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1.
Respir Res ; 25(1): 42, 2024 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-38238743

RESUMO

THE QUESTION ADDRESSED BY THE STUDY: Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. MATERIALS AND METHODS: We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. RESULTS: Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59-69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01-1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16-2.25] per each natural log of cf-nDNA copy number). ANSWER TO THE QUESTION: Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.


Assuntos
Ácidos Nucleicos Livres , Doença Pulmonar Obstrutiva Crônica , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Ácidos Nucleicos Livres/genética , DNA Mitocondrial , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Doença Pulmonar Obstrutiva Crônica/genética , Biomarcadores , Fenótipo , Progressão da Doença
3.
Am J Respir Cell Mol Biol ; 69(5): 556-569, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37487137

RESUMO

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive lung scarring and remodeling. Although treatments exist that slow disease progression, IPF is irreversible, and there is no cure. Cellular senescence, a major hallmark of aging, has been implicated in IPF pathogenesis, and mitochondrial dysfunction is increasingly recognized as a driver of senescence. Adenine nucleotide translocases (ANTs) are abundant mitochondrial ATP-ADP transporters critical for regulating cell fate and maintaining mitochondrial function. We sought to determine how alterations in ANTs influence cellular senescence in pulmonary fibrosis. We found that SLC25A4 (solute carrier family 25 member 4) (ANT1) and SLC25A5 (ANT2) expression is reduced in the lungs of patients with IPF, particularly within alveolar type II (AT2) cells, by single-cell RNA sequencing and tissue staining. Loss of ANT1 by siRNA in lung epithelial cells resulted in increased senescence markers such as ß-galactosidase and p21, with a reduction in the ratio of nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. Bleomycin-treated ANT1 knockdown cells also had increased senescence markers compared with bleomycin-treated control cells. Loss of ANT1 in AT2 cells resulted in a reduction in alveolar organoid growth, with an increase in p21 by staining. Global loss of ANT1 resulted in worse lung fibrosis and increased senescence in the bleomycin- and asbestos-induced mouse models of pulmonary fibrosis. In summary, loss of ANT1 contributes to IPF pathogenesis through mitochondrial dysfunction, increased senescence, and decreased regenerative capacity of AT2 cells, resulting in enhanced lung fibrosis. Modulation of ANTs presents a new therapeutic avenue that may alter cellular senescence pathways and limit pulmonary fibrosis.


Assuntos
Fibrose Pulmonar Idiopática , NAD , Animais , Humanos , Camundongos , Bleomicina/farmacologia , Senescência Celular , Células Epiteliais/metabolismo , Fibrose Pulmonar Idiopática/patologia , Pulmão/patologia , NAD/metabolismo
4.
Front Immunol ; 13: 882484, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35634304

RESUMO

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.


Assuntos
Células-Tronco Hematopoéticas , Hipóxia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação , Camundongos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo
5.
JCI Insight ; 7(6)2022 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-35315356

RESUMO

MD-PhD trainees constitute an important source of physician-scientists. Persistence on this challenging path is facilitated by success in garnering independent (R grant) support from the NIH. Published research tracks academic appointments and global R01 success for MD-PhD trainees but has not included information on future funding success of individual MD-PhD predoctoral grant holders. Here, we used data from the NIH RePORTER database to identify and track the funding trajectory of physician-scientists who received predoctoral grant support through the F30 mechanism, which is specific for dual-degree candidates. Male and female F30 awardees did not differ in their success in garnering K (postdoctoral training) grants, but, among F30 grant awardees, men were 2.6 times more likely than women to receive R funding. These results underscore the need for analysis of factors that contribute to the disproportionate loss of NIH-supported female physician-scientists between the predoctoral F30 and the independent R grant-supported stages.


Assuntos
Pesquisa Biomédica , Médicos , Bases de Dados Factuais , Feminino , Humanos , Masculino
7.
Acad Med ; 97(4): 492-496, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34292189

RESUMO

Substantial gender inequities persist across academic medicine. These issues are not new: Recent evidence still points to a chilly climate for women in academic medicine, including those in physician-scientist training. The discussion for how to address gender equity and issues of work-life integration typically centers around faculty and rarely includes trainees. The authors delineate specific strategies to address gender inequity in physician-scientist training by identifying key stakeholders for implementation and proposing areas to integrate these strategies with current training timelines. Strategies discussed include multiple-role mentoring, allyship training for trainees and faculty, early implementation of professional development sessions, incorporation of childcare and family-friendly policies, and additional policies for funding bodies to prioritize gender equity practices. The goal of this article is to equip trainees and the academic community with proactive strategies to create a more equitable environment for future generations of trainees in academic medicine.


Assuntos
Medicina , Médicos , Feminino , Equidade de Gênero , Humanos , Mentores , Políticas
8.
J Cell Sci ; 134(4)2021 02 25.
Artigo em Inglês | MEDLINE | ID: mdl-33526710

RESUMO

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.


Assuntos
Dictyostelium , Doença Pulmonar Obstrutiva Crônica , Dictyostelium/genética , Células Epiteliais/metabolismo , Humanos , Pulmão , Mitocôndrias , Translocases Mitocondriais de ADP e ATP/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/metabolismo
9.
ATS Sch ; 2(1): 19-28, 2020 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-33870320

RESUMO

The coronavirus disease (COVID-19) pandemic has created significant stressors for the academic and scientific community, with unique challenges for early-career physician-scientists. The pandemic-related disruptions have significantly affected research productivity, access to mentoring, professional development and networking opportunities, funding, and personal wellness. This is especially true for pulmonary and critical care medicine faculty because of the burden of specialized clinical care responsibilities that the COVID-19 pandemic has demanded. Departmental, institutional, and national leadership should foster open dialogue to identify and mitigate these challenges to promote ongoing career development of early-career physician-scientists. Implementation of thoughtful interventions to address these challenges will provide essential support for junior faculty and help retain a generation of physician-scientists.

10.
Am J Physiol Lung Cell Mol Physiol ; 317(5): L639-L652, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31461316

RESUMO

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.


Assuntos
Células Endoteliais/patologia , Hipóxia/complicações , Indóis/toxicidade , Pulmão/patologia , Mitocôndrias/patologia , Hipertensão Arterial Pulmonar/patologia , Pirróis/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Inibidores da Angiogênese/toxicidade , Animais , Cálcio/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Pulmão/metabolismo , Masculino , Mitocôndrias/metabolismo , Consumo de Oxigênio , Hipertensão Arterial Pulmonar/etiologia , Hipertensão Arterial Pulmonar/metabolismo , Ratos , Ratos Wistar , Remodelação Vascular
11.
Am J Physiol Lung Cell Mol Physiol ; 314(5): L893-L907, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29388466

RESUMO

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca2+]i) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca2]i in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca2+ entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca2]i, migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca2+ influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.


Assuntos
Cálcio/metabolismo , Endotélio Vascular/patologia , Hipertensão Pulmonar/patologia , Hipóxia/fisiopatologia , Indóis/toxicidade , Artéria Pulmonar/patologia , Pirróis/toxicidade , Canais de Cátion TRPV/metabolismo , Doenças Vasculares/patologia , Inibidores da Angiogênese/toxicidade , Animais , Células Cultivadas , Endotélio Vascular/metabolismo , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/metabolismo , Masculino , Microvasos/metabolismo , Microvasos/patologia , Artéria Pulmonar/metabolismo , Ratos , Ratos Wistar , Canais de Cátion TRPV/genética , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo
12.
Am J Respir Crit Care Med ; 195(12): 1651-1660, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28002683

RESUMO

RATIONALE: Estimating the probability of finding N2 or N3 (prN2/3) malignant nodal disease on endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with non-small cell lung cancer (NSCLC) can facilitate the selection of subsequent management strategies. OBJECTIVES: To develop a clinical prediction model for estimating the prN2/3. METHODS: We used the AQuIRE (American College of Chest Physicians Quality Improvement Registry, Evaluation, and Education) registry to identify patients with NSCLC with clinical radiographic stage T1-3, N0-3, M0 disease that had EBUS-TBNA for staging. The dependent variable was the presence of N2 or N3 disease (vs. N0 or N1) as assessed by EBUS-TBNA. Univariate followed by multivariable logistic regression analysis was used to develop a parsimonious clinical prediction model to estimate prN2/3. External validation was performed using data from three other hospitals. MEASUREMENTS AND MAIN RESULTS: The model derivation cohort (n = 633) had a 25% prevalence of malignant N2 or N3 disease. Younger age, central location, adenocarcinoma histology, and higher positron emission tomography-computed tomography N stage were associated with a higher prN2/3. Area under the receiver operating characteristic curve was 0.85 (95% confidence interval, 0.82-0.89), model fit was acceptable (Hosmer-Lemeshow, P = 0.62; Brier score, 0.125). We externally validated the model in 722 patients. Area under the receiver operating characteristic curve was 0.88 (95% confidence interval, 0.85-0.90). Calibration using the general calibration model method resulted in acceptable goodness of fit (Hosmer-Lemeshow test, P = 0.54; Brier score, 0.132). CONCLUSIONS: Our prediction rule can be used to estimate prN2/3 in patients with NSCLC. The model has the potential to facilitate clinical decision making in the staging of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Neoplasias Pulmonares/patologia , Linfonodos/patologia , Linfadenopatia/patologia , Idoso , Feminino , Humanos , Metástase Linfática , Masculino , Valor Preditivo dos Testes , Estudos Retrospectivos
13.
BMC Pulm Med ; 15: 134, 2015 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-26514822

RESUMO

BACKGROUND: Evidence suggests that individuals with interstitial lung abnormalities (ILA) on a chest computed tomogram (CT) may have an increased risk to develop a clinically significant interstitial lung disease (ILD). Although methods used to identify individuals with ILA on chest CT have included both automated quantitative and qualitative visual inspection methods, there has been not direct comparison between these two methods. To investigate this relationship, we created lung density metrics and compared these to visual assessments of ILA. METHODS: To provide a comparison between ILA detection methods based on visual assessment we generated measures of high attenuation areas (HAAs, defined by attenuation values between -600 and -250 Hounsfield Units) in >4500 participants from both the COPDGene and Framingham Heart studies (FHS). Linear and logistic regressions were used for analyses. RESULTS: Increased measures of HAAs (in ≥ 10 % of the lung) were significantly associated with ILA defined by visual inspection in both cohorts (P < 0.0001); however, the positive predictive values were not very high (19 % in COPDGene and 13 % in the FHS). In COPDGene, the association between HAAs and ILA defined by visual assessment were modified by the percentage of emphysema and body mass index. Although increased HAAs were associated with reductions in total lung capacity in both cohorts, there was no evidence for an association between measurement of HAAs and MUC5B promoter genotype in the FHS. CONCLUSION: Our findings demonstrate that increased measures of lung density may be helpful in determining the severity of lung volume reduction, but alone, are not strongly predictive of ILA defined by visual assessment. Moreover, HAAs were not associated with MUC5B promoter genotype.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Doenças Pulmonares Intersticiais/diagnóstico por imagem , Pulmão/diagnóstico por imagem , Enfisema Pulmonar/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Índice de Massa Corporal , Estudos de Coortes , Feminino , Volume Expiratório Forçado , Humanos , Modelos Lineares , Modelos Logísticos , Pulmão/fisiopatologia , Doenças Pulmonares Intersticiais/genética , Doenças Pulmonares Intersticiais/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mucina-5B/genética , Regiões Promotoras Genéticas , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/fisiopatologia , Espirometria , Tomografia Computadorizada por Raios X , Capacidade Pulmonar Total , Capacidade Vital
15.
J Leukoc Biol ; 98(2): 143-52, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25743626

RESUMO

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-ß-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.


Assuntos
Elastase de Leucócito/metabolismo , Pulmão/enzimologia , Miofibroblastos/enzimologia , Neutrófilos/enzimologia , Enfisema Pulmonar/enzimologia , Fibrose Pulmonar/enzimologia , Animais , Diferenciação Celular , Proliferação de Células , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/imunologia , Elastase de Leucócito/genética , Elastase de Leucócito/imunologia , Pulmão/imunologia , Pulmão/patologia , Camundongos , Miofibroblastos/imunologia , Miofibroblastos/patologia , Neutrófilos/imunologia , Neutrófilos/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Enfisema Pulmonar/genética , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/imunologia , Fibrose Pulmonar/patologia , Transdução de Sinais
16.
Int J Clin Exp Pathol ; 4(4): 349-55, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21577320

RESUMO

AIM: The purpose of this study was to develop an improved method for collagen and protein assessment of fibrotic lungs while decreasing animal use. METHODS: 8-10 week old, male C57BL/6 mice were given a single intratracheal instillation of crocidolite asbestos or control titanium dioxide. Lungs were collected on day 14 and dried as whole lung, or homogenized in CHAPS buffer, for hydroxyproline analysis. Insoluble and salt-soluble collagen content was also determined in lung homogenates using a modified Sirius red colorimetric 96-well plate assay. RESULTS: The hydroxyproline assay showed significant increases in collagen content in the lungs of asbestos-treated mice. Identical results were present between collagen content determined on dried whole lung or whole lung homogenates. The Sirius red plate assay showed a significant increase in collagen content in lung homogenates however, this assay grossly over-estimated the total amount of collagen and underestimated changes between control and fibrotic lungs, conclusions: The proposed method provides accurate quantification of collagen content in whole lungs and additional homogenate samples for biochemical analysis from a single animal. The Sirius-red colorimetric plate assay provides a complementary method for determination of the relative changes in lung collagen but the values tend to overestimate absolute values obtained by the gold standard hydroxyproline assay and underestimate the overall fibrotic injury.


Assuntos
Asbestose/metabolismo , Colágeno/metabolismo , Colorimetria , Pulmão/metabolismo , Fibrose Pulmonar/metabolismo , Análise de Variância , Animais , Asbesto Crocidolita , Asbestose/etiologia , Asbestose/patologia , Compostos Azo , Biomarcadores/metabolismo , Colorimetria/normas , Corantes , Modelos Animais de Doenças , Hidroxiprolina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Espectrofotometria , Regulação para Cima
17.
Int J Clin Exp Pathol ; 4(3): 241-54, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21487520

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a progressive disease with poor survival. The identification of therapeutic targets is essential to improving outcomes. Previous studies found that expression of the receptor for advanced glycation end products (RAGE) in the lung is significantly decreased in human IPF lungs and in two animal models of pulmonary fibrosis. In addition, RAGE-null mice spontaneously develop pulmonary fibrosis with age and more severe fibrosis when challenged with asbestos. In contrast to the findings that the lack of RAGE enhanced pulmonary fibrosis, He et al. found that RAGE null mice were protected from bleomycin-induced fibrosis and suggested the effect was due to a lack of HMGB1 induced RAGE signaling. The current study further tests this hypothesis by blocking RAGE signaling via administration of soluble RAGE, a decoy receptor, to determine if this will also protect against pulmonary fibrosis. Wild-type, RAGE(+/-), and RAGE(-/-) mice were treated with bleomycin and assessed for fibrosis. Wild-type mice were also treated with exogenous soluble RAGE or vehicle control. In addition, in vitro studies with primary alveolar epithelial cells from wild-type and RAGE null mice were used to investigate the effect of RAGE on cell viability and migration in response to injury. A lack of RAGE was found to be protective against bleomycin injury in both in vivo and in vitro studies. However, soluble RAGE administration was unable to ameliorate fibrosis. This study confirms paradoxical responses to two different models of pulmonary fibrosis and suggests a further role for RAGE in cellular migration.


Assuntos
Fibrose Pulmonar/metabolismo , Receptores Imunológicos/metabolismo , Análise de Variância , Animais , Bleomicina , Western Blotting , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibrose Pulmonar/induzido quimicamente , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Transdução de Sinais
18.
Free Radic Biol Med ; 50(9): 1075-80, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21334435

RESUMO

The extracellular matrix is a complex system that regulates cell function within a tissue. The antioxidant enzyme extracellular superoxide dismutase (EC-SOD) is bound to the matrix, and previous studies show that a lack of EC-SOD results in increased cardiac injury, fibrosis, and loss of cardiac function. This study tests the hypothesis that EC-SOD protects against cardiac fibrosis mechanistically by limiting oxidative stress and oxidant-induced shedding of syndecan-1 in the extracellular matrix. Wild-type and EC-SOD null mice were treated with a single dose of doxorubicin, 15 mg/kg, and evaluated on day 15. Serum and left-ventricle tissue were collected for biochemical assays, including Western blot, mRNA expression, and immunohistochemical staining for syndecan-1. The loss of EC-SOD and doxorubicin-induced oxidative injury led to increases in shed syndecan-1 in the serum, which originates from the endothelium of the vasculature. The shed syndecan-1 ectodomain induces proliferation of primary mouse cardiac fibroblasts. This study suggests that one mechanism by which EC-SOD protects the heart against cardiac fibrosis is the prevention of oxidative shedding of cardiovascular syndecan-1 and its subsequent induction of fibroblast proliferation. This study provides potential new targets for understanding and altering fibrosis progression in the heart.


Assuntos
Doxorrubicina/efeitos adversos , Matriz Extracelular/enzimologia , Fibroblastos/metabolismo , Superóxido Dismutase/metabolismo , Sindecana-1/metabolismo , Sindecana-1/farmacologia , Animais , Antioxidantes/metabolismo , Western Blotting , Técnicas de Cultura de Células , Proliferação de Células/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose/prevenção & controle , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
Hepatology ; 53(2): 587-95, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21274879

RESUMO

UNLABELLED: TCBOPOP (1,4-bis [2-(3,5-dichaloropyridyloxy)] benzene) an agonist of the constitutive androstane receptor (CAR), produces rapid hepatocyte hyperplasia and hepatomegaly in the absence of hepatic injury. In this study we demonstrate that integrin-linked kinase (ILK), which is involved in transmission of the extracellular matrix (ECM) signaling by way of integrin receptors, plays an important role in regulating TCPOBOP-induced proliferation of hepatocytes and hepatomegaly. Hepatocyte-specific ILK knockout mice (ILK/liver-/- mice) and wildtype mice (WT) were given a single dose of TCPOBOP (3 mg/kg) by oral gavage. Mice were sacrificed at days 1, 2, 5, and 7 after TCPOBOP administration. WT mice showed maximum proliferation on days 1 and 2, which came back to baseline levels by days 5 and 7 after TCPOBOP administration. The ILK/liver-/- mice, on the other hand, showed a prolonged and a sustained proliferative response as evident by an increased number of proliferative cell nuclear antigen assay (PCNA)-positive cells even at days 5 and 7 after TCPOBOP administration. At day 7 the WT mice showed close to a 2.5-fold increase in liver weight, whereas the ILK/liver-/- mice showed a 3.7-fold increase in liver weight. The prolonged proliferative response in the ILK/liver-/- mice seems to be due to sustained induction of CAR leading to sustained induction of c-Myc, which is known to be a key mediator of TCPOPOP-CAR induced direct liver hyperplasia. CONCLUSION: The data indicate that ECM-mediated signaling by way of ILK is essential for adjustment of final liver size and proper termination of TCPOBOP-induced proliferation of hepatocytes.


Assuntos
Hepatócitos/metabolismo , Hepatócitos/patologia , Hepatomegalia/induzido quimicamente , Hepatomegalia/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Piridinas/efeitos adversos , Animais , Peso Corporal/fisiologia , Ciclo Celular/fisiologia , Proliferação de Células , Modelos Animais de Doenças , Matriz Extracelular/fisiologia , Hepatomegalia/metabolismo , Camundongos , Camundongos Knockout , Tamanho do Órgão/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais/fisiologia
20.
Free Radic Biol Med ; 49(5): 707-17, 2010 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-20452419

RESUMO

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive fibrosis of the alveolar interstitium. The pathogenesis is thought to involve abnormal reepithelialization and dysregulated remodeling of the extracellular matrix after alveolar injury. There is growing evidence through human and animal studies that oxidative stress plays a role in this dysregulation. Markers of oxidative stress have been identified in the lungs of IPF patients and aberrant antioxidant activity exacerbates pulmonary fibrosis in animal models. In addition, the extracellular matrix is a critical component in regulating cellular homeostasis and appropriate wound healing. Recent investigations support that the matrix is a target of oxidative stress in the lung and IPF. Extracellular matrix degradation products, produced by reactive oxygen species, may promote fibrogenesis by influencing epithelial, mesenchymal, and inflammatory cell activity. The impact of the interactions of oxidative stress and the matrix of the lung remains unclear and may prove to be an important target for new therapies in IPF. Utilizing oxidative enzymes, antioxidants, or the matrix as therapeutic targets to control oxidative stress in IPF will continue be an area of active research and innovative discoveries in the coming years.


Assuntos
Proteínas da Matriz Extracelular/fisiologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/etiologia , Estresse Oxidativo/fisiologia , Animais , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Descoberta de Drogas/métodos , Proteínas da Matriz Extracelular/antagonistas & inibidores , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/metabolismo , Pulmão/fisiologia , Modelos Biológicos , Terapia de Alvo Molecular , Estresse Oxidativo/efeitos dos fármacos
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