RESUMO
Although it is well known that epidermal growth factor (EGF) accelerates corneal epithelial wound healing by stimulating mitosis, it is also believed that EGF may directly stimulate the motility of individual corneal epithelial cells. We employed three different experimental methods to determine if EGF does indeed enhance the motility of corneal epithelial cells (independent of mitotic effects). First, the effects of EGF on the motility of tissue-cultured rat and rabbit corneal epithelial cells were investigated by a Boyden chamber assay. In rat corneal epithelium, these effects were further investigated by a second method, the agarose drop assay. Both assay techniques demonstrated no increase in corneal epithelial cell motility in the presence of EGF. These findings were corroborated by a third method which consisted of measuring the closure rate of epithelial wounds in organ-cultured rat corneas in the presence and absence of EGF, while concurrently arresting mitosis with colchicine. The wound closure rate before addition of any drug was 0.46 +/- 0.03 mm2/hr. The wound closure rate with EGF (50 ng/ml) was 0.55 +/- 0.03 mm2/hr, significantly (P less than 0.005) more rapid than the drug-free controls. However, when EGF (50 ng/ml) and colchicine (40 micrograms/ml) were used simultaneously, the acceleration of wound closure by EGF was completely negated by the presence of colchicine, resulting in a wound closure rate (0.46 +/- 0.06 mm2/hr) that did not differ significantly (P greater than 0.50) from that of the drug-free control.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Córnea/citologia , Fator de Crescimento Epidérmico/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Colchicina/metabolismo , Córnea/efeitos dos fármacos , Células Epiteliais , Masculino , Mitose/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos , Cicatrização/efeitos dos fármacosRESUMO
FR3T3 cells transfected with either the Ha-ras oncogene or the epidermal-growth-factor (EGF) gene demonstrate the transformed phenotype as indicated by in vitro and in vivo criteria. We have examined non-transformed FR3T3 cells as well as Ha-ras-oncogene-transformed and EGF-gene-transformed cells for expression of cell surface fibronectin and cell surface laminin. Fibronectin was absent from the surface of the Ha-ras-oncogene-transformed cells but present on both the EGF-gene-transformed cells and the non-transformed FR3T3 cells. Laminin was present on the cell surface in all 3 lines. The lack of surface fibronectin on the Ha-ras-oncogene-transformed cells was associated with reduced fibronectin production as indicated by immunoblotting of whole cell extracts and by ELISA. Concomitantly, there was a significant reduction of fibronectin binding by the Ha-ras-oncogene-transformed cells was compared to their EGF-gene-transformed and non-transformed counterparts. The Ha-ras-oncogene-transformed cells demonstrated reduced cell-substrate adhesiveness relative to the other two cell lines, as indicated by rates of attachment and spreading on plastic culture dishes in the presence of bovine serum albumin. They also demonstrated reduced adhesiveness in response to fibronectin but not laminin. Taken together, our results suggest that aberrant expression of fibronectin/fibronectin receptors is associated with Ha-ras-oncogene-induced transformation. In contrast, transformation by the EGF gene does not appear to involve aberrant expression of fibronectin/fibronectin receptors.
Assuntos
Fator de Crescimento Epidérmico/genética , Fibronectinas/biossíntese , Genes ras , Laminina/biossíntese , Receptores Imunológicos/análise , Transfecção , Animais , Adesão Celular , Divisão Celular , Ratos , Receptores de Fibronectina , Receptores de LamininaRESUMO
Normal human melanocytes were separated from keratinocytes and maintained in culture using KGM medium supplemented with 12-O-tetradecanoylphorbol acetate and cholera toxin. The melanocytes were examined for the production of extracellular matrix molecules including fibronectin, laminin, and thrombospondin and for the utilization of these molecules in adhesion and motility assays. Melanocytes produced significant amounts of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay (ELISA). Fibronectin was expressed on the surface of these cells. Laminin was also produced by melanocytes and expressed on the cell surface. The amount of laminin produced was significantly less than the amount of fibronectin. In contrast, melanocytes did not produce measurable thrombospondin as indicated by biosynthetic labeling/immunoprecipitation. Only traces of thrombospondin were detected by ELISA and no surface fluorescence was observed. When examined in adhesion and motility assays, melanocytes were found to utilize fibronectin for both processes. Laminin also stimulated adhesion but it was much less effective than fibronectin. Thrombospondin did not stimulate either attachment and spreading or motility. The pattern of extracellular matrix molecule production and utilization by melanocytes is significantly different from that shown previously for human epidermal keratinocytes (J. Varani et al., 1988, J. Clin. Invest. 81, 1537). These differences may underlie the differences with which the two cell types interact with basement membranes in vivo.
Assuntos
Matriz Extracelular/metabolismo , Melanócitos/metabolismo , Células Cultivadas , Meios de Cultura , Matriz Extracelular/fisiologia , Fibronectinas/biossíntese , Fibronectinas/fisiologia , Glicoproteínas/biossíntese , Glicoproteínas/fisiologia , Humanos , Laminina/biossíntese , Laminina/fisiologia , Melanócitos/efeitos dos fármacos , Melanócitos/fisiologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/fisiologia , Trombospondinas , Fatores de Crescimento Transformadores/farmacologiaRESUMO
Normal cultured human epidermal melanocytes and melanoma cells derived from three different malignant melanomas were examined for synthesis of extracellular matrix components before and after treatment for one day with interferon-gamma, tumor necrosis factor-alpha, or both. Treatment of the cells with either cytokine individually had minimal effects on fibronectin levels. Treatment of the cells with the two agents in combination greatly stimulated fibronectin production as indicated by biosynthetic labeling and immunoprecipitation and by enzyme-linked immunosorbent assay. Synthesis of laminin was decreased slightly by the same treatment whereas thrombospondin production was stimulated slightly. The same treatment reduced melanocyte viability slightly but significantly inhibited proliferation and altered the morphology of the melanocytes. The treated cells became flattened and polygonal in shape while the untreated cells exhibited a bipolar shape with one or more long dendritic processes. These morphologic changes were not seen in cultures treated with interferon-gamma or tumor necrosis factor-alpha individually. The effects of interferon-gamma and tumor necrosis factor-alpha on fibronectin production by epidermal melanocytes are in contrast to the effects of the same treatments on fibronectin production by epidermal keratinocytes where fibronectin production is decreased but are similar to the effects of transforming growth factor-beta on a number of other cell types in which increased synthesis of fibronectin occurs and is associated with decreased growth.
Assuntos
Fibronectinas/biossíntese , Interferon gama/farmacologia , Melanócitos/metabolismo , Melanoma/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Células Cultivadas , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Fibronectinas/farmacologia , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/fisiologia , Células Tumorais CultivadasRESUMO
We studied the impact of an endoluminally placed stented aortic graft on the geometry of a surgically created abdominal aortic dilation (AAD) in nonatherosclerotic mongrel dogs. Patulous iliac vein patch infrarenal aortoplasty produced a fusiform AAD, doubling the aorta diameter. Lumbar and mesenteric aortic tributaries were preserved and no mural thrombus formed. AADs created in 23 dogs were endoluminally excluded through transfemoral placement of a thin-wall Dacron graft 4 +/- 2 months later. Balloon-expandable stents were used to anchor each end of the graft to the aorta. The graft was crimped radially in its body and longitudinally at its ends to provide longitudinal and radial expandability in these respective zones. Serial color duplex, angiography, and direct caliper measurements were made. Before graft placement, a 19% +/- 11% diameter growth was observed. At graft placement, flow arrest immediately occurred in the space between the graft and the AAD intima in all cases. Although microscopic recanalization of the thrombus in this space was seen at sacrifice 6 and 12 months later, no macroscopic duplex flow was imaged. A 10% +/- 11% reduction in AAD diameter was measured at 6 months (p < 0.001), with no further reduction at 12 months. Graft dimensions remained stable. No anastomotic leaks developed. AAD growth stopped during the first year after effective endoluminal exclusion in normotensive dogs despite patent side branches (< 1.5 mm internal diameter) and no mural thrombus at the time of graft placement. Whether microscopic recanalization of the thrombus that forms outside the graft has an impact after 1 year remains to be seen.