RESUMEN
Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.
Asunto(s)
Anticuerpos/inmunología , Tropoelastina/análisis , Animales , Especificidad de Anticuerpos , Sangre , Bovinos , Células Cultivadas , Medios de Cultivo/análisis , Elastina/inmunología , Ensayo de Inmunoadsorción Enzimática , Inmunoensayo , Inmunohistoquímica , Técnicas de Inmunoadsorción , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Solubilidad , Tropoelastina/genética , Tropoelastina/inmunologíaRESUMEN
Techniques are described for visualizing intracellular tropoelastin at the light level using immunofluorescence and immunogold techniques. Best results were obtained with B5 fixative on cells permeabilized with acetone. Using either formaldehyde or paraformaldehyde for fixation (instead of B5) resulted in both less reproducible and less intense intracellular staining, and permeabilization of the cells with ethanol resulted in relatively high background staining compared with that obtained with cold (-20 degrees C) acetone. Intracellular tropoelastin was seen most prominently in the perinuclear region, and the intensity of staining agreed with the reported rate of tropoelastin synthesis as assayed by enzyme-linked immunosorbent assay (ELISA) and RNA hybridization studies. The applicability of the intracellular staining technique for studying the elastin phenotype was tested by demonstrating increases in both the number of positive cells and in the intensity of elastin staining in cells treated with smooth muscle elastogenic factor (SMEF), an elastogenic factor known to stimulate elastin production.
Asunto(s)
Elastina/análogos & derivados , Elastina/metabolismo , Tropoelastina/metabolismo , Animales , Bovinos , Citoplasma/metabolismo , Elastina/inmunología , Fijadores , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica/métodos , Técnicas In Vitro , Tropoelastina/inmunologíaRESUMEN
The temporal expression of elastogenesis is unique among connective tissues in that elastin production occurs primarily during late fetal and early neonatal periods and is essentially fully repressed once fiber assembly is completed. To test whether elastin synthesis in adult nuchal ligament fibroblasts is permanently repressed or whether the cells retain the ability to reinitiate production upon proper stimulation, we examined in adult ligament cells various parameters known to be involved in the regulation of elastin production. Elastin synthetic capacity, as determined by the levels of steady-state tropoelastin mRNA, of adult tissue was significantly decreased relative to fetal tissue. Likewise, fibroblasts grown from explants of adult ligament had about a fourfold decrease in elastin production and elastin-specific mRNA levels. On the other hand, adult cells were similar to fetal ligament cells in that they were sensitive to glucocorticoid stimulation and demonstrated chemotactic responsiveness to elastin peptides. Since our previous studies have shown that the extracellular matrix (ECM) plays an important role in influencing elastin phenotypic expression, fetal and adult fibroblasts were grown on slices of nonviable adult ligament to test if repression of elastin production was directed by factors in ECM of adult tissues. No change in elastin synthesis was detected with either cell type grown on adult ligament, whereas both fetal and adult cells demonstrated increased elastin production in response to contact with fetal ligament. These results suggest that adult ligament ECM does not provide a metabolic signal to shut off the elastin gene and that adult cells remain responsive to external stimuli that may reinitiate high levels of elastin synthesis.
Asunto(s)
Elastina/biosíntesis , Ligamentos/fisiología , Animales , Bovinos , Diferenciación Celular , Células Cultivadas , Dexametasona/farmacología , Elastina/genética , Fibroblastos/efectos de los fármacos , Ligamentos/efectos de los fármacos , Fenotipo , ARN Mensajero/análisis , Tropoelastina/biosíntesis , Tropoelastina/genéticaRESUMEN
Abnormal accumulation of connective tissue in blood vessels contributes to alterations in vascular physiology associated with disease states such as hypertension and atherosclerosis. Elastin synthesis was studied in blood vessels from newborn calves with severe pulmonary hypertension induced by alveolar hypoxia in order to investigate the cellular stimuli that elicit changes in pulmonary arterial connective tissue production. A two- to fourfold increase in elastin production was observed in pulmonary artery tissue and medial smooth muscle cells from hypertensive calves. This stimulation of elastin production was accompanied by a corresponding increase in elastin messenger RNA consistent with regulation at the transcriptional level. Conditioned serum harvested from cultures of pulmonary artery smooth muscle cells isolated from hypertensive animals contained one or more low molecular weight elastogenic factors that stimulated the production of elastin in both fibroblasts and smooth muscle cells and altered the chemotactic responsiveness of fibroblasts to elastin peptides. These results suggest that connective tissue changes in the pulmonary vasculature in response to pulmonary hypertension are orchestrated by the medial smooth muscle cell through the generation of specific differentiation factors that alter both the secretory phenotype and responsive properties of surrounding cells.
Asunto(s)
Tejido Conectivo/fisiopatología , Hipertensión Pulmonar/fisiopatología , Músculo Liso Vascular/fisiopatología , Animales , Bovinos , Tejido Conectivo/patología , Modelos Animales de Enfermedad , Elastina/genética , Elastina/fisiología , Humanos , Hipertensión Pulmonar/patología , Hipoxia , Músculo Liso Vascular/patología , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
High resolution gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cell-free translation, and elastin-specific antibodies were used to identify three tropoelastin isoforms secreted by bovine tissue and cells. Tropoelastin isolated from nuchal ligament and from conditioned culture medium or cell-matrix extracts of ligament fibroblasts and auricular chondrocytes resolved as three distinct bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of approximately 67,500 (tropoelastin I), 65,000 (tropoelastin II), and 62,000 (tropoelastin III). Three tropoelastin polypeptides with molecular mass 2-3 kDa higher than their corresponding tissue forms were also evident in cell-free translation products of ligamentum nuchae RNA, suggesting that each tropoelastin species is encoded by a unique mRNA. The presence of cysteine in all three tropoelastin isoforms was demonstrated by the incorporation of [35S]cysteine into newly synthesized tropoelastin polypeptides and by immunoreactivity with an antibody raised against a synthetic peptide that defines the cysteine-containing carboxyl-terminal region of tropoelastin. Immunological co-localization of the carboxyl-terminal antibody with insoluble elastin in lung vasculature and parenchyma suggests that intact tropoelastin and not a processed form is incorporated into the elastin fiber.