Fibroblasts support migration of monocyte‐derived dendritic cells by secretion of PGE2 and MMP‐1

The outcome of a cutaneous immune response is critically dependent upon the ability of dendritic cells (DC) to migrate from skin to the draining lymph nodes – a process that is influenced by the cutaneous tissue microenvironment. Here, the role of fibroblasts – a major component of the dermal microenvironment – on the migratory capacity of monocyte‐derived DC (MoDC) was investigated in a 3D collagen I matrix. Indeed, dermal fibroblasts supported the migration of pre‐activated MoDC through a 3D collagen I matrix. Activation of human MoDC resulted in the release of TNFα and IL‐1β that in turn stimulated MMP‐1 (human collagenase) and PGE₂ secretion by human dermal fibroblasts. Transmigration assays confirmed the importance of fibroblast‐derived MMP‐1 and PGE₂ for the migration of MoDC through a 3D collagen I matrix. Finally, in mice initiation of inflammation by induction of an irritant contact dermatitis or a psoriasis‐like skin inflammation, the expression of the PGE₂ generating cox‐2 and the mouse collagen I degrading enzyme matrix metalloproteinases (MMP)‐13 was strongly up‐regulated. Our study indicates that MoDC are able to instruct dermal fibroblasts resulting in enhanced migratory capability of MoDC, thus highlighting the role of a crosstalk of DC with their stromal microenvironment for the control of cutaneous immune responses.     

Low‐dose 5‐fluorouracil induces cell cycle G2 arrest and apoptosis in keloid fibroblasts

Background Intralesional injection of low‐dose 5‐fluorouracil (5‐FU) has recently been used as an experimental modality for treating keloid scarring and has shown promising efficacy in improving scar appearance and preventing recurrence of the keloid. Objectives We sought to explore the cellular‐ and molecular‐based evidence for the observed clinical benefits. Methods Primary cell lines of keloid fibroblasts were treated with 5‐FU at a range of lower doses (～10 mg mL^?1) in monolayer culture and subjected to examination for cell viability, proliferative potential, apoptosis, cell cycle and associated proteins involved in cell cycle control. Results 5‐FU significantly inhibited cell proliferation of keloid fibroblasts in the full dose range used in this study. The DNA synthesis was completely inhibited by 5‐FU at 72 h, and significant cell apoptosis was observed at concentrations ≥ 1 mg mL^?1 for a period over 72 h. 5‐FU caused a significant delay in cell cycle progression and the G2/M phase arrest. 5‐FU induced p53 and p21 accumulation together with a decrease in cyclin B1 and Bcl‐2 levels in treated keloid fibroblasts. Conclusions Our data indicate that low‐dose 5‐FU (as low as 1 mg mL^?1) induces significant inhibition of proliferation, G2/M cell cycle arrest and apoptosis but not immediate cell death of keloid fibroblasts. The lack of tissue necrosis is a particular benefit as further scarring is likely to be prevented. These results support the use of low‐dose 5‐FU as a potential modality for treating keloid scars.     

Zeaxanthin inhibits PDGF‐BB‐induced migration in human dermal fibroblasts

Please cite this paper as: Zeaxanthin inhibits PDGF‐BB‐induced migration in human dermal fibroblasts. Experimental Dermatology 2010; 19 : e173–e181. Abstract: Zeaxanthin is the dihydroxy carotenoid and is distributed in our daily foods. Various natural carotenoids, including zeaxanthin, have been shown to inhibit proliferation of several types of cancer cells, but available data on the effect of zeaxanthin on skin fibroblasts and melanoma cells are limited. Platelet‐derived growth factor (PDGF) functions as a chemotactic factor for dermal fibroblasts and plays an important role in the progression of melanoma. In this study, we investigated the effects of zeaxanthin on the migration of skin fibroblasts induced by PDGF‐BB and melanoma cells. We demonstrated that zeaxanthin inhibited PDGF‐BB‐induced skin fibroblast migration on collagen and gelatin by a modified Boyden chamber system. The electric cell‐substrate impedance sensing (ECIS) method also showed similar inhibitory effects of zeaxanthin on the migration of fibroblasts. In functional studies, zeaxanthin decreased melanoma‐induced fibroblast migration in a non‐contact coculture system and also the migration stimulated by melanoma‐derived conditioned medium. Further analysis showed that zeaxanthin attenuated PDGF‐BB and melanoma‐conditioned medium induced phosphorylation of PDGFR‐β and MAP kinase in a concentration‐dependent manner in human skin fibroblasts. However, these effects did not result from direct interaction of zeaxanthin with PDGF‐BB. Thus, our results provide the first evidence showing that zeaxanthin is an effective inhibitor of migration of stromal fibroblasts induced by PDGF‐BB and melanoma cells and this effect may further support its antitumor potential.     

Effect of hematoporphyrin monomethyl ether-mediated photodynamic therapy on hypertrophic scar fibroblasts

Background: Clinical studies have demonstrated that photodynamic therapy (PDT) for hyperplastic dermatosis results in a beneficial outcome. Hypertrophic scar (HS) is a pathological process characterized by fibroblastic hyperproliferation. However, it is unclear whether photochemical interactions between PDT and fibroblasts contribute to a beneficial outcome. To investigate the primary photochemical effects of PDT, we studied the efficacy of 630 nm PDT on human fibroblasts from HS using hematoporphyrin monomethyl ether (HMME) as a photosensitizer. Methods: Fibroblasts were cultured from nontreated HSs, and cells at passage 4–6 were used for the experiments. Morphological and biochemical changes in fibroblasts were assessed by Hoechst 33258 staining and annexin V‐FITC/PI flow cytometry (FCM). Caspase‐3 activity assay and immunofluorescence staining were performed to investigate caspase‐3 expression in fibroblasts. Results: The morphological features of cell apoptosis were viewed under a fluorescent microscope by Hoechst 33258 staining. FCM indicated that the apoptotic rate was significantly increased after HMME–PDT, and caspase‐3 activity was observed. Conclusions: Low‐level exposure to 630 nm PDT mediated by HMME appears to induce fibroblast apoptosis and stimulate caspase‐3 activation. However, the effect of HMME–PDT on fibroblasts needs further investigation to determine its therapeutic potential for HS.     

Induction of matrix metalloproteinase‐1 by small interfering RNA targeting connective tissue growth factor in dermal fibroblasts from patients with systemic sclerosis

Please cite this paper as: Induction of matrix metalloproteinase‐1 by small interfering RNA targeting connective tissue growth factor in dermal fibroblasts from patients with systemic sclerosis. Experimental Dermatology 2010; 19 : e111–e116. Abstract: We aimed to evaluate the effect of small interfering RNA (siRNA) targeting CTGF on extracellular matrices (ECMs) metabolism in normal and SSc fibroblasts. Normal and SSc fibroblasts were transfected with CTGF‐specific siRNAs to silence CTGF synthesis. After silencing CTGF, production of type I collagen and matrix metalloproteinase (MMP)‐1 by fibroblasts stimulated with TGF‐β was examined. Then quantitative analyses of protein production or mRNA expression of type I collagen, MMP‐1,‐2,‐9 and tissue inhibitor of metalloproteinase (TIMP)‐1 with TGF‐β stimulation were carried out. Furthermore, after silencing CTGF, proliferations of normal and SSc fibroblasts were investigated. CTGF‐specific siRNA significantly reduced CTGF production. The production of type I collagen was significantly reduced by CTGF silencing in normal fibroblasts. The CTGF silencing significantly increased the production of MMP‐1 and decreased the production of TIMP‐1 in SSc fibroblasts. The mRNA expression of MMP‐1 was increased in CTGF‐silenced SSc fibroblasts, but not in normal fibroblasts. There were no significant changes in the production or mRNA expression of other ECM‐related genes in normal and SSc fibroblasts. Fibroblast proliferations were suppressed by CTGF silencing in normal and SSc fibroblasts. Our data showed that MMP‐1 was increased by CTGF‐specific siRNA transfection only in SSc fibroblasts. RNAi targeting CTGF could be a novel therapeutic strategy for SSc.     

Deciphering the pro‐fibrotic phenotype of fibroblasts in systemic sclerosis

LeRoy's seminal work on the phenotypic features of scleroderma fibroblasts has been directing fibrosis research in the field of systemic sclerosis (SSc, scleroderma) for the past 30 years. His principal experiment, to culture skin fibroblasts from patients with SSc and study their pro‐fibrotic phenotype in comparison with skin fibroblasts from healthy individuals, has been used by most basic and translational fibrosis studies in SSc. LeRoy's findings have revolutionized our understanding of the disease pathogenesis and guided the development of novel antifibrotic therapies towards fibroblast‐specific approaches.     

Ultraviolet B‐induced apoptosis of human skin fibroblasts involves activation of caspase‐8 and ‐3 with increased expression of vimentin

Background: After irradiation with a high dose of ultraviolet B (UVB), cells undergo apoptosis. Caspase‐8 and ‐3 are key mediators of apoptosis in many cells. Vimentin, an important cytoskeleton component, can be cleaved by caspase‐3, ‐6, ‐7 and ‐8. Cell apoptosis is promoted via caspase‐triggered proteolysis of vimentin. In this study, we explored the roles of caspase‐8 and ‐3 and the changes in vimentin expression in UVB‐induced apoptosis of human dermal fibroblasts. Methods: Skin fibroblasts were irradiated with 150 mJ/cm² UVB and cell death was monitored by the 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐diphenytetrazoliumromide assay and Hoechst staining. Caspase‐8 and ‐3 activities were detected by the caspase activity assay. Vimentin expression was assessed by immunofluorescence and Western blot. Results: Caspase‐8 and ‐3 were activated by 150 mJ/cm² UVB irradiation. Caspase‐8 and ‐3 activities changed in a time‐dependent way after UVB irradiation to induce apoptosis of fibroblasts, and caspase‐8 and ‐3 interacted with each other in this process. However, their substrate, vimentin, showed an enhanced expression over time after UVB irradiation. Conclusions: UVB‐triggered apoptosis of fibroblasts was dependent on the activation of caspase‐8 and ‐3 with an increased expression of vimentin.     

Subpopulations of dermal skin fibroblasts secrete distinct extracellular matrix: implications for using skin substitutes in the clinic

Chronic wounds affect 1–2% of the world's population at any given time. These can be as a result of burns, or ulceration, and are essentially wounds which do not close. To facilitate closure, there are a number of biological products available which can be used as temporary skin replacements, or to promote tissue repair. These products usually replicate the two main layers found in human skin: the epidermis and dermis. Within the skin dermis the most abundant cell type are fibroblasts, whose primary role is to secrete extracellular matrix and support growth of cells in the adjacent epidermal layer. As fibroblasts within the skin are highly varied, the extracellular matrix in distinct locations of the dermis is also different; however skin substitutes do not usually reflect this diversity. In this study, from the UK, the researchers isolated three fibroblast sub‐types from human scalp skin dermis, and set about to characterise the extracellular matrix which the different sub‐types of fibroblasts synthesised in culture (i.e. developed in the lab, rather than on living skin). They found that the different fibroblast sub‐types produced extracellular matrix in culture reflective of the extracellular matrix found in distinct dermal locations in vivo (in living skin). They also found that certain fibroblast sub‐types were more proficient at supporting adjacent epithelial cells than others, which reflected the sub‐anatomical location from which the fibroblast sub‐types were originally isolated. The authors concluded that inspiration should be taken from the extracellular matrix which fibroblasts secrete to improve the design of biomimetic skin substitutes with improved therapeutic potential for skin tissue engineering.     

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative‐senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age‐related phenotypic and functional alteration of fibroblasts from ‘young’ (<35 years) and ‘old’ (>50 years) donors. Our results brought evidence of the senescent status of ‘old’ fibroblasts by senescence associated β‐galactosidase (SA‐βgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in ‘old’ fibroblasts as well as a subset of upregulated genes involved in senescence features. In ‘old’ fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFβ. Old fibroblasts were also more sensitive to oxidative stress than ‘young’ ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of ‘old’ fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.     

Constitutive activation of c‐Abl/protein kinase C‐δ/Fli1 pathway in dermal fibroblasts derived from patients with localized scleroderma

Background A noncanonical pathway of transforming growth factor‐β signalling, the c‐Abl/protein kinase C‐δ (PKC‐δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts. Objectives To investigate the significance of the c‐Abl/PKC‐δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc). Methods The activation status of the c‐Abl/PKC‐δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet‐derived growth factor receptor inhibitor AG1296 and gene silencing of c‐Abl on the expression levels of type I collagen were evaluated by immunoblotting. Results The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c‐Abl were elevated compared with cultured normal fibroblasts and PKC‐δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c‐Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts. Conclusions Constitutive activation of the c‐Abl/PKC‐δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.     

4‐Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell‐derived factors

Background There is a close correlation between tumour progression and hyaluronan production, either by tumour cells or by stromal cells that are stimulated by tumour‐derived factors. Inhibition of tumour stimulation of fibroblast hyaluronan may suppress tumour growth and invasion. Objectives To examine the effect of the hyaluronan synthesis inhibitor 4‐methylumbelliferone (4‐MU) on the growth of and hyaluronan synthesis by fibroblasts and C8161 and MV3 melanoma cell lines, invasion, and inhibition of tumour cell‐derived factor activation of fibroblasts. Methods Effects of 4‐MU on growth and hyaluronan synthesis by fibroblasts and melanoma cells were examined in monolayer culture and fibroblast‐contracted collagen lattices, and their effects on the growth and invasion of tumour cells into collagen lattices were also studied. Results 4‐MU caused a dose‐dependent growth inhibition of fibroblast and melanoma cells with maximum inhibition at 0·5 mmol L^?1 4‐MU. At this dose, 4‐MU inhibited ³H‐glucosamine incorporation into fibroblast glycosaminoglycans by 52%, and hyaluronan synthesis by 64%. The relative inhibition was more pronounced when fibroblasts were stimulated with C8161 melanoma cell‐conditioned medium. 4‐MU reduced the level of hyaluronan in fibroblast‐contracted collagen lattices, and inhibited both the growth on and invasion into the lattices by melanoma cells. This growth inhibition appears to be predominantly independent of inhibition of hyaluronan synthesis. The effect on growth inhibition was reversible, and 4‐MU had no effect on apoptosis. Conclusions 4‐MU is a potent inhibitor of hyaluronan synthesis, induction of stromal hyaluronan accumulation by tumour cells, and fibroblast and melanoma cell proliferation, and results suggest that 4‐MU may have potential as a tumour cell anti‐invasive and antiproliferative agent.     

Evaluation of a near-senescent human dermal fibroblast cell line and effect of amelogenin

Background Fibroblast senescence may delay healing of chronic wounds. Objectives To characterize a chronic human dermal fibroblast cell line (CRL‐7815) with near‐senescent properties, cell proliferation and production of wound‐healing modulating cytokines, and biosynthesis and remodelling of collagen were compared with normal human dermal fibroblasts. Also, the response of CRL‐7815 fibroblasts to the extracellular matrix protein amelogenin that is beneficial in the treatment of stalled chronic wounds was studied. Methods Fibroblast proliferation was monitored by time‐resolved growth curves and factors secreted into the culture medium containing 10% fetal bovine serum were measured by enzyme‐linked immunosorbent assays. Fibroblast‐mediated reorganization was examined in three‐dimensional type I collagen matrices. Results Cell proliferation over 9 days was significantly (P < 0·01) slower for CRL‐7815 than for normal fibroblasts. Amelogenin at 1 mg mL^?1 increased (P < 0·01) CRL‐7815 proliferation to the level of the normal fibroblasts. The neutrophil chemoattractant interleukin (IL)‐8 was low while the constitutive production of monocyte chemoattractant protein (MCP)‐1 was highly elevated in medium from cultured CRL‐7815 fibroblasts. Amelogenin augmented IL‐8 but attenuated MCP‐1 secretion in CRL‐7815 fibroblasts. The elevated vascular endothelial growth factor production in CRL‐7815 fibroblasts was further increased with amelogenin while increased type I collagen synthesis by CRL‐7815 was reduced with 0·1 mg mL^?1 amelogenin. The dramatically impaired collagen matrix remodelling with CRL‐7815 fibroblasts (P < 0·001) was slightly improved with amelogenin (P = 0·0011). Conclusions The near‐senescent cell line CRL‐7815 shares functional anomalies with fibroblasts isolated from nonhealing chronic cutaneous wounds. Amelogenin has the capacity to switch chronic fibroblasts into an acute‐like phenotype.     

Long non‐coding RNA TSIX is upregulated in scleroderma dermal fibroblasts and controls collagen mRNA stabilization

Long non‐coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real‐time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real‐time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)‐β1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease‐related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half‐lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF‐β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.     

Proteome analysis of ultraviolet-B-induced protein expression in vitro human dermal fibroblasts

Background: Ultraviolet‐B (UVB) radiation can result in acute photodamage, photoaging and skin cancer through the induction of reactive oxygen species, DNA damage, activation of signaling pathways, and regulation of gene expression. In this study, we investigated UVB‐induced alterations in protein expression in human dermal fibroblasts. Methods: Skin fibroblasts were irradiated with 100 mJ/cm² UVB, and cell viability was monitored by the 3‐(4,5)‐dimethylthiahiazo(‐z‐y1)‐3,5‐diphenytetrazoliumromide assay. Two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionization time of flight mass spectroscopy were used to identify differentially expressed proteins. The mRNA and levels of identified proteins were detected using a quantitative real‐time polymerase chain reaction assay and Western blot. Results: UVB decreased the viability of skin fibroblasts. In UVB‐treated cells, eighteen differentially expressed proteins were identified. Among these proteins, the amounts of receptor‐interacting protein (RIP) and vimentin were significantly up‐regulated. However, their mRNA levels decreased and remained relatively stable, respectively. Conclusions: The differential expression of RIP and vimentin was validated in UVB‐irradiated fibroblasts. RIP may promote cell injury, and vimentin may contribute to the resistance of cells to UVB‐induced damage.     

The role of stem cell factor and c‐KIT in keloid pathogenesis: do tyrosine kinase inhibitors have a potential therapeutic role?

Background Keloids are fibroproliferative disorders characterized by increased deposition of extracellular matrix components. Stem cell factor (SCF) and its receptor c‐KIT are expressed in a wide variety of cells and have also been demonstrated to be important modulators of the wound healing process. Objectives To examine the role of the SCF/c‐KIT system in keloid pathogenesis. Methods Immunohistochemical staining and Western blot analyses were used to examine localization and expression of SCF and c‐KIT in keloid and normal skin tissue. This was followed by the detection of SCF and c‐KIT expression in fibroblasts cultured in vitro and fibroblasts exposed to serum. To investigate the effect of epithelial–mesenchymal interactions, a two‐chamber system was employed in which keratinocytes on membrane inserts were cocultured with the fibroblasts. SCF and c‐KIT expression levels in all cell extracts and conditioned media were assayed by Western blotting. In another set of experiments, the effect of imatinib (Glivec^®, Gleevec^®; Novartis Pharma AG, Basel, Switzerland) on keloid fibroblasts was examined. Results SCF and c‐KIT were upregulated in keloid scar tissue and in cultured fibroblasts stimulated with serum, highlighting their importance in the initial phase of wound healing. We further demonstrated that epithelial–mesenchymal interactions, mimicked by coculture of keratinocytes and fibroblasts in vitro, not only stimulated secretion of the soluble form of SCF in keloid cocultures but also brought about shedding of the extracellular domain of c‐KIT perhaps by upregulation of tumour necrosis factor‐α converting enzyme which was also upregulated in keloid scars in vivo and keloid cocultures in vitro. In addition keloid cocultures expressed increased levels of phosphorylated c‐KIT highlighting an activation of the SCF/c‐KIT system. Finally, we demonstrated that imatinib, a tyrosine kinase inhibitor, may be a possible therapeutic agent for keloids. Conclusion These data indicate that the SCF/c‐KIT system plays an important role in scar pathogenesis, and underscore the role of imatinib as a key therapeutic agent in keloid scars.