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1.
Int J Mol Sci ; 22(17)2021 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-34502098

RESUMO

Clinical effects induced by arrhythmogenic cardiomyopathy (ACM) originate from a large spectrum of genetic variations, including the missense mutation of the lamin A/C gene (LMNA), LMNA D192G. The aim of our study was to investigate the biophysical and biomechanical impact of the LMNA D192G mutation on neonatal rat ventricular fibroblasts (NRVF). The main findings in mutated NRVFs were: (i) cytoskeleton disorganization (actin and intermediate filaments); (ii) decreased elasticity of NRVFs; (iii) altered cell-cell adhesion properties, that highlighted a strong effect on cellular communication, in particular on tunneling nanotubes (TNTs). In mutant-expressing fibroblasts, these nanotubes were weakened with altered mechanical properties as shown by atomic force microscopy (AFM) and optical tweezers. These outcomes complement prior investigations on LMNA mutant cardiomyocytes and suggest that the LMNA D192G mutation impacts the biomechanical properties of both cardiomyocytes and cardiac fibroblasts. These observations could explain how this mutation influences cardiac biomechanical pathology and the severity of ACM in LMNA-cardiomyopathy.


Assuntos
Adesão Celular , Lamina Tipo A/metabolismo , Miofibroblastos/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Células Cultivadas , Lamina Tipo A/genética , Microscopia de Força Atômica , Mutação de Sentido Incorreto , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Miofibroblastos/fisiologia , Nanotubos/química , Pinças Ópticas , Ratos , Ratos Sprague-Dawley
2.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445337

RESUMO

In fibrotic diseases, myofibroblasts derive from a range of cell types including endothelial-to-mesenchymal transition (EndMT). Increasing evidence suggests that miRNAs are key regulators in biological processes but their profile is relatively understudied in EndMT. In human umbilical vein endothelial cells (HUVEC), EndMT was induced by treatment with TGFß2 and IL1ß. A significant decrease in endothelial markers such as VE-cadherin, CD31 and an increase in mesenchymal markers such as fibronectin were observed. In parallel, miRNA profiling showed that miR-126-3p was down-regulated in HUVECs undergoing EndMT and over-expression of miR-126-3p prevented EndMT, maintaining CD31 and repressing fibronectin expression. EndMT was investigated using lineage tracing with transgenic Cdh5-Cre-ERT2; Rosa26R-stop-YFP mice in two established models of fibrosis: cardiac ischaemic injury and kidney ureteric occlusion. In both cardiac and kidney fibrosis, lineage tracing showed a significant subpopulation of endothelial-derived cells expressed mesenchymal markers, indicating they had undergone EndMT. In addition, miR-126-3p was restricted to endothelial cells and down-regulated in murine fibrotic kidney and heart tissue. These findings were confirmed in patient kidney biopsies. MiR-126-3p expression is restricted to endothelial cells and is down-regulated during EndMT. Over-expression of miR-126-3p reduces EndMT, therefore, it could be considered for miRNA-based therapeutics in fibrotic organs.


Assuntos
Transdiferenciação Celular/genética , Rim/patologia , MicroRNAs/fisiologia , Miocárdio/patologia , Animais , Células Cultivadas , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Fibrose/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Rim/metabolismo , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia
3.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204432

RESUMO

Several transmembrane mucins have demonstrated that they contribute intracellularly to induce fibrotic processes. The extracellular domain of MUC16 is considered as a biomarker for disease progression and death in IPF patients. However, there is no evidence regarding the signalling capabilities of MUC16 that contribute to IPF development. Here, we demonstrate that MUC16 was overexpressed in the lung tissue of IPF patients (n = 20) compared with healthy subjects (n = 17) and localised in fibroblasts and hyperplastic alveolar type II cells. Repression of MUC16 expression by siRNA-MUC16 transfection inhibited the TGF-ß1-induced fibrotic processes such as mesenchymal/ myofibroblast transformations of alveolar type II A549 cells and lung fibroblasts, as well as fibroblast proliferation. SiRNA-MUC16 transfection also decreased the TGF-ß1-induced SMAD3 phosphorylation, thus inhibiting the Smad Binding Element activation. Immunoprecipitation assays and confocal immunofluorescence showed the formation of a protein complex between MUC16/p-SMAD3 in the cell membrane after TGF-ß1 stimulation. This study shows that MUC16 is overexpressed in IPF and collaborates with the TGF-ß1 canonical pathway to induce fibrotic processes. Therefore, direct or indirect targeting of MUC16 could be a potential drug target for human IPF.


Assuntos
Antígeno Ca-125/genética , Expressão Gênica , Fibrose Pulmonar Idiopática/etiologia , Fibrose Pulmonar Idiopática/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Idoso , Biomarcadores , Antígeno Ca-125/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Proliferação de Células , Suscetibilidade a Doenças , Feminino , Fibroblastos/metabolismo , Humanos , Fibrose Pulmonar Idiopática/diagnóstico , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Modelos Biológicos , Miofibroblastos/metabolismo , Fosforilação , Testes de Função Respiratória
4.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202024

RESUMO

Orbital fibrosis, a hallmark of tissue remodeling in Graves' ophthalmopathy (GO), is a chronic, progressive orbitopathy with few effective treatments. Orbital fibroblasts are effector cells, and transforming growth factor ß1 (TGF-ß1) acts as a critical inducer to promote myofibroblast differentiation and subsequent tissue fibrosis. Curcumin is a natural compound with anti-fibrotic activity. This study aims to investigate the effects of curcumin on TGF-ß1-induced myofibroblast differentiation and on the pro-angiogenic activities of orbital fibroblasts. Orbital fibroblasts from one healthy donor and three patients with GO were collected for primary cell culture and subjected to myofibroblast differentiation under the administration of 1 or 5 ng/mL TGF-ß1 for 24 h. The effects of curcumin on TGF-ß1-induced orbital fibroblasts were assessed by measuring the cellular viability and detecting the expression of myofibroblast differentiation markers, including connective tissue growth factor (CTGF) and α-smooth muscle actin (α-SMA). The pro-angiogenic potential of curcumin-treated orbital fibroblasts was evaluated by examining the transwell migration and tube-forming capacities of fibroblast-conditioned EA.hy926 and HMEC-1 endothelial cells. Treatment of orbital fibroblasts with curcumin inhibited the TGF-ß1 signaling pathway and attenuated the expression of CTGF and α-SMA induced by TGF-ß1. Curcumin, at the concentration of 5 µg/mL, suppressed 5 ng/mL TGF-ß1-induced pro-angiogenic activities of orbital fibroblast-conditioned EA hy926 and HMEC-1 endothelial cells. Our findings suggest that curcumin reduces the TGF-ß1-induced myofibroblast differentiation and pro-angiogenic activity in orbital fibroblasts. The results support the potential application of curcumin for the treatment of GO.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Curcumina/farmacologia , Miofibroblastos/citologia , Miofibroblastos/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Oftalmopatia de Graves/etiologia , Oftalmopatia de Graves/metabolismo , Oftalmopatia de Graves/patologia , Humanos , Miofibroblastos/metabolismo , Espécies Reativas de Oxigênio , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia
5.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202139

RESUMO

Systemic sclerosis (SSc) is a complex disorder resulting from dysregulated interactions between the three main pathophysiological axes: fibrosis, immune dysfunction, and vasculopathy, with no specific treatment available to date. Adipose tissue-derived mesenchymal stromal cells (ASCs) and their extracellular vesicles (EVs) have proved efficacy in pre-clinical murine models of SSc. However, their precise action mechanism is still not fully understood. Because of the lack of availability of fibroblasts isolated from SSc patients (SSc-Fb), our aim was to determine whether a TGFß1-induced model of human myofibroblasts (Tß-Fb) could reproduce the characteristics of SSc-Fb and be used to evaluate the anti-fibrotic function of ASCs and their EVs. We found out that Tß-Fb displayed the main morphological and molecular features of SSc-Fb, including the enlarged hypertrophic morphology and expression of several markers associated with the myofibroblastic phenotype. Using this model, we showed that ASCs were able to regulate the expression of most myofibroblastic markers on Tß-Fb and SSc-Fb, but only when pre-stimulated with TGFß1. Of interest, ASC-derived EVs were more effective than parental cells for improving the myofibroblastic phenotype. In conclusion, we provided evidence that Tß-Fb are a relevant model to mimic the main characteristics of SSc fibroblasts and investigate the mechanism of action of ASCs. We further reported that ASC-EVs are more effective than parental cells suggesting that the TGFß1-induced pro-fibrotic environment may alter the function of ASCs.


Assuntos
Tecido Adiposo/citologia , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Escleroderma Sistêmico/etiologia , Escleroderma Sistêmico/metabolismo , Animais , Biomarcadores , Comunicação Celular , Suscetibilidade a Doenças , Fibroblastos/metabolismo , Fibrose , Humanos , Imunofenotipagem , Células-Tronco Mesenquimais/citologia , Camundongos , Miofibroblastos/metabolismo , Escleroderma Sistêmico/patologia , Fator de Crescimento Transformador beta1/metabolismo
6.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200497

RESUMO

Left ventricular (LV) heart failure (HF) is a significant and increasing cause of death worldwide. HF is characterized by myocardial remodeling and excessive fibrosis. Transcriptional co-activator Yes-associated protein (Yap), the downstream effector of HIPPO signaling pathway, is an essential factor in cardiomyocyte survival; however, its status in human LV HF is not entirely elucidated. Here, we report that Yap is elevated in LV tissue of patients with HF, and is associated with down-regulation of its upstream inhibitor HIPPO component large tumor suppressor 1 (LATS1) activation as well as upregulation of the fibrosis marker connective tissue growth factor (CTGF). Applying the established profibrotic combined stress of TGFß and hypoxia to human ventricular cardiac fibroblasts in vitro increased Yap protein levels, down-regulated LATS1 activation, increased cell proliferation and collagen I production, and decreased ribosomal protein S6 and S6 kinase phosphorylation, a hallmark of mTOR activation, without any significant effect on mTOR and raptor protein expression or phosphorylation of mTOR or 4E-binding protein 1 (4EBP1), a downstream effector of mTOR pathway. As previously reported in various cell types, TGFß/hypoxia also enhanced cardiac fibroblast Akt and ERK1/2 phosphorylation, which was similar to our observation in LV tissues from HF patients. Further, depletion of Yap reduced TGFß/hypoxia-induced cardiac fibroblast proliferation and Akt phosphorylation at Ser 473 and Thr308, without any significant effect on TGFß/hypoxia-induced ERK1/2 activation or reduction in S6 and S6 kinase activities. Taken together, these data demonstrate that Yap is a mediator that promotes human cardiac fibroblast proliferation and suggest its possible contribution to remodeling of the LV, opening the door to further studies to decipher the cell-specific roles of Yap signaling in human HF.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proliferação de Células , Insuficiência Cardíaca/patologia , Miofibroblastos/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Estudos de Casos e Controles , Células Cultivadas , Feminino , Insuficiência Cardíaca/metabolismo , Humanos , Masculino , Miofibroblastos/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Fatores de Transcrição/genética , Ativação Transcricional
7.
Nat Commun ; 12(1): 4384, 2021 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-34282151

RESUMO

Skin and lung fibrosis in systemic sclerosis (SSc) is driven by myofibroblasts, alpha-smooth muscle actin expressing cells. The number of myofibroblasts in SSc skin correlates with the modified Rodnan skin score, the most widely used clinical measure of skin disease severity. Murine fibrosis models indicate that myofibroblasts can arise from a variety of different cell types, but their origin in SSc skin has remained uncertain. Utilizing single cell RNA-sequencing, we define different dermal fibroblast populations and transcriptome changes, comparing SSc to healthy dermal fibroblasts. Here, we show that SSc dermal myofibroblasts arise in two steps from an SFRP2hi/DPP4-expressing progenitor fibroblast population. In the first step, SSc fibroblasts show globally upregulated expression of transcriptome markers, such as PRSS23 and THBS1. A subset of these cells shows markers indicating that they are proliferating. Only a fraction of SFRP2hi SSc fibroblasts differentiate into myofibroblasts, as shown by expression of additional markers, SFRP4 and FNDC1. Bioinformatics analysis of the SSc fibroblast transcriptomes implicated upstream transcription factors, including FOSL2, RUNX1, STAT1, FOXP1, IRF7 and CREB3L1, as well as SMAD3, driving SSc myofibroblast differentiation.


Assuntos
Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo , Miofibroblastos/metabolismo , Escleroderma Sistêmico/metabolismo , Pele/patologia , Transcriptoma , Animais , Diferenciação Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Dipeptidil Peptidase 4 , Fibrose , Fatores de Transcrição Forkhead , Fator Regulador 7 de Interferon , Proteínas de Membrana/genética , Camundongos , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas , Fibrose Pulmonar/patologia , Proteínas Repressoras , Escleroderma Sistêmico/genética , Escleroderma Sistêmico/patologia , Serina Endopeptidases/metabolismo , Dermatopatias/patologia , Proteína Smad3
8.
Int J Mol Sci ; 22(14)2021 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-34299379

RESUMO

Myofibroblasts are contractile cells found in multiple tissues. They are physiological cells as in the human placenta and can be obtained from bone marrow mesenchymal stem cells after differentiation by transforming growth factor-ß (TGF-ß). They are also found in the stroma of cancerous tissues and can be located in non-muscle contractile tissues. When stimulated by an electric current or after exposure to KCl, these tissues contract. They relax either by lowering the intracellular Ca2+ concentration (by means of isosorbide dinitrate or sildenafil) or by inhibiting actin-myosin interactions (by means of 2,3-butanedione monoxime or blebbistatin). Their shortening velocity and their developed tension are dramatically low compared to those of muscles. Like sarcomeric and smooth muscles, they obey Frank-Starling's law and exhibit the Hill hyperbolic tension-velocity relationship. The molecular motor of the myofibroblast is the non-muscle myosin type IIA (NMIIA). Its essential characteristic is the extreme slowness of its molecular kinetics. In contrast, NMIIA develops a unitary force similar to that of muscle myosins. From a thermodynamic point of view, non-muscle contractile tissues containing NMIIA operate extremely close to equilibrium in a linear stationary mode.


Assuntos
Contração Muscular/fisiologia , Músculo Liso/metabolismo , Músculo Liso/fisiologia , Miofibroblastos/metabolismo , Miofibroblastos/fisiologia , Miosinas/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Humanos , Cinética , Termodinâmica
9.
Am J Physiol Gastrointest Liver Physiol ; 321(3): G280-G297, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34288735

RESUMO

Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs), contributing to tissue stiffening and luminal narrowing. Human nuclear receptor 4A 1 (NR4A1) was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling, cytosporone B (Csn-B) or 6-mercaptopurine (6-MP), could reduce fibrosis. We also used the dextran sulfate sodium (DSS) model of colitis and assessed the magnitude of colonic fibrosis in mouse nuclear receptor 4A 1 (Nr4a1-/-) and their wild-type littermates (Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-ß1 (TGF-ß1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen, and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-ß1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was antiproliferative in Nr4a1+/+ but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-ß1-induced collagen deposition and fibrosis-related gene expression. Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.NEW & NOTEWORTHY Fibrosis and increased muscle thickening contribute to stricture formation and intestinal obstruction, a complication that occurs in 30%-50% of patients with CD within 10 yr of disease onset. More than 50% of those who undergo surgery to remove the obstructed bowel will experience stricture recurrence. To date, there are no drug-based approaches approved to treat intestinal strictures. In the current submission, we identify NR4A1 as a novel target to treat inflammation-associated intestinal fibrosis.


Assuntos
Fibrose/metabolismo , Inflamação/metabolismo , Miofibroblastos/metabolismo , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Animais , Células Cultivadas , Humanos , Intestinos/patologia , Camundongos , Transdução de Sinais/fisiologia
10.
Am J Physiol Renal Physiol ; 321(2): F170-F178, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-34180718

RESUMO

Pericytes play an important role in the recovery process after ischemic injury of many tissues. Brain pericytes in the peri-infarct area express macrophage markers in response to injury stimuli and are involved in neovascularization. In the kidney, nerve/glial antigen 2 (NG2)+ pericytes have been found to accumulate after renal injury. These accumulated NG2+ cells are not involved in scar formation. However, the role of accumulated NG2+ cells in injured kidneys remains unknown. Here, using a reversible ischemia-reperfusion (I/R) model, we found that renal NG2+ cells were increased in injured kidneys and expressed macrophage markers (CD11b or F4/80) on day 3 after reperfusion. Isolated NG2+ cells from I/R kidneys also had phagocytic activity and expressed anti-inflammatory cytokine genes, including mannose receptor and IL-10. These macrophage-like NG2+ cells did not likely differentiate into myofibroblasts because they did not increase α-smooth muscle actin expression. Intravenous transfusion of renal NG2+ cells isolated from donor mice on day 3 after reperfusion into recipient mice on day 1 after I/R surgery revealed that NG2+ cell-injected mice had lower plasma blood urea nitrogen, reduced kidney injury molecule-1 mRNA expression, ameliorated renal damage, and reduced cellular debris accumulation compared with PBS-injected mice on day 5 after reperfusion. In conclusion, these data suggest that renal NG2+ cells have an M2 macrophage-like ability and play a novel role in facilitating the recovery process after renal I/R injury.NEW & NOTEWORTHY Brain pericytes have macrophage-like activities after injury. However, such properties of pericytes in peripheral tissues have not been investigated. Here, we provide evidence that nerve/glial antigen 2-positive cells increase after renal injury. The population of nerve/glial antigen 2-positive cells, which does not increase expression of myofibroblast-associated gene, express macrophage markers and anti-inflammatory cytokine genes, have phagocytic activity, and play a role in renal recovery after kidney injury.


Assuntos
Antígenos/metabolismo , Isquemia/metabolismo , Rim/metabolismo , Macrófagos/metabolismo , Proteoglicanas/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Isquemia/patologia , Rim/patologia , Macrófagos/patologia , Masculino , Camundongos , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Fagocitose/fisiologia , Fenótipo , Traumatismo por Reperfusão/patologia
11.
FASEB J ; 35(7): e21733, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34160846

RESUMO

Tendon injuries are common and heal poorly, due in part to a lack of understanding of fundamental tendon cell biology. A major impediment to the study of tendon cells is the absence of robust, well-characterized in vitro models. Unlike other tissue systems, current tendon cell models do not account for how differences in isolation methodology may affect the activation state of tendon cells or the presence of various tendon cell subpopulations. The objective of this study was to characterize how common isolation methods affect the behavior, fate, and lineage composition of tendon cell cultures. Tendon cells isolated by explant exhibited reduced proliferative capacity, decreased expression of tendon marker genes, and increased expression of genes associated with fibroblast activation compared to digested cells. Consistently, explanted cells also displayed an increased propensity to differentiate to myofibroblasts compared to digested cells. Explanted cultures from multiple different tendons were substantially enriched for the presence of scleraxis-lineage (Scx-lin+) cells compared to digested cultures, while the overall percentage of S100a4-lineage (S100a4-lin+) cells was dependent on both isolation method and tendon of origin. Neither isolation methods preserved the ratios of Scx-lin+ or S100a4-lin+ to non-lineage cells seen in tendons in vivo. Combined, these data indicate that further refinement of in vitro cultures models is required in order to more accurately understand the effects of various stimuli on tendon cell behavior. Statement of clinical significance: The development of informed in vitro tendon cell models will facilitate enhanced screening of potential therapeutic candidates to improve tendon healing.


Assuntos
Tendões/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/citologia , Miofibroblastos/metabolismo , Traumatismos dos Tendões/metabolismo , Traumatismos dos Tendões/terapia , Tendões/metabolismo , Cicatrização/fisiologia
12.
J Biol Chem ; 297(1): 100893, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34153319

RESUMO

Fibrosis is a pronounced feature of heart disease and the result of dysregulated activation of resident cardiac fibroblasts (CFs). Recent work identified stress-induced degradation of the cytoskeletal protein ßIV-spectrin as an important step in CF activation and cardiac fibrosis. Furthermore, loss of ßIV-spectrin was found to depend on Ca2+/calmodulin-dependent kinase II (CaMKII). Therefore, we sought to determine the mechanism for CaMKII-dependent regulation of ßIV-spectrin and CF activity. Computational screening and MS revealed a critical serine residue (S2250 in mouse and S2254 in human) in ßIV-spectrin phosphorylated by CaMKII. Disruption of ßIV-spectrin/CaMKII interaction or alanine substitution of ßIV-spectrin Ser2250 (ßIV-S2254A) prevented CaMKII-induced degradation, whereas a phosphomimetic construct (ßIV-spectrin with glutamic acid substitution at serine 2254 [ßIV-S2254E]) showed accelerated degradation in the absence of CaMKII. To assess the physiological significance of this phosphorylation event, we expressed exogenous ßIV-S2254A and ßIV-S2254E constructs in ßIV-spectrin-deficient CFs, which have increased proliferation and fibrotic gene expression compared with WT CFs. ßIV-S2254A but not ßIV-S2254E normalized CF proliferation, gene expression, and contractility. Pathophysiological targeting of ßIV-spectrin phosphorylation and subsequent degradation was identified in CFs activated with the profibrotic ligand angiotensin II, resulting in increased proliferation and signal transducer and activation of transcription 3 nuclear accumulation. While therapeutic delivery of exogenous WT ßIV-spectrin partially reversed these trends, ßIV-S2254A completely negated increased CF proliferation and signal transducer and activation of transcription 3 translocation. Moreover, we observed ßIV-spectrin phosphorylation and associated loss in total protein within human heart tissue following heart failure. Together, these data illustrate a considerable role for the ßIV-spectrin/CaMKII interaction in activating profibrotic signaling.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fibrose Endomiocárdica/metabolismo , Miofibroblastos/metabolismo , Espectrina/metabolismo , Substituição de Aminoácidos , Animais , Células COS , Proliferação de Células , Células Cultivadas , Chlorocebus aethiops , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Miocárdica , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/fisiologia , Fosforilação , Espectrina/genética
13.
Int J Mol Sci ; 22(11)2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067440

RESUMO

Myocardial infarction remains the most common cause of heart failure with adverse remodeling. MicroRNA (miR)155 is upregulated following myocardial infarction and represents a relevant regulatory factor for cardiac remodeling by engagement in cardiac inflammation, fibrosis and cardiomyocyte hypertrophy. Here, we investigated the role of miR155 in cardiac remodeling and dysfunction following myocardial infarction in a dyslipidemic mouse model. Myocardial infarction was induced in dyslipidemic apolipoprotein E-deficient (ApoE-/-) mice with and without additional miR155 knockout by ligation of the LAD. Four weeks later, echocardiography was performed to assess left ventricular (LV) dimensions and function, and mice were subsequently sacrificed for histological analysis. Echocardiography revealed no difference in LV ejection fractions, LV mass and LV volumes between ApoE-/- and ApoE-/-/miR155-/- mice. Histology confirmed comparable infarction size and unaltered neoangiogenesis in the myocardial scar. Notably, myofibroblast density was significantly decreased in ApoE-/-/miR155-/- mice compared to the control, but no difference was observed for total collagen deposition. Our findings reveal that genetic depletion of miR155 in a dyslipidemic mouse model of myocardial infarction does not reduce infarction size and consecutive heart failure but does decrease myofibroblast density in the post-ischemic scar.


Assuntos
MicroRNAs/genética , Infarto do Miocárdio/genética , Miofibroblastos/metabolismo , Função Ventricular Esquerda/genética , Animais , Modelos Animais de Doenças , Ecocardiografia/métodos , Fibrose/genética , Fibrose/metabolismo , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/metabolismo , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Volume Sistólico/genética , Remodelação Ventricular/genética
14.
Exp Cell Res ; 405(2): 112682, 2021 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-34118250

RESUMO

It has been demonstrated that the T-box family transcription factor 18 (Tbx18) -positive cells give rise to renal mesenchymal cells and contribute to the development of the urinary system. However, it is unclear whether Tbx18-positive cells are the origin of the myofibroblasts during renal fibrosis. The present study aimed to determine the contribution of Tbx18-positive cells in kidney fibrosis and their underlying mechanism. We show that Tbx18-positive cells contribute to the development of the urinary system, especially renal fibroblasts. Following unilateral ureteral obstruction (UUO), genetic fate tracing results demonstrated that Tbx18-positive cells not only proliferate but also expand and differentiate into fibroblasts and myofibroblasts, indicating that they may act as profibrotic progenitors. Cell culture results suggest that transforming growth factor (TGF)-ß promotes Tbx18-positive cells differentiation into myofibroblasts and assist their contribution to kidney fibrosis. Overall, the present study demonstrated that Tbx18-positive cells may act as profibrotic progenitor cells in a pathological condition of UUO-induced injury. Moreover, TGF-ß may play a role in differentiation of Tbx18-positive cells into myofibroblasts in kidney fibrosis. These findings may provide a potential target on Tbx18-positive myofibroblast progenitors in the treatment of renal fibrosis.


Assuntos
Fibrose/metabolismo , Miofibroblastos/metabolismo , Proteínas com Domínio T/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Nefropatias/metabolismo , Camundongos , Fatores de Crescimento Transformadores/metabolismo , Obstrução Ureteral/metabolismo
15.
BMC Cardiovasc Disord ; 21(1): 308, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34154526

RESUMO

BACKGROUND: Circular RNA (circRNA) have been reported to play important roles in cardiovascular diseases including myocardial infarction and heart failure. However, the role of circRNA in atrial fibrillation (AF) has rarely been investigated. We recently found a circRNA hsa_circ_0099734 was significantly differentially expressed in the AF patients atrial tissues compared to paired control. We aim to investigate the functional role and molecular mechanisms of mmu_circ_0005019 which is the homologous circRNA in mice of hsa_circ_0099734 in AF. METHODS: In order to investigate the effect of mmu_circ_0005019 on the proliferation, migration, differentiation into myofibroblasts and expression of collagen of cardiac fibroblasts, and the effect of mmu_circ_0005019 on the apoptosis and expression of Ito, INA and SK3 of cardiomyocytes, gain- and loss-of-function of cell models were established in mice cardiac fibroblasts and HL-1 atrial myocytes. Dual-luciferase reporter assays and RIP were performed to verify the binding effects between mmu_circ_0005019 and its target microRNA (miRNA). RESULTS: In cardiac fibroblasts, mmu_circ_0005019 showed inhibitory effects on cell proliferation and migration. In cardiomyocytes, overexpression of mmu_circ_0005019 promoted Kcnd1, Scn5a and Kcnn3 expression. Knockdown of mmu_circ_0005019 inhibited the expression of Kcnd1, Kcnd3, Scn5a and Kcnn3. Mechanistically, mmu_circ_0005019 exerted biological functions by acting as a miR-499-5p sponge to regulate the expression of its target gene Kcnn3. CONCLUSIONS: Our findings highlight mmu_circ_0005019 played a protective role in AF development and might serve as an attractive candidate target for AF treatment.


Assuntos
Potenciais de Ação , Comunicação Celular , Fibroblastos/metabolismo , Frequência Cardíaca , Miócitos Cardíacos/metabolismo , RNA Circular/metabolismo , Animais , Linhagem Celular , Movimento Celular , Proliferação de Células , Transdiferenciação Celular , Técnicas de Cocultura , Fibroblastos/patologia , Humanos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Miócitos Cardíacos/patologia , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , RNA Circular/genética , Canais de Potássio Shal/genética , Canais de Potássio Shal/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
16.
Methods Mol Biol ; 2299: 139-145, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028740

RESUMO

Fibroblast-to-myofibroblast transdifferentiation and the acquisition of a senescent phenotype are hallmarks of fibrotic diseases. The study of the localization of senescent myofibroblasts as well as their interactions with other cell types in the fibrotic tissue has been hindered by the lack of methods to detect these cells in vivo. Here, we describe methods to detect tissue localization of senescent myofibroblasts in precision-cut lung slices (PCLS) by combining ß-galactosidase staining with immunofluorescence techniques.


Assuntos
Fibroblastos/metabolismo , Pulmão/patologia , Miofibroblastos/metabolismo , Animais , Comunicação Celular , Transdiferenciação Celular , Senescência Celular , Fibroblastos/citologia , Fibrose , Humanos , Pulmão/metabolismo , Camundongos , Miofibroblastos/citologia , beta-Galactosidase/metabolismo
17.
Biochem Biophys Res Commun ; 561: 180-186, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34023784

RESUMO

Fibrosis is a condition characterized by the overproduction of extracellular matrix (ECM) components (e.g., collagen) in the myofibroblasts, causing tissue hardening and eventual organ dysfunction. Currently, the molecular mechanisms that regulate ECM production in the myofibroblasts are still obscure. In this study, we investigated the function of GPRC5B in the cardiac and lung myofibroblasts using real-time RT-PCR and siRNA-mediated knockdown. We discovered a significantly high expression of Gprc5b in the tissues of the fibrosis mice models and confirmed that Gprc5b was consistently expressed in the myofibroblasts of fibrotic hearts and lungs. We also found that Gprc5b expression was associated and may be dependent on the actin-MRTF-SRF signaling pathway. Notably, we observed that Gprc5b knockdown reduced the expression of collagen genes in the cardiac and lung myofibroblasts. Therefore, our findings reveal that GPRC5B enhances collagen production in the myofibroblasts, which directly promotes fibrosis in the tissues.


Assuntos
Colágeno/metabolismo , Fibrose/patologia , Coração/fisiopatologia , Fígado/metabolismo , Pulmão/metabolismo , Miofibroblastos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Fibrose/metabolismo , Fígado/patologia , Pulmão/patologia , Masculino , Camundongos , Miofibroblastos/patologia , Transdução de Sinais
18.
Environ Pollut ; 286: 117259, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33965804

RESUMO

As an environmental toxicant, arsenic causes damage to various organs and systems of the body and has attracted worldwide attention. It is well-known that exposure to arsenic can induce pulmonary fibrosis, but the molecular mechanisms are elusive. Glycolysis is involved in the process of various diseases, including pulmonary fibrosis. Extracellular vehicles (EVs) are mediators of cell communication through transporting miRNAs. The potential of miRNAs in EVs as liquid biopsy biomarkers for various diseases has been reported, and they have been applied in clinical diagnoses. In the present investigation, we focused on the roles and mechanisms of miR-21 in EVs on arsenic-induced glycolysis and pulmonary fibrosis through experiments with human populations, experimental animals, and cells. The results for arsenicosis populations showed that the serum levels of hydroxyproline, lactate, and EVs-miRNAs were elevated and that EVs-miR-21 levels were positively related to the levels of hydroxyproline and lactate. For mice, chronic exposure to arsenite led to high levels of miR-21, AKT activation, elevated glycolysis, and pulmonary fibrosis; however, these effects were blocked by the depletion of miR-21 in miR-21 knockout (miR-21KO) mice. After MRC-5 cells were co-cultured with arsenite-treated HBE cells, the levels of miR-21, AKT activation, glycolysis, and myofibroblast differentiation were enhanced, effects that were blocked by reducing miR-21 and by inhibiting the EVs in HBE cells. The down-regulation of PTEN in MRC-5 cells and primary lung fibroblasts (PLFs) reversed the blocking effect of inhibiting miR-21 in HBE cells. Thus, miR-21 down-regulates PTEN and promotes glycolysis via activating AKT, which is associated with arsenite-induced myofibroblast differentiation and pulmonary fibrosis. Our results provide a new approach for the construction of clinical diagnosis technology based on analysis of the mechanism of arsenite-induced pulmonary fibrosis.


Assuntos
Arsênio , MicroRNAs , Fibrose Pulmonar , Animais , Arsênio/toxicidade , Diferenciação Celular , Células Epiteliais/metabolismo , Glicólise , Pulmão/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Miofibroblastos/metabolismo , Fibrose Pulmonar/induzido quimicamente
19.
Methods Mol Biol ; 2299: 17-47, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028733

RESUMO

The identification of myofibroblasts is essential for mechanistic in vitro studies, cell-based drug tests, and to assess the level of fibrosis in experimental animal or human fibrosis. The name myo-fibroblast was chosen in 1971 to express that the formation of contractile features-stress fibers is the essential criterion to define these cells. Additional neo-expression of α-smooth muscle actin (α-SMA) in stress fibers has become the most widely used molecular marker. Here, we briefly introduce the concept of different myofibroblast activation states, of which the highly contractile α-SMA-positive phenotype represents a most advanced functional stage. We provide targeted immunofluorescence protocols to assess this phenotype, and publicly accessible image analysis tools to quantify the level of myofibroblast activation in culture and in tissues.


Assuntos
Actinas/metabolismo , Técnicas de Cultura de Células/métodos , Imunofluorescência/métodos , Miofibroblastos/citologia , Células 3T3 , Actinas/genética , Animais , Biomarcadores/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Camundongos , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Fenótipo , Ratos , Fator de Crescimento Transformador beta1/farmacologia
20.
Methods Mol Biol ; 2299: 85-97, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028735

RESUMO

Myofibroblasts form adhesions to their underlying extracellular matrices, which is an essential step in their formation and differentiation. These adhesions comprise protein-rich aggregates of a wide variety of signaling, cytoskeletal, cell adhesion, and matrix proteins that interact with one another to enable bidirectional flow of information between the cell and the surrounding extracellular matrix. The concentrated repertoire of the proteins in matrix adhesions of myofibroblasts (i.e., over 450 different proteins) and their important role in regulating the metabolic activities of myofibroblasts, has motivated in-depth analysis of their protein complement and how this repertoire is influenced by experimental conditions.In this protocol I describe in detail: (1) the method for isolating focal adhesion-associated proteins using matrix ligand-bound magnetite beads; (2) the method for eluting the proteins from the beads and their preparation for mass spectrometry (Fig. 1). I also briefly consider the mass spectrometry methods including the use of isobaric tags to enable multifactorial experiments and the analysis of the identified proteins. I consider the advantages of these approaches, and the challenges and pitfalls that are encountered with these methods.


Assuntos
Adesões Focais/metabolismo , Miofibroblastos/metabolismo , Proteômica/métodos , Adesão Celular , Células Cultivadas , Humanos , Espectrometria de Massas
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