RESUMEN
The term 'fibroblast' often serves as a catch-all for a diverse array of mesenchymal cells, including perivascular cells, stromal progenitor cells and bona fide fibroblasts. Although phenotypically similar, these subpopulations are functionally distinct, maintaining tissue integrity and serving as local progenitor reservoirs. In response to tissue injury, these cells undergo a dynamic fibroblast-myofibroblast transition, marked by extracellular matrix secretion and contraction of actomyosin-based stress fibres. Importantly, whereas transient activation into myofibroblasts aids in tissue repair, persistent activation triggers pathological fibrosis. In this Review, we discuss the roles of mechanical cues, such as tissue stiffness and strain, alongside cell signalling pathways and extracellular matrix ligands in modulating myofibroblast activation and survival. We also highlight the role of epigenetic modifications and myofibroblast memory in physiological and pathological processes. Finally, we discuss potential strategies for therapeutically interfering with these factors and the associated signal transduction pathways to improve the outcome of dysregulated healing.
Asunto(s)
Fibrosis , Miofibroblastos , Cicatrización de Heridas , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Animales , Fibrosis/metabolismo , Cicatrización de Heridas/fisiología , Transducción de Señal , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Epigénesis GenéticaRESUMEN
Lantibiotics are ribosomally synthesized and posttranslationally modified peptides (RiPPs) that are produced by bacteria. Interest in this group of natural products is increasing rapidly as alternatives to conventional antibiotics. Some human microbiome-derived commensals produce lantibiotics to impair pathogens' colonization and promote healthy microbiomes. Streptococcus salivarius is one of the first commensal microbes to colonize the human oral cavity and gastrointestinal tract, and its biosynthesis of RiPPs, called salivaricins, has been shown to inhibit the growth of oral pathogens. Herein, we report on a phosphorylated class of three related RiPPs, collectively referred to as salivaricin 10, that exhibit proimmune activity and targeted antimicrobial properties against known oral pathogens and multispecies biofilms. Strikingly, the immunomodulatory activities observed include upregulation of neutrophil-mediated phagocytosis, promotion of antiinflammatory M2 macrophage polarization, and stimulation of neutrophil chemotaxis-these activities have been attributed to the phosphorylation site identified on the N-terminal region of the peptides. Salivaricin 10 peptides were determined to be produced by S. salivarius strains found in healthy human subjects, and their dual bactericidal/antibiofilm and immunoregulatory activity may provide new means to effectively target infectious pathogens while maintaining important oral microbiota.
Asunto(s)
Bacteriocinas , Humanos , Bacteriocinas/farmacología , Bacteriocinas/química , Bacterias , Antibacterianos/farmacología , Antibacterianos/química , PéptidosRESUMEN
Myofibroblasts are the construction workers of wound healing and repair damaged tissues by producing and organizing collagen/extracellular matrix (ECM) into scar tissue. Scar tissue effectively and quickly restores the mechanical integrity of lost tissue architecture but comes at the price of lost tissue functionality. Fibrotic diseases caused by excessive or persistent myofibroblast activity can lead to organ failure. This review defines myofibroblast terminology, phenotypic characteristics, and functions. We will focus on the central role of the cell, ECM, and tissue mechanics in regulating tissue repair by controlling myofibroblast action. Additionally, we will discuss how therapies based on mechanical intervention potentially ameliorate wound healing outcomes. Although myofibroblast physiology and pathology affect all organs, we will emphasize cutaneous wound healing and hypertrophic scarring as paradigms for normal tissue repair versus fibrosis. A central message of this review is that myofibroblasts can be activated from multiple cell sources, varying with local environment and type of injury, to either restore tissue integrity and organ function or create an inappropriate mechanical environment.
Asunto(s)
Cicatriz , Miofibroblastos , Humanos , Miofibroblastos/metabolismo , Miofibroblastos/patología , Cicatriz/metabolismo , Cicatriz/patología , Cicatrización de Heridas/fisiología , Colágeno/metabolismo , Fibrosis , Matriz Extracelular/metabolismo , Diferenciación Celular/fisiologíaRESUMEN
Inflammation-driven foreign body reactions, and the frequently associated encapsulation by fibrogenic fibroblasts, reduce the functionality and longevity of implanted medical devices and materials. Anti-inflammatory drugs, such as dexamethasone, can suppress the foreign body reaction for a few days post-surgery, but lasting drug delivery strategies for long-term implanted materials remain an unmet need. We here establish a thin-coating strategy with novel low molecular weight corticosteroid dimers to suppress foreign body reactions and fibrotic encapsulation of subcutaneous silicone implants. The dimer coatings are >75% dexamethasone by mass and directly processable into conformal coatings using conventional solvent-based techniques, such as casting or spray coating without added polymers or binding agents. In vitro, surface erosion of the coating, and subsequent hydrolysis, provide controlled release of free dexamethasone. In a rat subcutaneous implantation model, the resulting slow and sustained release profile of dexamethasone is effective at reducing the number and activation of pro-fibrotic macrophages both acutely and at chronic time points. Consequently, fibroblast activation, collagen deposition and fibrotic encapsulation are suppressed at least 45 days post-implantation. Thus, our approach to protect implants from host rejection is advantageous over polymeric drug delivery systems, which typically have low drug loading capacity (<30%), initial burst release profiles, and unpredictable release kinetics.
Asunto(s)
Polímeros , Prótesis e Implantes , Corticoesteroides , Animales , Preparaciones de Acción Retardada , Dexametasona/química , Fibrosis , Reacción a Cuerpo Extraño/prevención & control , Peso Molecular , RatasRESUMEN
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.
Asunto(s)
Actinas/metabolismo , Técnicas de Cultivo de Célula/métodos , Técnica del Anticuerpo Fluorescente/métodos , Miofibroblastos/citología , Células 3T3 , Actinas/genética , Animales , Biomarcadores/metabolismo , Células Cultivadas , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Ratones , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Fenotipo , Ratas , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
RATIONALE: Gene expression profiles have been mainly determined by analysis of transcript abundance. However, these analyses cannot capture posttranscriptional gene expression control at the level of translation, which is a key step in the regulation of gene expression, as evidenced by the fact that transcript levels often poorly correlate with protein levels. Furthermore, genome-wide transcript profiling of distinct cell types is challenging due to the fact that lysates from tissues always represent a mixture of cells. OBJECTIVES: This study aimed to develop a new experimental method that overcomes both limitations and to apply this method to perform a genome-wide analysis of gene expression on the translational level in response to pressure overload. METHODS AND RESULTS: By combining ribosome profiling (Ribo-seq) with a ribosome-tagging approach (Ribo-tag), it was possible to determine the translated transcriptome in specific cell types from the heart. After pressure overload, we monitored the cardiac myocyte translatome by purifying tagged cardiac myocyte ribosomes from cardiac lysates and subjecting the ribosome-protected mRNA fragments to deep sequencing. We identified subsets of mRNAs that are regulated at the translational level and found that translational control determines early changes in gene expression in response to cardiac stress in cardiac myocytes. Translationally controlled transcripts are associated with specific biological processes related to translation, protein quality control, and metabolism. Mechanistically, Ribo-seq allowed for the identification of upstream open reading frames in transcripts, which we predict to be important regulators of translation. CONCLUSIONS: This method has the potential to (1) provide a new tool for studying cell-specific gene expression at the level of translation in tissues, (2) reveal new therapeutic targets to prevent cellular remodeling, and (3) trigger follow-up studies that address both, the molecular mechanisms involved in the posttranscriptional control of gene expression in cardiac cells, and the protective functions of proteins expressed in response to cellular stress.
Asunto(s)
Miocitos Cardíacos/metabolismo , Ribosomas/metabolismo , Análisis de Secuencia de ARN/métodos , Disfunción Ventricular/genética , Animales , Células Cultivadas , Ventrículos Cardíacos/citología , Hemodinámica , Masculino , Ratones , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/química , Estrés Fisiológico , Disfunción Ventricular/metabolismoRESUMEN
BACKGROUND: ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity. METHODS: To mimic ARID1A-deficient colorectal cancer, we used CRISPR/Cas9-mediated gene editing to inactivate the ARID1A gene in established colorectal cancer cell lines. We integrated gene expression analyses with genome-wide ARID1A occupancy and epigenomic mapping data to decipher ARID1A-dependent transcriptional regulatory mechanisms. RESULTS: Interestingly, we found that CRC cell lines harboring KRAS mutations are critically dependent on ARID1A function. In the absence of ARID1A, proliferation of these cell lines is severely impaired, suggesting an essential role for ARID1A in this context. Mechanistically, we showed that ARID1A acts as a co-factor at enhancers occupied by AP1 transcription factors acting downstream of the MEK/ERK pathway. Consistently, loss of ARID1A led to a disruption of KRAS/AP1-dependent enhancer activity, accompanied by a downregulation of expression of the associated target genes. CONCLUSIONS: We identify a previously unknown context-dependent tumor-supporting function of ARID1A in CRC downstream of KRAS signaling. Upon the loss of ARID1A in KRAS-mutated cells, enhancers that are co-occupied by ARID1A and the AP1 transcription factors become inactive, thereby leading to decreased target gene expression. Thus, targeting of the BAF complex in KRAS-mutated CRC may offer a unique, previously unknown, context-dependent therapeutic option in CRC.
Asunto(s)
Neoplasias Colorrectales/genética , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Factor de Transcripción AP-1/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Sistemas CRISPR-Cas , Proliferación Celular , Neoplasias Colorrectales/metabolismo , Elementos de Facilitación Genéticos , Edición Génica , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células HCT116 , Células HT29 , Humanos , Sistema de Señalización de MAP Quinasas , Mutación , Transducción de SeñalRESUMEN
Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.
Asunto(s)
Calcio/química , Celulasa/química , Dominios de Inmunoglobulinas/genética , Alicyclobacillus/enzimología , Sitios de Unión , Celulasa/genética , Estabilidad de Enzimas/efectos de los fármacos , Unión Proteica , Especificidad por SustratoRESUMEN
Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.