Effects of density and cellular aging on collagen synthesis and growth kinetics in keloid and normal skin fibroblasts

Summary Cell proliferation and collagen synthesis were analyzed in high, medium, and low density keloid and normal skin fibroblasts and also at various times during the course of in vitro aging to expand previous findings of increased collagen synthesis in keloid compared to normal skin fibroblasts. It was found that both keloid and normal fibroblasts (<20 population doublings) responded similarly to high, medium, and low initial plating densities; however, sparsely plated keloid fibroblasts exhibited a loss of replicative capacity earlier in their in vitro lifespan than did sparsely plated normal skin fibroblasts. When analyzed at population doubling levels 2 to 38, collagen synthesis was elevated in keloid compared to normal skin fibroblasts but decreased at the same rate in both cell types throughout this in vitro interval. Supported by NIH Grant GM-20298.     

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 syntehsis is inhibited 60% in normal cultures by hydrocortisone (0.5 μg/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 syntehsis 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.     

Biochemical and dynamic studies of collagen from human normal skin and keloid tissue

Keloids are exuberant scars, in which collagen, fibronectin and glycosaminoglycans are overdeposited. Biochemical analysis of the collagen isolated from normal skin and keloid tissue by pepsin treatment, indicated an increase in the type III and GAG content. Viscosity measurements of collagen from normal skin and keloid tissue were used in the present study to establish the interaction between collagen and GAG. Physico-chemical properties such as intrinsic viscosity, reduced viscosity and hydrated volume were computed from viscosity measurements. These measurements were also used to determine the denaturation temperature of collagen which was further confirmed by DSC measurements. Chondroitinase has been used in this study to probe the influence of GAG on the physico-chemical characteristic of keloid collagen.     

Quantitative assay of types I and III collagen synthesized by keloid biopsies and fibroblasts

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts, However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.     

Quantitative assay of types I and III collagen synthesized by keloid biopsyes and fibroblasts.

Molecular sieve column chromatography was used to determine the amount of type I and III collagen synthesized by normal dermis and keloid biopsies and fibroblasts derived from these tissues. After incubation with radioactive proline, the collagen was extracted and separated into types I and III and then quantitated. There was no significant difference in the percent type III collagen synthesized by fresh keloid biopsies compared to normal dermis. Likewise, there was no significant difference in the percent type III collagen synthesized by keloid fibroblasts compared to normal dermal fibroblasts. However, fibroblasts from both keloid and normal dermis synthesized a lower percentage of type III collagen in cell culture compared to the original biopsies. These findings demonstrate that keloid collagen has the same type distribution as normal dermis and suggest that increased collagen synthesis in these lesions is not related to altered collagen types.     

Differential expression of transforming growth factor-beta receptors I and II and activation of Smad 3 in keloid fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in an excessive deposition of extracellular matrix, especially collagen. However, the molecular mechanisms regulating this pathologic collagen deposition still remain to be elucidated. A previous study by this group demonstrated that transforming growth factor (TGF)-beta1 and -beta2 ligands were expressed at greater levels in keloid fibroblasts when compared with normal human dermal fibroblasts (NHDFs), suggesting that TGF-beta may play a fibrosis-promoting role in keloid pathogenesis.To explore the biomolecular mechanisms of TGF-beta in keloid formation, the authors first compared the expression levels of the type I and type II TGF-beta receptors in keloid fibroblasts and NHDFs. Next, they investigated the phosphorylation of Smad 3, an intracellular TGF-beta signaling molecule, in keloid fibroblasts and NHDFs. Finally, they examined the regulation of TGF-beta receptor II by TGF-beta1, TGF-beta2, and TGF-beta3 ligands.Our findings demonstrated an increased expression of TGF-beta receptors (types I and II) and increased phosphorylation of Smad 3 in keloid fibroblasts relative to NHDFs. These data support a possible role of TGF-beta and its receptors as fibrosis-inducing growth factors in keloids. In addition, all three isoforms of recombinant human TGF-beta proteins could further stimulate the expression of TGF-beta receptor II in both keloids and NHDFs. Taken together, these results substantiate the hypothesis that the elevated levels of TGF-beta ligands and receptors present in keloids may support increased signaling and a potential role for TGF-beta in keloid pathogenesis.     

The effect of superpulsed carbon dioxide laser energy on keloid and normal dermal fibroblast secretion of growth factors: a serum-free study

An in vitro model was used to determine the effect of superpulsed CO2 laser energy on normal dermal and keloid-producing fibroblast proliferation and release of growth factors. Growth factors assayed included basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). bFGF is mitogenic, inhibits collagen production, and stabilizes cellular phenotype. TGF-beta1 stimulates growth and collagen secretion and is thought to be integral to keloid formation. Growth in a serum-free medium allowed measurement of these growth factors without confounding variables. Keloid and normal dermal fibroblasts cell lines were established from facial skin samples using standard explant techniques. Samples consisted of three separate keloid and three separate normal dermal fibroblast cell lines. Cells were used at passage 4 to seed 24-well trays at a concentration of 6 x 10(4) cells per milliliter in serum-free medium. At 48 hours, 18.8 percent of each cell well was exposed to a fluence of 2.4, 4.7, and 7.3 J/cm2 using the superpulsed CO2 laser. Cell viability and counts were established at four time points: 0 (time of superpulsed CO2 laser treatment), 24, 72, and 120 hours. Supernatants were collected and assessed for bFGF and TGF-beta1 using a sandwich enzyme immunoassay. All cell lines demonstrated logarithmic growth through 120 hours (conclusion of experiment), with a statistically significant shorter population doubling time for keloid fibroblasts (p < 0.05). Use of the superpulsed CO2 laser shortened population doubling times relative to that of controls; the differences were statistically significant in keloid dermal fibroblasts when fluences of 2.4 and 4.7 J/cm2 were used (p < 0.05 and 0.01, respectively). bFGF was present in greater levels in normal dermal fibroblasts than in keloid dermal fibroblasts. Application of superpulsed CO2 demonstrated a trend toward increased bFGF secretion in both fibroblast types; the increase was significant in the keloid group at 4.7J/cm2. A consistent trend in suppression of TGF-beta1 was seen in both groups exposed to superpulsed CO2, with the maximal effect occurring at 4.7 J/cm2. Serum-free culture sustains logarithmic cell growth and allows growth factor measurement without confounding variables from serum-containing media. Superpulsed CO2 enhances fibroblast replication and seems to stimulate bFGF secretion and to inhibit TGF-beta1 secretion. Given the function of these growth factors, the application of superpulsed CO2 may support normalized wound healing. These findings may explain the beneficial effects of laser resurfacing on a cellular level and support the use of superpulsed CO2 in the management of keloid scar tissue.     

Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.     

Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid

Keloid is a dermal fibrotic disease characterized by excessive accumulation of mainly type I collagen in extracellular matrix of the dermis. We have studied the expression levels of collagen types I and III, and its molecular chaperone HSP47 in keloid lesions and surrounding unaffected skin using Northern and Western blotting and immunohistochemical analyses. Collagen types I and III mRNA levels were found to be upregulated 20-fold in keloid tissues, contradicting previous reports of nearly normal type III collagen levels in this disease. HSP47 expression in keloid lesions was also highly upregulated; eightfold at mRNA level and more than 16-fold at the protein level. Strong upregulation of these three proteins in keloid was confirmed by immunohistochemical staining. These results suggest that accumulation of both type I and type III collagen is important for the development of keloid lesions, and that HSP47 plays a role in the rapid and extensive synthesis of collagen in keloid tissues.     

Investigation of the influence of keloid-derived keratinocytes on fibroblast growth and proliferation in vitro

Keloids are disfiguring, proliferative scars that represent a pathological response to cutaneous injury. The overabundant extracellular matrix formation, largely from collagen deposition, is characteristic of these lesions and has led to investigations into the role of the fibroblast in its pathogenesis. Curiously, the role of the epidermis in extracellular matrix collagen deposition of normal skin has been established, but a similar hypothesis in keloids has not been investigated. The aim of this study was to investigate the influence of keloid epithelial keratinocytes on the growth and proliferation of normal fibroblasts in an in vitro serum-free co-culture system. A permeable membrane separated two chambers; the upper chamber contained a fully differentiated stratified epithelium derived from the skin of excised earlobe keloid specimens, whereas the lower chamber contained a monolayer of normal or keloid fibroblasts. Both cell types were nourished by serum-free medium from the lower chamber.Epithelial keratinocytes from five separate earlobe keloid specimens were investigated. Four sets of quadruplicates were performed for each specimen co-cultured with normal fibroblasts or keloid-derived fibroblasts. Controls consisted of (1) normal keratinocytes co-cultured with normal fibroblasts, and (2) fibroblasts grown in serum-free media in the absence of keratinocytes in the upper chamber. Fibroblasts were indirectly quantified by 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay, with results confirmed by DNA content measurement, at days 1 and 5 after the co- culture initiation.Significantly, increased proliferation was seen in fibroblasts co-cultured with keloid keratinocytes, as compared with the normal keratinocyte controls at day 5 (analysis of variance, p < 0.001). These results strongly suggest that the overlying epidermal keratinocytes of the keloid may have an important, previously unappreciated role in keloid pathogenesis using paracrine or epithelial-mesenchymal signaling.     

Immunoglobulin, complement, and histocompatibility antigen studies in keloid patients.

The increased collagen synthesis and deposition, which is characteristic of keloids, may be related to an immune response initiated by wounding. Therefore, we examined various systemic and localized immune parameters in keloid patients to establish if such factors are related to keloid pathogenesis. To determine if there is a systemic immune response, we compared the serum levels of IgG and IgM in keloid patients to those in a closely matched population. In addition, we measured complement levels (Clq, C3, and C4) and receptors for sheep (E), mouse erythrocytes (MRBC), and complement (EAC) on blood lymphocytes. All of these were in the normal range in the keloid patients. However, the extractable IgG from keloid tissue was significantly increased (compared to normal skin and normal scar controls), suggesting a localized immune response. To determine whether keloid formation is associated with a specific histocompatibility locus, human lymphocyte antigen (HLA) profiles of 45 keloid patients were analyzed; no significant differences in the incidence of HLA-A and B antigens were found (compared to 200 controls). These studies suggest that there is a localized immune response involved in keloid pathogenesis, one which is not related to either the HLA-A or B histocompatibility loci.     

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.     

Cellular signaling by tyrosine phosphorylation in keloid and normal human dermal fibroblasts

Keloids represent a dysregulated response to cutaneous wounding that results in disfiguring scars. Unique to humans, keloids are characterized by an accumulation of extracellular matrix components. The underlying molecular mechanisms of keloid pathogenesis, however, remain largely uncharacterized. Similarly, cellular signaling mechanisms, which may indicate inherent differences in the way keloid fibroblasts and normal human dermal fibroblasts interact with extracellular matrix or other cells, have not been investigated. As part of a fundamental assessment of cellular response to injury in keloid fibroblasts, phosphorylation studies were performed using three different keloid (n = 3) and normal human dermal (n = 3) fibroblast cell lines. These studies were undertaken to elucidate whether keloid and normal human dermal fibroblasts exhibit different tyrosine kinase activity. Initially, distinct tyrosine phosphorylation patterns of keloid and normal human dermal fibroblasts were demonstrated. Next, the phosphorylation patterns were correlated with known molecules that may be important to keloid pathogenesis. On the basis of molecular weight, it was hypothesized that the highly phosphorylated bands seen in keloid fibroblasts represented epidermal growth factor receptor (EGFR); discoidin domain receptor 1 (DDR1); and Shc, an adaptor protein known to bind many tyrosine kinases, including EGFR and DDR1. Individual immunoblotting using EGFR, DDR1, and Shc antibodies revealed greater expression in keloid fibroblasts compared with normal human dermal fibroblasts. These data substantiate for the first time the finding of greater phosphorylation by the above-mentioned molecules, which may be important in keloid pathogenesis.     

Fibronectin is overproduced by keloid fibroblasts during abnormal wound healing.

Wound healing in certain individuals leads to the development of keloid tumors which exhibit abnormal collagen metabolism and an increased abundance of extracellular matrix components. Comparison of fibronectin levels in fibroblasts derived from keloids and normal dermis revealed a relative increase in intracellular and extracellular fibronectin in the keloid-derived cells. While fibronectin was similarly processed, compartmentalized, and degraded by both cell types, fibronectin biosynthesis was found to be accelerated as much as fourfold in keloid fibroblasts due to a corresponding increase in the amount of accumulated fibronectin mRNA. These changes account for the elevated steady-state level of the molecule in keloid fibroblasts and suggest that increased fibronectin in keloid lesions is due to overproduction by the wound-healing fibroblasts. Glucocorticoid treatment stimulated fibronectin biosynthesis in both normal and keloid fibroblasts. However, the amount of stimulation was less for the keloid-derived cells, indicating a limitation on maximal rates of fibronectin biosynthesis. These observations suggest that separate mechanisms act to control basal and maximal rates of fibronectin production. Biosynthesis of the 140-kilodalton fibronectin receptor was also found to be increased in keloid fibroblasts, suggesting some level of coordinate regulation for fibronectin and fibronectin receptor expression.     

Fibronectin (FN) in hypertrophic scars and keloids

Summary Fibronectin (FN) distribution was compared among samples of normal human dermis, hypertrophic scar, keloid, and granulation tissues from deep injuries. Localization was established by use of fibronectin antibodies and the indirect immunofluorescence method. Fresh-frozen tissue was sectioned on a cryostat and examined by epifluorescence. Hypertrophic scar and keloid demonstrated heavy deposition of FN, which conformed to the nodular characteristics of the lesions. Intense localization occurred in granulation tissue over fibroblasts which were stellate and vesiculated, and over small blood vessels. FN-staining was weak in areas over fibroblasts which were more rounded and nonvesiculated. Staining for FN was also minimal over the collagen in normal dermis and the deeper, larger collagen fascicles in the lesions. Fibroblasts cultured from normal dermis, hypertrophic scar, and keloid for 5–6 weeks were intensely stained for FN. Extracellular matrix was heavily positive in cultures from the lesions compared with those from normal dermis.Supported in part by NIH Research Grant 1 R01GM 25159     

Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.     

Asiatic Acid Isolated From Centella Asiatica Inhibits TGF-β1-induced Collagen Expression in Human Keloid Fibroblasts via PPAR-γ Activation

Keloids are fibroproliferative disorders characterized by exuberant extracellular matrix deposition and transforming growth factor (TGF)-β/Smad pathway plays a pivotal role in keloid pathogenesis. Centella asiatica extract has been applied in scar management for ages. As one of its major components, asiatic acid (AA) has been recently reported to inhibit liver fibrosis by blocking TGF-β/Smad pathway. However, its effect on keloid remains unknown. In order to investigate the effects of AA on cell proliferation, invasion and collagen synthesis, normal and keloid fibroblasts were exposed to TGF-β1 with or without AA. Relevant experiments including 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, 5-ethynyl-2-deoxyuridine (EdU) incorporation assay, Transwell invasion assay, enzyme-linked immunosorbent assay, Western blot, quantitative polymerase chain reaction and RNA interference assay were conducted. As a result, keloid fibroblasts showed higher responsiveness to TGF-β1 stimulation than normal fibroblasts in terms of invasion and collagen synthesis. AA could suppress TGF-β1-induced expression of collagen type I, inhibit Smad 2/3 phosphorylation and plasminogen activator inhibitor-1 (PAI-1) expression, while elevate Smad 7 protein level. Noteworthy, the effects of AA on keloid fibroblasts could be abrogated by PPAR-γ antagonist GW9662 and by silencing of PPAR-γ. The present study demonstrated that AA inhibited TGF-β1-induced collagen and PAI-1 expression in keloid fibroblasts through PPAR-γ activation, which suggested that AA was one of the active constituents of C. asiatica responsible for keloid management, and could be included in the arsenal for combating against keloid.     

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.