Reduced hyaluronan in keloid tissue and cultured keloid fibroblasts.

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.

Glucocorticoid regulation of elastin synthesis in human fibroblasts: down-regulation in fibroblasts from normal dermis but not from keloids.

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.

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

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.

Differential glucocorticoid regulation of collagen mRNAs in human dermal fibroblasts. Keloid-derived and fetal fibroblasts are refractory to down-regulation.

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.

Reduced growth-factor requirement of keloid-derived fibroblasts may account for tumor growth.

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.

Glucocorticoid receptors in human fibroblasts derived from normal dermis and keloid tissue.

Hydrocortisone stimulates proliferation and System A amino acid transport in cultured human fibroblasts, while decreasing production of collagen. Fibroblasts isolated from keloid tissue have an unusual glucocorticoid response; they are hyporesponsive with regard to proliferation and collagen production but hyperresponsive with regard to the induction of System A amino acid transport (Russell, J. D., Russell, S. B., and Trupin, K. M. (1978) J. Cell. Physiol. 97, 221-229; Russell, S. B., Russell, J. D., and Trupin, J. S. (1982) J. Biol. Chem. 256, 9525-9531). We show here that despite these differences, the glucocorticoid receptors of keloid cells do not differ from those of normal dermal fibroblasts in steroid specificity, dissociation constant (Kd), total number of binding sites (Bmax), or nuclear binding of glucocorticoid-receptor complexes. A single glucocorticoid binding species of molecular weight 93,000 was found in both cell types. A monolayer assay for glucocorticoid receptor binding is described which facilitates analysis of multiple strains of cultured cells. This assay gives the same specificity and dissociation constants as the conventional cytosol assay, but it is more sensitive. The magnitude of induction of System A amino acid transport was found to be directly proportional to glucocorticoid receptor occupancy in both keloid-derived and normal fibroblasts. This induction requires serum, which can be replaced with 1 nM insulin.

Hydrocortisone induction of system A amino acid transport in human fibroblasts from normal dermis and keloid.

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.

Variation in prolyl hydroxylase activity of keloid-derived and normal human fibroblasts in response to hydrocortisone and ascorbic acid.

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.

Alteration of amino acid transport by hydrocortisone. Different effects in human fibroblasts derived from normal skin and keloid.

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.

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: genetic or epigenetic?

Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density.

Collagen synthesis in human fibroblasts: effects of ascorbic acid and regulation by hydrocortisone.

The effects of hydrocortisone and ascorbic acid on collagen and noncollagen protein synthesis, and on growth were examined in fibroblasts derived from normal human dermis. When the medium was supplemented with 0.28 mM ascorbic acid, the apparent rate of collagen production increased 2-3 fold over the culture cycle. Ascorbic acid also caused a small increase in the apparent rate of synthesis of noncollagen protein and an elevation in growth rate and maximum cell density. Growth was not required for the increase in collagen production since addition of ascorbate to confluent cultures induced a similar increase. Hydrocortisone (1.5 microM) blocked the ascorbate-related increase in collagen production during growth and in confluent cultures. The hormone simultaneously increased the apparent rate of noncollagen protein production and maximum cell density, suggesting that the effect on collagen synthesis was specific. Inhibition of collagen production by hydrocortisone was observed only in the presence of ascorbate, while the increase in growth and noncollagen protein production occurred in the presence and absence of the vitamin.

Differential effects of hydrocortisone on both growth and collagen metabolism of human fibroblasts from normal and keloid tissue.

Cultured fibroblasts isolated from normal and keloid tissue do not differ in their growth characteristics or in the rate of collagen synthesis under routine culture conditions. The addition of hydrocortisone to the culture media results in significant differences in both growth and collagen synthesis between these cell types. Collagen synthesis is inhibited 60% in normal cultures by hydrocortisone (0,5 micrograms/ml) and the population size at which density-dependent growth inhibition is achieved is increased. Keloid-derived fibroblasts grow to a lower maximum density in the presence of hydrocortisone, while their rate of collagen synthesis is not significantly reduced. The rate of non-collagen protein synthesis is increased significantly by hydrocortisone in both cell types. Comparison of normal and keloid-derived cultures obtained from a single individual suggests that the keloid phenotype with respect to both growth and collagen synthesis is restricted to the fibroblasts isolated from the keloid nodule.

The effect of histamine on the growth of cultured fibroblasts isolated from normal and keloid tissue.

Cultured fibroblasts derived from human keloid tissue are presented as a possible model system for studying the genetic regulation of cell growth. Histamine is shown to have a marked effect on the growth of cultured fibroblasts. A small increase in growth rate is seen during the log phase of the culture cycle and a 50% increase in cell number is observed during the plateau phase. Differences in the extent of growth stimulation are observed between strains isolated from different individuals. While most strains showed approximately 50% stimulation, a few were not stimulated and some strains gave a 100% or greater increase in cell number due to histamine. This phenotypic difference in extent of growth stimulation in response to histamine cannot be attributed to the gene or genes for keloid formation. However, elevated levels of histamine in vivo may be a contributing factor to the abnormal cell growth observed in this disorder. The extent of growth stimulation due to histamine decreases with repeated subculturing.

The microvasculature of the spleen.

A method for examining the microvasculature of the dog spleen by angiography is described and the findings are related to morphological studies. The marginal sinus of the lymphoid follicle has been shown to be an important part of the vascular pathway in the spleen. It allows intimate mixing of blood elements and spleen cells and it is suggested that this plays an important immunological role. The control of blood flow to the lymphoid follicle is discussed but requires further elucidation.

The mechanism of action of mercuric chloride on the isolated perfused rat kidney.

Studies on 80 rat kidneys, perfused at constant flow, showed that mercuric chloride produced a marked increase in perfusion pressure within five minutes of administration. Adrenergic blocking agents (phentolamine and propranolol), angiotensin sensitivity depletion, bradykinin and low concentrations of mannitol had no effect on this increase in resistance. Perfusion of the kidney with 5 percent mannitol solution, however, significantly reduced the increase of vascular resistance induced by mercuric chloride. Evidence is presented that mercuric chloride may evoke an increase in vascular resistance by inducing endothelial cell swelling, an action antagonised by the hypertonic effect of mannitol. Other possible contributory mechanisms are also discussed.