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.     

Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin

Abstract: Solar ultraviolet (UV) radiation, particularly its UVB (290–320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti‐tumor‐promoting properties. In this study, we determined the effect of pomegranate‐derived products – POMx juice, POMx extract and pomegranate oil (POMo) – against UVB‐mediated damage using reconstituted human skin (EpiDerm^TM FT‐200). EpiDerm was treated with POMx juice (1–2 μl/0.1 ml/well), POMx extract (5–10 μg/0.1 ml/well) and POMo (1–2 μl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm²) irradiation and was harvested 12 h post‐UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8‐dihydro‐2′‐deoxyguanosine (8‐OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) collagenase (MMP‐1), (ii) gelatinase (MMP‐2, MMP‐9), (iii) stromelysin (MMP‐3), (iv) marilysin (MMP‐7), (v) elastase (MMP‐12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate‐derived products inhibited UVB‐induced MMP‐2 and MMP‐9 activities. Pomegranate‐derived products also caused a decrease in UVB‐induced protein expression of c‐Fos and phosphorylation of c‐Jun. Collectively, these results suggest that all three pomegranate‐derived products may be useful against UVB‐induced damage to human skin.     

胎鼠真皮成纤维细胞皮内注射对UV诱导小鼠皮肤光老化的改善作用

目的: 探索体外培养胎鼠真皮成纤维细胞(fibroblasts,FBs)皮内注射对小鼠背部光老化皮肤的改善作用.方法: 分离培养10只第18.5天(E18.5)BALB/c胎鼠皮肤,体外培养FBs两天;用CM-Dil荧光染料进行活细胞标记,以示FBs注射后在体内存活情况.取BALB/c雄性小鼠20只,随机分为:未处理组...     

Reduced expression of the adhesion protein tensin1 in cultured human dermal fibroblasts affects collagen gel contraction

Abstract:  As revealed by immunohistochemistry and RT-QPCR, the focal adhesion protein tensin1 is expressed in cultured human dermal fibroblasts and reduced by 60% after transfection with tensin1 siRNA. Tensin1 silenced fibroblast exhibited a strongly reduced capacity to contract collagen gels. Aged fibroblasts, generated with the Hayflick replicative senescence model, exhibit as siRNA silences fibroblasts, a reduced tensin1 expression and an impaired gel contraction capacity. Based on these results, we speculate that in human dermal fibroblasts, tensin1 plays an important role in cell–matrix interaction and that a reduced expression might contribute to the dermal alterations observed during skin ageing.     

Fluorofenidone inhibits UV‐A induced senescence in human dermal fibroblasts via the mammalian target of rapamycin‐dependent SIRT1 pathway

The aim of this study was to investigate the protective effect of fluorofenidone (5‐methyl‐1‐[3‐fluorophenyl]‐2‐[1H]‐pyridone, AKF‐PD) on ultraviolet (UV)‐A‐induced senescence in human dermal fibroblasts (HDF) and examine the mechanisms involved. HDF were treated with AKF‐PD. Senescence‐associated (SA)‐β‐galactosidase level, cell viability and expression of p16 were evaluated. In addition, UV‐A‐irradiated HDF were treated with AKF‐PD, rapamycin and MHY1485; SA‐β‐galactosidase staining, 3‐(4 5‐dimethylthiazol‐2‐yl)‐2 5‐diphenyltetrazolium bromide assay and western blot for SIRT1 were performed; and phosphorylated mammalian target of rapamycin (p‐mTOR) expression and reactive oxygen species (ROS) levels were measured. Intracellular ROS was detected by the 2′,7′‐dichlorofluroescein diacetate probe. Our results showed that AKF‐PD substantially attenuated the changes of p16 expression, SA‐β‐galactosidase staining and cellular proliferation induced by UV‐A irradiation in HDF. AKF‐PD rescued the increased mTOR phosphorylation and reduced SIRT1 expression induced by UV‐A irradiation in HDF. AKF‐PD and rapamycin together had a synergistic effect on p‐mTOR reduction and SIRT1 increase. mTOR activator MHY1485 partly blocked the above effects. Moreover, intracellular ROS level induced by UV‐A irradiation could partly decrease by AKF‐PD, and MHY1485 could reduce this effect. Our results indicated that AKF‐PD could alleviate HDF senescence induced by UV‐A‐irradiation by inhibiting the p‐mTOR and increasing SIRT1. Moreover, AKF‐PD may be a potential treatment material for skin.     

Senescent dermal fibroblasts enhance stem cell migration through CCL2/CCR2 axis

During aging, increases in the number of senescent cells are seen in various tissues. On the other hand, stem cells play crucial roles in tissue repair and homeostasis. Therefore, it is likely that stem cells give rise to new cells that replace senescent cells. However, how stem cells contribute to homeostasis in the dermis has not been elucidated. Here, we investigated the effects of factors secreted from senescent fibroblasts on stem cells. We found that senescent human dermal fibroblast (HDF) conditioned medium (CM) significantly enhanced stem cell migration compared with young HDF CM. The senescent HDF CM strongly secreted chemokine ligand 2 (CCL2). Furthermore, CCL2 was found to enhance stem cell migration, and the inhibition of CCR2, a receptor for CCL2, reduced stem cell migration. These results suggest that senescent fibroblasts recruit stem cells by secreting various factors and that the CCL2/CCR2 axis is one of the mechanisms underlying this phenomenon.     

SIRT1 modulates expression of matrix metalloproteinases in human dermal fibroblasts

Background SIRT1, an NAD⁺‐dependent histone/protein deacetylase, controls a broad range of cellular functions. Objectives We examined if SIRT1 is involved in the regulation of matrix metalloproteinase (MMP) expression in human dermal fibroblasts. Methods We studied the effect of inhibition of SIRT1 by specific inhibitor and small interfering RNA (siRNA) on MMP‐1 and MMP‐3 expression in human dermal fibroblasts. Results Treatment with a potent and selective inhibitor of SIRT1, EX‐527, increased the basal expression levels of MMP‐1 and MMP‐3 proteins. Knockdown of endogenous SIRT1 by siRNA led to increased expression of MMP‐1 and MMP‐3 at both mRNA and protein levels. SIRT1 knockdown also upregulated MMP protein induction caused by an inflammatory cytokine, interleukin (IL)‐1β. Moreover, treatment with a SIRT1 activator, resveratrol, significantly suppressed IL‐1β‐mediated induction of MMP‐1, which was attenuated by pretreatment with EX‐527. Finally, MMP‐1 promoter activity was increased by EX‐527 in cells treated with or without IL‐1β. Conclusions Our findings suggest that SIRT1 exerts a negative regulatory role in the production of MMP‐1 and MMP‐3 in human dermal fibroblasts.     

Caspase-3在UVB诱导人皮肤成纤维细胞凋亡中的作用

目的：评价caspase-3在UVB诱导皮肤成纤维细胞凋亡中的作用。方法：皮肤成纤维细胞经150mJ／cm2 UVB照射后,用MTF法检测细胞活性,用Hoechst33258染色法检测细胞凋亡,用抑制剂Z-DEVD-FMK抑制caspase-3活性后,检测凋亡细胞数量。结果：UVB明显抑制成纤维细胞的活性,并导致细胞凋亡,并呈时间-效应关系;加入抑制剂Z-DEVD-FMK抑制了UVB导致的细胞凋亡。结论：Caspase-3在UVB照射诱导皮肤成纤维细胞凋亡中发挥重要作用。     

LL-37 suppresses sodium nitroprusside-induced apoptosis of systemic sclerosis dermal fibroblasts

The human cathelicidin antimicrobial peptide LL-37 regulates apoptosis of several cell types. Defective apoptosis of skin fibroblasts may contribute to systemic sclerosis (SSc). Here, we show that LL-37 inhibited apoptosis of SSc fibroblasts and identified the signalling pathways by which LL-37 mediates apoptosis. Immunohistochemistry showed that cathelicidin expression was enhanced in SSc patients compared with healthy controls. In addition, LL-37 decreased sodium nitroprusside (SNP)-induced apoptosis of SSc fibroblasts. LL-37 significantly increased expression of Bcl-2 and decreased levels of BAX protein. Pretreatment with LL-37 decreased activation of caspase-3 following SNP-treatment. Moreover, exposure of SSc fibroblasts to LL-37 resulted in increased expression of COX-2 and stimulation of prostaglandin E(2) (PGE(2)). Furthermore, LL-37 induced phosphorylation of ERK and the ERK inhibitor PD98059 blocked the inhibitory effect of LL-37 on apoptosis. Our data indicate that LL-37 may be associated with skin sclerosis by inhibiting apoptosis of dermal fibroblasts.     

Dermal adipocytes contribute to the metabolic regulation of dermal fibroblasts

Dermal fibroblasts are an essential population of skin cells. They are not only responsible for synthesis and remodelling of the extracellular matrix of the dermis, but also communicate with other skin cells via autocrine and paracrine interactions. Skin‐associated dermal adipocytes reside below the reticular dermis. Strong lipolysis is observed during the regression of dermal adipocytes. However, the nature of the local intercellular crosstalk in which lipids released by dermal adipocytes affecting the metabolism of adjacent skin fibroblasts has not yet been examined. With the use of a series of novel mouse models that allow us to manipulate adipocytes, we demonstrate that dermal adipocytes can modulate the structure of the dermis through regulating extracellular matrix production in dermal fibroblasts. Fatty acids released by dermal adipocytes are involved in this process. Our observations offer new in vivo insights into the role of dermal adipocyte‐derived lipids in influencing metabolism of adjacent local cells in the skin through a paracrine effect in the microenvironment of the dermal adipocyte.     

Heat-separation of normal human skin for epidermal and dermal prostaglandin analysis

Summary Heat-separation was introduced as a simple, reliable method of obtaining pure epidermis and dermis for prostaglandin (PG) analysis. Heating of normal human skin at 60°C for 1 min resulted in a distinct separation of the epidermis from the dermis. After heat-separation the mean concentration ±SEM of PGE₁ activity in normal epidermal tissue was 2906±281 pg/mg dry weight. The PGE₁ level in the corresponding dermal samples was 30±4 pg/mg dry weight and the mean leakage of PGE₁ from the tissue into the buffer used during heating was 426±54 pg/ml.This work was supported by grant 512-8125 from the Danish State Research Foundation     

Multilineage differentiation potential of human dermal skin-derived fibroblasts

Dermal skin-derived fibroblasts from rodent and human have been found to exhibit mesenchymal surface antigen immunophenotype and differentiation potential along the three main mesenchymal-derived tissues: bone, cartilage and fat. Human dermal skin-derived mesenchymal stem cells constitute a promising cell source in clinical applications. Therefore, we isolated fibroblastic mesenchymal stem-cell-like cells from human dermis derived from juvenile foreskins, which share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. We could show similar expression patterns for CD14(-), CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(-) and CD166/ALCAM(+) in well-established adipose tissue derived-stem cells and fibroblastic mesenchymal stem-cell-like cells by flow cytometry. Immunostainings showed that fibroblastic mesenchymal stem-cell-like cells expressed vimentin, fibronectin and collagen; they were less positive for alpha-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. When cultured under appropriate inducible conditions, both cell types could differentiate along the adipogenic and osteogenic lineages. Additionally, fibroblastic mesenchymal stem-cell-like cells demonstrated a high proliferation potential. These findings are of particular importance, because skin or adipose tissues are easily accessible for autologous cell transplantations in regenerative medicine. In summary, these data indicate that dermal fibroblasts with multilineage differentiation potential are present in human dermis and they might play a key role in cutaneous wound healing.     

Human adipose tissue-derived cells delay re-epithelialization in comparison with skin fibroblasts in organotypic skin culture

BACKGROUND: Wound healing of deep and extensive burns can induce hypertrophic scar formation. During the early steps of wound healing fibroblasts migrate into the wounded area. Fibroblastic cells present in tissues other than dermis may also migrate into the wounded area and participate in the wound healing process. OBJECTIVES: To examine the influence of human fibroblastic cells derived from subcutaneous fat or dermis on epidermal morphogenesis in vitro. METHODS: We prepared human skin equivalents (HSEs) made of a collagen type I matrix populated either with dermal fibroblasts or adipose tissue-derived cells (ADCs), on top of which keratinocytes were seeded and subsequently grown at the air-liquid interface. RESULTS: A fully differentiated epidermis was formed on matrices populated with ADCs. However, the HSE formed differed in a number of features from HSE generated with dermal fibroblasts. The major differences included: marked contraction of the dermal matrix, low lateral migration of keratinocytes, high keratin 17 expression indicating increased keratinocyte activation, delayed deposition of collagen IV at the epidermal/matrix junction, accumulation of alpha-smooth muscle actin-positive cells only underneath the epidermal compartment and positioning of these cells in a direction parallel to the epidermal compartment. The latter two phenomena have also been found in scar tissue. CONCLUSIONS: The possibility of generating HSEs with different cell types represents an attractive approach for in vitro studies focusing on the mechanism of wound healing.     

Effect of fibroblasts on epidermal regeneration

BACKGROUND: There is little information on specific interactions between dermal fibroblasts and epidermal keratinocytes. The use of engineered skin equivalents consisting of organotypic cocultures of keratinocytes and fibroblasts offers an attractive approach for such studies. OBJECTIVES: To examine the role fibroblasts play in generation and maintenance of reconstructed epidermis. METHODS: Human keratinocytes were seeded on collagen matrices populated with increasing numbers of fibroblasts and cultured for 2 weeks at the air-liquid interface. RESULTS: In the absence of fibroblasts, stratified epidermis with only three or four viable cell layers was formed. In the presence of fibroblasts, keratinocyte proliferation was stimulated and epidermal morphology was improved. Epidermal morphogenesis was also markedly improved in epidermis generated in organotypic keratinocyte monocultures grown in medium derived from dermal equivalents or from organotypic keratinocyte-fibroblast cocultures. These observations clearly indicate the proliferation-stimulating activity of soluble factors released from fibroblasts. Under all experimental conditions, onset of keratinocyte differentiation was shown by the expression of keratin 10 in all suprabasal cell layers. With increasing numbers of fibroblasts incorporated into the collagen matrix, the expression of markers associated with keratinocyte activation, e.g. keratins 6, 16 and 17 and the cornified envelope precursor SKALP decreased, and involucrin localization shifted toward the granulosum layer. This fibroblast-mediated effect was even more pronounced when the fibroblasts were precultured in the collagen matrices for 1 week instead of overnight. The basement membrane proteins collagen VII and laminin 5 were present at the epithelial-matrix border. The expression of integrin alpha 6 beta 4 and of E-cadherin was comparable with that seen in native skin and was not significantly modulated by fibroblasts. Under all experimental conditions the expression of integrin subunits alpha 2, alpha 3 and beta 1 was upregulated, indicating keratinocyte activation. CONCLUSIONS: Our results illustrate that numbers of fibroblasts in the collagen matrix and their functional state is a critical factor for establishment of normal epidermal morphogenesis.     

染料木素对UVA诱导成纤维细胞急性光损伤的防护作用

目的：明确染料木素对UVA诱导成纤维细胞急性光损伤的防护作用。方法：将体外培养的成纤维细胞分为空白对照组,UVA照射对照组及UVA+染料木素（0.01,0.1,1,10,100umol/L）组。采用CCK8法测定细胞增殖情况,流式细胞术检测细胞凋亡情况, RT-PCR检测Sirt1mRNA的表达水平。结果：与UVA组比较,UVA+0.01,0.1,1,10 μmol/L染料木素组细胞增值活性及Sirt1mRNA表达明显升高,细胞凋亡率降低（均P<0.05）;而UVA+100μmol/L染料木素,细胞凋亡率无明显差异（P>0.05）;结论：0.01～10μmol/L染料木素对UVA诱导成纤维细胞的急性光损伤有防护作用。     

Glycosaminoglycan content in the media of cultured dermal fibroblasts derived from burn scar and normal skin

The glycosaminoglycan (GAG) content of the extracellular matrix of burn scar in humans has been reported to differ from that of normal skin. In order to investigate whether the GAG content altered as a result of functional changes in fibroblasts, the GAG content was determined in culture media of fibroblasts derived from growing burn scar, mature scar, and normal skin tissue. No statistical differences were observed in the population doubling-times of scar and normal skin. Mature scar showed significantly higher values for all the concentrations of uronic acid, hexosamine, and sulfate measured in the glycosaminoglycan, as compared with normal skin values, and the concentrations from growing scar were slightly higher than those for normal skin. The above results may suggest an increase in glycosaminoglycan sulfate synthesis following the hyperplasia of the matrix in burn scar tissue.