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1.
Oncotarget ; 3(3): 267-85, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22408128

RESUMEN

Cancer-associated fibroblasts (CAFs), represent a pivotal compartment of solid cancers (desmoplasia), and are causatively implicated in cancer development and progression. CAFs are recruited by growth factors secreted by cancer cells and they present a myofibroblastic phenotype, similar to the one obtained by resident fibroblasts during wound healing. Paracrine signaling between cancer cells and CAFs results in a unique protein expression profile in areas of desmoplastic reaction, which is speculated to drive metastasis. In an attempt to decipher large-scale proteomic profiles of the cancer invasive margins, we developed an in vitro coculture model system, based on tumor-host cell interactions between colon cancer cells and CAFs. Proteomic analysis of conditioned media derived from these cocultures coupled to mass spectrometry and bioinformatic analysis was performed to uncover myofibroblastic signatures of the cancer invasion front. Our analysis resulted in the identification and generation of a desmoplastic protein dataset (DPD), consisting of 152 candidate proteins of desmoplasia. By using monoculture exclusion datasets, a secretome algorithm and gene-expression meta-analysis in DPD, we specified a 22-protein "myofibroblastic signature" with putative importance in the regulation of colorectal cancer metastasis. Of these proteins, we investigated collagen type XII by immunohistochemistry, a fibril-associated collagen with interrupted triple helices (FACIT), whose expression has not been reported in desmoplastic lesions in any type of cancer. Collagen type XII was highly expressed in desmoplastic stroma by and around alpha-smooth muscle actin (α-SMA) positive CAFs, as well as in cancer cells lining the invasion front, in a small cohort of colon cancer patients. Other stromal markers, such as collagen type III, were also expressed in stromal collagen, but not in cancer cells. In a complementary fashion, gene expression meta-analysis revealed that COL12A1 is also an upregulated gene in colorectal cancer. Our proteomic analysis identified previously documented markers of tumor invasion fronts and our DPD could serve as a pool for future investigation of the tumor microenvironment. Collagen type XII is a novel candidate marker of myofibroblasts, and/or cancer cells undergoing dedifferentiation.


Asunto(s)
Carcinoma/patología , Colágeno Tipo XII/fisiología , Neoplasias Colorrectales/patología , Fibroblastos/patología , Miofibroblastos/fisiología , Algoritmos , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Biomarcadores de Tumor/fisiología , Carcinoma/metabolismo , Diferenciación Celular , Técnicas de Cocultivo/métodos , Colágeno Tipo XII/análisis , Colágeno Tipo XII/metabolismo , Neoplasias Colorrectales/metabolismo , Fibroblastos/metabolismo , Humanos , Modelos Biológicos , Miofibroblastos/metabolismo , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteoma/análisis , Proteómica/métodos , Células Tumorales Cultivadas
2.
Clin Oral Implants Res ; 22(8): 815-9, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21198896

RESUMEN

OBJECTIVES: The purpose of the present study was to investigate the early peri-implant soft tissue healing on different titanium surface topographies. MATERIAL AND METHODS: Titanium implants with smooth or rough surfaces were placed in the extraction site at 1 month after the maxillary first molar extractions of 15 male Sprague-Dawley rats. At 4 and 7 days after implant surgery, the peri-implant oral mucosa was randomly harvested and analyzed for collagen fiber orientation, and expression of extracellular matrix genes. Descriptive and paired t-tests were performed where appropriate (α = 0.05). RESULTS: The laser scanning microscopic analysis of the Sirius red stained peri-implant connective tissue revealed a parallel and uniform collagen fiber orientation along the smooth implant at both 4 and 7 days. In contrast, the collagen fibers of the comparative peri-implant tissue from rough surface implants at 7 days were not arranged in parallel orientation but in an irregular a "cotton-ball-like." The levels of mRNA of types III and XII collagen and transforming growth factor-ß1 significantly increased in the smooth implant group compared with the rough implant group at both 4 and 7 days or either one of days. CONCLUSION: The results of this study suggest that implant surface characteristics may affect early events of soft tissue healing by influencing collagen fiber orientation and expression of key genes for initial healing.


Asunto(s)
Implantes Dentales , Materiales Dentales/química , Mucosa Bucal/patología , Titanio/química , Animales , Compuestos Azo , Colágeno/ultraestructura , Colágeno Tipo III/análisis , Colágeno Tipo XII/análisis , Colorantes , Tejido Conectivo/patología , Diseño de Prótesis Dental , Matriz Extracelular/patología , Colorantes Fluorescentes , Masculino , Microscopía Confocal , Modelos Animales , Diente Molar/cirugía , Ratas , Ratas Sprague-Dawley , Rodaminas , Propiedades de Superficie , Factores de Tiempo , Extracción Dental , Alveolo Dental/cirugía , Factor de Crecimiento Transformador beta1/análisis , Cicatrización de Heridas/fisiología
3.
J Periodontal Res ; 44(4): 425-33, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18973549

RESUMEN

BACKGROUND AND OBJECTIVE: Although periodontal ligament-derived cells are expected to be a useful source of cells for periodontal tissue engineering, the characteristic changes of primary cultured cells have not been well studied. Therefore, the aim of this study was to investigate the characteristics of periodontal ligament-derived cells and their changes during passage. MATERIAL AND METHODS: Human periodontal ligament tissue was obtained from extracted third molars. Cells were subcultured until passage 6 and the cell characteristics from early to late passages were evaluated using immunofluorescence microscopy, alkaline phosphatase activity analyses, reverse transcription-polymerase chain reaction and quantitative real-time polymerase chain reaction. To examine the function of periodontal ligament-derived cells further, cells were transplanted into the renal subcapsule of an immunocompromised rat. RESULTS: Immunofluorescence results showed relatively uniform expression of MSX-2 and osteonectin from passage 1 until passage 6. The STRO-1-positive fraction was 33.5% at passage 0, which was reduced to 14.7% at passage 3. Cultured cells at passage 1 expressed mRNA for collagen type I, collagen type XII, Runx2, alkaline phosphatase, osteonectin, osteopontin, scleraxis, tenomodulin, Msx2, GDF5 and GDF7 genes, but not for bone sialoprotein. The level of mRNA expression from tenomodulin and collagen type XII genes decreased after passage 3. Alkaline phosphatase activity decreased in cells at later passages. Osteogenic induction of periodontal ligament-derived cells resulted in a down-regulation of the tenomodulin gene. Transplanted cells from both early and late passages produced dense collagen fiber bundles without calcified tissue. CONCLUSION: Cultured periodontal ligament-derived cells were a morphologically homogeneous population, although expression of STRO-1 was limited in primary culture. Cultured cells showed de-differentiation during passage for both osteogenesis- and tendo/ligamentogenesis-related genes.


Asunto(s)
Ligamento Periodontal/citología , Ingeniería de Tejidos/métodos , Adulto , Fosfatasa Alcalina/análisis , Animales , Antígenos de Superficie/análisis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/análisis , Proteínas Morfogenéticas Óseas/análisis , Técnicas de Cultivo de Célula , Trasplante de Células , Colágeno Tipo I/análisis , Colágeno Tipo XII/análisis , Subunidad alfa 1 del Factor de Unión al Sitio Principal/análisis , Femenino , Factor 5 de Diferenciación de Crecimiento/análisis , Factores de Diferenciación de Crecimiento/análisis , Secuencias Hélice-Asa-Hélice , Proteínas de Homeodominio/análisis , Humanos , Riñón/cirugía , Masculino , Proteínas de la Membrana/análisis , Osteogénesis/fisiología , Osteonectina/análisis , Osteopontina/análisis , Ratas
4.
Differentiation ; 72(4): 113-22, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15157235

RESUMEN

In order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intra-muscular pre-adipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up-regulated during the adipogenic differentiation of BIP cells, and cloned cDNAs from a differentiated BIP-lambda ZAP II cDNA library. Two cDNA clones were highly homologous to the sequence of mouse and human type XII collagen alpha-1, determined by a BLAST homology search. As type XII collagen has been reported to have four types of splicing isoform, two clones were determined to be XII-1 and XII-2 splicing isoforms, respectively, because of a difference in the C-terminal NC1 domain. From the expression analysis of type XII collagen, the XIIA-2 isoform was mainly expressed in differentiated BIP cells and adipose tissues. Although the function of type XII collagen has not been established as yet, these results suggest that type XII collagen may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.


Asunto(s)
Adipocitos/metabolismo , Bovinos/genética , Colágeno Tipo XII/genética , Adipocitos/citología , Tejido Adiposo/química , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos/crecimiento & desarrollo , Bovinos/metabolismo , Diferenciación Celular , Línea Celular , Clonación Molecular , Colágeno Tipo XII/análisis , Colágeno Tipo XII/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Distribución Tisular
5.
J Anat ; 202(5): 411-20, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12739618

RESUMEN

Type XII collagen is a fibril-associated collagen with multiple functional domains. The purpose of this work was to determine its role in regulating tendon matrix assembly. The temporal and spatial expression patterns of both collagen and mRNA were analysed in developing chicken metatarsal tendons using immunofluorescence microscopy, in situ hybridization and real-time quantitative PCR. Temporally, type XII collagen was present during all stages of development (day 14-hatch). However, spatially, type XII collagen expression shifted from the entire tendon at day 14, when the tendon is immature and fascicles are not well developed, to the interfacial matrix (endotendinium) associated with developing fascicles. This shift was obvious beginning at day 17, becoming prominent at day 19. Associated with this shift was a gradual decrease in type XII collagen reactivity in the tendon proper (non-sheath). By hatching, the reactivity was sequestered almost exclusively to the sheaths with some reactivity remaining at the fibroblast-matrix interface within the fascicle. In situ hybridization indicated that fibroblasts in the tendon expressed type XII collagen mRNA homogeneously at day 14. However, by hatching, when the tendon matures, type XII collagen is restricted primarily to the sheath cells. Quantitative PCR analyses, of NC3 splice variants, demonstrated highest expression levels for the short splice variant mRNA at days 14-17, followed by a significant decrease at day 19 with levels remaining constant to adult. Long variant mRNA expression was highest at day 14 then decreased and was constant from day 17 to adult. These changing patterns may be related to the spatial shift in type XII collagen expression to the sheaths. Differential temporal and spatial expression patterns indicate that type XII collagen functions to integrate the developing tendon matrices and fascicles into a functional unit.


Asunto(s)
Pollos/crecimiento & desarrollo , Pollos/metabolismo , Colágeno Tipo XII/análisis , Metatarso , Tendones/química , Animales , Embrión de Pollo , Colágeno Tipo XII/genética , Fibroblastos/metabolismo , Procesamiento de Imagen Asistido por Computador , Hibridación in Situ/métodos , Microscopía Fluorescente/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Tendones/embriología , Tendones/crecimiento & desarrollo
6.
Am J Obstet Gynecol ; 187(6): 1679-85, 2002 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-12501083

RESUMEN

OBJECTIVE: It has been suggested that there is a decrease in the collagen content of the fetal membranes when there is premature rupture of the membranes before the onset of labor. This study was designed to determine whether decreased amniochorion collagen production (as measured by reduced amounts of messenger RNA) or alterations in relative production of different fibrillar and nonfibrillar collagens are associated with premature rupture of the membranes. STUDY DESIGN: Fetal membranes were collected after preterm (24-36 weeks of gestation) and term (> or =37 weeks of gestation) deliveries both with and without premature rupture of the membranes. Specimens with evidence of histologic chorioamnionitis were excluded. The messenger RNA levels for fibrillar collagen types I, III, and V and fibril-associated collagens with interrupted triple-helices types XII and XIV were measured with relative quantitative reverse transcriptase-polymerase chain reaction. RESULTS: The messenger RNA levels for fibrillar collagens decreased with advancing gestational age. Preterm premature rupture of membranes was associated with increased messenger RNA levels for fibrillar collagens and fibril-associated collagens with interrupted triple-helices collagen XII, but not type XIV. The greatest change in relative amounts of collagen messsenger RNA was demonstrated by an increased type I/XIV ratio, which was due to the up-regulation of type I levels, but not type XIV levels. CONCLUSION: A rise in fibrillar collagen production (messenger RNA) for types I, III, and V and fibril-associated collagens with interrupted triple-helices collagen type XII is observed with preterm premature rupture of the membranes. There is no evidence for a similar up-regulation of messenger RNA for fibril-associated collagens with interrupted triple-helices collagen type XIV. The rise in the collagen I/XIV messenger RNA ratio in preterm premature rupture of the membranes may result in collagen fibrils without enough stabilizing fibril-associated collagens with interrupted triple-helices type XIV on the fibril surface to maintain structural integrity.


Asunto(s)
Amnios/química , Corion/química , Colágeno/genética , Rotura Prematura de Membranas Fetales/metabolismo , Expresión Génica , ARN Mensajero/análisis , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo III/genética , Colágeno Tipo V/genética , Colágeno Tipo XII/análisis , Colágeno Tipo XII/genética , Femenino , Colágenos Asociados a Fibrillas/genética , Técnica del Anticuerpo Fluorescente , Edad Gestacional , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Inmunohistoquímica , Embarazo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
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