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Human Fibroblasts (human + fibroblast)
Kinds of Human Fibroblasts Terms modified by Human Fibroblasts Selected AbstractsCell Proliferation of Human Fibroblasts on Alumina and Hydroxyapatite-Based Ceramics with Different Surface Treatments,INTERNATIONAL JOURNAL OF APPLIED CERAMIC TECHNOLOGY, Issue 2 2010Juliana Marchi Biocompatibility is an important characteristic of dental implant material, and in vitro tests are required to elucidate the interaction between these materials and human tissues. Cell proliferation assays were done with fibroblasts plated on the surface of alumina and hydroxyapatite sintered samples, each with a different surface treatment (sintered, rectified, or polished). After 1, 2, and three days, the samples were prepared for scanning electron microscopy observations. The data were compared by analysis of variance followed by Tukey's test. It was concluded that neither the hydroxyapatite or alumina substrate is cytotoxic, and hydroxyapatite is more biocompatible than alumina. [source] Phototoxicity of Protoporphhyrin IX, Diarginine Diprotoporphyrinate and N,N-Deiphenylalanyl Protoporphyrin Toward Human Fibroblasts and ketratinocytes In vitro: Effect of 5-Methoxypsoralen,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 3 2004Andrzej Bugaj The phototoxicity of two new porphyrin photosensitizers, diarginine diprotoporphyrinate (PP(Arg)2) and N,N -diphenylalanyl protoporphyrin (PP(Phe)2), and the synergistic effect of 5-methaoxyposralen (5-MOP) have been studied in comparison with that of protoporphyrin IX (PPIX). Under ultraviolet A (UV-A) irradiation (,= 365 nm), the phototoxicity of the porphyrins toward cultured human fibroblasts and keratino-cytes decerases in the order: PPIX > PP(Arg)2 > PP(Phe)2. A synergistic efect of 5-MOP on the phototoxicity of PPIX, PP(Arg)2 and PP(Phe)2 has been observed. The combination of PPIX, PP(Arg)2 and PP(Phe)2 with 0.1,0.5 ,M 5-MOP significantly potentiates the phototoxicity of the three porphyrins. The most effective potentiation was observed with the water-soluble PP(Arg)2 and 5-MOP concentrations lowere than 0.75 ,M. Above this 5-MOP concnetration this potentiation is abolished. The intracellular concentration of PPIX and PP(Phe)2 is independent of the presence of 5-MOP. On the other hand, the intracellular contnet of PP(Arg)2 is decerased in concentration-dependent manner by the psoralen. Illumination with red light, not absorbed by 5-MOP, leads to a weak potentiation of the PP(Arg)2 phototoxic effect in the presence of 5-MOP, suggesting that dark interaction of 5-MOP with cell membranes aggravated by porphyrin photosensitization is involved in the observed phenomena. The results are tentatively explained by differences in hydrophobicity and molecular structure of the examined photosensitizers. PPIX, which is barely soluble in water, has a significantly higher affinity for cell membranes and simultaneously exerts a stronger phototoxic effect than PP(Arg)2 whose solubility in water is high. On the other hand, the weak phototoxicity of PP(Phe)2 could be explained by the steric hindrance brought by the phenylalanyl substituents on the pyrrole ring. The loss in the PP(Arg)2 cell content probably explains the inhibition of the synergistic effect of 5-MOP on the PP(Arg)2 phototoxicity at high 5-MOP concentration. This study suggests that PP(Arg)2 in combinatin with 5-MOP might reveal a strong phototoxic effect when applied to skin cancer treatment. [source] UV-enhanced Expression of a Reporter Gene is Induced at Lower UV Fluences in Transcription-coupled Repair Deficient Compared to Normal Human Fibroblasts, and is Absent in SV40-transformed Counterparts,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2000Murray A. Francis ABSTRACT UV irradiation enhances transcription of a number of cellular and viral genes. We have compared dose responses for alterations in expression from reporter constructs driven by the human and murine cytomegalovirus (CMV) immediate early (IE) promoters in cells from patients with deficiencies in nucleotide excision repair (complementation groups of xeroderma pigmentosum and Cockayne syndrome) following UV exposure, or infection with UV-damaged recombinant vectors. Results suggest that unrepaired damage in active genes triggers increased reporter activity from constructs driven by the CMV promoters in human fibroblasts. Similar to human fibroblasts, HeLa cells and cells from Li,Fraumeni syndrome patients (characterized by an inherited mutation in the p53 gene) also displayed an increase in reporter activity following UV exposure; however, this response was absent in all simian virus 40 (SV40)-transformed cell lines examined. This suggests that a pathway affected by SV40-transformation (other than p53) plays an essential role in UV-enhanced expression from the CMV IE promoter. [source] Photoprotection of bacterial-derived melanin against ultraviolet A,induced cell death and its potential application as an active sunscreenJOURNAL OF THE EUROPEAN ACADEMY OF DERMATOLOGY & VENEREOLOGY, Issue 7 2008J Geng Abstract Background, The increase in the incidence of non-melanoma skin tumours, photoaging, and immunosuppression demand for more effective sunscreen on ultraviolet A (UVA) irradiation. Objectives, The aim of the study is to evaluate the photoprotective effects of a bacterial-derived melanin against UVA-induced damages in vitro and in vivo. Methods, Human fibroblasts were used to assess the role of the bacterial-derived melanin on cell viability against UVA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and nuclear morphology were employed to evaluate the photoprotection at the cellular level. Fluorometric assays were performed to detect the formation of reactive oxygen species (ROS) in the cells. Evaluations of the bacterial-derived melanin as a sunscreen were measured by transmission test and persistent pigment darkening on human skin. Results, Bacterial-derived melanin efficiently scavenged ROS in the fibroblasts after UVA irradiation. The cell viability of xeroderma pigmentosum (XP) fibroblast treated with varied doses of melanin increased dramatically in comparison with untreated control and the treated XP fibroblasts became more resistant to UVA-induced apoptosis than normal fibroblasts. Although the relative transmission didn't change too much with different concentration of bacterial-derived melanin, this melanin could keep UVA-irradiated skin from pigment darkening and act as an active sunscreen on skin. Conclusions, The bacterial-derived melanin provided significant protection to fibroblast cell and human skin against the UVA radiation. It has the potential to be developed as an active sunscreen for the patients with photosensitivity skin to sun exposure. [source] Preparation and Characterization of Microwave-treated Carboxymethyl Chitin and Carboxymethyl Chitosan Films for Potential Use in Wound Care ApplicationMACROMOLECULAR BIOSCIENCE, Issue 10 2005Panya Wongpanit Abstract Summary: CM-chitin and CM-chitosan films were successfully crosslinked by microwave treatment. Crosslinking of the microwave-treated CM-chitin films involved mainly the carboxylate and the secondary alcohol groups, while crosslinking of microwave-treated CM-chitosan films involved the carboxylate and the amino groups. In addition, the crystallinity of CM-chitin increased with increasing microwave treatment time, whereas an increase in the crystallinity of the microwave-treated CM-chitosan films was not observed. At a similar percentage of weight loss, the crosslinking of either CM-chitin or CM-chitosan films by microwave treatment required much less stringent condition when compared with the crosslinking by autoclave treatment. Based on both direct and indirect cytotoxicity assays, the cytotoxicity of the microwave-treated CM-chitin films was negative, while that of the microwave-treated CM-chitosan films was positive. Human fibroblasts adhered on the surface of microwave-treated CM-chitosan films much better than on the surface of microwave-treated CM-chitin films. Total amount of protein synthesis of living NHGF cells that were cultured on chitin, microwave-treated CM-chitin, chitosan, microwave-treated CM-chitosan films. [source] Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan-binding protein 1 (HABP1)/p32/gC1qRFEBS JOURNAL, Issue 1 2002Babal Kant Jha Hyaluronan-binding protein 1 (HABP1), a ubiquitous multifunctional protein, interacts with hyaluronan, globular head of complement component 1q (gC1q), and clustered mannose and has been shown to be involved in cell signalling. In vitro, this recombinant protein isolated from human fibroblast exists in different oligomeric forms, as is evident from the results of various independent techniques in near-physiological conditions. As shown by size-exclusion chromatography under various conditions and glutaraldehyde cross-linking, HABP1 exists as a noncovalently associated trimer in equilibrium with a small fraction of a covalently linked dimer of trimers, i.e. a hexamer. The formation of a covalently-linked hexamer of HABP1 through Cys186 as a dimer of trimers is achieved by thiol group oxidation, which can be blocked by modification of Cys186. The gradual structural transition caused by cysteine-mediated disulfide linkage is evident as the fluorescence intensity increases with increasing Hg2+ concentration until all the HABP1 trimer is converted into hexamer. In order to understand the functional implication of these transitions, we examined the affinity of the hexamer for different ligands. The hexamer shows enhanced affinity for hyaluronan, gC1q, and mannosylated BSA compared with the trimeric form. Our data, analyzed with reference to the HABP1/p32 crystal structure, suggest that the oligomerization state and the compactness of its structure are factors that regulate its function. [source] Cosmeceutical properties of levan produced by Zymomonas mobilisINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 3 2006K. H. Kim Levan, a polysaccharide that can be produced by both plants and micro-organisms, is a sugar polymer composed of fructose, with-2,6 linkages. Here, we have attempted to assess the possible use of levan produced by Zymomonas mobilis as a cosmeceutical ingredient. In service of this goal, we assessed a host of levan's properties, including its moisturizing effects, cell cytotoxicity, cell proliferation effects and anti-inflammation effects. Levan exhibited a moisturizing effect that was almost exactly the same as that evidenced by hyaluronic acid, as well as a similar cell proliferation effect in human fibroblast and keratinocyte cell lines. Moreover, in our cell proliferation test, which was conducted using bio-artificial skin constructed via 3-dimensional (3-D) culture after the induction of primary skin inflammation with 0.05% sodium lauryl sulphate (SLS), cell viability in the presence of levan (0.01 and 0.05 mg mL,1) was determined to be higher than cell viability in the absence of levan. In our anti-inflammation test, which was also conducted using 3-D artificial skin, and which involved the measurement of a quantity of secreted interleukin-1 (IL-1), a pre-inflammatory mediator induced by SLS, we determined that the quantity of IL-1 in the 3-D artificial skin treated with 0.01 and 0.05 mg mL,1 of levan was less than that registered in a skin sample that had been treated only with SLS. In this study, we determined that levan exerted an anti-inflammatory effect against inflammatory reactions to skin irritants, and also that levan exerted a cell-proliferative effect in bio-artificial skin, thereby indicating its potential applicability as a cosmeceutical agent. [source] Sedum telephium L. Polysaccharide Content Affects MRC5 Cell Adhesion to Laminin and FibronectinJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2000L. RAIMONDI In traditional medicine the fresh leaves and juice of Sedum telephium L. are used as wound-healing promoters. Cell adhesion represents a primary event in wound repair and in tissue homeostasis, and therefore we have investigated the effect of Sedum juice and its main fractions, polysaccharides and flavonols, on human fibroblast (MRC5) adhesion to fibronectin and laminin. Our findings revealed that total Sedum juice strongly inhibited cell adhesion to laminin and fibronectin (EC50 1.03 ± 0.12 mg mL,1). This anti-adhesive feature was concentrated mainly in the two polysaccharide fractions (EC50 values comprised between 0.09 and 0.44 mg mL,1). The flavonol fractions did not seem to contribute to this effect. A first attempt to elucidate the polysaccharide-related anti-adhesive feature of Sedum juice was also performed. The results confirmed that natural polysaccharides, with chemical structures different from heparin, were able to interfere with integrin-mediated cell behaviour and they contributed to the outstanding effects of Sedum juice and to the role of polysaccharides in cell-matrix interaction. [source] Fibroblast elongation and dendritic extensions in constrained versus unconstrained microtissuesCYTOSKELETON, Issue 3 2009Dylan M. Dean Abstract Cytoskeletal tension is fundamental to many biological processes, including germ layer sorting during embryogenesis [Krieg et al., 2008]. In vitro, such tension influences cell sorting in self-assembled, 3D microtissues and can be of sufficient magnitude to cause complex-shaped microtissue failure [Dean et al., 2007]. To examine the process of failure under cell-derived tension, we subjected normal human fibroblasts (NHFs) to directed self-assembly [Dean et al., 2007] in micro-molds designed to yield self-constraining microtissues. As cells contracted in this assay, the constrained microtissues narrowed, thinned and ultimately failed at their midpoints. By adding small numbers of GFP+ cells, changes in cell movement and morphology were assessed and compared to those of unconstrained microtissues. We found that cells formed numerous dendritic extensions within an hour of self-assembly and retracted these extensions as they elongated up to 30 times their initial diameter (,600 ,m) just prior to failure. Surprisingly, significant coordination in cell motility was observed over large distances within microtissues. Pharmacologic interventions showed that failure was myosin II and Rho kinase dependent and inhibition of failure resulted in shorter cells with greater numbers of extensions. These findings further our understanding of cellular self-assembly and introduce the use of GFP+ cells with directed self-assembly as a scaffold-free analogue to fibroblast-populated collagen gels (FPCGs). Cell Motil. Cytoskeleton 2009. © 2009 Wiley-Liss, Inc. [source] Effects of oestrogen agonists on human dermal fibroblasts in an in vitro wounding assayEXPERIMENTAL DERMATOLOGY, Issue 11 2009Susan Stevenson Abstract:, Oestrogen and dehydroepiandrosterone (DHEA) improve wound healing, but circulating levels decline significantly with age. Recently, the selective oestrogen receptor modulators (SERMs) tamoxifen and raloxifene have been shown to improve age-associated impaired wound healing. Therefore, we have evaluated the effects of 17,-oestradiol, ER, and ER, agonists, tamoxifen, raloxifene and DHEA on human dermal fibroblasts using an in vitro wound assay. An ER, agonist, 17,-oestradiol and DHEA all significantly accelerated cell migration; the DHEA effect was blocked with an aromatase inhibitor. Tamoxifen, raloxifene and DHEA all significantly increased DNA synthesis; the DHEA stimulatory effect was reversed by an aromatase inhibitor. This study demonstrates that 17,-oestradiol, an ER, agonist, tamoxifen, raloxifene and DHEA (following conversion to oestrogen) all have significant effects on human fibroblasts, the key mesenchymal cell involved in the wound healing process. Further understanding of the mechanisms involved may have important implications for the management of age-related impaired wound healing. [source] In vitro interactions between sensory nerves, epidermis, hair follicles and capillaries in a tissue-engineered reconstructed skinEXPERIMENTAL DERMATOLOGY, Issue 9 2004V. Gagnon Recent findings have established that cutaneous nerves modulate both skin homeostasis and various skin diseases, by influencing cell growth and differentiation, inflammation and wound healing. In order to study the influence of epidermis, hair follicles and capillaries on sensory neurons, and vice-versa, we developed a tissue-engineered model of innervated endothelialized reconstructed skin (MIERS). Mouse dorsal root ganglia neurons were seeded on a collagen sponge populated with human fibroblasts and human endothelial cells. Keratinocytes or mice newborn immature hair follicle buds were then seeded on the opposite side of the MIERS to study their influence on sensory nerves growth, and vice versa. A vigorous neurite elongation was detected inside the reconstructed dermis after 14 and 31 days of neurons culture. The presence of endothelial cells induced a significant increase of the neurite elongation after 14 days of culture. The addition of human keratinocytes totally avoided the twofold decrease in the amount of neurites observed between 14 and 31 days in controls. We have successfully developed the MIERS that allowed us to study the effects of epidermis and capillaries on nerve growth. This model will be a useful tool to study the modulation of sensory nerves on wound healing, angiogenesis, hair growth and neurogenic inflammation in the skin. [source] ERK activation by mechanical strain is regulated by the small G proteins rac-1 and rhoAEXPERIMENTAL DERMATOLOGY, Issue 2 2004Julien Laboureau Abstract: Physical forces play an important role in regulating cell functions. We applied mechanical strain to human fibroblasts by magnetic attraction of superparamagnetic arginine-glycine-aspartic acid (RGD)-coated beads. We confirmed that the MAP kinases Erk and p38 are activated by mechanical strain, and went further by demonstrating the activation of Elk-1 by mechanical strain, mainly through a MEK-Erk pathway. Transfection of a dominant negative form of the G protein rac-1 (rac T17N), and inhibition of PI3K, an effector of rac-1, efficiently prevented Elk-1 activation by mechanical forces. Transfection with C3 transferase, known to inhibit rhoA, and inhibition of rock (a downstream effector of rhoA), gave similar results. However, contrary to the active form of rhoA (rho G14V), transfection of the active form of rac-1 (rac G12V) induced Elk activation and mimicked the effects of mechanical strain. These results point out that the two small G proteins rhoA and rac-1 participate in cell sensitivity to mechanical strain and lead to the modulation of the Erk pathway. [source] Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell linesFEBS JOURNAL, Issue 16 2004Georgia Xouri Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3,6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells. [source] Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based AssaysADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Steffen Cosson Abstract A versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady-state gradients of biotinylated or Fc-tagged fusion proteins that are captured and bound to the surface in less than 5,min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single-cell migration experiments using time-lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types. [source] Accumulation of multiple forms of lamin A with down-regulation of FACE-1 suppresses growth in senescent human cellsGENES TO CELLS, Issue 3 2007Ryo Ukekawa 5-Bromodeoxyuridine (BrdU) clearly induces a senescence-like phenomenon in every cell type. Proteome analysis revealed that lamin A and C were most highly increased in the nuclei of HeLa cells upon addition of BrdU. Immunoblot analysis also revealed marked accumulation of nuclear prelamin A. Consistently, farnesylated-proteins converting enzyme 1 (FACE-1) was markedly down-regulated in the same cells. Similar phenomena were also observed in normal human fibroblasts undergoing replicative senescence. Immunochemical analysis confirmed the above results. Lamin A is a major component of lamina and responsible for several genetic diseases. Thus, we ectopically expressed a wild-type, a mature type and a premature type of lamin in HeLa cells. All of these forms similarly inhibited colony formation and delayed cell cycle progression mainly through G2 phase. These results suggest that a change in the amount of lamin A, rather than appearance of its truncated form, is responsible for growth retardation in affected cells. [source] Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation,HEPATOLOGY, Issue 5 2007Philippe A. Lysy The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte-like morphology and acquired liver-specific markers on protein and gene expression levels. Furthermore, these fibroblast-derived hepatocyte-like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal-epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte-like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha-fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver-specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal-derived hepatocyte-like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels. (HEPATOLOGY 2007;46:1574,1585.) [source] Adenovirus-mediated small hairpin RNA targeting Bcl-XL as therapy for colon cancerINTERNATIONAL JOURNAL OF CANCER, Issue 6 2007Hongbo Zhu Abstract Bcl-XL, an anti-apoptotic protein of Bcl-2 family, is overexpressed in colon cancers. To determine Bcl-XL's potential feasibility as a therapeutic target, we constructed a recombinant adenovirus that expressed a U6 promoter-driven small hairpin RNA (shRNA) targeting Bcl-XL (Ad/Bcl-XL shRNA) and evaluated the vector's ability to induce RNA interference in vivo and alter apoptosis induction in colon cancer cells and tumours. Ad/Bcl-XL shRNA effectively knocked down Bcl-XL expression in colon cancer cells and decreased their viability. Treatment with Ad/Bcl-XL shRNA but not control vectors led to dramatically increased cleavage of cellular apoptosis-related enzymes caspase-9, caspase-3 and poly(ADP-ribose) polymerase. Ad/Bcl-XL shRNA also significantly suppressed the growth of subcutaneous tumours derived from DLD1 cells in a nude mouse model and did so without causing any obvious damage to normal tissues or normal human fibroblasts. Together, our results support the feasibility of using adenovirus-mediated RNA interference therapy targeting Bcl-XL against colon cancers and warrant further studies of its safety and efficacy. © 2007 Wiley-Liss, Inc. [source] Two new lipoaminoacids with complementary modes of action: new prospects to fight out against skin agingINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 1 2010S. Dumont Synopsis The mode of action of two cosmetic active ingredients (AIs), palmitoyl glycine (PG) and cocoyl alanine (CA) was studied with cDNA array experiments and quantitative PCR confirmations, which were performed on experimentally aged human fibroblasts. These preliminary studies revealed complementary profiles. Thus, specific supplementary investigations were then carried out for each AI. Protocols used were based either on in vitro models: (i) biochemical assays, (ii) monolayer cell culture (primary human fibroblasts and keratinocytes) and (iii) the model of capillary-like tube formation by human endothelial cells or on ex vivo models, i.e. topically treated skin explants and both immunohistochemical and ChromameterTM investigations. New prospects are proposed to fight out against skin aging. Indeed, PG and CA showed complementary properties and thus enabled a regulation or a restoration effect on main aging-associated disorders. Thus, they can not only act on tissue architecture, cell,cell interactions and extracellular matrix protection but also on inflammation, cell longevity, skin immune system protection, skin radiance and stem cell survey. Finally, a clinical trial performed on Caucasian women confirmed AI anti-wrinkle efficacy, which was superior to that of a market reference ingredient. In the future, complementary experiments enabling a better understanding of the aging-induced decline of epidermal stem cells would be of a great interest. Résumé Le mode d'action de deux actifs cosmétiques, Palmitoyl glycine (PG) et Cocoyl Alanine (CA), a été déterminéà l'aide d'expériences de cDNA arrays et de confirmations par qPCR, réalisées sur des fibroblastes humains vieillis expérimentalement. Ces études préliminaires ont révélé des modes d'action complémentaires. Des expériences supplémentaires spécifiques ont donc ensuite été réalisées pour chaque actif. Les protocoles utilisés étaient basés sur des modèles in vitro: i) études biochimiques, ii) cultures cellulaires en monocouches (cultures primaires de fibroblastes et de kératinocytes humains) et iii) modèle de formation de pseudo-tubules par des cellules endothéliales humaines; ou sur des modèles ex-vivo, i.e. des explants de peau traités de manière topique et analysés à l'aide d'études immuno-histochimiques et d'un ChromamètreTM. De nouvelles perspectives s'ouvrent pour combattre le vieillissement cutané. En effet, PG et CA montrent des propriétés complémentaires et permettent ainsi une régulation ou une restauration des principaux dysfonctionnements liés à l'âge. Ainsi, ils peuvent agir non-seulement sur l'architecture des tissus, l'interaction entre les cellules et la protection de la matrice extracellulaire mais aussi sur l'inflammation, la longévité cellulaire, la survie des cellules souches, le système de protection immunitaire et l'éclat de la peau. Finalement, des essais cliniques réalisés sur des femmes de type Caucasien ont confirmé l'efficacité antirides des actifs, laquelle était supérieure à celle d'une référence anti-âge du marché. Dans un futur proche, des tests complémentaires pourraient permettre une meilleure compréhension de la dégradation des cellules souches épidermiques au cours du vieillissement. [source] Cosmeceutical properties of polysaccharides from the root bark of Ulmus davidiana var. japonicaINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 2 2007Sang Yong Eom In Korea and China, Ulmus davidiana var. japonica has been used as a traditional oriental medicine for the treatment of difficulty in urination, skin inflammation, etc. In order to investigate the potential of a polysaccharide extract from Ulmus davidiana var. japonica as a cosmetic ingredient, we measured its moisturizing effect, photo-induced cytotoxicity, and anti-inflammatory effect. After hydrolysis, HPLC experiments showed that the composition of the polysaccharide extract was mainly rhamnose, galactose, and glucose. The molecular weight of the obtained Ulmus davidiana root extract was 20 000. The intrinsic viscosity was 90 dL/g. In a moisturizing test conducted through the measurement of water loss in a desiccator and of moisture content with a Corneometer CM820, Ulmus davidiana root extract showed almost the same moisturizing effect as hyaluronic acid. In an assay for inhibition of the H2O2-activated release of PGE2, IL-6, and IL-8 in normal human fibroblast cell lines, Ulmus davidiana root extract showed an inhibitory activity of PGE2 release in a dose-dependent manner (up to 85.9% at a concentration of 0.1%). The percent inhibition of the release of IL-6 was in the range of 45.6,64.5% (H2O2 was used as the positive control). Moreover, the release of IL-8 was completely inhibited in the entire concentration range (>0.0025%). In a test of recovery from photo-induced damage after UVA irradiation (3 J/cm2), the cell recovery of human fibroblasts increased to levels two times higher than that of the positive control, which was UVA-damaged cells in the absence of Ulmus davidiana root extract (up to 60.2% at 3.0% of Ulmus davidiana root extract). In a photo-induced cytotoxicity assay in the presence of promethazine as a photosensitizer, Ulmus davidiana root extract showed approximately 48% of the increased cell viability of the control. Therefore, Ulmus davidiana root extract may be useful for the development of a cosmetic ingredient. [source] Appearance of biomarkers of in vitro ageing after successive stimulation of WI-38 fibroblasts with IL-1, and TNF-,: senescence associated ,-galactosidase activity and morphotype transitionJOURNAL OF ANATOMY, Issue 4 2000PATRICK DUMONT Sublethal oxidative stresses increase the proportions of human fibroblasts positive for senescence associated ,-galactosidase activity and accelerate the transition in the fibroblast morphotypes characterising fibroblast ageing. Stimulation of fibroblasts with TNF-, or IL-1, transiently increases the production of reactive oxygen species (ROS) in human fibroblasts. Here we propose that repeated stimulation of WI-38 fibroblasts with TNF-, or IL-1, can generate enough ROS to accelerate the transition in the fibroblast morphotypes and increase the proportion of cells positive for senescence associated ,-galactosidase activity. The involvement of ROS is suggested by experiments where the stimulation of fibroblasts with TNF-, or IL-1, are performed in the presence of N-acetylcysteine which increases the intracellular antioxidant potential. It is proposed that the decrease in the proportions of morphotypes I and II, and the increase in the proportions of morphotypes III to VI observed after successive stimulation with TNF-, or IL1-, is attributed to an increased ROS production occurring during the stimulation. [source] Electrospinning of degradable elastomeric nanofibers with various morphology and their interaction with human fibroblastsJOURNAL OF APPLIED POLYMER SCIENCE, Issue 1 2008Erik Borg Abstract Artelon® (degradable poly(urethane urea) elastomer) was electrospun into scaffolds for tissue engineering. The diameter of the electrospun fibers, studied by scanning electron microscopy, ranged from 100 nm to a few ,m, with an average diameter of 750 nm. The molar mass of the polymer had a major influence on the morphology of the scaffold. Furthermore, aging of the polymer solution caused changes in viscosity, as measured by stress sweeps between 13.5,942 Pa that affected the morphology. The electrospun Artelon mats exhibited about the same elongations to break, both exceeding 200%, measured by tensile tests. The degradation study showed similar degradation behavior in electrospun mats and solids. In vitro study showed that human fibroblasts not only adhere to the surface but also migrate, proliferate, and produce components of an extracellular matrix. These results strongly support the use of electrospun Artelon as a scaffold in tissue engineering. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 [source] Chitosan-alginate films prepared with chitosans of different molecular weightsJOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 4 2001Xiao-Liang Yan Abstract Chitosan-alginate polyelectrolyte complex (CS-AL PEC) is water insoluble and more effective in limiting the release of encapsulated materials compared to chitosan or alginate. Coherent CS-AL PEC films have been prepared in our laboratory by casting and drying suspensions of chitosan-alginate coacervates. The objective of this study was to evaluate the properties of the CS-AL PEC films prepared with chitosans of different molecular weights. Films prepared with low-molecular-weight chitosan (Mv 1.30 × 105) were twice as thin and transparent, as well as 55% less permeable to water vapor, compared to films prepared with high-molecular-weight chitosan (Mv 10.0 × 105). It may be inferred that the low-molecular-weight chitosan reacted more completely with the sodium alginate (Mv 1.04 × 105) than chitosan of higher molecular weight. A threshold molecular weight may be required, because chitosans of Mv 10.0 × 105 and 5.33 × 105 yielded films with similar physical properties. The PEC films exhibited different surface properties from the parent films, and contained a higher degree of chain alignment with the possible formation of new crystal types. The PEC films exhibited good in vitro biocompatibility with mouse and human fibroblasts, suggesting that they can be further explored for biomedical applications. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res (Appl Biomater) 58: 358,365, 2001 [source] Role of D1 and E Cyclins in Cell Cycle Progression of Human Fibroblasts Adhering to Cementum Attachment Protein,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Takayoshi Yokokoji Abstract Cementum attachment protein (CAP) is a collagenous protein present in the matrix of tooth cementum that mediates preferential attachment of some mesenchymal cell types, and CAP binding capacity is related to mineralizing tissue-forming capacity in culture. We have examined if adhesion to surfaces containing CAP as the only attachment protein permits human fibroblasts to escape G1 arrest and synthesize DNA, and if adhesion to CAP modulates the levels of cyclins D1 and E. Human gingival fibroblasts (HGFs) were serum-starved, trypsinized, and added to plates coated with CAP or bovine serum albumin (BSA). Cells were then exposed to either 10% fetal bovine serum (FBS) or to cementum-derived growth factor (CGF), an insulin-like growth factor I (IGF-I)-like molecule sequestered in tooth cementum, plus epidermal growth factor (EGF). DNA synthesis was measured as [3H]thymidine uptake, and cyclin D1 and E levels were determined by Western analysis. Cyclin E-dependent kinase (Cdk) activity was assessed in terms of H1 kinase activity in immunoprecipitates of cyclin E. Cells adhering to CAP synthesized DNA, whereas on BSA they remained unattached and did not synthesize DNA. Protein levels of cyclin D1 were higher in cells adhering to CAP in the absence and presence of growth factors. Cyclin E levels were not affected by adhesion alone, but they increased in the presence of growth factors. Cyclin E-associated kinase activity was higher in cells adherent on CAP, and it increased further in the presence of growth factors. Our results indicate that adhesion to CAP increases cyclin D1 levels and cyclin E-associated Cdk activity, and that these increases contribute to cell cycle progression. We previously observed that the signaling reactions induced during adhesion are characteristic of the CAP; together these observations indicate that specific matrix components present in the local environment can contribute to recruitment and differentiation of specific cell types for normal homeostasis and wound healing. [source] PEDF from mouse mesenchymal stem cell secretome attracts fibroblastsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2008Harshini Sarojini Abstract Conditioned medium (secretome) derived from an enriched stem cell culture stimulates chemotaxis of human fibroblasts. These cells are classified as multipotent murine mesenchymal stromal cells (mMSC) by immunochemical analysis of marker proteins. Proteomic analysis of mMSC secretome identifies nineteen secreted proteins, including extracellular matrix structural proteins, collagen processing enzymes, pigment epithelium-derived factor (PEDF) and cystatin C. Immunodepletion and reconstitution experiments show that PEDF is the predominant fibroblast chemoattractant in the conditioned medium, and immunofluorescence microscopy shows strong staining for PEDF in the cytoplasm, at the cell surface, and in intercellular space between mMSCs. This stimulatory effect of PEDF on fibroblast chemotaxis is in contrast to the PEDF-mediated inhibition of endothelial cell migration, reported previously. These differential functional effects of PEDF toward fibroblasts and endothelial cells may serve to program an ordered temporal sequence of scaffold building followed by angiogenesis during wound healing. J. Cell. Biochem. 104: 1793,1802, 2008. © 2008 Wiley-Liss, Inc. [source] Nicotine inhibits human gingival fibroblast migration via modulation of Rac signalling pathwaysJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 12 2005Yiyu Fang Abstract Aim: Cigarette smoking is a risk factor in the development of periodontal diseases. In addition, a delayed healing process has been shown in smokers compared with non-smokers after periodontal treatment. Cell migration is a key process of wound healing and it is highly regulated by a variety of signalling pathways. The small G protein, Rac, is necessary for cell migration. Our aim was to determine if nicotine disrupted Rac and its downstream signalling proteins, p21-activated kinase 1/2 (PAK1/2), and p44/42 mitogen-activated protein kinase (MAPK) (extracellular regulated kinase 1/2). Material and Methods: Primary human fibroblasts from healthy gingival tissues were cultured and grown to confluence. Cells were serum starved for 24 h, and then treated with nicotine (0 or 0.5 ,M) prior to in vitro wounding. Cell migration was analysed in live cell assays following in vitro wounds. Rac activity, phosphorylation levels of PAK1/2, and p44/42 MAPK were assessed in cultures treated with or without nicotine after multiple wounds. Results: Nicotine decreased cell migration rates by 50% compared with controls. In addition, nicotine altered the activation patterns of Rac and PAK 1/2 and up-regulated p44/42 MAPK. Conclusion: Decreased cell migration in periodontal wounds exposed to nicotine may be mediated through the Rac and PAK1/2 signalling pathways. [source] Nicotinamide enhances mitochondria quality through autophagy activation in human cellsAGING CELL, Issue 4 2009Hyun Tae Kang Summary Nicotinamide (NAM) treatment causes a decrease in mitochondrial respiration and reactive oxygen species production in primary human fibroblasts and extends their replicative lifespan. In the current study, it is reported that NAM treatment induces a decrease in mitochondrial mass and an increase in membrane potential (,,m) by accelerating autophagic degradation of mitochondria. In the NAM-treated cells, the level of LC3-II as well as the number of LC3 puncta and lysosomes co-localizing with mitochondria substantially increased. Furthermore, in the NAM-treated cells, the levels of Fis1, Drp1, and Mfn1, proteins that regulate mitochondrial fission and fusion, increased and mitochondria experienced dramatic changes in structure from filaments to dots or rings. This structural change is required for the decrease of mitochondrial mass indicating that NAM accelerates mitochondrial autophagy, at least in part, by inducing mitochondrial fragmentation. The decrease in mitochondria mass was attenuated by treatment with cyclosporine A, which prevents the loss of mitochondrial membrane potential by blocking the mitochondrial permeability transition, suggesting autophagic degradation selective for mitochondria with low ,,m. All these changes were accompanied by and dependent on an increase in the levels of GAPDH, and are blocked by inhibition of the cellular conversion of NAM to NAD+. Taken together with our previous findings, these results suggest that up-regulation of GAPDH activity may prolong healthy lifespan of human cells through autophagy-mediated mitochondria quality maintenance. [source] Nuclear accumulation of glycogen synthase kinase-3 during replicative senescence of human fibroblastsAGING CELL, Issue 5 2004Jaroslaw W. Zmijewski Summary Activation of the tumor suppressor protein p53 contributes to cellular senescence. As glycogen synthase kinase-3 (GSK3) was recently found to interact with p53 and contribute to the actions of p53, this study examined whether GSK3 accumulated in the nucleus and associated with p53 in senescent cells. Compared with young and middle-aged human WI-38 fibroblasts, senescent cells were found to contain increased nuclear levels of GSK3,, and also tended to accumulate in the nucleus the other isoform of GSK3, GSK3,. Co-immunoprecipitation experiments demonstrated that GSK3, and p53 formed a complex in the nucleus. Further experiments tested whether inhibition of GSK3 altered the development of senescence using long-term treatment with the selective GSK3 inhibitor lithium. Lithium treatment reduced the senescence-associated accumulation of p53 and caused cells to enter a reversible quiescent state. These results indicate that a portion of the p53 that is activated in senescent cells is modulated by its association with GSK3, in the nucleus, an association that is known to facilitate the actions of p53 and that may contribute to senescence. [source] Pro-inflammatory cytokine production from normal human fibroblasts is induced by Tannerella forsythia detaching factorJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2008N. Tomi Background and Objective:,Tannerella forsythia is a periodontal pathogen. Recently, we have reported that the cytopathic component of T. forsythia contains two distinct factors. One arrests the cell cycle at the G2 phase and the other, named forsythia detaching factor, detaches adhesion-dependent immortalized human cells. In this study, we investigated the biological function of forsythia detaching factor using human normal fibroblasts. Material and Methods:, A recombinant forsythia detaching factor, reported previously, was used. TIG-3 cells, cultured in the absence or presence of forsythia detaching factor, were lysed and the supernatant was analyzed by western blotting with polyclonal forsythia detaching factor antibodies. The cells were subsequently fractionated to isolate the cytoplasmic, mitochondrial and remaining fractions. In order to measure the activity of mitochondria using nicotinamide adenine dinucleotide-linked reductase, the water-soluble tetrazolium method was used. The mitochondrial oxidative membrane potential was estimated by measuring the oxidization-dependent fluorogenic conversion of dihydrotetramethylrosamine using flow cytometry. The concentration of interleukin-8 in the culture supernatant was assayed using a Human IL-8 ELISA kit. Results:, Forsythia detaching factor-treated cells detached from the substratum and aggregated from 3 to 24 h. Then, the detached cells resumed adhesion and proliferated after 48 h. The western blot analysis revealed that most forsythia detaching factor trans -located into the mitochondrial fraction. Forsythia detaching factor suppressed the nicotinamide adenine dinucleotide-linked reductase activity in a dose-dependent manner and consequently increased the mitochondrial oxidative membrane potential. The production of interleukin-8 was reinforced in forsythia detaching factor-treated cells at 72 h through an increase of the mitochondrial oxidative membrane potential. Conclusion:, The forsythia detaching factor might be involved in the virulence of T. forsythia through induction of the pro-inflammatory cytokine interleukin-8. [source] Identification of marker genes distinguishing human periodontal ligament cells from human mesenchymal stem cells and human gingival fibroblastsJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2007T. Fujita Background and Objective:, Molecular gene markers, which can distinguish human bone marrow mesenchymal stem cells from human fibroblasts, have recently been reported. Messenger RNA levels of tissue factor pathway inhibitor-2, major histocompatibility complex-DR-,, major histocompatibility complex-DR-,, and neuroserpin are higher in human bone marrow mesenchymal stem cells than in human fibroblasts. However, human bone marrow mesenchymal stem cells express less apolipoprotein D mRNA than human fibroblasts. Periodontal ligament cells are a heterogeneous cell population including fibroblasts, mesenchymal stem cells, and progenitor cells of osteoblasts or cementoblasts. The use of molecular markers that distinguish human bone marrow mesenchymal stem cells from human fibroblasts may provide insight into the characteristics of human periodontal ligament cells. In this study, we compared the molecular markers of human periodontal ligament cells with those of human bone marrow mesenchymal stem cells and human gingival fibroblasts. Material and Methods:, The mRNA expression of the molecular gene markers was analyzed using real-time polymerase chain reaction. Statistical differences were determined with the two-sided Mann,Whitney U -test. Results:, Messenger RNA levels of major histocompatibility complex-DR-, and major histocompatibility complex-DR-, were lower and higher, respectively, in human periodontal ligament cells than in human bone marrow mesenchymal stem cells or human gingival fibroblasts. Human periodontal ligament cells showed the lowest apolipoprotein D mRNA levels among the three types of cells. Conclusion:, Human periodontal ligament cells may be distinguished from human bone marrow mesenchymal stem cells and human gingival fibroblasts by the genes for apolipoprotein D, major histocompatibility complex-DR-,, and major histocompatibility complex-DR-,. [source] The mechanisms underlying the anti-aging activity of the Chinese prescription Kangen-karyu in hydrogen peroxide-induced human fibroblastsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 10 2005Akiko Satoh Our previous study showed that Kangen-karyu extract protected against cellular senescence by reducing oxidative damage through the inhibition of reactive oxygen species generation and regulation of the antioxidative status. Although these findings suggest that Kangen-karyu could delay the aging process, the mechanisms responsible for protection against aging have rarely been elucidated. Therefore, this study was focussed on the mechanisms responsible for the anti-aging activity of Kangen-karyu extract using hydrogen peroxide (H2O2)-induced human diploid fibroblasts, a well-established experimental model of cellular aging. Kangen-karyu extract exerted a protective effect against the morphological changes induced by H2O2 treatment and inhibited senescence-associated ,-galactosidase activity. In addition, the beneficial effects of Kangen-karyu extract on cell viability and lifespan indicated that Kangen-karyu extract could delay the cellular aging process. The observation that Kangen-karyu extract prevented nuclear factor kappa B (NF-,B) translocation in response to oxidative stress suggested that Kangen-karyu exerted its anti-aging effect through NF-,B modulation and prevention of H2O2 -induced overexpression of haem oxygenase-1 protein. Moreover, pretreatment with Kangen-karyu extract reduced overexpression of bax protein and prevented the mitochondrial membrane potential decline, suggesting that Kangen-karyu extract may protect mitochondria from mitochondrial oxidative stress and dysfunction. These findings indicate that Kangen-karyu is a promising potential anti-aging agent that may delay, or normalize, the aging process by virtue of its protective activity against oxidative stress-related conditions. [source] | ||||||||||||||||||||||||||||||||||