RESUMEN
Asthma is characterized by a chronic inflammation and remodeling of the airways. Although inflammation can be controlled, therapeutic options to revert remodeling do not exist. Thus, there is a large and unmet need to understand the underlying molecular mechanisms to develop novel therapies. We previously identified a pivotal role for miR-142-3p in regulating airway smooth muscle (ASM) precursor cell proliferation during lung development by fine-tuning the Wingless/Integrase I (WNT) signaling. Thus, we here aimed to investigate the relevance of this interaction in asthma. We performed quantitative RT-PCR and immune staining in a murine model for ovalbumin-induced allergic airway inflammation and in bronchial biopsies from patients with asthma and isolated primary fibroblasts thereof. miR-142-3p was increased in hyperproliferative regions of lung in murine and human asthma, whereas this microRNA (miRNA) was excluded from regions with differentiated ASM cells. Increases in miR-142-3p were associated with a decrease of its known target Adenomatous polyposis coli. Furthermore, we observed a differential expression of miR-142-3p in bronchial biopsies from patients with early or late onset severe asthma, which coincided with a differential WNT signature. Our data suggest that miR-142-3p is involved in regulating the balance between proliferation and differentiation of ASM cells in asthma, possibly via controlling WNT signaling. Thus, this miRNA might be an interesting target to prevent ASM hyperproliferation in asthma.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias) , Asma/metabolismo , MicroARNs/biosíntesis , Miocitos del Músculo Liso/metabolismo , Vía de Señalización Wnt , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Animales , Asma/patología , Asma/fisiopatología , Proliferación Celular , Femenino , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/patología , Miocitos del Músculo Liso/patologíaRESUMEN
Pulmonary hypertension is a devastating disease characterized by excessive vascular muscularization. We previously demonstrated primed platelet-derived growth factor receptor ß+ (PDGFR-ß+)/smooth muscle cell (SMC) marker+ progenitors at the muscular-unmuscular arteriole border in the normal lung, and in hypoxia-induced pulmonary hypertension, a single primed cell migrates distally and expands clonally, giving rise to most of the pathological smooth muscle coating of small arterioles. Little is known regarding the molecular mechanisms underlying this process. Herein, we show that primed cell expression of Kruppel-like factor 4 and hypoxia-inducible factor 1-α (HIF1-α) are required, respectively, for distal migration and smooth muscle expansion in a sequential manner. In addition, the HIF1-α/PDGF-B axis in endothelial cells non-cell autonomously regulates primed cell induction, proliferation, and differentiation. Finally, myeloid cells transdifferentiate into or fuse with distal arteriole SMCs during hypoxia, and Pdgfb deletion in myeloid cells attenuates pathological muscularization. Thus, primed cell autonomous and non-cell autonomous pathways are attractive therapeutic targets for pulmonary hypertension.
Asunto(s)
Transdiferenciación Celular , Hipertensión Pulmonar/metabolismo , Músculo Liso Vascular/metabolismo , Mioblastos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/metabolismo , Animales , Femenino , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/patología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Linfocinas/genética , Linfocinas/metabolismo , Masculino , Ratones , Músculo Liso Vascular/patología , Mioblastos del Músculo Liso/patología , Miocitos del Músculo Liso/patología , Factor de Crecimiento Derivado de Plaquetas/genética , Factor de Crecimiento Derivado de Plaquetas/metabolismoRESUMEN
The vasculature plays an indispensible role in organ development and maintenance of tissue homeostasis, such that disturbances to it impact greatly on developmental and postnatal health. Although cell turnover in healthy blood vessels is low, it increases considerably under pathological conditions. The principle sources for this phenomenon have long been considered to be the recruitment of cells from the peripheral circulation and the re-entry of mature cells in the vessel wall back into cell cycle. However, recent discoveries have also uncovered the presence of a range of multipotent and lineage-restricted progenitor cells in the mural layers of postnatal blood vessels, possessing high proliferative capacity and potential to generate endothelial, smooth muscle, hematopoietic or mesenchymal cell progeny. In particular, the tunica adventitia has emerged as a progenitor-rich compartment with niche-like characteristics that support and regulate vascular wall progenitor cells. Preliminary data indicate the involvement of some of these vascular wall progenitor cells in vascular disease states, adding weight to the notion that the adventitia is integral to vascular wall pathogenesis, and raising potential implications for clinical therapies. This review discusses the current body of evidence for the existence of vascular wall progenitor cell subpopulations from development to adulthood and addresses the gains made and significant challenges that lie ahead in trying to accurately delineate their identities, origins, regulatory pathways, and relevance to normal vascular structure and function, as well as disease.
Asunto(s)
Enfermedades Cardiovasculares/patología , Células Progenitoras Endoteliales/patología , Músculo Liso Vascular/patología , Mioblastos del Músculo Liso/patología , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/cirugía , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/trasplante , Humanos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/trasplante , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/trasplante , Neovascularización Patológica , Neovascularización Fisiológica , Regeneración , Medicina Regenerativa/métodos , Nicho de Células MadreRESUMEN
OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). METHODS AND RESULTS: BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.
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Actinas/metabolismo , Traumatismos de las Arterias Carótidas/metabolismo , Mioblastos del Músculo Liso/metabolismo , Receptores de Trombina/antagonistas & inhibidores , Traslado Adoptivo , Animales , Antígenos CD34/metabolismo , Aorta/inmunología , Aorta/metabolismo , Aorta/patología , Aorta/trasplante , Traumatismos de las Arterias Carótidas/inmunología , Traumatismos de las Arterias Carótidas/patología , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mioblastos del Músculo Liso/inmunología , Mioblastos del Músculo Liso/patología , Neointima/inmunología , Neointima/metabolismo , Neointima/patología , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Receptores de Trombina/metabolismo , Transducción de Señal , Cicatrización de Heridas/fisiologíaRESUMEN
Double homeobox 4 (DUX4) is a candidate disease gene for facioscapulohumeral dystrophy (FSHD), one of the most common muscular dystrophies characterized by progressive skeletal muscle degeneration. Despite great strides in understanding precise genetics of FSHD, the molecular pathophysiology of the disease remains unclear. One of the major limitations has been the availability of appropriate molecular tools to study DUX4 protein. In the present study, we report the development of five new monoclonal antibodies targeted against the N- and C-termini of human DUX4, and characterize their reactivity using Western blot and immunofluorescence staining. Additionally, we show that expression of the canonical full coding DUX4 induces cell death in human primary muscle cells, whereas the expression of a shorter splice form of DUX4 results in no such toxicity. Immunostaining with these new antibodies reveals a differential effect of two DUX4 isoforms on human muscle cells. These antibodies will provide an excellent tool for investigating the role of DUX4 in FSHD pathogenesis.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Proteínas de Homeodominio/metabolismo , Células Musculares/metabolismo , Mioblastos del Músculo Liso/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Western Blotting , Técnicas de Cultivo de Célula , Clonación Molecular , Escherichia coli , Técnica del Anticuerpo Fluorescente , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Ratones , Células Musculares/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Músculo Esquelético/fisiopatología , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/patología , Distrofia Muscular Facioescapulohumeral/fisiopatología , Mioblastos del Músculo Liso/patología , Plásmidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Estructura Terciaria de Proteína/genética , Conejos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , TransfecciónRESUMEN
The classification of soft tissue tumors is based on their resemblance to normal non-neoplastic tissues and provides an indication of how the tumor will behave in the further disease course. The current article presents the principles to be considered when classifying tumors into categories and discusses additional findings to be taken into account in the diagnosis. The importance of considering combinations of findings when classifying a tumor is underscored; individual (in particular immunohistochemical) findings can be misleading. A statement on the grade of malignancy of a soft tissue tumor requires its identification as a known entity, otherwise incorrect prediction of its biological behaviour is possible. The category of "intermediate malignancy" has been added to the categories of "benign" and "malignant", whereby locally aggressive and incidentally metastasizing tumors have been included in this new category. The staging of soft tissue tumors according to the TNM system is explained, emphasizing that one important feature compared with carcinomas is the inclusion of depth localisation and grade of malignancy.
Asunto(s)
Sarcoma/patología , Neoplasias de los Tejidos Blandos/patología , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/patología , Condrosarcoma/clasificación , Condrosarcoma/genética , Condrosarcoma/patología , Tejido Conectivo/patología , Endotelio Vascular/patología , Fibroblastos/patología , Marcadores Genéticos/genética , Humanos , Liposarcoma/clasificación , Liposarcoma/genética , Liposarcoma/patología , Metástasis Linfática/patología , Mioblastos/patología , Mioblastos del Músculo Liso/patología , Estadificación de Neoplasias , Pericitos/patología , Pronóstico , Rabdomioma/clasificación , Rabdomioma/genética , Rabdomioma/patología , Rabdomiosarcoma/clasificación , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Sarcoma/clasificación , Sarcoma/genética , Neoplasias de los Tejidos Blandos/clasificación , Neoplasias de los Tejidos Blandos/genéticaRESUMEN
This study examined the efficacy and in vivo mechanism of action of the antifibrotic hormone, relaxin, in a mouse model of unilateral ureteric obstruction (UUO). Kidney fibrosis was assessed in recombinant human gene-2 relaxin-treated animals maintained for 3 and 9 d after UUO. Results were compared with untreated and unoperated animals (d 0). Total collagen, collagen subtypes (I, IV), TGF-ß2 production, mothers against decapentaplegic homolog 2 (Smad2) phosphorylation, myofibroblast differentiation, mitosis, and apoptosis were all progressively increased by UUO (all P<0.05 vs. d 0 group at d 3 and d 9), whereas TGF-ß1 production was increased and vascular endothelial growth factor expression (angiogenesis) decreased at d 9 (both P<0.05 vs. d 0). A progressive increase in matrix metalloproteinase (MMP)-2 after UUO suggested that it was reactive to the increased fibrogenesis. Conversely, MMP-9 was decreased at d 9, whereas its inhibitor tissue inhibitor of metalloproteinase-1 progressively decreased after UUO. Human gene-2 relaxin pretreatment of animals from 4 d prior to UUO ameliorated the increase in total collagen, collagen IV, Smad2 phosphorylation, and myofibroblasts at both time points (all P<0.05 vs. untreated groups) and inhibited TGF-ß2 production and cell proliferation (both P<0.05 vs. untreated groups) with a trend toward normalizing vascular endothelial growth factor expression at d 9, with no effect on TGF-ß1 production or apoptosis. The relaxin-mediated regulation of MMPs and tissue inhibitor of metalloproteinases in this model was not consistent with its antifibrotic properties. The beneficial effects of relaxin were lost when treatment was stopped. These findings establish that relaxin can inhibit both early and established phases of tubulointerstitial fibrosis, primarily by suppressing cell proliferation, myofibroblast differentiation, and collagen production. Not all of these effects paralleled changes to TGF-ß-Smad signaling.
Asunto(s)
Citoprotección/efectos de los fármacos , Enfermedades Renales/prevención & control , Túbulos Renales/efectos de los fármacos , Túbulos Renales/patología , Relaxina/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Colágeno/metabolismo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Femenino , Fibrosis/prevención & control , Gelatinasas/metabolismo , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Túbulos Renales/metabolismo , Ratones , Ratones Endogámicos C57BL , Mioblastos del Músculo Liso/efectos de los fármacos , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/patología , Relaxina/uso terapéutico , Obstrucción Ureteral/metabolismo , Obstrucción Ureteral/patología , Obstrucción Ureteral/prevención & controlRESUMEN
BACKGROUND: Endothelial dysfunction contributes to accelerated atherosclerosis in chronic kidney disease (CKD). Bone marrow-derived endothelial progenitor cells (EPC) constitute an endogenous vascular repair system protecting against atherosclerosis. Smooth muscle progenitor cells (SPC) may stimulate atherosclerosis development. We hypothesized that an imbalance in EPC and SPC occurs in CKD, which may contribute to the increased cardiovascular disease (CVD) risk. METHODS: EPC and SPC outgrowth from mononuclear cells (MNC), EPC migratory function and circulating CD34(+)KDR(+)-EPC were measured in 49 patients with varying degrees of CKD on regular therapy and 33 healthy volunteers. Renal function, CKD cause, CVD history and endothelial dysfunction parameters were determined as factors of influence on progenitor cells. RESULTS: Patients had reduced EPC outgrowth compared to controls [9 (2-22) vs 12 (1-38) cells/10(3) MNC, P = 0.026], independent of CKD cause and degree, whereas SPC outgrowth levels were higher in patients with more impaired kidney function (r = -0.397, P = 0.008). Patients had lower CD34(+)KDR(+)-EPC compared to controls [9 (0-52) vs 19 (4-110) cells/10(5) granulocytes, P = 0.004]. CVD history and increased endothelial dysfunction markers were related to lower EPC levels. Progenitor cell outgrowth was shifted towards SPC with progression of endothelial damage. Reduction in EPC could not be attributed to decreases in progenitor cell-mobilizing factors SDF-1 alpha and VEGF as levels increased with progressive kidney and endothelial dysfunction, while EPC remained low. CONCLUSIONS: Our data suggest that, already in mild CKD, EPC-mediated endogenous vascular regeneration is impaired, while SPC levels increase with declining kidney function.
Asunto(s)
Células Madre Adultas/patología , Células Endoteliales/patología , Fallo Renal Crónico/patología , Fallo Renal Crónico/fisiopatología , Insuficiencia Renal Crónica/patología , Insuficiencia Renal Crónica/fisiopatología , Adulto , Anciano , Anciano de 80 o más Años , Aterosclerosis/etiología , Células de la Médula Ósea/patología , Enfermedades Cardiovasculares/etiología , Estudios de Casos y Controles , Quimiocina CXCL12/sangre , Femenino , Humanos , Fallo Renal Crónico/complicaciones , Masculino , Persona de Mediana Edad , Modelos Biológicos , Mioblastos del Músculo Liso/patología , Regeneración , Insuficiencia Renal Crónica/complicaciones , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
BACKGROUND: Solitary fibrous tumor (SFT) is a rare ubiquitous mesenchymal neoplasm of probable fibroblastic type with a prominent hemangiopericytoma-like vascular pattern. Since their initial description as arising from the pleura, SFTs have been reported in many extraserosal sites. It is now accepted that this neoplasm is derived from mesenchymal cells but its histogenesis is still not known. METHODS: The authors gathered clinical data on 10 patients with SFT. Tissue microarrays were constructed to perform inmunohistochemical tests and we reviewed hematoxilin-eosin-stained slides. Electron-microscopically collected samples were fixed with formalin or Karnovsky reactive and embedded in epoxy resin. RESULTS: The histopathological review showed varying degrees of cell density and mitotic activity, which correlated with clinical behavior. Immunohistochemically most tumors stained positively for vimentin, CD99, and CD34. Ultrastructural study showed some degree of myofibroblastic differentiation in all cases and focal smooth muscle features. In addition, 9 cases showed perivascular undifferentiated cells. CONCLUSION: SFT is an uncommon neoplasm with different histological patterns and clinical behavior. The authors hypothesize that the perivascular undifferentiated cells that most cases showed might correspond to a quiescent stage of adult stem mesenchymal cell and could be the target of the molecular aberrations implied in its pathogenesis.
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Antígenos de Neoplasias/metabolismo , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Células Clonales/patología , Tumores Fibrosos Solitarios/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Células Clonales/inmunología , Femenino , Fibroblastos/inmunología , Fibroblastos/patología , Humanos , Inmunohistoquímica , Lactante , Masculino , Persona de Mediana Edad , Mioblastos del Músculo Liso/inmunología , Mioblastos del Músculo Liso/patología , Tumores Fibrosos Solitarios/inmunología , Tumores Fibrosos Solitarios/ultraestructura , Adulto JovenRESUMEN
The human peripheral blood contains a multipotent precursor that shows hematopoietic stem cell features and transiently expresses markers of the myeloid lineage. Under permissive conditions, this precursor gives rise to committed progenitors of various lineages, including a mesenchymal progenitor cell known by the name of fibrocyte. The fibrocytes still express some hematopoietic and myeloid antigens together with fibroblast markers. They constitutively release pro-fibrotic and angiogenic factors and can modulate ongoing inflammatory reactions through the release of a number of chemokines. Under appropriate stimulation, fibrocytes produce increased amounts of extracellular matrix components and acquire a contractile phenotype similar to that of activated fibroblasts (myofibroblasts). Fibrocytes synthesizing new collagen or acquiring myofibroblast markers have been detected in pulmonary diseases characterized by an extensive remodeling of the bronchial wall or progressive fibrosis, in the skin of patients affected by nephrogenic systemic fibrosis, in human hypertrophic scars, in proliferative vitreoretinopathies and atherosclerotic lesions. Similar cells also participate in the stromal reaction to tumor development. Prevention of detrimental tissue remodeling in fibrotic diseases may be achieved by inhibiting the accumulation of fibrocytes. In-vitro expanded fibrocytes may be used to improve ineffective tissue repair or may be engineered for the delivery of gene constructs in anti-cancer therapy.
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Antígenos de Diferenciación/metabolismo , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Mioblastos del Músculo Liso/metabolismo , Fibrosis Pulmonar/terapia , Remodelación de las Vías Aéreas (Respiratorias) , Animales , Humanos , Células Madre Mesenquimatosas/patología , Mioblastos del Músculo Liso/patología , Fibrosis Pulmonar/patología , Ingeniería de Tejidos , Cicatrización de HeridasRESUMEN
BACKGROUND: Haemodialysis vascular access dysfunction (due to venous stenosis and thrombosis) is a leading cause of hospitalization and morbidity. The aim of the current study was to identify the specific cell types present within stenotic tissue samples from patients with AV fistula and graft failure. METHODS: Discarded tissue segments were collected from the stenotic portions (usually near the graft-vein anastomosis or the AV anastomosis) of 23 dialysis grafts and 20 AV fistulae, and examined for expression of smooth muscle alpha actin, desmin, vimentin and a macrophage marker. RESULTS: The majority of cells within the venous neointima (both grafts and fistulae) were myofibroblasts, with a smaller number of desmin positive smooth muscle cells. The graft neointima had a similar cellular phenotype, albeit without any desmin positive contractile smooth muscle cells. The majority of cells within the PTFE graft material were macrophages. Analysis of sequential sections revealed the presence of fibroblasts within the venous neointima and intragraft region. CONCLUSIONS: Our results demonstrate that contractile smooth muscle cells, myofibroblasts, fibroblasts and macrophages all play a role in the pathogenesis of dialysis access dysfunction (grafts and fistulae). Targeting these specific cell types might result in the development of novel therapeutic paradigms for haemodialysis vascular access dysfunction.
Asunto(s)
Derivación Arteriovenosa Quirúrgica/efectos adversos , Diálisis Renal/efectos adversos , Actinas/metabolismo , Arterias/metabolismo , Arterias/patología , Arterias/cirugía , Prótesis Vascular/efectos adversos , Desdiferenciación Celular , Diferenciación Celular , Movimiento Celular , Constricción Patológica , Desmina/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/patología , Fenotipo , Politetrafluoroetileno , Túnica Íntima/metabolismo , Túnica Íntima/patología , Venas/metabolismo , Venas/patología , Venas/cirugía , Vimentina/metabolismoRESUMEN
Afferent signal transduction in the urinary bladder is still not clearly understood. An increasing body of evidence supports the view of complex interactions between urothelium, suburothelial myofibroblasts, and sensory nerves. Bladder tissue from tumour patients was used in this study. Methods included confocal immunofluorescence, polymerase chain reaction, calcium imaging, and fluorescence recovery after photobleaching (FRAP).Myofibroblasts express muscarinic and purinergic receptors. They show constitutive spontaneous activity in calcium imaging, which completely depends on extracellular calcium. Stimulation with carbachol and ATP-evoked intracellular calcium transients also depend on extracellular calcium. The intensive coupling between the cells is significantly diminished by incubation with TGF-beta 1. Myofibroblasts form an important cellular element within the afferent signalling of the urinary bladder. They possess all features required to take part in the complex interactions with urothelial cells and sensory nerves. Modulation of their function by cytokines may provide a pathomechanism for bladder dysfunction.
Asunto(s)
Fibroblastos/fisiología , Mioblastos del Músculo Liso/fisiología , Neuronas Aferentes/fisiología , Transducción de Señal/fisiología , Vejiga Urinaria/inervación , Urotelio/inervación , Canales de Calcio/fisiología , Células Cultivadas , Fibroblastos/patología , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Técnicas In Vitro , Microscopía Fluorescente , Mioblastos del Músculo Liso/patología , Neuronas Aferentes/patología , Nervios Periféricos/patología , Nervios Periféricos/fisiología , Receptores Muscarínicos/fisiología , Receptores Purinérgicos/fisiología , Células Receptoras Sensoriales/fisiología , Transmisión Sináptica/fisiología , Factor de Crecimiento Transformador beta1/fisiología , Vejiga Urinaria/patología , Urotelio/patologíaRESUMEN
Scar formation as a result of burn wounds leads to contraction of the formed granulation tissue, which causes both aesthetic and functional impairment for the patient. Currently, the main treatment methods focus on stretching to prevent tissue contraction. The myofibroblasts play a key role in the contraction of granulation tissue during scar formation, but their presence should normally decrease after wound re-epithelialization. In hypertrophic scars the myofibroblasts persist and is believed to cause further hypertrophy. Previous studies have shown that mechanical tension leads to increased myofibroblast numbers in granulation tissue. In order to evaluate the effect mechanical tension as a result of stretching has on the number of myofibroblasts in burn wound scars, an in vitro model was used. This model used human burn scar biopsies which were stretched and examined after 1 and 6 days to evaluate the effect on the number of myofibroblasts. The stretching caused an increase in the number of myofibroblasts after mechanical stimulation. This indicates that mechanical stimulation using stretching induces fibroblast to myofibroblast transdifferentiation, thus underlining the importance of further investigations of optimal methods of this regime for treating burn scars.
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Quemaduras/patología , Cicatriz Hipertrófica/patología , Fibroblastos/patología , Mioblastos del Músculo Liso/patología , Cicatrización de Heridas/fisiología , Actinas/análisis , Diferenciación Celular , Transdiferenciación Celular , Contractura , Humanos , Inmunohistoquímica , Estrés MecánicoRESUMEN
OBJECTIVE: TGF-beta plays a significant role in vascular injury-induced stenosis. This study evaluates the efficacy of a novel, small molecule inhibitor of ALK5/ALK4 kinase, in the rat carotid injury model of vascular fibrosis. METHODS AND RESULTS: The small molecule, SM16, was shown to bind with high affinity to ALK5 kinase ATP binding site using a competitive binding assay and biacore analysis. SM16 blocked TGF-beta and activin-induced Smad2/3 phosphorylation and TGF-beta-induced plasminogen activator inhibitor (PAI)-luciferase activity in cells. Good overall selectivity was demonstrated in a large panel of kinase assays, but SM16 also showed nanomolar inhibition of ALK4 and weak (micromolar) inhibition of Raf and p38. In the rat carotid injury model, SM16 dosed once daily orally at 15 or 30 mg/kg SM16 for 14 days caused significant inhibition of neointimal thickening and lumenal narrowing. SM16 also prevented induction of adventitial smooth muscle alpha-actin-positive myofibroblasts and the production of intimal collagen, but did not decrease the percentage of proliferative cells. CONCLUSIONS: These results are the first to demonstrate the efficacy of an orally active, small-molecule ALK5/ALK4 inhibitor in a vascular fibrosis model and suggest the potential therapeutic application of these inhibitors in vascular fibrosis.
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Compuestos de Azabiciclo/farmacología , Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Activinas Tipo I/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Administración Oral , Animales , Compuestos de Azabiciclo/administración & dosificación , Compuestos de Azabiciclo/metabolismo , Sitios de Unión , Traumatismos de las Arterias Carótidas/patología , Traumatismos de las Arterias Carótidas/fisiopatología , Línea Celular , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Fibrosis , Humanos , Masculino , Mioblastos del Músculo Liso/efectos de los fármacos , Mioblastos del Músculo Liso/patología , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Tipo I de Factor de Crecimiento Transformador beta , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Square skin wounds can heal to form a stellar scar with four protrusions at the four angles, whereas circular wounds can heal to form an ellipsoid scar. It is not clear why these differences occur and the aim of the present study was to clarify this phenomenon. Two square or circular full-thickness skin wounds were made on the dorsum of mice, and covered with hydrocolloid dressing. They were observed from day 0 to 15 after wounding, and used to prepare paraffin sections stained with anti-alpha-smooth muscle actin antibody to detect myofibroblasts. The square wound was transiently enlarged by edema and skin tension on day 3, at which time the angles became round, and thus the square form became more circular. Thereafter, the wound contracted rapidly and the circular form was maintained until day 11. On day 11 distinct angles appeared where the scar formation had progressed further, and there were fewer myofibroblasts than in any other section. A stellar scar with protrusions from the four angles was formed on day 15, when myofibroblasts almost disappeared in the protrusions. This indicates that due to the earlier disappearance of myofibroblasts and earlier scarring in the angles of the square wound, the scar angle cannot be pulled into the center of the wound but residual myofibroblasts on the side can pull the side into the center due to myofibroblastic contraction and consequently a stellar scar is formed. Thus, the earlier disappearance of myofibroblasts in the angles is very important for the formation of stellar scars.
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Cicatriz/patología , Fibroblastos/patología , Mioblastos del Músculo Liso/patología , Piel/lesiones , Cicatrización de Heridas , Actinas/análisis , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Mioblastos del Músculo Liso/químicaRESUMEN
BACKGROUND: Myofibroblasts play a pivotal role in numerous pathological alterations. Clarification of the structure and function and of the cellular plasticity of this cell type in the bladder may lead to new insights into the pathogenesis of lower urinary tract disorders. PATIENTS AND METHODS: Bladder biopsies from patients with bladder carcinoma and interstitial cystitis were used to analyse the morphology and receptor expression using confocal immunofluorescence and electron microscopy. Cytokine effects and coupling behavior were tested in cultured myofibroblasts and detrusor smooth muscle cells. RESULTS: Myofibroblasts are in close contact with the suburothelial capillary network. They express Cx43 and form functional syncytia. The expression of muscarinic and purinergic receptors is highly variable. Dye coupling experiments showed differences to detrusor myocytes. CONCLUSIONS: Upregulation of smooth muscle cell alpha-actin and/or transdifferentiation into smooth muscle cells may contribute to the etiology of urge incontinence. A multi-step model is presented as a working hypothesis.
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Mioblastos del Músculo Liso/fisiología , Enfermedades de la Vejiga Urinaria/fisiopatología , Urotelio/fisiopatología , Actinas/metabolismo , Anciano , Anciano de 80 o más Años , Membrana Basal/patología , Membrana Basal/fisiopatología , Biopsia , Conexina 43/metabolismo , Cistitis/patología , Cistitis/fisiopatología , Femenino , Fibroblastos/patología , Fibroblastos/fisiología , Fibronectinas/metabolismo , Humanos , Masculino , Microscopía Confocal , Microscopía Fluorescente , Persona de Mediana Edad , Membrana Mucosa/patología , Membrana Mucosa/fisiopatología , Mioblastos del Músculo Liso/patología , Receptores Muscarínicos/metabolismo , Receptores Purinérgicos/metabolismo , Enfermedades de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/fisiopatología , Incontinencia Urinaria de Urgencia/patología , Incontinencia Urinaria de Urgencia/fisiopatología , Urodinámica/fisiología , Urotelio/patologíaRESUMEN
Macrophages are an essential component of unstable atherosclerotic plaques and play a pivotal role in the destabilization process. We have demonstrated previously that local delivery of the mammalian target of rapamycin (mTOR) inhibitor everolimus selectively clears macrophages in rabbit plaques. Because mTOR controls mRNA translation, inhibition of protein synthesis might induce selective macrophage cell death. We therefore investigated in the present study the effect of the protein synthesis inhibitor cycloheximide on macrophage and smooth muscle cell (SMC) viability. In vitro studies with cultured macrophages and SMCs showed that cycloheximide induced selective apoptosis of macrophages in a concentration- and time-dependent manner. Moreover, macrophages could be selectively depleted in rabbit carotid artery rings with collar-induced atherosclerotic plaques after in vitro treatment with cycloheximide. Local in vivo administration of cycloheximide via osmotic minipumps to rabbit carotid arteries with collar-induced atherosclerotic plaques significantly reduced the macrophage but not the SMC content. Cycloheximide-treated plaques showed signs of apoptosis (increased terminal deoxynucleotidyl transferase end labeling and fluorescein isothiocyanate-Val-Ala-dl-Asp(O-methyl)-fluoromethylketone labeling) that did not colocalize with SMCs. Organ chamber studies demonstrated that the functionality of SMCs and the endothelium were not influenced by cycloheximide treatment. All together, these findings demonstrate that cycloheximide decreases the macrophage load in atherosclerotic plaques by induction of apoptosis without changing SMC content or contractility.
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Apoptosis/efectos de los fármacos , Enfermedades de las Arterias Carótidas/tratamiento farmacológico , Cicloheximida , Macrófagos/efectos de los fármacos , Inhibidores de la Síntesis de la Proteína , Animales , Western Blotting , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cicloheximida/administración & dosificación , Cicloheximida/farmacología , Cicloheximida/uso terapéutico , Relación Dosis-Respuesta a Droga , Inmunohistoquímica , Bombas de Infusión , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Ratones , Mioblastos del Músculo Liso/efectos de los fármacos , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/patología , Inhibidores de la Síntesis de la Proteína/administración & dosificación , Inhibidores de la Síntesis de la Proteína/farmacología , Inhibidores de la Síntesis de la Proteína/uso terapéutico , Conejos , Factores de TiempoRESUMEN
The muscularization of non-muscular pulmonary arterioles is an important pathological feature of hypoxic pulmonary vascular remodeling. However, the origin of the cells involved in this process is still not well understood. The present study was undertaken to test the hypothesis that transforming growth factor-beta1 (TGF-beta1) can induce transdifferentiation of fibroblasts into myofibroblasts, which might play a key role in the muscularization of non-muscular pulmonary arterioles. It was found that mean pulmonary arterial pressure increased significantly after 7 d of hypoxia. Pulmonary artery remodeling index and right ventricular hypertrophy became evident after 14 d of hypoxia. The distribution of nonmuscular, partially muscular, and muscular vessels was significantly different after 7 d of hypoxia. Immunocytochemistry results demonstrated that the expression of a-smooth muscle actin was increased in intra-acinar pulmonary arteries with increasing hypoxic time. TGF-beta1 mRNA expression in pulmonary arterial walls was increased significantly after 14 d of hypoxia, but showed no obvious changes after 3 or 7 d of hypoxia. In pulmonary tunica adventitia and tunica media, TGF-beta1 protein staining was poorly positive in control rats, but was markedly enhanced after 3 d of hypoxia, reaching its peak after 7 d of hypoxia. The myofibroblast phenotype was confirmed by electron microscopy, which revealed microfilaments and a well-developed rough endoplasmic reticulum. Taken together, our results suggested that TGF-beta1 induces transdifferentiation of fibroblasts into myofibroblasts, which is important in hypoxic pulmonary vascular remodeling.
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Fibroblastos/metabolismo , Fibroblastos/patología , Hipoxia/metabolismo , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/patología , Arteria Pulmonar/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Hipoxia/patología , Masculino , Arteria Pulmonar/patología , Ratas , Ratas WistarRESUMEN
Intestinal strictures are a common complication of Crohn's disease leading to serious consequences. With unknown etiology and cellular composition, strictures can be neither prevented nor reversed by current therapeutic strategies, and research has been limited by the lack of a well-developed animal model. We observed the sporadic occurrence of intestinal strictures at Day 35 in the TNBS rat model of colitis, which persisted beyond Day 90. Strictured tissue showed fusion, thickening, and disorganization of the smooth muscle layers. Immunocytochemistry revealed that all strictures were characterized by deficient innervation with a complete loss of intrinsic neurons, and a 92% loss of total axons per area. The number of alpha-smooth muscle actin-positive smooth muscle cells (SMC) increased in strictures, but immunolabeling showed phenotypic modulation of these cells, with the SMC phenotype (desmin-positive, vimentin-negative) entirely replaced by a myofibroblast phenotype (desmin-negative, vimentin-positive). Although cellular structure still predominated in the strictured regions, histochemistry showed increased extracellular matrix collagen, from 6 +/- 0.9% to 22 +/- 4% of total area. With previous evidence for neural loss in colitis, and in vitro studies showing neural regulation of smooth muscle cell (SMC) growth, we conclude that the regional loss of innervation may initiate tissue re-modeling that is characteristic of stricture formation.
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Colitis/patología , Sistema Nervioso Entérico/patología , Obstrucción Intestinal/patología , Músculo Liso/inervación , Miocitos del Músculo Liso/patología , Neuronas/patología , Animales , Axones/metabolismo , Axones/patología , Biomarcadores/metabolismo , Colitis/complicaciones , Colágeno/metabolismo , Constricción Patológica/metabolismo , Constricción Patológica/patología , Constricción Patológica/fisiopatología , Desmina/metabolismo , Modelos Animales de Enfermedad , Obstrucción Intestinal/etiología , Obstrucción Intestinal/metabolismo , Masculino , Músculo Liso/patología , Mioblastos del Músculo Liso/metabolismo , Mioblastos del Músculo Liso/patología , Miocitos del Músculo Liso/metabolismo , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Vimentina/metabolismoRESUMEN
Lung myofibroblasts play a major role in the pathophysiology of asthma, contributing not only to tissue remodelling but also to airway inflammation. Nevertheless, only recently, attention has been focused on these cells as potential targets for anti-allergic drugs. Herein, we analysed the pharmacological response of lung myofibroblasts to beta2-agonists associated or not to inhaled corticosteroids, investigating their effects on (i) the constitutive and transforming growth factor-beta (TGF-beta)-induced expression of alpha-smooth muscle actin (alpha-SMA), the main functional marker of myofibroblastic differentiation and contractility; (ii) isometric contraction and (iii) tumour necrosis factor-alpha (TNF-alpha)-induced nuclear translocation of the pro-inflammatory transcription factor nuclear factor-kappaB (NF-kappaB). The beta2-agonist salmeterol (SMl) has on human lung myofibroblasts new direct anti-contractile/anti-inflammatory effects that are amplified by the combined use of low concentrations of the glucocorticoid fluticasone propionate (FP). First, SMl and/or FP (10(-12) M) inhibits the constitutive and TGF-beta-induced expression of alpha-SMA. Second, the two drugs block the TNF-alpha-induced nuclear translocation of the pro-inflammatory transcription factor NF-kappaB. Finally, SMl decreases TNF- alpha-induced production of the inflammatory cytokine IL-6. The complementary anti-inflammatory/ anti-contractile effects displayed by SMl and FP on lung myofibroblasts in vitro may be related to the improvement in lung function and symptom control obtained in vivo by the early use of low doses of glucocorticoids in combination with long-acting beta2-agonists.