Increased vascular endothelial growth factor may account for elevated level of plasminogen activator inhibitor-1 via activating ERK1/2 in keloid fibroblasts

Keloids are characterized as an "overexuberant" healing response in which disequilibrium between production and catabolism of extracellular matrix (ECM) occurs. Previous studies from our laboratory and others demonstrate an intrinsically higher level of plasminogen activator inhibitor-1 (PAI-1) expression in keloid tissues and cultured fibroblasts compared with normal bordering skin. These findings support the concept that an altered balance of activator and inhibitor activities in the plasminogen system, in particular, an overexpression of PAI-1, may partly contribute to keloid formation and tissue fibrosis. Vascular endothelial growth factor (VEGF) has been implicated as a critical factor in regulating angiogenesis and inflammation under both physiological and pathological conditions. This study was designed to assess whether VEGF plays a role in keloid fibrosis. We report that VEGF was expressed at higher levels in keloid tissues and their derived fibroblasts compared with their associated normal skin. We have further demonstrated that VEGF stimulated the expression of PAI-1, but not urokinase plasminogen activator (uPA), in keloid fibroblasts at both mRNA and protein levels, in a dose- and time-dependent manner. However, treatment of normal skin fibroblasts with VEGF exerted little effects on PAI-1 gene expression. Additionally, we have characterized for the first time that the extracellular signal-regulated kinase (ERK)1/2 signaling pathway is mainly involved in VEGF-induced PAI-1 expression and have demonstrated its potential as a target molecule for modulation of scar fibrosis. These findings suggest that VEGF may play an important role in keloid formation by altering ECM homeostasis toward a state of impaired degradation and excessive accumulation. urokinase plasminogen activator; extracellular matrix; fibrosis     

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.     

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.     

Crosstalk of hypoxia-mediated signaling pathways in upregulating plasminogen activator inhibitor-1 expression in keloid fibroblasts

Keloids are skin fibrotic conditions characterized by an excess accumulation of extracellular matrix (ECM) components secondary to trauma or surgical injuries. Previous studies have shown that plasminogen activator inhibitor-1 (PAI-1) can be upregulated by hypoxia and may contribute to keloid pathogenesis. In this study we investigate the signaling mechanisms involved in hypoxia-mediated PAI-1 expression in keloid fibroblasts. Using Northern and Western blot analysis, transient transfections, and pharmacological agents, we demonstrate that hypoxia-induced upregulation of PAI-1 expression is mainly controlled by hypoxia inducible factors-1alpha (HIF-1alpha) and that hypoxia leads to a rapid and transient activation of phosphatidylinositol-3-kinase/Akt (PI3-K/Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2). Treatment of cells with PI-3K/Akt inhibitor (LY294002) and tyrosine protein kinase inhibitor (genistein) significantly attenuated hypoxia-induced PAI-1 mRNA and protein expression as well as promoter activation, apparently via an inhibition of the hypoxia-induced stabilization of HIF-1alpha protein, attenuation of the steady-state level of HIF-1alpha mRNA, and its DNA-binding activity. Even though disruption of ERK1/2 signaling pathway by PD98059 abolished hypoxia-induced PAI-1 promoter activation and mRNA/protein expression in keloid fibroblasts, it did not inhibit the hypoxia-mediated stabilization of HIF-1alpha protein and the steady-state level of HIF-1alpha mRNA nor its DNA binding activity. Our findings suggest that a combination of several signaling pathways, including ERK1/2, PI3-K/Akt, and protein tyrosine kinases (PTKs), may contribute to the hypoxia-mediated induction of PAI-1 expression via activation of HIF-1alpha in keloid fibroblasts.     

Methylation of secreted frizzled-related protein 1 (SFRP1) promoter downregulates Wnt/β-catenin activity in keloids

Keloid, a benign skin disorder, forms during wound healing in genetically susceptible individuals. To better control keloid and understand the molecular mechanisms, this study screened gene hypermethylations of GEO database microarray data on keloids and identified the hypermethylation of the secreted frizzled related protein-1 (SFRP1) promoter. Subsequently, hypermethylation and mRNA and protein levels were assessed in 57 cases of keloid vs. normal skin tissues. Fibroblasts from tissues were isolated for the assessment of gene regulation in vitro. The methods used were bioinformatic analysis, lentiviral infection carrying SFRP1 cDNA, qRT-PCR, western blot, immunohistochemistry, luciferase reporter assay, methylation-specific PCR and methylated DNA immunoprecipitation-qPCR, ELISA, and/or 5-Aza-2′-deoxycytidine treatment. The data revealed that the SFRP1 promoter was hypermethylated in keloid tissues, compared with that in normal skin tissues. The SFRP1 promoter methylation contributed to the downregulation of SFRP1 mRNA and protein in keloid tissues and keloid fibroblasts. The 5-Aza treatment significantly upregulated SFRP1 mRNA and protein level in keloid fibroblasts. Furthermore, the knockdown of DNMT1 expression, and not the expression of DNMT3a or DMNT3b, was responsible for the hypermethylation of the SFRP1 promoter and upregulation of SFRP1 mRNA and protein in keloid fibroblasts. In addition, the infection of lentivirus carrying SFRP1 cDNA significantly inhibited the signaling activity of Wnt/β-catenin and the mRNA and protein expression of β-catenin and α-SMA in keloid fibroblasts. In summary, the lost SFRP1 expression-induced Wnt/β-catenin signaling due to the hypermethylation of the SFRP1 promoter could associate with keloid development, suggesting that SFRP1 might be a therapeutic target for keloid treatment.     

瘢痕疙瘩中TIEG1的高表达

转化生长因子-β1(TGF-β1/Smads)信号转导通路的持续激活是瘢痕疙瘩形成的重要机制.研究发现这条通路重要的负反馈调节信号分子Smad7表达明显下调,Smad2/3的磷酸化水平和蛋白质量并无明显改变.但是,Smad7下调的机制尚不清楚.采用生物信息学方法对Smad7的启动子进行分析;用RT-PCR和蛋白质印迹分别检测了正常皮肤、正常瘢痕及瘢痕疙瘩组织中的Sp1样转录因子TIEG1mRNA及蛋白质的表达水平;体外培养正常皮肤、正常瘢痕及瘢痕疙瘩成纤维细胞,检测TIEG1 mRNA及蛋白的表达水平.研究结果显示,Smad7启动子上有Sp1的位点,TIEG1 mRNA及蛋白质水平在瘢痕疙瘩组织及瘢痕疙瘩成纤维细胞中表达明显高于正常瘢痕和正常皮肤(P<0.05).说明瘢痕疙瘩中TIEG1可能是Smad7下调的重要原因,有必要进一步研究TIEG1对Smad7的调控作用机制.     

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.     

Spatiotemporal expression of periostin during skin development and incisional wound healing: lessons for human fibrotic scar formation

Differentiation of fibroblasts to myofibroblasts and collagen fibrillogenesis are two processes essential for normal cutaneous development and repair, but their misregulation also underlies skin-associated fibrosis. Periostin is a matricellular protein normally expressed in adult skin, but its role in skin organogenesis, incisional wound healing and skin pathology has yet to be investigated in any depth. Using C57/BL6 mouse skin as model, we first investigated periostin protein and mRNA spatiotemporal expression and distribution during development and after incisional wounding. Secondarily we assessed whether periostin is expressed in human skin pathologies, including keloid and hypertrophic scars, psoriasis and atopic dermatitis. During development, periostin is expressed in the dermis, basement membrane and hair follicles from embryonic through neonatal stages and in the dermis and hair follicle only in adult. In situ hybridization demonstrated that dermal fibroblasts and basal keratinocytes express periostin mRNA. After incisional wounding, periostin becomes re-expressed in the basement membrane within the dermal-epidermal junction at the wound edge re-establishing the embryonic deposition pattern present in the adult. Analysis of periostin expression in human pathologies demonstrated that it is over-expressed in keloid and hypertrophic scars, atopic dermatitis, but is largely absent from sites of inflammation and inflammatory conditions such as psoriasis. Furthermore, in vitro we demonstrated that periostin is a transforming growth factor beta 1 inducible gene in human dermal fibroblasts. We conclude that periostin is an important ECM component during development, in wound healing and is strongly associated with pathological skin remodeling.     

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.     

Site-Specific Keloid Fibroblasts Alter the Behaviour of Normal Skin and Normal Scar Fibroblasts through Paracrine Signalling

Keloid disease (KD) is an abnormal cutaneous fibroproliferative disorder of unknown aetiopathogenesis. Keloid fibroblasts (KF) are implicated as mediators of elevated extracellular matrix deposition. Aberrant secretory behaviour by KF relative to normal skin fibroblasts (NF) may influence the disease state. To date, no previous reports exist on the ability of site-specific KF to induce fibrotic-like phenotypic changes in NF or normal scar fibroblasts (NS) by paracrine mechanisms. Therefore, the aim of this study was to investigate the influence of conditioned media from site-specific KF on the cellular and molecular behaviour of both NF and NS enabled by paracrine mechanisms. Conditioned media was collected from cultured primary fibroblasts during a proliferative log phase of growth including: NF, NS, peri-lesional keloid fibroblasts (PKF) and intra-lesional keloid fibroblasts (IKF). Conditioned media was used to grow NF, NS, PKF and IKF cells over 240 hrs. Cellular behavior was monitored through real time cell analysis (RTCA), proliferation rates and migration in a scratch wound assay. Fibrosis-associated marker expression was determined at both protein and gene level. PKF conditioned media treatment of both NF and NS elicited enhanced cell proliferation, spreading and viability as measured in real time over 240 hrs versus control conditioned media. Following PKF and IKF media treatments up to 240 hrs, both NF and NS showed significantly elevated proliferation rates (p<0.03) and migration in a scratch wound assay (p<0.04). Concomitant up-regulation of collagen I, fibronectin, α-SMA, PAI-1, TGF-β and CTGF (p<0.03) protein expression were also observed. Corresponding qRT-PCR analysis supported these findings (P<0.03). In all cases, conditioned media from growing marginal PKF elicited the strongest effects. In conclusion, primary NF and NS cells treated with PKF or IKF conditioned media exhibit enhanced expression of fibrosis-associated molecular markers and increased cellular activity as a result of keloid fibroblast-derived paracrine factors.     

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.     

Role of endothelin-1 in the skin fibrosis of systemic sclerosis

Endothelin-1 (ET-1) acts as a key regulator of vasoconstriction and fibrosis. Many previous studies have focused on the role of ET-1 in scleroderma (systemic sclerosis, SSc).We investigated the effects of ET-1 on the production of extracellular matrix in SSc and normal skin fibroblasts. Primary cultured dermal fibroblasts from SSc patients and healthy controls were treated with ET-1 (25 ng/mL) for 0 min, 15 min, 1 h, 24 h, 48 h and 72 h, respectively. Our results showed that, in SSc fibroblasts, ET-1 upregulated collagen type I, connective tissue growth factor (CTGF), type I plasminogen activator inhibitor (PAI-1) and pAkt in a time-dependent manner within 72 h; in normal fibroblasts, 25 ng/mL ET-1 stimulation correlated with high levels of CTGF, PAI-1 and pAkt. The secretion of fibronectin (FN), collagen type I, and PAI-1 is markedly increased in the supernatant of both SSc fibroblasts and normal fibroblasts. Furthermore, ET-1 phosphorylates Smad2 and Smad3 in normal fibroblasts, but not in SSc fibroblasts. In conclusion, our results demonstrated that ET-1 may induce fibrosis in dermal fibroblasts through Akt signals.     

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.     

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.     

Telomerase expression restores dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model

The lifespan of human fibroblasts and other primary cell strains can be extended by expression of the telomerase catalytic subunit (hTERT). Since replicative senescence is accompanied by substantial alterations in gene expression, we evaluated characteristics of in vitro-aged dermal fibroblast populations before and after immortalization with telomerase. The biological behavior of these populations was assessed by incorporation into reconstituted human skin. Reminiscent of skin in the elderly, we observed increased fragility and subepidermal blistering with increased passage number of dermal fibroblasts, but the expression of telomerase in late passage populations restored the normal nonblistering phenotype. DNA microarray analysis showed that senescent fibroblasts express reduced levels of collagen I and III, as well as increased levels of a series of markers associated with the destruction of dermal matrix and inflammatory processes, and that the expression of telomerase results in mRNA expression patterns that are substantially similar to early passage cells. Thus, telomerase activity not only confers replicative immortality to skin fibroblasts, but can also prevent or reverse the loss of biological function seen in senescent cell populations.     

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.