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1.
Mol Ther ; 32(2): 440-456, 2024 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-38213031

RESUMO

Here we introduce a first-in-class microRNA-sensitive oncolytic Zika virus (ZIKV) for virotherapy application against central nervous system (CNS) tumors. The described methodology produced two synthetic modified ZIKV strains that are safe in normal cells, including neural stem cells, while preserving brain tropism and oncolytic effects in tumor cells. The microRNA-sensitive ZIKV introduces genetic modifications in two different virus sites: first, in the established 3'UTR region, and secondly, in the ZIKV protein coding sequence, demonstrating for the first time that the miRNA inhibition systems can be functional outside the UTR RNA sites. The total tumor remission in mice bearing human CNS tumors, including metastatic tumor growth, after intraventricular and systemic modified ZIKV administration, confirms the promise of this virotherapy as a novel agent against brain tumors-highly deadly diseases in urgent need of effective advanced therapies.


Assuntos
Neoplasias do Sistema Nervoso Central , MicroRNAs , Terapia Viral Oncolítica , Vírus Oncolíticos , Infecção por Zika virus , Zika virus , Humanos , Camundongos , Animais , Vírus Oncolíticos/genética , Zika virus/genética , MicroRNAs/genética , Infecção por Zika virus/terapia , Terapia Viral Oncolítica/métodos
2.
FASEB J ; 37(7): e23005, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37289107

RESUMO

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/patologia
3.
Semin Cell Dev Biol ; 101: 104-110, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31879264

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fatal chronic lung disease characterized by progressive scarring of the lung tissue, leading to respiratory failure. There is no cure for IPF, and current anti-fibrotic treatments modestly arrest its further progression. IPF prevalence and incidence increase with age, which is a recognized risk factor. Intense clinical and basic research over the last fifteen years has shown that hallmarks of accelerated aging are present in the lungs of patients with IPF. Different cell types in IPF lungs exhibit premature hallmarks of aging, including telomere attrition and cellular senescence. In this Review, we discuss recent insights into the mechanisms behind these age-related alterations and their contribution to the development of lung fibrosis. We focus on the genetic and molecular basis of telomere attrition in alveolar type II epithelial cells, which promote cellular senescence and lung fibrosis. Mechanistically, senescent cells secrete pro-fibrotic factors that activate scar-forming myofibroblasts. Ultimately, senescent alveolar epithelial cells lose their regenerative capacity, impeding fibrosis resolution. In addition, mitochondrial dysfunction is strongly associated with the appearance of senescent epithelial cells and senescent myofibroblasts in IPF, which persist in the fibrotic tissue by adapting their metabolic pathways and becoming resistant to apoptosis. We discuss emerging novel therapeutic strategies to treat IPF by targeting cellular senescence with the so-called senotherapeutics.


Assuntos
Antifibrinolíticos/farmacologia , Senescência Celular/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Animais , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia
4.
Am J Pathol ; 191(7): 1193-1208, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33894177

RESUMO

Pulmonary fibrosis (PF) can arise from unknown causes, as in idiopathic PF, or as a consequence of infections, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current treatments for PF slow, but do not stop, disease progression. We report that treatment with a runt-related transcription factor 1 (RUNX1) inhibitor (Ro24-7429), previously found to be safe, although ineffective, as a Tat inhibitor in patients with HIV, robustly ameliorates lung fibrosis and inflammation in the bleomycin-induced PF mouse model. RUNX1 inhibition blunted fundamental mechanisms downstream pathologic mediators of fibrosis and inflammation, including transforming growth factor-ß1 and tumor necrosis factor-α, in cultured lung epithelial cells, fibroblasts, and vascular endothelial cells, indicating pleiotropic effects. RUNX1 inhibition also reduced the expression of angiotensin-converting enzyme 2 and FES Upstream Region (FURIN), host proteins critical for SARS-CoV-2 infection, in mice and in vitro. A subset of human lungs with SARS-CoV-2 infection overexpress RUNX1. These data suggest that RUNX1 inhibition via repurposing of Ro24-7429 may be beneficial for PF and to battle SARS-CoV-2, by reducing expression of viral mediators and by preventing respiratory complications.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , Subunidade alfa 2 de Fator de Ligação ao Core/antagonistas & inibidores , Furina/metabolismo , Pulmão/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Animais , Bleomicina , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Resultado do Tratamento
5.
Am J Respir Crit Care Med ; 204(12): 1433-1451, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34550870

RESUMO

Rationale: Mechanical signaling through cell-matrix interactions plays a major role in progressive vascular remodeling in pulmonary arterial hypertension (PAH). MMP-8 (matrix metalloproteinase-8) is an interstitial collagenase involved in regulating inflammation and fibrosis of the lung and systemic vasculature, but its role in PAH pathogenesis remains unexplored. Objectives: To evaluate MMP-8 as a modulator of pathogenic mechanical signaling in PAH. Methods: MMP-8 levels were measured in plasma from patients with pulmonary hypertension (PH) and controls by ELISA. MMP-8 vascular expression was examined in lung tissue from patients with PAH and rodent models of PH. MMP-8-/- and MMP-8+/+ mice were exposed to normobaric hypoxia or normoxia for 4-8 weeks. PH severity was evaluated by right ventricular systolic pressure, echocardiography, pulmonary artery morphometry, and immunostaining. Proliferation, migration, matrix component expression, and mechanical signaling were assessed in MMP-8-/- and MMP-8+/+ pulmonary artery smooth muscle cells (PASMCs). Measurements and Main Results: MMP-8 expression was significantly increased in plasma and pulmonary arteries of patients with PH compared with controls and induced in the pulmonary vasculature in rodent PH models. Hypoxia-exposed MMP-8-/- mice had significant mortality, increased right ventricular systolic pressure, severe right ventricular dysfunction, and exaggerated vascular remodeling compared with MMP-8+/+ mice. MMP-8-/- PASMCs demonstrated exaggerated proliferation and migration mediated by altered matrix protein expression, elevated integrin-ß3 levels, and induction of FAK (focal adhesion kinase) and downstream YAP (Yes-associated protein)/TAZ (transcriptional coactivator with PDZ-binding motif) activity. Conclusions: MMP-8 is a novel protective factor upregulated in the pulmonary vasculature during PAH pathogenesis. MMP-8 opposes pathologic mechanobiological feedback by altering matrix composition and disrupting integrin-ß3/FAK and YAP/TAZ-dependent mechanical signaling in PASMCs.


Assuntos
Metaloproteinase 8 da Matriz/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Artéria Pulmonar/metabolismo , Adulto , Idoso , Animais , Biomarcadores/metabolismo , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Metaloproteinase 8 da Matriz/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Hipertensão Arterial Pulmonar/patologia , Hipertensão Arterial Pulmonar/prevenção & controle , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Regulação para Cima , Remodelação Vascular
6.
Wound Repair Regen ; 29(4): 678-691, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34117675

RESUMO

In response to tissue injury, fibroblasts differentiate into professional repair cells called myofibroblasts, which orchestrate many aspects of the normal tissue repair programme including synthesis, deposition and contraction of extracellular matrix proteins, leading to wound closure. Successful tissue repair responses involve termination of myofibroblast activities in order to prevent pathologic fibrotic scarring. Here, we discuss the cellular and molecular mechanisms limiting myofibroblast activities during physiological tissue repair, including myofibroblast deactivation, apoptosis, reprogramming and immune clearance of senescent myofibroblasts. In addition, we summarize pathological mechanisms leading to myofibroblast persistence and survival, a hallmark of fibrotic diseases. Finally, we discuss emerging anti-fibrotic therapies aimed at targeting myofibroblast fate such as senolytics, gene therapy, cellular immunotherapy and CAR-T cells.


Assuntos
Miofibroblastos , Senoterapia , Apoptose , Diferenciação Celular , Fibrose , Humanos , Miofibroblastos/patologia , Cicatrização
7.
Curr Rheumatol Rep ; 21(6): 23, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30980212

RESUMO

PURPOSE OF REVIEW: Fibrosis is a pathological feature of many human diseases that affect multiple organs. The development of anti-fibrotic therapies has been a difficult endeavor due to the complexity of signaling pathways associated with fibrogenic processes, complicating the identification and modulation of specific targets. Evidence suggests that ephrin ligands and Eph receptors are crucial signaling molecules that contribute to physiological wound repair and the development of tissue fibrosis. Here, we discuss recent advances in the understanding of ephrin and Eph signaling in tissue repair and fibrosis. RECENT FINDINGS: Ephrin-B2 is implicated in fibrosis of multiple organs. Intercepting its signaling may help counteract fibrosis. Ephrins and Eph receptors are candidate mediators of fibrosis. Ephrin-B2, in particular, promotes fibrogenic processes in multiple organs. Thus, therapeutic strategies targeting Ephrin-B2 signaling could yield new ways to treat organ fibrosis.


Assuntos
Efrinas/metabolismo , Receptores da Família Eph/metabolismo , Transdução de Sinais/fisiologia , Cicatrização/fisiologia , Animais , Fibrose/metabolismo , Fibrose/patologia , Humanos
8.
Am J Respir Cell Mol Biol ; 58(4): 471-481, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29211497

RESUMO

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/genética
9.
Curr Rheumatol Rep ; 20(1): 2, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29349703

RESUMO

PURPOSE OF REVIEW: Organ fibrosis is a lethal component of scleroderma. The hallmark of scleroderma fibrosis is extensive extracellular matrix (ECM) deposition by activated myofibroblasts, specialized hyper-contractile cells that promote ECM remodeling and matrix stiffening. The purpose of this review is to discuss novel mechanistic insight into myofibroblast activation in scleroderma. RECENT FINDINGS: Matrix stiffness, traditionally viewed as an end point of organ fibrosis, is now recognized as a critical regulator of tissue fibrogenesis that hijacks the normal physiologic wound-healing program to promote organ fibrosis. Here, we discuss how matrix stiffness orchestrates fibrosis by controlling three fundamental pro-fibrotic mechanisms: (a) mechanoactivation of myofibroblasts, (b) integrin-mediated latent transforming growth factor beta 1 (TGF-ß1) activation, and (c) activation of non-canonical TGF-ß1 signaling pathways. We also summarize novel therapeutic targets for anti-fibrotic therapy based on the mechanobiology of scleroderma. Future research on mechanobiology of scleroderma may lead to important clinical applications such as improved diagnosis and treatment of patients with scleroderma and other fibrotic-related diseases.


Assuntos
Matriz Extracelular/patologia , Miofibroblastos/patologia , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/fisiopatologia , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibrose , Humanos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Escleroderma Sistêmico/tratamento farmacológico , Escleroderma Sistêmico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/fisiologia , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia
10.
Kidney Int ; 91(3): 628-641, 2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27927603

RESUMO

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/patologia
11.
FASEB J ; 30(6): 2435-50, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27006447

RESUMO

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 quimicamente
13.
Am J Respir Cell Mol Biol ; 54(6): 831-42, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26600305

RESUMO

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/farmacologia
14.
Am J Respir Cell Mol Biol ; 55(1): 105-16, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27082727

RESUMO

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.


Assuntos
Lisofosfolipídeos/metabolismo , Morfogênese , Alvéolos Pulmonares/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Animais , Contagem de Células , Movimento Celular , Tamanho Celular , Elasticidade , Elastina/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibidores Teciduais de Metaloproteinases/metabolismo , Tropoelastina/metabolismo
15.
J Am Soc Nephrol ; 26(1): 173-82, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25012164

RESUMO

In patients undergoing peritoneal dialysis (PD), chronic exposure to nonphysiologic PD fluids elicits low-grade peritoneal inflammation, leading to fibrosis and angiogenesis. Phenotype conversion of mesothelial cells into myofibroblasts, the so-called mesothelial-to-mesenchymal transition (MMT), significantly contributes to the peritoneal dysfunction related to PD. A number of factors have been described to induce MMT in vitro and in vivo, of which TGF-ß1 is probably the most important. The vasoconstrictor peptide endothelin-1 (ET-1) is a transcriptional target of TGF-ß1 and mediates excessive scarring and fibrosis in several tissues. This work studied the contribution of ET-1 to the development of peritoneal damage and failure in a mouse model of PD. ET-1 and its receptors were expressed in the peritoneal membrane and upregulated on PD fluid exposure. Administration of an ET receptor antagonist, either bosentan or macitentan, markedly attenuated PD-induced MMT, fibrosis, angiogenesis, and peritoneal functional decline. Adenovirus-mediated overexpression of ET-1 induced MMT in human mesothelial cells in vitro and promoted the early cellular events associated with peritoneal dysfunction in vivo. Notably, TGF-ß1-blocking peptides prevented these actions of ET-1. Furthermore, a positive reciprocal relationship was observed between ET-1 expression and TGF-ß1 expression in human mesothelial cells. These results strongly support a role for an ET-1/TGF-ß1 axis as an inducer of MMT and subsequent peritoneal damage and fibrosis, and they highlight ET-1 as a potential therapeutic target in the treatment of PD-associated dysfunction.


Assuntos
Endotelina-1/fisiologia , Diálise Peritoneal/efeitos adversos , Fibrose Peritoneal/patologia , Adenoviridae/genética , Animais , Células Cultivadas , Endotelina-1/metabolismo , Células Epiteliais/metabolismo , Epitélio/metabolismo , Feminino , Fibrose/metabolismo , Técnicas de Transferência de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peritônio/metabolismo , Peritônio/patologia , Fenótipo , Fator de Crescimento Transformador beta1/metabolismo
16.
Am J Physiol Lung Cell Mol Physiol ; 308(4): L344-57, 2015 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-25502501

RESUMO

Pathological fibrosis is driven by a feedback loop in which the fibrotic extracellular matrix is both a cause and consequence of fibroblast activation. However, the molecular mechanisms underlying this process remain poorly understood. Here we identify yes-associated protein (YAP) (homolog of drosophila Yki) and transcriptional coactivator with PDZ-binding motif (TAZ) (also known as Wwtr1), transcriptional effectors of the Hippo pathway, as key matrix stiffness-regulated coordinators of fibroblast activation and matrix synthesis. YAP and TAZ are prominently expressed in fibrotic but not healthy lung tissue, with particularly pronounced nuclear expression of TAZ in spindle-shaped fibroblastic cells. In culture, both YAP and TAZ accumulate in the nuclei of fibroblasts grown on pathologically stiff matrices but not physiologically compliant matrices. Knockdown of YAP and TAZ together in vitro attenuates key fibroblast functions, including matrix synthesis, contraction, and proliferation, and does so exclusively on pathologically stiff matrices. Profibrotic effects of YAP and TAZ operate, in part, through their transcriptional target plasminogen activator inhibitor-1, which is regulated by matrix stiffness independent of transforming growth factor-ß signaling. Immortalized fibroblasts conditionally expressing active YAP or TAZ mutant proteins overcome soft matrix limitations on growth and promote fibrosis when adoptively transferred to the murine lung, demonstrating the ability of fibroblast YAP/TAZ activation to drive a profibrotic response in vivo. Together, these results identify YAP and TAZ as mechanoactivated coordinators of the matrix-driven feedback loop that amplifies and sustains fibrosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fibroblastos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/metabolismo , Fosfoproteínas/metabolismo , Fibrose Pulmonar/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular , Feminino , Fibroblastos/patologia , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/patologia , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Endogâmicos NOD , Mutação , Células NIH 3T3 , Fosfoproteínas/genética , Inibidor 1 de Ativador de Plasminogênio/biossíntese , Inibidor 1 de Ativador de Plasminogênio/genética , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Serpina E2/biossíntese , Serpina E2/genética , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteínas de Sinalização YAP
17.
Ann Rheum Dis ; 74(7): 1432-40, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24651621

RESUMO

OBJECTIVES: Mammalian target of rapamycin (mTOR) (a serine/threonine protein kinase) is a major repressor of autophagy, a cell survival mechanism. The specific in vivo mechanism of mTOR signalling in OA pathophysiology is not fully characterised. We determined the expression of mTOR and known autophagy genes in human OA cartilage as well as mouse and dog models of experimental OA. We created cartilage-specific mTOR knockout (KO) mice to determine the specific role of mTOR in OA pathophysiology and autophagy signalling in vivo. METHODS: Inducible cartilage-specific mTOR KO mice were generated and subjected to mouse model of OA. Human OA chondrocytes were treated with rapamycin and transfected with Unc-51-like kinase 1 (ULK1) siRNA to determine mTOR signalling. RESULTS: mTOR is overexpressed in human OA cartilage as well as mouse and dog experimental OA. Upregulation of mTOR expression co-relates with increased chondrocyte apoptosis and reduced expression of key autophagy genes during OA. Subsequently, we show for the first time that cartilage-specific ablation of mTOR results in increased autophagy signalling and a significant protection from destabilisation of medial meniscus (DMM)-induced OA associated with a significant reduction in the articular cartilage degradation, apoptosis and synovial fibrosis. Furthermore, we show that regulation of ULK1/adenosine monophosphate-activated protein kinase (AMPK) signalling pathway by mTOR may in part be responsible for regulating autophagy signalling and the balance between catabolic and anabolic factors in the articular cartilage. CONCLUSIONS: This study provides a direct evidence of the role of mTOR and its downstream modulation of autophagy in articular cartilage homeostasis.


Assuntos
Autofagia/fisiologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Osteoartrite/metabolismo , Osteoartrite/prevenção & controle , Serina-Treonina Quinases TOR/deficiência , Regulação para Cima/fisiologia , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Apoptose/fisiologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Cartilagem Articular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Cães , Inativação Gênica , Humanos , Imunossupressores/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Osteoartrite/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/metabolismo
18.
Ann Rheum Dis ; 74(3): 569-78, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25573665

RESUMO

OBJECTIVES: We have previously shown that peroxisome proliferator-activated receptor gamma (PPARγ), a transcription factor, is essential for the normal growth and development of cartilage. In the present study, we created inducible cartilage-specific PPARγ knockout (KO) mice and subjected these mice to the destabilisation of medial meniscus (DMM) model of osteoarthritis (OA) to elucidate the specific in vivo role of PPARγ in OA pathophysiology. We further investigated the downstream PPARγ signalling pathway responsible for maintaining cartilage homeostasis. METHODS: Inducible cartilage-specific PPARγ KO mice were generated and subjected to DMM model of OA. We also created inducible cartilage-specific PPARγ/mammalian target for rapamycin (mTOR) double KO mice to dissect the PPARγ signalling pathway in OA. RESULTS: Compared with control mice, PPARγ KO mice exhibit accelerated OA phenotype with increased cartilage degradation, chondrocyte apoptosis, and the overproduction of OA inflammatory/catabolic factors associated with the increased expression of mTOR and the suppression of key autophagy markers. In vitro rescue experiments using PPARγ expression vector reduced mTOR expression, increased expression of autophagy markers and reduced the expression of OA inflammatory/catabolic factors, thus reversing the phenotype of PPARγ KO mice chondrocytes. To dissect the in vivo role of mTOR pathway in PPARγ signalling, we created and subjected PPARγ-mTOR double KO mice to the OA model to see if the genetic deletion of mTOR in PPARγ KO mice (double KO) can rescue the accelerated OA phenotype observed in PPARγ KO mice. Indeed, PPARγ-mTOR double KO mice exhibit significant protection/reversal from OA phenotype. SIGNIFICANCE: PPARγ maintains articular cartilage homeostasis, in part, by regulating mTOR pathway.


Assuntos
Cartilagem Articular/metabolismo , Osteoartrite do Joelho/metabolismo , PPAR gama/genética , Serina-Treonina Quinases TOR/genética , Animais , Modelos Animais de Doenças , Meniscos Tibiais/cirurgia , Camundongos , Camundongos Knockout , PPAR gama/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo
20.
JCI Insight ; 9(15)2024 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-38900587

RESUMO

Pathological deposition and crosslinking of collagen type I by activated myofibroblasts drives progressive tissue fibrosis. Therapies that inhibit collagen synthesis have potential as antifibrotic agents. We identify the collagen chaperone cyclophilin B as a major cellular target of the natural product sanglifehrin A (SfA) using photoaffinity labeling and chemical proteomics. Mechanistically, SfA inhibits and induces the secretion of cyclophilin B from the endoplasmic reticulum (ER) and prevents TGF-ß1-activated myofibroblasts from synthesizing and secreting collagen type I in vitro, without inducing ER stress or affecting collagen type I mRNA transcription, myofibroblast migration, contractility, or TGF-ß1 signaling. In vivo, SfA induced cyclophilin B secretion in preclinical models of fibrosis, thereby inhibiting collagen synthesis from fibrotic fibroblasts and mitigating the development of lung and skin fibrosis in mice. Ex vivo, SfA induces cyclophilin B secretion and inhibits collagen type I secretion from fibrotic human lung fibroblasts and samples from patients with idiopathic pulmonary fibrosis (IPF). Taken together, we provide chemical, molecular, functional, and translational evidence for demonstrating direct antifibrotic activities of SfA in preclinical and human ex vivo fibrotic models. Our results identify the cellular target of SfA, the collagen chaperone cyclophilin B, as a mechanistic target for the treatment of organ fibrosis.


Assuntos
Ciclofilinas , Animais , Humanos , Camundongos , Ciclofilinas/metabolismo , Ciclofilinas/antagonistas & inibidores , Colágeno Tipo I/metabolismo , Fibrose , Miofibroblastos/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/patologia , Fibrose Pulmonar Idiopática/metabolismo , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta1/metabolismo , Lactonas , Compostos de Espiro
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