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1.
Avian Dis ; 65(1): 10-17, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-34339116

RESUMO

Septicemia-toxemia (sep/tox) falls under U.S. Department of Agriculture (USDA) food safety Category 1 and is the most common and economically significant cause of broiler carcass condemnations. Hepatic lesions are considered a possible consequence of septicemia and associated bacterial contamination of the carcass. Thus, these lesions are considered an indicator of sep/tox (sep/tox hepatitis). This study was undertaken to analyze the histologic lesions preceding grossly visible liver lesions leading to condemnation because of sep/tox at the processing plant. Livers from carcasses of broilers condemned by USDA inspectors for sep/tox were used to establish microscopic and gross criteria of end-stage sep/tox hepatitis. Following the characterization of sep/tox hepatitis, broilers from a farm with a history of sep/tox condemnations were submitted for postmortem examination and bacteriologic investigation at four intervals during the final 20 days of production. Five healthy and five clinically ill chickens were submitted from four houses at 18, 25, 32, and 38 days of production (160 total). Microscopic lesions representing hepatic perisinusoidal myofibroblast proliferation (HPMP), periportal extramedullary granulopoiesis (PEMG), splenic follicular histiocytosis, and bone marrow cellularity (BMC) were graded subjectively for each bird, and subjective grading was evaluated with digital quantitative techniques. Perisinusoidal hepatic stellate cell morphology and progressive transformation of these cells into myofibroblasts was confirmed by immunohistochemistry for smooth muscle actin and desmin. Aerobic cultures of livers and gall bladders from sep/tox birds yielded no growth of bacteria associated with septicemia. Mild to severe HPMP was observed in all age groups, representing 28% of examined birds. Increases in inflammatory cells observed by PEMG and BMC were positively correlated with progressive HPMP and end-stage sep/tox hepatitis in broiler chickens.


Assuntos
Proliferação de Células , Galinhas , Hepatite Animal/patologia , Fígado/patologia , Miofibroblastos/fisiologia , Doenças das Aves Domésticas/patologia , Síndrome de Resposta Inflamatória Sistêmica/veterinária , Animais , Hepatite Animal/virologia , Doenças das Aves Domésticas/virologia , Sepse/veterinária , Sepse/virologia , Síndrome de Resposta Inflamatória Sistêmica/patologia , Síndrome de Resposta Inflamatória Sistêmica/virologia , Toxemia/veterinária , Toxemia/virologia
2.
FASEB J ; 35(9): e21799, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34339055

RESUMO

Cardiac fibroblasts (CFBs) support heart function by secreting extracellular matrix (ECM) and paracrine factors, respond to stress associated with injury and disease, and therefore are an increasingly important therapeutic target. We describe how developmental lineage of human pluripotent stem cell-derived CFBs, epicardial (EpiC-FB), and second heart field (SHF-FB) impacts transcriptional and functional properties. Both EpiC-FBs and SHF-FBs exhibited CFB transcriptional programs and improved calcium handling in human pluripotent stem cell-derived cardiac tissues. We identified differences including in composition of ECM synthesized, secretion of growth and differentiation factors, and myofibroblast activation potential, with EpiC-FBs exhibiting higher stress-induced activation potential akin to myofibroblasts and SHF-FBs demonstrating higher calcification and mineralization potential. These phenotypic differences suggest that EpiC-FBs have utility in modeling fibrotic diseases while SHF-FBs are a promising source of cells for regenerative therapies. This work directly contrasts regional and developmental specificity of CFBs and informs CFB in vitro model selection.


Assuntos
Linhagem da Célula/fisiologia , Miofibroblastos/fisiologia , Células-Tronco Pluripotentes/fisiologia , Diferenciação Celular/fisiologia , Células Cultivadas , Matriz Extracelular/fisiologia , Humanos , Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Fenótipo , Transcrição Genética/fisiologia
3.
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
4.
Aging (Albany NY) ; 13(13): 16957-16973, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253690

RESUMO

Many aging related diseases such as cancer implicate the myofibroblast in disease progression. Furthermore genesis of the myofibroblast is associated with manifestation of cellular senescence of unclear significance. In this study we investigated the role of a common regulator, namely telomerase reverse transcriptase (TERT), in order to evaluate the potential significance of this association between both processes. We analyzed the effects of TERT overexpression or deficiency on expression of CDKN2A and ACTA2 as indicators of senescence and differentiation, respectively. We assess binding of TERT or YB-1, a repressor of both genes, to their promoters. TERT repressed both CDKN2A and ACTA2 expression, and abolished stress-induced expression of both genes. Conversely, TERT deficiency enhanced their expression. Altering CDKN2A expression had no effect on ACTA2 expression. Both TERT and YB-1 were shown to bind the CDKN2A promoter but only YB-1 was shown to bind the ACTA2 promoter. TERT overexpression inhibited CDKN2A promoter activity while stimulating YB-1 expression and activation to repress ACTA2 gene. TERT repressed myofibroblast differentiation and senescence via distinct mechanisms. The latter was associated with TERT binding to the CDKN2A promoter, but not to the ACTA2 promoter, which may require interaction with co-factors such as YB-1.


Assuntos
Diferenciação Celular/fisiologia , Senescência Celular/fisiologia , Miofibroblastos/fisiologia , Telomerase/fisiologia , Actinas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Masculino , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Telomerase/biossíntese , Telomerase/genética
5.
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
6.
ACS Appl Mater Interfaces ; 13(22): 25589-25598, 2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34032413

RESUMO

The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 µm-1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 µm-1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.


Assuntos
Microambiente Celular , Células Endoteliais/fisiologia , Células-Tronco Mesenquimais/fisiologia , Miofibroblastos/fisiologia , Proteínas/química , Veias Umbilicais/fisiologia , Adesão Celular , Comunicação Celular , Movimento Celular , Células Endoteliais/citologia , Humanos , Células-Tronco Mesenquimais/citologia , Miofibroblastos/citologia , Propriedades de Superfície , Veias Umbilicais/citologia
7.
Methods Mol Biol ; 2299: 1-5, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028731

RESUMO

Myofibroblasts are key cells in mediating normal wound contraction and promoting connective tissue deformations characteristic of fibrosis and scarring. Five decades ago, myofibroblasts were discovered in electron micrographs of wound granulation tissue as fibroblastic cells containing microfilaments that are organized in bundles like those present in smooth muscle. The contractile function of myofibroblasts was demonstrated by measuring the contraction of strips of granulation tissue in response to smooth muscle agonists and in cell culture. Although formation of contractile bundles already defines the myofibroblast, neo-expression of α-smooth muscle actin (α-SMA) in fibroblastic cells has become the most widely used myofibroblast marker. Because α-SMA incorporation into stress fibers mediates enhanced fibroblast contraction, it has been proposed and successfully tested as a drug target in therapeutic approaches to reduce tissue contractures. Other anti-fibrosis strategies target growth factor-, extracellular matrix-, and mechanical stress-induced pathways of myofibroblast activation from various precursors or aim to induce myofibroblast apoptosis. To understand the involved mechanisms of myofibroblast formation and function, critical experimental tools and animal models have been developed, which are made available in this collection of protocols by experts in the field.


Assuntos
Actinas/metabolismo , Miofibroblastos/fisiologia , Animais , Fibrose , Humanos , Contração Muscular , Cicatrização
8.
Methods Mol Biol ; 2299: 9-15, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028732

RESUMO

Our understanding of myofibroblast biology has advanced over the past two decades, and the seemingly antagonistic roles of these cells in both normal tissue repair and fibrotic diseases are better reconciled. An age-related loss of cellular plasticity that results in impaired capacity for de-differentiation and apoptosis susceptibility may predispose individuals to non-resolving and progressive fibrotic disorders involving diverse organ systems.


Assuntos
Envelhecimento/fisiologia , Fibrose/fisiopatologia , Miofibroblastos/fisiologia , Cicatrização , Animais , Apoptose , Desdiferenciação Celular , Matriz Extracelular/fisiologia , Humanos
9.
Methods Mol Biol ; 2299: 159-169, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028742

RESUMO

Fibroblasts and myofibroblasts are found throughout mechanically loaded tissues, where they take primary responsibility for generating and maintaining the extracellular matrix scaffold upon which organ structure and function depends. They are thus tasked with creating the appropriate mechanical environment in which cells and tissues function optimally, and constantly adapting this environment as needed in response to changing environmental cues. To carry out these functions, fibroblasts must not only deposit and resorb the extracellular matrix, they must adhere to and sense its physical characteristics, and exert the forces necessary to shape, distort, and remodel it as desired. It is thus only through a constant reciprocal sensing and exertion of stress that fibroblasts can carry out their key functions. This introductory chapter will introduce these aspects of fibroblast stress sensing and matrix remodeling during tissue homeostasis, wound repair and fibrotic disease as a lead in to the detailed method chapters to follow on myofibroblast mechanobiology.


Assuntos
Matriz Extracelular/metabolismo , Fibroblastos/fisiologia , Miofibroblastos/fisiologia , Animais , Adesão Celular , Fibrose , Humanos , Fenômenos Mecânicos , Mecanotransdução Celular , Cicatrização
10.
Methods Mol Biol ; 2299: 181-195, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028744

RESUMO

Myofibroblasts play important roles in physiological processes such as wound healing and tissue repair. While high contractile forces generated by the actomyosin network enable myofibroblasts to physically contract the wound and bring together injured tissue, prolonged and elevated levels of contraction also drive the progression of fibrosis and cancer. However, quantitative mapping of these forces has been difficult due to their extremely low magnitude ranging from 100 pN/µm2 to 2 nN/µm2. Here, we provide a protocol to measure cellular forces exerted on two-dimensional compliant elastic hydrogels. We describe the fabrication of polyacrylamide hydrogels labeled with fluorescent fiducial markers, functionalization of substrates with ECM proteins, setting up the experiment, and imaging procedures. We demonstrate the application of this technique for quantitative analysis of traction forces exerted by myofibroblasts.


Assuntos
Actinas/metabolismo , Fibroblastos/citologia , Miofibroblastos/fisiologia , Resinas Acrílicas , Animais , Adesão Celular , Células Cultivadas , Matriz Extracelular/metabolismo , Humanos , Camundongos , Microscopia de Força Atômica , Contração Muscular , Miofibroblastos/citologia , Células NIH 3T3 , Estresse Mecânico
11.
Front Immunol ; 12: 633654, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33732255

RESUMO

Epigenetics plays an important role in the priming the dynamic response of airway epithelial cells to infectious and environmental stressors. Here, we examine the epigenetic role of the SWI/SNF Related, Matrix Associated, Actin Dependent Regulator of Chromatin A4 (SMARCA4) in the epithelial response to RSV infection. Depletion of SMARCA4 destabilized the abundance of the SMARCE1/ARID1A SWI/SNF subunits, disrupting the innate response and triggering a hybrid epithelial/mesenchymal (E/M) state. Assaying SMARCA4 complex-regulated open chromatin domains by transposase cleavage -next generation sequencing (ATAC-Seq), we observed that the majority of cleavage sites in uninfected cells have reduced chromatin accessibility. Paradoxically, SMARCA4 complex-depleted cells showed enhanced RSV-inducible chromatin opening and gene expression in the EMT pathway genes, MMP9, SNAI1/2, VIM, and CDH2. Focusing on the key MMP9, we observed that SMARCA4 complex depletion reduced basal BRD4 and RNA Polymerase II binding, but enhanced BRD4/Pol II binding in response to RSV infection. In addition, we observed that MMP9 secretion in SMARCA4 complex deficient cells contributes to mesenchymal transition, cellular fusion (syncytia) and subepithelial myofibroblast transition. We conclude the SMARCA4 complex is a transcriptional repressor of epithelial plasticity, whose depletion triggers a hybrid E/M state that affects the dynamic response of the small airway epithelial cell in mucosal remodeling via paracrine MMP9 activity.


Assuntos
Cromatina/genética , DNA Helicases/genética , Células Epiteliais/virologia , Células Gigantes/virologia , Miofibroblastos/fisiologia , Proteínas Nucleares/genética , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Fatores de Transcrição/genética , Células Cultivadas , Cromatina/classificação , Montagem e Desmontagem da Cromatina , Epigênese Genética , Células Gigantes/fisiologia , Humanos , Pulmão/citologia , Metaloproteinase 9 da Matriz/metabolismo , Miofibroblastos/virologia , Infecções por Vírus Respiratório Sincicial/patologia , Replicação Viral
12.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672311

RESUMO

Oral submucous fibrosis (OSF) is known as a potentially malignant disorder, which may result from chemical irritation due to areca nuts (such as arecoline). Emerging evidence suggests that fibrogenesis and carcinogenesis are regulated by the interaction of long noncoding RNAs (lncRNAs) and microRNAs. Among these regulators, profibrotic lncRNA H19 has been found to be overexpressed in several fibrosis diseases. Here, we examined the expression of H19 in OSF specimens and its functional role in fibrotic buccal mucosal fibroblasts (fBMFs). Our results indicate that the aberrantly overexpressed H19 contributed to higher myofibroblast activities, such as collagen gel contractility and migration ability. We also demonstrated that H19 interacted with miR-29b, which suppressed the direct binding of miR-29b to the 3'-untranslated region of type I collagen (COL1A1). We showed that ectopic expression of miR-29b ameliorated various myofibroblast phenotypes and the expression of α-smooth muscle actin (α-SMA), COL1A1, and fibronectin (FN1) in fBMFs. In OSF tissues, we found that the expression of miR-29b was downregulated and there was a negative correlation between miR-29b and these fibrosis markers. Lastly, we demonstrate that arecoline stimulated the upregulation of H19 through the transforming growth factor (TGF)-ß pathway. Altogether, this study suggests that increased TGF-ß secretion following areca nut chewing may induce the upregulation of H19, which serves as a natural sponge for miR-29b and impedes its antifibrotic effects.


Assuntos
Colágeno Tipo I/genética , MicroRNAs/genética , Fibrose Oral Submucosa/genética , RNA Longo não Codificante/genética , Arecolina/farmacologia , Biomarcadores , Transdiferenciação Celular/genética , Células Cultivadas , Regulação para Baixo , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Humanos , Mucosa Bucal/patologia , Mucosa Bucal/fisiologia , Miofibroblastos/patologia , Miofibroblastos/fisiologia , Fibrose Oral Submucosa/patologia , Lesões Pré-Cancerosas , Fator de Crescimento Transformador beta/metabolismo
13.
Endocrinology ; 162(7)2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33640969

RESUMO

CONTEXT: Arteriovenous fistula (AVF) maturation failure remains a clinical dilemma, and its pathobiology is largely unclear. Secondary hyperparathyroidism is a complication of chronic renal failure that is associated with cardiovascular disease. While parathyroid hormone (PTH) has a prosclerotic effect on vascular smooth muscle cells (VSMCs), its role in AVF maturation failure remained unknown. OBJECTIVE: This work aimed to investigate the association between plasma PTH and AVF maturation. METHODS: Patients receiving AVF creation were enrolled retrospectively. A mouse model of secondary hyperparathyroidism and aortocaval AVF was used to investigate the effect of PTH on an AVF lesion. A cell model of VSMCs treated with PTH in a pressurized culture system was used to disclose the signaling pathway underlying the effect of PTH on an AVF lesion. RESULTS: In patients receiving AVF creation, higher PTH was associated with an increased risk for maturation failure. In a mouse model, vascular wall thickness and myofibroblasts of AVF significantly increased with higher PTH. When the same mice were treated with cinacalcet, AVF lesions were attenuated by suppression of PTH. A cell model showed that PTH increased the marker of myofibroblasts, integrin ß6 subunit (ITGB6), via the phosphorylated protein kinase B pathway. Finally, in the same model of mice AVF, higher PTH also increased the expression of ITGB6 in the smooth muscle layer of AVF, suggesting the transition to myofibroblast. CONCLUSION: Overall, our results suggest that higher PTH increased the risk of AVF maturation failure through increasing the transition of VSMCs to myofibroblasts. Lowering PTH may be a strategy to enhance AVF maturation.


Assuntos
Derivação Arteriovenosa Cirúrgica , Miofibroblastos/fisiologia , Hormônio Paratireóideo/sangue , Falha de Tratamento , Adenina/administração & dosagem , Idoso , Animais , Biomarcadores , Técnicas de Cultura de Células , Modelos Animais de Doenças , Feminino , Humanos , Hiperparatireoidismo/complicações , Cadeias beta de Integrinas/análise , Falência Renal Crônica/induzido quimicamente , Falência Renal Crônica/complicações , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Miofibroblastos/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Estudos Retrospectivos , Fatores de Risco
14.
Int J Mol Sci ; 22(3)2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-33573365

RESUMO

The fascia can be defined as a dynamic highly complex connective tissue network composed of different types of cells embedded in the extracellular matrix and nervous fibers: each component plays a specific role in the fascial system changing and responding to stimuli in different ways. This review intends to discuss the various components of the fascia and their specific roles; this will be carried out in the effort to shed light on the mechanisms by which they affect the entire network and all body systems. A clear understanding of fascial anatomy from a microscopic viewpoint can further elucidate its physiological and pathological characteristics and facilitate the identification of appropriate treatment strategies.


Assuntos
Fáscia/citologia , Músculo Esquelético/citologia , Animais , Colágeno/metabolismo , Matriz Extracelular/fisiologia , Fáscia/inervação , Fáscia/fisiologia , Fibroblastos/fisiologia , Humanos , Ácido Hialurônico/metabolismo , Mecanotransdução Celular/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Miofibroblastos/fisiologia , Fibras Nervosas/fisiologia , Telócitos/fisiologia , Viscosidade
15.
J Immunol ; 206(7): 1549-1560, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33637617

RESUMO

Outside-in integrin signaling regulates cell fate decisions in a variety of cell types, including hematopoietic stem cells (HSCs). Our earlier published studies showed that interruption of periostin (POSTN) and integrin-αv (ITGAV) interaction induces faster proliferation in HSCs with developmental stage-dependent functional effects. In this study, we examined the role of POSTN-ITGAV axis in lymphohematopoietic activity in spleen that hosts a rare population of HSCs, the functional regulation of which is not clearly known. Vav-iCre-mediated deletion of Itgav in the hematopoietic system led to higher proliferation rates, resulting in increased frequency of primitive HSCs in the adult spleen. However, in vitro CFU-C assays demonstrated a poorer differentiation potential following Itgav deletion. This also led to a decrease in the white pulp area with a significant decline in the B cell numbers. Systemic deletion of its ligand, POSTN, phenocopied the effects noted in Vav-Itgav-/- mice. Histological examination of Postn-deficient spleen also showed an increase in the spleen trabecular areas. Importantly, these are the myofibroblasts of the trabecular and capsular areas that expressed high levels of POSTN within the spleen tissue. In addition, vascular smooth muscle cells also expressed POSTN. Through CFU-S12 assays, we showed that hematopoietic support potential of stroma in Postn-deficient splenic hematopoietic niche was defective. Overall, we demonstrate that POSTN-ITGAV interaction plays an important role in spleen lymphohematopoiesis.


Assuntos
Moléculas de Adesão Celular/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Integrina alfa5/metabolismo , Linfócitos/fisiologia , Miócitos de Músculo Liso/fisiologia , Miofibroblastos/fisiologia , Baço/imunologia , Animais , Moléculas de Adesão Celular/genética , Proliferação de Células , Técnicas de Silenciamento de Genes , Hematopoese , Integrina alfa5/genética , Camundongos , Camundongos Knockout , Transdução de Sinais , Nicho de Células-Tronco
16.
FASEB J ; 35(3): e21381, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33617091

RESUMO

Renal interstitial fibrosis (RIF) is a pathological process that fibrotic components are excessively deposited in the renal interstitial space due to kidney injury, resulting in impaired renal function and chronic kidney disease. The molecular mechanisms controlling renal fibrosis are not fully understood. In this present study, we identified Nuclear protein 1 (Nupr1), a transcription factor also called p8, as a novel regulator promoting renal fibrosis. Unilateral ureteral obstruction (UUO) time-dependently induced Nupr1 mRNA and protein expression in mouse kidneys while causing renal damage and fibrosis. Nupr1 deficiency (Nupr1-/- ) attenuated the renal tubule dilatation, tubular epithelial cell atrophy, and interstitial collagen accumulation caused by UUO. Consistently, Nupr1-/- significantly decreased the expression of type I collagen, myofibroblast markers smooth muscle α-actin (α-SMA), fibroblast-specific protein 1 (FSP-1), and vimentin in mouse kidney that were upregulated by UUO. These results suggest that Nupr1 protein was essential for fibroblast activation and/or epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Indeed, Nupr1 was indispensable for TGF-ß-induced myofibroblast activation of kidney interstitial NRK-49F fibroblasts, multipotent mesenchymal C3H10T1/2 cells, and the EMT of kidney epithelial NRK-52E cells. It appears that Nupr1 mediated TGF-ß-induced α-SMA expression and collagen synthesis by initiating Smad3 signaling pathway. Importantly, trifluoperazine (TFP), a Nupr1 inhibitor, alleviated UUO-induced renal fibrosis. Taken together, our results demonstrate that Nupr1 promotes renal fibrosis by activating myofibroblast transformation from both fibroblasts and tubular epithelial cells.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Transição Epitelial-Mesenquimal , Fibroblastos/fisiologia , Rim/patologia , Proteínas de Neoplasias/fisiologia , Animais , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/fisiologia , Ratos , Transdução de Sinais/fisiologia , Proteína Smad3/fisiologia , Fatores de Transcrição da Família Snail/fisiologia , Trifluoperazina/farmacologia
17.
Sci Rep ; 11(1): 63, 2021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33420132

RESUMO

Aberrant regeneration or fibrosis in muscle is the denouement of deregulated cellular and molecular events that alter original tissue architecture due to accumulation of excessive extracellular matrix. The severity of the insult to the skeletal muscle determines the nature of regeneration. Numerous attempts at deciphering the mechanism underlying fibrosis and the subsequent strategies of drug therapies have yielded temporary solutions. Our intent is to understand the interaction between the myofibroblasts (MFs) and the satellite cells (SCs), during skeletal muscle regeneration. We hypothesize that MFs contribute to the impairment of SCs function by exhibiting an antagonistic influence on their proliferation. A modified laceration based skeletal muscle injury model in mouse was utilized to evaluate the dynamics between the SCs and MFs during wound healing. We show that the decline in MFs' number through inhibition of PDGFRα signaling consequently promotes proliferation of the SCs and exhibits improved skeletal muscle remodeling. We further conclude that in situ administration of PDGFRα inhibitor prior to onset of fibrosis may attenuate aberrant regeneration. This opens new possibility for the early treatment of muscle fibrosis by specific targeting of MFs rather than transplantation of SCs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/farmacologia , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/efeitos dos fármacos , Animais , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/lesões , Miofibroblastos/fisiologia , Células Satélites de Músculo Esquelético/fisiologia , Cicatrização/efeitos dos fármacos
18.
Nat Rev Gastroenterol Hepatol ; 18(3): 151-166, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33128017

RESUMO

Chronic liver injury leads to liver inflammation and fibrosis, through which activated myofibroblasts in the liver secrete extracellular matrix proteins that generate the fibrous scar. The primary source of these myofibroblasts are the resident hepatic stellate cells. Clinical and experimental liver fibrosis regresses when the causative agent is removed, which is associated with the elimination of these activated myofibroblasts and resorption of the fibrous scar. Understanding the mechanisms of liver fibrosis regression could identify new therapeutic targets to treat liver fibrosis. This Review summarizes studies of the molecular mechanisms underlying the reversibility of liver fibrosis, including apoptosis and the inactivation of hepatic stellate cells, the crosstalk between the liver and the systems that orchestrate the recruitment of bone marrow-derived macrophages (and other inflammatory cells) driving fibrosis resolution, and the interactions between various cell types that lead to the intracellular signalling that induces fibrosis or its regression. We also discuss strategies to target hepatic myofibroblasts (for example, via apoptosis or inactivation) and the myeloid cells that degrade the matrix (for example, via their recruitment to fibrotic liver) to facilitate fibrosis resolution and liver regeneration.


Assuntos
Cirrose Hepática/etiologia , Cirrose Hepática/patologia , Células Estreladas do Fígado/fisiologia , Hepatócitos/fisiologia , Humanos , Miofibroblastos/fisiologia
19.
Exp Eye Res ; 201: 108272, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33010289

RESUMO

Myofibroblasts are alpha-smooth muscle actin (SMA)+ cells that have a critical role in the corneal stromal response to infections, injuries, and surgeries, and which produce corneal scarring fibrosis when they develop in excess. These contractile and opaque cells-produce large amounts of disordered extracellular matrix (ECM)-and develop from keratocyte-derived corneal fibroblasts or bone marrow-derived fibrocytes, and possibly other cell types, in response to TGFß1, TGFß2 and PDGF from the epithelium, tears, endothelium, and other stromal cells. Recent proteomic analyses have revealed that the myofibroblasts that develop from different progenitors aren't interchangeable, but have major differences in protein expression and functions. Absence or defective regeneration of the epithelial basement membrane (EBM) and/or Descemet's basement membrane (DBM) results in development and persistence of myofibroblasts in the corneal stroma. The functions of myofibroblasts in the cornea include production of volume-additive ECM, tissue contraction, production of various growth factors, cytokines and chemokines that regulate stromal cells, including other myofibroblasts, production of collagenases and metalloproteinases involved in tissue remodeling, and the expression of toll-like receptors that likely have critical roles in the clearance of bacteria and viruses causing corneal infections.


Assuntos
Córnea/patologia , Doenças da Córnea/patologia , Matriz Extracelular/metabolismo , Miofibroblastos/fisiologia , Animais , Doenças da Córnea/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Miofibroblastos/metabolismo
20.
Biomed Res Int ; 2020: 7176169, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33083482

RESUMO

Background: Bronchopulmonary dysplasia (BPD) is a common and serious complication in premature infants. Lung fibroblasts (LFs) are present in the extracellular matrix and participate in pulmonary development in response to BPD. The aim of this study was to investigate the effect of extracellular signal-regulated kinase (ERK) on LFs cultured from newborn rats. Material and Methods. Primary LFs were isolated and treated with epidermal growth factor (EGF, 20 ng/mL) in the presence or absence of an ERK inhibitor, PD98059 (10 µmol/L). Phosphorylated ERK1/2 (p-ERK1/2) protein levels were determined using immunocytochemistry, western blotting, and real-time reverse transcription quantitative (RT-q)PCR. LF proliferation was examined by flow cytometry and a cell counting kit-8 assay. LF transdifferentiation was examined by protein and mRNA expression of α-smooth muscle actin (α-SMA) by immunocytochemistry, western blotting, and RT-qPCR. LF migration was examined by the transwell method. Results: Phosphorylated ERK1/2, which was activated by EGF, promoted LF proliferation by accelerating cell-cycle progression from the G1 to S phase. After treatment with PD98059, the expression of p-ERK1/2 in LFs, cellular proliferation, and the percentage of cells in S phase were significantly decreased. Phosphorylated ERK1/2 also promoted the differentiation of LFs into myofibroblasts through increased α-SMA synthesis and migration. Conclusion: The activation of ERK promotes proliferation, transdifferentiation, and migration of lung fibroblasts from newborn rats.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Transdiferenciação Celular/genética , Fator de Crescimento Epidérmico/genética , Fibroblastos/fisiologia , Pulmão/fisiologia , Sistema de Sinalização das MAP Quinases/genética , Actinas/genética , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Matriz Extracelular/genética , Miofibroblastos/fisiologia , Fosforilação/genética , RNA Mensageiro/genética , Ratos
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