Epicatechin and its methylated metabolite attenuate UVA-induced oxidative damage to human skin fibroblasts

The ultraviolet A component of sunlight causes both acute and chronic damage to human skin. In this study the potential of epicatechin, an abundant dietary flavanol, and 3'-O-methyl epicatechin, one of its major in vivo metabolites, to protect against UVA-induced damage was examined using cultured human skin fibroblasts as an in vitro model. The results obtained clearly show that both epicatechin and its metabolite protect these fibroblasts against UVA damage and cell death. The hydrogen-donating antioxidant properties of these compounds are probably not the mediators of this protective response. The protection is a consequence of induction of resistance to UVA mediated by the compounds and involves newly synthesized proteins. The study provides clear evidence that this dietary flavanol has the potential to protect human skin against the deleterious effects of sunlight.

Effect of a calcium-channel blocker (verapamil) on the morphology, cytoskeleton and collagenase activity of human skin fibroblasts

The effects of verapamil modulating collagen biosynthesis have prompted us to study the role of this drug in cultured fibroblasts. In this article, we describe the effects of verapamil on fibroblast behaviour, with special emphasis to phenotypic modifications, reorganisation of actin filaments and secretion of MMP1. Human dermal fibroblasts treated with 50-mu M verapamil changed their normal spindle-shaped morphology to stellate. Treated cells showed discrete reorganisation of actin filaments, as revealed by fluorescein isothiocyanate (FITC)-phalloidin staining and confocal microscopy. We hypothesised that these effects would be associated to lower levels of cytosolic Ca(2+). Indeed, short time loading with calcium green confirmed that verapamil-treated fibroblasts exhibited lower intracellular calcium levels compared to controls. We also observed that verapamil increases the secretion of MMP1 in cultured fibroblasts, as demonstrated by zymography, specific substrate assays and immunoblot. The morphological alterations induced by verapamil are neither cytotoxic nor associated with other dramatic cytoskeleton alterations. Thus we may conclude that this drug enhances collagenase secretion and does not disrupt the major tracks necessary to deliver these enzymes in the extracellular space. The present results suggested that verapamil could be used at physiological levels to enhance collagen I breakdown, and maybe considered a potential candidate for intralesional therapy of wound healing and fibrocontractive diseases. (C) 2010 Elsevier Ltd and ISBI. All rights reserved.

Regulation of antigen processing and presentation molecules in West Nile virus-infected human skin fibroblasts

Infection of humans with the West Nile flavivirus principally occurs via tick and mosquito bites. Here, we document the expression of antigen processing and presentation molecules in West Nile virus (WNV)-infected human skin fibroblast (HFF) cells. Using a new Flavivirus-specific antibody, 4G4, we have analyzed cell surface human leukocyte antigen (HLA) expression on virus-infected cells at a single cell level. Using this approach, we show that West Nile Virus infection alters surface HLA expression on both infected HFF and neighboring uninfected HFF cells. Interestingly, increased surface HLA evident on infected HFF cultures is almost entirely due to virus-induced interferon (IFN)alpha/beta because IFNalpha/beta-neutralizing antibodies completely prevent increased surface HLA expression. In contrast, RT-PCR analysis indicates that WNV infection results in increased mRNAs for HLA-A, -B, and -C genes, and HLA-associated molecules low molecular weight polypeptide-2 (LMP-2) and transporter associated with antigen presentation-1 (TAP-1), but induction of these mRNAs is not diminished in HFF cells cultured with IFNalpha/beta-neutralizing antibodies. Taken together, these data support the idea that that both cytokine-dependent and cytokine-independent mechanisms account for WNV-induced HLA expression in human skin fibroblasts. (C) 2004 Elsevier Inc. All rights reserved.

Water-filtered infrared-A radiation (wIRA) is not implicated in cellular degeneration of human skin.

BACKGROUND: Excessive exposure to solar ultraviolet radiation is involved in the complex biologic process of cutaneous aging. Wavelengths in the ultraviolet-A and -B range (UV-A and UV-B) have been shown to be responsible for the induction of proteases, e. g. the collagenase matrix metalloproteinase 1 (MMP-1), which are related to cell aging. As devices emitting longer wavelengths are widely used in therapeutic and cosmetic interventions and as the induction of MMP-1 by water-filtered infrared-A (wIRA) had been discussed, it was of interest to assess effects of wIRA on the cellular and molecular level known to be possibly involved in cutaneous degeneration. OBJECTIVES: Investigation of the biological implications of widely used water-filtered infrared-A (wIRA) radiators for clinical use on human skin fibroblasts assessed by MMP-1 gene expression (MMP-1 messenger ribonucleic acid (mRNA) expression).Methods: Human skin fibroblasts were irradiated with approximately 88% wIRA (780-1400 nm) and 12% red light (RL, 665-780 nm) with 380 mW/cm(2) wIRA(+RL) (333 mW/cm(2) wIRA) on the one hand and for comparison with UV-A (330-400 nm, mainly UV-A1) and a small amount of blue light (BL, 400-450 nm) with 28 mW/cm(2) UV-A(+BL) on the other hand. Survival curves were established by colony forming ability after single exposures between 15 minutes and 8 hours to wIRA(+RL) (340-10880 J/cm(2) wIRA(+RL), 300-9600 J/cm(2) wIRA) or 15-45 minutes to UV-A(+BL) (25-75 J/cm(2) UV-A(+BL)). Both conventional Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and quantitative real-time RT-PCR techniques were used to determine the induction of MMP-1 mRNA at two physiologic temperatures for skin fibroblasts (30 degrees C and 37 degrees C) in single exposure regimens (15-60 minutes wIRA(+RL), 340-1360 J/cm(2) wIRA(+RL), 300-1200 J/cm(2) wIRA; 30 minutes UV-A(+BL), 50 J/cm(2) UV-A(+BL)) and in addition at 30 degrees C in a repeated exposure protocol (up to 10 times 15 minutes wIRA(+RL) with 340 J/cm(2) wIRA(+RL), 300 J/cm(2) wIRA at each time). RESULTS: Single exposure of cultured human dermal fibroblasts to UV-A(+BL) radiation yielded a very high increase in MMP-1 mRNA expression (11 +/-1 fold expression for RT-PCR and 76 +/-2 fold expression for real-time RT-PCR both at 30 degrees C, 75 +/-1 fold expression for real-time RT-PCR at 37 degrees C) and a dose-dependent decrease in cell survival. In contrast, wIRA(+RL) did not produce cell death and did not induce a systematic increase in MMP-1 mRNA expression (less than twofold expression, within the laboratory range of fluctuation) detectable with the sensitive methods applied. Additionally, repeated exposure of human skin fibroblasts to wIRA(+RL) did not induce MMP-1 mRNA expression systematically (less than twofold expression by up to 10 consecutive wIRA(+RL) exposures and analysis with real-time RT-PCR). CONCLUSIONS: wIRA(+RL) even at the investigated disproportionally high irradiances does not induce cell death or a systematic increase of MMP-1 mRNA expression, both of which can be easily induced by UV-A radiation. Furthermore, these results support previous findings of in vivo investigations on collagenase induction by UV-A but not wIRA and show that infrared-A with appropriate irradiances does not seem to be involved in MMP-1 mediated photoaging of the skin. As suggested by previously published studies wIRA could even be implicated in a protective manner.

Effect of green Coffea arabica L. seed oil on extracellular matrix components and water-channel expression in in vitro and ex vivo human skin models

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Laser-ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts.

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8 x 106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 microl) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 micros instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemming from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.

Laser-Ultraviolet-A induced ultra weak photon emission in human skin cells: A biophotonic comparison between keratinocytes and fibroblasts

Photons participate in many atomic and molecular interactions and processes. Recent biophysical research has discovered an ultraweak radiation in biological tissues. It is now recognized that plants, animal and human cells emit this very weak biophotonic emission which can be readily measured with a sensitive photomultiplier system. UVA laser induced biophotonic emission of cultured cells was used in this report with the intention to detect biophysical changes between young and adult fibroblasts as well as between fibroblasts and keratinocytes. With suspension densities ranging from 1-8x106 cells/ml, it was evident that an increase of the UVA-laser-light induced photon emission intensity could be observed in young as well as adult fibroblastic cells. By the use of this method to determine ultraweak light emission, photons in cell suspensions in low volumes (100 mu l) could be detected, in contrast to previous procedures using quantities up to 10 ml. Moreover, the analysis has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 mu s instead of more than 100 milliseconds. These significant changes lead to an improvement factor up to 106 in comparison to classical detection procedures. In addition, different skin cells as fibroblasts and keratinocytes stemining from the same donor were measured using this new highly sensitive method in order to find new biophysical insight of light pathways. This is important in view to develop new strategies in biophotonics especially for use in alternative therapies.

Nerve growth factor displays stimulatory effects on human skin and lung fibroblasts, demonstrating a direct role for this factor in tissue repair

Nerve growth factor (NGF) is a polypeptide which, in addition to its effect on nerve cells, is believed to play a role in inflammatory responses and in tissue repair. Because fibroblasts represent the main target and effector cells in these processes, to investigate whether NGF is involved in lung and skin tissue repair, we studied the effect of NGF on fibroblast migration, proliferation, collagen metabolism, modulation into myofibroblasts, and contraction of collagen gel. Both skin and lung fibroblasts were found to produce NGF and to express tyrosine kinase receptor (trkA) under basal conditions, whereas the low-affinity p75 receptor was expressed only after prolonged NGF exposure. NGF significantly induced skin and lung fibroblast migration in an in vitro model of wounded fibroblast and skin migration in Boyden chambers. Nevertheless NGF did not influence either skin or lung fibroblast proliferation, collagen production, or metalloproteinase production or activation. In contrast, culture of both lung and skin fibroblasts with NGF modulated their phenotype into myofibroblasts. Moreover, addition of NGF to both fibroblast types embedded in collagen gel increased their contraction. Fibrotic human lung or skin tissues displayed immunoreactivity for NGF, trkA, and p75. These data show a direct pro-fibrogenic effect of NGF on skin and lung fibroblasts and therefore indicate a role for NGF in tissue repair and fibrosis.

Sustained gene expression in transplanted skin fibroblasts in rats

Retrovirus-mediated gene transfer into adult skin fibroblasts has provided measurable amounts of therapeutic proteins in animal models. However, the major problem emerging from these experiments was a limited time of vector encoded gene expression once transduced cells were engrafted We hypothesized that sustained transduced gene expression in quiescent fibroblasts in vivo might be obtained by using a fibronectin (Fn) promoter. Fibronectin plays a key role in cell adhesion, migration and wound healing and is up-regulated in quiescent fibroblasts. Retroviral vectors containing human adenosine deaminase (ADA) cDNA linked to rat fibronectin promoter (LNFnA) or viral LTR promoter (LASN) were compared for their ability to express ADA from transduced primary rat skin fibroblasts in vivo. Skin grafts formed from fibroblasts transduced with LNFnA showed strong human ADA enzyme activity from 1 week to 3 months. In contrast, skin grafts containing LASN-transduced fibroblasts tested positive for human ADA for weeks 1 and 2, were faintly positive at week 3 and showed no human ADA expression at 1, 2 and 3 months. Thus, a fibronectin promoter provided sustained transduced gene expression at high levels for at least 3 months in transplanted rat skin fibroblasts, perhaps permitting the targeting of this tissue for human gene therapy.

Affected and non-affected skin fibroblasts from systemic sclerosis patients share a gene expression profile deviated from the one observed in healthy individuals

Objectives To evaluate the gene expression profile of fibroblasts from affected and non-affected skin of systemic sclerosis (SSc) patients and from controls. Materials and methods Labeled cDNA from fibroblast cultures from forearm (affected) and axillary (non-affected) skin from six diffuse SSc patients, from three normal controls, and from MOLT-4/HEp-2/normal fibroblasts (reference pool) was probed in microarrays generated with 4193 human cDNAs from the IMAGE Consortium. Microarray images were converted into numerical data and gene expression was calculated as the ratio between fibroblast cDNA (Cy5) and reference pool cDNA (Cy3) data and analyzed by R environment/Aroma, Cluster, Tree View, and SAM softwares. Differential expression was confirmed by real time PCR for a set of selected genes. Results Eighty-eight genes were up- and 241 genes down-regulated in SSc fibroblasts. Gene expression correlation was strong between affected and non-affected fibroblast samples from the same patient (r>0.8), moderate among fibroblasts from all patients (r=0.72) and among fibroblasts from all controls (r=0.70), and modest among fibroblasts from patients and controls (r=0.55). The differential expression was confirmed by real time PCR for all selected genes. Conclusions Fibroblasts from affected and non-affected skin of SSc patients shared a similar abnormal gene expression profile, suggesting that the widespread molecular disturbance in SSc fibroblasts is more sensitive than histological and clinical alterations. Novel molecular elements potentially involved in SSc pathogenesis were identified.

Effect of laser phototherapy on the release of fibroblast growth factors by human gingival fibroblasts

The effects of laser phototherapy on the release of growth factors by human gingival fibroblasts were studied in vitro. Cells from a primary culture were irradiated twice (6 h interval), with continuous diode laser [gallium-aluminum-arsenium (GaAlAs), 780 nm, or indium-gallium-aluminum-phosphide (InGaAlP),_660 nm] in punctual and contact mode, 40 mW, spot size 0.042 cm(2), 3 J/cm(2) and 5 J/cm(2) (3 s and 5 s, respectively). Positive [10% fetal bovine serum (FBS)] and negative (1%FBS) controls were not irradiated. Production of keratinocyte growth factor (KGF) and basic fibroblast growth factor (bFGF) was quantified by enzyme-linked immunosorbent assay (ELISA). The data were statistically compared by analysis of variance (ANOVA) followed by Tukey`s test (P a parts per thousand currency signaEuro parts per thousand 0.05). The characterization of the cell line indicated a mesenchymal nature. KGF release was similar in all groups, while that of bFGF was significantly greater (1.49-times) in groups treated with infra-red laser. It was concluded that increased production of bFGF could be one of the mechanisms by which infra-red laser stimulates wound healing.

Expression of intercellular adhesion molecule-1 in UVA-irradiated human skin cells in vitro and in vivo.

Ultraviolet A (UVA) radiation represents an important oxidative stress to human skin and certain forms of oxidative stress have been shown to modulate intercellular adhesion molecule-1 (ICAM-1) expression. ICAM-1 has been shown to play an important part in many immune reactions and the perturbations of this molecule by ultraviolet radiation could have implications in many inflammatory responses. An enhancement immunohistochemical method with avidin/biotin was used for analysing the early effects of UVA radiation on human cell cultures and human skin (340-400 nm). Both in vitro and in vivo data show that ICAM-1 staining in epidermal keratinocytes, which was expressed constitutively, decreased in a UVA dose-dependent manner. The decrease was most noted at 3-6 h following UVA radiation with some ICAM-1 staining returning by 48 h post-UVA. ICAM-1 positive staining in the dermis was specific for vascular structures and was increased 24 h after UVA radiation. Cultured dermal fibroblasts exhibited ICAM-1 staining which increased slightly within 6-48 h post-UVA radiation. As epidermal ICAM-1 expression is depleted following UVA radiation and dermal expression increases due to an increase in the vascular structures, ICAM-1 provides a valuable marker following UVA radiation in human skin that can be readily measured in situ.

Enhancement of DNA repair in human skin cells by thymidine dinucleotides: Evidence for a p53-mediated mammalian SOS response

Thymidine dinucleotide (pTpT) stimulates melanogenesis in mammalian pigment cells and intact skin, mimicking the effects of UV irradiation and UV-mimetic DNA damage. Here it is shown that, in addition to tanning, pTpT induces a second photoprotective response, enhanced repair of UV-induced DNA damage. This enhanced repair results in a 2-fold increase in expression of a UV-damaged chloramphenicol acetyltransferase expression vector transfected into pTpT-treated skin fibroblasts and keratinocytes, compared with diluent-treated cells. Direct measurement of thymine dimers and (6–4) photoproducts by immunoassay demonstrates faster repair of both of these UV-induced photoproducts in pTpT-treated fibroblasts. This enhanced repair capacity also improves cell survival and colony-forming ability after irradiation. These effects of pTpT are accomplished, at least in part, by the up-regulation of a set of genes involved in DNA repair (ERCC3 and GADD45) and cell cycle inhibition (SDI1). At least two of these genes (GADD45 and SDI1) are known to be transcriptionally regulated by the p53 tumor suppressor protein. Here we show that pTpT activates p53, leading to nuclear accumulation of this protein, and also increases the specific binding of this transcription factor to its DNA consensus sequence.

Sustained delivery of erythropoietin in mice by genetically modified skin fibroblasts.

We have examined whether the secretion of erythropoietin (Epo) from genetically modified cells could represent an alternative to repeated injections of the recombinant hormone for treating chronic anemias responsive to Epo. Primary mouse skin fibroblasts were transduced with a retroviral vector in which the murine Epo cDNA is expressed under the control of the murine phosphoglycerate kinase promoter. "Neo-organs" containing the genetically modified fibroblasts embedded into collagen lattices were implanted into the peritoneal cavity of mice. Increased hematocrit (> 80%) and elevated serum Epo concentration (ranging from 60 to 408 milliunits/ml) were observed in recipient animals over a 10-month observation period. Hematocrit values measured in recipient mice varied according to the number of implanted Epo-secreting fibroblasts (ranging from 2.5 to 20 x 10(6)). The implantation of neo-organs containing Epo-secreting fibroblasts appeared, therefore, as a convenient method to achieve permanent in vivo delivery of the hormone. We estimated that the biological efficacy of the approach may be relevant for the treatment of human hemoglobinopathies.