RESUMEN
Keloids are benign collagenous tumors that occur during dermal wound healing in genetically predisposed individuals. The lesions are characterized by over-proliferation of fibroblasts, some leukocyte infiltration, and prolonged high rates of collagen synthesis. To determine whether leukocyte chemoattractants or chemokines are participating in this disease process, immunohistochemical staining for the CXC chemokine, MGSA/GROalpha, and its receptor, CXCR2, was performed on tissue from keloids, hypertrophic scars and normal skin. Immunoreactive MGSA/GROalpha was not observed in hypertrophic scars or normal dermis, but was present in some myofibroblasts and lymphocytes in nodular areas of the keloid samples. This staining positively correlated with the degree of inflammatory infiltrate in the lesions. Keloids, but not hypertrophic scars or normal dermis, also exhibited intensive immunoreactivity for the CXCR2 receptor in endothelial cells and inflammatory infiltrates with occasional staining of myofibroblasts. In contrast, cultured fibroblasts from either keloids or normal skin did not express detectable amounts of mRNA for MGSA/GRO or CXCR2, although interleukin-1 strongly induced MGSA/GRO mRNA in both cell types. Interleukin-1 induction of MGSA/GRO was inhibited by glucocorticoid in normal and keloid fibroblasts, and the effect was more pronounced in keloid fibroblasts. This event was not correlated with inhibition of nuclear activation of NF-kappaB, AP-1 or Sp1, and might therefore be mediated by another mechanism such as decreased mRNA stability or transcriptional repression through the glucocorticoid response element in the MGSA/GRO promoter. Data from in vitro wounding experiments with cultured normal and keloid fibroblasts indicate that there were no significant differences in MGSA/GRO or CXCR2 receptor levels between normal and keloid fibroblasts. We also show that cultured keloid fibroblasts exhibit a delayed wound healing response. We postulate that the inflammatory component is important in development of keloid lesions and chemotactic cytokines may participate in this process.
Asunto(s)
Quimiocinas CXC , Quimiocinas/análisis , Quimiocinas/genética , Factores Quimiotácticos/análisis , Factores Quimiotácticos/genética , Cicatriz/patología , Fibroblastos/química , Expresión Génica/genética , Sustancias de Crecimiento/análisis , Sustancias de Crecimiento/genética , Péptidos y Proteínas de Señalización Intercelular , Queloide/patología , Receptores de Citocinas/análisis , Receptores de Citocinas/genética , Receptores de Interleucina-8B/análisis , Receptores de Interleucina-8B/genética , Northern Blotting , Estudios de Casos y Controles , Células Cultivadas , Quimiocina CXCL1 , Cicatriz/genética , Fibroblastos/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad/genética , Humanos , Inmunohistoquímica , Interleucina-1/farmacología , Queloide/genética , Receptores de Citocinas/efectos de los fármacos , Receptores de Interleucina-8B/efectos de los fármacos , Cicatrización de Heridas/fisiologíaRESUMEN
Extracellular matrix hyaluronan is prominent during wound healing, appearing at elevated levels early in the repair process. It is prevalent throughout the course of fetal wound healing, which is scar-free, but decreases late in adult wound repair, that is often marked by scarring. To determine whether aberrant hyaluronan metabolism is associated with the excessive scarring that characterizes keloids, cultured fibroblasts derived from keloids and from the dermis of normal human skin and scar were compared. Levels of hyaluronan in 48 h conditioned media of keloid-derived cultures were significantly lower than in cultures of normal skin and scar fibroblasts. Profiles of hyaluronan polymer size were comparable in these two cell types, suggesting that excessive hyaluronan degradation was not involved. Hydrocortisone decreased hyaluronan levels approximately 70% in the conditioned media of both keloid and normal fibroblasts. Diminished hyaluronan accumulation in keloid-derived cells compared with normal fibroblasts was also observed in an in vitro wound healing model. Histolocalization of hyaluronan in keloids, normal skin, and scar samples confirmed the biochemical observations that the dermis of keloids, which comprises most of the scar tissue, contained markedly diminished levels of hyaluronan. Alterations in hyaluronan in the epidermis overlying keloids, however, were also observed. A modest increase in hyaluronan staining intensity was observed in the epidermis of keloids, as well as changes in the patterns of distribution within the epidermis, compared with that in normal skin and scar. Increased hyaluronan was present in the granular and spinous layers of the keloid epidermis Abnormalities are present apparently in both the overlying epidermis as well as in the dermis of keloids. Aberrations in signaling between keloid stroma and keloid epidermis may underlie abnormalities that contribute to the excessive fibrosis characteristic of these lesions.
Asunto(s)
Ácido Hialurónico/análisis , Queloide/metabolismo , Adolescente , Adulto , Células Cultivadas , Femenino , Fibroblastos/química , Humanos , Ácido Hialurónico/biosíntesis , Hidrocortisona/farmacología , Masculino , Cicatrización de HeridasRESUMEN
Keloids arise as benign connective tissue masses at sites of injury in genetically predisposed individuals. In addition to excessive collagen accumulation, there is biochemical and histologic evidence of elastic tissue. Previous studies showed that glucocorticoid regulation of collagen synthesis differs in fibroblasts from normal adult dermis and keloids. To define further the abnormal regulation of matrix synthesis in keloid fibroblasts, we examined glucocorticoid regulation of elastin synthesis. The basal level of elastin synthesis was significantly higher in keloid than in normal cells, and hydrocortisone reduced synthesis of elastin and elastin mRNA in normal but not in keloid fibroblasts. We had shown previously that fibroblasts from fetal dermis resembled keloid fibroblasts in glucocorticoid regulation of growth and collagen synthesis. In this study, glucocorticoids failed to down-regulate elastin synthesis in fetal cells that had not differentiated to produce normal levels of elastin, whereas fetal cells with normal elastin production exhibited glucocorticoid down-regulation. Abnormal regulation in keloid cells was independent of cell density and was confined to fibroblasts cultured from the keloid nodule. These findings reinforce the conclusion that a matrix-regulatory pathway is deranged in these focal lesions. Coordinate down-regulation of collagen and elastin by hydrocortisone in normal adult dermal fibroblasts and the failure of hydrocortisone to down-regulate synthesis of either protein in keloid cells support the existence of common elements in the regulatory pathways of these two matrix proteins.
Asunto(s)
Elastina/biosíntesis , Fibroblastos/metabolismo , Hidrocortisona/farmacología , Adulto , Regulación hacia Abajo/efectos de los fármacos , Elastina/genética , Feto/metabolismo , Humanos , Hidrocortisona/fisiología , Lactante , ARN Mensajero/análisis , Piel/citología , Piel/embriologíaRESUMEN
To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.
Asunto(s)
Proteínas de Caenorhabditis elegans , ADN/efectos de los fármacos , Dinoprostona/metabolismo , Fibroblastos/metabolismo , Queloide/metabolismo , Forbol 12,13-Dibutirato/farmacología , Receptores de Droga/metabolismo , Sitios de Unión , Proteínas Portadoras , Células Cultivadas , ADN/biosíntesis , Dinoprostona/biosíntesis , Regulación hacia Abajo , Fibroblastos/efectos de los fármacos , Fibroblastos/enzimología , Humanos , Indometacina/farmacología , Isoenzimas/metabolismo , Forbol 12,13-Dibutirato/metabolismo , Proteína Quinasa C/metabolismo , Timidina/metabolismoRESUMEN
Abnormal regulation of collagen synthesis has been observed in fibroblasts from keloids, benign collagenous tumors that develop as a result of an inherited defect in dermal wound healing. Hydrocortisone reduces the rate of collagen synthesis in fibroblasts from normal adult dermis and scars, but fails to down regulate collagen synthesis in keloid-derived fibroblasts. We show here that loss of glucocorticoid control of collagen synthesis in keloid cells is due to an inability of hydrocortisone to reduce the levels of types I, III, and V collagen mRNA, whereas it coordinately lowers these RNAs in normal adult cells. The defective regulatory mechanism is expressed only in fibroblasts from the lesion. Fibroblasts from uninvolved dermis respond normally to hydrocortisone. Not all glucocorticoid-modulated matrix proteins are abnormally regulated in this disorder; fibronectin mRNA is induced to a similar extent in normal and keloid cells. The failure of hydrocortisone to reduce collagen gene expression is also seen in fibroblasts from fetal dermis. We have reported similarities between keloid and fetal cells with regard to growth factor requirements and growth response to hydrocortisone. Thus, keloids may be due to the inappropriate expression of a pattern of growth and matrix production that is developmentally regulated.
Asunto(s)
Colágeno/genética , Hidrocortisona/farmacología , ARN Mensajero/genética , Piel/metabolismo , Adulto , Northern Blotting , Línea Celular , Células Cultivadas , Cicatriz/metabolismo , Colágeno/biosíntesis , Sondas de ADN , Feto , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibronectinas/biosíntesis , Fibronectinas/genética , Humanos , Queloide/metabolismo , ARN Mensajero/efectos de los fármacos , Valores de ReferenciaRESUMEN
Keloids are benign dermal tumors that form during an abnormal wound-healing process in genetically susceptible individuals. Although growth of normal and keloid cells did not differ in medium containing 10% (vol/vol) fetal bovine serum, keloid cultures grew to significantly higher densities than normal cells in medium containing 5% (vol/vol) plasma or 1% fetal bovine serum. Conditioned medium from keloid cultures did not stimulate growth of normal cells in plasma nor did it contain detectable platelet-derived growth factor or epidermal growth factor. Keloid fibroblasts responded differently than normal adult fibroblasts to transforming growth factor beta. Whereas transforming growth factor beta reduced growth stimulation by epidermal growth factor in cells from normal adult skin or scars, it enhanced the activity of epidermal growth factor in cells from keloids. Normal and keloid fibroblasts also responded differently to hydrocortisone: growth was stimulated in normal adult cells and unaffected or inhibited in keloid cells. Fetal fibroblasts resembled keloid cells in their ability to grow in plasma and in their response to hydrocortisone. The ability of keloid fibroblasts to grow to higher cell densities in low-serum medium than cells from normal adult skin or from normal early or mature scars suggests that a reduced dependence on serum growth factors may account for their prolonged growth in vivo. Similarities between keloid and fetal cells suggest that keloids may result from the untimely expression of a growth-control mechanism that is developmentally regulated.
Asunto(s)
Queloide/patología , Factor de Necrosis Tumoral alfa/farmacología , Adulto , División Celular/efectos de los fármacos , Células Cultivadas , Cicatriz , Replicación del ADN/efectos de los fármacos , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Recién Nacido , Cinética , Masculino , Neoplasias/patología , Valores de Referencia , Piel/citología , Piel/efectos de los fármacosRESUMEN
The rate of proline transport increases when human dermal fibroblasts are grown in physiological levels of hydrocortisone. This response to hydrocortisone is significantly greater in fibroblasts derived from keloids, benign dermal tumors caused by an inherited abnormality in wound healing (Russell, S. B., Russell, J. D., and Trupin, J. S. (1982) J. Biol. Chem. 257, 9525-9531). We report here that increased transport activity is largely accounted for by an increase in Vmax of the System A component of proline uptake; this stimulation is 1- to 2-fold in normal fibroblasts and 5- to 10-fold in keloid-derived cells. Similar results are obtained with 2-(methylamino)isobutyric acid, a specific substrate of System A transport, and for the System A components of glycine and alanine uptake. The stimulatory effect of the hormone is blocked by cycloheximide and actinomycin D in both keloid and normal cells. Hydrocortisone did not alter the measured membrane potential in either cell type. These data suggest that hydrocortisone induces a protein specifically involved in System A amino acid transport. Keloid cells may provide a unique opportunity to study this protein.
Asunto(s)
Aminoácidos/metabolismo , Hidrocortisona/farmacología , Queloide/metabolismo , Piel/metabolismo , Ácidos Aminoisobutíricos/metabolismo , Transporte Biológico Activo , Fibroblastos/metabolismo , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Compuestos Onio/farmacología , Compuestos Organofosforados/farmacología , Prolina/metabolismo , Sodio/metabolismoRESUMEN
The effects of ascorbic acid and hydrocortisone on activity of prolyl hydroxylase in fibroblasts from keloid and normal human dermis were investigated and compared to the effects of these agents on collagen synthesis. Prolyl hydroxylase activity in normal fibroblasts grown to confluency in 1.5 microM hydrocortisone was approximately half that of cells grown without the steroid. The concentration of hydrocortisone effective in reducing enzyme activity was the same as that for reducing the rate of collagen synthesis; a half-maximal effect on both parameters was achieved at 10(-7) M. Hydrocortisone lowered enzyme activity through most of the culture cycle. Fibroblasts derived from keloids were significantly less subject to hydrocortisone-mediated reduction of prolyl hydroxylase activity and rate of collagen synthesis. This difference between keloid and normal cells was dependent on the simultaneous presence of ascorbic acid and hydrocortisone. These data suggest that the defect in wound healing that results in keloid formation is associated with a change in a regulatory mechanism that controls the rate of collagen synthesis and is sensitive to physiological levels of hydrocortisone. Continuous culture of fibroblasts in medium supplemented with ascorbic acid also lowered prolyl hydroxylase activity. Unlike the effect of hydrocortisone, growth in ascorbate increased the rate of collagen synthesis and affected keloid and normal strains equally.
Asunto(s)
Ácido Ascórbico/farmacología , Hidrocortisona/farmacología , Queloide/enzimología , Procolágeno-Prolina Dioxigenasa/metabolismo , Técnicas de Cultivo , ADN/metabolismo , Fibroblastos/metabolismo , Humanos , Factores de TiempoRESUMEN
The rate of proline transport increases significantly when human dermal fibroblasts are grown with 1.5 microM hydrocortisone. Fibroblasts derived from keloid tissue are significantly more stimulated than normal fibroblasts. An average increase of 41% is obtained with 8 normal strains, whereas uptake in 8 keloid-derived strains increases 210%. Similar results are obtained with the system A amino acid analogue 2-(methylamino)isobutyric acid, for which the uptake rate increases 87% and 329% in normal and keloid cells, respectively. The hydrocortisone-mediated increase in proline transport and the difference between keloid and normal fibroblasts are observed throughout the culture cycle and after depletion of amino acid pools. The uptake of nine other amino acids are differentially altered in normal and keloid cells. Competition experiments with 2-(methylamino)isobutyric acid indicate that the greatest differences occur with amino acids that are transported preferentially by the A system. Inhibition of the hydrocortisone-mediated increase by progesterone and a lag period of approximately 3 h indicate that hydrocortisone is regulating proline transport by a cytosolic receptor mechanism.