RESUMEN
Background Aortic dissection (AD) is one of the most life-threatening cardiovascular diseases that exhibit high genetic heterogeneity. However, it is unclear whether variants within the COL5A1 gene can cause AD. Therefore, we intend to determine whether COL5A1 is a causative gene of AD. Methods and Results We performed targeted sequencing in 702 patients with unrelated sporadic AD and 163 matched healthy controls using a predesigned panel with 152 vessel matrix-related genes. As a result, we identified that 11 variants in COL5A1 caused AD in 11 out of the 702 patients with AD. Furthermore, Col5a1 knockout (Col5a1+/-) rats were generated through the CRISPR/Cas9 system. Although there was no spontaneous AD, electron microscopy revealed a fracture of elastic fibers and disarray of collagenous fibers in 6-week-old Col5a1+/- rats, but not in WT rats (93.3% versus 0.0%, P<0.001). Three-week-old rats were used to induce the AD phenotype with ß-aminopropionitrile monofumarate for 4 weeks followed by angiotensin II for 72 hours. The ß-aminopropionitrile monofumarate and angiotensin II-treated rat model confirmed that Col5a1+/- rats had considerably higher AD incidence than WT rats. Subsequent mechanism analyses demonstrated that the transforming growth factor-ß-signaling pathway was significantly activated in Col5a1+/- rats. Conclusions Our findings, for the first time, revealed a relationship between variants in COL5A1 and AD via targeted sequencing in 1.57% patients with sporadic aortic dissection. The Col5a1 knockout rats exhibited AD after an intervention, indicating that COL5A1 is a causative gene of AD. Activation of the transforming growth factor-ß-signaling pathway may be implicated in the pathogenesis of this kind of AD.
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Aneurisma de la Aorta Torácica/genética , Disección Aórtica/genética , Colágeno Tipo V/genética , Factor de Crecimiento Transformador beta/genética , Disección Aórtica/diagnóstico , Disección Aórtica/metabolismo , Animales , Aorta Torácica/ultraestructura , Aneurisma de la Aorta Torácica/diagnóstico , Aneurisma de la Aorta Torácica/metabolismo , Western Blotting , Colágeno Tipo V/biosíntesis , ADN/genética , Modelos Animales de Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Microscopía Electrónica , Persona de Mediana Edad , Fenotipo , Ratas , Ratas Transgénicas , Estudios Retrospectivos , Transducción de Señal , Tomografía Computarizada por Rayos X , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
The antitumor properties of cruciferous vegetables are mainly due to their high content of isothiocyanates, and sulforaphane (SFA) is the most well-known compound. The aim of this study was to determine the mechanism of SFA inhibiting gastric cancer (GC) progression. After verifying SFA suppressing GC growth in vivo, we utilized the GSE79973 and GSE118916 datasets to identify the GC development signatures that overlap with the RNA-seq analysis in SFA-treated AGS cells. GSEA of the RNA-seq data indicated that SFA regulation of GC progression was related to extracellular matrix and collagens; thus, we identified COL3A1 and COL5A1 as the targets of SFA, which functioned as oncogenes. We found positive correlations between COL3A1 and COL5A1 expression in GC cells, and confirmed that miR-29a-3p is the common regulator of their expression. RNA immunoprecipitation assays based on Ago2, Dicer, and exportin-5 showed that SFA could promote mature miR-29a-3p generation. We also proved that SFA inactivated the Wnt/ß-catenin pathway in GC cells in a miR-29a-3p-dependent manner. Overall, SFA boosts miR-29a-3p maturation to downregulate COL3A1 and COL5A1 and inactivate the Wnt/ ß -catenin pathway to suppress GC progression.
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Anticarcinógenos/uso terapéutico , Colágeno Tipo III/biosíntesis , Colágeno Tipo V/biosíntesis , Isotiocianatos/uso terapéutico , MicroARNs/biosíntesis , Neoplasias Gástricas/metabolismo , Sulfóxidos/uso terapéutico , Animales , Anticarcinógenos/farmacología , Línea Celular Tumoral , Colágeno Tipo III/antagonistas & inhibidores , Colágeno Tipo III/genética , Colágeno Tipo V/antagonistas & inhibidores , Colágeno Tipo V/genética , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Isotiocianatos/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/genética , Neoplasias Gástricas/tratamiento farmacológico , Neoplasias Gástricas/genética , Sulfóxidos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Lung cancer leads to the largest number of cancer-related deaths worldwide and is usually accompanied with metastasis which is the primary cause of those death and correlated with poor prognosis. However, the mechanism of lung cancer metastasis is still lack of definition. METHODS: We compared the primary lung adenocarcinoma (AD) and its metastasis tissues induced by overexpression of KrasG12D and inactivation of P53 in mouse lungs by analyzing GSE40222 about the differentially expressed genes (DEGs), pathways and hub genes. And human lung AD databases are used to verify the conversed changes of identified key gene and then followed functional studies are performed to explore the functions of key gene. RESULTS: We identified 165 genes differentially expressed in lung AD metastasis compared to primary AD. Pathway analysis identified 649 GO biological processes and 8 KEGG pathways, such as ECM-receptor interaction. Biological network interaction identified the hub genes during lung adenocarcinoma metastasis, such as the up-regulated COL5A1, a novel gene in AD metastasis. We found it's also increased in human AD and advanced stage. Knockdown of COL5A1 in human AD metastatic cells inhibited cell growth and invasion, and induced cell apoptosis. Notably, higher expression of COL5A1 was observed in the lung AD patients with recurrence and short survive. CONCLUSION: By analyzing mouse lung AD and its metastases, we identified the potential key genes and pathways regulating lung AD metastasis, such as COL5A1. The following analysis of COL5A1 in human AD database and cells explores its functions, holding the implications of target therapy in AD metastasis and also providing more clues for future studies.
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Adenocarcinoma/metabolismo , Colágeno Tipo V/biosíntesis , Neoplasias Pulmonares/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Sustitución de Aminoácidos , Animales , Colágeno Tipo V/genética , Bases de Datos de Ácidos Nucleicos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Mutación Missense , Metástasis de la Neoplasia , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Curcumin is a potent anti-cancer drug in several types of human cancers. Despite of several preclinical and clinical studies of curcumin, the precise mechanism of curcumin in cancer prevention has remained unclear. In our study, we for the first time investigated whole transcriptome alteration in A549 non-small cell lung cancer (NSCLC) cell lines after treatment with curcumin using RNA sequencing. We found that lots of genes and signaling pathways were significantly altered after curcumin treatment in A549 cells. With bioinformatics approaches (gene ontology, Kyoto Encyclopedia of Genes and Genomes, and STRING), we found that those curcumin altered genes were not only the genes that induce cell death but also those extracellular matrix receptors and mitogen-activated protein kinase signaling pathway genes which regulate cell migration and proliferation. Among those significantly altered genes, eight genes ( COL1A1, COL4A1, COL5A1, LAMA5, ITGA3, ITGA2B, DDIT3, and DUSP1) were further examined by quantitative reverse transcription polymerase chain reaction and western blot analysis in four non-small cell lung cancer cell lines. Both in cell lines and in mouse model, the extracellular matrix receptors including the integrin ( ITGA3 and ITGA2B), collagen ( COL5A1), and laminin ( LAMA5) were significantly inhibited by curcumin at messenger RNA and protein levels. Functional studies confirmed that curcumin not only induced A549 cell death but also repressed cell proliferation and migration by regulating extracellular matrix receptors. Collectively, our study suggests that curcumin may be used as a promising drug candidate for intervening lung cancer in future studies.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Colágeno Tipo V/biosíntesis , Curcumina/administración & dosificación , Integrina alfa2/biosíntesis , Integrina alfa3/biosíntesis , Laminina/biosíntesis , Células A549 , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colágeno Tipo V/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Integrina alfa2/genética , Integrina alfa3/genética , Laminina/genética , Ratones , ARN Mensajero/biosíntesis , Receptores de Superficie Celular/antagonistas & inhibidores , Receptores de Superficie Celular/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cartilage canals (CCs) are microscopic structures involved in secondary ossification centers (SOCs) development. The features of CCs were investigated in the humeral and femoral proximal epiphyses of small-sized newborn dogs (from premature to 28 days after birth) with histochemical and immunohistochemical approaches. Masson's Trichrome revealed a ring-shaped area around CCs, which changes in colour from green (immature collagen) to red (mature collagen) as ossification progresses; perichondrium staining always matched the ring colour. Safranin-O was always negative. Immunohistochemical analysis revealed immunopositivity for both collagen type I and V around the CCs; collagen type II was negative. CCs count showed a tendency to be higher in the humerus than in the femur. This work enlightened for the first time changes in composition of CCs surrounding matrix during SOCs development in dogs, paving the way to further investigations.
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Colágeno Tipo I/biosíntesis , Colágeno Tipo V/biosíntesis , Fémur/citología , Fémur/metabolismo , Placa de Crecimiento/citología , Placa de Crecimiento/metabolismo , Animales , Animales Recién Nacidos , PerrosRESUMEN
To elucidate the complex molecular mechanisms of anaplastic thyroid carcinoma (ATC), the mRNA and miRNA expression profiles of ATC were systematically explored. A total of 55 common differentially expressed genes (DEGs) were obtained from two mRNA expression datasets including 23 ATC samples and 24 paired normal samples. Gene expression levels of three randomly selected DEGs, VCAN, COL5A1 and KCNJ16, were examined using RT-PCR in 10 ATC samples. Notably, the ATC and normal samples were clearly classified into two groups based on their common DEGs. Moreover 23 common DEGs, such as TG, NKX2-1, KCNJ16 and CTHRC1, were predicted to be the potential targets of 17 identified miRNAs in ATC. Meanwhile, several miRNA target genes were associated with biological processes related to tumor progression such as angiogenesis, cell migration or growth and potassium channel regulation. In summary, the poor prognosis of ATC is possibly caused via complex biological processes. Firstly, angiogenesis was activated by the high expression of CTHRC1, VCAN and POSTN, providing necessary nutrition for tumor cells. Then tumor distant metastasis was induced via stimulation of cell migration and cell growth or regulation of cell-cell interaction. Moreover, intracellular potassium concentration changes promoted ATC progression indirectly. Hence, identification of these critical DEGs was valuable in understanding the molecular mechanisms of ATC.
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Moléculas de Adhesión Celular/biosíntesis , Colágeno Tipo V/biosíntesis , Proteínas de la Matriz Extracelular/biosíntesis , Canales de Potasio de Rectificación Interna/biosíntesis , Carcinoma Anaplásico de Tiroides/genética , Versicanos/biosíntesis , Moléculas de Adhesión Celular/genética , Comunicación Celular , Movimiento Celular/genética , Proliferación Celular/genética , Colágeno Tipo V/genética , Proteínas de la Matriz Extracelular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/biosíntesis , MicroARNs/genética , Neovascularización Patológica/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Canales de Potasio de Rectificación Interna/genética , Pronóstico , Carcinoma Anaplásico de Tiroides/patología , Factor Nuclear Tiroideo 1 , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Versicanos/genéticaRESUMEN
Idiopathic pulmonary fibrosis is a devastating disease, with no good diagnostic biomarker and limited treatment options. Previous studies suggest that collagen V overexpression and collagen V-mediated immune response play roles in the pathogenesis of idiopathic pulmonary fibrosis. This study aimed to identify dysregulated miRNA-related collagen V overexpression during idiopathic pulmonary fibrosis. We found that the expression levels of miR-185 and miR-186 were decreased in the lungs of idiopathic pulmonary fibrosis patients. The levels of miR-185 and miR-186 were not correlated with disease severity of idiopathic pulmonary fibrosis. The direct regulation of COL5A1 by miR-185 and miR-186 was confirmed by a luciferase reporter assay. Furthermore, mimics of miR-185 and miR-186 blocked transforming growth factor-ß-induced collagen V overexpression and alleviated transforming growth factor-ß-induced epithelial-mesenchymal transition in A549 cells and HCC827 cells. Our findings suggest that attenuated expression of miR-185 and miR-186 may be responsible for collagen V overexpression during idiopathic pulmonary fibrosis, and these miRNAs may serve as pathogenesis-related biomarkers and treatment targets.
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Colágeno Tipo V/biosíntesis , Transición Epitelial-Mesenquimal/genética , Fibrosis Pulmonar Idiopática/patología , MicroARNs/metabolismo , Anciano , Western Blotting , Femenino , Regulación de la Expresión Génica/genética , Humanos , Fibrosis Pulmonar Idiopática/genética , Fibrosis Pulmonar Idiopática/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Aberrant proliferation and migration of vascular smooth muscle cells (VSMCs) play a crucial role in the pathogenesis of cardiovascular diseases including coronary heart disease, restenosis and atherosclerosis. MicroRNAs are a class of small, non-coding and endogenous RNAs that play critical roles in VSMCs function. In this study, we showed that PDGF-bb, as a stimulant, promoted VSMCs proliferation and suppressed the expression of miR-599. Moreover, overexpression of miR-599 inhibited VSMCs proliferation and also suppressed the PCNA and ki-67 expression. In addition, we demonstrated that ectopic expression of miR-599 repressed the VSMCs migration. We also showed that miR-599 inhibited type I collagen, type V collagen and proteoglycan expression. Furthermore, we identified TGFb2 as a direct target gene of miR-599 in VSMCs. Overexpression of TGFb2 reversed miR-599-induced inhibition of VSMCs proliferation and type I collagen, type V collagen and proteoglycan expression. In conclusion, our findings suggest miR-599 plays a crucial role in controlling VSMCs proliferation and matrix gene expression by regulating TGFb2 expression.
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Movimiento Celular/genética , Proliferación Celular/genética , MicroARNs/genética , Músculo Liso Vascular/citología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Factor de Crecimiento Transformador beta2/genética , Aterosclerosis/genética , Aterosclerosis/patología , Becaplermina , Línea Celular , Colágeno Tipo I/biosíntesis , Colágeno Tipo V/biosíntesis , Enfermedad Coronaria/genética , Enfermedad Coronaria/patología , Reestenosis Coronaria/genética , Reestenosis Coronaria/patología , Humanos , MicroARNs/biosíntesis , Proteoglicanos/biosíntesis , Factor de Crecimiento Transformador beta2/biosíntesisRESUMEN
Genome-wide association studies have implicated PLEXIN D1 (PLXND1) in body fat distribution and type 2 diabetes. However, a role for PLXND1 in regional adiposity and insulin resistance is unknown. Here we use in vivo imaging and genetic analysis in zebrafish to show that Plxnd1 regulates body fat distribution and insulin sensitivity. Plxnd1 deficiency in zebrafish induced hyperplastic morphology in visceral adipose tissue (VAT) and reduced lipid storage. In contrast, subcutaneous adipose tissue (SAT) growth and morphology were unaffected, resulting in altered body fat distribution and a reduced VAT:SAT ratio in zebrafish. A VAT-specific role for Plxnd1 appeared conserved in humans, as PLXND1 mRNA was positively associated with hypertrophic morphology in VAT, but not SAT. In zebrafish plxnd1 mutants, the effect on VAT morphology and body fat distribution was dependent on induction of the extracellular matrix protein collagen type V alpha 1 (col5a1). Furthermore, after high-fat feeding, zebrafish plxnd1 mutant VAT was resistant to expansion, and excess lipid was disproportionately deposited in SAT, leading to an even greater exacerbation of altered body fat distribution. Plxnd1-deficient zebrafish were protected from high-fat-diet-induced insulin resistance, and human VAT PLXND1 mRNA was positively associated with type 2 diabetes, suggesting a conserved role for PLXND1 in insulin sensitivity. Together, our findings identify Plxnd1 as a novel regulator of VAT growth, body fat distribution, and insulin sensitivity in both zebrafish and humans.
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Moléculas de Adhesión Celular Neuronal/fisiología , Colágeno Tipo V/biosíntesis , Insulina/metabolismo , Grasa Intraabdominal/patología , Glicoproteínas de Membrana/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Composición Corporal , Proliferación Celular , Células Endoteliales/citología , Matriz Extracelular/metabolismo , Femenino , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Lípidos/química , Ratones , Mutación , Obesidad , ARN Mensajero/metabolismo , Transducción de Señal , Pez CebraRESUMEN
Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.
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Animales , Masculino , Apoptosis/fisiología , Colágeno Tipo V/biosíntesis , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar/patología , Telomerasa/metabolismo , Hidroxitolueno Butilado , Proliferación Celular , Colágeno Tipo I/análisis , Colágeno Tipo II/análisis , Colágeno Tipo V/análisis , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Técnica del Anticuerpo Fluorescente , Fibroblastos/metabolismo , Fibroblastos/patología , Hidroxiprolina/análisis , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones Endogámicos BALB C , Microscopía Electrónica , Alveolos Pulmonares/patología , Alveolos Pulmonares/ultraestructura , Coloración y Etiquetado , Telomerasa/aislamiento & purificaciónRESUMEN
Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis.
Asunto(s)
Apoptosis/fisiología , Colágeno Tipo V/biosíntesis , Fibrosis Pulmonar Idiopática/patología , Fibrosis Pulmonar/patología , Telomerasa/metabolismo , Animales , Hidroxitolueno Butilado , Proliferación Celular , Colágeno Tipo I/análisis , Colágeno Tipo II/análisis , Colágeno Tipo V/análisis , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Fibroblastos/metabolismo , Fibroblastos/patología , Técnica del Anticuerpo Fluorescente , Hidroxiprolina/análisis , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones Endogámicos BALB C , Microscopía Electrónica , Alveolos Pulmonares/patología , Alveolos Pulmonares/ultraestructura , Coloración y Etiquetado , Telomerasa/aislamiento & purificaciónRESUMEN
Abnormal scarring results from the expression and composition of extracellular matrix molecules. The transcription and translation of collagens I and III, fibronectin, laminin, periostin, and tenascin are all increased in raised dermal scar tissue. However, human keloid development is not fully defined. In this study, we identified proteins expressed differentially between normal skin and keloid scar tissues and examined their function in keloid formation using fibroblasts. Skin specimens from normal volunteers and patients with keloids were obtained by skin biopsy. Whole proteins were isolated by two-dimensional electrophoresis, and differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry. Protein function was determined by proliferation assay using annexin A2-overexpressing keloid fibroblasts. The expression of 11 protein spots was altered by at least 1.5-fold in patients with keloids than in normal volunteers. Of these proteins, annexin A2, a pre-serum amyloid P component, serum albumin precursor, and tryptase-I, were down-regulated in keloid tissue compared to normal skin. Collagen alpha 1(V) chain precursor, collagen alpha 1(I) chain precursor, ferritin light subunit, alpha 1(III) collagen, 6-phosphogluconolactonase, and calponin 2 were up-regulated. Diminished expression of annexin A2 was confirmed by immunoblotting and immunohistochemistry. Treatment with the recombinant human epidermal growth factor increased proliferation of keloid fibroblasts, which was more inhibited in annexin A2-overexpressing fibroblasts than in non-transfected control cells. These results imply that annexin A2 may participate in keloid formation by inhibiting keloid fibroblast proliferation. Therefore, it is concluded that annexin A2 may be a valuable therapeutic target for keloid lesions.
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Anexina A2/biosíntesis , Proliferación Celular , Fibroblastos , Queloide/metabolismo , Piel/metabolismo , Adolescente , Adulto , Anciano , Apoferritinas/biosíntesis , Hidrolasas de Éster Carboxílico/biosíntesis , Células Cultivadas , Colágeno Tipo I/biosíntesis , Colágeno Tipo III/biosíntesis , Colágeno Tipo V/biosíntesis , Regulación hacia Abajo , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Proteínas de Microfilamentos/biosíntesis , Persona de Mediana Edad , Interferencia de ARN , ARN Interferente Pequeño , Albúmina Sérica/biosíntesis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triptasas/biosíntesis , Regulación hacia Arriba , Adulto JovenRESUMEN
Meningiomas are frequent, mostly benign intracranial or spinal tumors. A small subset of meningiomas is characterized by histological features of atypia or anaplasia that are associated with more aggressive biological behavior resulting in increased morbidity and mortality. Infiltration into the adjacent brain tissue is a major factor linked to higher recurrence rates. The molecular mechanisms of progression, including brain invasion are still poorly understood. We have studied the role of micro-RNA 145 (miR-145) in meningiomas and detected significantly reduced miR-145 expression in atypical and anaplastic tumors as compared with benign meningiomas. Overexpression of miR-145 in IOMM-Lee meningioma cells resulted in reduced proliferation, increased sensitivity to apoptosis, reduced anchorage-independent growth and reduction of orthotopic tumor growth in nude mice as compared with control cells. Moreover, meningioma cells with high miR-145 levels had impaired migratory and invasive potential in vitro and in vivo. PCR-array studies of miR145-overexpressing cells suggested that collagen type V alpha (COL5A1) expression is downregulated by miR-145 overexpression. Accordingly, COL5A1 expression was significantly upregulated in atypical and anaplastic meningiomas. Collectively, our data indicate an important anti-migratory and anti-proliferative function of miR-145 in meningiomas.
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Neoplasias Meníngeas/metabolismo , Meningioma/metabolismo , MicroARNs/fisiología , Invasividad Neoplásica/genética , ARN Neoplásico/fisiología , Animales , Adhesión Celular , Diferenciación Celular , División Celular , Movimiento Celular , Colágeno Tipo V/biosíntesis , Colágeno Tipo V/genética , Regulación hacia Abajo , Humanos , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/patología , Meningioma/genética , Meningioma/patología , Ratones , Ratones Desnudos , MicroARNs/biosíntesis , MicroARNs/genética , Clasificación del Tumor , Invasividad Neoplásica/fisiopatología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Ensayo de Tumor de Célula MadreRESUMEN
The progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) marks a critical step in the evolution of breast cancer. There is some evidence to suggest that dynamic interactions between the neoplastic cells and the tumour microenvironment play an important role. Using the whole-genome cDNA-mediated annealing, selection, extension and ligation assay (WG-DASL, Illumina), we performed gene expression profiling on 87 formalin-fixed paraffin-embedded (FFPE) samples from 17 patients consisting of matched IDC, DCIS and three types of stroma: IDC-S (<3 mm from IDC), DCIS-S (<3 mm from DCIS) and breast cancer associated-normal stroma (BC-NS; >10 mm from IDC or DCIS). Differential gene expression analysis was validated by quantitative real time-PCR, immunohistochemistry and immunofluorescence. The expression of several genes was down-regulated in stroma from cancer patients relative to normal stroma from reduction mammoplasties. In contrast, neoplastic epithelium underwent more gene expression changes during progression, including down regulation of SFRP1. In particular, we observed that molecules related to extracellular matrix (ECM) remodelling (e.g. COL11A1, COL5A2 and MMP13) were differentially expressed between DCIS and IDC. COL11A1 was overexpressed in IDC relative to DCIS and was expressed by both the epithelial and stromal compartments but was enriched in invading neoplastic epithelial cells. The contributions of both the epithelial and stromal compartments to the clinically important scenario of progression from DCIS to IDC. Gene expression profiles, we identified differential expression of genes related to ECM remodelling, and specifically the elevated expression of genes such as COL11A1, COL5A2 and MMP13 in epithelial cells of IDC. We propose that these expression changes could be involved in facilitating the transition from in situ disease to invasive cancer and may thus mark a critical point in disease development.
Asunto(s)
Neoplasias de la Mama/genética , Carcinoma Ductal de Mama/genética , Carcinoma Intraductal no Infiltrante/genética , Células Epiteliales/metabolismo , Perfilación de la Expresión Génica , Células del Estroma/metabolismo , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patología , Carcinoma Intraductal no Infiltrante/metabolismo , Carcinoma Intraductal no Infiltrante/patología , Colágeno Tipo V/biosíntesis , Colágeno Tipo V/genética , Colágeno Tipo XI/biosíntesis , Colágeno Tipo XI/genética , Progresión de la Enfermedad , Matriz Extracelular/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Péptidos y Proteínas de Señalización Intercelular/genética , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Microambiente TumoralRESUMEN
Accumulation of sub-rupture fatigue damage has been implicated in the development of tendinopathy. We previously developed an in vivo model of damage accumulation using the rat patellar tendon. Our model allows us to control the input loading parameters to induce fatigue damage in the tendon. Despite this precise control, the resulting induced damage could vary among animals because of differences in size or strength among their patellar tendons. In this study, we used number of applied cycles and initial (day-0) parameters that are indicative of induced damage to assess the molecular response 7 days after fatigue loading. We hypothesized that day-0 hysteresis, elongation, and stiffness of the loading and unloading load-displacement curves would be predictive of the 7-day molecular response. Results showed correlations between the 7-day molecular response and both day-0 elongation and unloading stiffness. Additionally, loading resulted in upregulation of several extracellular matrix genes that suggest adaptation; however, several of these genes (Col-I, -XII, MMP 2, and TIMP 3) shut down after a high level of damage was induced. We showed that evaluating the 7-day molecular profile in light of day-0 elongation provides important insight that is lost from comparing number of fatigue loading cycles only. Our data showed that loading generally results in an adaptive response. However, the tendon's ability to effectively respond deteriorates as greater damage is induced.
Asunto(s)
Fatiga/fisiopatología , Ligamento Rotuliano/fisiopatología , Tendinopatía/fisiopatología , Animales , Fenómenos Biomecánicos , Colágeno Tipo I/biosíntesis , Colágeno Tipo V/biosíntesis , Modelos Animales de Enfermedad , Femenino , Metaloproteinasa 14 de la Matriz , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratas , Inhibidor Tisular de Metaloproteinasa-1/biosíntesis , Inhibidor Tisular de Metaloproteinasa-2/biosíntesis , Regulación hacia ArribaRESUMEN
Collagen V, a fibrillar collagen with important functions in tissues, assembles into distinct chain associations. The most abundant and ubiquitous molecular form is the heterotrimer [alpha1(V)](2)alpha2(V). In the attempt to produce high levels of recombinant collagen V heterotrimer for biomedical device uses, and to identify key factors that drive heterotrimeric chain association, several cell expression systems (yeast, insect, and mammalian cells) have been assayed by cotransfecting the human proalpha1(V) and proalpha2(V) chain cDNAs. Suprisingly, in all recombinant expression systems, the formation of [alpha1(V)](3) homotrimers was considerably favored over the heterotrimer. In addition, pepsin-sensitive proalpha2(V) chains were found in HEK-293 cell media indicating that these cells lack quality control proteins preventing collagen monomer secretion. Additional transfection with Hsp47 cDNA, encoding the collagen-specific chaperone Hsp47, did not increase heterotrimer production. Double immunofluorescence with antibodies against collagen V alpha-chains showed that, contrary to fibroblasts, collagen V alpha-chains did not colocalized intracellularly in transfected cells. Monensin treatment had no effect on the heterotrimer production. The heterotrimer production seems to require specific machinery proteins, which are not endogenously expressed in the expression systems. The different constructs and transfected cells we have generated represent useful tools to further investigate the mechanisms of collagen trimer assembly.
Asunto(s)
Colágeno Tipo V/biosíntesis , Multimerización de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Animales , Línea Celular , Células Clonales , Colágeno Tipo V/química , Técnica del Anticuerpo Fluorescente , Proteínas del Choque Térmico HSP47/metabolismo , Humanos , Insectos/citología , Pichia/metabolismoRESUMEN
BACKGROUND: It has been demonstrated that hypertrophic scar fibroblasts (HSFs) overexpress collagen messenger ribonucleic acid (mRNA) and protein, especially alpha1 collagen. Antisense nucleic acids are effective in inhibiting harmful or uncontrolled gene expression, suggesting that antisense ribonucleic acid (RNA) can effectively downregulate the expression of alpha1 collagen gene and attenuate the scars. AIMS: This study was conducted to observe the effect of recombinant plasmid pREP9-COL1 on alpha1 collagen expression in HSFs and clarify the prospect of antisense RNA on scar treatment. METHODS: The alpha1 collagen gene fragment including the region of 5' UTR to exon (229 bp) was cloned in the eukaryotic expression plasmid pREP9 in the antisense orientation relative to the RSV-LTR promoter to reconstruct the pREP9- COL1 plasmid. Then it was transferred into HSFs through lipofectamine. The expression of alpha1 collagen was examined by immunostaining, reverse-transcriptase polymerase chain reaction, and Western blots. RESULTS: The recombinant plasmid pREP9-COL1 with a correct sequence was constructed successfully; pREP9-COL1 consistently inhibited human alpha1 collagen gene expression at both mRNA and protein levels. CONCLUSIONS: Antisense RNA was effective in downregulating alpha1 collagen expression of HSFs. Therefore, this approach offered a prospect of scar treatment by attenuation of alpha1 collagen production with antisense RNA.
Asunto(s)
Cicatriz Hipertrófica/patología , Colágeno Tipo V/antagonistas & inhibidores , Fibroblastos/metabolismo , Expresión Génica , ARN sin Sentido/farmacología , ARN Mensajero/genética , Biopsia , Western Blotting , Células Cultivadas , Cicatriz Hipertrófica/metabolismo , Colágeno Tipo V/biosíntesis , Colágeno Tipo V/genética , Fibroblastos/patología , Humanos , Microscopía Fluorescente , ARN sin Sentido/genética , ARN sin Sentido/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Retinoids are anti-proliferative and anti-inflammatory compounds. We had previously shown that retinoids alleviate kidney damage in acute models of renal disease. We now examined whether retinoids are also effective in a chronic renal ablation model. Subtotally nephrectomized rats (SNx; two-third ablation) were compared to sham-operated controls (sham). SNx rats were administered either 10 mg/kg b.w. (low dose, LD) or 40 mg/kg b.w. (high dose, HD) isotretinoin or vehicle (n = 10 per group). The experiment was terminated after 16 weeks. Systolic blood pressure was significantly higher after SNx compared to sham but lower in SNx with LD isotretinoin (vs. SNx + vehicle). Compared to SNx + vehicle, SNx + LD isotretinoin had lower glomerular cell numbers, less glomerular hypertrophy and sclerosis, and less interstitial expansion. Morphological improvement in SNx + LD isotretinoin was accompanied by improvement in creatinine clearance and reduced urinary albumin excretion. In contrast, HD isotretinoin caused aggravation of renal damage with fibrinoid necroses of vessels and elevated urinary albumin excretion despite lower blood pressure. The dichotomous effects of isotretinoin are at least in part due to time- and dose-dependent alterations of transforming growth factor beta1 and collagen IV gene expression as also suggested by cell-culture studies in vascular smooth muscle cells. In addition, isotretinoin affected the systemic and the renal renin-angiotensin system (which was further analyzed in a model of angiotensin II infusion of the rat). Isotretinoin failed to cumulate at LD but cumulated at HD in SNx. We conclude that LD isotretinoin attenuates progressive renal damage, whereas HD isotretinoin cumulates and aggravates renal damage independent of blood pressure reduction.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Isotretinoína/farmacología , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/metabolismo , Albuminuria/tratamiento farmacológico , Albuminuria/metabolismo , Animales , Presión Sanguínea/efectos de los fármacos , Células Cultivadas , Enfermedad Crónica , Colágeno Tipo V/biosíntesis , Fármacos Dermatológicos/farmacología , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Nefrectomía , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , Factor de Crecimiento Transformador beta1/biosíntesisRESUMEN
A cutaneous metaplastic synovial cyst is a rare entity that is probably caused by trauma or surgery. We report the second case of cutaneous metaplastic synovial cyst in a child with Ehlers-Danlos syndrome. His father is also affected with Ehlers-Danlos syndrome, and his diagnosis is substantiated by the demonstration of reduced synthesis of collagen type V.
Asunto(s)
Síndrome de Ehlers-Danlos/complicaciones , Quiste Sinovial/complicaciones , Quiste Sinovial/patología , Niño , Preescolar , Colágeno Tipo V/biosíntesis , Electroforesis en Gel de Poliacrilamida , Humanos , Lactante , Recién Nacido , Masculino , LinajeRESUMEN
BACKGROUND/AIM: Matrix production and degradation are critically important in chronic nephritis. Our aim was to investigate the precise expression of matrix-related molecules which is essential for understanding the pathogenesis of renal disease. METHODS: Chronic nephritis was induced by a single injection of anti-Thy1.1 antibody to unilaterally nephrectomized rats. RNA was extracted from renal cortex and isolated glomeruli 4, 7, and 10 weeks after the antibody injection. Matrix-related gene expressions were measured by polymerase chain reaction. The expression of alpha1(IV) and alpha3(IV) collagens was studied by immunohistochemistry. The gelatinolytic activity in the glomeruli was assayed by gelatin zymography. RESULTS: Polymerase chain reaction revealed an increase of alpha1(IV) in both glomeruli and renal cortex from nephritic rats. In contrast, the expression of alpha3(IV), normally a component of the glomerular basement membrane, was decreased in nephritic animals. Immunohistochemistry confirmed the finding that alpha1(IV) and alpha3(IV) were up- and downregulated, respectively, in the glomeruli. Gene expression and activity of matrix metalloproteinase 2 were enhanced, while those of matrix metalloproteinase 9 were clearly suppressed in nephritis. CONCLUSIONS: Downregulation of alpha3(IV) and enhancement of the matrix metalloproteinase-2 activity in the glomeruli may contribute to the glomerular damage by altering the glomerular basement membrane components. Impairment of the glomerular basement membrane integrity may possibly be implicated in irreversible renal dysfunction.