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1.
Nat Immunol ; 20(11): 1469-1480, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31591568

RESUMEN

Tissue-resident memory T cells (TRM cells) are crucial mediators of adaptive immunity in nonlymphoid tissues. However, the functional heterogeneity and pathogenic roles of CD4+ TRM cells that reside within chronic inflammatory lesions remain unknown. We found that CD69hiCD103lo CD4+ TRM cells produced effector cytokines and promoted the inflammation and fibrotic responses induced by chronic exposure to Aspergillus fumigatus. Simultaneously, immunosuppressive CD69hiCD103hiFoxp3+ CD4+ regulatory T cells were induced and constrained the ability of pathogenic CD103lo TRM cells to cause fibrosis. Thus, lung tissue-resident CD4+ T cells play crucial roles in the pathology of chronic lung inflammation, and CD103 expression defines pathogenic effector and immunosuppressive tissue-resident cell subpopulations in the inflamed lung.


Asunto(s)
Comunicación Celular/inmunología , Tolerancia Inmunológica , Memoria Inmunológica , Fibrosis Pulmonar/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD/metabolismo , Antígenos Fúngicos/inmunología , Aspergillus fumigatus/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Cadenas alfa de Integrinas/metabolismo , Pulmón/citología , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones Transgénicos , Fibrosis Pulmonar/patología , Linfocitos T Reguladores/metabolismo
2.
FASEB J ; 37(5): e22842, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-37000501

RESUMEN

Joint contracture causes distressing permanent mobility disorder due to trauma, arthritis, and aging, with no effective treatment available. A principal and irreversible cause of joint contracture has been regarded as the development of joint capsule fibrosis. However, the molecular mechanisms underlying contracture remain unclear. We established a mouse model of knee joint contracture, revealing that fibrosis in joint capsules causes irreversible contracture. RNA-sequencing of contracture capsules demonstrated a marked enrichment of the genes involved in the extracellular region, particularly periostin (Postn). Three-dimensional magnetic resonance imaging and immunohistological analysis of contracture patients revealed posterior joint capsule thickening with abundant type I collagen (Col1a2) and POSTN in humans. Col1a2-GFPTG ; Postn-/- mice and chimeric mice with Col1a2-GFPTG ; tdTomatoTG bone marrow showed fibrosis in joint capsules caused by bone marrow-derived fibroblasts, and POSTN promoted the migration of bone marrow-derived fibroblasts, contributing to fibrosis and contracture. Conversely, POSTN-neutralizing antibody attenuated contracture exacerbation. Our findings identified POSTN as a key inducer of fibroblast migration that exacerbates capsule fibrosis, providing a potential therapeutic strategy for joint contracture.


Asunto(s)
Médula Ósea , Contractura , Humanos , Ratones , Animales , Médula Ósea/patología , Rango del Movimiento Articular , Contractura/genética , Contractura/tratamiento farmacológico , Fibrosis , Fibroblastos/patología
3.
Biochem Biophys Res Commun ; 583: 93-99, 2021 Oct 27.
Artículo en Inglés | MEDLINE | ID: mdl-34735885

RESUMEN

Exosomes are secreted from a variety of cells and transmit parental cell-derived biomolecules, such as nucleic acids and proteins, to recipient cells in distant organs. In addition to their important roles in both physiological and pathological conditions, exosomes are expected to serve as natural drug carriers without any cytotoxicity, immunogenicity, or tumorigenicity. However, the use of exosomes as drug delivery tools is limited due to the low uptake efficiency of the target cells, insufficient release of the contents from the endosome to the cytosol, and possible adverse effects caused by the delivery to non-target cells. In the present study, we examined the effects of the modification of exosomes with carbonate apatite or a lactose-carrying polymer. Using newly generated monitoring exosomes that contain either firefly luciferase or fused mCherry/enhanced green fluorescent protein, we demonstrated that the modification of exosomes with carbonate apatite improved their release from the endosome into the cytosol in recipient cells. Meanwhile, the modification of exosomes with a lactose-carrying polymer enhanced the selective delivery to parenchymal hepatocytes. These modified exosomes may provide an efficient strategy for macromolecule therapy for incurable diseases that cannot be treated with conventional small-molecule compounds.

4.
Hepatology ; 71(4): 1437-1452, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31549421

RESUMEN

BACKGROUND AND AIMS: Hepatic stellate cells (HSCs), a key player in the progression of liver fibrosis, are activated by various inflammatory stimuli and converted to myofibroblast-like cells with excessive collagen production. Despite many attempts to suppress activation of HSCs or inhibit collagen production in activated HSCs, their clinical applications have not been established yet. Recently, the deactivation of HSCs has been reported as a mechanism underlying the reversibility of experimental liver fibrosis. In the present study, we sought for deactivation factors of HSCs that induce regression of established liver fibrosis. APPROACH AND RESULTS: We identified transcription factor 21 (Tcf21) as one of the transcription factors whose expression was up-regulated in parallel to the differentiation of fetal HSCs. Expression of Tcf21 in HSCs remarkably decreased during culture-induced activation in vitro and in murine and human fibrotic liver tissue in vivo. This reduced Tcf21 expression was recovered during the spontaneous regression of murine liver fibrosis. Tcf21 was also examined for its effects by adeno-associated virus serotype 6-mediated Tcf21 gene transfer into cultured activated HSCs and mice with carbon tetrachloride- or methionine-choline deficient diet-induced liver fibrosis. Overexpression of Tcf21 in activated HSCs not only suppressed fibrogenic gene expression but also restored cells, at least in part, to a quiescent phenotype both in vitro and in vivo. These phenotypic changes of HSCs were accompanied by the regression of steatohepatitis and fibrosis and improved hepatic architecture and function. CONCLUSIONS: Tcf21 has been identified as a deactivation factor of fibrogenic HSCs, providing insight into a treatment strategy for the otherwise intractable liver fibrosis.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica , Técnicas de Transferencia de Gen , Células Estrelladas Hepáticas/patología , Cirrosis Hepática/genética , Ratones , Ratones Endogámicos C57BL
5.
Biochem Biophys Res Commun ; 528(3): 420-425, 2020 07 30.
Artículo en Inglés | MEDLINE | ID: mdl-32505348

RESUMEN

Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/ß-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.


Asunto(s)
Hepatocitos/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Acetaminofén/toxicidad , Animales , Separación Celular , Células Cultivadas , Citometría de Flujo , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hígado/anatomía & histología , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Transgénicos , Mitocondrias Hepáticas/metabolismo , NADPH-Ferrihemoproteína Reductasa/genética , Oxígeno/metabolismo , Vía de Señalización Wnt/efectos de los fármacos
6.
Stem Cells ; 37(1): 89-101, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30270488

RESUMEN

Granulocyte colony stimulating factor (G-CSF) has been reported to ameliorate impaired liver function in patients with advanced liver diseases through mobilization and proliferation of hepatic progenitor cells (HPCs). However, the underlying mechanisms remain unknown. We previously showed that G-CSF treatment increased the number of bone marrow (BM)-derived cells migrating to the fibrotic liver following repeated carbon tetrachloride (CCl4 ) injections into mice. In this study, we identified opioid growth factor receptor-like 1 (OGFRL1) as a novel BM cell-derived accelerator of fibrotic liver regeneration in response to G-CSF treatment. Endogenous Ogfrl1 was highly expressed in the hematopoietic organs such as the BM and spleen, whereas the liver contained a relatively small amount of Ogfrl1 mRNA. Among the peripheral blood cells, monocytes were the major sources of OGFRL1. Endogenous Ogfrl1 expression in both the peripheral blood monocytes and the liver was decreased following repeated CCl4 injections. An intrasplenic injection of cells overexpressing OGFRL1 into CCl4 -treated fibrotic mice increased the number of HPC and stimulated proliferation of hepatic parenchymal cells after partial resection of the fibrotic liver. Furthermore, overexpression of OGFRL1 in cultured HPC accelerated their differentiation as estimated by increased expression of liver-specific genes such as hepatocyte nuclear factor 4α, cytochrome P450, and fatty acid binding protein 1, although it did not affect the colony forming ability of HPC. These results indicate a critical role of OGFRL1 in the mobilization and differentiation of HPC in the fibrotic liver, and administration of OGFRL1-expressing cells may serve as a potential regenerative therapy for advanced liver fibrosis. Stem Cells 2019;37:89-101.


Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Cirrosis Hepática/genética , Cirrosis Hepática/terapia , Regeneración Hepática/genética , Medicina Regenerativa/métodos , Células Madre/metabolismo , Animales , Diferenciación Celular , Humanos , Masculino , Ratones , Transfección
7.
Biochem Biophys Res Commun ; 514(3): 684-690, 2019 06 30.
Artículo en Inglés | MEDLINE | ID: mdl-31078262

RESUMEN

Pulmonary fibrosis is characterized by progressive and irreversible scarring of alveoli, which causes reduction of surface epithelial area and eventually respiratory failure. The precise mechanism of alveolar scarring is poorly understood. In this study, we explored transcriptional signatures of activated fibroblasts in alveolar airspaces by using intratracheal transfer in bleomycin-induced lung fibrosis. Lung fibroblasts transferred into injured alveoli upregulated genes related to translation and metabolism in the first two days, and upregulated genes related to extracellular matrix (ECM) production between day 2 and 7. Upstream analysis of these upregulated genes suggested possible contribution of hypoxia-inducible factors 1a (Hif1a) to fibroblast activation in the first two days, and possible contribution of kruppel-like factor 4 (Klf4) and glioma-associated oncogene (Gli) transcription factors to fibroblast activation in the following profibrotic phase. Fibroblasts purified based on high Acta2 expression after intratracheal transfer were also characterized by ECM production and upstream regulation by Klf4 and Gli proteins. Pharmacological inhibition of Gli proteins by GANT61 in bleomycin-induced lung fibrosis altered the pattern of scarring characterized by dilated airspaces and smaller fibroblast clusters. Activated fibroblasts isolated from GANT61-treated mice showed decreased migration capacity, suggesting that Gli signaling inhibition attenuated fibroblast activation. In conclusion, we revealed transcriptional signatures and possible upstream regulators of activated fibroblasts in injured alveolar airspaces. The altered scar formation by Gli signaling inhibition supports that activated fibroblasts in alveolar airspaces may play a critical role in scar formation.


Asunto(s)
Cicatriz/metabolismo , Cicatriz/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Transducción de Señal , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Cicatriz/genética , Fibroblastos/efectos de los fármacos , Factor 4 Similar a Kruppel , Ratones Endogámicos C57BL , Fibrosis Pulmonar/genética , Piridinas/farmacología , Pirimidinas/farmacología , Factores de Transcripción/metabolismo , Regulación hacia Arriba/genética
8.
Lab Invest ; 98(11): 1500-1511, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-29968852

RESUMEN

The adult liver performs many metabolic functions for maintaining homeostasis. There are several sex differences in liver function and disease pathogenesis. One important function of the liver is drug metabolism, where cytochrome p450s (CYPs) in hepatocytes are the main enzymes involved. The toxicity of various drugs and chemicals differs with sex due to differences in hepatocytic CYP expression. However, the molecular mechanism regulating sex-related differences in drug metabolism remains unknown. In this study, we identified transcriptional regulator B-cell lymphoma 6 (Bcl6) as an important factor in sex-biased differential CYP expression. Microarray analysis of livers derived from liver-specific Bcl6-knockout mice showed that Bcl6 is required for sex-biased CYP expression patterns in the liver. Additionally, quantitative PCR analysis revealed that hepatocytic expression of male-biased genes, such as Cyp2d9, Cyp2u1, Cyp4a12a/12b, and Cyp7b1, in liver-specific Bcl6-knockout male mice significantly decreased to levels similar to those observed in wild-type female mice. Conversely, hepatocytic expression of female-biased genes, such as Cyp2a4/2a5, Cyp2b9, Cyp3a41, and Cyp17a1, significantly increased in liver-specific Bcl6-knockout male mice. Deletion of Bcl6 caused female-like expression of CYPs in male livers. These results suggest that Bcl6 is a key regulator of sex-related differential regulation of drug metabolism. Moreover, serum sex hormone levels and fertility did not change in liver-specific, Bcl6-knockout mice. Hepatocytic Bcl6 regulates sex-related differential CYP expression in the liver without changing the sex of the whole body. Thus, this mouse model is useful for analyzing liver-specific sex-dependent regulation of drug metabolism and pathogenesis.


Asunto(s)
Inactivación Metabólica , Hígado/metabolismo , Modelos Animales , Proteínas Proto-Oncogénicas c-bcl-6/fisiología , Caracteres Sexuales , Animales , Femenino , Hormonas Esteroides Gonadales/sangre , Hepatocitos/metabolismo , Masculino , Ratones Noqueados
9.
Am J Pathol ; 187(12): 2831-2840, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28935572

RESUMEN

Ligamentum flavum (LF) hypertrophy causes lumbar spinal canal stenosis, leading to leg pain and disability in activities of daily living in elderly individuals. Although previous studies have been performed on LF hypertrophy, its pathomechanisms have not been fully elucidated. In this study, we demonstrated that infiltrating macrophages were a causative factor for LF hypertrophy. Induction of macrophages into the mouse LF by applying a microinjury resulted in LF hypertrophy along with collagen accumulation and fibroblasts proliferation at the injured site, which were very similar to the characteristics observed in the severely hypertrophied LF of human. However, we found that macrophage depletion by injecting clodronate-containing liposomes counteracted LF hypertrophy even with microinjury. For identification of fibroblasts in the LF, we used collagen type I α2 linked to green fluorescent protein transgenic mice and selectively isolated green fluorescent protein-positive fibroblasts from the microinjured LF using laser microdissection. A quantitative RT-PCR on laser microdissection samples revealed that the gene expression of collagen markedly increased in the fibroblasts at the injured site with infiltrating macrophages compared with the uninjured location. These results suggested that macrophage infiltration was crucial for LF hypertrophy by stimulating collagen production in fibroblasts, providing better understanding of the pathophysiology of LF hypertrophy.


Asunto(s)
Colágeno/biosíntesis , Fibroblastos/metabolismo , Ligamento Amarillo/patología , Macrófagos/metabolismo , Estenosis Espinal/patología , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Hipertrofia/metabolismo , Hipertrofia/patología , Región Lumbosacra , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Estenosis Espinal/metabolismo
10.
Am J Pathol ; 186(10): 2751-60, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27634343

RESUMEN

The onset and/or growth of breast tumor are controlled, at least in part, by estrogen. Therefore, to prevent the development of breast tumor, estrogen-dependent proliferation of mammary epithelial cells during pregnancy needs to be suppressed once the mammary gland is fully developed to enable lactation. However, the underlying molecular mechanisms remain unknown. Nrk is an X-linked protein serine/threonine kinase in the germinal center kinase family. Herein, we demonstrate a frequent occurrence of breast tumors in homozygous and heterozygous Nrk mutant mice that have experienced pregnancy/parturition. The tumors never developed in the mutant mice without a history of pregnancy/parturition. They exhibited histopathological features of noninvasive tubular adenocarcinoma, and expressed estrogen receptor α. At late gestation when estrogen receptor α expression was significantly reduced in the wild-type mammary gland, grossly normal mammary glands in the pregnant Nrk mutant mice occasionally contained hyperplastic foci continuously expressing the receptor. Consistently, Nrk expression was induced in the wild-type mammary gland at this period of pregnancy. On the other hand, the pregnant Nrk mutant mice also showed elevated blood estrogen levels at late gestation. We suggest that Nrk suppresses the excessive proliferation of mammary epithelial cells during pregnancy, and the impairment of this regulatory system leads to frequent occurrence of breast tumor in Nrk mutant mice.


Asunto(s)
Neoplasias de la Mama/enzimología , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Mamarias Experimentales/enzimología , Proteínas Serina-Treonina Quinasas/genética , Animales , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Regulación hacia Abajo , Células Epiteliales/metabolismo , Células Epiteliales/patología , Receptor alfa de Estrógeno/genética , Receptor alfa de Estrógeno/metabolismo , Estrógenos/sangre , Femenino , Genes Ligados a X/genética , Quinasas del Centro Germinal , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lactancia , Células MCF-7 , Masculino , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Transgénicos , Mutación , Embarazo , Proteínas Serina-Treonina Quinasas/metabolismo
11.
Biochem Biophys Res Commun ; 470(3): 504-509, 2016 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-26806309

RESUMEN

Synovial fibroblasts play crucial roles in inflammation and joint destruction in rheumatoid arthritis (RA). How they accumulate in the RA joints remains unclear. This study was conducted to discern whether cellular influx from the outside of the joints and local proliferation are responsible for synovial fibroblast accumulation in an animal model of RA. We found that synovial fibroblasts were identified as GFP+ cells using collagen type I alpha 2 (Col1a2)-GFP transgenic reporter mice. Then, bone marrow transplantation and parabiosis techniques were utilized to study the cellular influx. Irradiated wild-type mice were transplanted with bone marrow from Col1a2-GFP mice. Col1a2-GFP and wild-type mice were conjoined for parabiosis. The transplanted mice and the parabionts were subjected to collagen antibody-induced arthritis (CAIA). We found no GFP+ cells in the hyperplastic synovial tissues from the transplanted mice with CAIA and from the wild-type parabionts with CAIA. Furthermore, normal and CAIA synovial tissues from Col1a2-GFP mice and from fluorescent ubiquitination-based cell cycle indicator (Fucci) transgenic mice, in which cells in S/G2/M phases of the cell cycle express Azami-Green, were studied for Ki67, a cellular proliferation marker, and vimentin, a fibroblast marker, expression. The percentages of Ki67+/GFP+ and Azami-Green+/vimentin+ cells in the CAIA synovial tissues were higher than those in the untreated synovial tissues (34% vs. 0.40% and 19% vs. 0.26%, respectively). These findings indicate that local fibroblast proliferation but not cellular influx is responsible for the synovial hyperplasia in CAIA. Suppression of proliferation of the local synovial fibroblasts should be a promising treatment for RA.


Asunto(s)
Artritis Reumatoide/patología , Fibroblastos/patología , Membrana Sinovial/patología , Animales , Artritis Reumatoide/inmunología , Movimiento Celular/inmunología , Proliferación Celular , Fibroblastos/inmunología , Hiperplasia/inmunología , Hiperplasia/patología , Masculino , Ratones , Membrana Sinovial/inmunología
12.
Am J Pathol ; 185(11): 2939-48, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26456579

RESUMEN

Pulmonary fibrosis is a devastating disease for which there are few effective therapies. Activated fibroblasts form subepithelial clusters known as fibroblastic foci, which are characterized by excessive collagen deposition. The origin of activated fibroblasts is controversial and needs to be clarified to understand their pathogenicity. Here, using an intratracheal adoptive cell transfer method, we show that resident fibroblasts in alveolar walls have the highest profibrotic potential. By using collagen I(α)2-green fluorescent protein and neural/glial antigen 2-DsRed fluorescent reporter mice, we identified resident fibroblasts and pericytes in the alveolar walls based on surface marker expression and ultrastructural characteristics. In the early phase of bleomycin-induced pulmonary fibrosis, activated fibroblasts migrated into epithelium-denuded alveolar airspaces. Purified resident fibroblasts delivered into injured alveoli by an intratracheal route showed similar activated signatures as activated fibroblasts and formed fibroblastic foci. Neither pericytes nor epithelial cells had the same profibrotic potential. Transferred resident fibroblasts highly up-regulated profibrotic genes including α-smooth muscle actin and were a significant source of collagen deposition. These data provide insights into the cellular mechanisms of fibrogenesis and show intratracheal cell transfer to be a useful tool for exploring novel therapeutic targets against pulmonary fibrosis.


Asunto(s)
Colágeno/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Fibrosis Pulmonar/patología , Animales , Bleomicina/efectos adversos , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Epitelio/metabolismo , Epitelio/patología , Femenino , Fibroblastos/metabolismo , Genes Reporteros , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Fibrosis Pulmonar/metabolismo , Regulación hacia Arriba
13.
Am J Pathol ; 185(11): 2923-38, 2015 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-26456580

RESUMEN

Pulmonary fibrosis (PF) is an intractable disorder with a poor prognosis. Lung macrophages have been reported to regulate both progression and remission of bleomycin-induced diffuse PF. However, it remains unclear how macrophages contribute to silica-induced progressive nodular PF and the associated tissue cell responses in vivo. We found that lack of monocyte-derived macrophages results in the formation of diffuse PF after silica instillation. We found that the proportion and the number of monocyte-derived macrophages were persistently higher in silica-induced progressive PF compared with bleomycin-induced PF. Surprisingly, in Ccr2(-/-) mice, in which monocyte-derived macrophage infiltration is impaired, silica administration induced diffuse PF with loose nodule formation and greater activation of tissue cells. In the diffuse lesions, the distribution of epithelial cells, distribution of myofibroblasts, and architecture of the basement membrane were disrupted. Consistent with the development of diffuse lesions, genes that were differentially expressed in CD45(-) tissue cells from the lung of wild-type and Ccr2(-/-) mice were highly enriched in human diffuse, progressive PF. In gene ontology network analyses, many of these genes were associated with tissue remodeling and included genes not previously associated with PF, such as Mmp14, Thbs2, and Fgfr4. Overall, these results indicate that monocyte-derived macrophages prevent transition from nodular to diffuse silica-induced PF, potentially by regulating tissue cell responses.


Asunto(s)
Macrófagos Alveolares/patología , Fibrosis Pulmonar/patología , Receptores CCR2/metabolismo , Animales , Bleomicina/efectos adversos , Modelos Animales de Enfermedad , Células Epiteliales/patología , Perfilación de la Expresión Génica , Humanos , Hidroxiprolina/análisis , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Monocitos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fibrosis Pulmonar/inducido químicamente , Receptores CCR2/genética , Dióxido de Silicio/efectos adversos
14.
J Immunol ; 193(4): 1636-44, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-25024385

RESUMEN

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Ganglios Linfáticos/citología , Células del Estroma/inmunología , Virus Vaccinia/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/inmunología , Antígeno B7-1/biosíntesis , Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular/inmunología , Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase II/biosíntesis , Receptores de Hialuranos/biosíntesis , Tolerancia Inmunológica , Integrina beta1/biosíntesis , Ganglios Linfáticos/virología , Receptor beta de Linfotoxina/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Parabiosis , Receptores del Factor de Crecimiento Derivado de Plaquetas/biosíntesis , Transducción de Señal/inmunología , Vaccinia/inmunología , Molécula 1 de Adhesión Celular Vascular/biosíntesis
15.
Biochem Biophys Res Commun ; 468(4): 554-60, 2015 Dec 25.
Artículo en Inglés | MEDLINE | ID: mdl-26603932

RESUMEN

Intestinal fibrotic stricture is a major complication of inflammatory bowel disease. Despite its clinical importance, anti-fibrotic therapy has not been implemented. Transforming growth factor-ß (TGF-ß) is considered to be a major factor contributing to tissue fibrosis. We have previously shown that the administration of a small compound, HSc025, which promotes the nuclear translocation of YB-1 as a downstream effector of IFN-γ and antagonizes TGF-ß/Smad signaling, improves fibrosis in several murine tissues. In this study, we evaluated the anti-fibrotic effect of HSc025 on colorectal fibrosis in TNBS-induced murine chronic colitis. Daily oral administration of HSc025 (3, 15 and 75 mg/kg) suppressed collagen production and decreased the severity of colorectal fibrosis in a dose-dependent manner. In addition, the local production of TGF-ß was decreased after HSc025 treatment, whereas that of IL-13 and TNF-α was not affected. HSc025 administration maintained the level of IFN-γ production, even at a late stage when IFN-γ production was lost without the drug treatment. These results demonstrate that HSc025 could be a therapeutic candidate for intestinal fibrosis in inflammatory bowel disease that acts by altering the local production of cytokines, as well as by directly suppressing collagen production.


Asunto(s)
Alcadienos/administración & dosificación , Colitis/tratamiento farmacológico , Colitis/inmunología , Colon/inmunología , Colon/patología , Citocinas/inmunología , Animales , Antiinflamatorios/administración & dosificación , Colitis/inducido químicamente , Colon/efectos de los fármacos , Femenino , Fibrosis , Ratones , Ratones Endogámicos BALB C , Resultado del Tratamiento , Ácido Trinitrobencenosulfónico
16.
Am J Pathol ; 184(5): 1458-67, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24641900

RESUMEN

Alcohol-induced liver fibrosis and eventually cirrhosis is a leading cause of death. Acetaldehyde, the first metabolite of ethanol, up-regulates expression of the human α2(I) collagen gene (COL1A2). Early acetaldehyde-mediated effects involve phosphorylation and nuclear translocation of SMAD3/4-containing complexes that bind to COL1A2 promoter to induce fibrogenesis. We used human and mouse hepatic stellate cells to elucidate the mechanisms whereby acetaldehyde up-regulates COL1A2 by modulating the role of Ski and the expression of SMADs 3, 4, and 7. Acetaldehyde induced up-regulation of COL1A2 by 3.5-fold, with concomitant increases in the mRNA (threefold) and protein (4.2- and 3.5-fold) levels of SMAD3 and SMAD4, respectively. It also caused a 60% decrease in SMAD7 expression. Ski, a member of the Ski/Sno oncogene family, is colocalized in the nucleus with SMAD4. Acetaldehyde induces translocation of Ski and SMAD4 to the cytoplasm, where Ski undergoes proteasomal degradation, as confirmed by the ability of the proteasomal inhibitor lactacystin to blunt up-regulation of acetaldehyde-dependent COL1A2, but not of the nonspecific fibronectin gene (FN1). We conclude that acetaldehyde up-regulates COL1A2 by enhancing expression of the transactivators SMAD3 and SMAD4 while inhibiting the repressor SMAD7, along with promoting Ski translocation from the nucleus to cytoplasm. We speculate that drugs that prevent proteasomal degradation of repressors targeting COL1A2 may have antifibrogenic properties.


Asunto(s)
Acetaldehído/farmacología , Colágeno Tipo II/genética , Proteínas de Unión al ADN/metabolismo , Células Estrelladas Hepáticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Smad/metabolismo , Regulación hacia Arriba/genética , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacología , Animales , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Colágeno Tipo II/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Fibronectinas/genética , Fibronectinas/metabolismo , Genes Reporteros , Células Estrelladas Hepáticas/efectos de los fármacos , Humanos , Ratones , Complejo de la Endopetidasa Proteasomal/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Represoras/metabolismo , Elementos de Respuesta/genética , Proteínas Smad/genética , Proteína smad3/genética , Proteína smad3/metabolismo , Proteína Smad4/genética , Proteína Smad4/metabolismo , Proteína smad7/genética , Proteína smad7/metabolismo , Transcripción Genética/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos
17.
Hepatol Res ; 45(1): 29-37, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-24773763

RESUMEN

The liver comprises two stem/progenitor cell systems: fetal and adult liver stem/progenitor cells. Fetal hepatic progenitor cells, derived from foregut endoderm, differentiate into mature hepatocytes and cholangiocytes during liver development. Adult hepatic progenitor cells contribute to regeneration after severe and chronic liver injuries. However, the characteristics of these somatic hepatic stem/progenitor cells remain unknown. Culture systems that can be used to analyze these cells were recently established and hepatic stem/progenitor cell-specific surface markers including delta-like 1 homolog (DLK), cluster of differentiation (CD) 13, CD133, and LIV2 were identified. Cells purified using antibodies against these markers proliferate for an extended period and differentiate into mature cells both in vitro and in vivo. Methods to force the differentiation of human embryonic stem and induced pluripotent stem (iPS) cells into hepatic progenitor cells have been recently established. We demonstrated that the CD13(+) CD133(+) fraction of human iPS-derived cells contained numerous hepatic progenitor-like cells. These analyses of hepatic stem/progenitor cells derived from somatic tissues and pluripotent stem cells will contribute to the development of new therapies for severe liver diseases.

18.
19.
Am J Pathol ; 183(3): 758-73, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23886891

RESUMEN

Pulmonary fibrosis is characterized by accumulation of activated fibroblasts that produce excessive amounts of extracellular matrix components such as collagen type I. However, the dynamics and activation signatures of fibroblasts during fibrogenesis remain poorly understood, especially in vivo. We examined changes in lung tissue cell populations and in the phenotype of activated fibroblasts after acute injury in a model of bleomycin-induced pulmonary fibrosis. Despite clustering of collagen type I-producing fibroblasts in fibrotic regions, flow cytometry-based quantitative analysis of whole lungs revealed that the number of fibroblasts in the lungs remained constant. At the peak of inflammation, fibroblast proliferation and apoptosis were both increased, suggesting that the clustering was not merely a result of proliferation, but also of fibroblast migration from nearby alveolar walls. Parabiosis experiments demonstrated that fibroblasts were not supplied from the circulation. Comprehensive gene expression analysis of freshly isolated fibroblasts revealed a detailed activation signature associated with fibrogenesis, including changes in genes responsible for migration and extracellular matrix construction. The Spp1 gene, which encodes osteopontin, was highly up-regulated and was an identifying characteristic of activated fibroblasts present at the sites of remodeling. Osteopontin may serve as a useful marker of profibrotic fibroblasts. These results provide insights into the cellular and molecular mechanisms underlying pulmonary fibrosis and provide a foundation for development of specific antifibrotic therapies.


Asunto(s)
Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Animales , Apoptosis , Biomarcadores/metabolismo , Bleomicina , Células de la Médula Ósea/patología , Proliferación Celular , Colágeno Tipo I/metabolismo , Perfilación de la Expresión Génica , Ontología de Genes , Proteínas Fluorescentes Verdes/metabolismo , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Osteopontina/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia Arriba/genética
20.
J Biol Chem ; 286(6): 4485-92, 2011 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-21115500

RESUMEN

Transforming growth factor-ß (TGF-ß) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-ß/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-ß-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.


Asunto(s)
Alcadienos/farmacología , Núcleo Celular/metabolismo , Colágeno/biosíntesis , Regulación de la Expresión Génica/efectos de los fármacos , Cirrosis Hepática Experimental/tratamiento farmacológico , Cirrosis Hepática Experimental/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Animales , Tetracloruro de Carbono/toxicidad , Intoxicación por Tetracloruro de Carbono/tratamiento farmacológico , Intoxicación por Tetracloruro de Carbono/genética , Intoxicación por Tetracloruro de Carbono/metabolismo , Núcleo Celular/genética , Colágeno/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/genética , Humanos , Cirrosis Hepática Experimental/genética , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Proteína smad3/genética , Proteína smad3/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Proteína 1 de Unión a la Caja Y
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