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.     

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.     

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.     

microRNA deregulation in keloids: an opportunity for clinical intervention?

Keloids are defined as benign dermal scars invading adjacent healthy tissue, characterized by aberrant fibroblast dynamics and overproduction of extracellular matrix. However, the aetiology and molecular mechanism of keloid production remain poorly understood. Recent discoveries have shed new light on the involvement of a class of non‐coding RNAs known as microRNAs (miRNA), in keloid formation. A number of miRNAs have differential expression in keloid tissues and keloid‐derived fibroblasts. These miRNAs have been characterized as novel regulators of cellular processes pertinent to wound healing, including extracellular matrix deposition and fibroblast proliferation. Delineating the functional significance of miRNA deregulation may help us better understand pathogenesis of keloids, and promote development of miRNA‐directed therapeutics against this condition.     

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.     

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.     

Keloids in rural black South Africans. Part 3: a lipid model for the prevention and treatment of keloid formations

In the third part of this study a basic lipid model (regarding phospholipids, triglycerides, cholesterol esters and free fatty acids) for keloids (n=20), compared with normal skin of keloid prone and non-keloid prone patients (n=20 of each), was constructed according to standard methods, to serve as a sound foundation for essential fatty acid supplementation strategies in the prevention and treatment of keloid formations. Essential fatty acid deficiency (EFAD) of the omega-6 series (linoleic acid (LA), g-linolenic acid (GLA), and dihomo-g-linolenic acid (DGLA)) and the omega-3 series (a-linolenic acid (ALA) and eicosapentaenoic acid (EPA)), but enhanced arachidonic acid (AA) levels, were prevalent in keloid formations. Enhanced AA, but a deficiency of AA precursors (LA, GLA and DGLA) and inflammatory competitors (DGLA and EPA), are inevitably responsible for the overproduction of pro-inflammatory metabolites (prostaglandin E(2)(PGE(2))) participating in the pathogenesis of inflammation. Of particular interest was the extremely high free oleic acid (OA) levels present, apart from the high free AA levels, in the keloid formations. OA stimulates PKC activity which, in turn, activates PLA(2)activity for the release or further release of AA from membrane pools. Interactions between EFAs, eicosanoids, cytokines, growth factors and free radicals can modulate the immune response and the immune system in undoubtedly involved in keloid formation. The histopathology of keloids can be adequately explained by: persistence of inflammatory- and cytokine-mediated reactions in the keloid/dermal interface and peripheral areas, where fibroblast proliferation and continuous depletion of membrane linoleic acid occur; microvascular regeneration and circulation of sufficient EFAs in the interface and peripheral areas, where maintenance of metabolic active fibroblasts for collagen production occur; microvessel occlusion and hypoxia in the central areas, where deprivation of EFAs and oxygen with consequent fibroblast apoptosis occur, while excessive collagen remain. All these factors contribute to different fibroblast populations present in: the keloid / dermal interface and peripheral areas where increases in fibroblast proliferation and endogenous TGF-b occur, and these metabolic active fibroblast populations are responsible for enhanced collagen production: the central areas where fibroblast populations under hypoxic conditions occur, and these fibroblasts are responsible for excessive collagen production. It was concluded that: fibroblast membrane EFAD of AA precursors and inflammatory competitors, but prevailing enhanced AA levels, can contribute to a chain of reactions eventually responsible for keloid formations.     

TN-C与MDA-7在增生性瘢痕中表达与调控

增生性瘢痕是以皮肤损伤后成纤维细胞过度增殖为特征的一种病理改变,其发病机制尚不明确,目前没有有效的治疗方法。当皮肤组织损伤时,腱糖蛋白C(Tenascin-C,TN-C)具有多种不同的作用介导炎症和纤维化进程,并使组织有效修复。TN-C是细胞外基质中一个具有独特的六聚体结构的寡聚糖蛋白家族,TN-C一过性表达在器官形成期,在大多数成人组织不表达或表达极少。然而,在病理条件下TN-C表达增加,诸如炎症,伤口愈合和纤维化。TN-C参与胚胎形成、肿瘤发生及损伤修复过程有关,参与细胞黏附、增殖、迁徙、分化、细胞间相互作用以及细胞凋亡。黑色素瘤分化相关基因7/白介素24(MDA-7/IL-24)能选择性抑制瘢痕疙瘩中成纤维细胞的增殖,并诱导瘢痕疙瘩中成纤维细胞的凋亡,而对正常细胞无任何作用。MDA-7/IL-24很可能与瘢痕的形成有关。     

TWEAK/Fn14 signaling may function as a reactive compensatory mechanism against extracellular matrix accumulation in keloid fibroblasts

Overabundance of the extracellular matrix resulting from hyperproliferation of keloid fibroblasts (KFs) and dysregulation of apoptosis represents the main pathophysiology underlying keloids. TWEAK is a weak apoptosis inducer, and it plays a critical role in pathological tissue remodeling via its receptor, Fn14. However, the role of TWEAK/Fn14 signaling in the pathogenesis of keloids has not been investigated. In this study, we confirmed the overexpression levels of TWEAK and Fn14 in clinical keloid tissue specimens and primary KFs. The extracellular matrix (ECM)-related genes were also evaluated between primary KFs and their normal counterparts to determine the factors leading to the formation or development of keloids. Unexpectedly, exogenous TWEAK significantly reduced the levels of collagen I and collagen III, as well as alpha-smooth muscle actin (α-SMA). Additionally, TWEAK promoted MMPs expression and apoptosis activity of KFs. Furthermore, we verified that the inhibitory effect of TWEAK on KFs is through down-regulation of Polo-like kinase 5, which modulates cell differentiation and apoptosis. The TWEAK-Fn14 axis seems to be a secondary, although less effective, compensatory mechanism to increase the catabolic functions of fibroblasts in an attempt to further decrease the accumulation of collagen.Data AvailabilityAll data generated or analyzed during this study are included in this published article (and its Supporting Information files).     

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.     

Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. The keloid matrix contains excess collagen and glycosaminoglycans (GAGs), but lacks elastic fiber. However, the roles of these matrix components in the pathogenesis of keloid are largely unknown. Here, we show that elastin and DANCE (also known as fibulin-5), a protein required for elastic fiber formation, are not deposited in the extracellular matrix of keloids, due to excess accumulation of chondoitin sulfate (CS), although the expression of elastin and DANCE is not affected. Amount of CS accumulated in the keloid legion was 6.9-fold higher than in normal skin. Fibrillin-1, a scaffold protein for elastic fiber assembly, was abnormally distributed in the keloid matrix. Addition of purified CS to keloid fibroblast culture resulted in abnormal deposition of fibrillin-1, concomitant with significantly decreased accumulation of elastin and DANCE in the extracellular matrix. We propose that CS plays a crucial role in the development of keloid lesions through inhibition of elastic fiber assembly.     

Downregulation of PLK4 expression induces apoptosis and G0/G1‐phase cell cycle arrest in keloid fibroblasts

ObjectivesKeloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids.Materials and MethodsWe evaluated the expression of PLK4 in keloids and adjacent normal skin tissue samples. Then, we established PLK4 knockdown and overexpression cell lines in keloid fibroblasts (KFs) and normal skin fibroblasts (NFs), respectively, to investigate the roles of PLK4 in the regulation of proliferation, migration, invasion, apoptosis, and cell cycle in KFs. Centrinone B (Cen‐B), a highly selective PLK4 inhibitor, was used to inhibit PLK4 activity in KFs to evaluate the therapeutic effect on KFs.ResultsWe discovered that PLK4 was overexpressed in keloid dermal samples and KFs compared with adjacent normal skin samples and NFs derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by Cen‐B suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase in vitro.ConclusionsThese findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.

Keloids are benign fibroproliferative tumors that display many cancer‐like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo‐like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids. Here, we discovered that PLK4 is a potential target for the treatment of keloids. PLK4 was overexpressed in keloid dermal samples and keloid fibroblasts (KFs) compared with adjacent normal skin samples and normal skin fibroblasts derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by a highly selective inhibitor, centrinone B (Cen‐B), suppressed KF growth, induced KF apoptosis via the caspase‐9/3 pathway, and induced cell cycle arrest at the G0/G1 phase via the p53/p21/Cyclin D1 pathway in vitro. These findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen‐B is a promising candidate drug for keloid treatment.     

Anti-fas induces apoptosis and proliferation in human dermal fibroblasts: Differences between foreskin and adult fibroblasts

Apoptosis, or programmed cell death, is a naturally occurring process mediated by extracellular signals. We studied anti-Fas (CD95/Apo-1) antibody-induced apoptosis in cultured human foreskin and adult dermal fibroblasts. Induction of apoptosis was identified by fluorescence in situ DNA end-labeling. Anti-Fas antibody induced apoptosis in fibroblasts in a dose- and time-dependent manner. Adult dermal skin fibroblasts were more susceptible to anti-Fas antibody-induced apoptosis than foreskin fibroblasts, with 21–52% dead cells in different strains. In foreskin fibroblasts, anti-Fas antibody (1.0 μg/ml) predominantly induced proliferation ([³H]thymidine incorporation increased to 115–165% of control level) and only low levels of apoptotic cell death after 48 hours of treatment. No induction of proliferation by anti-Fas was found in the adult fibroblasts. Addition of tumor necrosis factor-α (TNF-α) slightly augmented the anti-Fas antibody-induced apoptosis in both cell types. When we examined the levels of Fas expression using flow cytometry, we found two- to threefold higher Fas expression in adult fibroblasts. C6-ceramide treatment, which induces Fas-independent apoptosis, gave similar levels of cell death in both foreskin and adult fibroblasts. No proliferation was observed in C6-ceramide-treated fibroblasts. Thus, this difference in apoptosis between adult dermal and foreskin fibroblasts appears to be related to the level of Fas expression. When clones of foreskin fibroblasts were examined, there was heterogeneity of anti-Fas antibody-induced apoptosis and proliferation in the cloned fibroblast subpopulations, but this was not correlated with differences in Fas expression. Alterations in fibroblast populations during the process of differentiation and aging may result from selective loss of apoptosis-susceptible populations. J. Cell. Physiol. 175:19–29, 1998. © 1998 Wiley-Liss, Inc.     

The role of the activin system in keloid pathogenesis

Keloid scars represent a pathological response to cutaneous injury under the regulation of many growth factors. Activin-A, a dimeric protein and a member of the transforming growth factor- superfamily, has been shown to regulate various aspects of cell growth and differentiation in the repair of the skin mesenchyme and the epidermis. Thus our aim was to study the role of activin and its antagonist, follistatin, in keloid pathogenesis. Increased mRNA expression for activin was observed in keloid scar tissue by performing RNase protection assay. Immunohistochemistry showed increased localization of both activin-A and follistatin in the basal layer of epidermis of keloid tissue compared with normal tissue. ELISA demonstrated a 29-fold increase in concentration of activin-A and an 5-fold increase in follistatin in conditioned media in keloid fibroblasts compared with normal fibroblasts. Although keloid keratinocytes produced 25% more follistatin than normal keratinocytes, the amounts of activin-A, in contrast, was 77% lower. Proliferation of fibroblasts was stimulated when treated with exogenous activin-A (46% increase in keloids fibroblasts) or following co-culture with hAHaCaT cells (66% increase). Activin-A upregulated key extracellular matrix components, namely collagen, fibronectin, and -smooth muscle actin, in normal and keloid fibroblasts. Co-treatment of follistatin with activin-A blocked the stimulatory effects of activin on extracellular matrix components. These findings emphasize the importance of the activin system in keloid biology and pathogenesis and suggest a possible therapeutic potential of follistatin in the prevention and treatment of keloids. collagen; fibroblasts; follistatin; keloid scar; keratinocytes; -smooth muscle actin; transforming growth factor-     

HETEROGENEITY IN RADIOSENSITIVITY OF HUMAN DIPLOID FIBROBLASTS FROM KELOIDS AND NORMAL SKINS

Colony-forming ability was employed in evaluating the susceptibility toin vitrogamma ionizing radiation in human diploid skin fibroblasts (HDF). Twelve pairs of HDF, each composed of fibroblasts from excised keloid lesion and local normal skin tissue as its control, were studied in patients with clinically persistent keloids. Parameters of radiosensitivity, both D₀and D₁₀, and growth kinetics were examined. The radiosensitivity in three of the 12 keloids (25%) were demonstrated significantly increased than their counterpart controls, even though no difference in growth kinetics in between. Moreover, a broad range in the radiosensitivity of fibroblast cells was demonstrated and it is suggested that there is a great heterogeneity of cellular response to radiation in HDF.     

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.     

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

Summary 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. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

Hypoxia-induced HIF-1 alpha accumulation is augmented in a co-culture of keloid fibroblasts and human mast cells: involvement of ERK1/2 and PI-3K/Akt

Keloids represent a prolonged inflammatory fibrotic state with areas that display distinctive histological features characterized by an abundant extracellular matrix stroma, a local infiltration of inflammatory cells including mast cells, and a milieu of enriched cytokines. Previous studies from our laboratory demonstrated an intrinsic higher level of HIF-1alpha and VEGF protein expression in keloid tissues compared with their adjacent unremarkable skins. To further investigate the mechanisms underlying the elevated expression of HIF-1alpha and VEGF in keloids, we exposed a co-culture of keloid fibroblasts and mast cells (HMC-1) to hypoxic conditions and studied the expression of HIF-1alpha and its target gene, VEGF. Our results showed that hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression is augmented in keloid fibroblasts when co-cultured with HMC-1 cells under the condition where direct cell-cell contact is allowed. But such augmentation is not observed in the transwell co-culture system whereas fibroblasts and HMC-1 cells were separated by a porous membrane. Our results also indicated that the enhancement of hypoxia-mediated activation of ERK1/2 and Akt requires direct cell-cell interaction between mast cells and keloid fibroblasts, and activation of both ERK1/2 and Akt is involved in the hypoxia-dependent HIF-1alpha protein accumulation and VEGF expression in the co-culture system. These findings suggest that under hypoxic conditions mast cells may contribute, at least in part, to an elevated expression of HIF-1alpha and VEGF protein in keloids via direct cell-cell interaction with fibroblasts.