RESUMEN
Fibroblasts are subjected to changes of the mechanical force balance during physiological as well as pathological situations, such as wound healing, development of hypertrophic scars, and fibrogenesis. However, the molecular response and the changes in fibroblast gene expression upon mechanical stimulation remain poorly understood. As an in vitro model, human dermal fibroblasts were cultured within a three-dimensional network of fibrillar collagen either under high (stressed) or low tension (relaxed). cDNA microarray technology in combination with Northern blot analysis led to identification of mechano-responsive genes coding for extracellular matrix proteins, fibrogenic growth factors, protease inhibitors, components of focal adhesions, and the cytoskeleton. Application of biaxial strain to fibroblasts cultured on flexible silicone membranes revealed that the type of strain as well as the properties of the substrate induced different patterns of gene regulation. The transcriptional profile of mechanically induced genes in collagen lattices suggests that mechanical stimuli lead to a "synthetic" fibroblast phenotype characterized by induction of connective tissue synthesis while simultaneously inhibiting matrix degradation.
Asunto(s)
Colágeno/química , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular , Actinas/biosíntesis , Northern Blotting , Adhesión Celular , Células Cultivadas , Colágeno/metabolismo , Factor de Crecimiento del Tejido Conjuntivo , ADN Complementario/metabolismo , Matriz Extracelular/metabolismo , Sustancias de Crecimiento/metabolismo , Humanos , Proteínas Inmediatas-Precoces/metabolismo , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , ARN Mensajero/metabolismo , Estrés Fisiológico , Factores de Tiempo , Factor de Crecimiento Transformador beta/metabolismo , Factor de Crecimiento Transformador beta1 , Factor de Crecimiento Transformador beta3 , Vinculina/biosíntesis , Cicatrización de HeridasRESUMEN
The phorbol ester, tetradecanoyl-phorbol 13-acetate (TPA), stimulates rapid proteolytic processing of the transmembrane, pro- form of heparin-binding epidermal growth factor-like growth factor (HB-EGF) at cell surfaces, suggesting the involvement of protein kinase C (PKC) isoforms in the HB-EGF secretion mechanism. To test this possibility, we expressed a chimeric protein, consisting of proHB-EGF fused to placental alkaline phosphatase (AP) near the amino terminus of processed HB-EGF, in NbMC-2 prostate epithelial cells. The proHB-EGF-AP chimera localized to plasma membranes and functioned as a diphtheria toxin receptor. Secreted HB-EGF-AP bound to heparin and exhibited potent growth factor activity. The presence of the AP moiety allowed highly quantitative measurements of cleavage-secretion responses of proHB-EGF to extracellular stimuli. As expected, rapid secretion of HB-EGF-AP was induced in a time- and dose-dependent manner by TPA. However, this was also observed with the Ca2+ ionophore, ionomycin, suggesting the involvement of extracellular Ca2+ ions in the secretion mechanism. Ionomycin-induced secretion was inhibited by extracellular calcium chelation but not by the PKC inhibitors, GF109203X, staurosporine, or chelerythrine. The TPA-mediated secretion effect was inhibited by staurosporine, GF109203X, and by pretreatment with TPA, but not by calcium chelation. A small secretion response was induced by thapsigargin, which releases Ca2+ from intracellular stores, but this was completely eliminated by extracellular calcium chelation. Ionomycin- and TPA-induced HB-EGF-AP secretion was not dependent on the presence of the proHB-EGF cytoplasmic domain and was specifically inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and tissue inhibitor of metalloproteinase-1 (TIMP-1). These data demonstrate that extracellular Ca2+ influx activates a membrane-associated metalloproteinase to process proHB-EGF by a pathway that does not require PKC.
Asunto(s)
Calcio/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Espacio Extracelular/enzimología , Heparina/metabolismo , Metaloendopeptidasas/metabolismo , Proteína Quinasa C/metabolismo , Fosfatasa Alcalina/genética , Animales , Calcio/fisiología , Células Cultivadas , Citoplasma/metabolismo , Inhibidores Enzimáticos/farmacología , Factor de Crecimiento Epidérmico/antagonistas & inhibidores , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Péptidos y Proteínas de Señalización Intercelular , Masculino , Metaloendopeptidasas/antagonistas & inhibidores , Precursores de Proteínas/metabolismo , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The proliferation of intimal smooth muscle cells (SMCs) in large muscular arteries and veins often occurs after surgical interventions such as angioplasty and bypass grafting, and may lead to restenosis and graft failure. Clinical observations suggest that increased pulsatile deformation of veins grated into an arterial position may play a role in intimal hyperplasia. Since intimal hyperplasia occurs at the vein/arterial interface of the graft, SMC hyperplasia could be due to the proliferation of either aortic or venous SMCs. Therefore, we compared the effects of in vitro mechanical deformation on the proliferation of aortic SMCs with venous SMCs. Using the Flexercell apparatus (Flexercell Corp., McKeesport, Pa., USA), aortic SMCs, stretched at 3 and 60 cpm did not lead to a significant increase in growth as compared to the nonstretched controls. In contrast, stretch of venous SMCs at 3 and 60 cpm led to a significant increase in growth as compared to the nonstretched controls. These results suggest that the SMC proliferation, as occurs in vein interposition grafts in vivo, may be partially due to a stimulatory response by venous SMCs to increased mechanical stimulation.
Asunto(s)
Arterias/citología , Oclusión de Injerto Vascular/etiología , Músculo Liso Vascular/citología , Estrés Mecánico , Venas/citología , Animales , Aorta/citología , Bovinos , División Celular , Células Cultivadas , Puente de Arteria Coronaria , Medios de Cultivo Condicionados/farmacología , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Oclusión de Injerto Vascular/patología , Humanos , Hiperplasia/etiología , Músculo Liso Vascular/patología , Vena Safena/citología , Cicatrización de HeridasRESUMEN
The signal transduction pathways through which growth factors regulate vascular cell growth are not fully understood. Recent studies suggest that metabolites of the lipoxygenase pathway may be involved in vascular cell growth. We have measured the effect of the lipoxygenase pathway inhibitors nordihydroguiaretic acid (NDGA), 5,6-dehydroarachidonic acid, and baicalein on bovine capillary endothelial cell (EC) and aortic smooth muscle cell (SMC) growth in the presence or the absence of growth factors. NDGA totally suppressed serum-stimulated EC and SMC growth as well as growth factor-stimulated proliferation over a 9-day time course. Removal of the inhibitor revealed that the inhibitory effect of NDGA was reversible and not due to cytotoxicity. The morphology of NDGA-treated EC was changed in a reversible manner from the characteristic polygonal to spindle shape. The 5-lipoxygenase inhibitor 5,6-dehydroarachidonic acid had no effect on vascular cell proliferation, but inhibition of 12-lipoxygenase with baicalein blocked both EC and SMC cell growth in a dose-dependent manner, in the presence and the absence of growth factors. Indomethacin, an inhibitor of the cyclooxygenase pathway, had no effect on EC and SMC proliferation. Quinacrine and oleyloxyethylphosphorycholine inhibition of the phospholipase A2-catalyzed release of arachidonic acid from membrane phospholipids blocked growth factor- and serum-stimulated proliferation of EC and SMC. These results suggested that arachidonic acid metabolites are critical intermediaries in the regulation of vascular cell growth.
Asunto(s)
Ácido Araquidónico/metabolismo , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Flavanonas , Músculo Liso Vascular/citología , Factor de Crecimiento Derivado de Plaquetas/farmacología , Animales , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , Inhibidores de la Ciclooxigenasa/farmacología , Flavonoides/farmacología , Técnicas In Vitro , Inhibidores de la Lipooxigenasa/farmacología , Masoprocol/farmacología , Fosfolipasa D/antagonistas & inhibidores , Fosfolipasas A/antagonistas & inhibidores , Fosfolipasas A2 , Propranolol/farmacología , Quinacrina/farmacología , Transducción de SeñalRESUMEN
To investigate the role of mast cells during the process of tumor angiogenesis, we compared the rates of tumor vascularization, growth and metastasis in control WBB6F1(-)+/+ mice and in their mast-cell-deficient WBB6F1-W/Wv littermates injected with MB49 murine bladder carcinoma cells. Our results demonstrate that in mast-cell-deficient mice injected with tumor cells, there is a decreased number of capillaries at the tumor periphery, reduced tumor size relative to control mice, and an absence of metastases. In contrast, when tumor cells were inoculated intravenously, both strains of mice showed high numbers of lung metastases. These results suggested that the reduction of blood vessels at the tumor periphery may lead to a reduction in the number of metastatic cells shed into the circulation in mast-cell-deficient mice.
Asunto(s)
Mastocitos/fisiología , Metástasis de la Neoplasia , Neovascularización Patológica/fisiopatología , Neoplasias de la Vejiga Urinaria/irrigación sanguínea , Neoplasias de la Vejiga Urinaria/patología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Trasplante de NeoplasiasRESUMEN
Using high-voltage and conventional electron microscopy of cell whole mounts, we have investigated the effects of tumor-conditioned medium and hypothalmus-derived growth factor on the structure of capillary endothelial cells during their attachment and spreading in tissue culture. Cells were cultured in A, Dulbecco's Modified Eagle's Medium (DMEM) and 10% calf serum; B, equal parts of A and 48 hr mouse sarcoma conditioned medium; and C, A containing 10 units of hypothalamus-derived growth factor. Cells cultured in all three media were fully spread, and to the same extent, by 4 hr after plating. While spreading, cells cultured in DMEM alone developed prominent stress fibers and contained numerous bundles of microtubules which formed radical tracts along which mitochondria and other organelles rapidly moved to the cell periphery. Stress fibers were thinner and microtubule tracts fewer in number in cells cultured in tumor-conditioned medium. In 4 hr, organelles moved only part of the distance to the cell margin. Stress fibers were rudimentary and microtubules randomly orientated in cells exposed to hypothalamus-derived growth factor. Most organelles remained near the cell nucleus. The dramatic decrease in stress fibers and microtubule tracts in cells grown in tumor-conditioned medium and hypothalamus-derived growth factor and the subsequent decreased capacity of the cells to move organelles toward their periphery could have some functional significance relative to the growth-promoting activity of these substances.
Asunto(s)
Capilares/ultraestructura , Citoesqueleto/ultraestructura , Endotelio/ultraestructura , Sustancias de Crecimiento/farmacología , Hipotálamo/fisiología , Organoides/ultraestructura , Sarcoma Experimental/patología , Glándulas Suprarrenales/irrigación sanguínea , Animales , Capilares/efectos de los fármacos , Bovinos , Medios de Cultivo , Citoesqueleto/efectos de los fármacos , Endotelio/efectos de los fármacos , Ratones , Microscopía Electrónica , Organoides/efectos de los fármacos , ProteínasRESUMEN
Hemangiomas, the most common tumors of infancy, are characterized by a postnatal period of rapid growth, followed by a phase of gradual involution. The proliferative phase is characterized by increased numbers of endothelial and mast cells and thickened basement membrane. Ultrastructural analysis of hemangiomas in the late proliferative phase showed that mast cells had numerous fingerlike processes aligned parallel to the outer lamina of the thickened basement membranes surrounding the vessels and to the surfaces of opposing cell membranes. We found evidence of different types of interactions between mast cells and adjacent connective tissue cells (fibroblasts, macrophages, multinucleated giant cells, and plasma cells) in the perivascular regions of the lesions. Intercellular contacts were observed in areas where these mast cell processes were in close association to the opposing cell membrane. Areas of membrane fusions were seen between cell types. Coated vesicles and pinocytotic vesicles were present along the periphery of cells adjacent to mast cells. Occasionally, cytoplasmic bridges were found between mast cells and fibroblasts. These ultrastructural findings suggest the proliferation and involution of hemangiomas are determined by interactions between the various types of cells found in the lesions.