RESUMO
Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403+/-) with Lpar1-ablated mice to create mice carrying both genetic changes (403+/- LPAR1 -/-) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT, 403+/- LPAR1 -/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a. Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.
Assuntos
Cardiomiopatia Hipertrófica , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/patologia , Proteínas de Transporte , Modelos Animais de Doenças , Células Endoteliais/patologia , Fibrose , Hipertrofia/patologia , CamundongosRESUMO
Fibroblast accumulation and extracellular matrix (ECM) deposition are common critical steps for the progression of organ fibrosis, but the precise molecular mechanisms remain to be fully investigated. We have previously demonstrated that lysophosphatidic acid contributes to organ fibrosis through the production of connective tissue growth factor (CTGF) via actin cytoskeleton-dependent signaling, myocardin-related transcription factor family (MRTF) consisting of MRTF-A and MRTF-B-serum response factor (SRF) pathway. In this study, we investigated the role of the MRTF-SRF pathway in the development of renal fibrosis, focusing on the regulation of ECM-focal adhesions (FA) in renal fibroblasts. Here we showed that both MRTF-A and -B were required for the expressions of ECM-related molecules such as lysyl oxidase family members, type I procollagen and fibronectin in response to transforming growth factor (TGF)-ß1 . TGF-ß1 -MRTF-SRF pathway induced the expressions of various components of FA such as integrin α subunits (αv , α2 , α11 ) and ß subunits (ß1 , ß3 , ß5 ) as well as integrin-linked kinase (ILK). On the other hand, the blockade of ILK suppressed TGF-ß1 -induced MRTF-SRF transcriptional activity, indicating a mutual relationship between MRTF-SRF and FA. Myofibroblast differentiation along with CTGF expression was also dependent on MRTF-SRF and FA components. Finally, global MRTF-A deficient and inducible fibroblast-specific MRTF-B deficient mice (MRTF-AKO BiFBKO mice) are protected from renal fibrosis with adenine administration. Renal expressions of ECM-FA components and CTGF as well as myofibroblast accumulation were suppressed in MRTF-AKO BiFBKO mice. These results suggest that the MRTF-SRF pathway might be a therapeutic target for renal fibrosis through the regulation of components forming ECM-FA in fibroblasts.
Assuntos
Fibroblastos , Nefropatias , Fatores de Transcrição , Animais , Camundongos , Actinas/metabolismo , Fibroblastos/metabolismo , Fibrose , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Nefropatias/metabolismo , Nefropatias/patologiaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease which leads to significant morbidity and mortality from respiratory failure. The two drugs currently approved for clinical use slow the rate of decline in lung function but have not been shown to halt disease progression or reverse established fibrosis. Thus, new therapeutic targets are needed. Endothelial injury and the resultant vascular permeability are critical components in the response to tissue injury and are present in patients with IPF. However, it remains unclear how vascular permeability affects lung repair and fibrosis following injury. Lipid mediators such as sphingosine-1-phosphate (S1P) are known to regulate multiple homeostatic processes in the lung including vascular permeability. We demonstrate that endothelial cell-(EC) specific deletion of the S1P receptor 1 (S1PR1) in mice (EC-S1pr1-/-) results in increased lung vascular permeability at baseline. Following a low-dose intratracheal bleomycin challenge, EC-S1pr1-/- mice had increased and persistent vascular permeability compared with wild-type mice, which was strongly correlated with the amount and localization of resulting pulmonary fibrosis. EC-S1pr1-/- mice also had increased immune cell infiltration and activation of the coagulation cascade within the lung. However, increased circulating S1P ligand in ApoM-overexpressing mice was insufficient to protect against bleomycin-induced pulmonary fibrosis. Overall, these data demonstrate that endothelial cell S1PR1 controls vascular permeability in the lung, is associated with changes in immune cell infiltration and extravascular coagulation, and modulates the fibrotic response to lung injury.
Assuntos
Permeabilidade Capilar , Células Endoteliais/metabolismo , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Bleomicina , Coagulação Sanguínea , Deleção de Genes , Fibrose Pulmonar Idiopática/sangue , Pulmão/irrigação sanguínea , Pulmão/patologia , Lisofosfolipídeos/sangue , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , RNA-Seq , Análise de Célula Única , Esfingosina/análogos & derivados , Esfingosina/sangueRESUMO
Idiopathic pulmonary fibrosis is a progressive lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. We previously identified HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme A) reductase inhibitors (statins) as YAP inhibitors based on a high-throughput small-molecule screen in primary human lung fibroblasts. Here we report that several Aurora kinase inhibitors were also identified from the top hits of this screen. MK-5108, a highly selective inhibitor for AURKA (Aurora kinase A), induced YAP phosphorylation and cytoplasmic retention and significantly reduced profibrotic gene expression in human lung fibroblasts. The inhibitory effect on YAP nuclear translocation and profibrotic gene expression is specific to inhibition of AURKA, but not Aurora kinase B or C, and is independent of the Hippo pathway kinases LATS1 and LATS2 (Large Tumor Suppressor 1 and 2). Further characterization of the effects of MK-5108 demonstrate that it inhibits YAP nuclear localization indirectly via effects on actin polymerization and TGFß (Transforming Growth Factor ß) signaling. In addition, MK-5108 treatment reduced lung collagen deposition in the bleomycin mouse model of pulmonary fibrosis. Our results reveal a novel role for AURKA in YAP-mediated profibrotic activity in fibroblasts and highlight the potential of small-molecule screens for YAP inhibitors for identification of novel agents with antifibrotic activity.
Assuntos
Aurora Quinase A , Fibrose Pulmonar Idiopática , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/patologia , Camundongos , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Sinalização YAPRESUMO
Rationale: Early, accurate diagnosis of interstitial lung disease (ILD) informs prognosis and therapy, especially in idiopathic pulmonary fibrosis (IPF). Current diagnostic methods are imperfect. High-resolution computed tomography has limited resolution, and surgical lung biopsy (SLB) carries risks of morbidity and mortality. Endobronchial optical coherence tomography (EB-OCT) is a low-risk, bronchoscope-compatible modality that images large lung volumes in vivo with microscopic resolution, including subpleural lung, and has the potential to improve the diagnostic accuracy of bronchoscopy for ILD diagnosis. Objectives: We performed a prospective diagnostic accuracy study of EB-OCT in patients with ILD with a low-confidence diagnosis undergoing SLB. The primary endpoints were EB-OCT sensitivity/specificity for diagnosis of the histopathologic pattern of usual interstitial pneumonia (UIP) and clinical IPF. The secondary endpoint was agreement between EB-OCT and SLB for diagnosis of the ILD fibrosis pattern. Methods: EB-OCT was performed immediately before SLB. The resulting EB-OCT images and histopathology were interpreted by blinded, independent pathologists. Clinical diagnosis was obtained from the treating pulmonologists after SLB, blinded to EB-OCT. Measurements and Main Results: We enrolled 31 patients, and 4 were excluded because of inconclusive histopathology or lack of EB-OCT data. Twenty-seven patients were included in the analysis (16 men, average age: 65.0 yr): 12 were diagnosed with UIP and 15 with non-UIP ILD. Average FVC and DlCO were 75.3% (SD, 18.5) and 53.5% (SD, 16.4), respectively. Sensitivity and specificity of EB-OCT was 100% (95% confidence interval, 75.8-100.0%) and 100% (79.6-100%), respectively, for both histopathologic UIP and clinical diagnosis of IPF. There was high agreement between EB-OCT and histopathology for diagnosis of ILD fibrosis pattern (weighted κ: 0.87 [0.72-1.0]). Conclusions: EB-OCT is a safe, accurate method for microscopic ILD diagnosis, as a complement to high-resolution computed tomography and an alternative to SLB.
Assuntos
Broncoscopia/métodos , Broncoscopia/normas , Confiabilidade dos Dados , Fibrose Pulmonar Idiopática/diagnóstico , Tomografia de Coerência Óptica/métodos , Tomografia de Coerência Óptica/normas , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Idiopathic pulmonary fibrosis is a lung disease with limited therapeutic options that is characterized by pathological fibroblast activation and aberrant lung remodeling with scar formation. YAP (Yes-associated protein) is a transcriptional coactivator that mediates mechanical and biochemical signals controlling fibroblast activation. In this study, we developed a high-throughput small-molecule screen for YAP inhibitors in primary human lung fibroblasts. Multiple HMG-CoA (hydroxymethylglutaryl-coenzyme A) reductase inhibitors (statins) were found to inhibit YAP nuclear localization via induction of YAP phosphorylation, cytoplasmic retention, and degradation. We further show that the mevalonate pathway regulates YAP activation, and that simvastatin treatment reduces fibrosis markers in activated human lung fibroblasts and in the bleomycin mouse model of pulmonary fibrosis. Finally, we show that simvastatin modulates YAP in vivo in mouse lung fibroblasts. Our results highlight the potential of small-molecule screens for YAP inhibitors and provide a mechanism for the antifibrotic activity of statins in idiopathic pulmonary fibrosis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fibrose Pulmonar/tratamento farmacológico , Acil Coenzima A/metabolismo , Animais , Biomarcadores/metabolismo , Bleomicina/farmacologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Ácido Mevalônico/metabolismo , Camundongos , Fosfoproteínas/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinvastatina/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Proteínas de Sinalização YAPRESUMO
BLT (bone marrow-liver-thymus) humanized mice, which reconstitute a functional human immune system, develop prototypic human virus-specific CD8+ T cell responses following infection with human immunodeficiency virus type 1 (HIV-1). We explored the utility of the BLT model for HIV-1 vaccine development by immunizing BLT mice against the conserved viral Gag protein, utilizing a rapid prime-boost protocol of poly(lactic-co-glycolic) acid microparticles and a replication-defective herpes simplex virus (HSV) recombinant vector. After HIV-1 challenge, the mice developed broad, proteome-wide gamma interferon-positive (IFN-γ+) T cell responses against HIV-1 that reached magnitudes equivalent to what is observed in HIV-1-infected individuals. The functionality of these responses was underscored by the consistent emergence of escape mutations in multiple CD8+ T cell epitopes during the course of infection. Although prechallenge vaccine-induced responses were largely undetectable, the Gag immunization increased both the magnitude and the kinetics of anamnestic Gag-specific T cell responses following HIV-1 infection, and the magnitude of these postchallenge Gag-specific responses was inversely correlated with acute HIV-1 viremia. Indeed, Gag immunization was associated with a modest but significant 0.5-log reduction in HIV-1 viral load when analyzed across four experimental groups of BLT mice. Notably, the HSV vector induced elevated plasma concentrations of polarizing cytokines and chemotactic factors, including interleukin-12p70 (IL-12p70) and MIP-1α, which were positively correlated with the magnitude of Gag-specific responses. Overall, these results support the ability of BLT mice to recapitulate human pathogen-specific T cell responses and to respond to immunization; however, additional improvements to the model are required to develop a robust system for testing HIV-1 vaccine efficacy.IMPORTANCE Advances in the development of humanized mice have raised the possibility of a small-animal model for preclinical testing of an HIV-1 vaccine. Here, we describe the capacity of BLT humanized mice to mount broadly directed HIV-1-specific human T cell responses that are functionally active, as indicated by the rapid emergence of viral escape mutations. Although immunization of BLT mice with the conserved viral Gag protein did not result in detectable prechallenge responses, it did increase the magnitude and kinetics of postchallenge Gag-specific T cell responses, which was associated with a modest but significant reduction in acute HIV-1 viremia. Additionally, the BLT model revealed immunization-associated increases in the plasma concentrations of immunomodulatory cytokines and chemokines that correlated with more robust T cell responses. These data support the potential utility of the BLT humanized mouse for HIV-1 vaccine development but suggest that additional improvements to the model are warranted.
Assuntos
Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Linfócitos T/imunologia , Linfócitos T/virologia , Viremia , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologia , Doença Aguda , Animais , Evolução Biológica , Linfócitos T CD4-Positivos , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Infecções por HIV/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunização , Camundongos , Camundongos Transgênicos , Linfócitos T/metabolismo , Carga ViralRESUMO
Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies. However, administration of purified bNAbs poses challenges in resource-poor settings, where the HIV-1 disease burden is greatest. In vivo vector-based production of bNAbs represents an alternative strategy. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121 in wild-type and immunocompromised C57BL/6 mice as well as in HIV-1-infected bone marrow-liver-thymus (BLT) humanized mice. Ad5.PGT121 and AAV1.PGT121 produced functional antibody in vivo Ad5.PGT121 produced PGT121 rapidly within 6 h, whereas AAV1.PGT121 produced detectable PGT121 in serum by 72 h. Serum PGT121 levels were rapidly reduced by the generation of anti-PGT121 antibodies in immunocompetent mice but were durably maintained in immunocompromised mice. In HIV-1-infected BLT humanized mice, Ad5.PGT121 resulted in a greater reduction of viral loads than did AAV1.PGT121. Ad5.PGT121 also led to more-sustained virologic control than purified PGT121 IgG. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice. Further evaluation of vector delivery of HIV-1 bNAbs is warranted, although approaches to prevent the generation of antiantibody responses may also be required.IMPORTANCE Broadly neutralizing antibodies (bNAbs) are being explored for HIV-1 prevention and cure strategies, but delivery of purified antibodies may prove challenging. We investigated adenovirus serotype 5 (Ad5) and adeno-associated virus serotype 1 (AAV1) vectors to deliver the HIV-1-specific bNAb PGT121. Ad5.PGT121 afforded more rapid, robust, and durable antiviral efficacy than AAV1.PGT121 and purified PGT121 IgG in HIV-1-infected humanized mice.
Assuntos
Adenoviridae , Terapia Genética/métodos , Vetores Genéticos , Infecções por HIV , HIV-1 , Transdução Genética/métodos , Animais , Feminino , Infecções por HIV/genética , Infecções por HIV/metabolismo , Infecções por HIV/patologia , Infecções por HIV/terapia , HIV-1/genética , HIV-1/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos KnockoutRESUMO
Background: Small-molecule CD4-mimetic compounds (CD4mc) inhibit human immunodeficiency virus (HIV-1) entry by blocking binding to the CD4 receptor and by premature triggering of the viral envelope glycoprotein (Env) spike. Methods: The efficacy of a CD4mc in protecting bone marrow-liver-thymus (BLT) humanized mice from vaginal HIV-1 challenge was evaluated. Results: Intravaginal application of the CD4mc JP-III-48, either before or simultaneously with virus challenge, protected BLT humanized mice from HIV-1JR-CSF infection in a dose- dependent manner. Conclusion: The direct antiviral effects of a CD4mc prevent HIV-1 infection in a murine model of sexual transmission.
Assuntos
Biomimética , Antígenos CD4/administração & dosagem , Inibidores da Fusão de HIV/administração & dosagem , Infecções por HIV/prevenção & controle , HIV-1/efeitos dos fármacos , Administração Intravaginal , Animais , Medula Óssea , Modelos Animais de Doenças , Feminino , Fígado , Camundongos SCID , Timo , Resultado do TratamentoRESUMO
Pulmonary fibrosis is thought to result from dysregulated wound repair after repetitive lung injury. Many cellular responses to injury involve rearrangements of the actin cytoskeleton mediated by the two isoforms of the Rho-associated coiled-coil-forming protein kinase (ROCK), ROCK1 and ROCK2. In addition, profibrotic mediators such as transforming growth factor-ß, thrombin, and lysophosphatidic acid act through receptors that activate ROCK. Inhibition of ROCK activation may be a potent therapeutic strategy for human pulmonary fibrosis. Pharmacological inhibition of ROCK using nonselective ROCK inhibitors has been shown to prevent fibrosis in animal models; however, the specific roles of each ROCK isoform are poorly understood. Furthermore, the pleiotropic effects of this kinase have raised concerns about on-target adverse effects of ROCK inhibition such as hypotension. Selective inhibition of one isoform might be a better-tolerated strategy. In the present study, we used a genetic approach to determine the roles of ROCK1 and ROCK2 in a mouse model of bleomycin-induced pulmonary fibrosis. Using ROCK1- or ROCK2-haploinsufficient mice, we found that reduced expression of either ROCK1 or ROCK2 was sufficient to protect them from bleomycin-induced pulmonary fibrosis. In addition, we found that both isoforms contribute to the profibrotic responses of epithelial cells, endothelial cells, and fibroblasts. Interestingly, ROCK1- and ROCK2-haploinsufficient mice exhibited similar protection from bleomycin-induced vascular leak, myofibroblast differentiation, and fibrosis; however, ROCK1-haploinsufficient mice demonstrated greater attenuation of epithelial cell apoptosis. These findings suggest that selective inhibition of either ROCK isoform has the potential to be an effective therapeutic strategy for pulmonary fibrosis.
Assuntos
Fibroblastos/enzimologia , Pulmão/enzimologia , Fibrose Pulmonar/prevenção & controle , Quinases Associadas a rho/metabolismo , Animais , Apoptose , Bleomicina , Permeabilidade Capilar , Diferenciação Celular , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Células Endoteliais/patologia , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Fibroblastos/patologia , Haploinsuficiência , Humanos , Pulmão/patologia , Camundongos Knockout , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Quinases Associadas a rho/deficiência , Quinases Associadas a rho/genéticaRESUMO
After host entry through mucosal surfaces, human immunodeficiency virus-1 (HIV-1) disseminates to lymphoid tissues to establish a generalized infection of the immune system. The mechanisms by which this virus spreads among permissive target cells locally during the early stages of transmission and systemically during subsequent dissemination are not known. In vitro studies suggest that the formation of virological synapses during stable contacts between infected and uninfected T cells greatly increases the efficiency of viral transfer. It is unclear, however, whether T-cell contacts are sufficiently stable in vivo to allow for functional synapse formation under the conditions of perpetual cell motility in epithelial and lymphoid tissues. Here, using multiphoton intravital microscopy, we examine the dynamic behaviour of HIV-infected T cells in the lymph nodes of humanized mice. We find that most productively infected T cells migrate robustly, resulting in their even distribution throughout the lymph node cortex. A subset of infected cells formed multinucleated syncytia through HIV envelope-dependent cell fusion. Both uncoordinated motility of syncytia and adhesion to CD4(+) lymph node cells led to the formation of long membrane tethers, increasing cell lengths to up to ten times that of migrating uninfected T cells. Blocking the egress of migratory T cells from the lymph nodes into efferent lymph vessels, and thus interrupting T-cell recirculation, limited HIV dissemination and strongly reduced plasma viraemia. Thus, we have found that HIV-infected T cells are motile, form syncytia and establish tethering interactions that may facilitate cell-to-cell transmission through virological synapses. Migration of T cells in lymph nodes therefore spreads infection locally, whereas their recirculation through tissues is important for efficient systemic viral spread, suggesting new molecular targets to antagonize HIV infection.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV/imunologia , Animais , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Células Gigantes , Infecções por HIV/transmissão , Humanos , Linfonodos/virologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos TransgênicosRESUMO
Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiled-coil forming protein kinase (ROCK) family of serine/threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond.
Assuntos
Desenho de Fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Pulmão/efeitos dos fármacos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Quinases Associadas a rho/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/patologia , Pulmão/enzimologia , Pulmão/patologia , Conformação Proteica , Inibidores de Proteínas Quinases/efeitos adversos , Relação Estrutura-Atividade , Quinases Associadas a rho/química , Quinases Associadas a rho/metabolismoRESUMO
Numerous compounds have shown efficacy in limiting development of pulmonary fibrosis using animal models, yet few of these compounds have replicated these beneficial effects in clinical trials. Given the challenges associated with performing clinical trials in patients with idiopathic pulmonary fibrosis (IPF), it is imperative that preclinical data packages be robust in their analyses and interpretations to have the best chance of selecting promising drug candidates to advance to clinical trials. The American Thoracic Society has convened a group of experts in lung fibrosis to discuss and formalize recommendations for preclinical assessment of antifibrotic compounds. The panel considered three major themes (choice of animal, practical considerations of fibrosis modeling, and fibrotic endpoints for evaluation). Recognizing the need for practical considerations, we have taken a pragmatic approach. The consensus view is that use of the murine intratracheal bleomycin model in animals of both genders, using hydroxyproline measurements for collagen accumulation along with histologic assessments, is the best-characterized animal model available for preclinical testing. Testing of antifibrotic compounds in this model is recommended to occur after the acute inflammatory phase has subsided (generally after Day 7). Robust analyses may also include confirmatory studies in human IPF specimens and validation of results in a second system using in vivo or in vitro approaches. The panel also strongly encourages the publication of negative results to inform the lung fibrosis community. These recommendations are for preclinical therapeutic evaluation only and are not intended to dissuade development of emerging technologies to better understand IPF pathogenesis.
Assuntos
Congressos como Assunto , Modelos Animais de Doenças , Fibrose Pulmonar/terapia , Sociedades Médicas , Animais , Determinação de Ponto Final , Feminino , Humanos , Masculino , Organismos Geneticamente Modificados , Reprodutibilidade dos TestesRESUMO
The expansion of the fibroblast pool is a critical step in organ fibrosis, but the mechanisms driving expansion remain to be fully clarified. We previously showed that lysophosphatidic acid (LPA) signaling through its receptor LPA1 expressed on fibroblasts directly induces the recruitment of these cells. Here we tested whether LPA-LPA1 signaling drives fibroblast proliferation and activation during the development of renal fibrosis. LPA1-deficient (LPA1-/-) or -sufficient (LPA1+/+) mice were crossed to mice with green fluorescent protein expression (GFP) driven by the type I procollagen promoter (Col-GFP) to identify fibroblasts. Unilateral ureteral obstruction-induced increases in renal collagen were significantly, though not completely, attenuated in LPA1-/-Col-GFP mice, as were the accumulations of both fibroblasts and myofibroblasts. Connective tissue growth factor was detected mainly in tubular epithelial cells, and its levels were suppressed in LPA1-/-Col-GFP mice. LPA-LPA1 signaling directly induced connective tissue growth factor expression in primary proximal tubular epithelial cells, through a myocardin-related transcription factor-serum response factor pathway. Proximal tubular epithelial cell-derived connective tissue growth factor mediated renal fibroblast proliferation and myofibroblast differentiation. Administration of an inhibitor of myocardin-related transcription factor/serum response factor suppressed obstruction-induced renal fibrosis. Thus, targeting LPA-LPA1 signaling and/or myocardin-related transcription factor/serum response factor-induced transcription could be promising therapeutic strategies for renal fibrosis.
Assuntos
Comunicação Celular/efeitos dos fármacos , Fator de Crescimento do Tecido Conjuntivo/metabolismo , Células Epiteliais/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Nefropatias/metabolismo , Rim/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Receptores de Ácidos Lisofosfatídicos/agonistas , Transdução de Sinais/efeitos dos fármacos , Obstrução Ureteral/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/genética , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Rim/metabolismo , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Nefropatias/prevenção & controle , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fenótipo , Regiões Promotoras Genéticas , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/deficiência , Receptores de Ácidos Lisofosfatídicos/genética , Receptores de Ácidos Lisofosfatídicos/metabolismo , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Transfecção , Obstrução Ureteral/genética , Obstrução Ureteral/patologiaRESUMO
Lysophosphatidic acid (LPA) is an important mediator of pulmonary fibrosis. In blood and multiple tumor types, autotaxin produces LPA from lysophosphatidylcholine (LPC) via lysophospholipase D activity, but alternative enzymatic pathways also exist for LPA production. We examined the role of autotaxin (ATX) in pulmonary LPA production during fibrogenesis in a bleomycin mouse model. We found that bleomycin injury increases the bronchoalveolar lavage (BAL) fluid levels of ATX protein 17-fold. However, the LPA and LPC species that increase in BAL of bleomycin-injured mice were discordant, inconsistent with a substrate-product relationship between LPC and LPA in pulmonary fibrosis. LPA species with longer chain polyunsaturated acyl groups predominated in BAL fluid after bleomycin injury, with 22:5 and 22:6 species accounting for 55 and 16% of the total, whereas the predominant BAL LPC species contained shorter chain, saturated acyl groups, with 16:0 and 18:0 species accounting for 56 and 14% of the total. Further, administration of the potent ATX inhibitor PAT-048 to bleomycin-challenged mice markedly decreased ATX activity systemically and in the lung, without effect on pulmonary LPA or fibrosis. Therefore, alternative ATX-independent pathways are likely responsible for local generation of LPA in the injured lung. These pathways will require identification to therapeutically target LPA production in pulmonary fibrosis.-Black, K. E., Berdyshev, E., Bain, G., Castelino, F. V., Shea, B. S., Probst, C. K., Fontaine, B. A., Bronova, I., Goulet, L., Lagares, D., Ahluwalia, N., Knipe, R. S., Natarajan, V., Tager, A. M. Autotaxin activity increases locally following lung injury, but is not required for pulmonary lysophosphatidic acid production or fibrosis.
Assuntos
Lesão Pulmonar/induzido quimicamente , Pulmão/metabolismo , Lisofosfolipídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Fibrose Pulmonar/metabolismo , Animais , Antibióticos Antineoplásicos/toxicidade , Benzoatos/farmacologia , Bleomicina/toxicidade , Regulação da Expressão Gênica/fisiologia , Lesão Pulmonar/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Diester Fosfórico Hidrolases/genética , Fibrose Pulmonar/induzido quimicamenteRESUMO
Humanized mice reconstituted with a human immune system can be mucosally infected with human immunodeficiency virus (HIV), opening up the possibility of studying HIV transmission in a small-animal model. Here we report that passive immunization with the broadly neutralizing antibody b12 protected humanized mice against repetitive intravaginal infection in a dose-dependent manner. In addition, treatment with the antibody PGT126, which is more potent in vitro, was more efficacious in vivo and provided sterilizing protection. Our results demonstrate that humanized mice can be used as a small-animal model to study the efficacy and mechanism of broadly neutralizing antibody protection against HIV acquisition.
Assuntos
Anticorpos Neutralizantes/administração & dosagem , Modelos Animais de Doenças , Anticorpos Anti-HIV/administração & dosagem , Infecções por HIV/prevenção & controle , Imunização Passiva/métodos , Animais , Relação Dose-Resposta Imunológica , Feminino , Camundongos , Camundongos SCID , Resultado do TratamentoRESUMO
Fibrogenesis is the active production of extracellular matrix in response to tissue injury. In many chronic diseases persistent fibrogenesis results in the accumulation of scar tissue, which can lead to organ failure and death. However, no non-invasive technique exists to assess this key biological process. All tissue fibrogenesis results in the formation of allysine, which enables collagen cross-linking and leads to tissue stiffening and scar formation. We report herein a novel allysine-binding gadolinium chelate (GdOA), that can non-invasively detect and quantify the extent of fibrogenesis using magnetic resonance imaging (MRI). We demonstrate that GdOA signal enhancement correlates with the extent of the disease and is sensitive to a therapeutic response.
Assuntos
Aminas/química , Quelantes/química , Imageamento por Ressonância Magnética , Sondas Moleculares/química , Fibrose Pulmonar/diagnóstico , Ácido 2-Aminoadípico/análogos & derivados , Ácido 2-Aminoadípico/química , Animais , Bleomicina , Gadolínio/química , Camundongos , Conformação Molecular , Fibrose Pulmonar/induzido quimicamenteRESUMO
Pathologic accumulation of fibroblasts in pulmonary fibrosis appears to depend on their invasion through basement membranes and extracellular matrices. Fibroblasts from the fibrotic lungs of patients with idiopathic pulmonary fibrosis (IPF) have been demonstrated to acquire a phenotype characterized by increased cell-autonomous invasion. Here, we investigated whether fibroblast invasion is further stimulated by soluble mediators induced by lung injury. We found that bronchoalveolar lavage fluids from bleomycin-challenged mice or patients with IPF contain mediators that dramatically increase the matrix invasion of primary lung fibroblasts. Further characterization of this non-cell-autonomous fibroblast invasion suggested that the mediators driving this process are produced locally after lung injury and are preferentially produced by fibrogenic (e.g., bleomycin-induced) rather than nonfibrogenic (e.g., LPS-induced) lung injury. Comparison of invasion and migration induced by a series of fibroblast-active mediators indicated that these two forms of fibroblast movement are directed by distinct sets of stimuli. Finally, knockdown of multiple different membrane receptors, including platelet-derived growth factor receptor-ß, lysophosphatidic acid 1, epidermal growth factor receptor, and fibroblast growth factor receptor 2, mitigated the non-cell-autonomous fibroblast invasion induced by bronchoalveolar lavage from bleomycin-injured mice, suggesting that multiple different mediators drive fibroblast invasion in pulmonary fibrosis. The magnitude of this mediator-driven fibroblast invasion suggests that its inhibition could be a novel therapeutic strategy for pulmonary fibrosis. Further elaboration of the molecular mechanisms that drive non-cell-autonomous fibroblast invasion consequently may provide a rich set of novel drug targets for the treatment of IPF and other fibrotic lung diseases.
Assuntos
Fibroblastos/patologia , Fibrose Pulmonar Idiopática/complicações , Fibrose Pulmonar Idiopática/patologia , Lesão Pulmonar/complicações , Lesão Pulmonar/patologia , Animais , Bleomicina , Líquido da Lavagem Broncoalveolar , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Solubilidade , Fator de Crescimento Transformador beta/farmacologiaRESUMO
Lysophosphatidic acid (LPA) signaling through one of its receptors, LPA1, contributes to both the development and the pathological remodeling after injury of many organs. Because we found previously that LPA-LPA1 signaling contributes to pulmonary fibrosis, here we investigated whether this pathway is also involved in lung development. Quantitative assessment of lung architecture of LPA1-deficient knock-out (KO) and wild-type (WT) mice at 3, 12, and 24 weeks of age using design-based stereology suggested the presence of an alveolarization defect in LPA1 KO mice at 3 weeks, which persisted as alveolar numbers increased in WT mice into adulthood. Across the ages examined, the lungs of LPA1 KO mice exhibited decreased alveolar numbers, septal tissue volumes, and surface areas, and increased volumes of the distal airspaces. Elastic fibers, critical to the development of alveolar septa, appeared less organized and condensed and more discontinuous in KO alveoli starting at P4. Tropoelastin messenger RNA expression was decreased in KO lungs, whereas expression of matrix metalloproteinases degrading elastic fibers was either decreased or unchanged. These results are consistent with the abnormal lung phenotype of LPA1 KO mice, being attributable to reduced alveolar septal formation during development, rather than to increased septal destruction as occurs in the emphysema of chronic obstructive pulmonary disease. Peripheral septal fibroblasts and myofibroblasts, which direct septation in late alveolarization, demonstrated reduced production of tropoelastin and matrix metalloproteinases, and diminished LPA-induced migration, when isolated from LPA1 KO mice. Taken together, our data suggest that LPA-LPA1 signaling is critically required for septation during alveolarization.