RESUMEN
Cancer-related inflammation impacts significantly on cancer development and progression. From early stages, neutrophils and macrophages are drawn to pre-neoplastic cells in the epidermis, but before directly interacting, they must first breach the underlying extracellular matrix barrier layer that includes the basement membrane. Using several different skin cancer models and a collagen I-GFP transgenic zebrafish line, we have undertaken correlative light and electron microscopy (CLEM) to capture the moments when immune cells traverse the basement membrane. We show evidence both for active proteolytic burrowing and for the opportunistic use of pre-existing weak spots in the matrix layer. We show that these small holes, as well as much larger, cancer cell-generated or wound-triggered gaps in the matrix barrier, provide portals for immune cells to access cancer cells in the epidermis and thus are rate limiting in cancer progression.
Asunto(s)
Membrana Basal/enzimología , Carcinogénesis/inmunología , Matriz Extracelular/metabolismo , Células Caliciformes/citología , Macrófagos/citología , Neutrófilos/citología , Neoplasias Cutáneas/inmunología , Animales , Animales Modificados Genéticamente , Membrana Basal/citología , Membrana Basal/metabolismo , Membrana Basal/ultraestructura , Carcinogénesis/genética , Carcinogénesis/ultraestructura , Proliferación Celular , Colágeno/metabolismo , Modelos Animales de Enfermedad , Epidermis/crecimiento & desarrollo , Epidermis/inmunología , Epidermis/patología , Matriz Extracelular/enzimología , Células Caliciformes/metabolismo , Células Caliciformes/ultraestructura , Macrófagos/enzimología , Macrófagos/inmunología , Macrófagos/ultraestructura , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/metabolismo , Microscopía Electrónica de Transmisión , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/ultraestructura , Proteolisis/efectos de los fármacos , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/ultraestructura , Pez CebraRESUMEN
Ischemia/reperfusion (I/R) injury is a severe inflammatory insult associated with numerous pathologies, such as myocardial infarction, stroke and acute kidney injury. I/R injury is characterized by a rapid influx of activated neutrophils secreting toxic free radical species and degrading enzymes that can irreversibly damage the tissue, thus impairing organ functions. Significant efforts have been invested in identifying therapeutic targets to suppress neutrophil recruitment and activation post-I/R injury. In this context, pharmacological targeting of neutrophil elastase (NE) has shown promising anti-inflammatory efficacy in a number of experimental and clinical settings of I/R injury and is considered a plausible clinical strategy for organ care. However, the mechanisms of action of NE, and hence its inhibitors, in this process are not fully understood. Here we conducted a comprehensive analysis of the impact of NE genetic deletion on neutrophil infiltration in four murine models of I/R injury as induced in the heart, kidneys, intestine and cremaster muscle. In all models, neutrophil migration into ischemic regions was significantly suppressed in NE-/- mice as compared with wild-type controls. Analysis of inflamed cremaster muscle and mesenteric microvessels by intravital and confocal microscopy revealed a selective entrapment of neutrophils within venular walls, most notably at the level of the venular basement membrane (BM) following NE deletion/pharmacological blockade. This effect was associated with the suppression of NE-mediated remodeling of the low matrix protein expressing regions within the venular BM used by transmigrating neutrophils as exit portals. Furthermore, whilst NE deficiency led to reduced neutrophil activation and vascular leakage, levels of monocytes and prohealing M2 macrophages were reduced in tissues of NE-/- mice subjected to I/R. Collectively our results identify a vital and non-redundant role for NE in supporting neutrophil breaching of the venular BM post-I/R injury but also suggest a protective role for NE in promoting tissue repair. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Asunto(s)
Elastasa de Leucocito/fisiología , Neutrófilos/fisiología , Daño por Reperfusión/enzimología , Migración Transendotelial y Transepitelial/fisiología , Remodelación Vascular/fisiología , Animales , Membrana Basal/enzimología , Membrana Basal/patología , Membrana Basal/fisiopatología , Modelos Animales de Enfermedad , Eliminación de Gen , Riñón/irrigación sanguínea , Riñón/patología , Elastasa de Leucocito/deficiencia , Elastasa de Leucocito/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Daño por Reperfusión Miocárdica/enzimología , Daño por Reperfusión Miocárdica/patología , Daño por Reperfusión Miocárdica/fisiopatología , Infiltración Neutrófila/fisiología , Neutrófilos/enzimología , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Vénulas/enzimología , Vénulas/patología , Vénulas/fisiopatologíaRESUMEN
RATIONALE: Giant cell arteritis (GCA)-a primary vasculitis of medium and large arteries-is associated with vessel wall damage, elastic membrane fragmentation, and vascular remodeling. Proteinases are believed to contribute to pathogenesis by degrading extracellular matrix and causing tissue injury. OBJECTIVE: The MMP (matrix metalloproteinase)-9-a type IV collagenase-is produced in the vasculitic lesions of GCA. It is unknown which pathogenic processes are MMP-9 dependent. METHODS AND RESULTS: The tissue transcriptome of GCA-affected temporal arteries contained high amounts of MMP-9 transcripts, and immunostaining for pro-MMP-9 localized the enzyme to wall-infiltrating macrophages. MMP-2 and MMP-9 transcripts were also abundant in monocytes and monocyte-derived macrophages from patients with GCA. Patient-derived monocytes outperformed healthy monocytes in passing through engineered basement membranes. GCA CD (cluster of differentiation) 4+ T cells required MMP-9-producing monocytes to penetrate through matrix built from type IV collagen. In vivo functions of MMP-9 were tested in a human artery-SCID (severe combined immunodeficiency) chimera model by blocking enzyme activity with a highly specific monoclonal antibody or by injecting rMMP-9 (recombinant MMP-9). Inhibiting MMP-9 activity profoundly suppressed vascular injury, decreased the density of inflammatory infiltrates ( P<0.001), reduced intramural neoangiogenesis ( P<0.001), and prevented intimal layer hyperplasia ( P<0.001). rMMP-9 amplified all domains of vasculitic activity, promoted assembly of T-cell infiltrates ( P<0.05), intensified formation of new microvessels ( P<0.001), and worsened intimal thickening ( P<0.001). Systemic delivery of N-acetyl-proline-glycine-proline-a matrikine produced by MMP-9-mediated gelatinolysis-had limited vasculitogenic effects. CONCLUSIONS: In large vessel vasculitis, MMP-9 controls the access of monocytes and T cells to the vascular wall. T cells depend on MMP-9-producing monocytes to pass through collagen IV-containing basement membrane. Invasion of vasculitogenic T cells and monocytes, formation of neoangiogenic networks, and neointimal growth all require the enzymatic activity of MMP-9, identifying this protease as a potential therapeutic target to restore the immunoprivilege of the arterial wall in large vessel vasculitis.
Asunto(s)
Arteria Axilar/enzimología , Linfocitos T CD4-Positivos/enzimología , Movimiento Celular , Arteritis de Células Gigantes/enzimología , Metaloproteinasa 9 de la Matriz/metabolismo , Monocitos/enzimología , Arterias Temporales/enzimología , Remodelación Vascular , Anciano , Anciano de 80 o más Años , Animales , Antiinflamatorios/farmacología , Anticuerpos Monoclonales/farmacología , Arteria Axilar/efectos de los fármacos , Arteria Axilar/inmunología , Arteria Axilar/patología , Membrana Basal/enzimología , Membrana Basal/patología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Estudios de Casos y Controles , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Femenino , Arteritis de Células Gigantes/inmunología , Arteritis de Células Gigantes/patología , Arteritis de Células Gigantes/prevención & control , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Monocitos/efectos de los fármacos , Monocitos/inmunología , Neointima , Neovascularización Patológica , Transducción de Señal , Arterias Temporales/efectos de los fármacos , Arterias Temporales/inmunología , Arterias Temporales/patología , Remodelación Vascular/efectos de los fármacosRESUMEN
Purpose: Basement membrane degradation and macrophage aggregation at the optic fissure margins are crucial to optic fissure closure during normal murine eye development. Basement membrane degradation is also an essential step in cancer development, and matrix metalloproteinases (MMPs) play an important role. In this study, we investigated MMP alteration at the degrading basement membrane of optic fissure margins in mice and attempted to clarify the relationship between MMP activity and macrophages. Methods: Serial coronal frozen sections of eyes from BALB/c fetuses were prepared and gelatinase activity was examined using in situ zymography techniques. The frozen sections were immunohistochemically stained with anti-F4/80, anti-MMP 2, and anti-MMP 9 antibodies. Serial coronal paraffin sections were also immunohistochemically stained with anti-type IV collagen and anti-F4/80, and basement membrane disintegration and macrophage aggregation at the optic fissure margins were examined. Results: The basement membrane of optic fissure margins was rapidly degraded during gestational days (GDs) 12.0 to 12.5. Meanwhile, gelatinase activity at F4/80-positive macrophages significantly increased during GDs 11.5 to 12.0 and declined thereafter; some of those were also positive for MMP2. The number of macrophages was also increased and decreased at nearly the same time. Conclusions: Intramacrophage MMPs may be responsible for basement membrane degradation at the optic fissure margins during normal eye development in mice.
Asunto(s)
Membrana Basal/embriología , Membrana Basal/enzimología , Ojo/embriología , Macrófagos/enzimología , Metaloproteinasas de la Matriz/metabolismo , Organogénesis , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Fagocitos/fisiologíaRESUMEN
OBJECTIVE: Oral lichen planus (OLP) is a chronic, inflammatory disorder that affects the oral mucous membrane. During an inflammatory response, several chemokines and cytokines are released by the cells of the immune system. Activation of MMPs, along with mast cell-derived chymase and tryptase, degrades the basement membrane structural proteins, resulting in basement membrane breaks. AIM: To investigate the association between the COX-2 expressions, presence of intact or degranulating mast cells within the connective tissue and the extent of basement membrane discontinuity in OLP cases. METHODS: This study included a total of 50 formalin-fixed paraffin-embedded specimens (FFPE) of histologically confirmed cases of idiopathic oral lichen planus. A retrospective cross-sectional analysis was carried out by immunohistochemistry to study the epithelial expression of COX-2 and by the use of special stains such as toluidine blue and periodic acid-Schiff (PAS) to study the mast cell count and basement membrane changes in the oral mucosal tissue, respectively. RESULTS: There was a significant (P = 0.03) association between the COX-2 expressions and mast cell count. As the intensity of COX-2 expression increased from mild to moderate or severe, the number of mast cell count almost doubled. CONCLUSION: Interaction between upregulation of COX-2, mast cell and basement membrane sets a vicious cycle which relates to the chronic nature of the disease. Inhibitors of COX-2 may reduce the inflammatory process preceding the immune dysregulation in OLP.
Asunto(s)
Ciclooxigenasa 2/metabolismo , Liquen Plano Oral/etiología , Adulto , Anciano , Anciano de 80 o más Años , Membrana Basal/enzimología , Membrana Basal/patología , Colorantes , Estudios Transversales , Femenino , Humanos , Liquen Plano Oral/enzimología , Liquen Plano Oral/patología , Masculino , Mastocitos/patología , Persona de Mediana Edad , Mucosa Bucal/enzimología , Mucosa Bucal/patología , Estudios Retrospectivos , Adulto JovenRESUMEN
Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.
Asunto(s)
Membrana Basal/enzimología , Membrana Basal/patología , Epidermólisis Ampollosa Distrófica/enzimología , Epidermólisis Ampollosa Distrófica/patología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/análisis , Membrana Basal/metabolismo , Trasplante de Médula Ósea , Células Cultivadas , Colágeno Tipo VII/análisis , Colágeno Tipo VII/metabolismo , Epidermólisis Ampollosa Distrófica/metabolismo , Epidermólisis Ampollosa Distrófica/terapia , Humanos , Queratinocitos/enzimología , Queratinocitos/metabolismo , Queratinocitos/patología , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Mapas de Interacción de Proteínas , Piel/enzimología , Piel/metabolismo , Piel/patologíaRESUMEN
Infection of the lepidopteran insect Trichoplusia ni with the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV) by the oral route stimulates activation of host matrix metalloproteases (MMP) and effector caspases, a process dependent on expression of the viral fibroblast growth factor (vFGF). This pathway leads to tracheal cell basal lamina remodelling, enabling virus escape from the primary site of infection, the midgut epithelium, and establishment of efficient systemic infection. In this study, we asked whether the MMP-caspase pathway was also activated following infection by intrahaemocoelic injection. We found that intrahaemocoelic infection did not lead to any observable tracheal cell or midgut epithelium basal lamina remodelling. MMP and caspase activities were not significantly stimulated. We conclude that the main role of the AcMNPV vFGF is in facilitating virus midgut escape.
Asunto(s)
Mariposas Nocturnas/virología , Nucleopoliedrovirus/fisiología , Animales , Membrana Basal/enzimología , Caspasas/genética , Caspasas/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Interacciones Huésped-Patógeno , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Metaloproteinasas de la Matriz/genética , Metaloproteinasas de la Matriz/metabolismo , Mariposas Nocturnas/enzimología , Mariposas Nocturnas/genética , Nucleopoliedrovirus/genética , Tráquea/enzimología , Tráquea/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Tumour vasculogenesis can occur by a process referred to as vasculogenic mimicry, whereby the vascular structures are derived from the tumour itself. These tumours are highly aggressive and do not respond well to anti-angiogenic therapy. Here, we use the well characterised ECV304 cell line, now known as the bladder cancer epithelial cell line T24/83 which shows both epithelial and endothelial characteristics, as a model of in vitro vasculogenic mimicry. Using optimised ratios of co-cultures of ECV304 and C378 human fibroblasts, tubular structures were identifiable after 8 days. The tubular structures showed high levels of TG2 antigen and TG in situ activity. Tubular structures and in situ activity could be blocked either by site-directed irreversible inhibitors of TG2 or by silencing the ECV304 TG2 by antisense transfection. In situ activity for TG2 showed co-localisation with both fibronectin and collagen IV. Deposition of these proteins into the extracellular matrix could be reduced by inclusion of non-cell penetrating TG inhibitors when analysed by Western blotting suggesting that the contribution of TG2 to tube formation is extracellular. Incubation of ECV304 cells with these same irreversible inhibitors reduced cell migration which paralleled a loss in focal adhesion assembly, actin cytoskeleton formation and fibronectin deposition. TG2 appears essential for ECV304 tube formation, thus representing a potential novel therapeutic target in the inhibition of vasculogenic mimicry.
Asunto(s)
Neovascularización Patológica/enzimología , Transglutaminasas/metabolismo , Citoesqueleto de Actina/metabolismo , Membrana Basal/enzimología , Vasos Sanguíneos/enzimología , Vasos Sanguíneos/patología , Cadaverina/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Movimiento Celular , Técnicas de Cocultivo , Colágeno Tipo IV/metabolismo , Matriz Extracelular/metabolismo , Fluoresceína-5-Isotiocianato/metabolismo , Colorantes Fluorescentes/metabolismo , Adhesiones Focales/metabolismo , Proteínas de Unión al GTP , Técnicas de Silenciamiento del Gen , Humanos , Microscopía Fluorescente , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transporte de Proteínas , Transglutaminasas/genética , Transglutaminasas/fisiologíaRESUMEN
The aim of this study was to compare the expressions of basal lamina (BL) collagen IV α chains and matrix metalloproteinases (MMP)-2 and MMP-9 in oral dysplasia (OED) and invasive carcinoma. Ten cases each of OEDs, carcinomas-in situ and oral squamous cell carcinomas (OSCCs) were examined by immunohistochemistry. Another 5 cases, each of normal and hyperplastic oral mucosa, served as controls. Results showed that α1(IV)/α2(IV) and α5(IV)/α6(IV) chains were intact in BLs of control and OEDs. In BLs of carcinoma-in situ, α1(IV)/α2(IV) chains preceded α5(IV)/α6(IV) chains in showing incipient signs of disruption. OSCCs exhibited varying degrees of collagen α(IV) chain degradation. MMP-2 and MMP-9 were absent in controls and OED, but weakly detectable in carcinoma-in situ. In OSCC, these proteolytic enzymes were expressed in areas corresponding to collagen α(IV) chain loss. Enzymatic activity was enhanced in higher grade OSCC, and along the tumor advancing front. Overall the present findings suggest that loss of BL collagen α(IV) chains coincided with gain of expression for MMP-2 and MMP-9, and that these protein alterations are crucial events during progression from OED to OSCC.
Asunto(s)
Carcinoma/enzimología , Colágeno Tipo IV/química , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Neoplasias de la Boca/enzimología , Adulto , Anciano , Anciano de 80 o más Años , Membrana Basal/enzimología , Carcinoma in Situ , Estudios de Casos y Controles , Colágeno Tipo IV/metabolismo , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica/métodos , Masculino , Persona de Mediana Edad , Mucosa Bucal/patología , Invasividad NeoplásicaRESUMEN
Matrix metalloproteinase 9 (MMP-9) is a member of a family of zinc-dependent endopeptidases capable of degrading extracellular matrix (ECM) proteins. A single bout of exercise increases levels of activated MMP-9 in skeletal muscle and in the circulation. However, whether the exercise-induced activation of MMP-9 is associated with ECM remodeling and the cellular source behind MMP-9 in the circulation is not known. In the present study ten healthy male subjects performed a single cycle exercise bout and arterial and venous femoral blood was collected. To test if exercise induces basal lamina degradation and if circulating levels of MMP-9 is related to a release from the exercising muscle, arteriovenous differences of collagen IV and MMP-9 were measured by ELISA and zymography, respectively. Furthermore, markers of neutrophil degranulation elastase and neutrophil gelatinase-associated lipocalin (NGAL) were measured by ELISA. Plasma levels of collagen IV increased during the exercise bout and an increased arteriovenous difference of collagen IV was noted at 27 min of exercise. Plasma levels of MMP-9 were increased at both 27 and 57 min of exercise but no arteriovenous difference was noted. No changes over time were detected for elastase and NGAL. The observed release of collagen IV from the exercising muscle indicate basal lamina turnover following a single bout of exercise. No detectable release of MMP-9 was observed, suggesting that the increase in plasma MMP-9 could come from a source other than the skeletal muscle.
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Ejercicio Físico , Metaloproteinasa 9 de la Matriz/sangre , Proteínas de Fase Aguda , Adolescente , Adulto , Membrana Basal/enzimología , Membrana Basal/metabolismo , Colágeno Tipo IV/sangre , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Matriz Extracelular/metabolismo , Humanos , Elastasa de Leucocito/sangre , Lipocalina 2 , Lipocalinas/sangre , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas/sangreRESUMEN
Genetic evidence indicates that the major gelatinases MMP-2 and MMP-9 are involved in mammalian craniofacial development. Since these matrix metalloproteinases are secreted as proenzymes that require activation, their tissue distribution does not necessarily reflect the sites of enzymatic activity. Information regarding the spatial and temporal expression of gelatinolytic activity in the head of the mammalian embryo is sparse. Sensitive in situ zymography with dye-quenched gelatin (DQ-gelatin) has been introduced recently; gelatinolytic activity results in a local increase in fluorescence. Using frontal sections of wild-type mouse embryo heads from embryonic day 14.5-15.5, we optimized and validated a simple double-labeling in situ technique for combining DQ-gelatin zymography with immunofluorescence staining. MMP inhibitors were tested to confirm the specificity of the reaction in situ, and results were compared to standard SDS-gel zymography of tissue extracts. Double-labeling was used to show the spatial relationship in situ between gelatinolytic activity and immunostaining for gelatinases MMP-2 and MMP-9, collagenase 3 (MMP-13) and MT1-MMP (MMP-14), a major activator of pro-gelatinases. Strong gelatinolytic activity, which partially overlapped with MMP proteins, was confirmed for Meckel's cartilage and developing mandibular bone. In addition, we combined in situ zymography with immunostaining for extracellular matrix proteins that are potential gelatinase substrates. Interestingly, gelatinolytic activity colocalized precisely with laminin-positive basement membranes at specific sites around growing epithelia in the developing mouse head, such as the ducts of salivary glands or the epithelial fold between tongue and lower jaw region. Thus, this sensitive method allows to associate, with high spatial resolution, gelatinolytic activity with epithelial morphogenesis in the embryo.
Asunto(s)
Membrana Basal/embriología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Coloración y Etiquetado/métodos , Animales , Membrana Basal/enzimología , Membrana Basal/ultraestructura , Investigaciones con Embriones , Femenino , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Ratones Endogámicos C57BL , Sensibilidad y EspecificidadRESUMEN
Colitis-associated colorectal cancers are an etiologically distinct subgroup of colon cancers that occur in individuals suffering from inflammatory bowel disease and arise as a consequence of persistent exposure of hyperproliferative epithelial stem cells to an inflammatory microenvironment. An intrinsic defect in the intestinal epithelial barrier has been proposed to be one of several factors that contribute to the inappropriate immune response to the commensal microbiota that underlies inflammatory bowel disease. Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that strengthens the intestinal epithelial barrier by promoting tight junction formation. Here, we show that intestinal epithelial-specific ablation of St14 in mice causes formation of colon adenocarcinoma with very early onset and high penetrance. Neoplastic progression is preceded by a chronic inflammation of the colon that resembles human inflammatory bowel disease and is promoted by the commensal microbiota. This study demonstrates that inflammation-associated colon carcinogenesis can be initiated and promoted solely by an intrinsic intestinal permeability barrier perturbation, establishes St14 as a critical tumor-suppressor gene in the mouse gastrointestinal tract and adds matriptase to the expanding list of pericellular proteases with tumor-suppressive functions.
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Adenocarcinoma/enzimología , Colitis/enzimología , Neoplasias del Colon/enzimología , Genes Supresores de Tumor , Serina Endopeptidasas/genética , Adenocarcinoma/patología , Adenoma/enzimología , Animales , Antibacterianos/farmacología , Membrana Basal/enzimología , Membrana Basal/metabolismo , Proliferación Celular , Transformación Celular Neoplásica/metabolismo , Colitis/microbiología , Colitis/patología , Colon/patología , Neoplasias del Colon/patología , Epitelio/enzimología , Epitelio/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/enzimología , Enfermedades Inflamatorias del Intestino/microbiología , Enfermedades Inflamatorias del Intestino/patología , Absorción Intestinal , Proteínas de la Membrana , Metagenoma/efectos de los fármacos , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Invasividad Neoplásica , Lesiones Precancerosas , Serina Endopeptidasas/metabolismo , Transducción de Señal , beta Catenina/metabolismoRESUMEN
AIM: Recent advances in quantitative methods and sensitive imaging techniques of trace elements provide opportunities to uncover and explain their biological roles. In particular, the distribution of selenium in tissues and cells under both physiological and pathological conditions remains unknown. In this work, we applied high-resolution synchrotron X-ray fluorescence microscopy (XFM) to map selenium distribution in mouse liver and kidney. RESULTS: Liver showed a uniform selenium distribution that was dependent on selenocysteine tRNA([Ser]Sec) and dietary selenium. In contrast, kidney selenium had both uniformly distributed and highly localized components, the latter visualized as thin circular structures surrounding proximal tubules. Other parts of the kidney, such as glomeruli and distal tubules, only manifested the uniformly distributed selenium pattern that co-localized with sulfur. We found that proximal tubule selenium localized to the basement membrane. It was preserved in Selenoprotein P knockout mice, but was completely eliminated in glutathione peroxidase 3 (GPx3) knockout mice, indicating that this selenium represented GPx3. We further imaged kidneys of another model organism, the naked mole rat, which showed a diminished uniformly distributed selenium pool, but preserved the circular proximal tubule signal. INNOVATION: We applied XFM to image selenium in mammalian tissues and identified a highly localized pool of this trace element at the basement membrane of kidneys that was associated with GPx3. CONCLUSION: XFM allowed us to define and explain the tissue topography of selenium in mammalian kidneys at submicron resolution.
Asunto(s)
Glutatión Peroxidasa/genética , Túbulos Renales Proximales/metabolismo , Selenio/metabolismo , Animales , Membrana Basal/enzimología , Membrana Basal/metabolismo , Microanálisis por Sonda Electrónica , Técnicas de Inactivación de Genes , Glutatión Peroxidasa/metabolismo , Riñón/metabolismo , Túbulos Renales Proximales/enzimología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas Topo , Aminoacil-ARN de Transferencia/genética , Selenoproteína P/genética , Espectrometría por Rayos XRESUMEN
OBJECTIVE: To observe the effect of salviandic acid B (SA-B) on MMP-2/9 and TIMP-2 of fibrotic cardiac tissues in rats and explore the action mechanism of SA-B anti-fibrosis of heart. METHOD: Ventricular remodeling model was induced by abdominal aortic banding (AAB) in rats. Rats were randomly divided into 6 groups: normal, model, SA-B high, SA-B middle, SA-B low and captopril control group. Histological changes of heart were observed with hemotoxylin and eosin (H&E) staining and Sirius red staining. Hydroxyproline (Hyp) content in heart tissue was measured by hydrolysis method. Expression of heart tissue collagen NIV, MMP-2/9 and TIMP-2 were analyzed with Western blot The activities of heart tissue MMP-2 were determined with gelatin zymography substrate degradation method. RESULT: SA-B treated groups had lower heart inflammation and lower heart Hyp content; decreased Collagen deposit and alleviated cardiac fibrosis. SA-B treated groups obviously decreased the expression of Collagen IV, MMP-2/9 and TIMP-2. The activity of MMP-2 was decreased in treated SA-B treated groups. CONCLUSION: The mechanism of SA-B action against cardiac fibrosis may be related to down-regulating the expression of TIMP -2 and the activity of MMP-2/9, thus protect the normal basal membrane.
Asunto(s)
Membrana Basal/enzimología , Cardiomegalia/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Animales , Membrana Basal/efectos de los fármacos , Cardiomegalia/enzimología , Cardiomegalia/genética , Modelos Animales de Enfermedad , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Ratas , Ratas Sprague-Dawley , Inhibidor Tisular de Metaloproteinasa-1/genéticaRESUMEN
Mouse mast cell protease-4 (mMCP-4) has been linked to autoimmune and inflammatory diseases, although the exact mechanisms underlying its role in these pathological conditions remain unclear. Here, we have found that mMCP-4 is critical in a mouse model of the autoimmune skin blistering disease bullous pemphigoid (BP). Mice lacking mMCP-4 were resistant to experimental BP. Complement activation, mast cell (MC) degranulation, and the early phase of neutrophil (PMN) recruitment occurred comparably in mMCP-4(-/-) and WT mice. However, without mMCP-4, activation of matrix metalloproteinase (MMP)-9 was impaired in cultured mMCP-4(-/-) MCs and in the skin of pathogenic IgG-injected mMCP-4(-/-) mice. MMP-9 activation was not fully restored by local reconstitution with WT or mMCP-4(-/-) PMNs. Local reconstitution with mMCP-4(+/+) MCs, but not with mMCP-4(-/-) MCs, restored blistering, MMP-9 activation, and PMN recruitment in mMCP-4(-/-) mice. mMCP-4 also degraded the hemidesmosomal transmembrane protein BP180 both in the skin and in vitro. These results demonstrate that mMCP-4 plays two different roles in the pathogenesis of experimental BP, by both activating MMP-9 and by cleaving BP180, leading to injury of the hemidesmosomes and extracellular matrix of the basement membrane zone.
Asunto(s)
Mastocitos/enzimología , Penfigoide Ampolloso/enzimología , Serina Endopeptidasas/metabolismo , Piel/enzimología , Animales , Autoantígenos/genética , Autoantígenos/metabolismo , Membrana Basal/enzimología , Membrana Basal/patología , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/fisiología , Modelos Animales de Enfermedad , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Hemidesmosomas/enzimología , Hemidesmosomas/genética , Hemidesmosomas/patología , Humanos , Inmunoglobulina G/toxicidad , Mastocitos/patología , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/enzimología , Neutrófilos/patología , Colágenos no Fibrilares/genética , Colágenos no Fibrilares/metabolismo , Penfigoide Ampolloso/inducido químicamente , Penfigoide Ampolloso/genética , Penfigoide Ampolloso/patología , Serina Endopeptidasas/genética , Piel/patología , Colágeno Tipo XVIIRESUMEN
OBJECTIVE: The aim of this study was to evaluate the immunohistochemical expression of collagen IV, matrix metalloproteinase (MMP) 9 and tissue inhibitor of MMP (TIMP) 2 in dentigerous cysts (DCs), radicular cysts (RCs), keratocystic odontogenic tumors (KOTs), and ameloblastomas. STUDY DESIGN: Twenty cases of DCs, 20 RCs, 20 KOTs, and 20 ameloblastomas were selected and analyzed by immunohistochemistry. RESULTS: Most DCs and RCs showed continuous and >50% staining for collagen IV in the basement membrane of the epithelium, whereas predominantly discontinuous thin and ≤ 50% staining was observed in KOTs and ameloblastomas, with a significant difference in staining percentage (P < .001). MMP-9 was diffusely distributed and localized in both epithelial and mesenchymal cells of all of the lesions analyzed. The staining percentage was higher in the epithelium (P = .058) and mesenchyme (P = .005) of KOTs and ameloblastomas. Moreover, the distribution pattern, location, and percentage of expression of TIMP-2 were similar in the lesions studied, except for ameloblastoma, with a significant difference in staining percentage (P < .001). CONCLUSION: These results demonstrate that the interaction between collagen IV, MMP-9, and TIMP-2 is an important factor for the establishment of differences in the biologic behavior of the odontogenic cysts and tumors studied.
Asunto(s)
Ameloblastoma/patología , Colágeno Tipo IV/análisis , Metaloproteinasa 9 de la Matriz/análisis , Quistes Odontogénicos/patología , Inhibidores de Proteasas/análisis , Inhibidor Tisular de Metaloproteinasa-2/análisis , Ameloblastoma/enzimología , Membrana Basal/enzimología , Membrana Basal/patología , Colorantes , Tejido Conectivo/enzimología , Tejido Conectivo/patología , Quiste Dentígero/enzimología , Quiste Dentígero/patología , Células Endoteliales/enzimología , Células Endoteliales/patología , Células Epiteliales/enzimología , Células Epiteliales/patología , Epitelio/enzimología , Epitelio/patología , Fibroblastos/enzimología , Fibroblastos/patología , Humanos , Inmunohistoquímica , Mesodermo/enzimología , Mesodermo/patología , Quistes Odontogénicos/enzimología , Quiste Radicular/enzimología , Quiste Radicular/patologíaRESUMEN
Glutathione peroxidase-3 (Gpx3), the extracellular glutathione peroxidase synthesized largely in the kidney, binds to basement membranes of renal cortical epithelial cells. The present study assessed extrarenal expression of Gpx3 using RT-PCR and presence of Gpx3 protein using immunocytochemistry. Gpx3 expression was higher in kidney and epididymis than in other tissues. Gpx3 bound to basement membranes of epithelial cells in the gastrointestinal tract, the efferent ducts connecting the seminiferous tubules with the epididymis, the bronchi, and type II pneumocytes. It was not detected on the basement membrane of type I pneumocytes. Gpx3 was also present in the lumen of the epididymis. Transplantation of Gpx3(+/+) kidneys into Gpx3(-/-) mice led to Gpx3 binding to the same basement membranes to which it bound in Gpx3(+/+) mice but not to its presence in the epididymal lumen. These results show that Gpx3 from the blood binds to basement membranes of specific epithelial cells and indicate that the cells modify their basement membranes to cause the binding. They further indicate that at least two Gpx3 compartments exist in the organism. In one compartment, kidney supplies Gpx3 through the blood to specific basement membranes in a number of tissues. In the other compartment, the epididymis provides Gpx3 to its own lumen. Tissues other than kidney and epididymis express Gpx3 at lower levels and may supply Gpx3 to other compartments.
Asunto(s)
Membrana Basal/enzimología , Tracto Gastrointestinal/enzimología , Glutatión Peroxidasa/metabolismo , Riñón/enzimología , Células Epiteliales Alveolares/enzimología , Animales , Bronquios/enzimología , Epidídimo/enzimología , Epidídimo/metabolismo , Células Epiteliales/enzimología , Glutatión Peroxidasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Túbulos Seminíferos/enzimologíaRESUMEN
Recently, we reported that heparanase plays important roles in barrier-disrupted skin, leading to increased interaction of growth factors between epidermis and dermis and facilitating various cutaneous changes, including epidermal hyperplasia and wrinkle formation. However, the role of heparanase in sun-exposed skin remains unknown. Here, we show that heparanase in human keratinocytes is activated by ultraviolet B (UVB) exposure and that heparan sulfate of perlecan is markedly degraded in UVB-irradiated human skin. The degradation of heparan sulfate resulted in a marked reduction of binding activity of the basement membrane for vascular endothelial growth factor, fibroblast growth factor-2 and -7 at the dermal-epidermal junction. Degradation of heparan sulfate was observed not only in acutely UVB-irradiated skin, but also in skin chronically exposed to sun. Interestingly, heparan sulfate was found to be degraded in sun-exposed skin, but not in sun-protected skin. These findings suggest that chronic UVB exposure activates heparanase, leading to degradation of heparan sulfate in the basement membrane and increased growth factor interaction between epidermis and dermis. These changes may facilitate photo-aging.
Asunto(s)
Membrana Basal/enzimología , Membrana Basal/efectos de la radiación , Dermis/efectos de la radiación , Epidermis/efectos de la radiación , Glucuronidasa/metabolismo , Heparitina Sulfato/metabolismo , Membrana Basal/patología , Western Blotting , Dermis/patología , Activación Enzimática/efectos de la radiación , Epidermis/patología , Femenino , Humanos , Masculino , Envejecimiento de la Piel/efectos de la radiación , Rayos UltravioletaRESUMEN
BACKGROUND AND AIMS: Collagen type IV and hyaluronic acid (HA) are the major components of basement membrane and extracellular matrix, respectively. Cathepsin D is an aspartyl lysosomal protease involved in the degradation of the basement membrane and extracellular matrix. The aim of this study is to investigate the clinical significance of collagen type IV and hyaluronic acid in gastric juice and serum in diagnosis of gastric cancer and the degrading effect of cathepsin D on collagen type IV and HA. METHODS: Fifty gastric cancer patients were enrolled in our study compared with 41 patients with precancerous lesion and 30 control subjects. Collagen type IV and HA in gastric juice and serum were analyzed by radioimmunoassay. Expression of cathepsin D and collagen type IV in tissue were analyzed by immunohistochemical staining with monoclonal antibodies. RESULTS: The contents of collagen type IV and HA in gastric juice and HA in serum were significantly higher in patients with gastric cancer than those in patients with precancerous lesion and control group (p < 0.05, p < 0.0001). Gastric cancer patients with lymph node metastasis had a higher level of collagen type IV and HA in gastric juice than those in patients without metastasis (p = 0.049, p = 0.043). The expression of cathepsin D had significantly increased in patients with gastric cancer compared to the control group (p < 0.0001). The continuous expression of collagen type IV in basement membrane in gastric cancer group was lower than that in the precancerous lesion group and control group (p < 0.0001). CONCLUSIONS: The analysis of collagen type IV and HA in gastric juice and serum may provide a simple aid in diagnosing gastric cancer and evaluating whether metastasis is occurring or not.