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1.
Am J Physiol Renal Physiol ; 318(2): F375-F387, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31813251

RESUMO

Protein arginine methyltransferase 1 (PRMT1), which primarily causes asymmetric arginine methylation of histone and nonhistone proteins, has been found to activate gene expression and mediate multiple pathological processes. Its role in renal fibrosis, however, remains unclear. In the present study, we observed that PRMT1 and its specific epigenetic marker, asymmetric di-methylated histone 4 arginine 3 (H4R3Me2a), were highly expressed in cultured renal interstitial fibroblasts. Treatment of PRMT1 with AMI-1, a selective inhibitor of PRMT1, or silencing PRMT1 with siRNA inhibited serum-induced and transforming growth factor (TGF)-ß1-induced expression of α-smooth muscle actin (α-SMA) and collagen type I, two hallmarks of renal fibroblast activation, in a dose-dependent and time-dependent manner. In a murine model of renal fibrosis induced by unilateral ureteral obstruction, PRMT1 expression and H4R3Me2a were also upregulated, which was coincident with increased expression of α-SMA, collagen type I, and fibronectin. Administration of AMI-1 reduced PRMT1 and H4R3Me2a expression, attenuated extracellular matrix protein deposition, and inhibited renal fibroblast activation and proliferation. Moreover, AMI-1 treatment inhibited Smad3 phosphorylation and TGF-ß receptor I expression but prevented Smad7 downregulation both in the kidney after unilateral ureteral obstruction injury and in cultured renal interstitial fibroblasts exposed to TGF-ß1. Collectively, these results demonstrate that PRMT1 may mediate renal fibroblast activation and renal fibrosis development through activation of the TGF-ß/Smad3 signaling pathway. They also suggest that PRMT1 inhibition may be a potential therapeutic approach for the treatment of fibrotic kidney disease.


Assuntos
Desdiferenciação Celular , Fibroblastos/enzimologia , Nefropatias/enzimologia , Rim/enzimologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteína Smad3/metabolismo , Animais , Desdiferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/etiologia , Nefropatias/patologia , Nefropatias/prevenção & controle , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Naftalenossulfonatos/farmacologia , Fosforilação , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia , Ureia/análogos & derivados , Ureia/farmacologia , Obstrução Ureteral/complicações
2.
Cardiovasc Res ; 116(5): 956-969, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31297506

RESUMO

AIMS: Cardiac fibrosis is a major cause of heart failure (HF), and mediated by the differentiation of cardiac fibroblasts into myofibroblasts. However, limited tools are available to block cardiac fibrosis. ADAMTS16 is a member of the ADAMTS superfamily of extracellular protease enzymes involved in extracellular matrix (ECM) degradation and remodelling. In this study, we aimed to establish ADAMTS16 as a key regulator of cardiac fibrosis. METHODS AND RESULTS: Western blot and qRT-PCR analyses demonstrated that ADAMTS16 was significantly up-regulated in mice with transverse aortic constriction (TAC) associated with left ventricular hypertrophy and HF, which was correlated with increased expression of Mmp2, Mmp9, Col1a1, and Col3a1. Overexpression of ADAMTS16 accelerated the AngII-induced activation of cardiac fibroblasts into myofibroblasts. Protein structural analysis and co-immunoprecipitation revealed that ADAMTS16 interacted with the latency-associated peptide (LAP)-transforming growth factor (TGF)-ß via a RRFR motif. Overexpression of ADAMTS16 induced the activation of TGF-ß in cardiac fibroblasts; however, the effects were blocked by a mutation of the RRFR motif to IIFI, knockdown of Adamts16 expression, or a TGF-ß-neutralizing antibody (ΝAb). The RRFR tetrapeptide, but not control IIFI peptide, blocked the interaction between ADAMTS16 and LAP-TGF-ß, and accelerated the activation of TGF-ß in cardiac fibroblasts. In TAC mice, the RRFR tetrapeptide aggravated cardiac fibrosis and hypertrophy by up-regulation of ECM proteins, activation of TGF-ß, and increased SMAD2/SMAD3 signalling, however, the effects were blocked by TGF-ß-NAb. CONCLUSION: ADAMTS16 promotes cardiac fibrosis, cardiac hypertrophy, and HF by facilitating cardiac fibroblasts activation via interacting with and activating LAP-TGF-ß signalling. The RRFR motif of ADAMTS16 disrupts the interaction between ADAMTS16 and LAP-TGF-ß, activates TGF-ß, and aggravated cardiac fibrosis and hypertrophy. This study identifies a novel regulator of TGF-ß signalling and cardiac fibrosis, and provides a new target for the development of therapeutic treatment of cardiac fibrosis and HF.


Assuntos
Proteínas ADAMTS/metabolismo , Cardiomegalia/enzimologia , Miocárdio/enzimologia , Miofibroblastos/enzimologia , Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Remodelação Ventricular , Proteínas ADAMTS/genética , Motivos de Aminoácidos , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Modelos Animais de Doenças , Fibrose , Células HeLa , Humanos , Masculino , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Miofibroblastos/patologia , Peptídeos/genética , Domínios e Motivos de Interação entre Proteínas , Precursores de Proteínas/genética , Transdução de Sinais , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Regulação para Cima
3.
Int J Cancer ; 145(11): 3064-3077, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31032902

RESUMO

Myofibroblasts are a population of highly contractile fibroblasts that express and require the activity of the transcription factor Snail1. Cancer-associated fibroblasts (CAFs) correlate with low survival of cancer patients when present in the stroma of primary tumors. Remarkably, the presence of myofibroblastic CAFs (which express Snail1) creates mechanical properties in the tumor microenvironment that support metastasis. However, therapeutic blockage of fibroblast activity in patients with cancer is a double-edged sword, as normal fibroblast activities often restrict tumor cell invasion. We used fibroblasts depleted of Snail1 or protein arginine methyltransferases 1 and 4 (PRMT1/-4) to identify specific epigenetic modifications induced by TGFß/Snail1. Furthermore, we analyzed the in vivo efficiency of methyltransferase inhibitors using mouse models of wound healing and metastasis, as well as fibroblasts isolated from patients with idiopathic pulmonary fibrosis (IPF). Mechanistically, TGFß-induced Snail1 promotes the epigenetic mark of asymmetrically dimethylated arginine. Critically, we found that inhibitors of methyltransferases prevent myofibroblast activity (but not regular fibroblast activity) in the extracellular matrix, both in cell culture and in vivo. In a mouse breast cancer model, the inhibitor sinefungin reduces both the myofibroblast activity in the tumor stroma and the metastatic burden in the lung. Two distinct inhibitors effectively blocked the exacerbated myofibroblast activity of patient-derived IPF fibroblasts. Our data reveal epigenetic regulation of myofibroblast transdifferentiation in both wound healing and in disease (fibrosis and breast cancer). Thus, methyltransferase inhibitors are good candidates as therapeutic reagents for these diseases.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/administração & dosagem , Fibrose Pulmonar Idiopática/tratamento farmacológico , Neoplasias Pulmonares/secundário , Metiltransferases/antagonistas & inibidores , Miofibroblastos/efeitos dos fármacos , Fatores de Transcrição da Família Snail/genética , Adenosina/administração & dosagem , Adenosina/análogos & derivados , Adenosina/farmacologia , Animais , Neoplasias da Mama/enzimologia , Fibroblastos Associados a Câncer/citologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Fibroblastos Associados a Câncer/metabolismo , Linhagem Celular Tumoral , Transdiferenciação Celular , Células Cultivadas , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Epigênese Genética , Feminino , Deleção de Genes , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Metiltransferases/genética , Camundongos , Miofibroblastos/citologia , Miofibroblastos/enzimologia , Fatores de Transcrição da Família Snail/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Am J Physiol Heart Circ Physiol ; 316(6): H1281-H1296, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30901279

RESUMO

MK5 is a protein serine/threonine kinase activated by p38, ERK3, and ERK4 MAPKs. MK5 mRNA and immunoreactivity are detected in mouse cardiac fibroblasts, and MK5 haplodeficiency attenuates the increase in collagen 1-α1 mRNA evoked by pressure overload. The present study examined the effect of MK5 haplodeficiency on reparative fibrosis following myocardial infarction (MI). Twelve-week-old MK5+/- and wild-type littermate (MK5+/+) mice underwent ligation of the left anterior descending coronary artery (LADL). Surviving mice were euthanized 8 or 21 days post-MI. Survival rates did not differ significantly between MK5+/+ and MK5+/- mice, with rupture of the LV wall being the primary cause of death. Echocardiographic imaging revealed similar increases in LV end-diastolic diameter, myocardial performance index, and wall motion score index in LADL-MK5+/+ and LADL-MK5+/- mice. Area at risk did not differ between LADL-MK5+/+ and LADL-MK5+/- hearts. In contrast, infarct size, scar area, and scar collagen content were reduced in LADL-MK5+/- hearts. Immunohistochemical analysis of mice experiencing heart rupture revealed increased MMP-9 immunoreactivity in the infarct border zone of LADL-MK5+/- hearts compared with LADL-MK5+/+. Although inflammatory cell infiltration was similar in LADL-MK5+/+ and LADL-MK5+/- hearts, angiogenesis was more pronounced in the infarct border zone of LADL-MK5+/- mice. Characterization of ventricular fibroblasts revealed reduced motility and proliferation in fibroblasts isolated from MK5-/- mice compared with those from both wild-type and haplodeficient mice. siRNA-mediated knockdown of MK5 in fibroblasts from wild-type mice also impaired motility. Hence, reduced MK5 expression alters fibroblast function and scar morphology but not mortality post-MI. NEW & NOTEWORTHY MK5/PRAK is a protein serine/threonine kinase activated by p38 MAPK and/or atypical MAPKs ERK3/4. MK5 haplodeficiency reduced infarct size, scar area, and scar collagen content post-myocardial infarction. Motility and proliferation were reduced in cultured MK5-null cardiac myofibroblasts.


Assuntos
Cicatriz/enzimologia , Colágeno/metabolismo , Haploinsuficiência , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Infarto do Miocárdio/enzimologia , Miocárdio/enzimologia , Miofibroblastos/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Cicatrização , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Cicatriz/patologia , Cicatriz/fisiopatologia , Modelos Animais de Doenças , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Miofibroblastos/patologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Função Ventricular Esquerda , Remodelação Ventricular
5.
Transl Res ; 208: 30-46, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30857762

RESUMO

B-type natriuretic peptide (BNP) was approved by the US Food and Drug Administration in 2001 for the treatment of heart failure. However, the effects of BNP in clinical applications are controversial and uncertain. Recently, study indicated that high BNP levels are associated with an increased risk of developing atrial fibrillation. In this study, we investigated the direct effects of BNP on TNF-α-induced atrial fibrosis mice, as well as its effects on human atrial myofibroblasts. We found that injecting TNF-α-induced mice with recombinant human BNP enhanced atrial fibrosis via matrix metalloproteinase-2 (MMP-2) expression and collagen accumulation. Furthermore, we found that BNP stimulated MMP-2 expression in human atrial myofibroblasts. Treatment of human atrial myofibroblasts with cycloheximide had no effect on this outcome; however, treatment of cells with MG132 enhanced BNP-induced MMP-2 expression, indicating that protein stability and inhibition of proteasome-mediated protein degradation pathways are potentially involved. Inhibition of SIRT1 significantly decreased BNP-induced MMP-2 expression. Additionally, confocal and coimmunoprecipitation data indicated that BNP-regulated MMP-2 expression are likely to be mediated through direct interaction with SIRT1, which is thought to deacetylate MMP-2 and to increase its protein stability in human atrial myofibroblasts. Finally, we confirmed that SIRT1 is expressed and cytoplasmically redistributed as well as colocalized with MMP-2 in mouse fibrotic atrial tissue. We suggest a possible fibrosis-promoting role of BNP in the atrium, although the antifibrotic properties of BNP in the ventricle have been reported in previous studies, and that the coordination between MMP-2 and SIRT1 in BNP-induced atrial myofibroblasts participates in atrial fibrosis.


Assuntos
Átrios do Coração/enzimologia , Metaloproteinase 2 da Matriz/metabolismo , Miofibroblastos/metabolismo , Peptídeo Natriurético Encefálico/fisiologia , Acetilação , Animais , Fibrose , Átrios do Coração/patologia , Humanos , Técnicas In Vitro , Camundongos , Miofibroblastos/enzimologia , Sirtuína 1/metabolismo
6.
J Cardiovasc Pharmacol ; 73(4): 248-256, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30801261

RESUMO

Pathological stimulus-triggered differentiation of cardiac fibroblasts plays a major role in the development of myocardial fibrosis. Aldehyde dehydrogenase 2 (ALDH2) was reported to exert a protective role in cardiovascular disease, and whether ALDH2 is involved in cardiac fibroblast differentiation remains unclear. In this study, we used transforming growth factor-ß1 (TGF-ß1) to induce the differentiation of human cardiac fibroblasts (HCFs) and adopted ALDH2 activator Alda-1 to verify the influence of ALDH2 on HCF differentiation. Results showed that ALDH2 activity was obviously impaired when treating HCFs with TGF-ß1. Activation of ALDH2 with Alda-1 inhibited the transformation of HCFs into myofibroblasts, demonstrated by the decreased smooth muscle actin (α-actin) and periostin expression, reduced HCF-derived myofibroblast proliferation, collagen production, and contractility. Moreover, application of Smad2/3 inhibitor alleviated TGF-ß1-induced HCF differentiation and improved ALDH2 activity, which was reversed by the application of ALDH2 inhibitor daidzin. Finally, Alda-1-induced HCF alterations alleviated neonatal rat cardiomyocyte hypertrophy, supported by the immunostaining of α-actin. To summarize, activation of ALDH2 enzymatic activity inhibited the differentiation of cardiac fibroblasts via the TGF-ß1/Smad signaling pathway, which might be a promising strategy to relieve myocardial fibrosis of various causes.


Assuntos
Aldeído-Desidrogenase Mitocondrial/metabolismo , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Plasticidade Celular/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Ventrículos do Coração/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Animais Recém-Nascidos , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Cardiomegalia/prevenção & controle , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno/metabolismo , Ativação Enzimática , Fibrose , Ventrículos do Coração/enzimologia , Ventrículos do Coração/patologia , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Comunicação Parácrina , Fenótipo , Fosforilação , Ratos , Transdução de Sinais
7.
Clin Sci (Lond) ; 133(2): 239-252, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30617188

RESUMO

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor ß (TGF-ß) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.


Assuntos
Benzazepinas/farmacologia , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Neprilisina/antagonistas & inibidores , Inibidores de Proteases/farmacologia , Obstrução Ureteral/tratamento farmacológico , Animais , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Fibrose , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/enzimologia , Rim/patologia , Nefropatias/enzimologia , Nefropatias/genética , Nefropatias/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Células NIH 3T3 , Neprilisina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Obstrução Ureteral/enzimologia , Obstrução Ureteral/genética , Obstrução Ureteral/patologia
8.
Histol Histopathol ; 34(7): 745-753, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30516266

RESUMO

HIPK2 is an evolutionarily conserved serine/threonine kinase and is considered a co-regulator of an increasing number of transcription factors modulating a variety of cellular processes, including inflammation, proliferation and fibrosis. Skeletal muscle injuries repair is an overlapping event between inflammation and tissue repair. There are no reports about HIPK2 expression in skeletal muscles after trauma. A foundational study on distribution and time-dependent expression of HIPK2 was performed by immunohistochemical staining, Western blotting and quantitative real-time PCR, which is expected to obtain a preliminary insight into the functions of HIPK2 during the repair of contused skeletal muscle in mice. An animal model of skeletal muscle contusion was established in 50 C57B6/L male mice. Samples were taken at 1, 3, 5, 7, 9, 14, 17, 21 and 28 days after contusion, respectively (5 mice at each posttraumatic interval). 5 mice were employed as control. No HIPK2-positive staining was detected in uninjured skeletal muscle. Intensive immunoreactivties of HIPK2 were observed in polymorphonuclear cells, round-shaped mononuclear cells, regenerated multinucleated myotubes and spindle-shaped fibroblastic cells in the contused tissue. The HIPK2-positive cells were identified as neutrophils, macrophages and myofibroblasts by double immunofluorescent procedure. HIPK2 protein and mRNA expression were remarkably up-regulated after contusion by Western blotting and qPCR analysis. The results demonstrated that the expression of HIPK2 is distributed in certain cell types and is time-dependently expressed in skeletal muscle after contusion, which suggested that HIPK2 may participate in the whole process of skeletal muscle wound healing, including inflammatory response, muscle regeneration and fibrogenesis.


Assuntos
Contusões/enzimologia , Músculo Esquelético/enzimologia , Músculo Esquelético/lesões , Proteínas Serina-Treonina Quinases/metabolismo , Cicatrização , Animais , Contusões/patologia , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibrose , Inflamação/enzimologia , Inflamação/patologia , Macrófagos/citologia , Macrófagos/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fibras Musculares Esqueléticas/enzimologia , Músculo Esquelético/patologia , Miofibroblastos/citologia , Miofibroblastos/enzimologia , Neutrófilos/citologia , Neutrófilos/enzimologia , Regeneração , Fatores de Tempo
9.
Am J Physiol Lung Cell Mol Physiol ; 316(1): L175-L186, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30358439

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disease, and fibroblast-myofibroblast differentiation (FMD) is thought to be a key event in the pathogenesis of IPF. Histone deacetylase-8 (HDAC8) has been shown to associate with α-smooth muscle actin (α-SMA; a marker of FMD) and regulates cell contractility in vascular smooth muscle cells. However, the role of HDAC8 in FMD or pulmonary fibrosis has never been reported. This study investigated the role of HDAC8 in pulmonary fibrosis with a focus on FMD. We observed that HDAC8 expression was increased in IPF lung tissue as well as transforming growth factor (TGF)ß1-treated normal human lung fibroblasts (NHLFs). Immunoprecipitation experiments revealed that HDAC8 was associated with α-SMA in TGFß1-treated NHLFs. HDAC8 inhibition with NCC170 (HDAC8-selective inhibitor) repressed TGFß1-induced fibroblast contraction and α-SMA protein expression in NHLFs cultured in collagen gels. HDAC8 inhibition with HDAC8 siRNA also repressed TGFß1-induced expression of profibrotic molecules such as fibronectin and increased expression of antifibrotic molecules such as peroxisome proliferator-activated receptor-γ (PPARγ). Chromatin immunoprecipitation quantitative PCR using an antibody against H3K27ac (histone H3 acetylated at lysine 27; a known HDAC8 substrate and a marker for active enhancers) suggested that HDAC8 inhibition with NCC170 ameliorated TGFß1-induced loss of H3K27ac at the PPARγ gene enhancer. Furthermore, NCC170 treatment significantly decreased fibrosis measured by Ashcroft score as well as expression of type 1 collagen and fibronectin in bleomycin-treated mouse lungs. These data suggest that HDAC8 contributes to pulmonary fibrosis and that there is a therapeutic potential for HDAC8 inhibitors to treat IPF as well as other fibrotic lung diseases.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Fibrose Pulmonar Idiopática/tratamento farmacológico , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Miofibroblastos/enzimologia , Proteínas Repressoras/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilases/biossíntese , Histonas/metabolismo , Humanos , Fibrose Pulmonar Idiopática/enzimologia , Fibrose Pulmonar Idiopática/patologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Miofibroblastos/patologia , PPAR gama/metabolismo , Proteínas Repressoras/biossíntese , Fator de Crescimento Transformador beta1/metabolismo
10.
Eur Urol ; 75(2): 329-340, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30344087

RESUMO

BACKGROUND: Peyronie's disease (PD) is a fibrotic disorder of the penile tunica albuginea, characterised by the formation of a localised fibrous plaque that can lead to deformity and erectile dysfunction. Nonsurgical therapeutic options for PD are limited in efficacy and safety. Myofibroblasts are key cells in the pathogenesis of PD, and inhibition of myofibroblast transformation has been suggested as a therapeutic option. OBJECTIVE: To identify potential drugs using a novel phenotypic assay and then to test them using in vitro and in vivo models of PD. DESIGN, SETTING, AND PARTICIPANTS: We have developed and validated a phenotypic screening assay that measures myofibroblast transformation, by which we tested 21 compounds that were suggested to be efficacious in treating PD. The successful hits from this assay were further tested using in vitro and in vivo models of PD. RESULTS AND LIMITATIONS: The new assay was able to detect transforming growth factor-ß1-induced myofibroblast transformation. Using this assay, phosphodiesterase type 5 inhibitors (PDE5i) and selective oestrogen receptor modulators (SERMs) were identified to significantly inhibit myofibroblast transformation. A PDE5i (vardenafil) and an SERM (tamoxifen) inhibited myofibroblast transformation, collagen gel contraction, and extracellular matrix production in a synergistic fashion. In a rat model of PD, the antifibrotic effect of the combination of vardenafil and tamoxifen was greater than that of each drug alone. This study is limited by not providing a molecular mechanism for the proposed synergy. CONCLUSIONS: This is the first demonstration of a synergistic activity between a PDE5i and an SERM discovered through a phenotypic screening approach. Future clinical trials using a combination of these drugs should be considered during the active phase of PD, given the early evidence of benefit in both in vitro and in vivo models. PATIENT SUMMARY: This report suggests that the combination of a phosphodiesterase type 5 inhibitor and a selective oestrogen receptor modulator may be efficacious in treating Peyronie's disease in its active phase.


Assuntos
Miofibroblastos/efeitos dos fármacos , Induração Peniana/tratamento farmacológico , Pênis/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Tamoxifeno/farmacologia , Dicloridrato de Vardenafila/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/enzimologia , Matriz Extracelular/patologia , Fibrose , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Induração Peniana/enzimologia , Induração Peniana/patologia , Pênis/enzimologia , Pênis/patologia , Fenótipo , Ratos Sprague-Dawley
11.
Med Sci Monit ; 24: 6264-6272, 2018 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-30194718

RESUMO

BACKGROUND Myocardial fibrosis is closely related to all types of cardiovascular diseases. Hirudin is widely used in the prevention and treatment of cardiovascular diseases and cancers. In this study, we examined the potential role(s) and mechanism of hirudin in angiotensin II (Ang II)-induced myocardial fibrosis. MATERIAL AND METHODS The viability of myocardial fibroblasts, and reactive oxygen species (ROS) rates were measured respectively using cell counting kit-8 (CCK-8) and flow cytometry. Malondialdehyde (MDA) content, the activities of lactate dehydrogenase (LDH), and superoxide dismutase (SOD) were detected by the respective kits. The mRNA and protein levels of fibrosis-related factors were separately assessed by qRT-PCR and western blot. RESULTS Our data revealed that hirudin suppressed the viability of myocardial fibroblasts, and that it relieved the proliferation induced by Ang II in a dose-dependent manner. We also found that hirudin reduced ROS production, LDH activity, and MDA content; however, it enhanced SOD activity. Moreover, while hirudin significantly downregulated the levels of matrix metalloproteinase-2 (MMP-2), MMP-9, fibronectin (FN), transforming growth factor beta 1 (TGF-ß1), collagen-I (COL-I), and COL-III, it upregulated the expression level of tissue inhibitor of metalloproteinases-2 (TIMP-2). Furthermore, phosphorylated extracellular signal-regulated kinase1/2 (p-ERK1/2) was decreased by hirudin, compared to the Ang-II group. CONCLUSIONS Hirudin depressed Ang II-induced myocardial fibroblasts via inhibiting oxidative stress, regulating fibrosis-related factors, and repressing the ERK1/2 pathway.


Assuntos
Angiotensina II/farmacologia , Fibrose Endomiocárdica/tratamento farmacológico , Hirudinas/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Células do Cúmulo , Fibrose Endomiocárdica/enzimologia , Fibrose Endomiocárdica/metabolismo , Fibrose Endomiocárdica/patologia , Fibronectinas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Miocárdio/citologia , Miocárdio/metabolismo , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/enzimologia , Miofibroblastos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Inibidor Tecidual de Metaloproteinase-2/metabolismo , Fator de Crescimento Transformador beta1/metabolismo
12.
Pharmacology ; 102(3-4): 142-153, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30016781

RESUMO

AIM: Previous studies have suggested that quercetin is effective for treating diverse chronic disorders including organ fibrosis and airway and cardiovascular disorders. To access the pharmacological background for its broad efficacy, we examined the ability of quercetin to modulate the inflammatory and fibrotic responses associated with organ injury that commonly underlie the pathogenesis of those disorders. METHODS: A cutaneous wound model on rabbit ear was used for in vivo study. Quercetin was topically applied to the wounds, and the number of macrophages and myofibroblasts and the size of the hypertrophic scar formed were estimated. An in vitro study examined the ability of quercetin to inhibit cell-signaling pathways that activate RAW264.7 macrophages and primary dermal fibroblasts and the tyrosine kinase activity of discoidin domain receptor 2. RESULTS: Quercetin reduced the population of macrophages and myofibroblasts and the scar formation in cutaneous wound healing. Quercetin suppressed the signaling pathways activating RAW264.7 macrophages and dermal fibroblasts, which is associated with its inhibition of multiple tyrosine kinases to regulate the pathways. This pharmacological activity of quercetin to simultaneously inhibit the inflammatory and fibrotic responses upon tissue damage by targeting multi-kinases could be the action mechanism to support its broad efficacy for various chronic disorders.


Assuntos
Receptor com Domínio Discoidina 2/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Miofibroblastos/efeitos dos fármacos , Quercetina/farmacologia , Ferimentos e Lesões/tratamento farmacológico , Animais , Anti-Inflamatórios/metabolismo , Ciclo-Oxigenase 2/metabolismo , Receptor com Domínio Discoidina 2/metabolismo , Regulação para Baixo/efeitos dos fármacos , Feminino , Fibrose/enzimologia , Fibrose/patologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Óxido Nítrico Sintase Tipo II/metabolismo , Cultura Primária de Células , Células RAW 264.7 , Coelhos , Ferimentos e Lesões/enzimologia , Ferimentos e Lesões/patologia
13.
Clin Sci (Lond) ; 132(6): 685-699, 2018 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-29540538

RESUMO

T-cell infiltration and the subsequent increased intracardial chronic inflammation play crucial roles in the development of cardiac hypertrophy and heart failure (HF). A77 1726, the active metabolite of leflunomide, has been reported to have powerful anti-inflammatory and T cell-inhibiting properties. However, the effect of A77 1726 on cardiac hypertrophy remains completely unknown. Herein, we found that A77 1726 treatment attenuated pressure overload or angiotensin II (Ang II)-induced cardiac hypertrophy in vivo, as well as agonist-induced hypertrophic response of cardiomyocytes in vitro In addition, we showed that A77 1726 administration prevented induction of cardiac fibrosis by inhibiting cardiac fibroblast (CF) transformation into myofibroblast. Surprisingly, we found that the protective effect of A77 1726 was not dependent on its T lymphocyte-inhibiting property. A77 1726 suppressed the activation of protein kinase B (AKT) signaling pathway, and overexpression of constitutively active AKT completely abolished A77 1726-mediated cardioprotective effects in vivo and in vitro Pretreatment with siRNA targetting Fyn (si Fyn) blunted the protective effect elicited by A77 1726 in vitro More importantly, A77 1726 was capable of blocking pre-established cardiac hypertrophy in mice. In conclusion, A77 1726 attenuated cardiac hypertrophy and cardiac fibrosis via inhibiting FYN/AKT signaling pathway.


Assuntos
Fibroblastos/efeitos dos fármacos , Ventrículos do Coração/efeitos dos fármacos , Hipertrofia Ventricular Esquerda/prevenção & controle , Leflunomida/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/enzimologia , Fibroblastos/patologia , Fibrose , Ventrículos do Coração/enzimologia , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
14.
Cardiovasc Res ; 114(5): 703-712, 2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29415171

RESUMO

Aims: Cardiac myofibroblasts (CMFs) play a crucial role in the progression of pathological fibrotic cardiac remodelling. The expression of osteoglycin (OGN) is increased in diseased hearts; however, the role of OGN in pathological cardiac remodelling is not understood. Here, we sought to determine the effect of OGN on cardiac interstitial fibrosis and investigate the molecular mechanisms of OGN in CMF activation and matrix production. Methods and results: We found that OGN expression was significantly upregulated in mouse hearts in response to chronic 14-day angiotensin II (Ang II) infusion. Mice lacking OGN (OGN-/-) exhibited enhanced cardiac interstitial fibrosis and significantly more severe cardiac dysfunction following Ang II infusion compared to wild-type mice. OGN deficiency did not alter blood pressure, nor had effect on transforming growth factor-beta signalling activation, but presented with increased proliferative activity in hearts. In vitro studies with isolated CMFs revealed that OGN deficiency significantly increased proliferation and migration and enhanced the transactivation of epidermal growth factor receptor (EGFR) signalling by Ang II. On the other hand, OGN overexpression in CMFs decreased their proliferation and migration via reducing EGFR activation. Overexpression of OGN also suppressed the shedding of membrane anchored EGFR ligand. Moreover, OGN was found to interact with a lysophosphatidic acid (LPA) receptor isoform 3 and thus to attenuate EGFR transactivation through blocking cell surface translocation of membrane type 1 matrix metalloproteinase (MT1-MMP) and subsequent pro-MMP-2 activation in a Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK)-dependent manner. Conclusion: These findings suggest that OGN negatively regulates cardiac fibrotic remodelling by attenuating CMF proliferation and migration through LPA3-mediated and Rho/ROCK-dependent inhibition of MT1-MMP translocation, MMP2 activation and EGFR transactivation.


Assuntos
Cardiomegalia/enzimologia , Proliferação de Células , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Miofibroblastos/enzimologia , Receptores de Ácidos Lisofosfatídicos/metabolismo , Remodelação Ventricular , Angiotensina II , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Movimento Celular , Células Cultivadas , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Fibrose , Hipertensão/induzido quimicamente , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Miofibroblastos/patologia , Receptor Cross-Talk , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
15.
Kidney Int ; 93(1): 81-94, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28739141

RESUMO

Chronic kidney disease is a major cause of death, and renal fibrosis is a common pathway leading to the progression of this disease. Although activated fibroblasts are responsible for the production of the extracellular matrix and the development of renal fibrosis, the molecular mechanisms underlying fibroblast activation are not fully defined. Here we examined the functional role of AMP-activated protein kinase (AMPK) in the activation of fibroblasts and the development of renal fibrosis. AMPKα1 was induced in the kidney during the development of renal fibrosis. Mice with global or fibroblast-specific knockout of AMPKα1 exhibited fewer myofibroblasts, developed less fibrosis, and produced less extracellular matrix protein in the kidneys following unilateral ureteral obstruction or ischemia-reperfusion injury. Mechanistically, AMPKα1 directly phosphorylated cofilin leading to cytoskeleton remodeling and myocardin-related transcription factor-A nuclear translocation resulting in fibroblast activation and extracellular matrix protein production. Thus, AMPK may be a critical regulator of fibroblast activation through regulation of cytoskeleton dynamics and myocardin-related transcription factor-A nuclear translocation. Hence, AMPK signaling may represent a novel therapeutic target for fibrotic kidney disease.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Fibroblastos/enzimologia , Rim/enzimologia , Miofibroblastos/enzimologia , Insuficiência Renal Crônica/enzimologia , Traumatismo por Reperfusão/enzimologia , Transativadores/metabolismo , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Transporte Ativo do Núcleo Celular , Animais , Cofilina 1/metabolismo , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibroblastos/patologia , Fibrose , Células HEK293 , Humanos , Rim/patologia , Masculino , Camundongos Knockout , Mutação , Miofibroblastos/patologia , Fosforilação , Insuficiência Renal Crônica/etiologia , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/patologia , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/patologia , Transdução de Sinais , Fatores de Transcrição/metabolismo , Obstrução Ureteral/complicações
16.
J Cell Physiol ; 233(1): 447-462, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28295277

RESUMO

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-ß, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4+ and the ratio of CD4+ CD25high /CD4+ CD25low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34+ and CD45+ cells, but decreased the frequency of CD33+ and CD14+ myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miofibroblastos/efeitos dos fármacos , Ligamento Periodontal/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Microambiente Celular , Condrogênese/efeitos dos fármacos , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Miofibroblastos/enzimologia , Miofibroblastos/imunologia , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , PPAR gama/genética , PPAR gama/metabolismo , Ligamento Periodontal/enzimologia , Ligamento Periodontal/imunologia , Fenótipo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/enzimologia , Células-Tronco/imunologia , Fatores de Tempo , Migração Transendotelial e Transepitelial/efeitos dos fármacos
17.
Kidney Int ; 93(1): 173-187, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29042082

RESUMO

Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-ß/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-ß1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases.


Assuntos
Transdiferenciação Celular , Cicatriz/enzimologia , Nefropatias/enzimologia , Rim/enzimologia , Macrófagos/enzimologia , Miofibroblastos/enzimologia , Quinases da Família src/metabolismo , Animais , Transdiferenciação Celular/efeitos dos fármacos , Transdiferenciação Celular/genética , Células Cultivadas , Cicatriz/genética , Cicatriz/patologia , Cicatriz/prevenção & controle , Modelos Animais de Doenças , Fibrose , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Rim/efeitos dos fármacos , Rim/patologia , Nefropatias/genética , Nefropatias/patologia , Nefropatias/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Inibidores de Proteínas Quinases/farmacologia , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Proteína Smad3/genética , Proteína Smad3/metabolismo , Obstrução Ureteral/tratamento farmacológico , Obstrução Ureteral/enzimologia , Obstrução Ureteral/genética , Quinases da Família src/genética
18.
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
19.
Invest Ophthalmol Vis Sci ; 58(12): 5217-5226, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29049722

RESUMO

Purpose: Fibroblast activation may play an important role in pterygium progression. Synthetic peroxisome proliferator-activated receptor γ (PPAR-γ) ligands have been shown to be effective antifibrotic agents against transforming growth factor ß1 (TGF-ß1) induced fibrosis in several tissues. We aimed to investigate the antifibrotic effects of the PPAR-γ ligand rosiglitazone in pterygium fibroblasts and the underlying mechanisms. Methods: Profibrotic activation was induced by TGF-ß1 in primary cultured human pterygium fibroblasts and the effect of rosiglitazone treatment on α-smooth muscle actin (α-SMA), and extra cellular matrix proteins synthesis was detected by western blotting, real-time PCR, immunostaining, and flow cytometry. Pharmaceutical inhibition of PPAR-γ receptor was used to determine the dependency or otherwise of rosiglitazone's action on PPAR-γ signaling. Major signaling pathways downstream of TGF-ß1 were investigated by western blotting to assess their possible association with rosiglitazone's effect. Cell viability and apoptosis were investigated to assess drug-induced cytotoxicity, and the effect of rosiglitazone treatment on cell migration was further determined. Results: α-SMA and fibronectin synthesis induced by TGF-ß1 were suppressed by rosiglitazone treatment in a dose-dependent manner. Rosiglitazone also inhibited intrinsic TGF-ß1 expression. Smad2/3, ERK1/2, and P38 pathways were activated in response to TGF-ß1. Rosiglitazone suppressed TGF-ß1-induced P38 MAPK activation, while ERK1/2 and Smad2/3 signaling remained unaffected. The observed antifibrotic effect of rosiglitazone was not affected by the PPAR-γ antagonist GW9662, indicating it is not PPAR-γ dependent. Rosiglitazone also inhibited the proliferation and migration of pterygium fibroblasts. Conclusions: Rosiglitazone suppresses TGF-ß1-induced myofibroblast activation and extra cellular matrix synthesis in pterygium fibroblasts at least partly through the modulation of the p38 MAPK pathway.


Assuntos
Hipoglicemiantes/farmacologia , Miofibroblastos/efeitos dos fármacos , PPAR gama/agonistas , Pterígio/tratamento farmacológico , Tiazolidinedionas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Actinas/metabolismo , Apoptose , Western Blotting , Células Cultivadas , Proteínas da Matriz Extracelular/metabolismo , Fibrose/prevenção & controle , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Miofibroblastos/enzimologia , Miofibroblastos/patologia , Pterígio/enzimologia , Pterígio/patologia , Reação em Cadeia da Polimerase em Tempo Real , Rosiglitazona , Transdução de Sinais , Fator de Crescimento Transformador beta1/farmacologia
20.
Am J Respir Cell Mol Biol ; 57(6): 702-710, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28787175

RESUMO

Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives HAS2 expression were generated. Mixed male and female α-SMA-HAS2 mice (HAS2+ mice, n = 16; HAS2- mice, n = 13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and α-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, α-SMA, and collagen deposition in OVA-challenged α-SMA-HAS2+ mice compared with α-SMA-HAS2- mice. Unexpectedly, OVA-challenged α-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated α-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged α-SMA-HAS2+ mice and α-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.


Assuntos
Asma/enzimologia , Regulação Enzimológica da Expressão Gênica , Hialuronan Sintases/biossíntese , Pulmão/enzimologia , Miócitos de Músculo Liso/enzimologia , Miofibroblastos/enzimologia , Actinas/biossíntese , Actinas/genética , Alérgenos/toxicidade , Animais , Asma/induzido quimicamente , Asma/genética , Broncoconstrição/efeitos dos fármacos , Broncoconstrição/genética , Doença Crônica , Humanos , Hialuronan Sintases/genética , Pulmão/patologia , Camundongos , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Miofibroblastos/patologia
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