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.     

Alginate oligosaccharides modulate cell morphology, cell proliferation and collagen expression in human skin fibroblasts in vitro

Abstract Effects of alginate oligosaccharides on cell proliferation and expression of collagen in cultured skin fibroblasts were studied. The oligosaccharides were found to suppress fibroblast proliferation to half the level in control cultures at a dose of 10 mg/ml during a period of 5 days. The inhibition was accompanied by a change in cell shape. The inhibition of cell proliferation was reversible, since depletion of these oligosaccharides led to a recovery of cell motility. Treatment of confluent cells with 10 mg/ml oligosaccharides for 5 days resulted in a reduction in collagen synthesis to one half of that in control cultures and inhibition of steady state levels of α₁(I), α₂(I), α₁(III) and α₁(VI) collagen mRNAs. These results suggest that alginate oligosaccharides are potential modulators of dermal fibroblasts and may provide a useful tool for the treatment of disorders related to abnormal collagen metabolism. Received: 16 October 1998 / Received after revision: 8 February 1999 / Accepted: 12 February 1999     

Replicative senescence of human dermal fibroblasts affects structural and functional aspects of the Golgi apparatus

It is well recognized that the world population is ageing rapidly. Therefore, it is important to understand ageing processes at the cellular and molecular levels to predict the onset of age‐related diseases and prevent them. Recent research has focused on the identification of ageing biomarkers, including those associated with the properties of the Golgi apparatus. In this context, Golgi‐mediated glycosylation of proteins has been well characterized. Additionally, other studies show that the secretion of many compounds, including pro‐inflammatory cytokines and extracellular matrix–degrading enzymes, is modified during ageing, resulting in physical and functional skin degradation. Since the Golgi apparatus is a central organelle of the secretory pathway, we investigated its structural organization in senescent primary human dermal fibroblasts using confocal and electron microscopy. In addition, we monitored the expression of Golgi‐related genes in the same cells. Our data showed a marked alteration in the Golgi morphology during replicative senescence. In contrast to its small and compact structure in non‐senescent cells, the Golgi apparatus exhibited a large and expanded morphology in senescent fibroblasts. Our data also demonstrated that the expression of many genes related to Golgi structural integrity and function was significantly modified in senescent cells, suggesting a relationship between Golgi apparatus function and ageing.     

Previous chronic exogenous glucocorticoid administration in vivo does not affect functional characteristics and cellular lifespan of human skin fibroblasts in vitro

Excess of glucocorticoids (GCs) has been reported to lead to skin atrophy and impaired wound healing. The present study investigates whether human skin fibroblasts suffer permanent damages due to a long-term exposure to GC excess. Fibroblasts obtained from patients being under GC treatment for periods over one year were cultured under standard conditions in vitro, and studied regarding pivotal parameters involved in skin homeostasis and aging, i.e. collagen production, cell proliferation, and cellular replicative lifespan. No statistical differences were observed regarding these functions compared to those of normal human skin fibroblasts. Furthermore, no differences between normal and patient-derived cells were observed regarding their sensitivity to a supra-physiological cortisol concentration. In conclusion, the prolonged exposure of human skin fibroblasts in vivo to high concentrations of exogenously-administered GC does not lead to persistent adverse effects on their physiology.     

咪喹莫特对人皮肤成纤维细胞活性和凋亡的影响

目的：观察咪喹莫特（imiquimod）溶液对人真皮成纤维细胞（FB）细胞毒性、细胞增殖及细胞凋亡率的影响，探讨其治疗瘢痕疙瘩的可能机制。方法：分离培养人真皮成纤维细胞，用四甲基偶氮唑蓝（MTF）法检测不同浓度咪喹莫特溶液对成纤维细胞毒性及细胞增殖的影响：用流式细胞技术检测咪喹莫特对成纤维细胞凋亡率的影响。结果：不同浓度咪喹莫特加入成纤维细胞培养体系孵育24h后，成纤维细胞形态均有不同程度受损，且细胞增殖活性下降，尤以20mg/L浓度时细胞活性下降明显；各浓度咪喹莫特处理组细胞凋亡率均高于对照组（P〈0．05），但在不同时间点检测的细胞凋亡率差异无统计学意义（P〉0．05）。结论：咪喹莫特可以降低成纤维细胞增殖活性并增加细胞凋亡率，这些可能是其促进皮肤瘢痕组织消退的相关机制。     

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.     

Positive photobiological investigation in reticular erythematous mucinosis syndrome

BACKGROUND: Reticular erythematous mucinosis (REM) syndrome is a rare disorder. Its clinical course is cyclic with remissions and exacerbations. In this disease, photosensitivity has previously been noticed but rarely demonstrated. We report three new cases with positive photobiological investigation. CASE REPORTS: Three patients (two males, one female) with a mean age 47 years were seen with reticular erythematous papules on the upper chest and or back. After sun exposure, the lesions were exacerbated. Skin biopsies showed dermal lymphocytic perivascular infiltration with mucin deposition between collagen bundles. Direct immunofluorescence was negative. Antinuclear antibodies were absent. In cabin, ultraviolet (UV)A exposure reproduced clinically and histologically REM lesions in our cases. UVA and UVB provocating phototests were negative. In all patients treatment with oral antimalarials and external photoprotection was effective. CONCLUSIONS: In our patients, we confirm the photosensitive feature of REM syndrome by provocative irradiation in UVA cabin. The mechanism of triggering is actually unclear. It is supposed that UV radiation, heat, and perspiration are necessary to reveal this affection.     

Functional characterization of cancer-associated fibroblasts of human cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (SCC) is the second most common type of skin cancer in the Caucasian population worldwide, having a propensity for invasion, local recurrence and metastasis. Stromal cancer-associated fibroblasts (CAFs) are suspected to play an important role in SCC carcinogenesis. In this study, we characterized CAFs isolated from primary cutaneous SCCs and compared them to normal fibroblasts (NFs) isolated from healthy dermis. Human skin CAFs in monolayers displayed different morphology, increased proliferation and migration compared to NFs. CAFs caused strong contraction of collagen matrices in which they were seeded and released high levels of the extracellular matrix component pro-collagen I. CAFs decreased proliferation and differentiation in the epidermis of human skin equivalents (HSEs) seeded with SCC cell lines, without affecting basement membrane composition. Finally, CAFs significantly increased invasion and dermal-epidermal detachment of SCC cell lines SCC-12B2 and SCC-13, respectively, when cultured in HSEs. These distinct features of CAFs point out a specific role in cutaneous SCC development.     

Enhancement of adenovirus-mediated gene transfer into dermal fibroblasts in vitro and in vivo by polyethylene glycol 6000

Gene therapy directed to the skin requires efficient transfer of the desired gene into cutaneous cells. In this study, we examined several chemical substances present in various ointments by enhancement of virus infectivity. The recombinant adenovirus vector, AxCALacZ, was used to infect dermal fibroblasts with some chemicals both in vitro and in vivo, and the expression of the LacZ gene was determined by X-Gal reaction. As the result, it was shown that PEG 6000 had the highest ability to enhance the exogenous gene expression in cultured fibroblasts with little toxicity. In vivo, it was also demonstrated that fibroblasts in mouse skin were efficiently gene transfer by adenovirus vector and 20% PEG 6000-treatment. These results suggest that this chemical treatment appears to be a simple, safe, convenient, and effective method for facilitating virus-mediated gene therapy in the skin.     

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。     

Heat shocks enhance procollagen type I and III expression in fibroblasts in ex vivo human skin

Background: The well‐known characteristics of aging skin are the development of fine lines and wrinkles, but changes in skin tone, skin texture, thickness and moisture content are also aspects of aging. Rejuvenation of the skin aims at reversing the signs of aging and can be established in the epidermis as well as in the dermis. Aged dermis, in fact, has a degenerated collagen matrix. To regenerate this matrix, fibroblasts need to be stimulated into synthesizing new collagen. Aims: In this study, the effects of heat shocks of different temperatures on human dermal fibroblasts in ex vivo skin on the expression of procollagen 1, procollagen 3, heat shock protein (hsp)27, hsp47, and hsp70 are investigated. Materials and methods: The heat shocks were applied on ex vivo skin samples by immersing the samples in heated phosphate‐buffered saline of 45 °C or 60 °C. Metabolic activity was measured and at similar time points propidium–iodide–calceine staining was performed to establish cell viability. Quantitative polymerase chain reaction (qPCR) was performed after the heat shock to determine gene expression levels relative to the reference temperature. Furthermore, PicroSirius Red and hematoxylin stainings were performed to visualize the collagen network and the cells. Results: The skin samples were shown to be viable and metabolically active. Histology indicated that the heat shocks did not influence the structure of the collagen network or cell appearance. qPCR results showed that in contrast to the 45 °C heat shock the 60 °C heat shock resulted in significant upregulations of procollagen type I and III, hsp70 and hsp47. Conclusion: A 60 °C, heat shock stimulates the human dermal fibroblasts in ex vivo skin to upregulate their procollagen type I and type III expression.     

Cryotherapy modifies synthetic activity and differentiation of keloidal fibroblasts in vitro

In order to obtain a persuasive explanation for the beneficial clinical effect of cryotherapy on keloids, we developed a reproducible model to apply freezing temperatures on cell cultures, and investigated their influence on proliferation, viability, synthetic activity and differentiation of dermal fibroblasts in vitro. Cell cultures were established from 13 untreated keloids and 10 healthy skin specimens matched for age and skin localization to the donors. No significant influence of cell freezing on the proliferation rates of both keloidal and normal fibroblasts was documented, but mechanical cell destruction with a wide variation in lethality rates (29% average lethal effect on keloidal fibroblasts and 41% on normal ones) was observed. When comparing specimens of keloidal and normal tissue derived from the same four donors, the keloidal fibroblasts were similar regarding their synthetic activity but presented enhanced tenascin-C expression compared with the normal fibroblasts. After cryotherapy, delayed collagen III increase was detected in both cell types (P = 0.03). The collagen II/collagen I ratio increased from 1.6 to 2.8 in the keloidal and only from 1.9 to 2.2 in the normal fibroblasts after subcultivation. Normal fibroblasts exhibited a significantly lasting increase in fibronectin synthesis after freezing (P = 0.03). The intensity of staining against tenascin-C was decreased in five of nine keloidal fibroblast cultures after cryotherapy (P < 0.05) but increased in four of five normal fibroblast cultures (P = 0.016), so that the intensity of tenascin-C staining after freezing became identical in both cell types. Immunoblot studies in four patients and two controls confirmed a temporary decrease of tenascin-C in keloidal but not in normal fibroblasts immediately after freezing. Significantly decreased staining with two markers of myogenic differentiation, myosin in keloidal fibroblasts (P = 0.002) and desmin (P = 0.007) in normal fibroblasts, could also be detected after treatment. In summary, with the help of a model for controlled cell freezing in vitro, cryotherapy was found to modify collagen synthesis and differentiation of keloidal fibroblasts.     

Induction of connective tissue growth factor expression by sphingosylphosphorylcholine in cultured human skin fibroblasts

Sphingosylphosphorylcholine (SPC) is a bioactive sphingolipid metabolite that can enhance wound healing. In an effort to find downstream effectors of SPC, we performed microarray analysis and found that the expression of the gene for connective tissue growth factor (CTGF) was significantly affected in human skin fibroblasts cultured in vitro. Northern blot analysis showed that SPC markedly induced CTGF mRNA expression in a dose- and time-dependent manner. Consistent with this result, Western blot analysis also showed that SPC significantly induced the CTGF production. Pretreatment with cycloheximide did not prevent the CTGF induction by SPC, indicating that SPC stimulates CTGF mRNA expression without the increased synthesis of a regulatory protein. Inhibition by pretreatment with Y27632, but not by PD98059 (a mitogen-activated protein kinase 1/2 inhibitor) and LY294002 (a phosphatidylinositol 3-kinase inhibitor), indicated that rho-kinase pathway was involved in SPC-induced CTGF expression. Together, these results reveal the potential importance of CTGF induction as a downstream event in SPC-induced cellular responses.     

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.     

Action spectra for 8-methoxypsoralen and 3-carbethoxypsoralen in skin fibroblasts in vitro

Summary 8-methoxypsoralen (8-MOP) and 3-Carbethoxypsoralen (3-CPs) are known photoreagents which have been employed in the treatment of psoriasis. Using skin fibroblasts grown in vitro we have determined the action spectra of both compounds in the long ultraviolet range. Narrow-band interference filters were applied to a Xenon lamp. Reduction in methyl-³H-thymidine (³H-TdR) uptake served as a parameter for photoinhibited cells. The results show a linear increate of 8-MOP-mediated photoinhibition with increasing wavelengths; 3-CPs showed comperatively weak inhibitory rates at the lower wavelength range (349–365 nm). Possibly this is due to the formation of 3-CPs photoproducts which are unreactive with DNA. Compared to 8-MOP, the monofunctional photoreagent 3-CPs is a weak photoinhibitor.This work was supported by Stiftung Volkswagenwerk