted fixed effect meta-analysis. In total, 4,561 cases (1,280 females and 2,281 males) and 23,500 controls (8,360 females and 14,528 males) of European genetic ancestry were analysed. We used polygenic risk scores (PRS) to assess differences in genetic risk prediction between males and females. Findings: Three independent genetic association signals were identified. All showed a consistent direction of effect across all individual IPF studies and an opposite direction of effect in IPF susceptibility between females and males. None had been previously identified in IPF susceptibility genome-wide association studies (GWAS). The predictive accuracy of the PRSs were similar between males and females, regardless of whether using combined or sex-specific GWAS results. Interpretation: We prioritised three genetic variants whose effect on IPF risk may be modified by sex, however these require further study. We found no evidence that the predictive accuracy of common SNP-based PRSs varies significantly between males and females.

11.
ERJ Open Res ; 10(1)2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38375425

RESUMO

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF, and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Owing to the important role that the human leukocyte antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising 5159 cases and 27 459 controls, including a prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold of p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

12.
Am J Respir Cell Mol Biol ; 49(3): 471-80, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23614789

RESUMO

Receptor-targeted nanocomplexes are nonviral vectors developed for gene delivery to the airway epithelium for the treatment of pulmonary disease associated with cystic fibrosis. The present study aimed to optimize the delivery of the nanocomplex by nebulization, and to monitor the in vivo deposition of radiolabeled vector in the airways of a large animal model by γ-camera scintigraphy. Large White weaner pigs were nebulized with nanocomplexes mixed with technetium-99m radiopharmaceuticals. The aerosol deposition scans suggested that the nebulized radiovectors were deposited mainly in the trachea-main bronchi and in the midregion of the lungs. The plasmid biodistribution, assessed by real-time PCR, correlated with the scintigraphy images. The highest plasmid copy numbers were found in the bronchial areas and in the tissues proximal to the main bronchi bifurcation. Immunohistochemistry detected transgene expression in the tracheal and bronchial ciliated epithelium. Histological analysis of lung tissue showed no evidence of inflammation, and no increase in inflammatory cytokines or inflammatory cells was detected in the bronchoalveolar lavage. The deposition of nebulized nanocomplexes coassociated with technetium-99m can be monitored by nuclear medicine techniques. The use of a noninvasive strategy to follow the delivery of the vector could improve the clinical management of patients undergoing cystic fibrosis gene therapy.


Assuntos
Técnicas de Transferência de Genes , Imagem Molecular/métodos , Compostos Radiofarmacêuticos/farmacocinética , Mucosa Respiratória/diagnóstico por imagem , Sistema Respiratório/diagnóstico por imagem , Tecnécio/farmacocinética , Animais , Fibrose Cística/diagnóstico por imagem , Feminino , Terapia Genética , Humanos , Injeções Intravenosas , Masculino , Nanoconjugados/administração & dosagem , Nanoconjugados/química , Nebulizadores e Vaporizadores , Plasmídeos , Cintilografia , Compostos Radiofarmacêuticos/administração & dosagem , Mucosa Respiratória/ultraestrutura , Sistema Respiratório/ultraestrutura , Suínos , Tecnécio/administração & dosagem
13.
Am J Pathol ; 180(4): 1398-412, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22322297

RESUMO

STAT3 is a latent transcription factor that plays a role in regulating fibroblast function in fibrotic lung diseases. To further understand the role of STAT3 in the phenotypic divergence and function of human lung fibroblasts (LFs), we investigated the effect of basal and cytokine-induced STAT3 activity on indices of LF differentiation and activation, including expression of α-smooth muscle actin (α-SMA), collagen, and adhesion molecules Thy-1/CD90 and α(v) ß(3) and ß(5) integrins. We identified a population of fibroblasts from usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) lungs characterized by constitutively phosphorylated STAT3, lower proliferation rates, and diminished expression of α-SMA, Thy-1/CD90, and ß(3) integrins compared with control LFs. Staining of UIP lung biopsy specimens demonstrated that phosphorylated STAT3 was not present in α-SMA-positive fibroblastic foci but was observed in the nuclei of cells located in the areas of dense fibrosis. STAT3 activation in LFs did not significantly influence basal or transforming growth factor ß(1)-induced collagen I expression but inhibited expression of α-SMA, Thy-1/CD90, and αv ß(3) integrins. Suppression of STAT3 signaling diminished resistance of IPF LFs to staurosporine-induced apoptosis and responsiveness to transforming growth factor ß(1) but increased basal α-SMA and restored ß(3) integrin expression in LFs via an ALK-5-dependent, SMAD3/7-independent mechanism. These data suggest that STAT3 activation regulates several pathways in human LFs associated with normal wound healing, whereas aberrant STAT3 signaling plays a critical role in UIP/IPF pathogenesis.


Assuntos
Fibroblastos/patologia , Fibrose Pulmonar Idiopática/patologia , Fator de Transcrição STAT3/fisiologia , Actinas/metabolismo , Apoptose/fisiologia , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Colágeno Tipo I/metabolismo , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Integrina alfaVbeta3/metabolismo , Interleucina-6/farmacologia , Pulmão/metabolismo , Pulmão/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Oncostatina M/farmacologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/fisiologia , Antígenos Thy-1/metabolismo , Transdução Genética , Fator de Crescimento Transformador beta1/farmacologia
14.
medRxiv ; 2023 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-37546732

RESUMO

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial pneumonia marked by progressive lung fibrosis and a poor prognosis. Recent studies have highlighted the potential role of infection in the pathogenesis of IPF and a prior association of the HLA-DQB1 gene with idiopathic fibrotic interstitial pneumonia (including IPF) has been reported. Due to the important role that the Human Leukocyte Antigen (HLA) region plays in the immune response, here we evaluated if HLA genetic variation was associated specifically with IPF risk. Methods: We performed a meta-analysis of associations of the HLA region with IPF risk in individuals of European ancestry from seven independent case-control studies of IPF (comprising a total of 5,159 cases and 27,459 controls, including the prior study of fibrotic interstitial pneumonia). Single nucleotide polymorphisms, classical HLA alleles and amino acids were analysed and signals meeting a region-wide association threshold p<4.5×10-4 and a posterior probability of replication >90% were considered significant. We sought to replicate the previously reported HLA-DQB1 association in the subset of studies independent of the original report. Results: The meta-analysis of all seven studies identified four significant independent single nucleotide polymorphisms associated with IPF risk. However, none met the posterior probability for replication criterion. The HLA-DQB1 association was not replicated in the independent IPF studies. Conclusion: Variation in the HLA region was not consistently associated with risk in studies of IPF. However, this does not preclude the possibility that other genomic regions linked to the immune response may be involved in the aetiology of IPF.

15.
Pulm Pharmacol Ther ; 24(5): 478-86, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21824523

RESUMO

Airway remodelling is a collective term for changes in the amount or organisation of the cellular and molecular constituents of the airway wall. Remodelling occurs in and is associated with the pathophysiology of airways diseases including asthma and chronic obstructive pulmonary disease. The remodelling that occurs in these diseases exhibits both shared and distinct features. Remodelling is generally considered to be deleterious to airway function but recent studies also indicate potential protective effects. However, the true impact of different aspects of the remodelling process on lung function, both negative and positive, is poorly understood. In addition, the genetic susceptibility and processes by which environmental insults drive the cell and molecular events which result in airway remodelling and the potential for therapeutic reversibility are also incompletely understood. The last 10-15 years has seen the development of animal models of airway remodelling which have been refined and modified as new factors such as exacerbations and early life influences have been recognised as being of importance. In addition, invertebrate models have been put forward and complex in vitro culture systems and lung slice preparations developed. In parallel, imaging technology has developed to an extent where it is feasible using a combination of techniques to image structural components, cells and proteins in the airway wall as well as to analyse biological processes, cell and receptor activation non-invasively over time. The integration of data from in vivo and in vitro models together with use of imaging techniques in man and animals should allow validation of models, further our understanding of the pathophysiology of airway remodelling and potentially improve predictive accuracy for the translation of novel therapeutic agents into the clinic.


Assuntos
Remodelação das Vias Aéreas , Asma/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Remodelação das Vias Aéreas/genética , Animais , Asma/genética , Técnicas de Cultura , Modelos Animais de Doenças , Predisposição Genética para Doença , Humanos , Modelos Biológicos , Doença Pulmonar Obstrutiva Crônica/genética
16.
Am J Respir Crit Care Med ; 182(1): 73-82, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20203246

RESUMO

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF), a progressive disease with a dismal prognosis, exhibit an unexplained disparity of increased alveolar epithelial cell (AEC) apoptosis but reduced fibroblast apoptosis. OBJECTIVES: To examine whether the failure of patients with IPF to up-regulate cyclooxygenase (COX)-2, and thus the antifibrotic mediator prostaglandin (PG)E(2), accounts for this imbalance. METHODS: Fibroblasts and primary type II AECs were isolated from control and fibrotic human lung tissue. The effects of COX-2 inhibition and exogenous PGE(2) on fibroblast and AEC sensitivity to Fas ligand (FasL)-induced apoptosis were assessed. MEASUREMENTS AND MAIN RESULTS: IPF lung fibroblasts are resistant to FasL-induced apoptosis compared with control lung fibroblasts. Inhibition of COX-2 in control lung fibroblasts resulted in an apoptosis-resistant phenotype. Administration of PGE(2) almost doubled the rate of FasL-induced apoptosis in fibrotic lung fibroblasts compared with FasL alone. Conversely, in primary fibrotic lung type II AECs, PGE(2) protected against FasL-induced apoptosis. In human control and, to a greater extent, fibrotic lung fibroblasts, PGE(2) inhibits the phosphorylation of Akt, suggesting that regulation of this prosurvival protein kinase is an important mechanism by which PGE(2) modulates cellular apoptotic responses. CONCLUSIONS: The observation that PGE(2) deficiency results in increased AEC but reduced fibroblast sensitivity to apoptosis provides a novel pathogenic insight into the mechanisms driving persistent fibroproliferation in IPF.


Assuntos
Apoptose/fisiologia , Ciclo-Oxigenase 2/fisiologia , Dinoprostona/fisiologia , Fibroblastos/fisiologia , Fibrose Pulmonar Idiopática/fisiopatologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Células Cultivadas , Células Epiteliais/fisiologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/fisiologia , Cicatrização/fisiologia
17.
Biomedicines ; 9(5)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946612

RESUMO

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5'-aza-2'-deoxycytidine' treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

18.
Am J Pathol ; 174(4): 1264-79, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19147812

RESUMO

Activation of latent transforming growth factor beta (TGF-beta) by alphavbeta6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes alphavbeta6 integrin-mediated TGF-beta activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce alphavbeta6 integrin-mediated TGF-beta activation is unknown; in addition, the alphavbeta6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces alphavbeta6-mediated TGF-beta activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced alphavbeta6 integrin-mediated TGF-beta activity is mediated via the LPA2 receptor, which signals via G alpha(q). Finally, we show that the expression levels of both the LPA2 receptor and alphavbeta6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and alphavbeta6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces alphavbeta6 integrin-mediated TGF-beta activation in epithelial cells via LPA2, G alpha(q), RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.


Assuntos
Antígenos de Neoplasias/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Integrinas/metabolismo , Lesão Pulmonar/metabolismo , Lisofosfolipídeos/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Western Blotting , Técnicas de Cocultura , Fibrose , Citometria de Fluxo , Imunofluorescência , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/fisiopatologia , Lisofosfolipídeos/farmacologia , Camundongos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
19.
Am J Respir Crit Care Med ; 179(5): 414-25, 2009 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-19060230

RESUMO

RATIONALE: Studies in patients and experimental animals provide compelling evidence of the involvement of the major thrombin receptor, proteinase-activated receptor-1 (PAR(1)), and the potent chemokine, chemokine (CC motif) ligand-2 (CCL2)/monocyte chemotactic protein-1, in the pathogenesis of idiopathic pulmonary fibrosis (IPF). PAR(1) knockout mice are protected from bleomycin-induced lung inflammation and fibrosis and this protection is associated with marked attenuation in CCL2 induction. OBJECTIVES: The aim of this study was to determine which cell types represent the major source of PAR(1)-inducible CCL2 in the fibrotic lung. METHODS: Using immunohistochemistry and dual immunofluorescence, we examined PAR(1) and CCL2 expression in the bleomycin model and human IPF lung. PAR(1) and CCL2 gene expression was also assessed in laser-captured alveolar septae from patients with IPF. The ability of PAR(1) to induce CCL2 production by lung epithelial cells was also examined in vitro. MEASUREMENTS AND MAIN RESULTS: We report for the first time that PAR(1) and CCL2 are coexpressed and co-up-regulated on the activated epithelium in fibrotic areas in IPF. Similar observations were found in bleomycin-induced lung injury. Furthermore, we show that thrombin is a potent inducer of CCL2 gene expression and protein release by cultured lung epithelial cells via a PAR(1)-dependent mechanism. CONCLUSIONS: These data support the notion that PAR(1) activation on lung epithelial cells may represent an important mechanism leading to increased local CCL2 release in pulmonary fibrosis. Targeting PAR(1) on the pulmonary epithelium may offer a unique opportunity for therapeutic intervention in pulmonary fibrosis and other inflammatory and fibroproliferative conditions associated with excessive local generation of thrombin and CCL2 release.


Assuntos
Quimiocina CCL2/metabolismo , Fibrose Pulmonar/metabolismo , Receptor PAR-1/metabolismo , Sequência de Aminoácidos , Animais , Bleomicina , Estudos de Casos e Controles , Quimiocina CCL2/biossíntese , Quimiocina CCL2/genética , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptor PAR-1/biossíntese , Receptor PAR-1/genética , Receptores CCR2/metabolismo , Trombina/farmacologia
20.
Mol Ther ; 16(5): 907-15, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18388925

RESUMO

Synthetic vectors for cystic fibrosis (CF) gene therapy are required that efficiently and safely transfect airway epithelial cells, rather than alveolar epithelial cells or macrophages, and that are nonimmunogenic, thus allowing for repeated delivery. We have compared several vector systems against these criteria including GL67, polyethylenimine (PEI) 22 and 25 kd and two new, synthetic vector formulations, comprising a cationic, receptor-targeting peptide K(16)GACSERSMNFCG (E), and the cationic liposomes (L) DHDTMA/DOPE or DOSEP3/DOPE. The lipid and peptide formulations self assemble into receptor-targeted nanocomplexes (RTNs) LED-1 and LED-2, respectively, on mixing with plasmid (D). LED-1 transfected airway epithelium efficiently, while LED-2 and GL67 preferentially transfected alveolar cells. PEI transfected airway epithelial cells with high efficiency, but was more toxic to the mice than the other formulations. On repeat dosing, LED-1 was equally as effective as the single dose, while GL67 was 30% less effective and PEI 22 kd displayed a 90% reduction of efficiency on repeated delivery. LED-1 thus was the only formulation that fulfilled the criteria for a CF gene therapy vector while GL67 and LED-2 may be appropriate for other respiratory diseases. Opportunities for PEI depend on a solution to its toxicity problems. LED-1 formulations were stable to nebulization, the most appropriate delivery method for CF.


Assuntos
Fibrose Cística/terapia , Técnicas de Transferência de Genes , Terapia Genética/métodos , Nanotecnologia/métodos , Animais , Caspase 3/metabolismo , Cátions , Feminino , Terapia Genética/instrumentação , Vetores Genéticos , Humanos , Pulmão/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos
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