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Sample records for ageing human fibroblasts

  1. AGE-RELATED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS INDUCED BY MMS

    EPA Science Inventory

    Age-Related Gene Expression Changes In Human Skin Fibroblasts Induced By methyl methanesulfonate. Geremy W. Knapp, Alan H. Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Prote...

  2. AGE-RELATED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS INDUCED BY MMS

    EPA Science Inventory

    Age-Related Gene Expression Changes In Human Skin Fibroblasts Induced By methyl methanesulfonate. Geremy W. Knapp, Alan H. Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Prote...

  3. Collagen Fragmentation Promotes Oxidative Stress and Elevates Matrix Metalloproteinase-1 in Fibroblasts in Aged Human Skin

    PubMed Central

    Fisher, Gary J.; Quan, Taihao; Purohit, Trupta; Shao, Yuan; Cho, Moon Kyun; He, Tianyuan; Varani, James; Kang, Sewon; Voorhees, John J.

    2009-01-01

    Aged human skin is fragile because of fragmentation and loss of type I collagen fibrils, which confer strength and resiliency. We report here that dermal fibroblasts express increased levels of collagen-degrading matrix metalloproteinases-1 (MMP-1) in aged (>80 years old) compared with young (21 to 30 years old) human skin in vivo. Transcription factor AP-1 and α2β1 integrin, which are key regulators of MMP-1 expression, are also elevated in fibroblasts in aged human skin in vivo. MMP-1 treatment of young skin in organ culture causes fragmentation of collagen fibrils and reduces fibroblast stretch, consistent with reduced mechanical tension, as observed in aged human skin. Limited fragmentation of three-dimensional collagen lattices with exogenous MMP-1 also reduces fibroblast stretch and mechanical tension. Furthermore, fibroblasts cultured in fragmented collagen lattices express elevated levels of MMP-1, AP-1, and α2β1 integrin. Importantly, culture in fragmented collagen raises intracellular oxidant levels and treatment with antioxidant MitoQ10 significantly reduces MMP-1 expression. These data identify positive feedback regulation that couples age-dependent MMP-1-catalyzed collagen fragmentation and oxidative stress. We propose that this self perpetuating cycle promotes human skin aging. These data extend the current understanding of the oxidative theory of aging beyond a cellular-centric view to include extracellular matrix and the critical role that connective tissue microenvironment plays in the biology of aging. PMID:19116368

  4. Specific Age-Associated DNA Methylation Changes in Human Dermal Fibroblasts

    PubMed Central

    Lin, Qiong; Bork, Simone; Goergens, Maria; Joussen, Sylvia; Pallua, Norbert; Ho, Anthony D.; Zenke, Martin; Wagner, Wolfgang

    2011-01-01

    Epigenetic modifications of cytosine residues in the DNA play a critical role for cellular differentiation and potentially also for aging. In mesenchymal stromal cells (MSC) from human bone marrow we have previously demonstrated age-associated methylation changes at specific CpG-sites of developmental genes. In continuation of this work, we have now isolated human dermal fibroblasts from young (<23 years) and elderly donors (>60 years) for comparison of their DNA methylation profiles using the Infinium HumanMethylation27 assay. In contrast to MSC, fibroblasts could not be induced towards adipogenic, osteogenic and chondrogenic lineage and this is reflected by highly significant differences between the two cell types: 766 CpG sites were hyper-methylated and 752 CpG sites were hypo-methylated in fibroblasts in comparison to MSC. Strikingly, global DNA methylation profiles of fibroblasts from the same dermal region clustered closely together indicating that fibroblasts maintain positional memory even after in vitro culture. 75 CpG sites were more than 15% differentially methylated in fibroblasts upon aging. Very high hyper-methylation was observed in the aged group within the INK4A/ARF/INK4b locus and this was validated by pyrosequencing. Age-associated DNA methylation changes were related in fibroblasts and MSC but they were often regulated in opposite directions between the two cell types. In contrast, long-term culture associated changes were very consistent in fibroblasts and MSC. Epigenetic modifications at specific CpG sites support the notion that aging represents a coordinated developmental mechanism that seems to be regulated in a cell type specific manner. PMID:21347436

  5. The hallmarks of fibroblast ageing.

    PubMed

    Tigges, Julia; Krutmann, Jean; Fritsche, Ellen; Haendeler, Judith; Schaal, Heiner; Fischer, Jens W; Kalfalah, Faiza; Reinke, Hans; Reifenberger, Guido; Stühler, Kai; Ventura, Natascia; Gundermann, Sabrina; Boukamp, Petra; Boege, Fritz

    2014-06-01

    Ageing is influenced by the intrinsic disposition delineating what is maximally possible and extrinsic factors determining how that frame is individually exploited. Intrinsic and extrinsic ageing processes act on the dermis, a post-mitotic skin compartment mainly consisting of extracellular matrix and fibroblasts. Dermal fibroblasts are long-lived cells constantly undergoing damage accumulation and (mal-)adaptation, thus constituting a powerful indicator system for human ageing. Here, we use the systematic of ubiquitous hallmarks of ageing (Lopez-Otin et al., 2013, Cell 153) to categorise the available knowledge regarding dermal fibroblast ageing. We discriminate processes inducible in culture from phenomena apparent in skin biopsies or primary cells from old donors, coming to the following conclusions: (i) Fibroblasts aged in culture exhibit most of the established, ubiquitous hallmarks of ageing. (ii) Not all of these hallmarks have been detected or investigated in fibroblasts aged in situ (in the skin). (iii) Dermal fibroblasts aged in vitro and in vivo exhibit additional features currently not considered ubiquitous hallmarks of ageing. (iv) The ageing process of dermal fibroblasts in their physiological tissue environment has only been partially elucidated, although these cells have been a preferred model of cell ageing in vitro for decades. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  6. CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Robichaud, Patrick; Voorhees, John J; Fisher, Gary J

    2011-08-01

    Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482-90, 2006, J Invest Dermatol 130:1697-706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an "Age-Associated Secretory Phenotype", in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.

  7. Vascular endothelial growth factor receptor-1 (VEGFR-1) expression in human corneal fibroblast decreased with age

    PubMed Central

    Berthaut, Alexandre; Mirshahi, Pezhman; Benabbou, Nadia; Azzazene, Dalel; Bordu, Camille; Therwath, Amu; Legeais, Jean-marc

    2009-01-01

    Purpose Mechanisms by which fibroblast networks between stromal lamellae are laid in the corneal stroma are far from clear. We have investigated the role of vascular endothelial growth factor receptors (VEGFRs) by in vitro studies in the human corneal network formation obtained from donors whose ages ranged from 19 to 89 years. Methods Corneal fibroblasts were prepared from cornea donations. The functional properties of these cells to form networks were analyzed using a semi solid matrix (substratum) of Matrigel™. The presence of VEGF receptor-1 (VEGFR-1) and the functionality in these fibroblasts were investigated using immunofluorescence, molecular analysis (gene microarray, reverse transcription polymerase chain reaction [RT–PCR] and VEGFR siRNA transfections), and cell culture. Results Corneal fibroblasts from 61 donors were classified into two groups according to whether they formed (82%) a reticulum on Matrigel™ or not (18%). By RT–PCR and immunofluorescence analysis, we showed that corneal fibroblasts expressed VEGFR-1 (mRNA and protein). Further, cell culture analysis revealed that only the network (reticulum) forming corneal fibroblast expressed VEGFR-1 in contrast to non network-forming fibroblasts. Use of inhibitors such as VEGFR-1 siRNA transfection or neutralizing antibody (Avastin™) indicated that VEGFR-1 was essential to the formation of the corneal network in vitro. Conclusions The cell reticulum formation seemed to be directly related to the expression of VEGFR-1 in the corneal fibroblast, and this expression decreased with age. The decrease in VEGFR-1 expression is probably related to the diminution of autocrine functions, which may alter the overall tissular homeostasis. This may culminate in the gradual development of poor vision, which is observed in certain pathologies and in aging individuals. PMID:19816604

  8. Reduced host cell reactivation of oxidatively damaged DNA in ageing human fibroblasts.

    PubMed

    Rainbow, Andrew J; Zacal, Natalie J; Leach, Derrik M

    2013-06-01

    Many reports have linked oxidative damage to DNA and the associated avoidance and/or repair processes to carcinogenesis, ageing and neurodegeneration. Cancer incidence increases with age and there is evidence that oxidative stress plays a role in human ageing and neurodegeneration. Several reports have suggested that the accumulation of unrepaired DNA lesions plays a causal role in mammalian ageing. Since base excision repair (BER) is the main pathway for the repair of oxidative DNA lesions, the relationship of BER to human ageing and carcinogenesis is of considerable interest. The aim of the present study was to examine the relationship between donor age and increasing time of cells in tissue culture and the repair of oxidative DNA damage in primary human skin fibroblasts. Methylene blue (MB) acts as a photosensitizer and after excitation by visible light (VL) produces reactive oxygen species that result in oxidative damage to DNA. MB+VL produce predominantly 8-hydroxyguanine as well as other single base modifications in DNA that are repaired by BER. We used host cell reactivation (HCR) of a non-replicating recombinant human adenovirus, Ad5CMVlacZ, which expresses the β-galactosidase (β-gal) reporter gene, to measure BER of MB+VL-damaged DNA. HCR of β-gal activity for the MB+VL-treated reporter gene was examined in 10 fibroblast strains from normal donors of ages 2 to 82. The effect of cell passage number on HCR was also examined in human skin fibroblasts from 2 normal donors. We found a significant reduction in HCR with increasing cell passage number, indicating that BER decreases with increasing time of cells grown in tissue culture. We also found a significant correlation of donor age with HCR of the MB+VL-treated reporter gene for high passage number, but not for low passage number fibroblasts. The present study provides evidence that a decrease in BER of oxidatively damaged DNA may play a role in carcinogenesis, ageing and neurodegeneration.

  9. Maintenance of age in human neurons generated by microRNA-based neuronal conversion of fibroblasts

    PubMed Central

    Huh, Christine J; Zhang, Bo; Victor, Matheus B; Dahiya, Sonika; Batista, Luis FZ; Horvath, Steve; Yoo, Andrew S

    2016-01-01

    Aging is a major risk factor in many forms of late-onset neurodegenerative disorders. The ability to recapitulate age-related characteristics of human neurons in culture will offer unprecedented opportunities to study the biological processes underlying neuronal aging. Here, we show that using a recently demonstrated microRNA-based cellular reprogramming approach, human fibroblasts from postnatal to near centenarian donors can be efficiently converted into neurons that maintain multiple age-associated signatures. Application of an epigenetic biomarker of aging (referred to as epigenetic clock) to DNA methylation data revealed that the epigenetic ages of fibroblasts were highly correlated with corresponding age estimates of reprogrammed neurons. Transcriptome and microRNA profiles reveal genes differentially expressed between young and old neurons. Further analyses of oxidative stress, DNA damage and telomere length exhibit the retention of age-associated cellular properties in converted neurons from corresponding fibroblasts. Our results collectively demonstrate the maintenance of age after neuronal conversion. DOI: http://dx.doi.org/10.7554/eLife.18648.001 PMID:27644593

  10. Proteome-wide analysis reveals an age-associated cellular phenotype of in situ aged human fibroblasts

    PubMed Central

    Waldera-Lupa, Daniel M.; Kalfalah, Faiza; Florea, Ana-Maria; Sass, Steffen; Kruse, Fabian; Rieder, Vera; Tigges, Julia; Fritsche, Ellen; Krutmann, Jean; Busch, Hauke; Boerries, Melanie; Meyer, Helmut E.; Boege, Fritz; Theis, Fabian

    2014-01-01

    We analyzed an ex vivo model of in situ aged human dermal fibroblasts, obtained from 15 adult healthy donors from three different age groups using an unbiased quantitative proteome-wide approach applying label-free mass spectrometry. Thereby, we identified 2409 proteins, including 43 proteins with an age-associated abundance change. Most of the differentially abundant proteins have not been described in the context of fibroblasts’ aging before, but the deduced biological processes confirmed known hallmarks of aging and led to a consistent picture of eight biological categories involved in fibroblast aging, namely proteostasis, cell cycle and proliferation, development and differentiation, cell death, cell organization and cytoskeleton, response to stress, cell communication and signal transduction, as well as RNA metabolism and translation. The exhaustive analysis of protein and mRNA data revealed that 77% of the age-associated proteins were not linked to expression changes of the corresponding transcripts. This is in line with an associated miRNA study and led us to the conclusion that most of the age-associated alterations detected at the proteome level are likely caused post-transcriptionally rather than by differential gene expression. In summary, our findings led to the characterization of novel proteins potentially associated with fibroblast aging and revealed that primary cultures of in situ aged fibroblasts are characterized by moderate age-related proteomic changes comprising the multifactorial process of aging. PMID:25411231

  11. RNA and protein synthesis in cultured human fibroblasts derived from donors of various ages.

    PubMed

    Chen, J J; Brot, N; Weissbach, H

    1980-07-01

    RNA synthesis in human fibroblasts from donors of various ages was studied in fibroblasts made permeable to nucleoside triphosphates with the nonionic detergent Nonidet P40. Cells from donors of 11 years and older showed a 30-40% decline in total RNA synthesis. The decrease in RNA synthesis was primarily due to a lowering of RNA polymerase II activity (alpha-amanitin sensitive). Studies on the incorporation of leucine into protein also showed a 30-40% decrease in cells from older donors.

  12. Consequences of aging on mitochondrial respiratory chain enzymes in cultured human fibroblasts treated with ascorbate.

    PubMed

    Sharma, P; Rupar, C A; Rip, J W

    1998-01-01

    The activities of mitochondrial respiratory chain enzymes with and without ascorbate pretreatment were assayed in 10- to 20-week-old cultures of human fibroblasts. Aging was associated with a significant loss of respiratory chain enzyme activities. The presence of ascorbate in the medium reduced the rate of loss of these enzymes. Free radical-mediated injuries may also contribute to aging since the changes seen in respiratory chain enzyme activities are similar to those seen in oxidatively stressed cells. This study demonstrates an age-related decline in mitochondrial respiratory chain activity as well as a protective role for ascorbate in aging.

  13. Donor's age and replicative senescence favour the in-vitro mineralization potential of human fibroblasts.

    PubMed

    Boraldi, Federica; Bartolomeo, Angelica; Di Bari, Caterina; Cocconi, Andrea; Quaglino, Daniela

    2015-12-01

    Aberrant mineralization of soft connective tissues (ectopic calcification) may occur as a frequent age-related complication. Still, it remains unclear the role of mesenchymal cell donor's age and of replicative senescence on ectopic calcification. Therefore, the ability of cells to deposit in-vitro hydroxyapatite crystals and the expression of progressive ankylosis protein homolog (ANKH), ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), tissue non specific alkaline phosphatase (TNAP) and osteopontin (OPN) have been evaluated in human dermal fibroblasts derived from neonatal (nHDF) and adult (aHDF) donors (ex-vivo ageing model) or at low and high cumulative population doublings (CPD) up to replicative senescence (in-vitro ageing model). This study demonstrates that: 1) replicative senescence favours hydroxyapatite formation in cultured fibroblasts; 2) donor's age acts as a major modulator of the mineralizing potential of HDF, since nHDF are less prone than aHDF to induce calcification; 3) donor's age and replicative senescence play in concert synergistically increasing the calcification process; 4) the ANKH+ENPP1/TNAP ratio, being crucial for pyrophosphate/inorganic phosphate balance, is greatly influenced by donor's age, as well as by replicative senescence, and regulates mineral deposition; 5) OPN is only modulated by replicative senescence. Copyright © 2015. Published by Elsevier Inc.

  14. Scopoletin has a potential activity for anti-aging via autophagy in human lung fibroblasts.

    PubMed

    Nam, Hyang; Kim, Moon-Moo

    2015-03-15

    Autophagy was known to be associated with aging in addition to cancer and neurodegeneration. The effects of scopoletin on autophagy and anti-aging were investigated in human lung fibroblast cell line, IMR 90. Here we show that scopoletin induces autophagy. It is also identified that the modulation of p53 by scopoletin are related to the induction of autophagy. Moreover, the level of SA-β-Gal staining, an aging marker, is reduced by scopoletin. In addition, while the expression levels of histone deacetylases such as HDAC1, SIRT1 and SIRT6 are increased in IMR 90 cells in the presence of scopoletin, the expression levels of histone acetyltransferases are decreased. Furthermore, scopoletin enhances the level of transcription factors such as Nrf-2and p-FoxO1 related to anti-aging. In addition, scopoletin modulates the reprogramming proteins. Therefore, these findings suggest that scopoletin could exert a positive effect on anti-aging related to autophagy through modulation of p53 in human lung fibroblasts.

  15. The chromene sargachromanol E inhibits ultraviolet A-induced ageing of skin in human dermal fibroblasts.

    PubMed

    Kim, J-A; Ahn, B-N; Kong, C-S; Kim, S-K

    2013-05-01

    Skin ageing is influenced by environmental factors such as ultraviolet (UV) radiation. The effects of UV radiation on skin functions should be investigated using human in vitro models to understand the mechanisms of skin ageing. Additionally, marine algae provide a valuable source for identifying and extracting biologically active substances. In this study, sargachromanol E was isolated from a marine brown alga, Sargassum horneri, and its inhibitory effect on skin ageing was investigated using UVA-irradiated dermal fibroblasts. Formation of intracellular reactive oxygen species (ROS), lipid peroxidation and protein oxidation induced by UVA irradiation were investigated in UVA-irradiated human dermal fibroblasts. The levels of matrix metalloproteinases (MMPs) were determined by reverse-transcriptase polymerase chain reaction and Western blot analysis. Sargachromanol E did not exhibit any significant cytotoxicity or phototoxicity in UVA-exposed dermal fibroblasts. Additionally, sargachromanol E suppressed intracellular formation of ROS, membrane protein oxidation, lipid peroxidation and expression of collagenases such as MMP-1, MMP-2 and MMP-9, all of which are caused by UVA exposure. It was further found that these inhibitions were related to an increase in the expression of the tissue inhibitor of metalloproteinase (TIMP) genes, TIMP1 and TIMP2. Moreover, we have shown that the transcriptional activation of activator protein 1 (AP-1) signalling caused by UVA irradiation was inhibited by treatment with sargachromanol E. This study suggests that UVA irradiation modulates MMP expression via the transcriptional activation of AP-1 signalling, whereas treatment with sargachromanol E protected cell damage caused by UVA irradiation. © 2013 The Authors. BJD © 2013 British Association of Dermatologists.

  16. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    PubMed

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

  17. Investigate correlation between mechanical property and aging biomarker in passaged human dermal fibroblasts.

    PubMed

    Kim, Kyung Sook; Park, Hun-Kuk; Lee, Jin-Woo; Kim, Young Il; Shin, Min Kyung

    2015-04-01

    Skin aging is associated with changes in both the mechanical properties of the skin and extracellular matrix (ECM) components. In this study, we investigated the relationships between mechanical property and aging biomarkers in passaged human dermal fibroblasts (HDFs). The stiffness of HDFs from passages 5-20 was assessed by atomic force microscopy. The ECM components including collagen, elastin, and fibrillin-1 and that of signaling molecules (SIRTs) were determined from each passage of cells. The stiffness of HDFs increased linearly from passages 5-15 and then became saturated: the average stiffness was 0.356 N/m at passages 5 and 1.186 N/m at passages 15, respectively. Expression of all aging biomarkers, including pro-collagen I and VII, elastin, fibrillin-1, and SIRT1 and SIRT6, were down-regulated by passaging. In particular, a change in the level of procollagen Type I was significantly associated with early aging, while a change in the level of fibrillin-1 was associated with late aging. All biomarkers except elastin showed a strong correlation with the cellular stiffness of HDFs. © 2015 Wiley Periodicals, Inc.

  18. Lidocaine Impairs Proliferative and Biosynthetic Functions of Aged Human Dermal Fibroblasts.

    PubMed

    Bentov, Itay; Damodarasamy, Mamatha; Spiekerman, Charles; Reed, May J

    2016-09-01

    The aged are at increased risk of postoperative wound healing complications. Because local anesthetics are infiltrated commonly into the dermis of surgical wounds, we sought to determine whether local anesthetics adversely affect proliferative and biosynthetic functions of dermal fibroblasts. We also evaluated the effect of local anesthetics on insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1), growth factors that are important regulators of wound healing. Human dermal fibroblasts (HFB) from aged and young donors were exposed to local anesthetic agents at clinically relevant concentrations. We screened the effects of lidocaine, bupivacaine, mepivacaine, and ropivacaine on proliferation of HFB. Lidocaine was most detrimental to proliferation in HFB. We then evaluated the effect of lidocaine on expression and function of the growth factors, IGF-1 and TGF-β1. Lastly, concurrent exposure to lidocaine and IGF-1 or TGF-β1 was evaluated for their effects on proliferation and expression of dermal collagens, respectively. Lidocaine and mepivacaine inhibited proliferation in aged HFB (for lidocaine 88% of control, 95% confidence interval [CI], 80%-98%, P = .009 and for mepivacaine 90% of control, 95% CI, 81%-99%, P = .032) but not in young HFB. Ropivacaine and bupivacaine did not inhibit proliferation. Because of the clinical utility of lidocaine relative to mepivacaine, we focused on lidocaine. Lidocaine decreased proliferation in aged HFB, which was abrogated by IGF-1. Lidocaine inhibited transcripts for IGF-1 and insulin-like growth factor-1 receptor (IGF1R) in fibroblasts from aged donors (IGF-1, log2 fold-change -1.25 [42% of control, 95% CI, 19%-92%, P = .035] and IGF1R, log2 fold-change -1.00 [50% of control, 95% CI, 31%-81%, P = .014]). In contrast, lidocaine did not affect the expression of IGF-1 or IGF1R transcripts in the young HFB. Transcripts for collagen III were decreased after lidocaine exposure in aged and young HFB (log2

  19. Gamma-tocotrienol modulation of senescence-associated gene expression prevents cellular aging in human diploid fibroblasts.

    PubMed

    Makpol, Suzana; Zainuddin, Azalina; Chua, Kien Hui; Yusof, Yasmin Anum Mohd; Ngah, Wan Zurinah Wan

    2012-01-01

    Human diploid fibroblasts undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular aging. The beneficial effects of vitamin E in aging have been established, but studies to determine the mechanisms of these effects are ongoing. This study determined the molecular mechanism of γ-tocotrienol, a vitamin E homolog, in the prevention of cellular aging in human diploid fibroblasts using the expression of senescence-associated genes. Primary cultures of young, pre-senescent, and senescent fibroblast cells were incubated with γ-tocotrienol for 24 h. The expression levels of ELN, COL1A1, MMP1, CCND1, RB1, and IL6 genes were determined using the quantitative real-time polymerase chain reaction. Cell cycle profiles were determined using a FACSCalibur Flow Cytometer. The cell cycle was arrested in the G(0)/G(1) phase, and the percentage of cells in S phase decreased with senescence. CCND1, RB1, MMP1, and IL6 were upregulated in senescent fibroblasts. A similar upregulation was not observed in young cells. Incubation with γ-tocotrienol decreased CCND1 and RB1 expression in senescent fibroblasts, decreased cell populations in the G(0)/G(1) phase and increased cell populations in the G(2)/M phase. γ-Tocotrienol treatment also upregulated ELN and COL1A1 and downregulated MMP1 and IL6 expression in young and senescent fibroblasts. γ-Tocotrienol prevented cellular aging in human diploid fibroblasts, which was indicated by the modulation of the cell cycle profile and senescence-associated gene expression.

  20. Marked aging-related decline in efficiency of oxidative phosphorylation in human skin fibroblasts.

    PubMed

    Greco, Marilena; Villani, Gaetano; Mazzucchelli, Franca; Bresolin, Nereo; Papa, Sergio; Attardi, Giuseppe

    2003-09-01

    An extensive analysis has been carried out of mitochondrial biochemical and bioenergetic properties of fibroblasts, mostly skin-derived, from a large group of subjects ranging in age between 20 wk fetal and 103 yr. A striking age-related change observed in a fundamental process underlying mitochondrial biogenesis and function was the very significant decrease in rate of mitochondrial protein synthesis in individuals above 40 yr. The analysis of endogenous respiration rate revealed a significant decrease in the age range from 40 to 90 yr and a tendency to uncoupling in the samples from subjects above 60 yr. A surprising finding was the occurrence of a subgroup of individuals >or=90 yr old whose skin fibroblasts exhibited an exceptionally high respiration rate. This high rate was not due to respiration uncoupling, rather pointing to a compensatory phenomenon, not involving an increase in mtDNA content, in the corresponding skin fibroblast populations, or, possibly, to a selection of a different cell type secondary to more extensive dermal atrophy. The most important aging-related phenotypic effects observed were those that affected the cell oxidative phosphorylation (OX-PHOS) capacity. These were, in particular, the very significant reduction in the ratio of uncoupled to oligomycin-inhibited endogenous respiration observed in intact fibroblasts, which pointed to a decrease with donor's age in the control of respiration by the mitochondrial membrane potential, the very significant decrease in efficiency of OX-PHOS, as determined by novel in situ measurements of P:O ratios, and, consistent with these results, the very significant reduction in the respiratory control ratios. These findings clearly pointed to a dramatic mitochondrial dysfunction, which would lead to a decrease in ATP synthesis rate, with the observed decline in mitochondrial protein synthesis rate being a likely contributing factor. These observations have important implications for understanding the

  1. Basal level of autophagy is increased in aging human skin fibroblasts in vitro, but not in old skin.

    PubMed

    Demirovic, Dino; Nizard, Carine; Rattan, Suresh I S

    2015-01-01

    Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.

  2. Expression of catalytically active Matrix Metalloproteinase-1 in dermal fibroblasts induces collagen fragmentation and functional alterations that resemble aged human skin

    PubMed Central

    Xia, Wei; Hammerberg, Craig; Li, Yong; He, Tianyuan; Quan, Taihao; Voorhees, John J; Fisher, Gary J

    2013-01-01

    Summary Increased expression of matrix metalloproteinase-1 (MMP-1) and reduced production of type I collagen by dermal fibroblasts are prominent features of aged human skin. We have proposed that MMP-1-mediated collagen fibril fragmentation is a key driver of age-related decline of skin function. To investigate this hypothesis, we constructed, characterized, and expressed constitutively active MMP-1 mutant (MMP-1 V94G) in adult human skin in organ culture and fibroblasts in three dimensional collagen lattice cultures. Expression of MMP-1 V94G in young skin in organ culture caused fragmentation and ultrastructural alterations of collagen fibrils similar to those observed in aged human skin in vivo. Expression of MMP-1 V94G in dermal fibroblasts cultured in three-dimensional collagen lattices caused substantial collagen fragmentation, which was markedly reduced by MMP-1 siRNA-mediated knockdown or MMP inhibitor MMI270. Importantly, fibroblasts cultured in MMP-1 V94G-fragmented collagen lattices displayed many alterations observed in fibroblasts in aged human skin, including reduced cytoplasmic area, disassembled actin cytoskeleton, impaired TGF-β pathway, and reduced collagen production. These results support the concept that MMP-1-mediated fragmentation of dermal collagen fibrils alters the morphology and function of dermal fibroblasts, and provide a foundation for understanding specific mechanisms that link collagen fibril fragmentation to age-related decline of fibroblast function. PMID:23601157

  3. Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro.

    PubMed

    Jørgensen, Peter; Milkovic, Lidija; Zarkovic, Neven; Waeg, Georg; Rattan, Suresh I S

    2014-02-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

  4. The Effects of Chronological Age on the Cellular Mechanics of Human Dermal Fibroblasts

    NASA Astrophysics Data System (ADS)

    Pan, Z.; Hung, V.; Kambhampati, S.; Ge, S. R.; Rafailovich, M.; Ghosh, K.; Clark, R.; Liu, Y. J.; Nakamura, T.; Shu, X. Z.; Prestwich, G.

    2006-03-01

    It is often observed that older people display diminished wound healing abilities. Understanding of this phenomenon is important for many in vivo applications of tissue engineering. In this study, the cell mechanics of dermal fibroblasts from 25, 40 and 84 years old female subjects were compared. These cells were cultured on functionalized hyaluronic acid hydrogel substrates which emulated physiological conditions in dermal tissue. The deformation of the substrate caused by cellular traction forces was detected by tracing the displacement of fluorescent beads embedded in the substrate using Digital Image Speckle Correlation. Then cellular traction forces were quantitatively determined by Finite Element Method in a linear elastic model with a high spatial resolution. These results were correlated with auxiliary measurements of substrate modulus, cell modulus and migration. We found that with increasing age, the magnitude of the cellular traction forces diminished. Similarly, the ability of the cells to adapt to changes in the mechanical properties of their environment and migrate was also impaired. The interrelationship between these factors and wound healing will be discussed. This work is supported by NSF- MRSEC program.

  5. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice.

    PubMed

    Godin, Lindsay M; Sandri, Brian J; Wagner, Darcy E; Meyer, Carolyn M; Price, Andrew P; Akinnola, Ifeolu; Weiss, Daniel J; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases.

  6. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

    PubMed Central

    Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258

  7. Age-Related Changes in the Mechanical Properties of Human Fibroblasts and Its Prospective Reversal After Anti-Wrinkle Tripeptide Treatment.

    PubMed

    Dulińska-Molak, Ida; Pasikowska, Monika; Pogoda, Katarzyna; Lewandowska, Małgorzata; Eris, Irena; Lekka, Małgorzata

    2014-01-01

    One of an essential characteristic of human skin are time dependent mechanical properties. Here, we demonstrate that stiffness of human dermal fibroblast correlates with age and it can be restored after anti-wrinkle tripeptide treatment. The stiffness of human fibroblasts isolated from donors of 30-, 40- and 60 years old were examined. Additionally the effect of anti- wrinkle tripeptide of latter cells was investigated. The atomic force microscopy measurements were performed on untreated fibroblast as well as on treated with the peptide. The Young's modulus for two indentation depths 200 and 600 nm of each cell type was determined. The Young's modulus increases with age of the cells. The highest values of Young's modulus were obtained for fibroblasts collected from 60 years old donors, for indentation depth of ~200 nm. For larger indentation depth of 600 nm there are no significant differences in stiffness between cells. Fibroblasts treated with the anti-wrinkle tripeptide exhibit lower Young's modulus. The cells derived from 40- and 60-years old donors restored stiffness characteristic to the level of 30 years old subjects. The results show correlation between stiffness and age of the human fibroblast as well as impact of anti-wrinkle tripeptide on the mechanical properties of skin cells.

  8. Pentosidine in advanced glycation end-products (AGEs) during UVA irradiation generates active oxygen species and impairs human dermal fibroblasts.

    PubMed

    Okano, Y; Masaki, H; Sakurai, H

    2001-08-01

    Our previous study reported that advanced glycation end-products (AGE)-modified BSA produced active oxygen species, *O2-, H2O2, and *OH under UVA irradiation and enhanced the cytotoxicity of UVA light. We examined whether pentosidine in AGE-modified BSA was involved in one of the mechanisms generating the active oxygen species. In biological investigations, fibroblasts exposed to UVA (20 J/cm2) in the presence of pentosidine-rich compounds (PRCs), which were prepared with L-arginine, L-lysine and glucose, showed a time-dependent leakage of the cytosolic enzyme LDH. In addition, release of LDH was suppressed by addition of DMSO and deferoxamine under UVA irradiation. From these results, it was determined that PRCs exposed to UVA damaged the plasma membrane of human dermal fibroblasts due to the conversion of *OH from H2O2 via a Fenton-like reaction. These features of PRCs exposed to UVA were consistent with those of AGE-modified BSA. In an ESR study, PRCs under UVA irradiation yielded DMPO-OH (DMPO-OH adduct) using DMPO as a spin-trapping reagent. *O2- generation from UVA-irradiated PRCs was also indicated by the combination of NBT reduction and SOD. When PRCs were exposed to UVA light controlled with a long-pass filter, WG-360, it was found that their production of *O2- was prohibited less than 50% in the NBT reduction assay. The *O2- production profile of PRCs depending on the wavelength of UVA light was similar to that of AGE-modified BSA. Furthermore, it was found that the H2O2 level was increased by PRCs exposed to UVA. These results indicated that pentosidine is an important factor of AGE-modified BSA in active oxygen generation under UVA irradiation.

  9. Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age.

    PubMed

    Ivanov, Nikolay A; Tao, Ran; Chenoweth, Joshua G; Brandtjen, Anna; Mighdoll, Michelle I; Genova, John D; McKay, Ronald D; Jia, Yankai; Weinberger, Daniel R; Kleinman, Joel E; Hyde, Thomas M; Jaffe, Andrew E

    2016-02-01

    Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), "block" (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts-83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation--while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p = 0

  10. Curcumin induces heme oxygenase-1 in normal human skin fibroblasts through redox signaling: relevance for anti-aging intervention.

    PubMed

    Lima, Cristovao F; Pereira-Wilson, Cristina; Rattan, Suresh I S

    2011-03-01

    Curcumin, a component of the spice turmeric, was tested for its potential hormetic anti-aging effects as an inducer of mild stress. Early passage young human skin fibroblasts treated with low doses of curcumin (below 20 μM) showed a time- and concentration-dependent induction of heme oxygenase-1 (HO-1), followed by compensatory increase in glutathione-S-transferase activity, GSH levels and GSH/GSSG ratio. These effects were preceded by induction of oxidative stress (increased levels of reactive oxygen species and DNA damage) and impairment of cells' GSH redox state. Curcumin also induced nuclear factor-erythroid-2-related factor 2 accumulation in the nuclei. The use of the antioxidant N-acetyl cysteine prevented the induction of HO-1 by curcumin. Pharmacological inhibition of phosphatidylinositol 3-kinase, but not other kinases, significantly prevented curcumin-induced HO-1 levels, which was corroborated by the induction of phospho-Akt levels by curcumin. Late passage senescent cells already had higher HO-1 levels, and further induction of HO-1 by curcumin was considerably impaired. The induction of stress responses by curcumin in human cells led to protective hormetic effects to further oxidant challenge. Curcumin induces cellular stress responses in normal human skin fibroblasts through phosphatidylinositol 3-kinase/Akt pathway and redox signaling, supporting the view that curcumin-induced hormetic stimulation of cellular antioxidant defenses can be a useful approach toward anti-aging intervention. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age

    PubMed Central

    Ivanov, Nikolay A.; Tao, Ran; Chenoweth, Joshua G.; Brandtjen, Anna; Mighdoll, Michelle I.; Genova, John D.; McKay, Ronald D.; Jia, Yankai; Weinberger, Daniel R.; Kleinman, Joel E.; Hyde, Thomas M.; Jaffe, Andrew E.

    2016-01-01

    Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), “block” (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts—83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation—while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p

  12. Age-associated increase of skin fibroblast-derived prostaglandin E2 contributes to reduced collagen levels in elderly human skin

    PubMed Central

    Li, Yong; Lei, Dan; Swindell, William R; Xia, Wei; Weng, Shinuo; Fu, Jianping; Worthen, Christal A; Okubo, Toru; Johnston, Andrew; Gudjonsson, Johann E; Voorhees, John J; Fisher, Gary J

    2015-01-01

    Production of type I collagen declines during aging, leading to skin thinning and impaired function. Prostaglandin E2 (PGE2) is a pleiotropic lipid mediator that is synthesized from arachidonic acid by the sequential actions of cyclooxygenases (COX) and PGE synthases (PTGES). PGE2 inhibits collagen production by fibroblasts in vitro. We report that PTGES1 and COX2 progressively increase with aging in sun-protected human skin. PTGES1 and COX2 mRNA was increased 3.4-fold and 2.7-fold, respectively, in the dermis of elderly (>80 years) versus young (21-30 years) individuals. Fibroblasts were the major cell source of both enzymes. PGE2 levels were increased 70% in elderly skin. Fibroblasts in aged skin display reduced spreading due to collagen fibril fragmentation. To investigate the relationship between spreading and PGE2 synthesis, fibroblasts were cultured on micropost arrays or hydrogels of varying mechanical compliance. Reduced spreading/mechanical force resulted in increased expression of both PTGES1 and COX2 and elevated levels of PGE2. Inhibition of PGE2 synthesis by diclofenac enhanced collagen production in skin organ cultures. These data suggest that reduced spreading/mechanical force of fibroblasts in aged skin elevates PGE2 production, contributing to reduced collagen production. Inhibition of PGE2 production may be therapeutically beneficial for combating age-associated collagen deficit in human skin. PMID:25905589

  13. Reduction of fibroblast size/mechanical force down-regulates TGF-β type II receptor: implications for human skin aging.

    PubMed

    Fisher, Gary J; Shao, Yuan; He, Tianyuan; Qin, Zhaoping; Perry, Daniel; Voorhees, John J; Quan, Taihao

    2016-02-01

    The structural integrity of human skin is largely dependent on the quality of the dermal extracellular matrix (ECM), which is produced, organized, and maintained by dermal fibroblasts. Normally, fibroblasts attach to the ECM and thereby achieve stretched, elongated morphology. A prominent characteristic of dermal fibroblasts in aged skin is reduced size, with decreased elongation and a more rounded, collapsed morphology. Here, we show that reduced size of fibroblasts in mechanically unrestrained three-dimensional collagen lattices coincides with reduced mechanical force, measured by atomic force microscopy. Reduced size/mechanical force specifically down-regulates TGF-β type II receptor (TβRII) and thus impairs TGF-β/Smad signaling pathway. Both TβRII mRNA and protein were decreased, resulting in 90% loss of TGF-β binding to fibroblasts. Down-regulation of TβRII was associated with significantly decreased phosphorylation, DNA-binding, and transcriptional activity of its key downstream effector Smad3 and reduced expression of Smad3-regulated essential ECM components type I collagen, fibronectin, and connective tissue growth factor (CTGF/CCN2). Restoration of TβRII significantly increased TGF-β induction of Smad3 phosphorylation and stimulated expression of ECM components. Reduced expression of TβRII and ECM components in response to reduced fibroblast size/mechanical force was fully reversed by restoring size/mechanical force. Reduced fibroblast size was associated with reduced expression of TβRII and diminished ECM production, in aged human skin. Taken together, these data reveal a novel mechanism that provides a molecular basis for loss of dermal ECM, with concomitant increased fragility, which is a prominent feature of human skin aging.

  14. Curcumin induces stress response and hormetically modulates wound healing ability of human skin fibroblasts undergoing ageing in vitro.

    PubMed

    Demirovic, Dino; Rattan, Suresh I S

    2011-10-01

    Wound healing becomes impaired in several diseases and during ageing. A commonly used model for the study of wound healing is a scratched monolayer of cells in vitro, which is convenient for the analysis of the cellular and molecular changes occurring during the two phases of wound healing, namely cell migration and cell proliferation. Cell migration, which is the primary event to occur during initial wound healing, is inversely dependent on the number of focal adhesions (FA) that attach cells to the extracellular matrix. Here we report that the number of FA, measured by determining the levels of FA-proteins paxillin and talin, increase with increasing population doubling level of the serially passaged normal adult skin fibroblasts, and that this increase may account for the age-related slowing down of wound healing in vitro. We also report that curcumin, a component of the widely used spice turmeric, modulates wound healing in vitro in a biphasic dose response manner, being stimulatory at low doses (between 1 and 5 μM), and inhibitory at higher doses. Furthermore, our results show that the hormetic effects of low levels of curcumin are achieved by virtue of it being a hormetin in terms of the induction of stress response pathways, including Nrf2 and HO-1 in human cells.

  15. Gastrodia elata Blume Extract Modulates Antioxidant Activity and Ultraviolet A-Irradiated Skin Aging in Human Dermal Fibroblast Cells.

    PubMed

    Song, Eunju; Chung, Haeyon; Shim, Eugene; Jeong, Jung-Ky; Han, Bok-Kyung; Choi, Hyuk-Joon; Hwang, Jinah

    2016-11-01

    Gastrodia elata Blume (GEB), a traditional herbal medicine, has been used to treat a wide range of neurological disorders (e.g., paralysis and stroke) and skin problems (e.g., atopic dermatitis and eczema) in oriental medicine. This study was designed to investigate the antioxidant ability of GEB and its antiaging effect on human dermal fibroblast cells (HDF). The total phenolic and flavonoid contents of GEB were 21.8 and 0.43 mg/g dry weight (DW), respectively. The ergothioneine content of GEB was 0.41 mg/mL DW. The DPPH and ABTS radical scavenging activities of GEB at 5 and 10 mg/mL approximately ranged between 31% and 44%. The superoxide dismutase activity of GEB at 10 and 25 mg/mL was 57% and 76%, respectively. GEB increased procollagen type 1 (PC1) production and inhibited matrix metalloproteinase-1 (MMP-1) production and elastase-1 activity in UVA-irradiated HDF. PC1 messenger RNA (mRNA) levels decreased upon UVA irradiation, but recovered in response to high doses of GEB in HDF. On the contrary, GEB significantly decreased MMP-1 and elastase-1 mRNA levels, which were markedly induced in UVA-irradiated HDF. Collectively, these results suggest that GEB has sufficient antioxidant ability to prevent the signs of skin aging in UVA-irradiated human skin cells, suggesting its potential as a natural antiaging product.

  16. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts.

    PubMed

    Marthandan, Shiva; Priebe, Steffen; Baumgart, Mario; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin.

  17. Similarities in Gene Expression Profiles during In Vitro Aging of Primary Human Embryonic Lung and Foreskin Fibroblasts

    PubMed Central

    Marthandan, Shiva; Priebe, Steffen; Groth, Marco; Cellerino, Alessandro; Guthke, Reinhard; Hemmerich, Peter; Diekmann, Stephan

    2015-01-01

    Replicative senescence is of fundamental importance for the process of cellular aging, since it is a property of most of our somatic cells. Here, we elucidated this process by comparing gene expression changes, measured by RNA-seq, in fibroblasts originating from two different tissues, embryonic lung (MRC-5) and foreskin (HFF), at five different time points during their transition into senescence. Although the expression patterns of both fibroblast cell lines can be clearly distinguished, the similar differential expression of an ensemble of genes was found to correlate well with their transition into senescence, with only a minority of genes being cell line specific. Clustering-based approaches further revealed common signatures between the cell lines. Investigation of the mRNA expression levels at various time points during the lifespan of either of the fibroblasts resulted in a number of monotonically up- and downregulated genes which clearly showed a novel strong link to aging and senescence related processes which might be functional. In terms of expression profiles of differentially expressed genes with age, common genes identified here have the potential to rule the transition into senescence of embryonic lung and foreskin fibroblasts irrespective of their different cellular origin. PMID:26339636

  18. Protective effects of adipose-derived stem cells secretome on human dermal fibroblasts from ageing damages.

    PubMed

    Wang, Ting; Guo, Shu; Liu, Xuehui; Xv, Nan; Zhang, Shuangyi

    2015-01-01

    Combined effects of intrinsic and extrinsic ageing factors on skin tissue and the therapies have been rarely studied before. ADSCs have gained popularity in anti-ageing field, which may provide promising methods to fight against skin ageing. To find out the fate of HDFs exposed to intrinsic or extrinsic ageing factors or both of them and further examine the impacts of ADSC-CM on the damaged HDFs. We irradiated HDFs with UVB at different senescent levels, and then treated them with ADSC-CM. After 48 h, we detected cellular proliferative activity, morphology, SA-β-Gal expression, apoptosis, mRNA expression of collagen I, collagen III and elastin. Intrinsic ageing factors inhibited cellular proliferation, increased senescent ratio and reduced mRNA expression of collagen I, collagen III and elastin, so did UVB, except for its induction of elastin mRNA expression. Furthermore, ADSC-CM treatment can slightly or significantly improve cellular proliferative activity and restore functions both in irradiated and non-irradiated HDFs. Besides, ADSC-CM treatment decreased cellular apoptosis and senescence induced by UVB but had no obvious effect on cellular senescence induced by intrinsic ageing factors. The results were similar in three generations of HDFs, yet in different degrees. The results suggest that ADSCs secretome protect HDFs from ageing damages but with some limitations.

  19. Protective effects of adipose-derived stem cells secretome on human dermal fibroblasts from ageing damages

    PubMed Central

    Wang, Ting; Guo, Shu; Liu, Xuehui; Xv, Nan; Zhang, Shuangyi

    2015-01-01

    Background: Combined effects of intrinsic and extrinsic ageing factors on skin tissue and the therapies have been rarely studied before. ADSCs have gained popularity in anti-ageing field, which may provide promising methods to fight against skin ageing. Objective: To find out the fate of HDFs exposed to intrinsic or extrinsic ageing factors or both of them and further examine the impacts of ADSC-CM on the damaged HDFs. Methods: We irradiated HDFs with UVB at different senescent levels, and then treated them with ADSC-CM. After 48 h, we detected cellular proliferative activity, morphology, SA-β-Gal expression, apoptosis, mRNA expression of collagen I, collagen III and elastin. Results: Intrinsic ageing factors inhibited cellular proliferation, increased senescent ratio and reduced mRNA expression of collagen I, collagen III and elastin, so did UVB, except for its induction of elastin mRNA expression. Furthermore, ADSC-CM treatment can slightly or significantly improve cellular proliferative activity and restore functions both in irradiated and non-irradiated HDFs. Besides, ADSC-CM treatment decreased cellular apoptosis and senescence induced by UVB but had no obvious effect on cellular senescence induced by intrinsic ageing factors. The results were similar in three generations of HDFs, yet in different degrees. Conclusions: The results suggest that ADSCs secretome protect HDFs from ageing damages but with some limitations. PMID:26884843

  20. Autophagy in human skin fibroblasts: Comparison between young and aged cells and evaluation of its cellular rhythm and response to Ultraviolet A radiation.

    PubMed

    Pernodet, Nadine; Dong, Kelly; Pelle, Edward

    2016-01-01

    Autophagic mechanisms play critical roles in cell maintenance. Damaged organelles that are not removed by autophagosomes, which act by engulfing and degrading these cellular components, have been linked to various pathologies. Recently, the progression of aging has also been correlated to a compromised autophagic response. Here, we report for the first time a significant reduction in autophagic levels in synchronized aged normal human skin fibroblasts as compared to young fibroblasts. We measured a 77.9% reduction in autophagy as determined by reverse transcription-polymerase chain reaction for LC3B expression, a microtubule-associated protein correlated to late stage autophagosome formation. In addition, we visualized these same changes by immunocytofluorescence with antibodies directed against LC3B. By harvesting synchronized, as well as unsynchronized cells over time, we were also able to measure for the first time a nighttime peak in autophagy that was present in young but absent in aged fibroblasts. Finally, since human skin is constantly subjected to environmentally induced oxidative stress from sunlight, we exposed fibroblasts to 10 J/cm2 ultraviolet A and found, in good agreement with current literature, not only that irradiation could partially reactivate autophagy in the aged cells, but also that this increase was phase shifted earlier from its endogenous temporal pattern because of its loss of synchronization with circadian rhythm.

  1. γ-Tocotrienol prevents cell cycle arrest in aged human fibroblast cells through p16(INK4a) pathway.

    PubMed

    Zainuddin, Azalina; Chua, Kien-Hui; Tan, Jen-Kit; Jaafar, Faizul; Makpol, Suzana

    2017-02-01

    Human diploid fibroblasts (HDFs) proliferation in culture has been used as a model of aging at the cellular level. Growth arrest is one of the most important mechanisms responsible for replicative senescence. Recent researches have been focusing on the function of vitamin E in modulating cellular signaling and gene expression. Therefore, the aim of this study was to elucidate the effect of palm γ-tocotrienol (vitamin E) in modulating cellular aging through p16(INK4a) pathway in HDF cells. Primary culture of senescent HDFs was incubated with 70 μM of palm γ-tocotrienol for 24 hours. Silencing of p16(INK4a) was carried out by siRNA transfection. RNA was extracted from the different treatment groups and gene expression analysis was carried out by real-time reverse transcription polymerase chain reaction. Proteins that were regulated by p16(INK4a) were determined by western blot technique. The finding of this study showed that p16(INK4a) mRNA was overexpressed in senescent HDFs, and hypophosphorylated-pRb and cyclin D1 protein expressions were increased (p < 0.05). However, downregulation of p16(INK4a) and hypophosphorylated-pRb and cyclin D1 protein expressions (p < 0.05) by γ-tocotrienol led to modulation of the cell cycle regulation during cellular aging. In conclusion, senescent HDFs showed change in biological process specifically in cell cycle regulation with elevated expression of genes and proteins which may contribute to cell cycle arrest. Palm γ-tocotrienol may delay cellular senescence of HDFs by regulating cell cycle through downregulation of p16(INK4a) and hypophosphorylated-pRb and cyclin D1 protein expressions.

  2. No donor age effect of human serum on collagen synthesis signaling and cell proliferation of human tendon fibroblasts.

    PubMed

    Bayer, Monika L; Schjerling, Peter; Biskup, Edyta; Herchenhan, Andreas; Heinemeier, Katja M; Doessing, Simon; Krogsgaard, Michael; Kjaer, Michael

    2012-05-01

    The aging process of tendon tissue is associated with decreased collagen content and increased risk for injuries. An essential factor in tendon physiology is transforming growth factor-β1 (TGF-β1), which is presumed to be reduced systemically with advanced age. The aim of this study was to investigate whether human serum from elderly donors would have an inhibiting effect on the expression of collagen and collagen-related genes as well as on cell proliferative capacity in tendon cells from young individuals. There was no difference in systemic TGF-β1 levels in serum obtained from young and elderly donors, and we found no difference in collagen expression when cells were subjected to human serum from elderly versus young donors. In addition, tendon cell proliferation was similar when culture medium was supplemented with serum of different donor age. These findings suggest that factors such as the cell intrinsic capacity or the tissue-specific environment rather than systemic circulating factors are important for functional capacity throughout life in human tendon cells. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Age dependency of the metabolic conversion of polyamines into amino acids in IMR-90 human embryonic lung diploid fibroblasts

    SciTech Connect

    Chen, K.Y.; Chang, Z.

    1986-07-01

    When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000-dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000-dalton protein. The authors have investigated these two polyamine-associated biochemical events in IMR-90 human diploid fibroblasts as a function of their population doubling level (PDL). They found that (1) the metabolic labeling of the 18,000-dalton protein was about two-fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six-fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4-fold higher in the young cells as compared to the old cells, their data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR-90 cells was about eight-fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000-dalton protein were also observed in cell strains derived from patients with premature aging disease.

  4. Interplay of IGF-I and 17beta-estradiol at age-specific levels in human sebocytes and fibroblasts in vitro.

    PubMed

    Makrantonaki, Evgenia; Vogel, Kim; Fimmel, Sabine; Oeff, Marina; Seltmann, Holger; Zouboulis, Christos C

    2008-10-01

    In order to obtain greater insights into the molecular mechanisms accompanying hormonal aging the effects of growth hormone (GH), insulin-like growth factor-I (IGF-I), 17beta-estradiol, progesterone and dehydroepiandrosterone were tested as single agents in concentrations corresponding to 20- and 60-year-old females on human SZ95 sebocytes and fibroblasts. Cell proliferation and viability were measured by 4-methylumbelliferyl heptanoate and lactate dehydrogenase microassays, respectively, whereas lipid accumulation was documented via nile red microassay and fluorescence microscopy. mRNA and protein expression were evaluated via real-time RT-PCR and Western blotting or ELISA, accordingly. Our results depict the importance of IGF-I for lipid synthesis in SZ95 sebocyte and demonstrate the lack of 17beta-estradiol, dehydroepiandrosterone and progesterone activity on lipid synthesis and SZ95 sebocyte proliferation suggesting that the action of these hormones in vivo may be implemented through indirect pathways. Fibroblast showed to be more susceptible to 17beta-estradiol treatment, while IGF-I could significantly stimulate fibroblast proliferation in a dose-dependent manner. Furthermore, an interplay between the 17beta-estradiol and IGF-I signaling pathway was documented in both cell types. In conclusion, IGF-I is a key regulator of human skin aging and declining IGF-I levels with age may play a significant role in the reduction of skin surface lipids and thickness.

  5. Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts

    PubMed Central

    Park, Jinny; Song, Hwa-Ryung; Lee, Minok; Hong, On-Yu; Whang, Pyoung H.; Han, Myung-Kwan; Kwon, Kang-Beom

    2016-01-01

    Reactive oxygen species (ROS) play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3) metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC) or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts. PMID:28003865

  6. Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts.

    PubMed

    Noh, Eun-Mi; Park, Jinny; Song, Hwa-Ryung; Kim, Jeong-Mi; Lee, Minok; Song, Hyun-Kyung; Hong, On-Yu; Whang, Pyoung H; Han, Myung-Kwan; Kwon, Kang-Beom; Kim, Jong-Suk; Lee, Young-Rae

    2016-01-01

    Reactive oxygen species (ROS) play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3) metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC) or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts.

  7. Anti-ageing properties of Khelma Longevity™: Treatment of human fibroblasts increases proteasome levels and decreases the levels of oxidized proteins.

    PubMed

    Voutetakis, Konstantinos; Delitsikou, Vasiliki; Magouritsas, Michel Georges; Gonosa, Efstathios S

    2017-03-05

    We have determined the putative anti-ageing properties of Khelma Longevity™, a formula based on various natural compounds from the Mediterranean area. Human primary fibroblast cultures were treated with a wide range of concentrations of Khelma Longevity™ for 1 day or 3 consecutive days. Following these treatments, two major and complementary biomarkers of ageing where measured, namely, the proteasome and the amount of oxidized proteins. It was observed that 24hours of treatment with Khelma Longevity™ resulted in a maximum increase of about 41% of the total protein levels of 20S proteasome. Levels of oxidized proteins were reduced by almost 6.5-fold following longer treatments. Specifically we have observed a maximum decrease of protein carbonyls to 84.7% in comparison with nontreated control cells following 3 days of continuous treatment with Khelma Longevity™. These results support the notion that formulas rich in natural compounds from the Mediterranean area possess anti-ageing properties.

  8. Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts.

    PubMed

    Makpol, Suzana; Yeoh, Thong Wei; Ruslam, Farah Adilah Che; Arifin, Khaizurin Tajul; Yusof, Yasmin Anum Mohd

    2013-08-16

    Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs.

  9. Comparative effect of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction on antioxidant enzymes activity in cellular ageing of human diploid fibroblasts

    PubMed Central

    2013-01-01

    Background Human diploid fibroblasts (HDFs) undergo a limited number of cellular divisions in culture and progressively reach a state of irreversible growth arrest, a process termed cellular ageing. Even though beneficial effects of Piper betle, Chlorella vulgaris and tocotrienol-rich fraction (TRF) have been reported, ongoing studies in relation to ageing is of interest to determine possible protective effects that may reverse the effect of ageing. The aim of this study was to evaluate the effect of P. betle, C. vulgaris and TRF in preventing cellular ageing of HDFs by determining the activity of antioxidant enzymes viz.; catalase, superoxide dismutase (SOD) and glutathione peroxidase. Methods Different passages of HDFs were treated with P. betle, C. vulgaris and TRF for 24 h prior to enzymes activity determination. Senescence-associated beta-galactosidase (SA β-gal) expression was assayed to validate cellular ageing. Results In cellular ageing of HDFs, catalase and glutathione peroxidase activities were reduced, but SOD activity was heightened during pre-senescence. P. betle exhibited the strongest antioxidant activity by reducing SA β-gal expression, catalase activities in all age groups, and SOD activity. TRF exhibited a strong antioxidant activity by reducing SA β-gal expression, and SOD activity in senescent HDFs. C. vulgaris extract managed to reduce SOD activity in senescent HDFs. Conclusion P. betle, C. vulgaris, and TRF have the potential as anti-ageing entities which compensated the role of antioxidant enzymes in cellular ageing of HDFs. PMID:23948056

  10. HETEROGENIC SERUM, AGE, AND MULTIPLICATION OF FIBROBLASTS

    PubMed Central

    Carrel, Alexis; Ebeling, Albert H.

    1922-01-01

    The presence in a culture medium of heterogenic serum of various concentrations exerts a definite influence on the rate of multiplication of fibroblasts. Dog serum does not inhibit the growth of See PDF for Structure chicken fibroblasts markedly until its concentration reaches 15 per cent. Beyond this figure, each increase of the concentration brings about a rapid decrease in the rate of cell multiplication. When the concentration reaches from 30 to 45 per cent, no growth takes place. The inhibiting action of cat serum begins to manifest itself at a concentration of 25 per cent and prevents cell proliferation completely at a concentration of 55 and 60 per cent. The ratio, See PDF for Equation can be taken as expressing the action of the serum on fibroblast multiplication; that is, as the growth index of the serum. See PDF for Structure The inhibiting influence of heterogenic serum was found to vary in direct ratio to the age of the animal from which it was obtained. The rate of proliferation of chicken fibroblasts was studied comparatively in media containing varied concentrations of serum from young and old animals. For each concentration of serum, the rate of growth in the serum of the old animal was expressed in relation to the rate of growth in the serum of the young animal. When cat serum was used, the curve obtained in plotting this ratio in ordinates and the serum concentration in abscissæ showed a rapid increase in the inhibiting action of the old serum as soon as the concentration reached 30 per cent. The same tests were repeated with the serum from young and old dogs. The general results were identical, although See PDF for Structure the quantitative inhibiting action of both sera was greater than that of cat serum. It may be concluded that under the conditions of the experiments: 1. Heterogenicsera inhibit and prevent the growth of chicken fibroblasts when their concentration is made to vary within certain limits. 2. A relation exists between the rate

  11. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    SciTech Connect

    Tashiro, Kanae; Shishido, Mayumi; Fujimoto, Keiko; Hirota, Yuko; Yo, Kazuyuki; Gomi, Takamasa; Tanaka, Yoshitaka

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  12. Age-dependent response of primary human dermal fibroblasts to oxidative stress: cell survival, pro-survival kinases, and entrance into cellular senescence.

    PubMed

    Gurjala, Anandev N; Liu, W Robert; Mogford, Jon E; Procaccini, Piero S A; Mustoe, Thomas A

    2005-01-01

    A central question in cell biology is how cells become senescent. After a finite number of cell divisions, normal cultured human cells enter a state of irreversible growth arrest, termed "replicative senescence." Alternatively, oxidative stress in the form of hydrogen peroxide (H(2)O(2)) can render human dermal fibroblasts (HDFs) nonproliferative and quiescent, a phenomenon known as stress-induced premature senescence (SIPS). Although critical to the understanding of the pathophysiological basis of many diseases, there is no research to date that has simultaneously examined the interactions between age, oxidative stress, and SIPS. Therefore, the goals of this study were to examine in concert the interactions between these three factors in primary HDFs, and to test our central hypothesis that aging lowers the ability of primary HDFs to respond to oxidative stress. Our data provide, for the first time, evidence that aging dramatically reduces the capacity of primary HDFs to respond to the challenge of hydrogen peroxide. Specifically, aged HDFs showed decreased cell viability, decreased phosphorylation (activation) of pro-survival kinases (Akt and ERK 1/2), and increased entrance into a senescent state when compared with their younger counterparts. Another important conclusion of this study is that blockade of transforming growth factor-beta1 had a pronounced "rescue effect" in the aged, preventing entrance of HDFs into cellular senescence.

  13. Nanoscale gelatinase A (MMP-2) inhibition on human skin fibroblasts of Longkong (Lansium domesticum Correa) leaf extracts for anti-aging.

    PubMed

    Manosroi, Aranya; Kumguan, Kulthida; Chankhampan, Charinya; Manosroi, Worapaka; Manosroi, Jiradej

    2012-09-01

    Leaves of Longkong which collected from Chantaburi in Thailand were extracted by the hot and cold processes using three different solvents including water, chloroform and methanol. The crude extracts were tested for antioxidative activities, tyrosinase inhibition and in vitro cytotoxicity as well as the MMP-2 inhibition activity on human skin fibroblasts for anti-aging evaluation. The hot water crude extract showed the highest antioxidative activities (DPPH radical scavenging, metal ion chelating and lipid peroxidation inhibition) with the SC50, CC50 and IPC50 values of 5.40 +/- 1.23, 32.31 +/- 0.84 and 3.29 +/- 0.30 mg/ml, respectively, and the highest tyrosinase inhibition activity with the IC50 value of 0.49 +/- 0.23 mg/ml. The extract also showed no cytotoxicity on human skin fibroblasts with the cell viability of 80.52 +/- 15.16%. It demonstrated the anti-aging potential by having the pro and active MMP-2 inhibition activity, but lower than ascorbic acid of 1.28 and 1.12 times, respectively. The semi-purified extracts were prepared from this crude extract by solvent-solvent partition. The ethyl acetate soluble fraction showed higher activities (DPPH radical scavenging, metal ion chelating and tyrosinase inhibition) than the crude extract of 23.48, 71.80 and 2.58 times, respectively. This fraction exhibited similar pro and active MMP-2 inhibitory effect to the crude extract. The results from this study have indicated the possible application of the ethyl acetate fraction of the hot water crude extract from leaves of Longkong to be developed as an anti-aging product.

  14. Role of human pulp fibroblasts in angiogenesis.

    PubMed

    Tran-Hung, L; Mathieu, S; About, I

    2006-09-01

    After pulp amputation, complete pulp healing requires not only reparative dentin production but also fibroblast proliferation, nerve fiber growth, and neoangiogenesis. This study was designed to investigate the role of pulp fibroblasts in angiogenesis. Human pulp fibroblasts from third molars co-cultured with human umbilical vein endothelial cells induced the organization of endothelial cells and the formation of tubular structures corresponding to capillaries in vivo. The direct contact between both cells was not necessary to induce angiogenesis, and the observed effect was due to soluble factors. This was confirmed with neutralizing antibodies against FGF-2 and VEGF, which decreased the angiogenic effects of these soluble factors. Immunohistochemistry showed that both FGF-2 and VEGF were expressed in human dental pulp fibroblasts, and this expression increased after injury. These results suggest that the pulp fibroblasts secrete angiogenic factors, which are necessary for complete pulp healing, particularly at the pulp injury site.

  15. Evaluation of Anti-aging Compounds Using the Promoters of Elastin and Fibrillin-1 Genes Combined with a Secreted Alkaline Phosphatase Reporter in Normal Human Fibroblasts.

    PubMed

    Lin, Chih-Chien; Yang, Chao-Hsun; Kuo, Wan-Ting; Chen, Cheng-Yu

    2015-01-01

    Elastic fibers are major constituents of the extracellular matrix (ECM) in dynamic tissues in the human body, and regulation of elastin and fibrillin-1 expression mediates the formation of these fibers. Traditional assays for the measurement of elastin and fibrillin-1, such as western blotting, Luna staining and immunostaining, are relatively complex and time-consuming. Thus, a relatively simple assay system that also provides rational results is urgently needed. In the study, we aimed to develop a human cell-based assay system that can be used to analyze functional compounds using the promoters of elastin (ELN) and fibrillin-1 (FBN1) genes integrated with a secreted alkaline phosphatase (SEAP) reporter in normal human fibroblast cells. We used this system to assess anti-aging compounds. We used several regulators of elastinogenesis, including retinol, coenzyme Q10, deoxyArbutin and Elestan(TM) (Manilkara multinervis leaf extract), to verify the efficacy of this assay system. Our results demonstrate that this assay system can be used as a fast and realistic method for identifying anti-aging components for future use in foods, cosmetics and drugs.

  16. Age related changes in steroid receptors on cultured lung fibroblasts

    SciTech Connect

    Barile, F.A.; Bienkowski, R.S.

    1986-03-05

    The number of high affinity glucocorticoid receptors (Ro) on human fetal lung fibroblasts decreases as the cells age in vitro, and it has been suggested that these cell systems may be useful models of age-related changes in vivo. They examined the relation between change in Ro with in vitro aging and donor age. Confluent monolayers of lung fibroblasts at various population doubling levels (PDL), were incubated with (/sup 3/H)-dexamethasone ((/sup 3/H)Dex) either alone or with excess (.01 mM) Dex. Specific binding was calculated as the difference between radioactivity in cells incubated with and without unlabeled Dex; Scatchard plots were used to analyze the data. Ro, measured as fmol (/sup 3/H)Dex/10/sup 6/ cells, for two lines of human fetal cells (HFL-1 and MRC-5) decreased with increasing age in vitro. However, human newborn (CRL-1485) and adult (CCL-201) cells and fetal rabbit cells (FAB-290), showed increases in Ro with continuous passage. For each cell line, the affinity constant (K/sub d/) did not change significantly with passage. They conclude that the direction of changes in steroid receptor levels on cells aging in vitro is influenced by donor age and species. Caution should be used in applying results obtained from model systems to aging organisms.

  17. The mechanisms underlying the anti-aging activity of the Chinese prescription Kangen-karyu in hydrogen peroxide-induced human fibroblasts.

    PubMed

    Satoh, Akiko; Yokozawa, Takako; Kim, Young Ae; Cho, Eun Ju; Okamoto, Takuya; Sei, Yasuo

    2005-10-01

    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 (H(2)O(2))-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 H(2)O(2) treatment and inhibited senescence-associated beta-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-kappaB) translocation in response to oxidative stress suggested that Kangen-karyu exerted its anti-aging effect through NF-kappaB modulation and prevention of H(2)O(2)-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.

  18. Biological characterization of human fibroblast-derived mitogenic factors for human melanocytes.

    PubMed Central

    Imokawa, G; Yada, Y; Morisaki, N; Kimura, M

    1998-01-01

    To clarify the paracrine linkage between human fibroblasts and melanocytes in cutaneous pigmentation, we studied the effects of human fibroblast-derived factors on the proliferation of human melanocytes. In medium conditioned for 4 days with human fibroblast culture, factors were produced that markedly stimulated DNA synthesis of human melanocytes. The stimulatory effect was higher in medium conditioned with fibroblasts from aged skin than in medium conditioned with fibroblasts from young skin, and was interrupted by inhibitors of tyrosine kinase, such as tyrphostin, genistein and herbimycin, but not by inhibitors of protein kinases C and A, such as H-7 and phloretin. The conditioned medium was also capable of activating mitogen-activated protein kinase of human melanocytes, with old fibroblasts being more effective than young ones. Analysis of factors released into the conditioned medium revealed that levels of hepatocyte growth factor (HGF) and stem cell factor (SCF) were increased in old-fibroblast-conditioned medium compared with young-fibroblast-conditioned medium. In contrast, levels of basic fibroblast growth factor (bFGF) were similar in both media. When the conditioned medium was treated with HGF antibody with or without SCF antibody, the increase in DNA synthesis by human melanocytes was decreased to 20% of the elevated level, whereas antibodies to bFGF had no effect. Analysis of the medium conditioned for 4 days after cytokine application demonstrated that, of the cytokines tested, interleukin 1alpha and tumour necrosis factor alpha are highly effective in stimulating HGF secretion by old fibroblasts. HGF and SCF, but not bFGF, were markedly increased in culture medium in the presence of IL-1alpha, and this stimulatory effect was confined to young human fibroblasts. These findings suggest that SCF and HGF derived from human fibroblasts may play a part in regulating cutaneous pigmentation during inflammation and aging. PMID:9494091

  19. Cell culture condition-dependent impact of AGE-rich food extracts on kinase activation and cell survival on human fibroblasts.

    PubMed

    Nass, Norbert; Weissenberg, Kristian; Somoza, Veronika; Ruhs, Stefanie; Silber, Rolf-Edgar; Simm, Andreas

    2014-03-01

    Advanced glycation end products (AGEs) are stable end products of the Maillard reaction. Effects of food extracts are often initially analysed in cellular test systems and it is not clear how different cell culture conditions might influence the results. Therefore, we compared the effects of two models for AGE-rich food, bread crust and coffee extract (CE) on WI-38 human lung fibroblasts under different cell culture conditions (sub-confluent versus confluent cells, with and without serum). WI-38 cells responded to coffee and bread crust extract (BCE) with a rapid phosphorylation of PKB (AKT), p42/44 MAPK (ERK 1/2) and p38 MAPK, strongly depending on culture conditions. BCE resulted in increased cell numbers, whereas CE appeared to be cytotoxic. When cell numbers under all culture conditions and treatments were correlated with kinase phosphorylation, the relation between phospho-p38 MAPK and phospho-AKT represented a good, cell culture condition-independent predictor of cell survival.

  20. Senescent human fibroblasts show increased glycolysis and redox homeostasis with extracellular metabolomes that overlap with those of irreparable DNA damage, aging, and disease.

    PubMed

    James, Emma L; Michalek, Ryan D; Pitiyage, Gayani N; de Castro, Alice M; Vignola, Katie S; Jones, Janice; Mohney, Robert P; Karoly, Edward D; Prime, Stephen S; Parkinson, Eric Kenneth

    2015-04-03

    Cellular senescence can modulate various pathologies and is associated with irreparable DNA double-strand breaks (IrrDSBs). Extracellular senescence metabolomes (ESMs) were generated from fibroblasts rendered senescent by proliferative exhaustion (PEsen) or 20 Gy of γ rays (IrrDSBsen) and compared with those of young proliferating cells, confluent cells, quiescent cells, and cells exposed to repairable levels of DNA damage to identify novel noninvasive markers of senescent cells. ESMs of PEsen and IrrDSBsen overlapped and showed increased levels of citrate, molecules involved in oxidative stress, a sterol, monohydroxylipids, tryptophan metabolism, phospholipid, and nucleotide catabolism, as well as reduced levels of dipeptides containing branched chain amino acids. The ESM overlaps with the aging and disease body fluid metabolomes, supporting their utility in the noninvasive detection of human senescent cells in vivo and by implication the detection of a variety of human pathologies. Intracellular metabolites of senescent cells showed a relative increase in glycolysis, gluconeogenesis, the pentose-phosphate pathway, and, consistent with this, pyruvate dehydrogenase kinase transcripts. In contrast, tricarboxylic acid cycle enzyme transcript levels were unchanged and their metabolites were depleted. These results are surprising because glycolysis antagonizes senescence entry but are consistent with established senescent cells entering a state of low oxidative stress.

  1. Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice

    PubMed Central

    Lee, Hyunji; Hong, Youngeun; Kwon, So Hee; Park, Jongsun; Park, Jisoo

    2016-01-01

    Background Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Objective Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. Methods PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student’s unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. Results PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. Conclusion This study

  2. Anti-aging effects of Piper cambodianum P. Fourn. extract on normal human dermal fibroblast cells and a wound-healing model in mice.

    PubMed

    Lee, Hyunji; Hong, Youngeun; Kwon, So Hee; Park, Jongsun; Park, Jisoo

    2016-01-01

    Aging of skin is associated with environmental factors such as ultraviolet rays, air pollution, gravity, and genetic factors, all of which can lead to wrinkling of skin. Previous reports suggest that the wound repair is impaired by the aging process and strategies to manipulate the age-related wound healing are necessary in order to stimulate repair. Several traditional plant extracts are well-known for their properties of skin protection and care. Piper cambodianum P. Fourn. (PPF), a member of Piperacecae, is a plant found in Vietnam that might have therapeutic properties. Therefore, the effects of PPF stem and leaf extract on aging process were investigated in vitro and in vivo. PPF extract dissolved in methanol was investigated using Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, flow cytometry, and cell wound-healing assays. We assessed the anti-aging effect of PPF in mouse using the wound-healing assay. The results were analyzed by Student's unpaired t-test; *P<0.05 and **P<0.01 were considered to indicate significant and highly significant values, respectively, compared with corresponding controls. PPF treatment demonstrated in vitro and in vivo anti-aging activity. Western blot analysis of PPF-treated normal human dermal fibroblast cells showed a dose-dependent increase in the expression of extracellular matrix genes such as collagen and elastin, but decreased expression of the aging gene matrix metalloproteinase-3. Quantitative polymerase chain reaction showed that PPF-treated cells displayed dose-dependent increase in messenger RNA expression levels of collagen, elastin, and hyaluronan synthase-2 and decreased expression levels of matrix metalloproteinase-1 aging gene. PPF treatment led to decreased production of reactive oxygen species in cells subjected to ultraviolet irradiation. Furthermore, PPF extract showed positive wound-healing effects in mice. This study demonstrated the anti-aging and wound

  3. NF-κB accumulation associated with COL1A1 transactivators defects during chronological aging represses type I collagen expression through a -112/-61-bp region of the COL1A1 promoter in human skin fibroblasts.

    PubMed

    Bigot, Nicolas; Beauchef, Gallic; Hervieu, Magalie; Oddos, Thierry; Demoor, Magali; Boumediene, Karim; Galéra, Philippe

    2012-10-01

    The aging process, especially of the skin, is governed by changes in the epidermal, dermo-epidermal, and dermal compartments. Type I collagen, which is the major component of dermis extracellular matrix (ECM), constitutes a prime target for intrinsic and extrinsic aging-related alterations. In addition, under the aging process, pro-inflammatory signals are involved and collagens are fragmented owing to enhanced matrix metalloproteinase activities, and fibroblasts are no longer able to properly synthesize collagen fibrils. Here, we demonstrated that low levels of type I collagen detected in aged skin fibroblasts are attributable to an inhibition of COL1A1 transcription. Indeed, on one hand, we observed decreased binding activities of specific proteins 1 and 3, CCAAT-binding factor, and human collagen-Krüppel box, which are well-known COL1A1 transactivators acting through the -112/-61-bp promoter sequence. On the other hand, the aging process was accompanied by elevated amounts and binding activities of NF-κB (p65 and p50 subunits), together with an increased number of senescent cells. The forced expression of NF-κB performed in young fibroblasts was able to establish an old-like phenotype by repressing COL1A1 expression through the short -112/-61-bp COL1A1 promoter and by elevating the senescent cell distribution. The concomitant decrease of transactivator functions and increase of transinhibitor activity is responsible for ECM dysfunction, leading to aging/senescence in dermal fibroblasts.

  4. Epidermal growth factor improves the migration and contractility of aged fibroblasts cultured on 3D collagen matrices.

    PubMed

    Kim, Daehwan; Kim, So Young; Mun, Seog Kyun; Rhee, Sangmyung; Kim, Beom Joon

    2015-04-01

    Epidermal growth factor (EGF) plays a critical role in fibroblasts by stimulating the production of collagen and supports cell renewal through the interaction between keratinocytes and fibroblasts. It is well known that the contractile activity of fibroblasts is required for the remodeling of the extracellular matrix (ECM), which contributes to skin elasticity. However, the role of EGF in the contraction of aged fibroblasts under 3-dimensional (3D) culture conditions is not yet fully understood. In the present study, we demonstrated that young fibroblasts spread and proliferated more rapidly than aged fibroblasts under 2-dimensional (2D) culture conditions. Cell migration assay using a nested collagen matrix revealed that the migration of young fibroblasts was also greater than that of aged fibroblasts under 3D culture conditions. However, the addition of recombinant human EGF (rhEGF) resulted in the enhanced migration of aged fibroblasts; the migration rate was similar to that of the young fibroblasts. The aged fibroblasts showed decreased cluster formation compared with the young fibroblasts on the collagen matrix, which was improved by the addition of rhEGF. Furthermore, cell contraction assay revealed that the basal contractility of the aged fibroblasts was lower than that of the young fibroblasts; however, following treatment with rhEGF, the contractility was restored to levels similar or even higher to those of the young fibroblasts. Taken together, our results suggest that rhEGF is a potential renewal agent that acts to improve the migration and contraction of aged fibroblasts more efficiently than young fibroblasts under 3D culture conditions; thus, EGF may have valuable regenerative effects on aged skin.

  5. Stromal-epithelial interactions in aging and cancer: Senescent fibroblasts alter epithelial cell differentiation

    SciTech Connect

    Parrinello, Simona; Coppe, Jean-Philippe; Krtolica, Ana; Campisi, Judith

    2004-07-14

    Cellular senescence suppresses cancer by arresting cells at risk for malignant tumorigenesis. However, senescent cells also secrete molecules that can stimulate premalignant cells to proliferate and form tumors, suggesting the senescence response is antagonistically pleiotropic. We show that premalignant mammary epithelial cells exposed to senescent human fibroblasts in mice irreversibly lose differentiated properties, become invasive and undergo full malignant transformation. Moreover, using cultured mouse or human fibroblasts and non-malignant breast epithelial cells, we show that senescent fibroblasts disrupt epithelial alveolar morphogenesis, functional differentiation, and branching morphogenesis. Further, we identify MMP-3 as the major factor responsible for the effects of senescent fibroblasts on branching morphogenesis. Our findings support the idea that senescent cells contribute to age-related pathology, including cancer, and describe a new property of senescent fibroblasts--the ability to alter epithelial differentiation--that might also explain the loss of tissue function and organization that is a hallmark of aging.

  6. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells.

    PubMed

    Miyoshi, Keiko; Horiguchi, Taigo; Tanimura, Ayako; Hagita, Hiroko; Noma, Takafumi

    2015-01-01

    Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming.

  7. Gene Signature of Human Oral Mucosa Fibroblasts: Comparison with Dermal Fibroblasts and Induced Pluripotent Stem Cells

    PubMed Central

    Miyoshi, Keiko; Horiguchi, Taigo; Tanimura, Ayako; Hagita, Hiroko; Noma, Takafumi

    2015-01-01

    Oral mucosa is a useful material for regeneration therapy with the advantages of its accessibility and versatility regardless of age and gender. However, little is known about the molecular characteristics of oral mucosa. Here we report the first comparative profiles of the gene signatures of human oral mucosa fibroblasts (hOFs), human dermal fibroblasts (hDFs), and hOF-derived induced pluripotent stem cells (hOF-iPSCs), linking these with biological roles by functional annotation and pathway analyses. As a common feature of fibroblasts, both hOFs and hDFs expressed glycolipid metabolism-related genes at higher levels compared with hOF-iPSCs. Distinct characteristics of hOFs compared with hDFs included a high expression of glycoprotein genes, involved in signaling, extracellular matrix, membrane, and receptor proteins, besides a low expression of HOX genes, the hDFs-markers. The results of the pathway analyses indicated that tissue-reconstructive, proliferative, and signaling pathways are active, whereas senescence-related genes in p53 pathway are inactive in hOFs. Furthermore, more than half of hOF-specific genes were similarly expressed to those of hOF-iPSC genes and might be controlled by WNT signaling. Our findings demonstrated that hOFs have unique cellular characteristics in specificity and plasticity. These data may provide useful insight into application of oral fibroblasts for direct reprograming. PMID:26339586

  8. Gastrointestinal Fibroblasts Have Specialized, Diverse Transcriptional Phenotypes: A Comprehensive Gene Expression Analysis of Human Fibroblasts

    PubMed Central

    Ishii, Genichiro; Aoyagi, Kazuhiko; Sasaki, Hiroki; Ochiai, Atsushi

    2015-01-01

    Background Fibroblasts are the principal stromal cells that exist in whole organs and play vital roles in many biological processes. Although the functional diversity of fibroblasts has been estimated, a comprehensive analysis of fibroblasts from the whole body has not been performed and their transcriptional diversity has not been sufficiently explored. The aim of this study was to elucidate the transcriptional diversity of human fibroblasts within the whole body. Methods Global gene expression analysis was performed on 63 human primary fibroblasts from 13 organs. Of these, 32 fibroblasts from gastrointestinal organs (gastrointestinal fibroblasts: GIFs) were obtained from a pair of 2 anatomical sites: the submucosal layer (submucosal fibroblasts: SMFs) and the subperitoneal layer (subperitoneal fibroblasts: SPFs). Using hierarchical clustering analysis, we elucidated identifiable subgroups of fibroblasts and analyzed the transcriptional character of each subgroup. Results In unsupervised clustering, 2 major clusters that separate GIFs and non-GIFs were observed. Organ- and anatomical site-dependent clusters within GIFs were also observed. The signature genes that discriminated GIFs from non-GIFs, SMFs from SPFs, and the fibroblasts of one organ from another organ consisted of genes associated with transcriptional regulation, signaling ligands, and extracellular matrix remodeling. Conclusions GIFs are characteristic fibroblasts with specific gene expressions from transcriptional regulation, signaling ligands, and extracellular matrix remodeling related genes. In addition, the anatomical site- and organ-dependent diversity of GIFs was also discovered. These features of GIFs contribute to their specific physiological function and homeostatic maintenance, and create a functional diversity of the gastrointestinal tract. PMID:26046848

  9. Advanced glycation end products (AGEs) and their receptor (RAGE) induce apoptosis of periodontal ligament fibroblasts.

    PubMed

    Li, D X; Deng, T Z; Lv, J; Ke, J

    2014-12-01

    Diabetics have an increased prevalence of periodontitis, and diabetes is one of the causative factors of severe periodontitis. Apoptosis is thought to be involved in this pathogenic relationship. The aim of this study was to investigate apoptosis in human periodontal ligament (PDL) fibroblasts induced by advanced glycation end products (AGEs) and their receptor (RAGE). We examined the roles of apoptosis, AGEs, and RAGE during periodontitis in diabetes mellitus using cultured PDL fibroblasts that were treated by AGE-modified bovine serum albumin (AGE-BSA), bovine serum albumin (BSA) alone, or given no treatment (control). Microscopy and real-time quantitative PCR indicated that PDL fibroblasts treated with AGE-BSA were deformed and expressed higher levels of RAGE and caspase 3. Cell viability assays and flow cytometry indicated that AGE-BSA reduced cell viability (69.80 ± 5.50%, P<0.01) and increased apoptosis (11.31 ± 1.73%, P<0.05). Hoechst 33258 staining and terminal-deoxynucleotidyl transferase-mediated nick-end labeling revealed that AGE-BSA significantly increased apoptosis of PDL fibroblasts. The results showed that the changes in PDL fibroblasts induced by AGE-BSA may explain how AGE-RAGE participates in and exacerbates periodontium destruction.

  10. Altered chromosome 6 in immortal human fibroblasts.

    PubMed

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  11. Altered chromosome 6 in immortal human fibroblasts.

    PubMed Central

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-01-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. Images PMID:1373811

  12. Microrna-217 modulates human skin fibroblast senescence by directly targeting DNA methyltransferase 1.

    PubMed

    Wang, Ben; Du, Rui; Xiao, Xiao; Deng, Zhi-Li; Jian, Dan; Xie, Hong-Fu; Li, Ji

    2017-05-16

    DNA methyltransferase 1 (DNMT1) is a major epigenetic regulator associated with many biological processes. However, the roles and mechanisms of DNMT1 in skin aging are incompletely understood. Here we explored the role of DNMT1 in human skin fibroblasts senescence and its related regulatory mechanisms. DNMT1 expression decreased in passage-aged fibroblasts and DNMT1 silencing in young fibroblasts induced the senescence phenotype. MiR-217 is predicted to target DNMT1 mRNA and miR-217 expression increased in passage-aged fibroblasts. MiR-217 directly targeted the 3'-untranslated region (3'-UTR) of DNMT1 in HEK 293T cells and inhibited DNMT1 expression in fibroblasts. MiR-217 overexpression induced a senescence phenotype in young fibroblasts, and miR-217 downregulation in old HSFs partially reversed the senescence phenotype. However, these effects could be significantly rescued by regulating DNMT1 expression in fibroblasts. After regulating miR-217 levels, we analyzed changes in the promoter methylation levels of 24 senescent-associated genes, finding that 6 genes were significantly altered, and verified p16 and phosphorylated retinoblastoma (pRb) protein levels. Finally, an inverse correlation between DNMT1 and miR-217 expression was observed in skin tissues and different-aged fibroblasts. Together, these findings revealed that miR-217 promotes fibroblasts senescence by suppressing DNMT1-mediated methylation of p16 and pRb by targeting the DNMT1 3'-UTR.

  13. Cytosolic malate dehydrogenase regulates senescence in human fibroblasts.

    PubMed

    Lee, Seung-Min; Dho, So Hee; Ju, Sung-Kyu; Maeng, Jin-Soo; Kim, Jeong-Yoon; Kwon, Ki-Sun

    2012-10-01

    Carbohydrate metabolism changes during cellular senescence. Cytosolic malate dehydrogenase (MDH1) catalyzes the reversible reduction of oxaloacetate to malate at the expense of reduced nicotinamide adenine dinucleotide (NADH). Here, we show that MDH1 plays a critical role in the cellular senescence of human fibroblasts. We observed that the activity of MDH1 was reduced in old human dermal fibroblasts (HDFs) [population doublings (PD) 56], suggesting a link between decreased MDH1 protein levels and aging. Knockdown of MDH1 in young HDFs (PD 20) and the IMR90 human fibroblast cell line resulted in the appearance of significant cellular senescence features, including senescence-associated β-galactosidase staining, flattened and enlarged morphology, increased population doubling time, and elevated p16(INK4A) and p21(CIP1) protein levels. Cytosolic NAD/NADH ratios were decreased in old HDFs to the same extent as in MDH1 knockdown HDFs, suggesting that cytosolic NAD depletion is related to cellular senescence. We found that AMP-activated protein kinase, a sensor of cellular energy, was activated in MDH1 knockdown cells. We also found that sirtuin 1 (SIRT1) deacetylase, a controller of cellular senescence, was decreased in MDH1 knockdown cells. These results indicate that the decrease in MDH1 and subsequent reduction in NAD/NADH ratio, which causes SIRT1 inhibition, is a likely carbohydrate metabolism-controlled cellular senescence mechanism.

  14. Plasminogen activator inhibitor 1, fibroblast apoptosis resistance, and aging-related susceptibility to lung fibrosis.

    PubMed

    Huang, Wen-Tan; Akhter, Hasina; Jiang, Chunsun; MacEwen, Mark; Ding, Qiang; Antony, Veena; Thannickal, Victor John; Liu, Rui-Ming

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is a fatal lung disorder with unknown cause and no effective treatment. The incidence of and mortality from IPF increase with age, suggesting that advanced age is a major risk factor for IPF. The mechanism underlying the increased susceptibility of the elderly to IPF, however, is unknown. In this study, we show for the first time that the protein level of plasminogen activator inhibitor 1 (PAI-1), a protease inhibitor which plays an essential role in the control of fibrinolysis, was significantly increased with age in mouse lung homogenate and lung fibroblasts. Upon bleomycin challenge, old mice experienced augmented PAI-1 induction and lung fibrosis as compared to young mice. Most interestingly, we show that fewer (myo)fibroblasts underwent apoptosis and more (myo)fibroblasts with increased level of PAI-1 accumulated in the lung of old than in young mice after bleomycin challenge. In vitro studies further demonstrate that fibroblasts isolated from lungs of old mice were resistant to H2O2 and tumor necrosis factor alpha-induced apoptosis and had augmented fibrotic responses to TGF-β1, compared to fibroblasts isolated from young mice. Inhibition of PAI-1 activity with a PAI-1 inhibitor, on the other hand, eliminated the aging-related apoptosis resistance and TGF-β1 sensitivity in isolated fibroblasts. Moreover, we show that knocking down PAI-1 in human lung fibroblasts with PAI-1 siRNA significantly increased their sensitivity to apoptosis and inhibited their responses to TGF-β1. Together, the results suggest that increased PAI-1 expression may underlie the aging-related sensitivity to lung fibrosis in part by protecting fibroblasts from apoptosis. Published by Elsevier Inc.

  15. Nucleus transfer efficiency of ear fibroblast cells isolated from Bama miniature pigs at various ages.

    PubMed

    Wang, Qing-Hua; Peng, Yun; Cai, Xin-Yong; Wan, Meng; Liu, Yu; Wei, Hong

    2015-08-01

    Somatic cell nucleus transfer (SCNT) has been considered the most effective method for conserving endangered animals and expanding the quantity of adult animal models. Bama miniature pigs are genetically stable and share similar biological features to humans. These pigs have been used to establish animal models for human diseases, and for many other applications. However, there is a paucity of studies on the effect of ear fibroblasts derived from different age of adult Bama miniature pigs on nucleus transfer (NT). The present study examined the NT efficiency of ear fibroblasts from fetal, newborn, 1-, 2-, 4-, 6-, 12-month-old miniature pigs by using trypan blue staining, flow cytometry and NT technique, etc., and the cell biological function and SCNT efficiency were compared between groups. The results showed that ear fibroblasts grew well after passage in each group. Spindle-shaped cells initially predominated, and gradually declined with increase of culture time and replaced by polygonal cells. Irregular cell growth occurred in the 2-month-old group and the elder groups. The growth curves of the ear fibroblasts were "S-shaped" in different age groups. The cell proliferation of postnatal ear fibroblasts, especially those from 2-, 4-, 6-, 12-month-old miniature pigs was significantly different from that of fetus ear fibroblasts (P<0.05 or P<0.01). Two-month- and 4-month-old ear fibroblasts had a significantly higher proportion of G1 stage cells (85% to 91%) than those at 6 and 12 months (66% to 74%, P<0.01). The blastocyst rate of reconstructed embryos originating from newborn, 1-, 2-, 4-month-old donor pigs was 6.06% to 7.69% with no significant difference from that in fetus fibroblast group (8.06%). It was concluded that <4-month-old adult Bama miniature pigs represent a better donor cell resource than elder pigs.

  16. Age-Related Changes in FGF-2, Fibroblast Growth Factor Receptors and β-Catenin Expression in Human Mesenchyme-Derived Progenitor Cells.

    PubMed

    Hurley, Marja M; Gronowicz, Gloria; Zhu, Li; Kuhn, Liisa T; Rodner, Craig; Xiao, Liping

    2016-03-01

    FGF-2 stimulates preosteoblast replication, and knockout of the FGF-2 gene in mice resulted in osteopenia with age, associated with decreased Wnt-β-Catenin signaling. In addition, targeted expression of FGF-2 in osteoblast progenitors increased bone mass in mice via Wnt-β-Catenin signaling. We posited that diminution of the intrinsic proliferative capacity of human mesenchyme-derived progenitor cells (HMDPCs) with age is due in part to reduction in FGF-2. To test this hypothesis HMDPCs from young (27-38), middle aged (47-56), and old (65-76) female human subjects were isolated from bone discarded after orthopedic procedures. HMDPCs cultures were mostly homogeneous with greater than 90% mesenchymal progenitor cells, determined by fluorescence-activated cell sorting. There was a progressive decrease in FGF-2 and FGFR1 mRNA and protein in HMDPCs with age. Since FGF-2 activates β-catenin, which can enhance bone formation, we also assessed its age-related expression in HMDPCs. An age-related decrease in total-β-Catenin mRNA and protein expression was observed. However there were increased levels of p-β-Catenin and decreased levels of activated-β-Catenin in old HMDSCs. FGF-2 treatment increased FGFR1 and β-Catenin protein, reduced the level of p-β-Catenin and increased activated-β-Catenin in aged HMDPCs. In conclusion, reduction in FGF-2 expression could contribute to age-related impaired function of HMDPCs via modulation of Wnt-β-catenin signaling.

  17. Stable and unstable forms of human fibroblast interferon.

    PubMed Central

    Edy, V G; Desmyter, J; Billiau, A; De Somer, P

    1977-01-01

    There is a minor fraction of human fibroblast interferon that resembles human leukocyte interferon in being renaturable after treatment with guanidine hydrochloride. However, antigenically and in its low activity on heterologous cells, it resembles the bulk of human fibroblast interferon. Since the production of this stable interferon fraction is not greatly inhibited by glucosamine at concentrations that significantly reduce total interferon production, it is suggested that it differs from the bulk of human fibroblast interferon in the extent or nature of glycosylation. PMID:863511

  18. The effect of vitamin E on basic fibroblast growth factor level in human fibroblast cell culture.

    PubMed

    Rashid, S A Harun Nor; Halim, A S; Muhammad, N A

    2008-07-01

    Basic fibroblast growth factor (bFGF) is angiogenic and effective in down-regulating excess collagen production. The aim of this study is to evaluate the effectiveness of vitamin E (Tocotrienol Rich Fraction) in altering the level of bFGF, a cytokine involved in the scar formation process. In this model, normal human fibroblasts were treated with various concentrations of vitamin E at different time frames. The levels of bFGF were determined by Enzyme-Linked Immunosorbant Assay (ELISA). This study demonstrated that Tocotrienol Rich Fraction (TRF) stimulated bFGF production by fibroblast and postulate that vitamin E may decrease aberrant scar formation.

  19. Methionine restriction slows down senescence in human diploid fibroblasts.

    PubMed

    Kozieł, Rafał; Ruckenstuhl, Christoph; Albertini, Eva; Neuhaus, Michael; Netzberger, Christine; Bust, Maria; Madeo, Frank; Wiesner, Rudolf J; Jansen-Dürr, Pidder

    2014-12-01

    Methionine restriction (MetR) extends lifespan in animal models including rodents. Using human diploid fibroblasts (HDF), we report here that MetR significantly extends their replicative lifespan, thereby postponing cellular senescence. MetR significantly decreased activity of mitochondrial complex IV and diminished the accumulation of reactive oxygen species. Lifespan extension was accompanied by a significant decrease in the levels of subunits of mitochondrial complex IV, but also complex I, which was due to a decreased translation rate of several mtDNA-encoded subunits. Together, these findings indicate that MetR slows down aging in human cells by modulating mitochondrial protein synthesis and respiratory chain assembly. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  20. Human dermal fibroblasts in psychiatry research.

    PubMed

    Kálmán, S; Garbett, K A; Janka, Z; Mirnics, K

    2016-04-21

    In order to decipher the disease etiology, progression and treatment of multifactorial human brain diseases we utilize a host of different experimental models. Recently, patient-derived human dermal fibroblast (HDF) cultures have re-emerged as promising in vitro functional system for examining various cellular, molecular, metabolic and (patho)physiological states and traits of psychiatric disorders. HDF studies serve as a powerful complement to postmortem and animal studies, and often appear to be informative about the altered homeostasis in neural tissue. Studies of HDFs from patients with schizophrenia (SZ), depression, bipolar disorder (BD), autism, attention deficit and hyperactivity disorder and other psychiatric disorders have significantly advanced our understanding of these devastating diseases. These reports unequivocally prove that signal transduction, redox homeostasis, circadian rhythms and gene*environment (G*E) interactions are all amenable for assessment by the HDF model. Furthermore, the reported findings suggest that this underutilized patient biomaterial, combined with modern molecular biology techniques, may have both diagnostic and prognostic value, including prediction of response to therapeutic agents.

  1. ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase.

    PubMed Central

    Vaziri, H; West, M D; Allsopp, R C; Davison, T S; Wu, Y S; Arrowsmith, C H; Poirier, G G; Benchimol, S

    1997-01-01

    Telomere loss has been proposed as a mechanism for counting cell divisions during aging in normal somatic cells. How such a mitotic clock initiates the intracellular signalling events that culminate in G1 cell cycle arrest and senescence to restrict the lifespan of normal human cells is not known. We investigated the possibility that critically short telomere length activates a DNA damage response pathway involving p53 and p21(WAF1) in aging cells. We show that the DNA binding and transcriptional activity of p53 protein increases with cell age in the absence of any marked increase in the level of p53 protein, and that p21(WAF1) promoter activity in senescent cells is dependent on both p53 and the transcriptional co-activator p300. Moreover, we detected increased specific activity of p53 protein in AT fibroblasts, which exhibit accelerated telomere loss and undergo premature senescence, compared with normal fibroblasts. We investigated the possibility that poly(ADP-ribose) polymerase is involved in the post-translational activation of p53 protein in aging cells. We show that p53 protein can associate with PARP and inhibition of PARP activity leads to abrogation of p21 and mdm2 expression in response to DNA damage. Moreover, inhibition of PARP activity leads to extension of cellular lifespan. In contrast, hyperoxia, an activator of PARP, is associated with accelerated telomere loss, activation of p53 and premature senescence. We propose that p53 is post-translationally activated not only in response to DNA damage but also in response to the critical shortening of telomeres that occurs during cellular aging. PMID:9312059

  2. Chromosomal analysis in young vs. senescent human fibroblasts by FISH

    SciTech Connect

    Mukheriee, A.B.; Thomas, S.

    1994-09-01

    Almost all previous studies on chromosomal analysis related to in vitro aging of human fibroblasts were done using only metaphase chromosomes. However, this procedure might provide only partial information since the aneuploidy presumably hidden in interphase cells would remain undetected. We, therefore, have analyzed aneuploidy both at interphase and at metaphase. Female (IMR-90) and male (IMR-91) cells were grown from the lowest to the highest population doubling levels and aneuploidy analysis was done by FISH with {alpha}-satellite DNA probes of 15 autosomes and 2 sex chromosomes. Our data on total aneuploidy in young cells indicate that significantly higher proportions of cells with aneuploidy are detected at interphase as opposed to metaphase. This presumably indicates that during active division of young cells, a greater proportion of cells with aneuploidy than diploidy is selected against entry to mitosis. In contrast, both cell strains at senescence exhibit significantly lower proportions of nuclei with aneuploidy at interphase as compared to that of young cells. This probably indicates that during senescense, a greater proportion of cells with aneuploidy than diploidy is selected against prolonged survival in culture. Our study shows that cellular dynamics with respect to aneuploidy involving various chromosomes differs significantly at interphase and at mitosis during in vitro aging of human fibroblasts.

  3. Influence of three laser wavelengths on human fibroblasts cell culture.

    PubMed

    Crisan, Bogdan; Soritau, Olga; Baciut, Mihaela; Campian, Radu; Crisan, Liana; Baciut, Grigore

    2013-02-01

    Although experimental studies in vitro and vivo have been numerous, the effect of laser wavelength irradiation on human fibroblast cell culture is poorly understood. This emphasizes the need of additional cellular and molecular research into laser influence with low energy and power. The aim of this study was to assess the influence of three different laser wavelengths on the human skin fibroblasts cell culture. We wanted to evaluate if near infrared lasers had any influence in healing of wounds by stimulating mitochondrial activity of fibroblasts. The cells were irradiated using 830-, 980- and 2,940-nm laser wavelengths. The irradiated cells were incubated and their mitochondrial activity was assessed by the MTT assay at 24, 48 and 72 h. Simultaneously, an apoptosis assay was assessed on the irradiated fibroblasts. It can be concluded that laser light of the near-infrared region (830 and 980 nm) influences fibroblasts mitochondrial activity compared to the 2,940-nm wavelength which produces apoptosis.

  4. Periodicity of nuclear morphology in human fibroblasts

    PubMed Central

    Seaman, Laura; Meixner, Walter; Snyder, John; Rajapakse, Indika

    2015-01-01

    Motivation: Morphology of the cell nucleus has been used as a key indicator of disease state and prognosis, but typically without quantitative rigor. It is also not well understood how nuclear morphology varies with time across different genetic backgrounds in healthy cells. To help answer these questions we measured the size and shape of nuclei in cell-cycle-synchronized primary human fibroblasts from 6 different individuals at 32 time points over a 75 hour period. Results: The nucleus was modeled as an ellipsoid and its dynamics analyzed. Shape and volume changed significantly over this time. Two prominent frequencies were found in the 6 individuals: a 17 hour period consistent with the cell cycle and a 26 hour period. Our findings suggest that the shape of the nucleus changes over time and thus any time-invariant shape property may provide a misleading characterization of cellular populations at different phases of the cell cycle. The proposed methodology provides a general method to analyze morphological change using multiple time points even for non-live-cell experiments. PMID:26734724

  5. Reprogramming of human fibroblasts toward a cardiac fate

    PubMed Central

    Nam, Young-Jae; Song, Kunhua; Luo, Xiang; Daniel, Edward; Lambeth, Kaleb; West, Katherine; Hill, Joseph A.; DiMaio, J. Michael; Baker, Linda A.; Bassel-Duby, Rhonda; Olson, Eric N.

    2013-01-01

    Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4–11 wk, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of nonmyocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach. PMID:23487791

  6. Super-telomeres in transformed human fibroblasts.

    PubMed

    Chiodi, Ilaria; Belgiovine, Cristina; Zongaro, Samantha; Ricotti, Roberta; Horard, Beatrice; Lossani, Andrea; Focher, Federico; Gilson, Eric; Giulotto, Elena; Mondello, Chiara

    2013-08-01

    Telomere length maintenance is critical for organisms' long-term survival and cancer cell proliferation. Telomeres are kept within species-specific length ranges by the interplay between telomerase activity and telomeric chromatin organization. In this paper, we exploited telomerase immortalized human fibroblasts (cen3tel) that gradually underwent neoplastic transformation during culture propagation to study telomere composition and length regulation during the transformation process. Just after telomerase catalytic subunit (hTERT) expression, cen3tel telomeres shortened despite the presence of telomerase activity. At a later stage and concomitantly with transformation, cells started elongating telomeres, which reached a mean length greater than 100kb in about 900 population doublings. Super-telomeres were stable and compatible with cell growth and tumorigenesis. Telomere extension was associated with increasing levels of telomerase activity that were linked to the deregulation of endogenous telomerase RNA (hTERC) and exogenous telomerase reverse transcriptase (hTERT) expression. Notably, the increase in hTERC levels paralleled the increase in telomerase activity, suggesting that this subunit plays a role in regulating enzyme activity. Telomeres ranging in length between 10 and more than 100kb were maintained in an extendible state although TRF1 and TRF2 binding increased with telomere length. Super-telomeres neither influenced subtelomeric region global methylation nor the expression of the subtelomeric gene FRG1, attesting the lack of a clear-cut relationship between telomere length, subtelomeric DNA methylation and expression in human cells. The cellular levels of the telomeric proteins hTERT, TRF1, TRF2 and Hsp90 rose with transformation and were independent of telomere length, pointing to a role of these proteins in tumorigenesis.

  7. DNA excision repair in permeable human fibroblasts

    SciTech Connect

    Kaufmann, W.K.; Bodell, W.J.; Cleaver, J.E.

    1983-01-01

    U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the (3H)DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells.

  8. Differential effects of planktonic and biofilm MRSA on human fibroblasts.

    PubMed

    Kirker, Kelly R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin-resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell-culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin-resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin-resistant S. aureus, namely limited migration followed by death. Enzyme-linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm-conditioned medium. Planktonic-conditioned medium induced more interleukin-6, interleukin-8, vascular endothelial growth factor, transforming growth factor-β1, heparin-bound epidermal growth factor, matrix metalloproteinase-1, and metalloproteinase-3 production in fibroblasts than the biofilm-conditioned medium. Biofilm-conditioned medium induced more tumor necrosis factor-α production in fibroblasts compared with planktonic-conditioned medium, and suppressed metalloproteinase-3 production compared with controls.

  9. Copper ability to induce premature senescence in human fibroblasts.

    PubMed

    Matos, Liliana; Gouveia, Alexandra; Almeida, Henrique

    2012-08-01

    Human diploid fibroblasts (HDFs) exposed to subcytotoxic concentrations of oxidative or stressful agents, such as hydrogen peroxide, tert-butylhydroperoxide, or ethanol, undergo stress-induced premature senescence (SIPS). This condition is characterized by the appearance of replicative senescence biomarkers such as irreversible growth arrest, increase in senescence-associated β-galactosidase (SA β-gal) activity, altered cell morphology, and overexpression of several senescence-associated genes. Copper is an essential trace element known to accumulate with ageing and to be involved in the pathogenesis of some age-related disorders. Past studies using either yeast or human cellular models of ageing provided evidence in favor of the role of intracellular copper as a longevity modulator. In the present study, copper ability to cause the appearance of senescent features in HDFs was assessed. WI-38 fibroblasts exposed to a subcytotoxic concentration of copper sulfate presented inhibition of cell proliferation, cell enlargement, increased SA β-gal activity, and mRNA overexpression of several senescence-associated genes such as p21, apolipoprotein J (ApoJ), fibronectin, transforming growth factor β-1 (TGF β1), insulin growth factor binding protein 3, and heme oxygenase 1. Western blotting results confirmed enhanced intracellular p21, ApoJ, and TGF β1 in copper-treated cells. Thus, similar to other SIPS-inducing agents, HDF exposure to subcytotoxic concentration of copper results in premature senescence. Further studies will unravel molecular mechanisms and the biological meaning of copper-associated senescence and lead to a better understanding of copper-related disorder establishment and progression.

  10. 7,8-Dihydroxyflavone attenuates TNF-α-induced skin aging in Hs68 human dermal fibroblast cells via down-regulation of the MAPKs/Akt signaling pathways.

    PubMed

    Choi, Ji Won; Lee, Jisun; Park, Yong Il

    2017-09-22

    7,8-Dihydroxyflavone (7,8-DHF, 7,8-dihydroxy-2-phenyl-4H-chromen-4-one) is a natural flavone found in plants and has been frequently reported to show anti-inflammatory and anti-oxidant properties. Skin aging is induced mainly by oxidative stress. In the present study, we evaluated 7,8-DHF for its potential anti-aging effects for skin using Hs68 human dermal fibroblast cells. To establish aged skin cell model, Hs68 cells were treated with tumor necrosis factor-α (TNF-α) for 18h 7,8-DHF (0-10μM) induced collagen synthesis and suppressed the expression of matrix metalloproteinase 1 (MMP 1) in a dose-dependent manner. 7,8-DHF also significantly reduced the generation of intracellular reactive oxygen species (ROS), induced the expression of anti-oxidant enzymes, such as catalase, manganese superoxide dismutase (Mn-SOD), and heme oxygenase-1 (HO-1), and scavenged DPPH free radicals. 7,8-DHF also disturbed the mitogen-activated protein kinases (MAPKs) and Akt signaling pathways that participate in the aging process. 7,8-DHF exerted potent anti-aging effects by inhibiting MMP 1 expression and inducing Type I collagen synthesis in Hs68 cells. 7,8-DHF effectively attenuated oxidative stress by up-regulating the anti-oxidant enzymes catalase, Mn-SOD, and HO-1, and reducing activation of the Akt and MAPKs signaling pathways in aged skin cells. These results suggest that 7,8-DHF can be used as a potent facultative ingredient in health-beneficial agents to prevent or treat the skin aging or inflammatory skin disorders. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. [Blood vessels in human dermis during aging].

    PubMed

    Gunin, A G; Petrov, V V; Vasil'eva, O V; Golubtsova, N N

    2014-01-01

    A factor that potentially influences on skin aging is blood supply which determines global conditions for an organ or a tissue functioning, including skin. Scientific data on conditions of blood supply in the skin during aging are insufficient and contradictory. Therefore, this work was aimed to the study of age-related changes in the number of blood vessels in the human dermis. Blood vessels were visualized with immunohistochemical technique to two endothelial markers, as von Willebrand factor and antigen CD31. The results showed that von Willebrand factor and antigen CD31 are present in endothelial cells of blood vessels of dermis in all examined age periods, from 20 weeks of pregnancy to 85 yeas. Intensity of immunohistochemical staining to von Willebrand factor is enhanced during age. Intensity of staining to CD31 is not changed with age. The number of blood vessels positively stained either to von Willebrand factor or to CD31 in dermis was decreased gradually with age. A total number of fibroblasts in dermis decreased with age. The number of PCNA+ fibroblasts in dermis showing their proliferative activity was decreased with the progression of age. The decrease in the number of blood vessels is statistically associated with that in the general number of fibroblasts and proliferating fibroblasts. Hence, a factor that leads to aged decrease in the number of dermal fibroblasts is diminished blood supply, and actions targeted to enhancement of blood supply are to be in the basis of clinical approaches to prophylaxis and treatment aging changes of the skin.

  12. Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

    PubMed Central

    Sato, Yuki; Mii, Akiko; Hamazaki, Yoko; Fujita, Harumi; Nakata, Hirosuke; Masuda, Kyoko; Nishiyama, Shingo; Shibuya, Shinsuke; Haga, Hironori; Ogawa, Osamu; Shimizu, Akira; Narumiya, Shuh; Kaisho, Tsuneyasu; Arita, Makoto; Yanagisawa, Masashi; Sharma, Kumar; Minato, Nagahiro; Kawamoto, Hiroshi

    2016-01-01

    Acute kidney injury (AKI) is a common clinical condition defined as a rapid decline in kidney function. AKI is a global health burden, estimated to cause 2 million deaths annually worldwide. Unlike AKI in the young, which is reversible, AKI in the elderly often leads to end-stage renal disease, and the mechanism that prevents kidney repair in the elderly is unclear. Here we demonstrate that aged but not young mice developed multiple tertiary lymphoid tissues (TLTs) in the kidney after AKI. TLT size was associated with impaired renal function and increased expression of proinflammatory cytokines and homeostatic chemokines, indicating a possible contribution of TLTs to sustained inflammation after injury. Notably, resident fibroblasts from a single lineage diversified into p75 neurotrophin receptor+ (p75NTR+) fibroblasts and homeostatic chemokine–producing fibroblasts inside TLTs, and retinoic acid–producing fibroblasts around TLTs. Deletion of CD4+ cells as well as late administration of dexamethasone abolished TLTs and improved renal outcomes. Importantly, aged but not young human kidneys also formed TLTs that had cellular and molecular components similar to those of mouse TLTs. Therefore, the inhibition of TLT formation may offer a novel therapeutic strategy for AKI in the elderly. PMID:27699223

  13. Preclinical safety studies on autologous cultured human skin fibroblast transplantation.

    PubMed

    Zeng, Wei; Zhang, Shuying; Liu, Dai; Chai, Mi; Wang, Jiaqi; Zhao, Yuming

    2014-01-01

    Recently, FDA approved the clinical use of autologous fibroblasts (LAVIV™) for the improvement of nasolabial fold wrinkles in adults. The use of autologous fibroblasts for the augmentation of dermal and subcutaneous defects represents a potentially exciting natural alternative to the use of other filler materials for its long-term corrective ability and absence of allergic adverse effects proved by clinical application. However, compared to the clinical evidence, preclinical studies are far from enough. In this study, human skin-derived fibroblasts were cultured and expanded for both in vitro and in vivo observations. In vitro, the subcultured fibroblasts were divided into two groups. One set of cells underwent cell cycle and karyotype analysis at passages 5 and 10. The second group of cells was cocultured in medium with different concentrations of human skin extract D for the measurement of collagen concentration and cell count. In vivo, the subcultured fibroblasts were injected into nude mice subcutaneously. Biopsies were taken for morphology observation and specific collagen staining at 1, 2, and 3 months after injection. The results in vitro showed no significant differences in cell cycle distribution between passages 5 and 10. Cell proliferation and secretion were inhibited as the concentration of extract D increased. In vivo, the fibroblasts were remarkably denser on the experimental side with no dysplastic cells. Mitotic cells were easily observed at the end of the first month but were rare at the end of the third month. Type III collagen was detected at the end of the first month, while collagen type I was positive at the end of the second month. The content of both collagens increased as time passed. The above results indicated that the use of the autologous fibroblasts was safe, providing a basic support for clinical use of fibroblasts.

  14. Fibroblast-mediated contraction in actinically exposed and actinically protected aging skin

    SciTech Connect

    Marks, M.W.; Morykwas, M.J.; Wheatley, M.J. )

    1990-08-01

    The changes in skin morphology over time are a consequence of both chronologic aging and the accumulation of environmental exposure. Through observation, we know that actinic radiation intensifies the apparent aging of skin. We have investigated the effects of aging and actinic radiation on the ability of fibroblasts to contract collagen-fibroblast lattices. Preauricular and postauricular skin samples were obtained from eight patients aged 49 to 74 undergoing rhytidectomy. The samples were kept separate, and the fibroblasts were grown in culture. Lattices constructed with preauricular fibroblasts consistently contracted more than lattices containing postauricular fibroblasts. The difference in amount of contraction in 7 days between sites was greatest for the younger patients and decreased linearly as donor age increased (r = -0.96). This difference may be due to preauricular fibroblasts losing their ability to contract a lattice as aging skin is exposed to more actinic radiation.

  15. Characterization of Multiple Ion Channels in Cultured Human Cardiac Fibroblasts

    PubMed Central

    Li, Gui-Rong; Sun, Hai-Ying; Chen, Jing-Bo; Zhou, Yuan; Tse, Hung-Fat; Lau, Chu-Pak

    2009-01-01

    Background Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts. Methods and Findings A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca2+-activated K+ current (BKCa) in most (88%) human cardiac fibroblasts, a delayed rectifier K+ current (IKDR) and a transient outward K+ current (Ito) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K+ current (IKir) in 24% of cells, and a chloride current (ICl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na+ currents (INa) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (INa.TTX, IC50 = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (INa.TTXR, IC50 = 1.8 µM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BKCa), Kv1.5, Kv1.6 (responsible for IKDR), Kv4.2, Kv4.3 (responsible for Ito), Kir2.1, Kir2.3 (for IKir), Clnc3 (for ICl), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX), and NaV1.5 (for INa.TTXR). Conclusions These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling. PMID:19806193

  16. Characterization of multiple ion channels in cultured human cardiac fibroblasts.

    PubMed

    Li, Gui-Rong; Sun, Hai-Ying; Chen, Jing-Bo; Zhou, Yuan; Tse, Hung-Fat; Lau, Chu-Pak

    2009-10-06

    Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts. A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca(2+)-activated K(+) current (BK(Ca)) in most (88%) human cardiac fibroblasts, a delayed rectifier K(+) current (IK(DR)) and a transient outward K(+) current (I(to)) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K(+) current (I(Kir)) in 24% of cells, and a chloride current (I(Cl)) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na(+) currents (I(Na)) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (I(Na.TTX), IC(50) = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (I(Na.TTXR), IC(50) = 1.8 microM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BK(Ca)), Kv1.5, Kv1.6 (responsible for IK(DR)), Kv4.2, Kv4.3 (responsible for I(to)), Kir2.1, Kir2.3 (for I(Kir)), Clnc3 (for I(Cl)), Na(V)1.2, Na(V)1.3, Na(V)1.6, Na(V)1.7 (for I(Na.TTX)), and Na(V)1.5 (for I(Na.TTXR)). These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling.

  17. SV40-mediated immortalization of human fibroblasts.

    PubMed

    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  18. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    PubMed

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes.

  19. Cytotoxicity of halothane on human gingival fibroblast cultures in vitro.

    PubMed

    Chang, Y C; Chou, M Y

    2001-02-01

    Recently halothane has been reported to be the most suitable alternative to chloroform in dissolving gutta-percha. Periapical tissue toxicity of halothane is not completely known. In this study gutta-percha dissolved by halothane was evaluated with the almar blue dye assay using human gingival fibroblast cultures. The cytotoxic effects of halothane on human gingival fibroblasts depended on the exposure dose, frequency, and duration. A reduced concentration and smaller amount of gutta-percha solvents may minimize the cytotoxic effects on host tissues.

  20. Excessive Cellular Proliferation Negatively Impacts Reprogramming Efficiency of Human Fibroblasts

    PubMed Central

    Gupta, Manoj K.; Teo, Adrian Kee Keong; Rao, Tata Nageswara; Bhatt, Shweta; Kleinridders, Andre; Shirakawa, Jun; Takatani, Tomozumi; Hu, Jiang; De Jesus, Dario F.; Windmueller, Rebecca; Wagers, Amy J.

    2015-01-01

    The impact of somatic cell proliferation rate on induction of pluripotent stem cells remains controversial. Herein, we report that rapid proliferation of human somatic fibroblasts is detrimental to reprogramming efficiency when reprogrammed using a lentiviral vector expressing OCT4, SOX2, KLF4, and cMYC in insulin-rich defined medium. Human fibroblasts grown in this medium showed higher proliferation, enhanced expression of insulin signaling and cell cycle genes, and a switch from glycolytic to oxidative phosphorylation metabolism, but they displayed poor reprogramming efficiency compared with cells grown in normal medium. Thus, in contrast to previous studies, our work reveals an inverse correlation between the proliferation rate of somatic cells and reprogramming efficiency, and also suggests that upregulation of proteins in the growth factor signaling pathway limits the ability to induce pluripotency in human somatic fibroblasts. Significance The efficiency with which human cells can be reprogrammed is of interest to stem cell biology. In this study, human fibroblasts cultured in media containing different concentrations of growth factors such as insulin and insulin-like growth factor-1 exhibited variable abilities to proliferate, with consequences on pluripotency. This occurred in part because of changes in the expression of proteins involved in the growth factor signaling pathway, glycolysis, and oxidative phosphorylation. These findings have implications for efficient reprogramming of human cells. PMID:26253715

  1. Human Cytomegalovirus Induces JC Virus DNA Replication in Human Fibroblasts

    NASA Astrophysics Data System (ADS)

    Heilbronn, Regine; Albrecht, Ingrid; Stephan, Sonja; Burkle, Alexander; Zur Hausen, Harald

    1993-12-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML.

  2. [The comparison of biologic character between mouse embryonic fibroblast and human embryonic fibroblast].

    PubMed

    Zhang, Yi; Zhao, Liansan; Wang, Chengxiao; Lei, Binjun

    2003-06-01

    To evaluate the feasibility of using human embryonic fibroblast(HEF) as feeder layer in the culture of human embryonic stem(ES) cells in vitro, we investigated the morphology, the sensitivity to 0.25% trypsin, the growth curve and cell cycle of HEF with DMEM(low glucose) +10% FBS used as culture medium, and then we compared HEF with mouse embryonic fibroblast (MEF). The results showed that both HEF and MEF are adherent cells in vitro, and HEF has longer life span and better growth ability than MEF. In room temperature, HEF is more sensitive to 0.25% trypsin. Our research suggested that HEF can be used as feeder layer in culture of ES cells. HEF has longer service life than MEF and is worthy to be studied further.

  3. CD44 and hyaluronan expression in human cutaneous scar fibroblasts.

    PubMed Central

    Messadi, D. V.; Bertolami, C. N.

    1993-01-01

    Fibrotic disorders of skin and other organs are typically associated with an abnormal accumulation of extracellular matrix. This study focuses on a matrix constituent, hyaluronan-which is known to be altered in fibrotic disorders of skin- and on CD44, a cell adhesion molecule and putative receptor for hyaluronan. Tissue samples were obtained from biopsies of human normal skin, normal cutaneous scar; and hypertrophic cutaneous scar. After culturing, cells were studied by single- and double-labeling immunohistochemistry using the two anti-CD44 monoclonal antibodies, BU-52 and J173, and a biotinylated hyaluronan binding complex probe, b-HABR. Certain cultures were pretreated with Streptomyces hyaluronidase to assess the dependency of CD44 expression on the presence of endogenous hyaluronan. CD44 expression, both in the presence and the absence of exogenous hyaluronan, was quantitated by radioimmunobinding assay. Overall glycosaminoglycan synthesis and identification of hyaluronan were accomplished by precursor incorporation assays and by quantitative cellulose acetate electrophoresis. CD44 was found to be a normal human adult fibroblastic antigen whose expression is markedly increased for hypertrophic scar fibroblasts compared with normal skin fibroblasts. Although hyaluronan was found to be the predominant glycosaminoglycan constituent of the pericellular matrix for these fibroblasts, CD44 attachment to the cell surface is neither mediated by hyaluronan nor is the presence of hyaluronan a prerequisite for CD44 expression. Exogenous hyaluronan induced a decline in measurable CD44 expression for normal skin fibroblasts but not for hypertrophic scar fibroblasts. These observations are compatible with current understanding of the way cells manage the hyaluronan economy of the extracellular matrix and emphasize phenotypic heterogeneities between fibroblasts derived from normal versus scar tissues. Images Figure 1 Figure 4 PMID:8475990

  4. Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser.

    PubMed

    Illescas-Montes, Rebeca; Melguizo-Rodríguez, Lucía; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción; Ramos-Torrecillas, Javier

    2017-07-13

    Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2-1 W and energy density: 1-7 J/cm²) using different transmission modes (continuous or pulsed). The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm²; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing.

  5. Cultured Human Fibroblast Biostimulation Using a 940 nm Diode Laser

    PubMed Central

    Illescas-Montes, Rebeca; Melguizo-Rodríguez, Lucía; Manzano-Moreno, Francisco Javier; García-Martínez, Olga; Ruiz, Concepción

    2017-01-01

    Background: Fibroblasts are the main cells involved in regeneration during wound healing. The objective was to determine the effect of 940 nm diode laser on cultured human fibroblasts using different irradiation regimens. Methods: The CCD-1064Sk human epithelial fibroblast cell line was treated with a 940 nm diode laser at different energy doses (power: 0.2–1 W and energy density: 1–7 J/cm2) using different transmission modes (continuous or pulsed). The effect on cell growth at 24 and 72 h post-treatment was examined by measuring the proliferative capacity, the impact on the cell cycle, and the effect on cell differentiation. Results: fibroblast proliferative capacity was increased at 24 and 72 h post-treatment as a function of the energy dose. The greatest increase was observed with a power of 0.2 or 0.5 W and energy density between 1 and 4 J/cm2; no difference was observed between continuous and pulsed modes. There were no significant differences in cell cycle between treated groups and controls. α-actin expression was increased by treatment, indicating enhanced cell differentiation. Conclusion: The 940 nm diode laser has biostimulating effects on fibroblasts, stimulating proliferative capacity and cell differentiation without altering the cell cycle. Further researches are necessary to explore its potential clinical usefulness in wound healing. PMID:28773152

  6. Standardized Kaempferia parviflora Extract Inhibits Intrinsic Aging Process in Human Dermal Fibroblasts and Hairless Mice by Inhibiting Cellular Senescence and Mitochondrial Dysfunction

    PubMed Central

    2017-01-01

    Intrinsic skin aging is a complex biological phenomenon mainly caused by cellular senescence and mitochondrial dysfunction. This study evaluated the inhibitory effect of Kaempferia parviflora Wall ex. Baker ethanol extract (KPE) on H2O2-stimulated cellular senescence and mitochondrial dysfunction both in vitro and in vivo. KPE significantly increased cell growth and suppressed senescence-associated β-galactosidase activation. KPE inhibited the expression of cell-cycle inhibitors (p53, p21, p16, and pRb) and stimulated the expression of cell-cycle activators (E2F1 and E2F2). H2O2-induced hyperactivation of the phosphatidylinositol 3-kinase/protein kinase B (AKT) signaling pathway was suppressed by KPE through regulated expression of forkhead box O3a (FoxO3a) and mammalian target of rapamycin (mTOR). KPE attenuated inflammatory mediators (interleukin-6 (IL-6), IL-8, nuclear factor kappa B (NF-κB), and cyclooxygenase-2 (COX-2)) and increased the mRNA expression of PGC-1α, ERRα, NRF1, and Tfam, which modulate mitochondrial biogenesis and function. Consequently, reduced ATP levels and increased ROS level were also reversed by KPE treatment. In hairless mice, KPE inhibited wrinkle formation, skin atrophy, and loss of elasticity by increasing the collagen and elastic fibers. The results indicate that KPE prevents intrinsic aging process in hairless mice by inhibiting cellular senescence and mitochondrial dysfunction, suggesting its potential as a natural antiaging agent. PMID:28831286

  7. Standardized Kaempferia parviflora Extract Inhibits Intrinsic Aging Process in Human Dermal Fibroblasts and Hairless Mice by Inhibiting Cellular Senescence and Mitochondrial Dysfunction.

    PubMed

    Park, Ji-Eun; Woo, Seon Wook; Kim, Mi-Bo; Kim, Changhee; Hwang, Jae-Kwan

    2017-01-01

    Intrinsic skin aging is a complex biological phenomenon mainly caused by cellular senescence and mitochondrial dysfunction. This study evaluated the inhibitory effect of Kaempferia parviflora Wall ex. Baker ethanol extract (KPE) on H2O2-stimulated cellular senescence and mitochondrial dysfunction both in vitro and in vivo. KPE significantly increased cell growth and suppressed senescence-associated β-galactosidase activation. KPE inhibited the expression of cell-cycle inhibitors (p53, p21, p16, and pRb) and stimulated the expression of cell-cycle activators (E2F1 and E2F2). H2O2-induced hyperactivation of the phosphatidylinositol 3-kinase/protein kinase B (AKT) signaling pathway was suppressed by KPE through regulated expression of forkhead box O3a (FoxO3a) and mammalian target of rapamycin (mTOR). KPE attenuated inflammatory mediators (interleukin-6 (IL-6), IL-8, nuclear factor kappa B (NF-κB), and cyclooxygenase-2 (COX-2)) and increased the mRNA expression of PGC-1α, ERRα, NRF1, and Tfam, which modulate mitochondrial biogenesis and function. Consequently, reduced ATP levels and increased ROS level were also reversed by KPE treatment. In hairless mice, KPE inhibited wrinkle formation, skin atrophy, and loss of elasticity by increasing the collagen and elastic fibers. The results indicate that KPE prevents intrinsic aging process in hairless mice by inhibiting cellular senescence and mitochondrial dysfunction, suggesting its potential as a natural antiaging agent.

  8. Iron Accumulation During Cellular Senescence in Human Fibroblasts In Vitro

    PubMed Central

    KILLILEA, DAVID W.; ATAMNA, HANI; LIAO, CHARLES; AMES, BRUCE N.

    2015-01-01

    Iron accumulates as a function of age in several tissues in vivo and is associated with the pathology of numerous age-related diseases. The molecular basis of this change may be due to a loss of iron homeostasis at the cellular level. Therefore, changes in iron content in primary human fibroblast cells (IMR-90) were studied in vitro as a model of cellular senescence. Total iron content increased exponentially during cellular senescence, resulting in 10-fold higher levels of iron compared with young cells. Low-dose hydrogen peroxide (H2O2) induced early senescence in IMR-90s and concomitantly accelerated iron accumulation. Furthermore, senescence-related and H2O2-stimulated iron accumulation was attenuated by N-tert-butylhydroxylamine (NtBHA), a mitochondrial antioxidant that delays senescence in vitro. However, SV40-transformed, immortalized IMR-90s showed no time-dependent changes in metal content in culture or when treated with H2O2 and/or NtBHA. These data indicate that iron accumulation occurs during normal cellular senescence in vitro. This accumulation of iron may contribute to the increased oxidative stress and cellular dysfunction seen in senescent cells. PMID:14580305

  9. Highly efficient direct conversion of human fibroblasts to neuronal cells by chemical compounds.

    PubMed

    Dai, Ping; Harada, Yoshinori; Takamatsu, Tetsuro

    2015-05-01

    Direct conversion of mammalian fibroblasts into induced neuronal (iN) cells has been attained by forced expression of pro-neural transcriptional factors, or by combining defined factors with either microRNAs or small molecules. Here, we show that neuronal cells can be converted from postnatal human fibroblasts into cell populations with neuronal purities of up to >80% using a combination of six chemical compounds. The chemical compound-induced neuronal cells (CiNCs) express neuron-specific proteins and functional neuron markers. The efficiency of CiNCs is unaffected by either the donor's age or cellular senescence (passage number). We propose this chemical direct converting strategy as a potential approach for highly efficient generation of neuronal cells from human fibroblasts for such uses as in neural disease modeling and regenerative medicine.

  10. Short exposure to collagenase and coculture with mouse embryonic pancreas improve human dermal fibroblast culture.

    PubMed

    Pandamooz, Sareh; Hadipour, Abbas; Akhavan-Niaki, Haleh; Pourghasem, Mohsen; Abedian, Zeinab; Ardekani, Ali Motevallizadeh; Golpour, Monireh; Hassan, Zuhair Mohammad; Mostafazadeh, Amrollah

    2012-01-01

    The dermal fibroblast as a major component of connective tissue has attracted much attention in the past few years, and application of these very fast growing cells in several fields has been intensively studied. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. Although using a dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical because a large number of cells can be lost over prolonged exposure to collagenase. This study was performed to increase the number of viable cells after digestion of fresh human foreskin of donors aged from 1 to 3 months with collagenase and also by to design a coculture system for resuscitation of the injured fibroblast. Our results demonstrate that we can maximize cell yield while maintaining cell viability by cutting the specimens into very small pieces (1-2 mm³) after removing the epidermal layer with dispase II and also by collecting released cells every 20 Min subsequent to digesting the dermal layer with collagenase. Moreover, our data strongly indicate that coculturing of isolated fibroblasts with embryonic pancreas explants can enhance the rate of proliferation in cultured fibroblasts.

  11. TGFβ functionalized starPEG-heparin hydrogels modulate human dermal fibroblast growth and differentiation.

    PubMed

    Watarai, Akira; Schirmer, Lucas; Thönes, Stephan; Freudenberg, Uwe; Werner, Carsten; Simon, Jan C; Anderegg, Ulf

    2015-10-01

    Hydrogels are promising biomaterials that can adapt easily to complex tissue entities. Furthermore, chemical modifications enable these hydrogels to become an instructive biomaterial to a variety of cell types. Human dermal fibroblasts play a pivotal role during wound healing, especially for the synthesis of novel dermal tissue replacing the primary fibrin clot. Thus, the control of growth and differentiation of dermal fibroblasts is important to modulate wound healing. In here, we utilized a versatile starPEG-heparin hydrogel platform that can be independently adjusted with respect to mechanical and biochemical properties for cultivating human dermal fibroblasts. Cell-based remodeling of the artificial matrix was ensured by using matrix metalloprotease (MMP) cleavable crosslinker peptides. Attachment and proliferation of fibroblasts on starPEG-heparin hydrogels of differing stiffness, density of pro-adhesive RGD peptides and MMP cleavable peptide linkers were tested. Binding and release of human TGFβ1 as well as biological effect of the pre-adsorbed growth factor on fibroblast gene expression and myofibroblast differentiation were investigated. Hydrogels containing RGD peptides supported fibroblast attachment, spreading, proliferation matrix deposition and remodeling compared to hydrogels without any modifications. Reversibly conjugated TGFβ1 was demonstrated to be constantly released from starPEG-heparin hydrogels for several days and capable of inducing myofibroblast differentiation of fibroblasts as determined by induction of collagen type I, ED-A-Fibronectin expression and incorporation of alpha smooth muscle actin and palladin into F-actin stress fibers. Taken together, customized starPEG-heparin hydrogels could be of value to promote dermal wound healing by stimulating growth and differentiation of human dermal fibroblasts. The increasing number of people of advanced age within the population results in an increasing demand for the treatment of non

  12. Engineering human neo-tendon tissue in vitro with human dermal fibroblasts under static mechanical strain.

    PubMed

    Deng, Dan; Liu, Wei; Xu, Feng; Yang, Yang; Zhou, Guangdong; Zhang, Wen Jie; Cui, Lei; Cao, Yilin

    2009-12-01

    Proper cell source is one of the key issues for tendon engineering. Our previous study showed that dermal fibroblasts could be used to successfully engineer tendon in vivo and tenocytes could engineer neo-tendon in vitro with static strain. This study further investigated the possibility of engineering human neo-tendon tissue in vitro using dermal fibroblasts. Human dermal fibroblasts were seeded on polyglycolic acid (PGA) fibers pre-fixed on a U-shape as a mechanical loading group, or simply cultured in a dish as a tension-free group. In addition, human tenocytes were also seeded on PGA fibers with tension as a comparison to human dermal fibroblasts. The results showed that human neo-tendon tissue could be generated using dermal fibroblasts during in vitro culture under static strain and the tissue structure became more mature with the increase of culture time. Longitudinally aligned collagen fibers and spindle shape cells were observed histologically and collagen fibril diameter and tensile strength increased with time and reached a peak at 14 weeks. In contrast, the dermal fibroblast-PGA constructs failed to form neo-tendon, but formed disorganized fibrous tissue in tension-free condition with significantly weaker strength and poor collagen fiber formation. Interestingly, neo-tendon tissues generated with human dermal fibroblasts were indistinguishable from the counterpart engineered with human tenocytes, which supports the viewpoint that human dermal fibroblasts is likely to replace tenocytes for future tendon graft development in vitro with dynamic mechanical loading in a bioreactor system.

  13. Protective effect of oat bran extracts on human dermal fibroblast injury induced by hydrogen peroxide.

    PubMed

    Feng, Bing; Ma, Lai-ji; Yao, Jin-jing; Fang, Yun; Mei, Yan-ai; Wei, Shao-min

    2013-02-01

    Oat contains different components that possess antioxidant properties; no study to date has addressed the antioxidant effect of the extract of oat bran on the cellular level. Therefore, the present study focuses on the investigation of the protective effect of oat bran extract by enzymatic hydrolysates on human dermal fibroblast injury induced by hydrogen peroxide (H(2)O(2)). Kjeldahl determination, phenol-sulfuric acid method, and high-performance liquid chromatography (HPLC) analysis indicated that the enzymatic products of oat bran contain a protein amount of 71.93%, of which 97.43% are peptides with a molecular range from 438.56 to 1301.01 Da. Assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity indicate that oat peptide-rich extract has a direct and concentration-dependent antioxidant activity. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay and the TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) assay for apoptosis showed that administration of H(2)O(2) in human dermal fibroblasts caused cell damage and apoptosis. Pre-incubation of human dermal fibroblasts with the Oatp for 24 h markedly inhibited human dermal fibroblast injury induced by H(2)O(2), but application oat peptides with H(2)O(2) at same time did not. Pre-treatment of human dermal fibroblasts with Oatp significantly reversed the H(2)O(2)-induced decrease of superoxide dismutase (SOD) and the inhibition of malondialdehyde (MDA). The results demonstrate that oat peptides possess antioxidant activity and are effective against H(2)O(2)-induced human dermal fibroblast injury by the enhanced activity of SOD and decrease in MDA level. Our results suggest that oat bran will have the potential to be further explored as an antioxidant functional food in the prevention of aging-related skin injury.

  14. Cytotoxicity and arecoline mechanisms in human gingival fibroblasts in vitro.

    PubMed

    Chang, Y C; Hu, C C; Lii, C K; Tai, K W; Yang, S H; Chou, M Y

    2001-03-01

    Betel nut chewing, like cigarette smoking, is a popular oral habit which impinges on the daily lives of a population of approximately 200 million. People who chew betel nuts have a higher prevalence of periodontal diseases than those who do not. Many of the undesirable effects of betel nuts have been attributed to arecoline, a major component of the particular alkaloid in betel nuts. In this in vitro study, we have focused on the effects of arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts. Human gingival fibroblasts were derived from three healthy individuals undergoing crown-lengthening procedures. We found that arecoline is cytotoxic to human gingival fibroblasts at a concentration higher than 50 micrograms/ml by depleting intracellular thiols and inhibiting mitochondrial activity (P < 0.05). In addition, the cells displayed a marked arrest at G2/M phase in a dose-dependent manner. Repeated and long-term exposure to arecoline could impair the gingival fibroblast functions. As they are cytotoxic, the use of betel nut products in conjunction with periodontal therapy may interfere with optimal healing and/or lead to further periodontal breakdown.

  15. A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging.

    PubMed

    Schauer, Marianne; Kottek, Tina; Schönherr, Madeleine; Bhattacharya, Animesh; Ibrahim, Saleh M; Hirose, Misa; Köhling, Rüdiger; Fuellen, Georg; Schmitz, Ulf; Kunz, Manfred

    2015-04-20

    Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways.

  16. A mutation in the NADH-dehydrogenase subunit 2 suppresses fibroblast aging

    PubMed Central

    Schauer, Marianne; Kottek, Tina; Schönherr, Madeleine; Bhattacharya, Animesh; Ibrahim, Saleh M; Hirose, Misa; Köhling, Rüdiger; Fuellen, Georg; Schmitz, Ulf; Kunz, Manfred

    2015-01-01

    Mutations of mitochondrial (mt)DNA cause a variety of human diseases and are implicated in premature aging syndromes. Here we investigated a single nucleotide exchange (leucine to methionine) at position nt4738 in the mitochondrial NADH dehydrogenase subunit 2 (Nd2) gene of the respiratory chain. Primary fibroblasts derived from the conplastic mouse strain C57BL/6J-mtALR/LTJ with mutant enzyme, possessed high enzyme activity and ATP production and low ROS production. Furthermore, Nd2-mutant fibroblasts expressed lower senescence markers. Transcriptome analysis revealed that the members of the p38MAPK pathway were significantly downregulated in Nd2-mutant mice. In agreement, inhibition of p38MAPK with SB203580 enhanced proliferation and reduced cytokine secretion in fibroblasts. In Nd2-mutant mouse skin, the amount of Ki67-positive cells was significantly higher than in control skin. The higher amount of Ki67-positive cells and the thicker epidermis in Nd2-mutant mice strongly supported the in vitro data. In conclusion, Nd2 is a mitochondrial gene, involved in age-related signaling pathways. PMID:25839158

  17. Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation

    PubMed Central

    Esnault, Stephane; Torr, Elizabeth E.; Bernau, Ksenija; Johansson, Mats W.; Kelly, Elizabeth A.; Sandbo, Nathan; Jarjour, Nizar N.

    2017-01-01

    Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF). Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore

  18. Generation of functional human serotonergic neurons from fibroblasts.

    PubMed

    Vadodaria, K C; Mertens, J; Paquola, A; Bardy, C; Li, X; Jappelli, R; Fung, L; Marchetto, M C; Hamm, M; Gorris, M; Koch, P; Gage, F H

    2016-01-01

    The brain's serotonergic system centrally regulates several physiological processes and its dysfunction has been implicated in the pathophysiology of several neuropsychiatric disorders. While in the past our understanding of serotonergic neurotransmission has come mainly from mouse models, the development of pluripotent stem cell and induced fibroblast-to-neuron (iN) transdifferentiation technologies has revolutionized our ability to generate human neurons in vitro. Utilizing these techniques and a novel lentiviral reporter for serotonergic neurons, we identified and overexpressed key transcription factors to successfully generate human serotonergic neurons. We found that overexpressing the transcription factors NKX2.2, FEV, GATA2 and LMX1B in combination with ASCL1 and NGN2 directly and efficiently generated serotonergic neurons from human fibroblasts. Induced serotonergic neurons (iSNs) showed increased expression of specific serotonergic genes that are known to be expressed in raphe nuclei. iSNs displayed spontaneous action potentials, released serotonin in vitro and functionally responded to selective serotonin reuptake inhibitors (SSRIs). Here, we demonstrate the efficient generation of functional human serotonergic neurons from human fibroblasts as a novel tool for studying human serotonergic neurotransmission in health and disease.

  19. Novel anti-adipogenic activity produced by human fibroblasts

    PubMed Central

    Lehmann, Geniece M.; Woeller, Collynn F.; Pollock, Stephen J.; O'Loughlin, Charles W.; Gupta, Shikha; Feldon, Steven E.

    2010-01-01

    Fatty tissue is generally found in distinct “depots” distributed throughout the human body. Adipocytes from each of the various depots differ in their metabolic capacities and their responses to environmental stimuli. Although a general understanding of the factors responsible for adipogenic transformation has been achieved, much is not understood about the mechanisms of adipose tissue deposition and the phenotypes of the adipocytes found within each depot. A clue to the factors regulating fat deposition may come from studies of adipogenesis using primary human orbital fibroblasts from patients with thyroid eye disease, a condition in which intense inflammation leads to expansion of orbital adipose tissue via differentiation of fibroblasts to adipocytes. We have previously demonstrated that adipogenesis of orbital fibroblasts is negatively correlated with cellular expression of the Thy-1 surface marker. In this study, we developed a novel imaging flow cytometric approach for the assessment of adipogenesis to test the hypothetical dependence of adipogenic potential on lack of Thy-1 expression. Using this technique, we learned that Thy-1-positive fibroblasts are, in fact, capable of differentiating into adipocytes but are less likely to do so because they secrete a paracrine anti-adipogenic factor. It is possible that such a factor plays an important role in the prevention of excess fat deposition in the normal orbit and may even be exploited as a therapy for the treatment of obesity, a major worldwide health concern. PMID:20554910

  20. Calcium pantothenate modulates gene expression in proliferating human dermal fibroblasts.

    PubMed

    Wiederholt, Tonio; Heise, Ruth; Skazik, Claudia; Marquardt, Yvonne; Joussen, Sylvia; Erdmann, Kati; Schröder, Henning; Merk, Hans F; Baron, Jens Malte

    2009-11-01

    Topical application of pantothenate is widely used in clinical practice for wound healing. Previous studies identified a positive effect of pantothenate on migration and proliferation of cultured fibroblasts. However, these studies were mainly descriptive with no molecular data supporting a possible model of its action. In this study, we first established conditions for an in vitro model of pantothenate wound healing and then analysed the molecular effects of pantothenate. To test the functional effect of pantothenate on dermal fibroblasts, cells were cultured and in vitro proliferation tests were performed using a standardized scratch test procedure. For all three donors analysed, a strong stimulatory effect of pantothenate at a concentration of 20 microg/ml on the proliferation of cultivated dermal fibroblasts was observed. To study the molecular mechanisms resulting in the proliferative effect of pantothenate, gene expression was analysed in dermal fibroblasts cultivated with 20 microg/ml of pantothenate compared with untreated cells using the GeneChip Human Exon 1.0 ST Array. A number of significantly regulated genes were identified including genes coding for interleukin (IL)-6, IL-8, Id1, HMOX-1, HspB7, CYP1B1 and MARCH-II. Regulation of these genes was subsequently verified by quantitative real-time polymerase chain reaction analysis. Induction of HMOX-1 expression by pantothenol and pantothenic acid in dermal cells was confirmed on the protein level using immunoblots. Functional studies revealed the enhanced suppression of free radical formation in skin fibroblasts cultured with panthenol. In conclusion, these studies provided new insight in the molecular mechanisms linked to the stimulatory effect of pantothenate and panthenol on the proliferation of dermal fibroblasts.

  1. Direct conversion of human fibroblasts to dopaminergic neurons

    PubMed Central

    Pfisterer, Ulrich; Kirkeby, Agnete; Torper, Olof; Wood, James; Nelander, Jenny; Dufour, Audrey; Björklund, Anders; Lindvall, Olle; Jakobsson, Johan; Parmar, Malin

    2011-01-01

    Recent reports demonstrate that somatic mouse cells can be directly converted to other mature cell types by using combined expression of defined factors. Here we show that the same strategy can be applied to human embryonic and postnatal fibroblasts. By overexpression of the transcription factors Ascl1, Brn2, and Myt1l, human fibroblasts were efficiently converted to functional neurons. We also demonstrate that the converted neurons can be directed toward distinct functional neurotransmitter phenotypes when the appropriate transcriptional cues are provided together with the three conversion factors. By combining expression of the three conversion factors with expression of two genes involved in dopamine neuron generation, Lmx1a and FoxA2, we could direct the phenotype of the converted cells toward dopaminergic neurons. Such subtype-specific induced neurons derived from human somatic cells could be valuable for disease modeling and cell replacement therapy. PMID:21646515

  2. Direct reprogramming of human fibroblasts to functional and expandable hepatocytes.

    PubMed

    Huang, Pengyu; Zhang, Ludi; Gao, Yimeng; He, Zhiying; Yao, Dan; Wu, Zhitao; Cen, Jin; Chen, Xiaotao; Liu, Changcheng; Hu, Yiping; Lai, Dongmei; Hu, Zhenlei; Chen, Li; Zhang, Ying; Cheng, Xin; Ma, Xiaojun; Pan, Guoyu; Wang, Xin; Hui, Lijian

    2014-03-06

    The generation of large numbers of functional human hepatocytes for cell-based approaches to liver disease is an important and unmet goal. Direct reprogramming of fibroblasts to hepatic lineages could offer a solution to this problem but so far has only been achieved with mouse cells. Here, we generated human induced hepatocytes (hiHeps) from fibroblasts by lentiviral expression of FOXA3, HNF1A, and HNF4A. hiHeps express hepatic gene programs, can be expanded in vitro, and display functions characteristic of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Upon transplantation into mice with concanavalin-A-induced acute liver failure and fatal metabolic liver disease due to fumarylacetoacetate dehydrolase (Fah) deficiency, hiHeps restore the liver function and prolong survival. Collectively, our results demonstrate successful lineage conversion of nonhepatic human cells into mature hepatocytes with potential for biomedical and pharmaceutical applications.

  3. Signaling pathway activation drift during aging: Hutchinson-Gilford Progeria Syndrome fibroblasts are comparable to normal middle-age and old-age cells.

    PubMed

    Aliper, Alexander M; Csoka, Antonei Benjamin; Buzdin, Anton; Jetka, Tomasz; Roumiantsev, Sergey; Moskalev, Alexy; Zhavoronkov, Alex

    2015-01-01

    For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our analysis reveals that signaling pathway activation states in cells derived from chronologically young patients with HGPS strongly resemble cells taken from normal middle-aged and old individuals. This clearly indicates that HGPS may truly represent accelerated aging, rather than being just a simulacrum. Our data also points to potential pathways that could be targeted to develop drugs and drug combinations for both HGPS and normal aging.

  4. Signaling pathway activation drift during aging: Hutchinson-Gilford Progeria Syndrome fibroblasts are comparable to normal middle-age and old-age cells

    PubMed Central

    Aliper, Alexander M.; Csoka, Antonei Benjamin; Buzdin, Anton; Jetka, Tomasz; Roumiantsev, Sergey; Moskalev, Alexey; Zhavoronkov, Alex

    2015-01-01

    For the past several decades, research in understanding the molecular basis of human aging has progressed significantly with the analysis of premature aging syndromes. Progerin, an altered form of lamin A, has been identified as the cause of premature aging in Hutchinson-Gilford Progeria Syndrome (HGPS), and may be a contributing causative factor in normal aging. However, the question of whether HGPS actually recapitulates the normal aging process at the cellular and organismal level, or simply mimics the aging phenotype is widely debated. In the present study we analyzed publicly available microarray datasets for fibroblasts undergoing cellular aging in culture, as well as fibroblasts derived from young, middle-age, and old-age individuals, and patients with HGPS. Using GeroScope pathway analysis and drug discovery platform we analyzed the activation states of 65 major cellular signaling pathways. Our analysis reveals that signaling pathway activation states in cells derived from chronologically young patients with HGPS strongly resemble cells taken from normal middle-aged and old individuals. This clearly indicates that HGPS may truly represent accelerated aging, rather than being just a simulacrum. Our data also points to potential pathways that could be targeted to develop drugs and drug combinations for both HGPS and normal aging. PMID:25587796

  5. Transcription abnormalities potentiate apoptosis of normal human fibroblasts.

    PubMed Central

    Andera, L.; Wasylyk, B.

    1997-01-01

    BACKGROUND: Apoptosis is a natural process by which damaged and potentially tumorigenic cells are removed. Induction of apoptosis is important in chemotherapy aimed at eliminating cancer cells. We address the mechanisms by which this process can be triggered in cells that are recalcitrant to cell death induced by DNA-damaging agents. MATERIALS AND METHODS: Normal human fibroblasts and lymphoblasts, and fibroblasts with defined genetic changes, were treated with DNA-damaging agents and inhibitors of transcription. Western blotting was used to study the expression of some of the key factors involved in the response to DNA damage and the induction of apoptosis, namely, p53, p21WAFI,Cip1, Mdm2, Bax, and CD95 (Fas/APO1). Apoptosis was followed by various criteria, including DNA fragmentation, specific proteolysis, cell morphology, viability, and FACS scan for sub-G1 cells. RESULTS: Normal human fibroblasts were more resistant than lymphoblasts to DNA damage-induced apoptosis. The DNA-damaging agents mitomycin C and cisplatin induced rapid apoptosis of fibroblasts with defects in the repair of transcribed DNA, compared with wild-type cells or those with defects in overall genome repair. Short-term treatment with inhibitors of RNA polymerase II transcription, actinomycin D, and alpha-amanitin induced rapid cell death of normal fibroblasts. These results show that there is a link between defective transcription and apoptosis. Treatments and genetic backgrounds that favored apoptosis were associated with efficient and prolonged induction of p53 and often altered or imbalanced expression of its downstream effectors p21WAFI,Cip1 and Mdm2, whereas there were no changes in Bax or CD95 (Fas/APO1). CONCLUSION: Transcription inhibitors increase p53 levels and are better inducers of apoptosis than DNA-damaging agents in some cell types. Apoptosis might be triggered by blocked polymerases and/or faulty expression of downstream effectors. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID

  6. Degradation of type IV collagen by neoplastic human skin fibroblasts

    SciTech Connect

    Sheela, S.; Barrett, J.C.

    1985-02-01

    An assay for the degradation of type IV (basement membrane) collagen was developed as a biochemical marker for neoplastic cells from chemically transformed human skin fibroblasts. Type IV collagen was isolated from basement membrane of Syrian hamster lung and type I collagen was isolated from rat tails; the collagens were radioactively labelled by reductive alkylation. The abilities of normal (KD) and chemically transformed (Hut-11A) human skin fibroblasts to degrade the collagens were studied. A cell-associated assay was performed by growing either normal or transformed cells in the presence of radioactively labelled type IV collagen and measuring the released soluble peptides in the medium. This assay also demonstrated that KD cells failed to synthesize an activity capable of degrading type IV collagen whereas Hut-11A cells degraded type IV collagen in a linear manner for up to 4 h. Human serum at very low concentrations, EDTA and L-cysteine inhibited the enzyme activity, whereas protease inhibitors like phenylmethyl sulfonyl fluoride, N-ethyl maleimide or soybean trypsin inhibitor did not inhibit the enzyme from Hut-11A cells. These results suggest that the ability to degrade specifically type IV collagen may be an important marker for neoplastic human fibroblasts and supports a role for this collagenase in tumor cell invasion.

  7. Effect of a volatile smoke component (acrolein) on human gingival fibroblasts: An in vitro study

    PubMed Central

    Anand, Nithya; Emmadi, Pamela; Ambalavanan, N.; Ramakrishnan, T.

    2011-01-01

    Aim: Tobacco and some of its volatile and non-volatile components have been found to affect many types of cells including human gingival fibroblasts. The aim of this present study was to estimate the effect of acrolein, a volatile fraction of cigarette smoke on the attachment, proliferation and ultra structure of human gingival fibroblasts in culture. Materials and Methods: Human gingival fibroblasts strains obtained from healthy subjects aged 20-30 years, were grown to confluency and utilized between 3rd -6th passages. The cell cultures seeded in 96 well microtitration plates at a density of 45,000 cells/well were incubated with acrolein at concentrations of 10-4, 3×10-5 and 10-5 . Attachment ability was evaluated after three hours using Neubauer hemocytometer. For the proliferation assay cell cultures seeded at a density of 10,000 cells/well were incubated at concentrations of 10-4, 3×10-5, 10-5, 3×10-6, 10-6 and cell count determined after 5 days using a hemocytometer. Cell morphology was examined under phase contrast microscope. Results: Acrolein produced a dose-dependent cytotoxic effect on human gingival fibroblasts with complete inhibition of attachment and proliferation at higher concentrations. Conclusion: This supports the hypothesis that cigarette smoke is a great risk factor in the development and progression of periodontal disease. PMID:22368362

  8. Recombinant Human Acidic Fibroblast Growth Factor (aFGF) Expressed in Nicotiana benthamiana Potentially Inhibits Skin Photoaging.

    PubMed

    Ha, Jang-Ho; Kim, Ha-Neul; Moon, Ki-Beom; Jeon, Jae-Heung; Jung, Dai-Hyun; Kim, Su-Jung; Mason, Hugh S; Shin, Seo-Yeon; Kim, Hyun-Soon; Park, Kyung-Mok

    2017-07-01

    Responding to the need for recombinant acidic fibroblast growth factor in the pharmaceutical and cosmetic industries, we established a scalable expression system for recombinant human aFGF using transient and a DNA replicon vector expression in Nicotiana benthamiana. Recombinant human-acidic fibroblast growth factor was recovered following Agrobacterium infiltration of N. benthamiana. The optimal time point at which to harvest recombinant human acidic fibroblast growth factor expressing leaves was found to be 4 days post-infiltration, before necrosis was evident. Commassie-stained SDS-PAGE gels of His-tag column eluates, concentrated using a 10 000 molecular weight cut-off column, showed an intense band at the expected molecular weight for recombinant human acidic fibroblast growth factor. An immunoblot confirmed that this band was recombinant human acidic fibroblast growth factor. Up to 10 µg recombinant human-acidic fibroblast growth factor/g of fresh leaves were achieved by a simple affinity purification protocol using protein extract from the leaves of agroinfiltrated N. benthamiana. The purified recombinant human acidic fibroblast growth factor improved the survival rate of UVB-irradiated HaCaT and CCD-986sk cells approximately 89 and 81 %, respectively. N. benthamiana-derived recombinant human acidic fibroblast growth factor showed similar effects on skin cell proliferation and UVB protection compared to those of Escherichia coli-derived recombinant human acidic fibroblast growth factor. Additionally, N. benthamiana-derived recombinant human acidic fibroblast growth factor increased type 1 procollagen synthesis up to 30 % as well as reduced UVB-induced intracellular reactive oxygen species generation in fibroblast (CCD-986sk) cells.UVB is a well-known factor that causes various types of skin damage and premature aging. Therefore, the present study demonstrated that N. benthamiana-derived recombinant human acidic fibroblast growth factor

  9. Involvement of the mitochondrial compartment in human NCL fibroblasts

    SciTech Connect

    Pezzini, Francesco; Gismondi, Floriana; Tessa, Alessandra; Tonin, Paola; Carrozzo, Rosalba; Mole, Sara E.; Santorelli, Filippo M.; Simonati, Alessandro

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. Black-Right-Pointing-Pointer Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. Black-Right-Pointing-Pointer Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

  10. Extracellular depolymerization of hyaluronic acid in cultured human skin fibroblasts

    SciTech Connect

    Nakamura, T.; Takagaki, K.; Kubo, K.; Morikawa, A.; Tamura, S.; Endo, M. )

    1990-10-15

    The chain length of ({sup 3}H)hyaluronic acid synthesized by cultivating human skin fibroblasts in the presence of ({sup 3}H)glucosamine was investigated. ({sup 3}H)Hyaluronic acid obtained from the matrix fraction was excluded from a Sepharose CL-2B column irrespective of the incubation period, whereas that from the medium was depolymerized into a constant chain length (Mr = 40,000). The reducing and non-reducing terminals of the depolymerized hyaluronic acid were N-acetylglucosamine and glucuronic acid, respectively. Prolonged incubation produced no oligosaccharides as shown by examination of hyaluronidase digests, suggesting the presence of a novel endo-beta-N-acetylglucosaminidase in cultured human skin fibroblasts.

  11. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast.

    PubMed

    Seol, Ja Young; Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami; Choi, Soon Young

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin's elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity.

  12. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast

    PubMed Central

    Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin’s elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity. PMID:27194933

  13. Cysteine-rich protein 61 (CCN1) mediates replicative senescence-associated aberrant collagen homeostasis in human skin fibroblasts.

    PubMed

    Quan, Taihao; Qin, Zhaoping; Voorhees, John J; Fisher, Gary J

    2012-09-01

    Dermal fibroblasts produce a collagen-rich extracellular matrix, which confers mechanical strength and resiliency to human skin. During aging, collagen production is reduced and collagen fragmentation is increased, which is initiated by matrix metalloproteinase-1 (MMP-1). This aberrant collagen homeostasis results in net collagen deficiency, which impairs the structural integrity and function of skin. Cysteine-rich protein 61 (CCN1), a member of the CCN family, negatively regulates collagen homeostasis, in primary human skin dermal fibroblasts. As replicative senescence is a form of cellular aging, we have utilized replicative senescent dermal fibroblasts to further investigate the connection between elevated CCN1 and aberrant collagen homeostasis. CCN1 mRNA and protein levels were significantly elevated in replicative senescent dermal fibroblasts. Replicative senescent dermal fibroblasts also expressed significantly reduced levels of type I procollagen and increased levels of MMP-1. Knockdown of elevated CCN1 in senescent dermal fibroblasts partially normalized both type I procollagen and MMP-1 expression. These data further support a key role of CCN1 in regulation of collagen homeostasis. Elevated expression of CCN1 substantially increased collagen lattice contraction and fragmentation caused by replicative senescent dermal fibroblasts. Atomic force microscopy (AFM) further revealed collagen fibril fragmentation and disorganization were largely prevented by knockdown of CCN1 in replicative senescent dermal fibroblasts, suggesting CCN1 mediates MMP-1-induced alterations of collagen fibrils by replicative senescent dermal fibroblasts. Given the ability of CCN1 to regulate both production and degradation of type I collagen, it is likely that elevated-CCN1 functions as an important mediator of collagen loss, which is observed in aged human skin.

  14. Insulin stimulation of glycogen synthase in cultured human diploid fibroblasts.

    PubMed

    Hidaka, H; Howard, B V; Kosmakos, F C; Fields, R M; Craig, J W; Bennett, P H; Larner, J

    1980-10-01

    The effect of insulin on glycogen synthase activity in human diploid fibroblasts has been studied. As little as 2 X 10(-10) M insulin increased the glycogen synthase / activity without changing the total activity. Stimulation occurred within 5 min and became maximal in 30 min. A half-maximal increase of / activity was achieved at 3 X 10(-9) M insulin. Glucose starvation increased the magnitude of response of glycogen synthase to insulin but did not change the insulin concentration necessary to give a half-maximal stimulation. Glucose increased the basal level of / activity in human diploid fibroblasts; the effect of insulin was additive. During in vitro senescence the total glycogen synthase activity declined, but the concentration of insulin that produced a half-maximal stimulation remained unchanged. These data indicate that regulation of glycogen synthase activity in human diploid fibroblasts is responsive to physiologic insulin levels and that the system provides a useful model for the in vitro study of insulin sensitivity.

  15. Effect of periodontal dressings on human gingiva fibroblasts in vitro

    SciTech Connect

    Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E. )

    1989-08-01

    In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings.

  16. Human cleft lip and palate fibroblasts and normal nicotine-treated fibroblasts show altered in vitro expressions of genes related to molecular signaling pathways and extracellular matrix metabolism.

    PubMed

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Lumare, Eleonora; Palmieri, Annalisa; Stabellini, Giordano; Bodo, Maria

    2010-03-01

    Nonsyndromic cleft lip with or without cleft palate (CLP) is a frequent craniofacial malformation caused by both genetic and environmental factors. Maternal smoking during pregnancy is a known risk factor, due to the teratogenic role of nicotine. To assess and compare the impact of CLP and nicotine, we studied the quantitative expression of genes involved in signaling pathways and extracellular matrix (ECM) metabolism in human normal nicotine-treated (NicN) and CLP fibroblasts compared to normal control (CTRL) cells. Palatal fibroblast cultures from seven CLP children and seven age-matched CTRL subjects were established and subconfluent cells incubated for 24 h without (CTRL and CLP fibroblasts) or with (NicN fibroblasts) 0.6 mM nicotine. Gene expressions were analyzed by real-time quantitative PCR. For the first time, a regulated cholinergic signaling in our human fibroblasts in vitro was demonstrated. Members of TGF-beta, retinoic acid (RA), and GABA-ergic signaling systems were also differently regulated. Among the ECM genes, fibronectin, syndecan, integrin alpha2, and MMP13 genes were concordantly modulated, while integrin beta5, and decorin genes were discordantly modulated. Interestingly, nicotine treatment regulated gene expressions of CD44 and CLPTM1, two candidate genes for CLP. Our findings show a positive association between nicotine treatment and CLP phenotype. Results suggest that nicotine deranges normal palate development, which might contribute to the development of a CLP malformative phenotype, through the impairment of some important signaling systems and ECM composition.

  17. Aminoguanidine delays the replicative senescence of human diploid fibroblasts.

    PubMed

    Wang, Pei-chang; Zhang, Jian; Zhang, Zong-yu; Tong, Tan-jun

    2007-11-20

    The accumulation of free radicals and advanced glycation end products (AGEs) in cell plays a very important role in replicative senescence. Aminoguanidine (AG) has potential antioxidant effects and decreases AGE levels. This study aimed to investigate its effect on replicative senescence in vitro. The effects of aminoguanidine on morphology, replicative lifespan, cell growth and proliferation, AGEs, DNA damage, DNA repair ability and telomere length were observed in human fetal lung diploid fibroblasts (2BS). Aminoguanidine maintained the non-senescent phenotype of 2BS cells even at late population doubling (PD) and increased cumulative population doublings by at least 17 - 21 PDs. Aminoguanidine also improved the potentials of growth and proliferation of 2BS cells as detected by the MTT assay. The AGE levels of late PD cells grown from early PD in DMEM containing aminiguanidine decreased significantly compared with those of late PD control cells and were similar to those of young control cells. In addition, the cells pretreated with aminoguanidine had a significant reduction in DNA strand breaks when they were exposed to 200 micromol/L H(2)O(2) for 5 minutes which indicated that the compound had a strong potential to protect genomic DNA against oxidative stress. And most of the cells exposed to 100 micromol/L H(2)O(2) had much shorter comet tails and smaller tail areas after incubation with aminoguanidine-supplemented DMEM, which indicated that the compound strongly improved the DNA repair abilities of 2BS cells. Moreover, PD55 cells grown from PD28 in 2 mmol/L or 4 mmol/L aminoguanidine retain telomere lengths of 7.94 kb or 8.12 kb, which was 0.83 kb or 1.11 kb longer than that of the control cells. Aminoguanidine delays replicative senescence of 2BS cells and the senescence-delaying effect of aminoguanidine appear to be due to its many biological properties including its potential for proliferation improvement, its inhibitory effect of AGE formation, antioxidant

  18. Cultured human foreskin fibroblasts produce a factor that stimulates their growth with properties similar to basic fibroblast growth factor

    SciTech Connect

    Story, M.T. )

    1989-05-01

    To determine if fibroblasts could be a source of fibroblast growth factor (FGF) in tissue, cells were initiated in culture from newborn human foreskin. Fibroblast cell lysates promoted radiolabeled thymidine uptake by cultured quiescent fibroblasts. Seventy-nine percent of the growth-promoting activity of lysates was recovered from heparin-Sepharose. The heparin-binding growth factor reacted on immunoblots with antiserum to human placenta-derived basic FGF and competed with iodinated basic FGF for binding to antiserum to (1-24)bFGF synthetic peptide. To confirm that fibroblasts were the source of the growth factor, cell lysates were prepared from cells incubated with radiolabeled methionine. Heparin affinity purified material was immunoprecipitated with basic FGF antiserum and electrophoresed. Radiolabeled material was detected on gel autoradiographs in the same molecular weight region as authentic iodinated basic FGF. The findings are consistant with the notion that cultured fibroblasts express basic FGF. As these cells also respond to the mitogen, it is possible that the regulation of their growth is under autocrine control. Fibroblasts may be an important source of the growth factor in tissue.

  19. Altered age-related changes in bioenergetic properties and mitochondrial morphology in fibroblasts from sporadic amyotrophic lateral sclerosis patients.

    PubMed

    Allen, Scott P; Duffy, Lynn M; Shaw, Pamela J; Grierson, Andrew J

    2015-10-01

    Mitochondria play a key role in aging, which is a well-established risk factor in amyotrophic lateral sclerosis (ALS). We have previously modeled metabolic dysregulation in ALS using fibroblasts isolated from sporadic ALS (SALS) and familial ALS patients. In the present study, we show that fibroblasts from SALS patients have an altered metabolic response to aging. Control fibroblasts demonstrated increased mitochondrial network complexity and spare respiratory capacity with age which was not seen in the SALS cases. SALS cases displayed an increase in uncoupled mitochondrial respiration, which was not evident in control cases. Unlike SALS cases, controls showed a decrease in glycolysis and an increase in the oxygen consumption rate/extracellular acidification rate ratio, indicating an increased reliance on mitochondrial function. Switching to a more oxidative state by removing glucose with in the culture media resulted in a loss of the mitochondrial interconnectivity and spare respiratory capacity increases observed in controls grown in glucose. Glucose removal also led to an age-independent increase in glycolysis in the SALS cases. This study is, to the best our knowledge, the first to assess the effect of aging on both mitochondrial and glycolytic function simultaneously in intact human fibroblasts and demonstrates that the SALS disease state shifts the cellular metabolic response to aging to a more glycolytic state compared with age-matched control fibroblasts. This work highlights that ALS alters the metabolic equilibrium even in peripheral tissues outside the central nervous system. Elucidating at a molecular level how this occurs and at what stage in the disease process is crucial to understanding why ALS affects cellular energy metabolism and how the disease alters the natural cellular response to aging.

  20. A voltage-activated proton current in human cardiac fibroblasts

    SciTech Connect

    El Chemaly, Antoun; Guinamard, Romain; Demion, Marie; Fares, Nassim; Jebara, Victor; Faivre, Jean-Francois; Bois, Patrick . E-mail: patrick.bois@univ-poitiers.fr

    2006-02-10

    A voltage-activated proton current in human cardiac fibroblasts, measured using the whole-cell recording configuration of the patch-clamp technique, is reported. Increasing the pH of the bathing solution shifted the current activation threshold to more negative potentials and increased both the current amplitude and its rate of activation. Changing the pH gradient by one unit caused a 51 mV shift in the reversal potential of the current, demonstrating a high selectivity for protons of the channel carrying the current. Extracellularly applied Zn{sup 2+} reversibly inhibited the current. Activation of the current contributes to the resting membrane conductance under conditions of intracellular acidosis. It is proposed that this current in cardiac fibroblasts is involved in the regulation of the intracellular pH and the membrane potential under physiological conditions as well as in response to pathological conditions such as ischemia.

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

    PubMed

    Damante, Carla Andreotti; De Micheli, Giorgio; Miyagi, Sueli Patrícia Harumi; Feist, Ilíria Salomão; Marques, Márcia Martins

    2009-11-01

    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

  2. Characterization of human vocal fold fibroblasts derived from chronic scar.

    PubMed

    Jetté, Marie E; Hayer, Supriya D; Thibeault, Susan L

    2013-03-01

    In vitro modeling of cell-matrix interactions that occur during human vocal fold scarring is uncommon, as primary human vocal fold scar fibroblast cell lines are difficult to acquire. The purpose of this study was to characterize morphologic features, growth kinetics, contractile properties, α-smooth muscle actin (α-SMA) protein expression and gene expression profile of human vocal fold fibroblasts derived from scar (sVFF) relative to normal vocal fold fibroblasts (nVFF). In vitro. We successfully cultured human vocal fold fibroblasts from tissue explants of scarred vocal folds from a 56-year-old female and compared these to normal fibroblasts from a 59-year-old female. Growth and proliferation were assessed by daily cell counts, and morphology was compared at 60% confluence for 5 days. Gel contraction assays were evaluated after seeding cells within a collagen matrix. α-SMA was measured using western blotting and immunocytochemistry (ICC). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to assess differential extracellular matrix gene expression between the two cell types. sVFF were morphologically indistinguishable from nVFF. sVFF maintained significantly lower proliferation rates relative to nVFF on days 3 to 6 (day 3: P = .0138; days 4, 5, and 6: P < .0001). There were no significant differences in contractile properties between the two cell types at any time point (0 hours: P = .70, 24 hours: P = .79, 48 hours: P = .58). ICC and western blot analyses revealed increased expression of α-SMA in sVFF as compared with nVFF at passages 4 and 5, but not at passage 6 (passage 4: P = .006, passage 5: P = .0015, passage 6: P = .8860). Analysis of 84 extracellular matrix genes using qRT-PCR revealed differential expression of 15 genes (P < .01). nVFF and sVFF displayed differences in proliferation rates, α-SMA expression, and gene expression, whereas no differences were observed in contractile properties or morphology. Further

  3. Aggregatibacter actinomycetemcomitans lipopolysaccharide affects human gingival fibroblast cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Contreras-Marmolejo, Luis Arturo; Román-Alvárez, Patricia; Barajas-Torres, Carolina

    2008-04-01

    The cytoskeleton is a dynamic structure that plays a key role in maintaining cell morphology and function. This study investigates the effect of bacterial wall lipopolysaccharide (LPS), a strong inflammatory agent, on the dynamics and organization of actin, tubulin, vimentin, and vinculin proteins in human gingival fibroblasts (HGF). A time-dependent study showed a noticeable change in actin architecture after 1.5 h of incubation with LPS (1 microg/ml) with the formation of orthogonal fibers and further accumulation of actin filament at the cell periphery by 24 h. When 0.01-10 microg/ml of LPS was added to human gingival fibroblast cultures, cells acquired a round, flat shape and gradually developed cytoplasmic ruffling. Lipopolysaccharides extracted from Aggregatibacter actinomycetemcomitans periodontopathogenic bacteria promoted alterations in F-actin stress fibres of human gingival cells. Normally, human gingival cells have F-actin fibres that are organized in linear distribution throughout the cells, extending along the cell's length. LPS-treated cells exhibited changes in cytoskeletal protein organization, and F-actin was reorganized by the formation of bundles underneath and parallel to the cell membrane. We also found the reorganization of the vimentin network into vimentin bundling after 1.5 h of treatment. HGF cells exhibited diffuse and granular gamma-tubulin stain. There was no change in LPS-treated HGF. However, vinculin plaques distributed in the cell body diminished after LPS treatment. We conclude that the dynamic and structured organization of cytoskeletal filaments and actin assembly in human gingival fibroblasts is altered by LPS treatment and is accompanied by a decrease in F-actin pools.

  4. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts.

    PubMed

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G

    2012-11-23

    The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

  5. Protective Effects of Triphala on Dermal Fibroblasts and Human Keratinocytes

    PubMed Central

    Varma, Sandeep R.; Sivaprakasam, Thiyagarajan O.; Mishra, Abheepsa; Kumar, L. M. Sharath; Prakash, N. S.; Prabhu, Sunil; Ramakrishnan, Shyam

    2016-01-01

    Human skin is body’s vital organ constantly exposed to abiotic oxidative stress. This can have deleterious effects on skin such as darkening, skin damage, and aging. Plant-derived products having skin-protective effects are well-known traditionally. Triphala, a formulation of three fruit products, is one of the most important rasayana drugs used in Ayurveda. Several skin care products based on Triphala are available that claim its protective effects on facial skin. However, the skin protective effects of Triphala extract (TE) and its mechanistic action on skin cells have not been elucidated in vitro. Gallic acid, ellagic acid, and chebulinic acid were deduced by LC-MS as the major constituents of TE. The identified key compounds were docked with skin-related proteins to predict their binding affinity. The IC50 values for TE on human dermal fibroblasts (HDF) and human keratinocytes (HaCaT) were 204.90 ± 7.6 and 239.13 ± 4.3 μg/mL respectively. The antioxidant capacity of TE was 481.33 ± 1.5 mM Trolox equivalents in HaCaT cells. Triphala extract inhibited hydrogen peroxide (H2O2) induced RBC haemolysis (IC50 64.95 μg/mL), nitric oxide production by 48.62 ± 2.2%, and showed high reducing power activity. TE also rescued HDF from H2O2-induced damage; inhibited H2O2 induced cellular senescence and protected HDF from DNA damage. TE increased collagen-I, involucrin and filaggrin synthesis by 70.72 ± 2.3%, 67.61 ± 2.1% and 51.91 ± 3.5% in HDF or HaCaT cells respectively. TE also exhibited anti-tyrosinase and melanin inhibition properties in a dose-dependent manner. TE increased the mRNA expression of collagen-I, elastin, superoxide dismutase (SOD-2), aquaporin-3 (AQP-3), filaggrin, involucrin, transglutaminase in HDF or HaCaT cells, and decreased the mRNA levels of tyrosinase in B16F10 cells. Thus, Triphala exhibits protective benefits on skin cells in vitro and can be used as a potential ingredient in skin care formulations. PMID:26731545

  6. Microarray analysis of radiation response genes in primary human fibroblasts

    SciTech Connect

    Kis, Enikoe; Szatmari, Tuende; Keszei, Marton; Farkas, Robert; Esik, Olga; Lumniczky, Katalin; Falus, Andras; Safrany, Geza . E-mail: safrany@hp.osski.hu

    2006-12-01

    Purpose: To identify radiation-induced early transcriptional responses in primary human fibroblasts and understand cellular pathways leading to damage correction. Methods and Materials: Primary human fibroblast cell lines were irradiated with 2 Gy {gamma}-radiation and RNA isolated 2 h later. Radiation-induced transcriptional alterations were investigated with microarrays covering the entire human genome. Time- and dose dependent radiation responses were studied by quantitative real-time polymerase chain reaction (RT-PCR). Results: About 200 genes responded to ionizing radiation on the transcriptional level in primary human fibroblasts. The expression profile depended on individual genetic backgrounds. Thirty genes (28 up- and 2 down-regulated) responded to radiation in identical manner in all investigated cells. Twenty of these consensus radiation response genes were functionally categorized: most of them belong to the DNA damage response (GADD45A, BTG2, PCNA, IER5), regulation of cell cycle and cell proliferation (CDKN1A, PPM1D, SERTAD1, PLK2, PLK3, CYR61), programmed cell death (BBC3, TP53INP1) and signaling (SH2D2A, SLIC1, GDF15, THSD1) pathways. Four genes (SEL10, FDXR, CYP26B1, OR11A1) were annotated to other functional groups. Many of the consensus radiation response genes are regulated by, or regulate p53. Time- and dose-dependent expression profiles of selected consensus genes (CDKN1A, GADD45A, IER5, PLK3, CYR61) were investigated by quantitative RT-PCR. Transcriptional alterations depended on the applied dose, and on the time after irradiation. Conclusions: The data presented here could help in the better understanding of early radiation responses and the development of biomarkers to identify radiation susceptible individuals.

  7. Autophagy impairment induces premature senescence in primary human fibroblasts.

    PubMed

    Kang, Hyun Tae; Lee, Ki Baek; Kim, Sung Young; Choi, Hae Ri; Park, Sang Chul

    2011-01-01

    Recent studies have demonstrated that activation of autophagy increases the lifespan of organisms from yeast to flies. In contrast to the lifespan extension effect in lower organisms, it has been reported that overexpression of unc-51-like kinase 3 (ULK3), the mammalian homolog of autophagy-specific gene 1 (ATG1), induces premature senescence in human fibroblasts. Therefore, we assessed whether the activation of autophagy would genuinely induce premature senescence in human cells. Depletion of ATG7, ATG12, or lysosomal-associated membrane protein 2 (Lamp2) by transfecting siRNA or infecting cells with a virus containing gene-specific shRNA resulted in a senescence-like state in two strains of primary human fibroblasts. Prematurely senescent cells induced by autophagy impairment exhibited the senescent phenotypes, similar to the replicatively senescent cells, such as increased senescence associated β-galactosidase (SA-β-gal) activity, reactive oxygen species (ROS) generation, and accumulation of lipofuscin. In addition, expression levels of ribosomal protein S6 kinase1 (S6K1), p-S6K1, p-S6, and eukaryotic translation initiation factor 4E (eIF4E) binding protein 1 (4E-BP1) in the mammalian target of rapamycin (mTOR) pathway and beclin-1, ATG7, ATG12-ATG5 conjugate, and the sequestosome 1 (SQSTM1/p62) monomer in the autophagy pathway were decreased in both the replicatively and the autophagy impairment-induced prematurely senescent cells. Furthermore, it was found that ROS scavenging by N-acetylcysteine (NAC) and inhibition of p53 activation by pifithrin-α or knockdown of p53 using siRNA, respectively, delayed autophagy impairment-induced premature senescence and restored the expression levels of components in the mTOR and autophagy pathways. Taken together, we concluded that autophagy impairment induces premature senescence through a ROS- and p53-dependent manner in primary human fibroblasts.

  8. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  9. Tocotrienols have potent antifibrogenic effects in human intestinal fibroblasts.

    PubMed

    Luna, Jeroni; Masamunt, Maria Carme; Rickmann, Mariana; Mora, Rut; España, Carolina; Delgado, Salvadora; Llach, Josep; Vaquero, Eva; Sans, Miquel

    2011-03-01

    Excessive fibroblast expansion and extracellular matrix (ECM) deposition are key events for the development of bowel stenosis in Crohn's disease (CD) patients. Tocotrienols are vitamin E compounds with proven in vitro antifibrogenic effects on rat pancreatic fibroblasts. We aimed at investigating the effects of tocotrienols on human intestinal fibroblast (HIF) proliferation, apoptosis, autophagy, and synthesis of ECM. HIF isolated from CD, ulcerative colitis (UC), and normal intestine were treated with tocotrienol-rich fraction (TRF) from palm oil. HIF proliferation was quantified by (3) H-thymidine incorporation, apoptosis was studied by DNA fragmentation, propidium iodide staining, caspase activation, and poly(ADP-ribose) polymerase cleavage, autophagy was analyzed by quantification of LC3 protein and identification of autophagic vesicles by immunofluorescence and production of ECM components was measured by Western blot. TRF significantly reduced HIF proliferation and prevented basic fibroblast growth factor-induced proliferation in CD and UC, but not control HIF. TRF enhanced HIF death by promoting apoptosis and autophagy. HIF apoptosis, but not autophagy, was prevented by the pan-caspase inhibitor zVAD-fmk, whereas both types of cell death were prevented when the mitochondrial permeability transition pore was blocked by cyclosporin A, demonstrating a key role of the mitochondria in these processes. TRF diminished procollagen type I and laminin γ-1 production by HIF. Tocotrienols exert multiple effects on HIF, reducing cell proliferation, enhancing programmed cell death through apoptosis and autophagy, and decreasing ECM production. Considering their in vitro antifibrogenic properties, tocotrienols could be useful to treat or prevent bowel fibrosis in CD patients. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

  10. Altered MCM Protein Levels and Autophagic Flux in Aged and Systemic Sclerosis Dermal Fibroblasts

    PubMed Central

    Dumit, Verónica I.; Küttner, Victoria; Käppler, Jakob; Piera-Velazquez, Sonsoles; Jimenez, Sergio A.; Bruckner-Tuderman, Leena; Uitto, Jouni; Dengjel, Jörn

    2014-01-01

    Aging is a common risk factor of many disorders. With age, the level of insoluble extracellular matrix increases leading to increased stiffness of a number of tissues. Matrix accumulation can also be observed in fibrotic disorders, such as systemic sclerosis (SSc). Although the intrinsic aging process in skin is phenotypically distinct from SSc, here we demonstrate similar behavior of aged and SSc skin fibroblasts in culture. We have used quantitative proteomics to characterize the phenotype of dermal fibroblasts from healthy subjects of various ages and from patients with SSc. Our results demonstrate that proteins involved in DNA and RNA processing decrease with age and in SSc, while those involved in mitochondrial and other metabolic processes behave the opposite. Specifically, mini-chromosome maintenance (MCM) helicase proteins are less abundant with age and SSc, and they exhibit an altered subcellular distribution. We observed that lower levels of MCM7 correlate with reduced cell proliferation, lower autophagic capacity and higher intracellular protein expression phenotypes of aged and SSc cells. Additionally, we show that SSc fibroblasts exhibit higher levels of senescence than their healthy counterparts, suggesting further similarities between the fibrotic disorder and the aging process. Hence, at the molecular level, SSc fibroblasts exhibit intrinsic characteristics of fibroblasts from aged skin. PMID:24496236

  11. Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq

    PubMed Central

    Marthandan, S.; Baumgart, M.; Priebe, S.; Groth, M.; Schaer, J.; Kaether, C.; Guthke, R.; Cellerino, A.; Platzer, M.; Diekmann, S.; Hemmerich, P.

    2016-01-01

    Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains. PMID:27140416

  12. Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.

    PubMed

    Marthandan, S; Baumgart, M; Priebe, S; Groth, M; Schaer, J; Kaether, C; Guthke, R; Cellerino, A; Platzer, M; Diekmann, S; Hemmerich, P

    2016-01-01

    Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.

  13. Caffeine protects human skin fibroblasts from acute reactive oxygen species-induced necrosis.

    PubMed

    Silverberg, Jonathan I; Patel, Mital; Brody, Neil; Jagdeo, Jared

    2012-11-01

    Oxidative damage by reactive oxygen species (ROS) plays a major role in aging and carcinogenesis. Little is known about either the effects of acute ROS in necrosis and inflammation of skin or the therapeutic agents for prevention and treatment. Previously, our laboratory identified caffeine as an inhibitor of hydrogen peroxide (H2O2)-generated lipid peroxidation products in human skin fibroblasts. Here, we study effects of caffeine on acute ROS-mediated necrosis. Human skin fibroblasts were incubated with caffeine, followed by H2O2 challenge. Flow cytometry was used to analyze cell morphology, counts, apoptosis and necrosis, and ROS. We found that caffeine protects from H2O2 cell damage at lower (0.01 mM) and intermediate (0.1 mM) doses. The beneficial effects of caffeine appear to be mediated by a mechanism other than antioxidant function.

  14. Luteolin inhibits lipopolysaccharide actions on human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Kawasaki-Cárdenas, Perla; Arroyo-Cruz, Santa Rita; Maldonado-Frías, Silvia

    2006-07-10

    Periodontal disease comprises a group of infections that lead to inflammation of the gingiva, periodontal tissue destruction, and in severe cases is accompanied by alveolar bone loss with tooth exfoliation. Actinobacillus actinomycetemcomitans is a Gram-negative microorganism, which possesses and produces lipopolysaccharide (LPS) molecules that play a key role in disease development. Human gingival fibroblasts are the major constituents of gingival connective tissue and may interact directly with bacteria and bacterial products including LPS. Flavonoids possess antioxidant and anti-inflammatory properties that reduce inflammatory molecule expression in macrophages and monocytes. In this study, we evaluated the ability of diverse flavonoids to regulate nitric oxide production of LPS-stimulated human gingival fibroblasts, and studied the effect of luteolin on diminish phosphorylation in mitogen-activated protein kinase (MAPK) family members as well as in protein kinase B (Akt), nuclear factor kappa B (NF-kappaB) activation, inducible nitric oxide synthase (NOS) expression, and nitric oxide (NO) synthesis. We also found that pretreatment with three flavonoids, including quercetin, genistein, and luteolin, blocked nitric oxide synthesis in a dose-dependent fashion. Luteolin exerted the strongest blocking action on expression of this inflammatory mediator. Luteolin pretreatment attenuated LPS-induced extracellular signal-regulated kinase, p38, and Akt phosphorylation. LPS treatment of human gingival fibroblasts resulted in NF-kappaB translocation. Cell pretreatment with luteolin abolished LPS effects on NF-kappaB translocation. In addition, luteolin treatment blocked LPS-induced cellular proliferation inhibition without affecting genetic material integrity. We concluded that luteolin interferes with LPS signaling pathways, reducing activation of several mitogen-activated protein kinase family members, and inhibits inflammatory mediator expression.

  15. Effect of microemulsions on cell viability of human dermal fibroblasts

    NASA Astrophysics Data System (ADS)

    Li, Juyi; Mironava, Tatsiana; Simon, Marcia; Rafailovich, Miriam; Garti, Nissim

    Microemulsions are optically clear, thermostable and isotropic mixture consisting of water, oil and surfactants. Their advantages of ease preparation, spontaneous formation, long-term stability and enhanced solubility of bioactive materials make them great potentials as vehicles in food and pharmaceutical applications. In this study, comparative in vitro cytotoxicity tests were performed to select a best formulation of microemulsion with the least toxicity for human dermal fibroblasts. Three different kinds of oils and six different kinds of surfactants were used to form microemulsions by different ratios. The effect of oil type and surfactant type as well as their proportions on cell proliferation and viability were tested.

  16. Isolation of the pericellular matrix of human fibroblast cultures

    PubMed Central

    1979-01-01

    The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix. PMID:383722

  17. Production of Procollagen by Human Fibroblasts in Culture

    PubMed Central

    Smith, Barbara D.; Byers, Peter H.; Martin, George R.

    1972-01-01

    Three hydroxyproline-containing proteins secreted into the medium by human fibroblasts in culture were isolated and characterized. A minor fraction was identical to the collagen monomer. The major fraction was a form of procollagen, which contained, in addition to pro α and α chains, a component estimated to have a molecular weight of 250,000. This component was a dimer of pro α chains joined by disulfide bonds. The third fraction, much lower in hydroxyproline and hydroxylysine content, was of still greater size. Pro α chains were released upon denaturation and reduction, indicating that this fraction may contain pro α chains linked by disulfide bonds to noncollagenous material. PMID:4508318

  18. Evaluating biotoxicity with fibroblasts derived from human embryonic stem cells.

    PubMed

    Wang, Xiaoying; Li, Shenglin; Cao, Tong; Fu, Xin; Yu, Guangyan

    2012-09-01

    To investigate the use of differentiated fibroblasts from human embryonic stem cells as a cellular model for cytotoxicity and genotoxicity screening. The EBf-H9 cells were derived from human embryonic stem cells (H9) via embryonic body (EB) and treated with Sodium fluoride (NaF) and Formaldehyde (FA). Proliferation, specific gene and protein expression and karyotype of cells were analyzed by MTT assay, RT-PCR, immunocytochemistry and karyotype analysis, respectively. Cytotoxicity was detected by MTT assay and flow cytometry, and genotoxicity was studied by micronucleus test (MNT), sister chromatid exchange (SCE) and comet assay. EBf-H9s were spindle-shaped with a diploid karyotype. They expressed the fibroblast markers prolyl 4-hydroxylase β and vimentin but did not express Oct-4 and Sox-2, and decreased expression of Nanog. The proliferation of EBf-H9 and murine L929 cells was inhibited by sodium fluoride (NaF) and formaldehyde (FA), and the cell cycle was arrested in different phases with the treatments. In genotoxicity assays with NaF and FA, positive responses were detected in human EBf-H9s comparable to those in the murine L929 cell line. EBf-H9 may be a suitable new cell source for toxicity research on biomaterials and other agents. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. C-reactive protein modulates human lung fibroblast migration.

    PubMed

    Kikuchi, Kazuhiko; Kohyama, Tadashi; Yamauchi, Yasuhiro; Kato, Jun; Takami, Kazutaka; Okazaki, Hitoshi; Desaki, Masashi; Nagase, Takahide; Rennard, Stephen I; Takizawa, Hajime

    2009-02-01

    C-reactive protein (CRP) has been classically used as a marker of inflammation. The aim of this study was to investigate the effect of CRP on migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn). Using the blindwell chamber technique, CRP inhibited HFL-1 migration in a dose-dependent fashion (at 1 microg/mL, inhibition: 32.5% +/- 7.1%; P < .05). Western blot analysis showed that CRP inhibited the p38 mitogen-activated protein kinase (MAPK) activity in the presence of HFn. Moreover, the MAPK inhibitors SB202190 (25 microM) and SB203580 (25 microM) inhibited HFn-induced cell migration, suggesting an important role of p38 MAPK in HFn-induced migration. Taken together, these results suggest that the inhibitory effect of CRP is mediated by blocking MAPK. In summary, this study demonstrates that CRP directly modulates human lung fibroblasts migration. Thus, CRP may contribute to regulation of wound healing and may be endogenous antifibrotic factor acting on lung fibrosis.

  20. Conversion of human fibroblasts into monocyte-like progenitor cells

    PubMed Central

    Vitaloni, Marianna; Guenechea, Guillermo; Xia, Yun; Kurian, Leo; Dubova, Ilir; Bueren, Juan; Laricchia-Robbio, Leopoldo; Belmonte, Juan Carlos Izpisua

    2014-01-01

    Reprogramming technologies have emerged as a promising approach for future regenerative medicine. Here we report on the establishment of a novel methodology allowing for the conversion of human fibroblasts into Hematopoietic Progenitor-like Cells (HPC) with macrophage differentiation potential. SOX2 overexpression in human fibroblasts, a gene found to be upregulated during hematopoietic reconstitution in mice, induced the rapid appearance of CD34+ cells with a concomitant upregulation of mesoderm-related markers. Profiling of Cord Blood hematopoietic progenitor cell populations identified miR-125b as a factor facilitating commitment of SOX2-generated CD34+ cells to immature hematopoietic-like progenitor cells with grafting potential. Further differentiation towards the monocytic lineage resulted in the appearance of CD14+ cells with functional phagocytic capacity. In vivo transplantation of SOX2/miR-125b-generated CD34+ cells facilitated the maturation of the engrafted cells towards CD45+ cells and ultimately the monocytic/macrophage lineage. Altogether, our results indicate that strategies combining lineage conversion and further lineage specification by in vivo or in vitro approaches could help to circumvent long-standing obstacles for the reprogramming of human cells into hematopoietic cells with clinical potential. PMID:25175072

  1. CEMP1 Induces Transformation in Human Gingival Fibroblasts

    PubMed Central

    Bermúdez, Mercedes; Imaz-Rosshandler, Ivan; Rangel-Escareño, Claudia; Zeichner-David, Margarita; Arzate, Higinio; Mercado-Celis, Gabriela E.

    2015-01-01

    Cementum Protein 1 (CEMP1) is a key regulator of cementogenesis. CEMP1 promotes cell attachment, differentiation, deposition rate, composition, and morphology of hydroxyapatite crystals formed by human cementoblastic cells. Its expression is restricted to cementoblasts and progenitor cell subpopulations present in the periodontal ligament. CEMP1 transfection into non-osteogenic cells such as adult human gingival fibroblasts results in differentiation of these cells into a “mineralizing” cell phenotype. Other studies have shown evidence that CEMP1 could have a therapeutic potential for the treatment of bone defects and regeneration of other mineralized tissues. To better understand CEMP1’s biological effects in vitro we investigated the consequences of its expression in human gingival fibroblasts (HGF) growing in non-mineralizing media by comparing gene expression profiles. We identified several mRNAs whose expression is modified by CEMP1 induction in HGF cells. Enrichment analysis showed that several of these newly expressed genes are involved in oncogenesis. Our results suggest that CEMP1 causes the transformation of HGF and NIH3T3 cells. CEMP1 is overexpressed in cancer cell lines. We also determined that the region spanning the CEMP1 locus is commonly amplified in a variety of cancers, and finally we found significant overexpression of CEMP1 in leukemia, cervix, breast, prostate and lung cancer. Our findings suggest that CEMP1 exerts modulation of a number of cellular genes, cellular development, cellular growth, cell death, and cell cycle, and molecules associated with cancer. PMID:26011628

  2. Diphenylhydantoin affects glycosaminoglycans and collagen production by human fibroblasts from cleft palate patients.

    PubMed

    Bosi, G; Evangelisti, R; Valeno, V; Carinci, F; Pezzetti, F; Calastrini, C; Bodo, M; Carinci, P

    1998-08-01

    During embryonic development, the proper production of extracellular matrix molecules mediates morphogenetic processes involved in palatogenesis. In the present study, we investigated whether any differences exist in glycosaminoglycan (GAG) and collagen synthesis between palate fibroblasts from infants, with or without cleft palate, in two age ranges. Subsequently, the effects of diphenylhydantoin (PHT), a teratogen known to induce cleft palate in human and mammalian newborns, on extracellular matrix (ECM) production were studied. We found that cleft palate fibroblasts (CPFs) synthesize greater amounts of GAG and collagen than normal fibroblasts (NFs). CPFs produced less cellular hyaluronic acid (HA) and more sulphated GAG. HA was the principal GAG species in the medium, and its percentage was lower in one- to three-year-old CPFs. Cleft palate fibroblasts produced more extracellular chondroitin 4- and 6-sulphate (CS) and dermatan sulphate (DS). Associated with a higher production of sulphated GAG, we observed a higher synthesis of type III and type I collagen with a normal ratio of alpha2(I) to alpha1(I) chains. PHT treatment of NFs reduced collagen and GAG synthesis, with a marked effect on sulphated GAG. The drug changed collagen synthesis, whereas it did not affect GAG production in CPFs whose phenotype may already be impaired. These findings indicate that, in CPFs, modifications in the pattern of ECM components, which are most likely responsible for the anomalous development, persist in infants. In addition, NFs and CPFs with a different phenotype respond differently to PHT treatment.

  3. Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains.

    PubMed

    Tsai, Ching-Wen; Kao, Yu-Ting; Chiang, I-Ni; Wang, Jyh-Horng; Young, Tai-Horng

    2015-01-01

    Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55-60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70-75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications.

  4. Chitosan Treatment Delays the Induction of Senescence in Human Foreskin Fibroblast Strains

    PubMed Central

    Tsai, Ching-Wen; Kao, Yu-Ting; Chiang, I-Ni; Wang, Jyh-Horng; Young, Tai-Horng

    2015-01-01

    Fibroblasts have been extensively used as a model to study cellular senescence. The purpose of this study was to investigate whether the human foreskin fibroblast aging process could be regulated by using the biomaterial chitosan. Fibroblasts cultured on commercial tissue culture polystyrene (TCPS) entered senescence after 55–60 population doublings (PDs), and were accompanied by larger cell shape, higher senescence-associated β-galactosidase (SA β-gal) activity, lower proliferation capacity, and upregulation of senescence-associated molecular markers p21, p53, retinoblastoma (pRB), and p16. Before senescence was reached, PD48 cells were collected from TCPS and seeded on chitosan for three days (PD48-Cd3) to form multicellular spheroids. The protein expression of senescence-associated secretory phenotypes (SASPs) and senescence-associated molecular markers of these cells in PD48-Cd3 spheroids were downregulated significantly. Following chitosan treatment, fibroblasts reseeded on TCPS showed lower SA β-gal activity, increased cellular motility, and a higher proliferation ability of 70–75 PDs. These phenotypic changes were not accompanied by colonies forming in soft agar and a continuous decrease in the senescence-associated proteins p53 and pRB which act as a barrier to tumorigenesis. These results demonstrate that chitosan treatment could delay the induction of senescence which may be useful and safe for future tissue engineering applications. PMID:26465338

  5. Pressure effects on the growth of human scar fibroblasts.

    PubMed

    Chang, Liang-Wey; Deng, Win-Ping; Yeong, Eng-Kean; Wu, Ching-Yuan; Yeh, Shih-Wei

    2008-01-01

    Although pressure therapy is the mainstay of treatment for hypertrophic scars, its actual mechanism remains unknown. An in vitro study was designed to investigate the effects of positive pressure on the growth of human scar-derived fibroblasts through its transforming growth factor beta1 (TGF-beta1) secretion. A pneumatic pressure system connecting to a cell culture chamber was designed. Six-well cultured plates with fibroblasts implanted were treated with different pressure settings. Cells were treated with constant pressure 20 mm Hg above atmosphere pressure (group A n = 18) or with 40 mm Hg above atmosphere pressure (group B n = 18) daily for nine successive days. Cells without pressure were treated as the control study (group C n = 6). Each experimental group was divided into daily pressure applied at 24 hours (n = 6), 18 hours (n = 6), and 12 hours (n = 6). Cell counting was performed on the 2nd, 4th, 7th, 9th, 11th, and 14th day after implantation. On day 4, the concentration of transforming growth factor beta1 was measured, and cell doubling time was calculated. Compared with the control group, there was a significant decrease in cell count and the concentration in the 18-hour and 24-hour 20 mm Hg or 40 mm Hg pressure treated group. The cell doubling time was significantly increased in the 24-hour 20 mm Hg or 40 mm Hg pressure treated groups, and the 18-hour 40 mm Hg pressure treated group. (P < .05) Pressure inhibits the growth and activity of human scar fibroblasts, and a higher pressure application can shorten the daily application period. There should be an optimal pressure level corresponding to a daily application period to achieve the most effective results on pressure therapy for scars.

  6. Characterization of Mesenchymal Stem Cells from Human Vocal Fold Fibroblasts

    PubMed Central

    Hanson, Summer; Kim, Jaehyup; Quinchia Johnson, Beatriz H.; Bradley, Bridget; Breunig, Melissa; Hematti, Peiman; Thibeault, Susan L.

    2009-01-01

    Objective/Hypothesis Mesenchymal stem cells (MSCs) originally isolated from bone marrow, are fibroblast-looking cells that are now assumed to be present in the stromal component of many tissues. MSCs are characterized by a certain set of criteria including their growth culture characteristics, a combination of cell surface markers, and the ability to differentiate along multiple mesenchymal tissue lineages. We hypothesized that human vocal fold fibroblasts (hVFF) isolated from the lamina propria meet the criteria established to define MSCs and are functionally similar to MSCs derived from BM and adipose tissue. Study Design In vitro study Methods HVFF were previously derived from human vocal fold tissues. MSCs were derived from adipose tissue (AT), and BM of healthy donors, based on their attachment to culture dishes and their morphology, and expanded in culture. Cells were analyzed for standard cell surface markers identified on BM-derived MSCs as well as the ability to differentiate into cells of mesenchymal lineage, i.e. fat, bone and cartilage. We investigated the immunophenotype of these cells before and after interferon-γ (INF- γ) stimulation. Results HVFF displayed cell surface markers and multipotent differentiation capacity characteristic of MSCs. Furthermore, these cells exhibited similar patterns of expression of HLA and co-stimulatory molecules, after stimulation with INF- γ compared to MSCs derived from BM and AT. Conclusions Based on our findings hVFF derived from lamina propria have the same cell surface markers, immunophenotypic characteristics, and differentiation potential as BM- and AT-derived MSCs. We propose VF fibroblasts are MSCs resident in the vocal fold lamina propria. PMID:20131365

  7. Functional changes induced by chronic UVA irradiation to cultured human dermal fibroblasts.

    PubMed

    Naru, E; Suzuki, T; Moriyama, M; Inomata, K; Hayashi, A; Arakane, K; Kaji, K

    2005-12-01

    Ultraviolet (UV) irradiation induces damage of the skin, and in particular, photoageing is known to be the result of chronic UV irradiation. Many investigations have attempted to clarify the mechanisms of photoageing induced by chronic UVA irradiation, but consensus has not been achieved yet by in vivo experiments, mostly due to differences among UV sources and animals used for experiments. In vitro experiments have shown that a single exposure to UVA irradiation causes overexpression of matrix metalloproteinases and denaturation of collagen, but the mechanisms of the photoageing effects of chronic UVA irradiation are still unclear. To examine the effects of chronic UVA irradiation, we used an in vitro fibroblast cellular ageing system as a model of photoageing. Chronic UVA irradiation of normal human fibroblasts induced shortening of the cellular life span and an increase of cellular diameter, in parallel with expression of senescence-associated beta-galactosidase. Extracellular degradation enzyme, matrix metalloproteinase 1 (MMP-1) was overexpressed after repeated UVA irradiation, but tissue inhibitor of metalloproteinase 1 (TIMP-1) expression was hardly changed by chronic UVA irradiation. We conclude that chronic UVA irradiation of normal human fibroblasts induces cellular functional changes, leading to accelerated cellular ageing and MMP-1 overexpression.

  8. Proinflammatory cytokines induce amelotin transcription in human gingival fibroblasts.

    PubMed

    Nakayama, Yohei; Takai, Hideki; Matsui, Sari; Matsumura, Hiroyoshi; Zhou, Liming; Kato, Ayako; Ganss, Bernhard; Ogata, Yorimasa

    2014-12-01

    Amelotin (AMTN) is a secreted protein transcribed predominantly during the maturation stage of enamel formation and localized in the junctional epithelium. We investigated differences in the levels of AMTN gene expression between non-inflamed gingiva and inflamed gingiva from patients with chronic periodontitis. Total RNAs were isolated from these tissues and their gene expression profiles were monitored by DNA microarray. The observed induction of AMTN mRNA in inflamed gingiva and cultured human gingival fibroblasts (HGF) was confirmed by real-time PCR. Transient transfection assays were performed using chimeric constructs of mouse AMTN gene promoter fragments linked to a luciferase reporter gene. Immunohistochemical localization of AMTN in inflamed and non-inflamed gingiva was assessed by immunohistochemistry. Among many differentially expressed genes, the level of AMTN mRNA was significantly increased in inflamed gingiva. Treatment of HGF with interleukin-1β (IL-1β), IL-6 and tumor necrosis factor-α (TNF-α) induced the expression of AMTN mRNA, and increased the luciferase activities of the AMTN promoter constructs. AMTN protein was detected in inflamed gingival connective tissue and junctional epithelium. These findings demonstrate that proinflammatory cytokines induce AMTN gene expression in human gingival fibroblasts and suggest a role for AMTN in gingival inflammation.

  9. Proliferative Effects of Histamine on Primary Human Pterygium Fibroblasts

    PubMed Central

    Fu, Qiuli; Zhang, Lifang; Yin, Houfa; Jin, Xiuming; Tang, Qiaomei; Lyu, Danni

    2016-01-01

    Purpose. It has been confirmed that inflammatory cytokines are involved in the progression of pterygium. Histamine can enhance proliferation and migration of many cells. Therefore, we intend to investigate the proliferative and migratory effects of histamine on primary culture of human pterygium fibroblasts (HPFs). Methods. Pterygium and conjunctiva samples were obtained from surgery, and toluidine blue staining was used to identify mast cells. 3-[4, 5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was performed to evaluate the proliferative rate of HPFs and human conjunctival fibroblasts (HCFs); ki67 expression was also measured by immunofluorescence analysis. Histamine receptor-1 (H1R) antagonist (Diphenhydramine Hydrochloride) and histamine receptor-2 (H2R) antagonist (Nizatidine) were added to figure out which receptor was involved. Wound healing model was used to evaluate the migratory ability of HPFs. Results. The numbers of total mast cells and degranulated mast cells were both higher in pterygium than in conjunctiva. Histamine had a proliferative effect on both HPFs and HCFs, the effective concentration (10 μmol/L) on HPFs was lower than on HCFs (100 μmol/L), and the effect could be blocked by H1R antagonist. Histamine showed no migratory effect on HPFs. Conclusion. Histamine may play an important role in the proliferation of HPFs and act through H1R. PMID:27872516

  10. Small molecules increase direct neural conversion of human fibroblasts

    PubMed Central

    Pfisterer, Ulrich; Ek, Fredrik; Lang, Stefan; Soneji, Shamit; Olsson, Roger; Parmar, Malin

    2016-01-01

    The generation of human induced neurons (hiNs) via exogenous delivery of neural transcription factors represents a novel technique to obtain disease and patient specific neurons. These cells have the potential to be used for disease modeling, diagnostics and drug screening, and also to be further developed for brain repair. In the present study, we utilized hiNs to develop an unbiased screening assay for small molecules that increase the conversion efficiency. Using this assay, we screened 307 compounds from five annotated libraries and identified six compounds that were very potent in potentiating the reprogramming process. When combined in an optimal combination and dose, these compounds increased the reprogramming efficiency of human fibroblasts more than 6-fold. Global gene expression and CellNet analysis at different timepoints during the reprogramming process revealed that neuron-specific genes and gene regulatory networks (GRNs) became progressively more activated while converting cells shut down fibroblast-specific GRNs. Further bioinformatics analysis revealed that the addition of the six compound resulted in the accelerated upregulation of a subset of neuronal genes, and also increased expression of genes associated with transcriptional activity and mediation of cellular stress response. PMID:27917895

  11. Effect of saccharin on metabolic cooperation between human fibroblasts

    SciTech Connect

    Mosser, D.D.; Bols, N.C.

    1983-01-01

    Autoradiography was used to study the effect of saccharin on metabolic cooperation between human diploid fibroblasts. When the donors, HGPRT+ cells, and recipients, HGPRT- cells, were plated together in the presence of saccharin, all the interactions that developed in 4 and 24 h were positive for metabolic cooperation. When saccharin was added after donor cells and recipient cells had made contact, the proportion of interactions that were positive for metabolic cooperation was unchanged but the number of grains over primary recipients was reduced. However, in donor cells saccharin caused a reduction in (/sup 3/H)hypoxanthine incorporation into both acid-soluble and acid-insoluble fractions, although the relative distribution of radioactivity between these two fractions and between the phosphorylated and non-phosphorylated derivatives of (/sup 3/H)hypoxanthine was unchanged. Metabolic cooperation was studied under conditions in which the number of grains over the nuclei of both the primary recipient and the primary recipient's donor could be counted. The change in the number of grains over these two cell types in response to saccharin was compared and found to be the same. Thus in normal human fibroblasts saccharin does not appear to affect metabolic cooperation, which is a measure of cell-to-cell communication.

  12. Biodemography of human ageing

    PubMed Central

    Vaupel, James W.

    2014-01-01

    Human senescence has been delayed by a decade. This finding, documented in 1994 and bolstered since, is a fundamental discovery about the biology of human ageing, and one with profound implications for individuals, society and the economy. Remarkably, the rate of deterioration with age seems to be constant across individuals and over time: it seems that death is being delayed because people are reaching old age in better health. Research by demographers, epidemiologists and other biomedical researchers suggests that further progress is likely to be made in advancing the frontier of survival — and healthy survival — to even greater ages. PMID:20336136

  13. Inhibitory effects of (-)-loliolide on cellular senescence in human dermal fibroblasts.

    PubMed

    Yang, Hyo Hyun; Hwangbo, Kyoung; Zheng, Ming Shan; Cho, Jung Hee; Son, Jong-Keun; Kim, Hwa Young; Baek, Suk Hwan; Choi, Hyung Chul; Park, So Young; Kim, Jae-Ryong

    2015-01-01

    Cellular senescence influences tumor suppression and progress, tissue repair and regeneration, tissue and organismal aging, and age-related diseases. Aging intervention might be an advantageous target for prevention and treatment of diverse age-related diseases. In this study, we investigated whether (-)-loliolide purified from the crude extract of Polygonum aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (HDFs). (-)-Loliolide diminished senescence-associated β-galactosidase activity (SA-β-gal), the level of p21 protein, and the level of reactive oxygen species in senescent cells induced by adriamycin treatment. (-)-Loliolide also attenuated SA-β-gal activity in HDFs under replicative senescence. These findings imply that (-)-loliolide rescues cellular senescence in HDFs and might be useful for the development of dietary supplements or cosmetics that ameliorate tissue aging or age-associated diseases.

  14. Inhibitory effects of juglanin on cellular senescence in human dermal fibroblasts.

    PubMed

    Yang, Hyo Hyun; Hwangbo, Kyoung; Zheng, Ming Shan; Son, Jong-Keun; Kim, Hwa Young; Baek, Suk Hwan; Choi, Hyung Chul; Park, So Young; Kim, Jae-Ryong

    2014-07-01

    Cellular senescence contributes to tissue and organismal aging, tumor suppression and progress, tissue repair and regeneration, and age-related diseases. Thus, aging intervention might be a promising target for treatment and prevention of diverse age-related diseases. In the present study, we investigated whether juglanin purified from the crude extract of Polygonum aviculare exerted inhibitory activity against cellular senescence in human dermal fibroblasts (HDFs). Juglanin decreased senescence-associated β-galactosidase activity (SA-β-gal) and the level of reactive oxygen species in senescent cells induced by adriamycin treatment. Juglanin also repressed SA-β-gal activity in HDFs under replicative senescence. These results suggest that juglanin represses cellular senescence in HDFs and might be useful for the development of dietary supplements or cosmetics that alleviate tissue aging or age-related diseases.

  15. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

    PubMed Central

    Saini, Natalie; Chan, Kin; Grimm, Sara A.; Dai, Shuangshuang; Fargo, David C.; Kaufmann, William K.; Taylor, Jack A.; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J.; Schurman, Shepherd H.; Malc, Ewa P.; Mieczkowski, Piotr A.

    2016-01-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307 PMID:27788131

  16. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts.

    PubMed

    Saini, Natalie; Roberts, Steven A; Klimczak, Leszek J; Chan, Kin; Grimm, Sara A; Dai, Shuangshuang; Fargo, David C; Boyer, Jayne C; Kaufmann, William K; Taylor, Jack A; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J; Schurman, Shepherd H; Malc, Ewa P; Mieczkowski, Piotr A; Gordenin, Dmitry A

    2016-10-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration: ClinicalTrials.gov NCT01087307.

  17. Phenotypic modulations of human umbilical vein endothelial cells and human dermal fibroblasts using two angiogenic assays.

    PubMed

    Bikfalvi, A; Cramer, E M; Tenza, D; Tobelem, G

    1991-01-01

    Different angiogenic assays in vitro have helped to define various events underlying angiogenesis. In this report we have compared the phenotypic modifications of human umbilical vein endothelial cells (HUVE cells) and human dermal fibroblasts using Matrigel and collagen gels. Both HUVE cells and human dermal fibroblasts form a network of anastomosing cords that apparently resemble blood capillaries when grown on Matrigel. The whole network was formed by several cellular aggregates joined to each other by cellular cords. Lumen formation was not observed in this angiogenic system. In opposite, considerable differences between HUVE cells and human dermal fibroblasts were observed in the three-dimensional angiogenic assay on collagen gels described by Montesano et al [14]. These results indicate that data obtained with angiogenic systems using Matrigel must be interpreted with caution and that the assay described by Montesano et al [14], is more reliable to describe angiogenesis.

  18. Stress responses of human dermal fibroblasts exposed to zinc pyrithione.

    PubMed

    Rudolf, Emil; Cervinka, Miroslav

    2011-07-28

    Zinc pyrithione is used as a topical agent in a range of medicinal and cosmetic applications. Despite its extensive use and reported beneficial effects in treatment of various dermal problems, its potential toxicity towards skin cells remains relatively underexplored. In this work we investigated effects of nM zinc pyrithione on cell stress response pathways of primary human skin fibroblasts during 24h of exposure. We demonstrate that zinc pyrithione-induced cytotoxity in dermal fibroblasts is dose-dependent and it associates with increased intracellular zinc concentrations and activated stress response pathways including p53 and stress kinase p38. Higher zinc pyrithione concentrations (500nM and above) stimulate oxidative stress and moderate DNA damage which occur in the presence of activated p38 kinase. Cells further upregulate the expression of p53 which increases its transcriptional activity while mitogenic signaling exemplified by mTOR (mammalian target of rapamycin) expression is suppressed and these steps lead to mitochondrial, caspase-dependent apoptosis. Conversely, lower zinc concentrations (125nM) fail to induce oxidative stress and significant DNA damage; however, treated cells still activate p38 and upregulate the expression and transcriptional activity of p53 and its target gene p21 as well as the expression of p16 in the presence of active mTOR pathway and a changed DNA methylation pattern. The end result is premature senescence phenotype. Specific pharmacological inhibitors as well as gene knockdown technology prove that an interaction between p38, p53 and mTOR might be responsible for these observed endpoints. Taken together, exposure of dermal fibroblasts to varying concentrations of zinc pyrithione may result in either cell death-apoptosis or cellular premature senescence which attests to the ability of this compound to affect this type of cells in an in vitro model system.

  19. Metallic nanoparticles reduce the migration of human fibroblasts in vitro

    NASA Astrophysics Data System (ADS)

    Vieira, Larissa Fernanda de Araújo; Lins, Marvin Paulo; Viana, Iana Mayane Mendes Nicácio; dos Santos, Jeniffer Estevão; Smaniotto, Salete; Reis, Maria Danielma dos Santos

    2017-03-01

    Nanoparticles have extremely wide applications in the medical and biological fields. They are being used in biosensors, local drug delivery, diagnostics, and medical therapy. However, the potential effects of nanoparticles on target cell and tissue function, apart from cytotoxicity, are not completely understood. Thus, the aim of this study was to investigate the in vitro effects of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) on human fibroblasts with respect to their interaction with the extracellular matrix and in cell migration. Immunofluorescence analysis revealed that treatment with AgNPs or AuNPs decreased collagen and laminin production at all the concentrations tested (0.1, 1, and 10 μg/mL). Furthermore, cytofluorometric analysis showed that treatment with AgNPs reduced the percentage of cells expressing the collagen receptor very late antigen 2, α2β1 integrin (VLA-2) and the laminin receptor very late antigen 6, α6β1 integrin (VLA-6). In contrast, AuNP treatment increased and decreased the percentages of VLA-2-positive and VLA-6-positive cells, respectively, as compared to the findings for the controls. Analysis of cytoskeletal reorganization showed that treatment with both types of nanoparticles increased the formation of stress fibres and number of cell protrusions and impaired cell polarity. Fibroblasts exposed to different concentrations of AuNPs and AgNPs showed reduced migration through transwell chambers in the functional chemotaxis assay. These results demonstrated that metal nanoparticles may influence fibroblast function by negatively modulating the deposition of extracellular matrix molecules (ECM) and altering the expression of ECM receptors, cytoskeletal reorganization, and cell migration.

  20. Response of human fibroblasts to low dose rate gamma irradiation

    SciTech Connect

    Dritschilo, A.; Brennan, T.; Weichselbaum, R.R.; Mossman, K.L.

    1984-11-01

    Cells from 11 human strains, including fibroblasts from patients with the genetic diseases of ataxia telangiectasia (AT), xeroderma pigmentosum (XP), and Fanconi's anemia (FA), were exposed to ..gamma.. radiation at high (1.6-2.2 Gy/min) and at low (0.03-0.07 Gy/min) dose rates. Survival curves reveal an increase inthe terminal slope (D/sub 0/) when cells are irradiated at low dose rates compared to high dose rates. This was true for all cell lines tested, although the AT, FA, and XP cells are reported or postulated to have radiation repair deficiencies. From the response of these cells, it is apparent that radiation sensitivities differ; however, at low dose rate, all tested human cells are able to repair injury.

  1. Antioxidant effects of the sarsaparilla via scavenging of reactive oxygen species and induction of antioxidant enzymes in human dermal fibroblasts.

    PubMed

    Park, Gunhyuk; Kim, Tae-mi; Kim, Jeong Hee; Oh, Myung Sook

    2014-07-01

    Ultraviolet (UV) radiation from sunlight causes distinct changes in collagenous skin tissues as a result of the breakdown of collagen, a major component of the extracellular matrix. UV irradiation downregulates reactive oxygen species (ROS)-elimination pathways, thereby promoting the production of ROS, which are implicated in skin aging. Smilax glabra Roxb (sarsaparilla) has been used in folk medicine because of its many effects. However, no study on the protective effects of sarsaparilla root (SR) on human dermal fibroblasts has been reported previously. Here, we investigated the protective effect of SR against oxidative stress in dermal fibroblasts. SR significantly inhibited oxidative damage and skin-aging factor via mitogen-activated protein kinase signaling pathways. Also, SR decreased Ca(2+) and ROS, mitochondrial membrane potential, dysfunction, and increased glutathione, NAD(P)H dehydrogenase and heme oxygenase-1. These results demonstrate that SR can protect dermal fibroblasts against UVB-induced skin aging via antioxidant effects.

  2. Schisandrin B protects against solar irradiation-induced oxidative injury in BJ human fibroblasts.

    PubMed

    Chiu, Po Yee; Lam, Philip Y; Yan, Chung Wai; Ko, Kam Ming

    2011-06-01

    The effects of schisandrin B (Sch B) and its analogs on solar irradiation-induced oxidative injury were examined in BJ human fibroblasts. Sch B and schisandrin C (Sch C) increased cellular reduced glutathione (GSH) level and protected against solar irradiation-induced oxidative injury. The photoprotection was paralleled by decreases in the elastases-type protease activity and matrix-metalloproteinases-1 expression in solar-irradiated fibroblasts. The cytochrome P-450-mediated metabolism of Sch B or Sch C caused ROS production. The results suggest that by virtue of its pro-oxidant action and the subsequent glutathione antioxidant response, Sch B or Sch C may offer the prospect of preventing skin photo-aging.

  3. Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

    PubMed

    Fujiwara, Toshihiro; Dohi, Teruyuki; Maan, Zeshaan N; Rustad, Kristine C; Kwon, Sun Hyung; Padmanabhan, Jagannath; Whittam, Alexander J; Suga, Hirotaka; Duscher, Dominik; Rodrigues, Melanie; Gurtner, Geoffrey C

    2017-07-04

    Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. MicroRNA-22 increases senescence and activates cardiac fibroblasts in the aging heart.

    PubMed

    Jazbutyte, Virginija; Fiedler, Jan; Kneitz, Susanne; Galuppo, Paolo; Just, Annette; Holzmann, Angelika; Bauersachs, Johann; Thum, Thomas

    2013-06-01

    MicroRNAs (miRs) are small non- coding RNA molecules controlling a plethora of biological processes such as development, cellular survival and senescence. We here determined miRs differentially regulated during cardiac postnatal development and aging. Cardiac function, morphology and miR expression profiles were determined in neonatal, 4 weeks, 6 months and 19 months old normotensive male healthy C57/Bl6N mice. MiR-22 was most prominently upregulated during cardiac aging. Cardiac expression of its bioinformatically predicted target mimecan (osteoglycin, OGN) was gradually decreased with advanced age. Luciferase reporter assays validated mimecan as a bona fide miR-22 target. Both, miR-22 and its target mimecan were co- expressed in cardiac fibroblasts and smooth muscle cells. Functionally, miR-22 overexpression induced cellular senescence and promoted migratory activity of cardiac fibroblasts. Small interference RNA-mediated silencing of mimecan in cardiac fibroblasts mimicked the miR-22-mediated effects. Rescue experiments revealed that the effects of miR-22 on cardiac fibroblasts were only partially mediated by mimecan. In conclusion, miR-22 upregulation in the aging heart contributed at least partly to accelerated cardiac fibroblast senescence and increased migratory activity. Our results suggest an involvement of miR-22 in age-associated cardiac changes, such as cardiac fibrosis.

  5. Differentiation of Human Embryonic Stem Cells on Periodontal Ligament Fibroblasts.

    PubMed

    Elçin, Y Murat; İnanç, Bülend; Elçin, A Eser

    2016-01-01

    Human embryonic stem cells' (hESCs) unlimited proliferative potential and differentiation capability to all somatic cell types makes them one of the potential cell sources in cell-based tissue engineering strategies as well as various experimental applications in fields such as developmental biology, pharmacokinetics, toxicology, and genetics. Periodontal tissue engineering is an approach to reconstitute the ectomesenchymally derived alveolar bone, periodontal ligament apparatus, and cementum tissues lost as a result of periodontal diseases. Cell-based therapies may offer potential advantage in overcoming the inherent limitations associated with contemporary regenerative procedures, such as dependency on defect type and size and the pool and capacity of progenitor cells resident in the wound area. Further elucidation of developmental mechanisms associated with tooth formation may also contribute to valuable knowledge based upon which the future therapies can be designed. Protocols for the differentiation of pluripotent hESCs into periodontal ligament fibroblastic cells (PDLF) as common progenitors for ligament, cementum, and alveolar bone tissue represent an initial step in developing hESC-based experimental and tissue engineering strategies. The present protocol describes methods associated with the guided differentiation of hESCs by the use of coculture with adult PDLFs and the resulting change of morphotype and phenotype of the pluripotent embryonic stem cells toward fibroblastic and osteoblastic lineages.

  6. Mesenchymal stem cell-derived inflammatory fibroblasts promote monocyte transition into myeloid fibroblasts via an IL-6-dependent mechanism in the aging mouse heart.

    PubMed

    Cieslik, Katarzyna A; Trial, JoAnn; Entman, Mark L

    2015-08-01

    Fibrosis in the old mouse heart arises partly as a result of aberrant mesenchymal fibroblast activation. We have previously shown that endogenous mesenchymal stem cells (MSCs) in the aged heart are markedly resistant to TGF-β signaling. Fibroblasts originating from these MSCs retain their TGF-β unresponsiveness and become inflammatory. In current studies, we found that these inflammatory fibroblasts secreted higher levels of IL-6 (3-fold increase, P < 0.05) when compared with fibroblasts derived from the young hearts. Elevated IL-6 levels in fibroblasts derived from old hearts arose from up-regulated expression of Ras protein-specific guanine nucleotide releasing factor 1 (RasGrf1), a Ras activator (5-fold, P < 0.01). Knockdown of RasGrf1 by gene silencing or pharmacologic inhibition of farnesyltransferase (FTase) or ERK caused reduction of IL-6 mRNA (more than 65%, P < 0.01) and decreased levels of secreted IL-6 (by 44%, P < 0.01). In vitro, IL-6 markedly increased monocyte chemoattractant protein-1-driven monocyte-to-myeloid fibroblast formation after transendothelial migration (TEM; 3-fold, P < 0.01). In conclusion, abnormal expression of RasGrf1 promoted production of IL-6 by mesenchymal fibroblasts in the old heart. Secreted IL-6 supported conversion of monocyte into myeloid fibroblasts. This process promotes fibrosis and contributes to the diastolic dysfunction in the aging heart. © FASEB.

  7. Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    PubMed Central

    Sklirou, Aimilia D.; Ralli, Marianna; Dominguez, Maria; Papassideri, Issidora; Skaltsounis, Alexios-Leandros; Trougakos, Ioannis P.

    2015-01-01

    Despite the fact that several natural products (e.g. crude extracts or purified compounds) have been found to activate cell antioxidant responses and/or delay cellular senescence the effect(s) of small peptides on cell viability and/or modulation of protective mechanisms (e.g. the proteostasis network) remain largely elusive. We have thus studied a hexapeptide (Hexapeptide-11) of structure Phe–Val–Ala–Pro–Phe–Pro (FVAPFP) originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity. We show herein that Hexapeptide-11 exhibits no significant toxicity in normal human diploid lung or skin fibroblasts. Exposure of fibroblasts to Hexapeptide-11 promoted dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. Moreover, it promoted increased nuclear accumulation of Nrf2; higher expression levels of proteasomal protein subunits and increased proteasome peptidase activities. In line with these findings we noted that Hexapeptide-11 conferred significant protection in fibroblasts against oxidative-stress-mediated premature cellular senescence, while at in vivo skin deformation assays in human subjects it improved skin elasticity. Finally, Hexapeptide-11 was found to induce the activity of extracellular MMPs and it also suppressed cell migration. Our presented findings indicate that Hexapeptide-11 is a promising anti-ageing agent. PMID:25974626

  8. Characterization of human fibroblastic reticular cells as potential immunotherapeutic tools.

    PubMed

    Valencia, Jaris; Jiménez, Eva; Martínez, Víctor G; Del Amo, Beatriz G; Hidalgo, Laura; Entrena, Ana; Fernández-Sevilla, Lidia M; Del Río, Francisco; Varas, Alberto; Vicente, Ángeles; Sacedón, Rosa

    2017-05-01

    Fibroblastic reticular cells (FRCs) are essential players during adaptive immune responses not only as a structural support for the encounter of antigen-presenting cells and naive T lymphocytes but also as a source of modulatory signals. However, little is known about this cell population in humans. To address the phenotypical and functional analysis of human FRCs here we established splenic (SP) and mesenteric lymph node (LN) CD45(-)CD31(-)CD90(+)podoplanin(+) myofibroblastic cell cultures. They shared the phenotypical characteristics distinctive of FRCs, including the expression of immunomodulatory factors and peripheral tissue antigens. Nevertheless, human FRCs also showed particular features, some differing from mouse FRCs, like the lack of nitric oxide synthase (NOS2) expression after interferon (IFN)γstimulation. Interestingly, SP-FRCs expressed higher levels of interleukin (IL)-6, BMP4, CCL2, CXCL12 and Notch molecules, and strongly adapted their functional profile to lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly I:C) and IFNγ stimulation. In contrast, we found higher expression of transforming growth factor (TGF)β and Activin A in LN-FRCs that barely responded via Toll-Like Receptor (TLR)3 and constitutively expressed retinaldehyde dehydrogenase 1 enzyme, absent in SP-FRCs. This study reveals human FRCs can be valuable models to increase our knowledge about the physiology of human secondary lymphoid organs in health and disease and to explore the therapeutic options of FRCs.

  9. Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts.

    PubMed

    Opländer, Christian; Wetzel, Wiebke; Cortese, Miriam M; Pallua, Norbert; Suschek, Christoph V

    2008-05-01

    Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

  10. Hydrogen peroxide-induced apoptosis in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Guadarrama-Solís, Adriana; Muñoz-Seca, Carmen; Arreguín-Cano, Juan Antonio

    2015-01-01

    In the process of bleaching vital, discolored teeth, low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2. However, interest has been expressed in the assessment of pathological effects of long-term exposure to bleaching agents such as irritation and ulceration of the gingival or other soft tissues. The aim of the present study was to determine the effect of hydrogen peroxide on apoptosis in human gingival fibroblasts (HGF). Cytochrome c, Bcl-2, Bax, Bid and caspase-3 protein expression were detected by Western blotting. HGF cell apoptosis induced by H2O2 was both dose and time dependent. The addition of H2O2 resulted in the release of cytochrome c to the cytosol, and an increase of Caspase-3 cleavage. Data suggest that oxidative stress-induced apoptosis in HGF is intrinsic pathway involved the release of apoptotic signal from mitochondria.

  11. Bradykinin promotes TLR2 expression in human gingival fibroblasts.

    PubMed

    Gutiérrez-Venegas, Gloria; Arreguín-Cano, Juan Antonio

    2011-12-01

    Bradykinin (BK) is implicated in the sensation of pain, vasodilation, increases in vascular permeability and pathogenic processes associated with inflammation. Studies have shown that BK promotes the intracellular movement of calcium in human gingival fibroblasts by binding to the B2 receptor. In this study we investigated the effect of BK on regulation of Toll-like receptor 2 (TLR2) expression. Our results show that BK stimulates TLR2 receptor transcription and translation by activation of protein kinase C as well as AKT. Our study contributes important information on the regulation and expression of molecules that promote chronic inflammatory processes, which lead to periodontitis and consequently to loss of the dental organ. Copyright © 2011 Elsevier B.V. All rights reserved.

  12. Hydrogen peroxide-induced apoptosis in human gingival fibroblasts

    PubMed Central

    Gutiérrez-Venegas, Gloria; Guadarrama-Solís, Adriana; Muñoz-Seca, Carmen; Arreguín-Cano, Juan Antonio

    2015-01-01

    In the process of bleaching vital, discolored teeth, low concentrations of hydrogen peroxide (H2O2) are effective alternatives to heat-activated 30% H2O2. However, interest has been expressed in the assessment of pathological effects of long-term exposure to bleaching agents such as irritation and ulceration of the gingival or other soft tissues. The aim of the present study was to determine the effect of hydrogen peroxide on apoptosis in human gingival fibroblasts (HGF). Cytochrome c, Bcl-2, Bax, Bid and caspase-3 protein expression were detected by Western blotting. HGF cell apoptosis induced by H2O2 was both dose and time dependent. The addition of H2O2 resulted in the release of cytochrome c to the cytosol, and an increase of Caspase-3 cleavage. Data suggest that oxidative stress-induced apoptosis in HGF is intrinsic pathway involved the release of apoptotic signal from mitochondria. PMID:26884825

  13. Genotoxicity of alpha particles in human embryonic skin fibroblasts

    SciTech Connect

    Chen, D.J.; Strniste, G.F.; Tokita, N.

    1984-11-01

    Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to ..cap alpha.. particles from /sup 238/ Pu and 250 kVp X rays. The survival curves resulting from exposure to ..cap alpha.. particles are exponential. The mean lethal dose, D/sub 0/, is approximately 1.3 Gy for X rays and 0.25 Gy for ..cap alpha.. particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for ..cap alpha.. particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to ..cap alpha.. particles than to X rays.

  14. 17β-estradiol protects human skin fibroblasts and keratinocytes against oxidative damage.

    PubMed

    Bottai, G; Mancina, R; Muratori, M; Di Gennaro, P; Lotti, T

    2013-10-01

    Reactive oxygen species (ROS) cause severe damage to extracellular matrix and to molecular structure of DNA, proteins and lipids. Accumulation of these molecular changes apparently constitutes the basis of cell ageing. 17b-estradiol (E2) has a key role in skin ageing homeostasis as evidenced by the accelerated decline in skin appearance seen in the perimenopausal years. Oestrogens improve many aspects of the skin such as skin thickness, vascularization, collagen content and quality. Despite these clinical evidences, the effects of oestrogens on skin at the cellular level need further clarification. HaCaT and human fibroblasts were cultured under various conditions with E2 and H2 O2 ; then were subjected to immunofluorescence and western blot analysis. Lipoperoxidation was investigated using BODIPY. In human fibroblasts oxidative stress decreases procollagen-I synthesis, while E2 significantly increases it. Fibroblasts and HaCaT cells viability in the presence of E2 demonstrates a notably increased resistance to H2 O2 effects. Furthermore E2 is able to counteract H2 O2 -mediated lipoperoxidation and DNA oxidative damage in skin cells. In this study we highlight that the menopause-associated oestrogens decline is involved in reduced collagen production and that E2 could counteract the detrimental effects of oxidative stress on the dermal compartment during skin aging. Furthermore, our data show that physiological concentrations of oestrogens are able to interfere with ROS-mediated cell viability reduction and to protect human skin cells against oxidative damage to cellular membranes and nucleic acids structure. Our experimental data show that the presence of 17β-estradiol may protect skin cells against oxidative damage and that the dramatic lowering of oestrogen levels during menopause, could render skin more susceptible to oxidative damage. © 2012 The Authors. Journal of the European Academy of Dermatology and Venereology © 2012 European Academy of Dermatology

  15. Demonstration of adenosine deaminase activity in human fibroblast lysosomes.

    PubMed Central

    Lindley, E R; Pisoni, R L

    1993-01-01

    Human fibroblast lysosomes, purified on Percoll density gradients, contain an adenosine deaminase (ADA) activity that accounts for approximately 10% of the total ADA activity in GM0010A human fibroblasts. In assays of lysosomal ADA, the conversion of [3H]adenosine into [3H]inosine was proportional to incubation time and the amount of lysosomal material added to reaction mixtures. Maximal activity was observed between pH 7 and 8, and lysosomal ADA displayed a Km of 37 microM for adenosine at 25 degrees C and pH 5.5. Lysosomal ADA was completely inhibited by 2.5 mM Cu2+ or Hg2+ salts, but not by other bivalent cations (Ba2+, Cd2+, Ca2+, Fe2+, Mg2+, Mn2+ and Zn2+). Coformycin (2.5 mM), deoxycoformycin (0.02 mM), 2'-deoxyadenosine (2.5 mM), 6-methylaminopurine riboside (2.5 mM), 2'-3'-isopropylidene-adenosine (2.5 mM) and erythro-9-(2-hydroxy-3-nonyl)adenine (0.2 mM) inhibited lysosomal ADA by > 97%. In contrast, 2.5 mM S-adenosyl-L-homocysteine and cytosine were poor inhibitors. Nearly all lysosomal ADA activity is eluted as a high-molecular-mass protein (> 200 kDa) just after the void volume on a Sephacryl S-200 column, and is very heat-stable, retaining 70% of its activity after incubation at 65 degrees C for 80 min. We speculate that compartmentalization of ADA within lysosomes would allow deamination of adenosine to occur without competition by adenosine kinase, which could assist in maintaining cellular energy requirements under conditions of nutritional deprivation. PMID:8452534

  16. Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures.

    PubMed

    Mammone, Thomas; Gan, David; Foyouzi-Youssefi, Reyhaneh

    2006-11-01

    Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

  17. Newborn human skin fibroblasts senesce in vitro without acquiring adult growth factor requirements

    SciTech Connect

    Wharton, W.

    1984-01-01

    Cultures of human fibroblasts were prepared from chest skin obtained either from newborns (less than 3 months old) or adults (more than 35 years old) and maintained in vitro until they senesced. Adult cells grew logarithmically in medium supplemented with whole blood serum but not with platelet-poor plasma. Early passage cells obtained from newborns grew equally well in either plasma- or serum-supplemented medium. The difference in growth factor requirements between adult and newborn cells persisted through the lifespan of the cells; i.e., newborn cells did not develop adult hormonal requirements when maintained in culture. Thus, in vitro cellular aging can be distinguished from some types of differentiation.

  18. Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

    PubMed

    Sobel, Katrin; Tham, Marius; Stark, Hans-Jürgen; Stammer, Hermann; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-06-15

    Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

  19. Small Molecule Inhibition of p38 MAP Kinase Extends the Replicative Life Span of Human ATR-Seckel Syndrome Fibroblasts

    PubMed Central

    2013-01-01

    Ataxia-telangiectasia and rad3 (ATR)-related Seckel syndrome is associated with growth retardation and premature aging features. ATR-Seckel fibroblasts have a reduced replicative capacity in vitro and an aged morphology that is associated with activation of stress-associated p38 mitogen-activated protein kinase and phosphorylated HSP27. These phenotypes are prevented using p38 inhibitors, with replicative capacity restored to the normal range. However, this stressed phenotype is retained in telomerase-immortalized ATR-Seckel fibroblasts, indicating that it is independent of telomere erosion. As with normal fibroblasts, senescence in ATR-Seckel is bypassed by p53 abrogation. Young ATR-Seckel fibroblasts show elevated levels of p21WAF1, p16INK4A, phosphorylated actin-binding protein cofilin, and phosphorylated caveolin-1, with small molecule drug inhibition of p38 reducing p16INK4A and caveolin-1 phosphorylation. In conclusion, ATR-Seckel fibroblasts undergo accelerated aging via stress-induced premature senescence and p38 activation that may underlie certain clinical features of Seckel syndrome, and our data suggest a novel target for pharmacological intervention in this human syndrome. PMID:23401567

  20. Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

    SciTech Connect

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo; Kim, So Young; Jang, Hwan-Hee; Ryu, Sung Ho; Kim, Beom Joon; Lee, Taehoon G.

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. Black-Right-Pointing-Pointer YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. Black-Right-Pointing-Pointer There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. Black-Right-Pointing-Pointer The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. Black-Right-Pointing-Pointer The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929-933 sequence of the {beta}1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate

  1. Molecular cloning and characterization of plastin, a human leukocyte protein expressed in transformed human fibroblasts.

    PubMed Central

    Lin, C S; Aebersold, R H; Kent, S B; Varma, M; Leavitt, J

    1988-01-01

    The phosphoprotein plastin was originally identified as an abundant transformation-induced polypeptide of chemically transformed neoplastic human fibroblasts. This abundant protein is normally expressed only in leukocytes, suggesting that it may play a role in hemopoietic cell differentiation. Protein microsequencing of plastin purified from leukemic T lymphocytes by high-resolution two-dimensional gel electrophoresis produced eight internal oligopeptide sequences. An oligodeoxynucleotide probe corresponding to one of the oligopeptides was used to clone cDNAs from transformed human fibroblasts that encoded the seven other oligopeptides predicted for human plastin. Sequencing and characterization of two cloned cDNAs revealed the existence of two distinct, but closely related, isoforms of plastin--l-plastin, which is expressed in leukocytes and transformed fibroblasts, and t-plastin, which is expressed in normal cells of solid tissues and transformed fibroblasts. The leukocyte isoform l-plastin is expressed in a diverse variety of human tumor cell lines, suggesting that it may be involved in the neoplastic process of some solid human tumors. Images PMID:3211125

  2. TGF-β1 induces differentiation of papillary fibroblasts to reticular fibroblasts in monolayer culture but not in human skin equivalents.

    PubMed

    Janson, David; Saintigny, Gaëlle; Zeypveld, Jeroen; Mahé, Christian; El Ghalbzouri, Abdoelwaheb

    2014-01-01

    Fibroblasts isolated from the papillary and reticular dermis are different from each other in vitro. If papillary fibroblasts are subjected to prolonged serial passaging they will differentiate into reticular fibroblasts. Reticular fibroblasts have been shown to resemble myofibroblasts in several ways. TGF-β1 is the most important factor involved in myofibroblast differentiation. we investigated if TGF-β1 can induce differentiation of papillary fibroblasts into reticular fibroblasts, in monolayer cultures and in human skin equivalents. Monolayer cultures of and human skin equivalents generated with papillary fibroblasts were stimulated with TGF-β1. The expression of markers specific for reticular and papillary fibroblasts was measured by qPCR and immunohistochemical analysis in monolayer cultures. In human skin equivalents, the morphology and the expression of several markers was analysed and compared to untreated papillary and reticular human skin equivalents. Monolayer cultures of papillary fibroblasts started to express a reticular marker profile after stimulation with TGF-β1. Human skin equivalents generated with papillary fibroblast and stimulated with TGF-β1 were similar to papillary control equivalents and did not obtain reticular characteristics. Expression of reticular markers was only found in the lower layers of TGF-β1-stimulated papillary skin equivalents. TGF-β1 can induce differentiation to reticular fibroblasts in monolayer cultures of papillary fibroblasts. In skin equivalents no such effects were found. The major difference between these experiments is the presence of extracellular matrix in skin equivalents. Therefore, we hypothesize that the matrix secreted by papillary fibroblasts protects them from TGF-β1 induced differentiation.

  3. In vitro cytotoxicity of hydrothermally synthesized ZnO nanoparticles on human periodontal ligament fibroblast and mouse dermal fibroblast cells.

    PubMed

    Seker, Sükran; Elçin, A Eser; Yumak, Tuğrul; Sınağ, Ali; Elçin, Y Murat

    2014-12-01

    The use of metal oxide nanoparticles (NPs) in industrial applications has been expanding, as a consequence, risk of human exposure increases. In this study, the potential toxic effects of zinc oxide (ZnO) NPs on human periodontal ligament fibroblast cells (hPDLFs) and on mouse dermal fibroblast cells (mDFs) were evaluated in vitro. We synthesized ZnO NPs (particle size; 7-8 nm) by the hydrothermal method. Characterization assays were performed with atomic force microscopy, Braun-Emmet-Teller analysis, and dynamic light scattering. The hPDLFs and mDFs were incubated with the NPs with concentrations of 0.1, 1, 10, 50 and 100 μg/mL for 6, 24 and 48h. Under the control and NP-exposed conditions, we have made different types of measurements for cell viability and morphology, membrane leakage and intracellular reactive oxygen species generation. Also, we monitored cell responses to ZnO NPs using an impedance measurement system in real-time. While the morphological changes were visualized using scanning electron microscopy, the subcellular localization of NPs was investigated by transmission electron microscopy. Results indicated that ZnO NPs have significant toxic effects on both of the primary fibroblastic cells at concentrations of ∼50-100 μg/mL. The cytotoxicity of ZnO NPs on fibroblasts depended on concentration and duration of exposure.

  4. Metformin Alleviates Aging Cellular Phenotypes in Hutchinson-Gilford Progeria Syndrome Dermal Fibroblasts.

    PubMed

    Park, Seul-Ki; Shin, Ok Sarah

    2017-02-13

    Metformin is a popular antidiabetic biguanide, which has been considered as a candidate drug for cancer treatment and aging prevention. Hutchinson-Gilford progeria syndrome (HGPS) is a devastating disease characterized by premature aging and severe age-associated complications leading to death. The effects of metformin on HGPS dermal fibroblasts remain largely undefined. In this study, we investigated whether metformin could exert a beneficial effect on nuclear abnormalities and delay senescence in fibroblasts derived from HGPS patients. Metformin treatment partially restored normal nuclear phenotypes, delayed senescence, activated the phosphorylation of AMP-activated protein kinase, and decreased reactive oxygen species formation in HGPS dermal fibroblasts. Interestingly, metformin reduced the number of phosphorylated histone variant H2AX-positive DNA damage foci and suppressed progerin protein expression, compared to the control. Furthermore, metformin-supplemented aged mice showed higher splenocyte proliferation and mRNA expression of the antioxidant enzyme, superoxide dismutase 2 than the control mice. Collectively, our results show that metformin treatment alleviates the nuclear defects and premature aging phenotypes in HGPS fibroblasts. Thus, metformin can be considered a promising therapeutic approach for life extension in HGPS. This article is protected by copyright. All rights reserved.

  5. Sleep and Human Aging.

    PubMed

    Mander, Bryce A; Winer, Joseph R; Walker, Matthew P

    2017-04-05

    Older adults do not sleep as well as younger adults. Why? What alterations in sleep quantity and quality occur as we age, and are there functional consequences? What are the underlying neural mechanisms that explain age-related sleep disruption? This review tackles these questions. First, we describe canonical changes in human sleep quantity and quality in cognitively normal older adults. Second, we explore the underlying neurobiological mechanisms that may account for these human sleep alterations. Third, we consider the functional consequences of age-related sleep disruption, focusing on memory impairment as an exemplar. We conclude with a discussion of a still-debated question: do older adults simply need less sleep, or rather, are they unable to generate the sleep that they still need?

  6. Key Regulatory Role of Dermal Fibroblasts in Pigmentation as Demonstrated Using a Reconstructed Skin Model: Impact of Photo-Aging

    PubMed Central

    Duval, Christine; Cohen, Catherine; Chagnoleau, Corinne; Flouret, Virginie; Bourreau, Emilie; Bernerd, Françoise

    2014-01-01

    To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is

  7. Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging.

    PubMed

    Duval, Christine; Cohen, Catherine; Chagnoleau, Corinne; Flouret, Virginie; Bourreau, Emilie; Bernerd, Françoise

    2014-01-01

    To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3) was compared to that of tissues containing natural photo-aged fibroblasts (n = 3) which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance) and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the hyperpigmentation that is

  8. Cultured human synovial fibroblasts rapidly metabolize kinins and neuropeptides.

    PubMed Central

    Bathon, J M; Proud, D; Mizutani, S; Ward, P E

    1992-01-01

    Kinins and substance P have been implicated in the pathogenesis of inflammatory arthritis by virtue of their abilities to induce vasodilation, edema, and pain. The relative biological potencies of these peptides in vivo would depend at least in part upon their rates of catabolism in the joint. We hypothesized that human synovial lining cells may regulate intraarticular levels of kinins and neuropeptides via degradation by cell surface-associated peptidases. We exposed intact human synovial fibroblasts to kinins and substance P, in the presence or absence of specific peptidase inhibitors, and measured the amount of intact substrate remaining and degradation product(s) generated over time. Aminopeptidase M (AmM; EC 3.4.11.2), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and dipeptidyl(amino)peptidase IV (DAP IV; EC 3.4.14.5) were identified on the cell surface of synovial cells. Bradykinin degradation was due entirely to NEP-24.11 (1.39 +/- 0.29 nmol/min per well). Lysylbradykinin was also degraded by NEP-24.11 (0.80 +/- 0.19 nmol/min per well); however, in the presence of phosphoramidon, AmM-mediated conversion to bradykinin (3.74 +/- 0.46 nmol/min per well) could be demonstrated. The combined actions of NEP-24.11 (0.93 +/- 0.15 nmol/min per well) and DAP IV (0.84 +/- 0.18 nmol/min per well) were responsible for the degradation of substance P. AmM (2.44 +/- 0.33 nmol/min per well) and NEP-24.11 (1.30 +/- 0.45 nmol/min per well) were responsible for the degradation of the opioid peptide, [Leu5]enkephalin. The identity of each of the three peptidases was confirmed via synthetic substrate hydrolysis, inhibition profile, and immunological identification. The profiles of peptidase enzymes identified in cells derived from rheumatoid and osteoarthritic joints were identical. These data demonstrate the human synovial fibroblast to be a rich source of three specific peptidases and suggest that it may play a prominent role in regulating peptide levels in the joint

  9. Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

    PubMed Central

    Janmaat, C. J.; de Rooij, K. E; Locher, H; de Groot, S. C.; de Groot, J. C. M. J.; Frijns, J. H. M.; Huisman, M. A.

    2015-01-01

    In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality. PMID:26678612

  10. Bryostatin and its synthetic analog, picolog rescue dermal fibroblasts from prolonged stress and contribute to survival and rejuvenation of human skin equivalents.

    PubMed

    Khan, Tapan K; Wender, Paul A; Alkon, Daniel L

    2017-06-07

    Skin health is associated with the day-to-day activity of fibroblasts. The primary function of fibroblasts is to synthesize structural proteins, such as collagen, extracellular matrix proteins, and other proteins that support the structural integrity of the skin and are associated with younger, firmer, and more elastic skin that is better able to resist and recover from injury. At sub-nanomolar concentrations (0.03-0.3 nM), bryostatin-1 and its synthetic analog, picolog (0.1-10 nM) sustained the survival and activation of human dermal fibroblasts cultured under the stressful condition of prolonged serum deprivation. Bryostatin-1 treatment stabilized human skin equivalents (HSEs), a bioengineered combination of primary human skin cells (keratinocytes and dermal fibroblasts) on an extracellular matrix composed of mainly collagen. Fibroblasts activated by bryostatin-1 protected the structural integrity of HSEs. Bryostatin-1 and picolog prolonged activation of Erk in fibroblasts to promote cell survival. Chronic stress promotes the progression of apoptosis. Dermal fibroblasts constitutively express all components of Fas associated apoptosis, including caspase-8, an initiator enzyme of apoptosis. Prolong bryostatin-1 treatment reduced apoptosis by decreasing caspase-8 and protected dermal fibroblasts. Our data suggest that bryostatin-1 and picolog could be useful in anti-aging skincare, and could have applications in tissue engineering and regenerative medicine. © 2017 Wiley Periodicals, Inc.

  11. Catalytically inactive human cathepsin D triggers fibroblast invasive growth

    PubMed Central

    Laurent-Matha, Valérie; Maruani-Herrmann, Sharon; Prébois, Christine; Beaujouin, Mélanie; Glondu, Murielle; Noël, Agnès; Alvarez-Gonzalez, Marie Luz; Blacher, Sylvia; Coopman, Peter; Baghdiguian, Stephen; Gilles, Christine; Loncarek, Jadranka; Freiss, Gilles; Vignon, Françoise; Liaudet-Coopman, Emmanuelle

    2005-01-01

    The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial–fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D–deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras–MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity. PMID:15668295

  12. Telomeres on chromosome 21 and aging in lymphocytes and gingival fibroblasts from individuals with Down syndrome.

    PubMed

    de Arruda Cardoso Smith, Marília; Borsatto-Galera, Bianca; Feller, Roger Israel; Gonçalves, Alaíde; Oyama, Rosa Sayoto Kawasaki; Segato, Rosemeire; Chen, Elizabeth; Carvalheira, Gianna Maria Griz; Filho, Antonio Santos Clemente; Burbano, Rommel Rodríguez; Payão, Spencer Luiz Marques

    2004-09-01

    Progressive chromosome 21 loss in individuals with trisomy 21 or Down syndrome (DS) is supposedly related to their premature senescence. In addition, the telomere hypothesis of cellular aging involving telomere shortening in normal and accelerated aging in vivo and in vitro is well documented. This study investigated the integrity of two chromosome 21 regions (the 21q telomere and the 21q22.13-q22.2 region) and their relationship with aging by means of fluorescence in situ hybridization (FISH) in lymphocytes and gingival fibroblasts cells. The use of tissues from different germ layers allows detection of mosaicism. Chromosome variations in tissue from the neuroectoderm layer could explain the variable phenotype of DS. This approach is original in the literature. Lymphocyte and gingival fibroblast nuclei from 18 affected individuals aged 5-54 years were analyzed. Although not significant (P = 0.06), analysis from 11 tissue-matched individuals as well as the comparison between lymphocytes and fibroblasts from different subjects (P = 0.05) suggested that lymphocyte cells are more likely to miss 21q telomere signals. Hence, gingival fibroblasts are probably capable of more efficient cell repair, and the occurrence of mosaicism is more related to cell proliferation than to germ layer origin. Investigation of the 21q22.13-q22.2 region from six tissue-matched individuals and from different DS patients revealed no significant differences between the tissues.

  13. Human cytomegalovirus replicates in gamma-irradiated fibroblasts

    SciTech Connect

    Shanley, J.D.

    1986-12-01

    Because of the unique interdependence of human cytomegalovirus (HCMV) and the physiological state of the host cell, we evaluated the ability of human foreskin fibroblasts (HFF), exposed to gamma radiation, to support HCMV growth. Irradiation of HFF with 2,500 rADS prevented cellular proliferation and suppressed cellular DNA, but not RNA or protein synthesis. Treatment of HFF cells with 2,500 rADS 6 or 48 hours prior to infection did not alter the time course or virus yield during HCMV replication. Virus plaquing efficiency in irradiated cells was comparable to that of nonirradiated cells. As judged by thymidine incorporation and BUdR inhibition of virus replication, HCMV infection induced both thymidine kinase activity and host cell DNA synthesis in irradiated cells. In addition, virus could be recovered from HFF exposed to radiation 0-2 days after infection with HCMV. These studies indicate that the damage to cells by gamma irradiation does not alter the capacity of host cells to support HCMV replication.

  14. Nerve regeneration by human corneal stromal keratocytes and stromal fibroblasts

    PubMed Central

    Yam, Gary Hin-Fai; Williams, Geraint P.; Setiawan, Melina; Yusoff, Nur Zahirah Binte M.; Lee, Xiao-wen; Htoon, Hla Myint; Zhou, Lei; Fuest, Matthias; Mehta, Jodhbir S.

    2017-01-01

    Laser refractive surgeries reshape corneal stroma to correct refractive errors, but unavoidably affect corneal nerves. Slow nerve regeneration and atypical neurite morphology cause desensitization and neuro-epitheliopathy. Following injury, surviving corneal stromal keratocytes (CSKs) are activated to stromal fibroblasts (SFs). How these two different cell types influence nerve regeneration is elusive. Our study evaluated the neuro-regulatory effects of human SFs versus CSKs derived from the same corneal stroma using an in vitro chick dorsal root ganglion model. The neurite growth was assessed by a validated concentric circle intersection count method. Serum-free conditioned media (CM) from SFs promoted neurite growth dose-dependently, compared to that from CSKs. We detected neurotrophic and pro-inflammatory factors (interleukin-8, interleukin-15, monocyte chemoattractant protein-1, eotaxin, RANTES) in SFCM by Bio-Plex Human Cytokine assay. More than 130 proteins in SFCM and 49 in CSKCM were identified by nanoLC-MS/MS. Proteins uniquely present in SFCM had reported neuro-regulatory activities and were predicted to regulate neurogenesis, focal adhesion and wound healing. Conclusively, this was the first study showing a physiological relationship between nerve growth and the metabolically active SFs versus quiescent CSKs from the same cornea source. The dose-dependent effect on neurite growth indicated that nerve regeneration could be influenced by SF density. PMID:28349952

  15. Lactic Acid Bacteria Convert Human Fibroblasts to Multipotent Cells

    PubMed Central

    Ohta, Kunimasa; Kawano, Rie; Ito, Naofumi

    2012-01-01

    The human gastrointestinal tract is colonized by a vast community of symbionts and commensals. Lactic acid bacteria (LAB) form a group of related, low-GC-content, gram-positive bacteria that are considered to offer a number of probiotic benefits to general health. While the role of LAB in gastrointestinal microecology has been the subject of extensive study, little is known about how commensal prokaryotic organisms directly influence eukaryotic cells. Here, we demonstrate the generation of multipotential cells from adult human dermal fibroblast cells by incorporating LAB. LAB-incorporated cell clusters are similar to embryoid bodies derived from embryonic stem cells and can differentiate into endodermal, mesodermal, and ectodermal cells in vivo and in vitro. LAB-incorporated cell clusters express a set of genes associated with multipotency, and microarray analysis indicates a remarkable increase of NANOG, a multipotency marker, and a notable decrease in HOX gene expression in LAB-incorporated cells. During the cell culture, the LAB-incorporated cell clusters stop cell division and start to express early senescence markers without cell death. Thus, LAB-incorporated cell clusters have potentially wide-ranging implications for cell generation, reprogramming, and cell-based therapy. PMID:23300571

  16. Mitochondrial oxidative stress in cancer-associated fibroblasts drives lactate production, promoting breast cancer tumor growth: understanding the aging and cancer connection.

    PubMed

    Balliet, Renee M; Capparelli, Claudia; Guido, Carmela; Pestell, Timothy G; Martinez-Outschoorn, Ubaldo E; Lin, Zhao; Whitaker-Menezes, Diana; Chiavarina, Barbara; Pestell, Richard G; Howell, Anthony; Sotgia, Federica; Lisanti, Michael P

    2011-12-01

    Increasing chronological age is the most significant risk factor for cancer. Recently, we proposed a new paradigm for understanding the role of the aging and the tumor microenvironment in cancer onset. In this model, cancer cells induce oxidative stress in adjacent stromal fibroblasts. This, in turn, causes several changes in the phenotype of the fibroblast including mitochondrial dysfunction, hydrogen peroxide production, and aerobic glycolysis, resulting in high levels of L-lactate production. L-lactate is then transferred from these glycolytic fibroblasts to adjacent epithelial cancer cells and used as "fuel" for oxidative mitochondrial metabolism.  Here, we created a new pre-clinical model system to directly test this hypothesis experimentally. To synthetically generate glycolytic fibroblasts, we genetically-induced mitochondrial dysfunction by knocking down TFAM using an sh-RNA approach.  TFAM is mitochondrial transcription factor A, which is important in functionally maintaining the mitochondrial respiratory chain. Interestingly, TFAM-deficient fibroblasts showed evidence of mitochondrial dysfunction and oxidative stress, with the loss of certain mitochondrial respiratory chain components, and the over-production of hydrogen peroxide and L-lactate. Thus, TFAM-deficient fibroblasts underwent metabolic reprogramming towards aerobic glycolysis.  Most importantly, TFAM-deficient fibroblasts significantly promoted tumor growth, as assayed using a human breast cancer (MDA-MB-231) xenograft model. These increases in glycolytic fibroblast driven tumor growth were independent of tumor angiogenesis. Mechanistically, TFAM-deficient fibroblasts increased the mitochondrial activity of adjacent epithelial cancer cells in a co-culture system, as seen using MitoTracker. Finally, TFAM-deficient fibroblasts also showed a loss of caveolin-1 (Cav-1), a known breast cancer stromal biomarker. Loss of stromal fibroblast Cav-1 is associated with early tumor recurrence, metastasis

  17. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    SciTech Connect

    Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  18. Human gingival fibroblasts display a non-fibrotic phenotype distinct from skin fibroblasts in three-dimensional cultures.

    PubMed

    Mah, Wesley; Jiang, Guoqiao; Olver, Dylan; Cheung, Godwin; Kim, Ben; Larjava, Hannu; Häkkinen, Lari

    2014-01-01

    Scar formation following skin injury can be a major psychosocial and physiological problem. However, the mechanisms of scar formation are still not completely understood. Previous studies have shown that wound healing in oral mucosa is faster, associates with a reduced inflammatory response and results to significantly reduced scar formation compared with skin wounds. In the present study, we hypothesized that oral mucosal fibroblasts from human gingiva are inherently distinct from fibroblasts from breast and abdominal skin, two areas prone to excessive scar formation, which may contribute to the preferential wound healing outcome in gingiva. To this end, we compared the phenotype of human gingival and skin fibroblasts cultured in in vivo-like three-dimensional (3D) cultures that mimic the cells' natural extracellular matrix (ECM) niche. To establish 3D cultures, five parallel fibroblast lines from human gingiva (GFBLs) and breast skin (SFBLs) were seeded in high density, and cultured for up to 21 days in serum and ascorbic acid containing medium to induce expression of wound-healing transcriptome and ECM deposition. Cell proliferation, morphology, phenotype and expression of wound healing and scar related genes were analyzed by real-time RT-PCR, Western blotting and immunocytochemical methods. The expression of a set of genes was also studied in three parallel lines of human abdominal SFBLs. Findings showed that GFBLs displayed morphologically distinct organization of the 3D cultures and proliferated faster than SFBLs. GFBLs expressed elevated levels of molecules involved in regulation of inflammation and ECM remodeling (MMPs) while SFBLs showed significantly higher expression of TGF-β signaling, ECM and myofibroblast and cell contractility-related genes. Thus, GFBLs display an inherent phenotype conducive for fast resolution of inflammation and ECM remodeling, characteristic for scar-free wound healing, while SFBLs have a profibrotic, scar-prone phenotype.

  19. MiRNA profile associated with replicative senescence, extended cell culture, and ectopic telomerase expression in human foreskin fibroblasts.

    PubMed

    Bonifacio, Laura N; Jarstfer, Michael B

    2010-09-01

    Senescence is a highly regulated process that limits cellular replication by enforcing a G1 arrest in response to various stimuli. Replicative senescence occurs in response to telomeric DNA erosion, and telomerase expression can offset replicative senescence leading to immortalization of many human cells. Limited data exists regarding changes of microRNA (miRNA) expression during senescence in human cells and no reports correlate telomerase expression with regulation of senescence-related miRNAs. We used miRNA microarrays to provide a detailed account of miRNA profiles for early passage and senescent human foreskin (BJ) fibroblasts as well as early and late passage immortalized fibroblasts (BJ-hTERT) that stably express the human telomerase reverse transcriptase subunit hTERT. Selected miRNAs that were differentially expressed in senescence were assayed for expression in quiescent cells to identify miRNAs that are specifically associated with senescence-associated growth arrest. From this group of senescence-associated miRNAs, we confirmed the ability of miR-143 to induce growth arrest after ectopic expression in young fibroblasts. Remarkably, miR-143 failed to induce growth arrest in BJ-hTERT cells. Importantly, the comparison of late passage immortalized fibroblasts to senescent wild type fibroblasts reveals that miR-146a, a miRNA with a validated role in regulating the senescence associated secretory pathway, is also regulated during extended cell culture independently of senescence. The discovery that miRNA expression is impacted by expression of ectopic hTERT as well as extended passaging in immortalized fibroblasts contributes to a comprehensive understanding of the connections between telomerase expression, senescence and processes of cellular aging.

  20. [Protein oxidation in the aging of skin fibroblasts].

    PubMed

    Grune, T

    2003-09-01

    The ageing process is accompanied by enhanced oxidative damage. All cellular components including proteins are affected by oxidation. Within the cell, the proteasome is responsible for the degradation of these oxidised proteins. During the ageing process this function of the proteasome is increasingly diminished, therefore oxidised proteins accumulate. Furthermore lipofuscin, a highly cross-linked and modified protein aggregate, is formed. This aggregate accumulates within cells and is able to inhibit the proteasome. The nucleus of the cells is less affected by these changes due to the lack of intranuclear lipofuscin accumulation.

  1. Mesenchymal stem cell-derived inflammatory fibroblasts mediate interstitial fibrosis in the aging heart.

    PubMed

    Trial, JoAnn; Entman, Mark L; Cieslik, Katarzyna A

    2016-02-01

    Pathologic fibrosis in the aging mouse heart is associated with dysregulated resident mesenchymal stem cells (MSC) arising from reduced stemness and aberrant differentiation into dysfunctional inflammatory fibroblasts. Fibroblasts derived from aging MSC secrete higher levels of 1) collagen type 1 (Col1) that directly contributes to fibrosis, 2) monocyte chemoattractant protein-1 (MCP-1) that attracts leukocytes from the blood and 3) interleukin-6 (IL-6) that facilitates transition of monocytes into myeloid fibroblasts. The transcriptional activation of these proteins is controlled via the farnesyltransferase (FTase)-Ras-Erk pathway. The intrinsic change in the MSC phenotype acquired by advanced age is specific for the heart since MSC originating from bone wall (BW-MSC) or fibroblasts derived from them were free of these defects. The potential therapeutic interventions other than clinically approved strategies based on findings presented in this review are discussed as well. This article is a part of a Special Issue entitled "Fibrosis and Myocardial Remodeling". Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Effects of Calendula officinalis on human gingival fibroblasts.

    PubMed

    Saini, Pragtipal; Al-Shibani, Nouf; Sun, Jun; Zhang, Weiping; Song, Fengyu; Gregson, Karen S; Windsor, L Jack

    2012-04-01

    Calendula officinalis is commonly called the marigold. It is a staple topical remedy in homeopathic medicine. It is rich in quercetin, carotenoids, lutein, lycopene, rutin, ubiquinone, xanthophylls, and other anti-oxidants. It has anti-inflammatory properties. Quercetin, one of the active components in Calendula, has been shown to inhibit recombinant human matrix metalloproteinase (MMP) activity and decrease the expression of tumor necrosis factor-α, interleukin-1β (IL), IL-6 and IL-8 in phorbol 12-myristate 13-acetate and calcium ionophore-stimulated human mast cells. To examine the effects of Calendula on human gingival fibroblast (HGF) mediated collagen degradation and MMP activity. Lactate dehydrogenate assays were performed to determine the non-toxic concentrations of Calendula, doxycycline and quercetin. Cell-mediated collagen degradation assays were performed to examine the inhibitory effect on cell-mediated collagen degradation. Gelatin zymography was performed to examine their effects on MMP-2 activity. The experiments were repeated three times and ANOVA used for statistical analyses. Calendula at 2-3% completely inhibited the MMP-2 activity in the zymograms. Doxycycline inhibited HGF-mediated collagen degradation at 0.005, 0.01, 0.02 and 0.05%, and MMP-2 activity completely at 0.05%. Quercetin inhibited HGF-mediated collagen degradation at 0.005, 0.01 and 0.02%, and MMP-2 activity in a dose-dependent manner. Calendula inhibited HGF-mediated collagen degradation and MMP-2 activity more than the same correlated concentration of pure quercetin. Calendula inhibits HGF-mediated collagen degradation and MMP-2 activity more than the corresponding concentration of quercetin. This may be attributed to additional components in Calendula other than quercetin. Published by Elsevier Ltd.

  3. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee.

    PubMed

    Advani, Alexander S; Chen, Annie Y; Babbitt, Courtney C

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors.

  4. Human fibroblasts display a differential focal adhesion phenotype relative to chimpanzee

    PubMed Central

    Advani, Alexander S.; Chen, Annie Y.; Babbitt, Courtney C.

    2016-01-01

    There are a number of documented differences between humans and our closest relatives in responses to wound healing and in disease susceptibilities, suggesting a differential cellular response to certain environmental factors. In this study, we sought to look at a specific cell type, fibroblasts, to examine differences in cellular adhesion between humans and chimpanzees in visualized cells and in gene expression. We have found significant differences in the number of focal adhesions between primary human and chimpanzee fibroblasts. Additionally, we see that adhesion related gene ontology categories are some of the most differentially expressed between human and chimpanzee in normal fibroblast cells. These results suggest that human and chimpanzee fibroblasts may have somewhat different adhesive properties, which could play a role in differential disease phenotypes and responses to external factors. PMID:26971204

  5. Kinetics of staurosporine-arrest of human diploid fibroblasts

    SciTech Connect

    Stevenson, A.P.; Bustos, L.D.; Dickson, J.A.; Habbersett, R.C.; Crissman, H.A. )

    1993-01-01

    The authors have previously shown that the general protein kinase inhibitor, staurosporine (Stsp), has a differential effect on the progression of normal and transformed cells. Low levels of Stsp (1.0 ng/ml) reversibly arrested normal diploid cells in early G1 phase, whereas concentrations of Stsp as high as 50-75 ng/ml had no effect on G1 progression in transformed cells. High concentrations of Stsp arrested normal cells in G1 and G2 phases but blocked transformed cells only in G2 phase of the cell cycle. To follow the accumulation of cells in G1 and/or G2 phases, asynchronous cultures of human diploid fibroblasts were treated with 0, 1, 5, 10, 20, 30 or 50 ng/ml staurosporine for 18 hours in the presence of bromodeoxyuridine. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that monitored emission of fluorescence from DNA-species stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Bivariate plots of Hoechst/mithramycin fluorescence signal ratio vs. Hoechst fluorescence identified the arrested cells as cohorts of cells in different positions across cell cycle at the time of Stsp addition.

  6. Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity.

    PubMed

    Breslin, Loretta; Prosser, Suzanna L; Cuffe, Sandra; Morrison, Ciaran G

    2014-01-01

    Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence.

  7. Ciliary abnormalities in senescent human fibroblasts impair proliferative capacity

    PubMed Central

    Breslin, Loretta; Prosser, Suzanna L; Cuffe, Sandra; Morrison, Ciaran G

    2014-01-01

    Somatic cells senesce in culture after a finite number of divisions indefinitely arresting their proliferation. DNA damage and senescence increase the cellular number of centrosomes, the 2 microtubule organizing centers that ensure bipolar mitotic spindles. Centrosomes also provide the basal body from which primary cilia extend to sense and transduce various extracellular signals, notably Hedgehog. Primary cilium formation is facilitated by cellular quiescence a temporary cell cycle exit, but the impact of senescence on cilia is unknown. We found that senescent human fibroblasts have increased frequency and length of primary cilia. Levels of the negative ciliary regulator CP110 were reduced in senescent cells, as were levels of key elements of the Hedgehog pathway. Hedgehog inhibition reduced proliferation in young cells with increased cilium length accompanying cell cycle arrest suggesting a regulatory function for Hedgehog in primary ciliation. Depletion of CP110 in young cell populations increased ciliation frequencies and reduced cell proliferation. These data suggest that primary cilia are potentially novel determinants of the reduced cellular proliferation that initiates senescence. PMID:25486364

  8. Conformation and activity of recombinant human fibroblast interferon-beta.

    PubMed

    Boublik, M; Moschera, J A; Wei, C; Kung, H F

    1990-04-01

    Conformation of highly purified recombinant human fibroblast interferon-beta (rHuIFN-beta) was correlated with its biological activity. The extent of ordered secondary structure was determined by circular dichroic (CD) spectroscopy in various buffer conditions to establish conditions of protein stability and its potential for helix formation. The highest "helicity" (about 50 +/- 5% of alpha-helices) and the highest antiviral activities (4-10 x 10(7) units/mg) were found in 50% ethylene glycol, 1 M NaCl and 0.05 M Na3PO4, pH 7.2 (Buffer I); 80 mM citric acid, 20 mM Na2HPO4, pH 2.9 (Buffer II); and 25 mM NH4OAc, 125 mM NaCl, pH 5.1 (Buffer III). Both helicity and antiviral activity of the IFN-beta decrease in parallel with denaturation by urea, heat, and/or by repeated cycles of freezing and thawing. Low pH (pH 2.9 Buffer II) exhibits a distinct stabilizing effect on the structure and antiviral activity of IFN-beta against heat denaturation.

  9. The Nature of the Collagen Synthesized by Cultured Human Fibroblasts

    PubMed Central

    Layman, Don L.; McGoodwin, Ermona B.; Martin, George R.

    1971-01-01

    The hydroxyproline-containing proteins (hyproproteins) synthesized by cultured human fibroblasts have been partially characterized. The hyproprotein extracted from the cell layer was found to be similar to the collagen extracted from skin in the ratio of hydroxyproline to proline, chain composition, solubility, and resistance to proteolytic digestion. The hyproproteins isolated from the medium were different. About 20% of the peptide-bound hydroxyproline was found in randomly coiled chains. The α2 chains were present in considerable excess over the α1 chains, suggesting that the α2 chain may be synthesized in quantities greater than required to form a collagen molecule with a chain composition (α1)2α2. The remaining medium hyproprotein appeared to be an unusual form of native collagen which, unlike typical native collagen, was soluble under physiological conditions. This hyproprotein did not yield α chains when denatured and contained material that had a molecular weight greater than α chains. A similar size distribution was observed in the protein synthesized in the presence of β-aminopropionitrile, a specific inhibitor of collagen cross-linking. After treatment with pepsin, typical α1 and α2 chains were obtained from the protein in a 2:1 ratio. Since the medium protein is soluble and has properties different from the typical collagen molecule, it may represent a modified form that functions in the transport of collagen from the cell to the fiber. PMID:5277100

  10. PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

    PubMed

    Khamaisi, Mogher; Katagiri, Sayaka; Keenan, Hillary; Park, Kyoungmin; Maeda, Yasutaka; Li, Qian; Qi, Weier; Thomou, Thomas; Eschuk, Danielle; Tellechea, Ana; Veves, Aris; Huang, Chenyu; Orgill, Dennis Paul; Wagers, Amy; King, George L

    2016-03-01

    Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients.

  11. PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts

    PubMed Central

    Khamaisi, Mogher; Katagiri, Sayaka; Keenan, Hillary; Park, Kyoungmin; Maeda, Yasutaka; Li, Qian; Qi, Weier; Thomou, Thomas; Eschuk, Danielle; Tellechea, Ana; Veves, Aris; Huang, Chenyu; Orgill, Dennis Paul; Wagers, Amy; King, George L.

    2016-01-01

    Abnormal fibroblast function underlies poor wound healing in patients with diabetes; however, the mechanisms that impair wound healing are poorly defined. Here, we evaluated fibroblasts from individuals who had type 1 diabetes (T1D) for 50 years or more (Medalists, n = 26) and from age-matched controls (n = 7). Compared with those from controls, Medalist fibroblasts demonstrated a reduced migration response to insulin, lower VEGF expression, and less phosphorylated AKT (p-AKT), but not p-ERK, activation. Medalist fibroblasts were also functionally less effective at wound closure in nude mice. Activation of the δ isoform of protein kinase C (PKCδ) was increased in postmortem fibroblasts from Medalists, fibroblasts from living T1D subjects, biopsies of active wounds of living T1D subjects, and granulation tissues from mice with streptozotocin-induced diabetes. Diabetes-induced PKCD mRNA expression was related to a 2-fold increase in the mRNA half-life. Pharmacologic inhibition and siRNA-mediated knockdown of PKCδ or expression of a dominant-negative isoform restored insulin signaling of p-AKT and VEGF expression in vitro and improved wound healing in vivo. Additionally, increasing PKCδ expression in control fibroblasts produced the same abnormalities as those seen in Medalist fibroblasts. Our results indicate that persistent PKCδ elevation in fibroblasts from diabetic patients inhibits insulin signaling and function to impair wound healing and suggest PKCδ inhibition as a potential therapy to improve wound healing in diabetic patients. PMID:26808499

  12. Alleviation of behavioral deficits in aged rodents following implantation of encapsulated GDNF-producing fibroblasts.

    PubMed

    Emerich, D F; Plone, M; Francis, J; Frydel, B R; Winn, S R; Lindner, M D

    1996-10-14

    The present study examined the effects of encapsulated cells which were genetically modified to secrete human glail-derived neurotrophic factor (hGDNF) on the motor deficits in aged rodents. Prior to implantation, animals were tested on a battery of motor tasks. Spontaneous locomotion and motor coordination was evaluated in young (5 month) and aged (20 months) rats. Aged animals tested for spontaneous locomotor activity were found to be hypoactive relative to young animals. Compared to the young animals the aged animals also: (1) were impaired on a bar pressing task, (2) were unable to descend a wooden pole covered with wire mesh in a coordinated manner, (3) fell more rapidly from a rotating rod and (4) were unable to maintain their balance on a series of wooden beams of varying widths. Following baseline testing, aged animals received either no implant, encapsulated baby hamster kidney fibroblast cells that were modified to produce hGDNF (BHK-hGDNF) or encapsulated BHK cells which were not modified to produce hGDNF (BHK-Control) implanted bilaterally into the striatum. Following surgery, a significant increase in locomotor activity and bar pressing was observed in those aged animals receiving BHK-hGDNF implants. Bar pressing in aged animals receiving BHK-Control cells was improved to a lesser extent and reached the level of performance seen in young rats. No recovery was observed in the animals receiving BHK-Control cell-loaded capsules on any of the other motor tasks. Histological analysis revealed that implants of hGDNF-producing cells produced a marked increase in the density of tyrosine hydroxylase staining in the striatum adjacent to the implant site. This increased staining was not seen in animals receiving BHK-Control cells. Histological analysis also revealed the presence of viable BHK-hGDNF cells within the capsules that continued to produce hGDNF as measured by ELISA. These results indicate that polymer-encapsulated hGDNF-secreting cells survive

  13. Human skeletal muscle fibroblasts stimulate in vitro myogenesis and in vivo muscle regeneration.

    PubMed

    Mackey, Abigail L; Magnan, Mélanie; Chazaud, Bénédicte; Kjaer, Michael

    2017-08-01

    Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. The extent of cross-talk between fibroblasts, as the source of matrix protein, and satellite cells in humans is unknown. We studied this in human muscle biopsies and cell-culture studies. We observed a strong stimulation of myogenesis by human fibroblasts in cell culture. In biopsies collected 30 days after a muscle injury protocol, fibroblast number increased to four times control levels, where fibroblasts were found to be preferentially located immediately surrounding regenerating muscle fibres. These novel findings indicate an important role for fibroblasts in supporting the regeneration of muscle fibres, potentially through direct stimulation of satellite cell differentiation and fusion, and contribute to understanding of cell-cell cross-talk during physiological and pathological muscle remodelling. Accumulation of skeletal muscle extracellular matrix is an unfavourable characteristic of many muscle diseases, muscle injury and sarcopenia. In addition to the indispensable role satellite cells play in muscle regeneration, there is emerging evidence in rodents for a regulatory influence on fibroblast activity. However, the influence of fibroblasts on satellite cells and muscle regeneration in humans is unknown. The purpose of this study was to investigate this in vitro and during in vivo regeneration in humans. Following a muscle injury protocol in young healthy men (n = 7), the number of fibroblasts (TCF7L2+), satellite cells (Pax7+), differentiating myogenic cells (myogenin+) and regenerating fibres (neonatal/embryonic myosin+) was determined from biopsy cross-sections. Fibroblasts and myogenic precursor cells (MPCs) were also isolated from human skeletal muscle (n = 4) and co-cultured using different cell ratios, with the two cell populations either in direct contact with each other or separated by a permeable

  14. Monoamine oxidase activity in cultured human skin fibroblasts.

    PubMed

    Groshong, R; Gibson, D A; Baldessarini, R J

    1977-10-01

    Skin fibroblast cultures were prepared from 21 men, and found to contain types A and B activity of monoamine oxidase, with a possible slight predominance of type A, as evaluated by substrate preferences and differential inhibition by clorgyline and deprenyl. Three women had similar activities. There was a close correlation of activities with different substrates, but there was no quantitative correlation between fibroblast and blood platelet enzyme (type B) activities. The fibroblasts also contained catechol-O-methyltransferase activity exceeding, but poorly correlated with, that in erythrocytes. Fibroblasts may be advantageous in studies of monoamine oxidase in man by providing both types of enzyme as found, for example, in the central nervous system, and by providing a means of removing many in vivo chemical influences from the cells in culture. Nevertheless, great caution must be exercised in generalizing results of this "model" to other tissues, since activities of both enzymes correlated poorly with those in blood cells of the same individuals.

  15. TRPA1 Channels Mediate Human Gingival Fibroblast Response to Phenytoin.

    PubMed

    López-González, M J; Luis, E; Fajardo, O; Meseguer, V; Gers-Barlag, K; Niñerola, S; Viana, F

    2017-07-01

    Drug-induced gingival enlargement (GE) is a frequent adverse effect observed in patients treated with anticonvulsant, immunosuppressant, and some antihypertensive medications-the antiepileptic phenytoin being the main drug associated with GE due to its high incidence (around 50%). The molecular mechanisms behind drug-induced gingival overgrowth are still unknown. By reverse transcription polymerase chain reaction, we demonstrate that the calcium-permeable ion channels TRPA1, TRPV1, and its capsaicin-insensitive isoform TRPV1b are expressed in human gingival fibroblasts (HGFs), the most abundant cellular type in periodontal tissue. Cultured HGFs responded with intracellular calcium elevations to phenytoin and to the canonical TRPA1 agonist allyl isothiocyanate. Application of phenytoin activated a nonselective cationic current in HGFs with a typical signature for TRPA1 channels. Moreover, this activation was blocked by HC030031, a specific TRPA1 blocker. Similarly, the use of shRNAs against hTRPA1 in HGFs reduced TRPA1 expression and activation by phenytoin. In addition, we show that phenytoin increased intracellular calcium levels in cells transfected with mouse or human TRPA1 channels. Responses to phenytoin were not observed in untransfected cells or cells expressing TRPM8 or TRPV1. The activation of HGFs by phenytoin was markedly reduced in the presence of antioxidant vitamins: ascorbic acid, folic acid, and α-tocopherol. By performing cell proliferation assays, we found that phenytoin did not augment the proliferation rate of HGFs. In contrast, alcian blue and picrosirius red staining of long-term HGFs cultures indicated that phenytoin induces extracellular matrix accumulation of collagen. Collectively, these findings support an important role of TRPA1 channels in phenytoin-induced GE, provide insight into the pathophysiologic mechanism, and offer novel therapeutic opportunities for its treatment.

  16. [Effects of culture supernatant of human amnion mesenchymal stem cells on biological characteristics of human fibroblasts].

    PubMed

    Wu, Qi'er; Lyu, Lu; Xin, Haiming; Luo, Liang; Tong, Yalin; Mo, Yongliang; Yue, Yigang

    2016-06-01

    To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts. (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post

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

    PubMed

    Pratsinis, Harris; Dimozi, Anastasia; Pilichos, Konstantinos; Tsagarakis, Stylianos; Yiacoumettis, Andreas M; Kletsas, Dimitris

    2011-06-01

    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.

  18. Expanding Our Understanding of Human Skin Aging.

    PubMed

    Chang, Anne Lynn S

    2016-05-01

    Two very different studies expand our understanding of human skin aging. In the first study, Hüls et al. show an association between nitrogen dioxide levels in outdoor air and number of lentigines on the cheek. In the second study, Bowman and Birch-Machin show that mitochondrial complex II activity in human skin fibroblasts decreases with age. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

  19. Extracellular superoxide dismutase deficiency impairs wound healing in advanced age by reducing neovascularization and fibroblast function

    PubMed Central

    Fujiwara, Toshihiro; Duscher, Dominik; Rustad, Kristine C.; Kosaraju, Revanth; Rodrigues, Melanie; Whittam, Alexander J.; Januszyk, Michael; Maan, Zeshaan N.; Gurtner, Geoffrey C.

    2016-01-01

    Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing. PMID:26663425

  20. Regeneration and control of human fibroblast cell density by intermittently delivered pulsed electric fields.

    PubMed

    Golberg, Alexander; Bei, Marianna; Sheridan, Robert L; Yarmush, Martin L

    2013-06-01

    Proliferative scarring is a human disease with neither available effective treatment nor relevant animal model. One of the hypotheses for scar formation involves deregulation of fibroblast signaling and delayed apoptosis. Here, we introduce a new chemical-free method for fibroblast density control in culture by intermittently delivered pulsed electric fields (IDPEF), which cause irreversible damage to cell membranes. Using 5-100 pulses with electric field strength of 150 V/mm, pulse duration 70 µs, and frequency of 1 Hz, we investigated the effects of PEF application on growth, death, and regeneration of normal human dermal fibroblasts in culture. We found that the fraction of fibroblasts that survive depends on the number of pulses applied and follows a Weibull distribution. We have successfully developed an IDPEF protocol that controls fibroblasts density in culture. Specifically, through application of IDPEF every 72 h for 12 days, we maintain a normal human dermal fibroblast density in the 3.1 ± 0.2 × 10(5) -1.4 ± 0.2 × 10(5)  cell/mL range. Our results suggest that IDPEFs may prove useful as a non-chemical method for fibroblast density control in human wound healing.

  1. METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS

    EPA Science Inventory

    METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS. Geremy W. Knapp, Alan Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, Re...

  2. METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS

    EPA Science Inventory

    METHYL METHANESULFONATE-INDUCED GENE EXPRESSION CHANGES IN HUMAN SKIN FIBROBLASTS. Geremy W. Knapp, Alan Tennant, and Russell D. Owen. Environmental Carcinogenesis Division, National Health and Environmental Effects Research Laboratory, U. S. Environmental Protection Agency, Re...

  3. HJURP regulates cellular senescence in human fibroblasts and endothelial cells via a p53-dependent pathway.

    PubMed

    Heo, Jong-Ik; Cho, Jung Hee; Kim, Jae-Ryong

    2013-08-01

    Holliday junction recognition protein (HJURP), a centromere protein-A (CENP-A) histone chaperone, mediates centromere-specific assembly of CENP-A nucleosome, contributing to high-fidelity chromosome segregation during cell division. However, the role of HJURP in cellular senescence of human primary cells remains unclear. We found that the expression levels of HJURP decreased in human dermal fibroblasts and umbilical vein endothelial cells in replicative or premature senescence. Ectopic expression of HJURP in senescent cells partially overcame cell senescence. Conversely, downregulation of HJURP in young cells led to premature senescence. p53 knockdown, but not p16 knockdown, abolished senescence phenotypes caused by HJURP reduction. These data suggest that HJURP plays an important role in the regulation of cellular senescence through a p53-dependent pathway and might contribute to tissue or organismal aging and protection of cellular transformation.

  4. Accelerated Telomere Shortening and Replicative Senescence in Human Fibroblasts Overexpressing Mutant and Wild Type Lamin A

    PubMed Central

    Huang, Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectible WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. PMID:17870066

  5. Constituents from the roots of Taraxacum platycarpum and their effect on proliferation of human skin fibroblasts.

    PubMed

    Warashina, Tsutomu; Umehara, Kaoru; Miyase, Toshio

    2012-01-01

    A MeOH extract from the roots of Taraxacum platycarpum has shown significant effects on the proliferation of normal human skin fibroblasts. Chemical analysis of the extract resulted in the isolation of 26 compounds, including eight new triterpenes, one new sesquiterpene glycoside, and seventeen known compounds. The structure of each new compound was established using NMR spectroscopy. Some triterpenes had a significant effect on the proliferation of normal human skin fibroblasts.

  6. Binding, uptake, and release of nicotine by human gingival fibroblasts

    SciTech Connect

    Hanes, P.J.; Schuster, G.S.; Lubas, S. )

    1991-02-01

    Previous studies of the effects of nicotine on fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to (1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non-specific in nature; (2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; (3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and (4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4{degree}C using a mixture of {sup 3}H-nicotine and unlabeled nicotine. Specific binding was calculated as the difference between {sup 3}H-nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (+/- 0.42) pmols/10(6) cells in the presence of unlabeled nicotine and 1.66 (+/- 0.55) pmols/10(6) cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37{degree}C after treating cells with {sup 3}H-nicotine for time periods up to 4 hours. Uptake in pmols/10(6) cells was 4.90 (+/- 0.34) at 15 minutes, 8.30 (+/- 0.75) at 30 minutes, 12.28 (+/- 2.62) at 1 hour and 26.31 (+/- 1.15) at 4 hours.

  7. Antiaging effects of the mixture of Panax ginseng and Crataegus pinnatifida in human dermal fibroblasts and healthy human skin.

    PubMed

    Hwang, Eunson; Park, Sang-Yong; Yin, Chang Shik; Kim, Hee-Taek; Kim, Yong Min; Yi, Tae Hoo

    2017-01-01

    Human skin undergoes distinct changes throughout the aging process, based on both intrinsic and extrinsic factors. In a process called photoaging, UVB irradiation leads to upregulation of matrix metalloproteinase-1, which then causes collagen degradation and premature aging. Mixtures of medicinal plants have traditionally been used as drugs in oriental medicine. Based on the previously reported antioxidant properties of Panax ginseng Meyer and Crataegus pinnatifida, we hypothesized that the mixture of P. ginseng Meyer and C. pinnatifida (GC) would have protective effects against skin aging. Anti-aging activity was examined both in human dermal fibroblasts under UVB irradiation by using Western blot analysis and in healthy human skin by examining noninvasive measurements. In vitro studies showed that GC improved procollagen type I expression and diminished matrix metalloproteinase-1 secretion. Based on noninvasive measurements, skin roughness values, including total roughness (R1), maximum roughness (R2), smoothness depth and average roughness (R3), and global photodamage scores were improved by GC application. Moreover, GC ameliorated the high values of smoothness depth (R4), which means that GC reduced loss of skin moisture. These results suggest that GC can prevent aging by inhibiting wrinkle formation and increasing moisture in the human skin.

  8. Retinoic acid, GABA-ergic, and TGF-beta signaling systems are involved in human cleft palate fibroblast phenotype.

    PubMed

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Becchetti, Ennio; Carinci, Paolo; Stabellini, Giordano; Calvitti, Mario; Lumare, Eleonora; Bodo, Maria

    2006-01-01

    During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-beta (TGF-beta), retinoic acid (RA), and gamma-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-beta binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA--which, at pharmacologic doses, induces cleft palate in newborns of many species--were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-beta3 mRNA expression and TGF-beta receptor number were higher and RA receptor-alpha (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-beta3 mRNA expression but reduced the number of TGF-beta receptors. TGF-beta receptor type I mRNA expression was decreased, TGF-beta receptor type II was increased, and TGF-beta receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-beta signaling systems could be

  9. Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype

    PubMed Central

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Becchetti, Ennio; Carinci, Paolo; Stabellini, Giordano; Calvitti, Mario; Lumare, Eleonora; Bodo, Maria

    2006-01-01

    During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-β (TGF-β), retinoic acid (RA), and γ-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-β binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA—which, at pharmacologic doses, induces cleft palate in newborns of many species—were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-β3 mRNA expression and TGF-β receptor number were higher and RA receptor-α (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-β3 mRNA expression but reduced the number of TGF-β receptors. TGF-β receptor type I mRNA expression was decreased, TGF-β receptor type II was increased, and TGF-β receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-β signaling systems could be involved in human cleft

  10. L-Lactate Protects Skin Fibroblasts against Aging-Associated Mitochondrial Dysfunction via Mitohormesis.

    PubMed

    Zelenka, Jaroslav; Dvořák, Aleš; Alán, Lukáš

    2015-01-01

    A moderate elevation of reactive oxygen species (ROS) production and a mild inhibition of mitochondrial respiratory chain have been associated with a health promotion and a lifespan extension in several animal models of aging. Here, we tested whether this phenomenon called mitohormesis could be mediated by L-lactate. The treatment with 5 mM L-lactate significantly increased H2O2 production and slightly inhibited the respiration in cultured skin fibroblasts and in isolated mitochondria. The L-lactate exposure was associated with oxidation of intracellular glutathione, phosphorylation of 5'AMP-activated protein kinase (AMPK), and induction of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α) transcription. A replicative aging of fibroblasts (L0) with a constant (LC), or intermittent 5 mM L-lactate (LI) in media showed that the high-passage LI fibroblasts have higher respiration, lower H2O2 release, and lower secretion of L-lactate compared to L0 and LC. This protection against mitochondrial dysfunction in LI cells was associated with lower activity of mechanistic target of rapamycin complex 1 (mTORC1), less signs of cellular senescence, and increased autophagy compared to L0 and LC. In conclusion, we demonstrated that intermittent but not constant exposure to L-lactate triggers mitohormesis, prevents aging-associated mitochondrial dysfunction, and improves other markers of aging.

  11. Optimal Viscosity and Particle Shape of Hyaluronic Acid Filler as a Scaffold for Human Fibroblasts.

    PubMed

    Kim, Deok-Yeol; Namgoong, Sik; Han, Seung-Kyu; Won, Chang-Hoon; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2015-07-01

    The authors previously reported that cultured human fibroblasts suspended in a hyaluronic acid filler can produce human dermal matrices with extended in vivo stability in animal and clinical studies. The present study was undertaken to determine the optimal viscosity and particle shape of hyaluronic acid filler as a scaffold for cultured human dermal fibroblasts to enhance the maximal viability of injected cells. The fibroblasts were suspended in either 1 of 3 hyaluronic acid viscosities at 2 different particle shapes. The viscosities used in this study were low (600,000-800,000 centipoises), moderate (2,000,000-4,000,000 centipoises), and high (8,000,000-12,000,000 centipoises). The particle shape was evaluated by testing round and irregular shapes. The fibroblast mixed bioimplants were injected into the back of individual athymic nude mice. The levels of type I collagen were measured using fluorescent-activated cell sorting (FACS) and immunohistochemical staining at 16 weeks after the injections. Results of FACS demonstrated that the mean cell ratio with human collagens in the moderate viscosity group was greater than those of control, low, and high viscosity groups. An immunohistochemical study showed similar results. The moderate viscosity group demonstrated the highest positive staining of human collagens. However, there were no significant differences between groups of irregular and round shape particles. A hyaluronic acid bioimplant with moderate viscosity is superior to that with low or high viscosity in the viability for human fibroblasts. However, the particle shape does not influence the viability of the fibroblasts.

  12. Mediated calcium transport by isolated human fibroblast lysosomes

    SciTech Connect

    Lemons, R.M.; Thoene, J.G. )

    1991-08-05

    Lysosomes purified by Percoll gradient from normal human fibroblasts (GM0010A) show uptake of Ca2+ in a mediated manner. The uptake is linear over the first 1.5 min and approaches a steady state by 10 min. Uptake is saturable, displaying a Vmax of about 10 pmol/min/unit hexosaminidase at 20 mM Ca2+ (7 nmol/min/mg protein), and a Km of 5.7 mM. Ca2+ uptake increases with increasing extralysosomal pH from 5.0 to 8.5. The Q10 is 1.6, and Ea 8.7 kcal/mol. Uptake of 0.1 mM Ca2+ was inhibited to the extent indicated by 1.0 mM of the following: Cd2+, 100%; Hg2+, 100%; Zn2+, 89%; Mg2+, 77%; Ba2+, 60%; Sr2+, 37%; Fe2+, 20%; Cu2+, 0%. Mono- and trivalent cations had no effect. ATP (1.0 mM) inhibited uptake by 80%, and chloroquine (0.1 mM) inhibited by 60%, as did 1.0 mM L-cystine. Cysteamine, N-ethylmaleimide, and the anions Cl-, SO(2-)4, and acetate had no effect. The calcium ionophore A23187 augmented uptake by 10-fold at 10 microM. Surprisingly, Pb2+ greatly augmented lysosomal Ca2+ uptake in a concentration-dependent manner. Pb2+, however, adversely affected lysosomal latency. Lysosomal calcium uptake was not affected by inositol 1,4,5-triphosphate, and calcium-induced calcium release from lysosomes was not observed. A role for lysosomes in cellular calcium homeostasis has not been previously suggested. This work shows that Ca2+ can be transported into and out of lysosomes and could assist in lysosomal proteolysis. The extent of further lysosomal participation in cellular calcium regulation is unclear.

  13. Tomography studies of human foreskin fibroblasts on polymer yarns

    NASA Astrophysics Data System (ADS)

    Thurner, Philipp; Müller, Bert; Beckmann, Felix; Weitkamp, Timm; Rau, Christoph; Müller, Ralph; Hubbell, Jeffrey A.; Sennhauser, Urs

    2003-01-01

    Cell culture experiments are usually performed as in vitro studies based on 2D seeding and characterization (light microscopy). With respect to the in vivo situation, however, 2D studies are often inappropriate due to the 3D character of living tissue in nature. Textiles with their versatile 3D structures are chosen as suitable scaffolds in tissue engineering for 3D in vitro studies. Micro-computed tomography using X-rays (μCT) belongs to the most promising techniques for isotropic, noninvasive 3D characterization. Using synchrotron radiation (SRμCT) the spatial resolution can be extended to the sub-micrometer range well below cell size. μCT does not need vacuum conditions making experiments in the hydrated state possible, as we show by data from SRμCT acquired at second and third-generation synchrotron sources. We seeded human foreskin fibroblasts on polymer multifilament yarns. These composites, embedded in a hydrogel or fluid, are held in thin-walled glass capillaries. Since the composites consist of light elements, the cells have to be labeled for visualization by the use of highly absorptive agents, osmium and gold. In order to hold the label concentration as low as possible, we present a way to choose the photon energy for which the minimum concentration is reached. Differences in threshold selection for second- and third-generation synchrotron sources are pointed out, revealing the advantages of both types with respect to quantitative analysis. The study is based on appropriate staining methods and protocols developed in our laboratory. With the results we demonstrate that SRμCT yields images similar to established electron and light microscopy but uncovers also the microstructure in 3D space.

  14. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    SciTech Connect

    Fan, Rong-hui; Zhu, Xiu-mei; Sun, Yao-wen; Peng, Hui-zi; Wu, Hang-li; Gao, Wen-jie

    2016-07-08

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  15. Leptin and Pro-Inflammatory Stimuli Synergistically Upregulate MMP-1 and MMP-3 Secretion in Human Gingival Fibroblasts

    PubMed Central

    Williams, Rachel C.; Skelton, Andrew J.; Todryk, Stephen M.; Rowan, Andrew D.; Preshaw, Philip M.; Taylor, John J.

    2016-01-01

    Introduction Gingival fibroblast-mediated extracellular matrix remodelling is implicated in the pathogenesis of periodontitis, yet the stimuli that regulate this response are not fully understood. The immunoregulatory adipokine leptin is detectable in the gingiva, human gingival fibroblasts express functional leptin receptor mRNA and leptin is known to regulate extracellular matrix remodelling responses in cardiac fibroblasts. We therefore hypothesised that leptin would enhance matrix metalloproteinase secretion in human gingival fibroblasts. Methods and Results We used in vitro cell culture to investigate leptin signalling and the effect of leptin on mRNA and protein expression in human gingival fibroblasts. We confirmed human gingival fibroblasts expressed cell surface leptin receptor, found leptin increased matrix metalloproteinase-1, -3, -8 and -14 expression in human gingival fibroblasts compared to unstimulated cells, and observed that leptin stimulation activated MAPK, STAT1/3 and Akt signalling in human gingival fibroblasts. Furthermore, leptin synergised with IL-1 or the TLR2 agonist pam2CSK4 to markedly enhance matrix metalloproteinase-1 and -3 production by human gingival fibroblasts. Signalling pathway inhibition demonstrated ERK was required for leptin-stimulated matrix metalloproteinase-1 expression in human gingival fibroblasts; whilst ERK, JNK, p38 and STAT3 were required for leptin+IL-1- and leptin+pam2CSK4-induced matrix metalloproteinase-1 expression. A genome-wide expression array and gene ontology analysis confirmed genes differentially expressed in leptin+IL-1-stimulated human gingival fibroblasts (compared to unstimulated cells) were enriched for extracellular matrix organisation and disassembly, and revealed that matrix metalloproteinase-8 and -12 were also synergistically upregulated by leptin+IL-1 in human gingival fibroblasts. Conclusions We conclude that leptin selectively enhances the expression and secretion of certain matrix

  16. The Cytotoxicity and Genotoxicity of Hexavalent Chromium in Steller Sea Lion Lung Fibroblasts Compared to Human Lung Fibroblasts

    PubMed Central

    Wise, John Pierce; Wise, Sandra S.; Holmes, Amie L.; LaCerte, Carolyne; Shaffiey, Fariba; Aboueissa, AbouEl-Makarim

    2010-01-01

    In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes. PMID:20211760

  17. Dermal Aged and Fetal Fibroblasts Realign in Response to Mechanical Strain

    NASA Technical Reports Server (NTRS)

    Sawyer, Christine; Grymes, Rose; Alvarez, Teresa (Technical Monitor)

    1994-01-01

    Integrins specifically recognize and bind extracellular matrix components, providing physical anchor points and functional setpoints. Focal adhesion complexes, containing integrin and cytoskeletal proteins, are potential mechanoreceptors, poised to distribute applied forces through the cytoskeleton. Pursuing the hypothesis that cells both perceive and respond to external force, we applied a stretch/relaxation regimen to normal human fetal and aged dermal fibroblast monolayers cultured on flexible membranes. The frequency and magnitude of the applied force is precisely controlled by the Flexercell Unit(Trademark). A protocol of stretch (20% elongation of the monolayer) at a frequency of 6 cycles/min caused a progressive change from a randomly distributed pattern of cells to a symmetric, radial distribution with cells aligned parallel to the applied force. We have coined the term 'orienteering' as the process of active alignment of cells in response to applied force. Cytochalasin D was added in graded doses to investigate the role of the actin cytoskeleton in force perception and transmission. A clear dose response was found; at high concentrations orienteering was abolished; and the drug's impact was reversible. The two cell strains used were similar in their alignment behavior and in their responses to cytochalasin D. Orienteering was influenced by cell density, and the cell strains studied differed in this respect. Fetal cells, unlike their aged counterparts, failed to orient at high cell density. In both cell strains, mid-density cultures aligned rapidly and sparse cultures lagged. These results indicate that both cell-cell adhesion and cytoskeleton integrity are critical in mediating the orienteering response. Differences between these two cell strains may relate to their expression of extracellular matrix molecules (fibronectin, collagen type 1) integrins and their relative binding affinities.

  18. Dermal Aged and Fetal Fibroblasts Realign in Response to Mechanical Strain

    NASA Technical Reports Server (NTRS)

    Sawyer, Christine; Grymes, Rose; Alvarez, Teresa (Technical Monitor)

    1994-01-01

    Integrins specifically recognize and bind extracellular matrix components, providing physical anchor points and functional setpoints. Focal adhesion complexes, containing integrin and cytoskeletal proteins, are potential mechanoreceptors, poised to distribute applied forces through the cytoskeleton. Pursuing the hypothesis that cells both perceive and respond to external force, we applied a stretch/relaxation regimen to normal human fetal and aged dermal fibroblast monolayers cultured on flexible membranes. The frequency and magnitude of the applied force is precisely controlled by the Flexercell Unit(Trademark). A protocol of stretch (20% elongation of the monolayer) at a frequency of 6 cycles/min caused a progressive change from a randomly distributed pattern of cells to a symmetric, radial distribution with cells aligned parallel to the applied force. We have coined the term 'orienteering' as the process of active alignment of cells in response to applied force. Cytochalasin D was added in graded doses to investigate the role of the actin cytoskeleton in force perception and transmission. A clear dose response was found; at high concentrations orienteering was abolished; and the drug's impact was reversible. The two cell strains used were similar in their alignment behavior and in their responses to cytochalasin D. Orienteering was influenced by cell density, and the cell strains studied differed in this respect. Fetal cells, unlike their aged counterparts, failed to orient at high cell density. In both cell strains, mid-density cultures aligned rapidly and sparse cultures lagged. These results indicate that both cell-cell adhesion and cytoskeleton integrity are critical in mediating the orienteering response. Differences between these two cell strains may relate to their expression of extracellular matrix molecules (fibronectin, collagen type 1) integrins and their relative binding affinities.

  19. IgG from patients with systemic sclerosis bind to DNA antitopoisomerase 1 in normal human fibroblasts extracts

    PubMed Central

    Tamby, Mathieu C; Servettaz, Amélie; Tamas, Nicolas; Reinbolt, Joseph; Caux, Frédéric; Meyer, Olivier; Allanore, Yannick; Kahan, André; Guillevin, Loïc; Mouthon, Luc

    2008-01-01

    By using a semi-quantitative immunoblotting technique, we have analyzed serum immunoglobulin G (IgG) reactivities of patients with limited cutaneous systemic sclerosis and anticentromere antibodies, patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies, patients with diffuse systemic sclerosis without antitopoisomerase 1 or anticentromere antibodies and age- and gender-matched healthy controls with normal human skin fibroblasts and HEp-2 cells antigens. Serum IgG reactivities of patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies differed significantly from those of healthy controls or systemic sclerosis patients in other groups for reactivity with fibroblast proteins. IgG from patients with antitopoisomerase 1 antibodies bound to a 90 kDa fibroblast band and to a 100 kDa protein band in a HEp-2 cell protein extract. These two bands were further identified as DNA topoisomerase 1. Our results indicate that IgG from patients with diffuse systemic sclerosis bind DNA topoisomerase 1 in normal human fibroblasts extracts. PMID:19707389

  20. Suppression of human fibroblast proliferation by D-penicillamine and copper sulfate in vitro.

    PubMed

    Matsubara, T; Hirohata, K

    1988-08-01

    We examined the effect of D-penicillamine (DP) and copper sulfate (CuSO4) on human fibroblast proliferation in vitro. DP plus CuSO4 inhibited both basal and interleukin-1 (IL-1)-induced tritiated thymidine incorporation into fibroblasts in a dose- and time-dependent manner. Significant inhibition was observed at the level of 60 microM in the presence of 4 microM CuSO4. At this range of concentrations, which is attained in serum and in tissues of treated patients, DP alone or CuSO4 alone did not affect fibroblast proliferation. Similar inhibition was observed with various thiols in the presence of copper, but not with disulfides such as DP disulfide or oxidized glutathione. Inhibition of fibroblast DNA synthesis induced by DP and CuSO4 was reversed by the simultaneous addition of catalase or horseradish peroxidase, but not by boiled catalase or superoxide dismutase. The inhibition by DP and CuSO4, therefore, may be attributable to hydrogen peroxide produced by these 2 agents. DP, in the presence of CuSO4, did not affect IL-1 secretion from human peripheral mononuclear phagocytes. These observations indicate that hydrogen peroxide produced by DP plus CuSO4 inhibits IL-1-induced fibroblast proliferation by directly affecting fibroblasts, without alteration of IL-1 secretion from mononuclear phagocytes. Thus, DP may play a role in inhibiting the growth of rheumatoid pannus and excessive collagenation in scleroderma by direct inhibition of fibroblast proliferation.

  1. Solar-simulated radiation and heat treatment induced metalloproteinase-1 expression in cultured dermal fibroblasts via distinct pathways: implications on reduction of sun-associated aging.

    PubMed

    Lan, Cheng-Che E; Wu, Ching-Shang; Yu, Hsin-Su

    2013-12-01

    Sun exposure is an important environmental factor affecting human beings. Most knowledge regarding solar aging focused on light radiation (photoaging), and little emphasis has been placed on heat, a factor that is also closely associated with sun exposure. This study was launched to evaluate the effects of simulated solar radiation (SSR) and environmental heat on skin fibroblasts in terms of dermal aging. Cultured human dermal fibroblasts were treated with moderate amount of SSR (200J/cm(2)) and heat (+2°C). The metalloproteinase-1 (MMP-1) expression was used as a surrogate marker for dermal aging and the involved regulatory mechanisms were explored. Both treatment conditions did not affect viability but significantly increased the expressions of MMP-1. In parallel, both treatments increased the intracellular levels of reactive oxygen species (ROS), but the increase induced by SSR is much greater than heat. In contrast, transient receptor potential vanilloid 1 (TRPV-1), the sensor of environmental heat, was upregulated by heat but not SSR treatment. Pretreating fibroblasts with antioxidant abrogated the SSR-induced MMP-1 but has limited effect on heat-induced MMP-1. On the other hand, TRPV-1 antagonist pretreatment reduced heat-induced MMP-1 in fibroblasts but not their SSR-treated counterparts. Both SSR and heat induced MMP-1 expression in dermal fibroblasts but through different pathways. As current strategies for reducing sun-related aging focused on filtering of light and use of antioxidants, future strategies design to reduce solar aging should also incorporate heat-induced aging into consideration. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  2. Influence of caffeine and hyaluronic acid on collagen biosynthesis in human skin fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Głuszuk, Katarzyna; Surażyński, Arkadiusz

    2014-01-01

    Aim The aim of this study was to evaluate the effect of caffeine on collagen biosynthesis in human skin fibroblasts and the influence of hyaluronic acid (HA) on this process. Materials and methods Collagen, [3H]-thymidine incorporation, and prolidase activity were measured in confluent human skin fibroblast cultures that had been treated with 1, 2, and 5 mM caffeine and with caffeine and 500 μg/mL HA. Western immunoblot analysis was performed to evaluate expression of β1-integrin receptor, insulin-like growth factor receptor phospho-Akt protein and mitogen-activated protein kinase (phospho-extracellular signal-regulated kinase). Results Caffeine inhibited collagen biosynthesis in a dose-dependent manner. The mechanism of this process was found at the level of prolidase activity. Caffeine significantly inhibited the enzyme activity. The addition of HA had no effect on collagen biosynthesis or prolidase activity in fibroblasts incubated with caffeine. Caffeine also had an inhibitory effect on DNA biosynthesis. HA, however, did not have any significant effect on this process. The inhibition of the expression of β1-integrin and insulin-like growth factor receptor in fibroblasts incubated with the caffeine indicates a possible mechanism of inhibition of collagen biosynthesis. Conclusion Caffeine reduces collagen synthesis in human cultured skin fibroblasts. HA did not have any significant protective effect on this process. This is the first study to our knowledge that reports caffeine-induced inhibition of collagen synthesis in human skin fibroblasts. PMID:25342885

  3. Repeated exposure of human fibroblasts to UVR induces secretion of stem cell factor and senescence.

    PubMed

    Shin, J; Kim, J-H; Kim, E K

    2012-12-01

    Some of chronic hyperpigmentary diseases, such as melasma, induced by multiple factors including chronic sunlight exposure, can recur even after chemical epidermal removal. Dermal factors may be involved in the pathogenesis of melasma. Changes in dermal fibroblasts resulting from chronic sun exposure might cause melanocytes to synthesize melanin in the epidermis. This study aimed at determining the effects of repetitive ultraviolet (UV) radiation on cultured fibroblasts and the secretion of melanogenic factors. Cultured human fibroblasts were exposed to ultraviolet A (UVA) or ultraviolet B (UVB) for five consecutive days. After each irradiation, the supernatant medium was isolated from each dish and measured for levels of stem cell factor (SCF) and hepatocyte growth factor using an ELISA kit assay. To assess the effect of the keratinocyte-derived factors on fibroblast-secretion of SCF and hepatocyte growth factor, we added supernatants of the UV-irradiated keratinocytes to the non-irradiated fibroblasts. Finally, the irradiated fibroblasts were stained with senescence associated-β-galactosidase to assess their senescent change. Fibroblasts irradiated with UVA or UVB for five consecutive days, secreted SCF at levels that increased with repeated UVA or UVB exposure. Conditioned culture medium from UV-irradiated keratinocytes also induced SCF release from fibroblasts, depending on the number of UV exposures. UVA- or UVB-irradiated fibroblasts stained positive for senescence associated-β-galactosidase, and the staining intensity increased with repeated exposure. These results suggest that fibroblast senescence and increased SCF secretion after repeated UV irradiation may be related to the pathogenesis of recurring hyperpigmentation disorders induced by chronic sun exposure. © 2011 The Authors. Journal of the European Academy of Dermatology and Venereology © 2011 European Academy of Dermatology and Venereology.

  4. Anti-Photoaging Effects of Angelica acutiloba Root Ethanol Extract in Human Dermal Fibroblasts

    PubMed Central

    Park, Min Ah; Sim, Mi Ja; Kim, Young Chul

    2017-01-01

    The effects that ultraviolet rays elicit on collagen synthesis and degradation are the most common causes of wrinkle formation and photo-aging in skin. The objectives of this study were to evaluate the effects of Angelica acutiloba root ethanol extract (AAEE) to promote collagen synthesis and inhibit collagen degradation in human dermal fibroblasts. By examining total polyphenol and flavonoid contents, electron donating ability, radical scavenging activity, and superoxide dismutase-like activity, we found that AAEE exhibited fairly good antioxidant activity. Treatment with AAEE significantly increased type I procollagen production by cultured fibroblasts, as well as reduced ultraviolet-induced matrix metalloproteinase-1 (MMP-1) expression and MMP-2 activity in a dose-dependent manner (p < 0.05). In addition, AAEE significantly increased TIMP-1 mRNA expression (p < 0.05), although without an associated dose-dependent increase in TIMP-1 protein expression. In summary, we suggest that AAEE may be a potentially effective agent for the prevention or alleviation of skin-wrinkle formation induced by ultraviolet rays. PMID:28503261

  5. Proliferative response patterns of human fibroblasts after photoinjury with 4,5',8-trimethylpsoralen

    SciTech Connect

    Cohen, S.R.; Carter, D.M.; Gala, M.

    1981-01-01

    The extent of growth suppression and recovery following exposure to 4,5',8-trimethylpsoralen plus uv-A irradiation was studied in 3 diploid human fibroblast strains. Inhibition of cellular proliferation was dose-dependent within the concentration range of TMP that was tested, using a constant level of uv-A. The population generation times for all cell strains were progressively lengthened under these conditions while maximal cell densities were reduced. At 2 to 4 x 10(-7) M TMP in the presence of uv-A, there was a triphasic pattern of growth which consisted of proliferative activity during the first 24 to 36 h, followed by complete growth inhibition for variable periods of time and a recovery period of log phase proliferation that was not as vigorous as measured for untreated cells. There were also declines in the percentage of cells labeled with 3H-Tdr at various times after TMP-uv-A treatment. These measurements were essentially identical for the three fibroblast strains evaluated. In that the cells employed for these investigations were derived from embryonic pulmonary tissue, neonatal foreskin and the buttock skin of an adult male, it seems unlikely that donor age and tissue source were important variables in determining growth response patterns after TMP-uv-A exposure. Because proliferative recovery was attenuated after this photochemical injury, researchers conclude that the biologic effect(s) of TMP-uv-A extend beyond the known period of psoralen-DNA cross-link removal.

  6. Effect of alpha-tocopherol and silibin dihemisuccinate on the proliferation of human skin fibroblasts.

    PubMed

    Onat, D; Boscoboinik, D; Azzi, A; Basaga, H

    1999-06-01

    Cell proliferation is a complex and important event in atherosclerosis, aging and cancer, and is under the control of signalling pathways. These signalling pathways in turn are effected by the presence of a number of chemicals. For this purpose, we have checked the effect of two chemicals on the proliferation of skin fibroblasts. alpha-Tocopherol and silibin dihemisuccinate (SDH) negatively regulate proliferation of human skin fibroblasts. To check the cell-cycle time intervals, a [3H]thymidine incorporation assay was performed, showing DNA replication at around 24 h; this indicated the time required for the incubation with the chemicals. When alpha-tocopherol was added to the growth medium at a physiological concentration of 50 microM, cell proliferation was inhibited by 40% in 72 h. A similar inhibitory effect of cell proliferation was achieved when 500 microM SDH was used (39% inhibition in 72 h). From the dose-response curves obtained it was concluded that both duration of treatment and the concentration of the chemicals are important parameters. The actual mechanism of the inhibition of cell proliferation may be due to the anti-oxidative potential of these chemicals as well as another mechanism effecting signal transduction pathways.

  7. Aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by hyperproliferation and increased apoptosis.

    PubMed

    Bridger, Joanna M; Kill, Ian R

    2004-05-01

    Hutchinson-Gilford progeria syndrome is a rare genetic disorder that mimics certain aspects of aging prematurely. Recent work has revealed that mutations in the lamin A gene are a cause of the disease. We show here that cellular aging of Hutchinson-Gilford progeria syndrome fibroblasts is characterised by a period of hyperproliferation and terminates with a large increase in the rate of apoptosis. The occurrence of cells with abnormal nuclear morphology reported by others is shown to be a result of cell division since the fraction of these abnormalities increases with cellular age. Similarly, the proportion of cells with an abnormal or absent A-type lamina increases with age. These data provide clues as to the cellular basis for premature aging in HGPS and support the view that cellular senescence and tissue homeostasis are important factors in the normal aging process.

  8. Rapid flow cytometric method for measuring senescence associated beta-galactosidase activity in human fibroblasts.

    PubMed

    Noppe, Gerard; Dekker, Pim; de Koning-Treurniet, Corine; Blom, Joke; van Heemst, Diana; Dirks, Roeland W; Tanke, Hans J; Westendorp, Rudi G J; Maier, Andrea B

    2009-11-01

    Senescence associated-beta-galactosidase (SA-beta-gal) activity is a widely used marker for cellular senenescence. SA-beta-gal activity is routinely detected cytochemically, manually discriminating negative from positive cells. This method is time-consuming, subjective and therefore prone to operator-error. We aimed to optimize a flow cytometric method described by other workers using endothelial cells to better differentiate between populations of fibroblasts in degrees of SA-beta-gal activity. Skin fibroblasts were isolated from young (mean age +/- SD: 25.5 +/- 1.8) and very old (age 90.2 +/- 0.3) subjects. Different pH modulators were tested for toxicity. To induce stress-induced senescence, fibroblasts were exposed to rotenone. Senescence was assessed measuring SA-beta-gal activity by cytochemistry (X-gal) and by flow cytometry (C(12)FDG). The pH modulator Bafilomycin A1 (Baf A1) was found to be least toxic for fibroblasts and to differentiate best between nonstressed and stressed fibroblast populations. Under nonstressed conditions, fibroblasts from very old subjects showed higher SA-beta-gal activity than fibroblasts from young subjects. This difference was found for both the flow cytometric and cytochemical methods (P = 0.013 and P = 0.056 respectively). Under stress-induced conditions the flow cytometric method but not the cytochemical method revealed significant higher SA-beta-gal activity in fibroblasts from very old compared to young subjects (P = 0.004 and P = 0.635 respectively). We found the modified flow cytometric method measuring SA-beta-gal activity superior in discriminating between degrees of senescence in different populations of fibroblasts. Copyright 2009 International Society for Advancement of Cytometry.

  9. Activation of the Innate Immune Response against DENV in Normal Non-Transformed Human Fibroblasts

    PubMed Central

    Bustos-Arriaga, José; García-Machorro, Jazmín; León-Juárez, Moisés; García-Cordero, Julio; Santos-Argumedo, Leopoldo; Flores-Romo, Leopoldo; Méndez-Cruz, A. René; Juárez-Delgado, Francisco J.; Cedillo-Barrón, Leticia

    2011-01-01

    Background When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times (“probing”) before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. Methodology/Principal Findings Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts. Conclusions/Significance In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral

  10. Circadian clocks in rat skin and dermal fibroblasts: differential effects of aging, temperature and melatonin.

    PubMed

    Sandu, Cristina; Liu, Taole; Malan, André; Challet, Etienne; Pévet, Paul; Felder-Schmittbuhl, Marie-Paule

    2015-06-01

    As a peripheral tissue localized at the interface between internal and external environments, skin performs functions which are critical for the preservation of body homeostasis, in coordination with environmental changes. Some of these functions undergo daily variations, such as temperature or water loss, suggesting the presence of time-keeping mechanisms. Rhythmic functions are controlled by a network of circadian oscillators present virtually in every cell and coordinated by the central clock located in the suprachiasmatic nuclei. At the molecular level, circadian rhythms are generated by conserved transcriptional-translational feedback loops involving several clock genes, among which Per1 and Per2 play a central role. Here we characterize clock activity in skin of the transgenic Per1-luciferase rat during postnatal development and adulthood, by real-time recording of bioluminescence in explants and primary dermal fibroblasts, and report marked transformation in circadian properties, from early life to aging. Using primary dermal fibroblast cultures we provide evidence that melatonin treatment phase dependently increases the amplitude of circadian oscillations and that ambient temperature impacts on their period, with slight overcompensation. Together, these findings demonstrate that skin contains a self-sustained circadian clock undergoing age-dependent changes. Dermal fibroblasts, one of the major skin cell types, also exhibit robust, yet specific, circadian rhythmicity which can be fine-tuned by both internal (melatonin) and external (temperature) factors.

  11. Aging adult porcine fibroblasts can support nuclear transfer and transcription factor-mediated reprogramming.

    PubMed

    Li, Xia; Zhang, Pengfei; Jiang, Shaoshuai; Ding, Biao; Zuo, Xiaoyuan; Li, Yunsheng; Cao, Zubing; Zhang, Yunhai

    2017-10-03

    Somatic cell nuclear transfer (SCNT) and induced pluripotent stem cells (iPSCs) technology are two classical reprogramming methods. Donor cell types can affect the reprogramming results in the above two methods. We here used porcine embryonic fibroblasts (PEFs) and adult porcine ear skin fibroblasts (APEFs) and adipose-derived stem cells (ADSCs) as donor cells for SCNT and source cells for iPSCs to study their in vitro developmental capability and colony-formation efficiency, respectively. For SCNT, fusion and cleavage rate has no significant difference among PEFs, ADSCs and APEFs. The rate and total cell number of blastocysts in the APEF group were significant lower than that in PEFs and ADSCs. For transcription factor-mediated reprogramming, the reprogramming efficiency of ADSCs were significantly higher than PEFs and APEFs and there is no significant difference between PEFs and APEFs. Furthermore, PEFs, APEFs and ADSCs can be used to generate iPSCs. Fianlly, somatic cloned pigs could still be successfully generated from APEFs, suggesting terminally differentiated aging adult somatic cells could be reprogrammed into a totipotent state. Considering the easy availability of animal tissue and the costs of establishing cell lines, aging porcine ear fibroblasts can support nuclear transfer-mediated and transcription factor-based reprogramming. © 2017 Japanese Society of Animal Science.

  12. FGF2-induced effects on transcriptome associated with regeneration competence in adult human fibroblasts

    PubMed Central

    2013-01-01

    Background Adult human fibroblasts grown in low oxygen and with FGF2 supplementation have the capacity to tip the healing outcome of skeletal muscle injury – by favoring regeneration response in vivo over scar formation. Here, we compare the transcriptomes of control adult human dermal fibroblasts and induced regeneration-competent (iRC) fibroblasts to identify transcriptional changes that may be related to their regeneration competence. Results We identified a unique gene-expression profile that characterizes FGF2-induced iRC fibroblast phenotype. Significantly differentially expressed genes due to FGF2 treatment were identified and analyzed to determine overrepresented Gene Ontology terms. Genes belonging to extracellular matrix components, adhesion molecules, matrix remodelling, cytoskeleton, and cytokines were determined to be affected by FGF2 treatment. Conclusions Transcriptome analysis comparing control adult human fibroblasts with FGF2-treated fibroblasts identified functional groups of genes that reflect transcriptional changes potentially contributing to their regeneration competence. This comparative transcriptome analysis should contribute new insights into genes that characterize cells with greater regenerative potential. PMID:24066673

  13. Cell senescence in human aging and disease.

    PubMed

    Fossel, Michael

    2002-04-01

    The most common causes of death and suffering, even in most underdeveloped nations, are age-related diseases. These diseases share fundamental and often unappreciated pathology at the cellular and genetic levels, through cell senescence. In cancer, enforcing cell senescence permits us to kill cancer cells without significantly harming normal cells. In other age-related diseases, cell senescence plays a direct role, and we may be able to prevent and reverse much of the pathology. While aging is attributed to "wear and tear," genetic studies show that these effects are avoidable (as is the case in germ cell lines) and occur only when cells down-regulate active (and sufficient) repair mechanisms, permitting degradation to occur. Aging occurs when cells permit accumulative damage by wear and tear, by altering their gene expression rather than vice versa. Using telomerase in laboratory settings, we can currently reset this pattern and its consequences both within cells and between cells. Doing so resets not only cell behavior but the pathological consequences within tissues comprising such cells. We can currently grow histologically young, reconstituted human skin using old human skin cells (keratinocytes and fibroblasts). Technically we could now test this approach in joints, vessels, the immune system, and other tissues. This model is consistent with all available laboratory data and known aging pathology. Within the next decade, we will be able to treat age-related diseases more effectively than ever before.

  14. Anti-scarring effects of butaprost on human subconjunctival Tenon's fibroblasts

    PubMed Central

    Shin, Jong Hoon; Seo, Je Hyun; Jung, Jae Ho; Kim, Tae Woo

    2017-01-01

    AIM To investigate the toxicity of the E-prostanoid 2 (EP2) receptor agonist, butaprost against human subconjunctival (Tenon's capsule) fibroblasts, and to determine the underlying mechanism. METHODS We isolated Tenon's fibroblasts from the subconjunctival area of healthy subjects and evaluated the types of EP receptors expressed using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). The toxicity of butaprost against the fibroblasts was evaluated using methyl thiazolyl tetrazolium and lactic dehydrogenase assays. The inhibition of conjunctival fibroblast proliferation by butaprost was assessed by measuring α-actin levels. The underlying mechanism was assessed by measuring intracellular cyclic adenosine monophosphate (cAMP) levels. Intergroup differences were statistically analyzed using an independent t-test. Densitometry of the Western blot bands was performed using the Image J software. RESULTS Quantitative real-time RT-PCR revealed that the fibroblast EP2 receptor levels were higher than those of the other EP receptors. Butaprost did not show toxicity against Tenon's tissue, but inhibited conjunctival fibroblast proliferation by reducing collagen synthesis. EP2 receptor activation enhanced the cAMP cascade, which might be an important mechanism underlying this effect. CONCLUSION Butaprost effectively reduces the subconjunctival scarring response. Given the significance of wound healing modulation in blebs, butaprost's inhibitory effect on subconjunctival Tenon's fibroblasts may be beneficial in managing postoperative scarring in glaucoma surgery. PMID:28730102

  15. Oxidative damage to human parametrial ligament fibroblasts induced by mechanical stress.

    PubMed

    Hong, Shasha; Li, Hong; Wu, Debin; Li, Bingshu; Liu, Cheng; Guo, Wenjun; Min, Jie; Hu, Ming; Zhao, Yang; Yang, Qing

    2015-10-01

    The aim of the present study was to explore the underlying mechanisms of the roles of mechanical factors in the pathogenesis of pelvic organ prolapse (POP). The experiments were performed on fibroblasts derived from uterosacral ligaments and cardinal ligaments of patients who received total hysterectomy due to benign disease excluding POP. Fibroblasts were cultured after collagenase digestion and identified by morphological observation and immunocytochemical methods. A four‑point bending device was used to subject fibroblasts at passage 4‑6 to strains of 0, 1,333 µ (1 mm), 2,666 µ (2 mm) or 5,333 µ (4 mm) at a frequency of 0.1 Hz for 4 h. Intracellular reactive oxygen species (ROS) were quantified using the fluorescent probe 2',7'‑dichlorodihydrofluorescein diacetate. Changes in the mitochondrial membrane potential were verified using the fluorescent dye JC‑1, and apoptosis was detected using Annexin V/propidium iodide staining and flow cytometric analysis. Mechanical strain changed the morphology and adherence ability of parametrial ligament fibroblasts. Furthermore, the production of ROS was significantly increased and the mitochondrial membrane potential obviously declined with the enhancement of mechanical stress loading. In addition, the apoptotic rate of fibroblasts subjected to high mechanical strain was significantly increased compared with that in fibroblast under low‑intensity strain. In conclusion, the present study showed that mechanical strain enhanced intracellular ROS levels, decreased the mitochondrial membrane potential and increased the apoptotic rate in human parametrial ligament fibroblasts, which may contribute to POP.

  16. Asymmetric Migration of Human Keratinocytes under Mechanical Stretch and Cocultured Fibroblasts in a Wound Repair Model

    PubMed Central

    Lü, Dongyuan; Liu, Xiaofeng; Gao, Yuxin; Huo, Bo; Kang, Yingyong; Chen, Juan; Sun, Shujin; Chen, Li; Luo, Xiangdong; Long, Mian

    2013-01-01

    Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment. PMID:24086354

  17. Interaction between human lung fibroblasts and T-lymphocytes prevents activation of CD4+ cells

    PubMed Central

    Vancheri, Carlo; Mastruzzo, Claudio; Trovato-Salinaro, Elisa; Gili, Elisa; Lo Furno, Debora; Pistorio, Maria P; Caruso, Massimo; La Rosa, Cristina; Crimi, Claudia; Failla, Marco; Crimi, Nunzio

    2005-01-01

    Background T lymphocytes are demonstrated to play an important role in several chronic pulmonary inflammatory diseases. In this study we provide evidence that human lung fibroblasts are capable of mutually interacting with T-lymphocytes leading to functionally significant responses by T-cells and fibroblasts. Methods Human lung fibroblast were co-cultured with PMA-ionomycin activated T-CD4 lymphocytes for 36 hours. Surface as well as intracellular proteins expression, relevant to fibroblasts and lymphocytes activation, were evaluated by means of flow cytometry and RT-PCR. Proliferative responses of T lymphocytes to concanavalin A were evaluated by the MTT assay. Results In lung fibroblasts, activated lymphocytes promote an increase of expression of cyclooxygenase-2 and ICAM-1, expressed as mean fluorescence intensity (MFI), from 5.4 ± 0.9 and 0.7 ± 0.15 to 9.1 ± 1.5 and 38.6 ± 7.8, respectively. Fibroblasts, in turn, induce a significant reduction of transcription and protein expression of CD69, LFA-1 and CD28 in activated lymphocytes and CD3 in resting lymphocytes. In activated T lymphocytes, LFA-1, CD28 and CD69 expression was 16.6 ± 0.7, 18.9 ± 1.9 and 6.6 ± 1.3, respectively, and was significantly reduced by fibroblasts to 9.4 ± 0.7, 9.4 ± 1.4 and 3.5 ± 1.0. CD3 expression in resting lymphocytes was 11.9 ± 1.4 and was significantly reduced by fibroblasts to 6.4 ± 1.1. Intracellular cytokines, TNF-alpha and IL-10, were evaluated in T lymphocytes. Co-incubation with fibroblasts reduced the number of TNF-alpha positive lymphocytes from 54,4% ± 6.12 to 30.8 ± 2.8, while IL-10 positive cells were unaffected. Finally, co-culture with fibroblasts significantly reduced Con A proliferative response of T lymphocytes, measured as MTT absorbance, from 0.24 ± 0.02 nm to 0.16 ± 0.02 nm. Interestingly, while the activation of fibroblasts is mediated by a soluble factor, a cognate interaction ICAM-1 mediated was demonstrated to be responsible for the modulation

  18. Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts.

    PubMed

    Cao, Kan; Blair, Cecilia D; Faddah, Dina A; Kieckhaefer, Julia E; Olive, Michelle; Erdos, Michael R; Nabel, Elizabeth G; Collins, Francis S

    2011-07-01

    Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process.

  19. Progerin and telomere dysfunction collaborate to trigger cellular senescence in normal human fibroblasts

    PubMed Central

    Cao, Kan; Blair, Cecilia D.; Faddah, Dina A.; Kieckhaefer, Julia E.; Olive, Michelle; Erdos, Michael R.; Nabel, Elizabeth G.; Collins, Francis S.

    2011-01-01

    Hutchinson-Gilford progeria syndrome (HGPS), a devastating premature aging disease, is caused by a point mutation in the lamin A gene (LMNA). This mutation constitutively activates a cryptic splice donor site, resulting in a mutant lamin A protein known as progerin. Recent studies have demonstrated that progerin is also produced at low levels in normal human cells and tissues. However, the cause-and-effect relationship between normal aging and progerin production in normal individuals has not yet been determined. In this study, we have shown in normal human fibroblasts that progressive telomere damage during cellular senescence plays a causative role in activating progerin production. Progressive telomere damage was also found to lead to extensive changes in alternative splicing in multiple other genes. Interestingly, elevated progerin production was not seen during cellular senescence that does not entail telomere shortening. Taken together, our results suggest a synergistic relationship between telomere dysfunction and progerin production during the induction of cell senescence, providing mechanistic insight into how progerin may participate in the normal aging process. PMID:21670498

  20. Elastin hydrolysate derived from fish enhances proliferation of human skin fibroblasts and elastin synthesis in human skin fibroblasts and improves the skin conditions.

    PubMed

    Shiratsuchi, Eri; Nakaba, Misako; Yamada, Michio

    2016-03-30

    Recent studies have shown that certain peptides significantly improve skin conditions, such as skin elasticity and the moisture content of the skin of healthy woman. This study aimed to investigate the effects of elastin hydrolysate on human skin. Proliferation and elastin synthesis were evaluated in human skin fibroblasts exposed to elastin hydrolysate and proryl-glycine (Pro-Gly), which is present in human blood after elastin hydrolysate ingestion. We also performed an ingestion test with elastin hydrolysate in humans and evaluated skin condition. Elastin hydrolysate and Pro-Gly enhanced the proliferation of fibroblasts and elastin synthesis. Maximal proliferation response was observed at 25 ng mL(-1) Pro-Gly. Ingestion of elastin hydrolysate improved skin condition, such as elasticity, number of wrinkles, and blood flow. Elasticity improved by 4% in the elastin hydrolysate group compared with 2% in the placebo group. Therefore, elastin hydrolysate activates human skin fibroblasts and has beneficial effects on skin conditions. © 2015 Society of Chemical Industry.

  1. Adipose-derived stem cells promote human dermal fibroblast function and increase senescence-associated β‑galactosidase mRNA expression through paracrine effects.

    PubMed

    Shen, Xiao; Du, Yunpeng; Shen, Weimin; Xue, Bin; Zhao, Yu

    2014-12-01

    Adipose‑derived stem cells (ADSCs) are known to secrete various cytokines, which affect fibroblast function through paracrine effects. In the present study, the paracrine effects of ADSCs on the function and senescence of young and aged human dermal fibroblasts (HDFs) were investigated in vitro. ADSCs and HDFs were isolated from healthy donors and flow cytometry was used for immunophenotype identification. ADSCs were co‑cultured with young or aged human dermal fibroblasts in Transwell plates, and control groups were established accordingly. Cellular proliferation was measured by an MTT assay. Type I collagen, matrix metalloproteinase‑1 (MMP‑1) and senescence-associated β‑galactosidase (SA‑β‑GAL) mRNA expression were measured by quantitative polymerase chain reaction. It was identified that ADSCs promoted proliferation of co‑cultured HDFs and induced increased expression of type I collagen and decreased expression of MMP‑1. The co‑cultured HDFs exhibited increased expression of SA‑β‑GAL. These results demonstrated that ADSCs improve fibroblast function through paracrine effects. The increased expression of SA‑β‑GAL indicated an accelerated aging process. It is proposed that ADSCs may improve fibroblast function, but not reverse the age status in vitro.

  2. Exposure of human lung fibroblasts to ozone: cell mortality and hyaluronan metabolism

    SciTech Connect

    Mayer, D.; Branscheid, D. )

    1992-04-01

    Exposure of cultures of human lung fibroblasts to 0.5 ppm ozone for 20 h resulted in a significant increase in cellular mortality by 29%; after exposure to 2.5 ppm ozone for 4 h, the increase amounted to 74%. A marked difference in sensitivity to ozone was observed between fibroblast lines from different individuals. This variability in resistance to ozone was more evident after exposure to 0.5 ppm ozone for 20 h, when compared with 2.5 ppm ozone for 4 h. In one fibroblast line, synthesis of hyaluronan was enhanced by exposure to 0.5 ppm ozone for 20 h. The concentrations of hyaluronan in culture media increased in experiments using different fibroblast cell lines, a phenomenon that was obvious both if cell numbers and combined protein concentrations of cells and media are selected as references for hyaluronan concentrations.

  3. Preferential attachment of human gingival fibroblasts to the resin ionomer Geristore.

    PubMed

    Al-Sabek, Fuwad; Shostad, Sandra; Kirkwood, Keith L

    2005-03-01

    The resin ionomer Geristore has been used extensively for root perforation repairs. The purpose of this study was to evaluate oral in vitro biocompatibility of the resin ionomer Geristore compared to two other dental perforation repair materials, Ketac-Fil and Immediate Restorative Material (IRM). Growth and morphology of human gingival fibroblasts (HGFs) was determined using scanning electron microscopy (SEM) of HGFs cells grown on test materials as well as cytotoxicity assays using eluates from test materials. SEM analysis showed that HGFs attached and spread well over Geristore with relatively normal morphology. SEM showed that fibroblasts did not attach and spread well over Ketac-Fil or IRM as cells appeared much fewer with rounded and different morphology than fibroblasts grown on Geristore. Cytotoxicity assays indicated that HGFs proliferated in the presence of Geristore eluates and not in the presence of Ketac-Fil or IRM eluates. In vitro interpretation indicates that Geristore is less cytotoxic to gingival fibroblasts.

  4. Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

    PubMed

    Shukla, Vasudha; Barnhouse, Victoria; Ackerman, William E; Summerfield, Taryn L; Powell, Heather M; Leight, Jennifer L; Kniss, Douglas A; Ghadiali, Samir N

    2017-09-22

    The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1β, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1β or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1β induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1β inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

  5. Inherited human IRAK-1 deficiency selectively impairs TLR signaling in fibroblasts

    PubMed Central

    Borghesi, Alessandro; Zhou, Hao; Bougarn, Salim; Boughorbel, Sabri; Israel, Laura; Meloni, Ilaria; Chrabieh, Maya; Ling, Yun; Itan, Yuval; Renieri, Alessandra; Mazzucchelli, Iolanda; Basso, Sabrina; Pavone, Piero; Falsaperla, Raffaele; Ciccone, Roberto; Cerbo, Rosa Maria; Stronati, Mauro; Picard, Capucine; Zuffardi, Orsetta; Abel, Laurent; Chaussabel, Damien; Marr, Nico; Li, Xiaoxia; Casanova, Jean-Laurent; Puel, Anne

    2017-01-01

    Most members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) families transduce signals via a canonical pathway involving the MyD88 adapter and the interleukin-1 receptor-associated kinase (IRAK) complex. This complex contains four molecules, including at least two (IRAK-1 and IRAK-4) active kinases. In mice and humans, deficiencies of IRAK-4 or MyD88 abolish most TLR (except for TLR3 and some TLR4) and IL-1R signaling in both leukocytes and fibroblasts. TLR and IL-1R responses are weak but not abolished in mice lacking IRAK-1, whereas the role of IRAK-1 in humans remains unclear. We describe here a boy with X-linked MECP2 deficiency-related syndrome due to a large de novo Xq28 chromosomal deletion encompassing both MECP2 and IRAK1. Like many boys with MECP2 null mutations, this child died very early, at the age of 7 mo. Unlike most IRAK-4– or MyD88-deficient patients, he did not suffer from invasive bacterial diseases during his short life. The IRAK-1 protein was completely absent from the patient’s fibroblasts, which responded very poorly to all TLR2/6 (PAM2CSK4, LTA, FSL-1), TLR1/2 (PAM3CSK4), and TLR4 (LPS, MPLA) agonists tested but had almost unimpaired responses to IL-1β. By contrast, the patient’s peripheral blood mononuclear cells responded normally to all TLR1/2, TLR2/6, TLR4, TLR7, and TLR8 (R848) agonists tested, and to IL-1β. The death of this child precluded long-term evaluations of the clinical consequences of inherited IRAK-1 deficiency. However, these findings suggest that human IRAK-1 is essential downstream from TLRs but not IL-1Rs in fibroblasts, whereas it plays a redundant role downstream from both TLRs and IL-1Rs in leukocytes. PMID:28069966

  6. Outcome of regenerative therapy for age-related vocal fold atrophy with basic fibroblast growth factor.

    PubMed

    Ohno, Satoshi; Hirano, Shigeru; Yasumoto, Akiyoshi; Ikeda, Hiroki; Takebayashi, Shinji; Miura, Makoto

    2016-08-01

    Age-related vocal fold atrophy has become a significant voice disorder as the elderly population grows. However, several therapeutic challenges have limited attempts to improve voice quality. We reported that basic fibroblast growth factor (bFGF) stimulates fibroblasts to produce extracellular matrices such as hyaluronic acid in the lamina propria, leading to a regeneration of pliable vocal folds in animal models. The aim of this study was to determine the efficacy of bFGF for the treatment of age-related vocal fold atrophy. Prospective study. Six patients with age-related vocal fold atrophy underwent injection of bFGF in their vocal folds. Vocal outcomes and stroboscopic examinations were evaluated 1, 3, and 6 months after the injection. The outcome measures included the Voice Handicap Index-10 (VHI-10), GRBAS (grade, roughness, breathiness, asthenia, strain) scale, maximum phonation time (MPT), the amplitude perturbation quotient (APQ), and the pitch perturbation quotient (PPQ). The VHI-10 was significantly improved 6 months after bFGF injection. The GRBAS scale, MPT, APQ, and PPQ were also improved. Stroboscopic examinations showed significant improvement of glottic closure and better mucosal wave. This is the first study to evaluate the regenerative effects of bFGF injection for the treatment of age-related vocal fold atrophy using the VHI-10. Injection of bFGF significantly improved VHI-10 scores and glottal insufficiency for at least 6 months. 4. Laryngoscope, 126:1844-1848, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  7. Adverse fibrosis in the aging heart depends on signaling between myeloid and mesenchymal cells; role of inflammatory fibroblasts.

    PubMed

    Cieslik, Katarzyna A; Trial, JoAnn; Crawford, Jeffrey R; Taffet, George E; Entman, Mark L

    2014-05-01

    Aging has been associated with adverse fibrosis. Here we formulate a new hypothesis and present new evidence that unresponsiveness of mesenchymal stem cells (MSC) and fibroblasts to transforming growth factor beta (TGF-β), due to reduced expression of TGF-β receptor I (TβRI), provides a foundation for cardiac fibrosis in the aging heart via two mechanisms. 1) TGF-β promotes expression of Nanog, a transcription factor that retains MSC in a primitive state. In MSC derived from the aging heart, Nanog expression is reduced and therefore MSC gradually differentiate and the number of mesenchymal fibroblasts expressing collagen increases. 2) As TGF-β signaling pathway components negatively regulate transcription of monocyte chemoattractant protein-1 (MCP-1), a reduced expression of TβRI prevents aging mesenchymal cells from shutting down their own MCP-1 expression. Elevated MCP-1 levels that originated from MSC attract transendothelial migration of mononuclear leukocytes from blood to the tissue. MCP-1 expressed by mesenchymal fibroblasts promotes further migration of monocytes and T lymphocytes away from the endothelial barrier and supports the monocyte transition into macrophages and finally into myeloid fibroblasts. Both myeloid and mesenchymal fibroblasts contribute to fibrosis in the aging heart via collagen synthesis. This article is part of a Special Issue entitled "Myocyte-Fibroblast Signalling in Myocardium ". © 2013. Published by Elsevier Ltd. All rights reserved.

  8. Role of postreplication repair in transformation of human fibroblasts to anchorage independence

    SciTech Connect

    Boyer, J.C.; Kaufmann, W.K.; Cordeiro-Stone, M. )

    1991-06-01

    Cellular capacity for postreplication repair (PRR) and sensitivity to transformation to anchorage independence (AI) were quantified in normal foreskin and xeroderma pigmentosum (XP) variant fibroblasts after treatment with UV or benzo(a)pyrene-diol-epoxide I (BPDE-I). PRR is defined here as a collection of pathways that facilitate the replication of DNA damaged by genotoxic agents. It is recognized biochemically as the process by which nascent DNA grows longer than the average distance between two lesions in the DNA template. PRR refers more directly to the elimination of gaps in the daughter-strand DNA by mechanisms which remain to be determined for human cells, but which may include translesion replication and recombination. PRR was measured in diploid human fibroblasts by analysis of the dose kinetics for inhibition of DNA strand growth in carcinogen-treated cells. Logarithmically growing foreskin fibroblasts (NHF1) displayed D0 values of 4.3 J/m{sup 2} and 0.14 microM for the inhibition of DNA synthesis in active replicons by UV and BPDE-I, respectively. XP variant cells (CRL1162) exhibited corresponding D0 values of 1.5 J/m{sup 2} and 0.16 microM. The increased sensitivity to inhibition of DNA replication by UV in these XP variant fibroblasts (2.9-fold greater than normal) was mirrored by an enhanced frequency of transformation to AI. XP variant fibroblasts (CRL1162) were 3.2 times more sensitive to transformation to AI by UV than were the normal foreskin fibroblasts. As predicted by the PRR studies, both cell types exhibited similar frequencies of AI colonies induced by BPDE-I. Apparent thresholds were observed for induction of AI by UV (normal fibroblasts, 2.7 J/m{sup 2}; XP variant fibroblasts, 0.3 J/m{sup 2}) and BPDE-I (both, 0.05 microM).

  9. Generation of iPSC line HEL24.3 from human neonatal foreskin fibroblasts.

    PubMed

    Trokovic, Ras; Weltner, Jere; Otonkoski, Timo

    2015-07-01

    Human iPSC line HEL24.3 was generated from healthy human foreskin fibroblasts using non-integrative reprogramming method. Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using Sendai viruses.

  10. In vitro effects of ascorbic acid and β-glycerophosphate on human gingival fibroblast cells.

    PubMed

    Martinez, Elizabeth F; Donato, Tatiani A G; Arana-Chavez, Victor E

    2012-10-01

    Ascorbic acid (AA) and β-glycerophosphate (βG) are considered in vitro osteogenic factors important to the differentiation of osteoblastic progenitor and dental pulp cells into mineralized tissue-forming cells. So, the present study investigated in vitro if these mineralizing inducible factors (AA and βG) could influence differentiation of human gingival fibroblasts when compared with human pulp cells and osteogenic cells derived from rat calvaria cultured. The expression of osteopontin (OPN) and osteoadherin (OSAD) was analyzed by indirect immunofluorescence, immunocytochemistry as well as Western-blotting. In addition, the main ultrastructural aspects were also investigated. No mineralized matrix formation occurred on gingival fibroblasts induced with AA+βG. On these cells, no expression of OPN and OSAD was observed when compared with pulp cells, pulp cells induced with AA+βG as well as osteogenic cells. Ultrastructure analysis additionally showed that gingival fibroblasts exhibited typical fibroblast morphology with no nodule formation. The present findings showed that AA and βG could not promote a mineralized cell differentiation of human gingival fibroblasts and confirm that human dental pulp cells, as the osteogenic cells, are capable to form a mineralized extracellular.

  11. Accelerated aging syndromes, are they relevant to normal human aging?

    PubMed

    Dreesen, Oliver; Stewart, Colin L

    2011-09-01

    Hutchinson-Gilford Progeria (HGPS) and Werner syndromes are diseases that clinically resemble some aspects of accelerated aging. HGPS is caused by mutations in theLMNA gene resulting in post-translational processing defects that trigger Progeria in children. Werner syndrome, arising from mutations in the WRN helicase gene, causes premature aging in young adults. What are the molecular mechanism(s) underlying these disorders and what aspects of the diseases resemble physiological human aging? Much of what we know stems from the study of patient derived fibroblasts with both mutations resulting in increased DNA damage, primarily at telomeres. However, in vivo patients with Werner's develop arteriosclerosis, among other pathologies. In HGPS patients, including iPS derived cells from HGPS patients, as well as some mouse models for Progeria, vascular smooth muscle (VSM) appears to be among the most severely affected tissues. Defective Lamin processing, associated with DNA damage, is present in VSM from old individuals, indicating processing defects may be a factor in normal aging. Whether persistent DNA damage, particularly at telomeres, is the root cause for these pathologies remains to be established, since not all progeroid Lmna mutations result in DNA damage and genome instability.

  12. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

    PubMed

    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  13. Heme Oxygenase 1 Mediates an Adaptive Response to Oxidative Stress in Human Skin Fibroblasts

    NASA Astrophysics Data System (ADS)

    Vile, G. F.; Basu-Modak, S.; Waltner, C.; Tyrrell, R. M.

    1994-03-01

    Oxidative stress of human skin fibroblasts by treatment with ultraviolet A (UVA) radiation has been shown to lead to an increase in levels of the heme catabolizing enzyme heme oxygenase 1 [heme, hydrogen-donor:oxygen oxidoreductase (α-methene-oxidizing, hydroxylating), EC 1.14.99.3] and the iron storage protein ferritin. Here we show that human skin fibroblasts, preirradiated with UVA, sustain less membrane damage during a subsequent exposure to UVA radiation than cells that had not been preirradiated. Pretreating cells with heme oxygenase 1 antisense oligonucleotide inhibited the irradiation-dependent induction of both the heme oxygenase 1 enzyme and ferritin and abolished the protective effect of preirradiation. Inhibition of the UVA preirradiation-dependent increase in ferritin, but not heme oxygenase, with desferrioxamine also abolished the protection. This identifies heme oxygenase 1 as a crucial enzymatic intermediate in an oxidant stress-inducible antioxidant defense mechanism, involving ferritin, in human skin fibroblasts.

  14. Possible identity of IL-8 converting enzyme in human fibroblasts as a cysteine protease.

    PubMed

    Ohashi, Kensaku; Sano, Emiko; Nakaki, Toshio; Naruto, Masanobu

    2003-04-01

    A converting activity was characterized in human diploid fibroblasts, which secrete 72IL-8 and 77IL-8 in treatment with IFN-beta and poly I: poly C. 77IL-8 was significantly converted to 72IL-8 by a partially purified fraction of the culture supernatant of human diploid fibroblasts. The converting activity, which was temperature-dependent and optimal at pH 6, was completely inhibited by cysteine protease inhibitors, antipain dihydrochloride and E-64, but not by other types of protease inhibitors. These data clearly show that human diploid fibroblasts are capable of processing IL-8 to produce a mature IL-8 and that the putative converting enzyme appears to be a cysteine protease.

  15. Reactive oxygen species production in mitochondria of human gingival fibroblast induced by blue light irradiation.

    PubMed

    Yoshida, Ayaka; Yoshino, Fumihiko; Makita, Tetsuya; Maehata, Yojiro; Higashi, Kazuyoshi; Miyamoto, Chihiro; Wada-Takahashi, Satoko; Takahashi, Shun-suke; Takahashi, Osamu; Lee, Masaichi Chang-il

    2013-12-05

    In recent years, it has become well known that the production of reactive oxygen species (ROS) induced by blue-light irradiation causes adverse effects of photo-aging, such as age-related macular degeneration of the retina. Thus, orange-tinted glasses are used to protect the retina during dental treatment involving blue-light irradiation (e.g., dental resin restorations or tooth bleaching treatments). However, there are few studies examining the effects of blue-light irradiation on oral tissue. For the first time, we report that blue-light irradiation by quartz tungsten halogen lamp (QTH) or light-emitting diode (LED) decreased cell proliferation activity of human gingival fibroblasts (HGFs) in a time-dependent manner (<5 min). Additionally, in a morphological study, the cytotoxic effect was observed in the cell organelles, especially the mitochondria. Furthermore, ROS generation induced by the blue-light irradiation was detected in mitochondria of HGFs using fluorimetry. In all analyses, the cytotoxicity was significantly higher after LED irradiation compared with cytotoxicity after QTH irradiation. These results suggest that blue light irradiation, especially by LED light sources used in dental aesthetic treatment, might have adverse effects on human gingival tissue. Hence, this necessitates the development of new dental aesthetic treatment methods and/or techniques to protect HGFs from blue light irradiation during dental therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro

    PubMed Central

    Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology. PMID:27536196

  17. Effects of chlorhexidine, essential oils and herbal medicines (Salvia, Chamomile, Calendula) on human fibroblast in vitro.

    PubMed

    Wyganowska-Swiatkowska, Marzena; Urbaniak, Paulina; Szkaradkiewicz, Anna; Jankun, Jerzy; Kotwicka, Malgorzata

    2016-01-01

    Antiseptic rinses have been successfully used in inflammatory states of the gums and oral cavity mucosa. Antibacterial effects of chlorhexidine, essential oils and some herbs are well documented. Reaction of host tissue to these substances has much poorer documentation. The aim of the study was to analyse the influence of chlorhexidine (CHX), essential oil (EO: thymol, 0.064%; eucalyptol, 0.092%; methyl salicylate, 0.060%; menthol, 0.042%) mouth rinses and salvia, chamomile and calendula brews on fibroblast biology in vitro. The human fibroblast CCD16 line cells were cultured in incubation media which contained the examined substances. After 24 and 48 hours, the cell morphology, relative growth and apoptosis were evaluated. Exposure of fibroblasts to CHX, EO or salvia caused various changes in cell morphology. Cells cultured for 48 hours with CHX revealed a noticeably elongated shape of while cells cultured in high EO concentration or with salvia were considerably smaller and contracted with fewer projections. Chlorhexidine, EO and salvia reduced the fibroblast proliferation rate and stimulated cell death. Both reactions to EO were dose dependent. Cells exposure to chamomile or calendula brews did not change morphology or proliferation of fibroblasts. The results of this in vitro study showed that in contrast to chamomile and calendula, the brews of EO, CHX or salvia had a negative influence on fibroblast biology.

  18. Proteomic analysis of the soluble fraction from human corneal fibroblasts with reference to ocular transparency.

    PubMed

    Karring, Henrik; Thøgersen, Ida B; Klintworth, Gordon K; Enghild, Jan J; Møller-Pedersen, Torben

    2004-07-01

    The transparent corneal stroma contains a population of corneal fibroblasts termed keratocytes, which are interspersed between the collagen lamellae. Under normal conditions, the keratocytes are quiescent and transparent. However, after corneal injury the keratocytes become activated and transform into backscattering wound-healing fibroblasts resulting in corneal opacification. At present, the most popular hypothesis suggests that particular abundant water-soluble proteins called enzyme-crystallins are involved in maintaining corneal cellular transparency. Specifically, corneal haze development is thought to be related to low levels of cytoplasmic enzyme-crystallins in reflective corneal fibroblasts. To further investigate this hypothesis, we have used a proteomic approach to identify the most abundant water-soluble proteins in serum-cultured human corneal fibroblasts that represent an in vitro model of the reflective wound-healing keratocyte phenotype. Densitometry of one-dimensional gels revealed that no single protein isoform exceeded 5% of the total water-soluble protein fraction, which is the qualifying property of a corneal enzyme-crystallin according to the current definition. This result indicates that wound-healing corneal fibroblasts do not contain enzyme-crystallins. A total of 254 protein identifications from two-dimensional gels were performed representing 118 distinct proteins. Proteins protecting against oxidative stress and protein misfolding were prominent, suggesting that these processes may participate in the generation of cytoplasmic light-scattering from corneal fibroblasts.

  19. Effect of three commercial mouth rinses on cultured human gingival fibroblast: an in vitro study.

    PubMed

    Flemingson; Emmadi, Pamela; Ambalavanan, N; Ramakrishnan, T; Vijayalakshmi, R

    2008-01-01

    To examine the effect of three commercial mouth rinses (Hexidine 0.2%, Listerine Cool Mint, Betadine 1%) upon cultured human gingival fibroblast proliferation. Human gingival fibroblasts were cultured and incubated in Dulbecco's Minimum Eagle's Medium containing Chlorhexidine, Listerine, Povidone-Iodine at varying concentrations (1%, 2%, 5%, 10%, 20% and 100% of the given solution) at 37 degrees C for 1, 5 and 15 min. Control cells received an equal volume of Dulbecco's Minimum Eagle's Medium without adding mouth rinses, for similar duration of exposure at 37 degrees C. Following incubation the media were removed, cells were washed twice with medium, supplemented with 10% Fetal Bovine Serum, and fibroblasts in the test and control group were allowed to recover in the same media for 24 h. In all the three groups, the proliferation inhibition was dependent on the concentration of solublized mouth rinses in the cell culture but independent of the duration of exposure to all three mouth rinses. The results showed that all three solutions were toxic to cultured human gingival fibroblasts, Chlorhexidine being the most cytotoxic. It was seen that at dilute concentrations (1% and 2% of given solutions) Listerine was more cytotoxic than Chlorhexidine and Povidone-Iodine. These results suggest that Chlorhexidine, Listerine and Povidone-Iodine are capable of inducing a dose-dependent reduction in cellular proliferation of fibroblasts. The results presented are interesting, but to know the clinical significance, further studies are needed.

  20. Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

    PubMed

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

    2015-01-01

    Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

  1. KL-6, a human MUC1 mucin, promotes proliferation and survival of lung fibroblasts

    SciTech Connect

    Ohshimo, Shinichiro; Yokoyama, Akihito . E-mail: yokoyan@hiroshima-u.ac.jp; Hattori, Noboru; Ishikawa, Nobuhisa; Hirasawa, Yutaka; Kohno, Nobuoki

    2005-12-30

    The serum level of KL-6, a MUC1 mucin, is a clinically useful marker for various interstitial lung diseases. Previous studies demonstrated that KL-6 promotes chemotaxis of human fibroblasts. However, the pathophysiological role of KL-6 remains poorly understood. Here, we further investigate the functional aspects of KL-6 in proliferation and apoptosis of lung fibroblasts. KL-6 accelerated the proliferation and inhibited the apoptosis of all human lung fibroblasts examined. An anti-KL-6 monoclonal antibody counteracted both of these effects induced by KL-6 on human lung fibroblasts. The pro-fibroproliferative and anti-apoptotic effects of KL-6 are greater than and additive to those of the maximum effective concentrations of platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor-{beta}. These findings indicate that increased levels of KL-6 in the epithelial lining fluid may stimulate fibrotic processes in interstitial lung diseases and raise the possibility of applying an anti-KL-6 antibody to treat interstitial lung diseases.

  2. [Elaboration of biodegradable polymer substrate for cultivation of human dermal fibroblasts].

    PubMed

    Shved, Iu A; Kukhareva, L V; Zorin, I M; Solov'ev, A Iu; Blinova, M I; Bilibin, A Iu; Pinaev, G P

    2006-01-01

    The influence of polylactic acid (PLA) surface films on the pattern of cell behavior was studied. The human dermal fibroblasts were cultivated on PLA covered glasses. The hydrophobic nature of PLA films depends on the availability of polymer solvent in the film preparation. PLA films obtained from a more polar solvent--aceton--appeared to be more hydrophilic than those obtained from methylene chloride. More hydrophilic polymer films also appeared to be more preferable for cell cultivation, and human dermal fibroblasts demonstrated a better adhesion and proliferation on hydrophilic rather than on hydrophobic PLA films.

  3. Attenuation of Inhibitory Prostaglandin E2 Signaling in Human Lung Fibroblasts Is Mediated by Phosphodiesterase 4

    PubMed Central

    Michalski, Joel; Kanaji, Nobuhiro; Liu, Xiangde; Nogel, Steve; Wang, Xingqi; Basma, Hesham; Nakanishi, Masanori; Sato, Tadashi; Gunji, Yoko; Fahrid, Maha; Nelson, Amy; Muller, Kai-Christian; Holz, Olaf; Magnussen, Helgo; Rabe, Klaus F.; Toews, Myron L.

    2012-01-01

    The etiology of chronic obstructive pulmonary disease (COPD) is complex and involves an aberrant inflammatory response. Prostaglandin (PG)E2 is elevated in COPD, is a key modulator of lung fibroblast functions, and may influence COPD progression. Most studies evaluating the effects of PGE2 on lung fibroblasts have used acute exposures. The current study evaluated whether longer-term exposure would induce attenuation of PGE2 signaling as part of an autoregulatory pathway. Human fetal lung fibroblasts were pretreated with PGE2 for 24 hours, and migration and cAMP accumulation in response to acute stimulation with PGE2 were assessed. Fibroblasts from adults with and without COPD were pretreated, and migration was assessed. PGE2 pretreatment attenuated subsequent PGE2-mediated inhibition of chemotaxis and cAMP stimulation. This attenuation was predominantly due to an increase in phosphodiesterase (PDE)4-mediated degradation of cAMP rather than to decreased activation of PGE2 receptors (receptor desensitization). Albuterol- and iloprost-mediated signaling were also attenuated after PGE2 pretreatment, suggesting that activation of PDE4 was able to broadly modulate multiple cAMP-coupled pathways. Lung fibroblasts from adult control subjects pretreated with PGE2 also developed attenuation of PGE2-mediated inhibition of chemotaxis. In contrast, fibroblasts obtained from patients with COPD maintained inhibitory PGE2 signaling after PGE2 pretreatment. These data identify a PDE4-mediated attenuation of PGE2 inhibitory signaling in normal fibroblasts that appears to be altered in COPD fibroblasts. These alterations may contribute to COPD pathogenesis and could provide novel therapeutic targets. PMID:23043089

  4. microRNA-141 regulates BMI1 expression and induces senescence in human diploid fibroblasts.

    PubMed

    Dimri, Manjari; Carroll, Jeremy D; Cho, Joon-Ho; Dimri, Goberdhan P

    2013-11-15

    Polycomb group protein BMI1 is an important regulator of senescence, aging, and cancer. On one hand, it is overexpressed in cancer cells and is required for self-renewal of stem cells. On the other hand, it is downregulated during senescence and aging. MicroRNAs have emerged as major regulators of almost every gene associated with cancer, aging, and related pathologies. At present, very little is known about the miRNAs that regulate the expression of BMI1. Here, we report that miR-141 posttranscriptionally downregulates BMI1 expression in human diploid fibroblasts (HDFs) via a miR-141 targeting sequence in the 3' untranslated region of BMI1 mRNA. We also show that overexpression of miR-141 induces premature senescence in HDFs via targeting of BMI1 in normal but not in exogenous BMI1-overexpressing HDFs. Induction of premature senescence in HDFs was accompanied by upregulation of p16INK4a, an important downstream target of BMI1 and a major regulator of senescence. Our results suggest that miR-141-based therapies could be developed to treat pathologies where BMI1 is deregulated.

  5. Modulation of Cell Cycle Profile by Chlorella vulgaris Prevents Replicative Senescence of Human Diploid Fibroblasts.

    PubMed

    Saberbaghi, Tayyebeh; Abbasian, Firouz; Mohd Yusof, Yasmin Anum; Makpol, Suzana

    2013-01-01

    In this study, the effects of Chlorella vulgaris (CV) on replicative senescence of human diploid fibroblasts (HDFs) were investigated. Hot water extract of CV was used to treat HDFs at passages 6, 15, and 30 which represent young, presenescence, and senescence ages, respectively. The level of DNA damage was determined by comet assay while apoptosis and cell cycle profile were determined using FACSCalibur flow cytometer. Our results showed direct correlation between increased levels of damaged DNA and apoptosis with senescence in untreated HDFs (P < 0.05). Cell cycle profile showed increased population of untreated senescent cells that enter G0/G1 phase while the cell population in S phase decreased significantly (P < 0.05). Treatment with CV however caused a significant reduction in the level of damaged DNA and apoptosis in all age groups of HDFs (P < 0.05). Cell cycle analysis showed that treatment with CV increased significantly the percentage of senescent HDFs in S phase and G2/M phases but decreased the population of cells in G0/G1 phase (P < 0.05). In conclusion, hot water extract of Chlorella vulgaris effectively decreased the biomarkers of ageing, indicating its potential as an antiageing compound.

  6. Malvidin Protects WI-38 Human Fibroblast Cells Against Stress-induced Premature Senescence

    PubMed Central

    Seo, Hye Rin; Choi, Mi Jin; Choi, Ji Myung; Ko, Jong Cheol; Ko, Jee Yeon; Cho, Eun Ju

    2016-01-01

    Background: Malvidin is one of the most abundant components in red wines and black rice. The effects of malvidin on aging and lifespan under oxidative stress have not been fully understood. This study focused on the anti-aging effect of malvidin on stress-induced premature senescence (SIPS) in WI-38 human lung-derived diploid fibroblasts. Methods: In order to determine the viability of WI-38 cells, MTT assay was conducted, and malondialdehyde level was determined using thiobarbituric acid-reactive substance assay. Protein expression of inflammation-related factors was also evaluated by Western blot analysis. Results: Acute and chronic oxidative stress via hydrogen peroxide (H2O2) treatment led to SIPS in WI-38 cells, which showed decreased cell viability, increased lipid peroxidation, and a shortened lifespan in comparison with non-H2O2-treated WI-38 cells. However, malvidin treatment significantly attenuated H2O2-induced oxidative stress by inhibiting lipid peroxidation and increasing cell viability. Furthermore, the lifespan of WI-38 cells was prolonged by malvidin treatment. In addition, malvidin downregulated the expression of oxidative stress-related proteins, including NF-κB, COX-2, and inducible nitric oxide synthase. Furthermore, protein expression levels of p53, p21, and Bax were also regulated by malvidin treatment in WI-38 cells undergoing SIPS. Conclusions: Malvidin may potentially inhibit the aging process by controlling oxidative stress. PMID:27051647

  7. Protective effect of porphyra-334 on UVA-induced photoaging in human skin fibroblasts.

    PubMed

    Ryu, Jina; Park, Su-Jin; Kim, In-Hye; Choi, Youn Hee; Nam, Taek-Jeong

    2014-09-01

    The significant increase in life expectancy is closely related to the growing interest in the impact of aging on the function and appearance of the skin. Skin aging is influenced by several factors, and solar ultraviolet (UV) irradiation is considered one of the most important causes of skin photoaging. The aim of this study was to examine the anti-photoaging role of porphyra-334 from Porphyra (P.) yezoensis, a mycosporine-like amino acid (MAA), using high-performance liquid chromatography (HPLC), and electrospray ionization‑mass spectrometry (ESI-MS). In the present study, extracted UV‑absorbing compounds from P. yezoensis included palythine, asterina-330 and porphyra-334. Porphyra-334 was the most abundant MAA in P. yezoensis, and it was therefore used for conducting antiphotoaging experiments. The effect of porphyra-334 on the prevention of photoaging was investigated by measuring reactive oxygen species (ROS) production and matrix metalloproteinase (MMP) levels, as well as extracellular matrix (ECM) components and protein expression in UVA‑irradiated human skin fibroblasts. Porphyra-334 suppressed ROS production and the expression of MMPs following UVA irradiation, while increasing levels of ECM components, such as procollagen, type I collagen, elastin. These results suggest that porphyra-334 has various applications in cosmetics and toiletries because of its anti‑photoaging activities and may serve as a novel anti-aging agent.

  8. Gene expression in response to cyclic mechanical stretch in primary human dermal fibroblasts.

    PubMed

    Reichenbach, Maria; Reimann, Kerstin; Reuter, Hendrik

    2014-12-01

    The human dermal skin is permanently exposed to mechanical stress, for instance during facial expression, which might cause wrinkles with age. Cyclic mechanical stretching of cells results in cellular and cytoskeleton alignment perpendicular to the stretch direction regulating cellular response. With gene expression profiling it was aimed to identify the differentially expressed genes associated with the regulation of the cytoskeleton to investigate the stretch-induced cell alignment mechanism. Here, the transcription activity of the genome in response to cyclic mechanical stress was measured using DNA microarray technology with Agilent SurePrint G3 Human GE 8x60k Microarrays, based on the overall measurement of the mRNA. Gene expression was measured at the beginning of the alignment process showing first reoriented cells after 5 h stretching and at the end after 24 h, where nearly all cells are aligned. Gene expression data of control vs. stretched primary human dermal fibroblasts after 5 h and 24 h demonstrated the regulation of differentially expressed genes associated with metabolism, differentiation and morphology and were deposited at http://www.ncbi.nlm.nih.gov/geo with the accession number GSE58389.

  9. Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

    SciTech Connect

    Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S. . E-mail: hcheung@med.miami.edu

    2005-05-13

    Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKC{alpha}-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis.

  10. ALK5 inhibition blocks TGFβ-induced CCN1 expression in human foreskin fibroblasts.

    PubMed

    Thompson, Katherine; Murphy-Marshman, Hannah; Leask, Andrew

    2014-03-01

    The potent profibrotic cytokine TGFβ induces connective tissue growth factor (CCN2/CTGF) is induced in fibroblasts in a fashion sensitive to SB-431542, a specific pharmacological inhibitor of TGFβ type I receptor (ALK5). In several cell types, TGFβ induces CCN1 but suppresses CCN3, which opposes CCN1/CCN2 activities. However, whether SB-431542 alters TGFβ-induced CCN1 or CCN3 in human foreskin fibroblasts in unclear. Here we show that TGFβ induces CCN1 but suppresses CCN3 expression in human foreskin fibroblasts in a SB-431542-sensitive fashion. These results emphasize that CCN1/CCN2 and CCN3 are reciprocally regulated and support the notion that blocking ALK5 or addition of CCN3 may be useful anti-fibrotic approaches.

  11. Efficient Generation of Chemically Induced Mesenchymal Stem Cells from Human Dermal Fibroblasts

    PubMed Central

    Lai, Pei-Lun; Lin, Hsuan; Chen, Shang-Fu; Yang, Shang-Chih; Hung, Kuo-Hsuan; Chang, Ching-Fang; Chang, Hsiang-Yi; Lu, Frank Leigh; Lee, Yi-Hsuan; Liu, Yu-Chuan; Huang, Hsiao-Chun; Lu, Jean

    2017-01-01

    Human mesenchymal stromal/stem cells (MSCs) are multipotent and currently undergoing hundreds of clinical trials for disease treatments. To date, no studies have generated induced MSCs from skin fibroblasts with chemicals or growth factors. Here, we established the first chemical method to convert primary human dermal fibroblasts into multipotent, induced MSC-like cells (iMSCs). The conversion method uses a defined cocktail of small molecules and growth factors, and it can achieve efficient conversion with an average rate of 38% in 6 days. The iMSCs have much higher clonogenicity than fibroblasts, and they can be maintained and expanded in regular MSC medium for at least 8 passages and further differentiated into osteoblasts, adipocytes, and chondrocytes. Moreover, the iMSCs can suppress LPS-mediated acute lung injury as effectively as bone marrow-derived mesenchymal stem cells. This finding may greatly benefit stem cell biology, cell therapy, and regenerative medicine. PMID:28303927

  12. Transcriptomic study of high‑glucose effects on human skin fibroblast cells.

    PubMed

    Pang, Lingxia; Wang, Youpei; Zheng, Meiqin; Wang, Qing; Lin, Hong; Zhang, Liqing; Wu, Lingjian

    2016-03-01

    Skin ulcers are a common complication of diabetes mellitus (DM). Fibroblasts are located within the dermis of skin tissue and can be damaged by diabetes. However, the underlying mechanism of how DM affects fibroblasts remains elusive. To understand the effects of DM on fibroblasts, the current study mimicked DM by high‑glucose (HG) supplementation in the culture medium of human foreskin primary fibroblast cells, and the analysis of transcriptomic changes was conducted. RNA sequencing‑based transcriptome analysis identified that, upon HG stress, 463 genes were upregulated and 351 genes downregulated (>1.5‑fold changes; P<0.05). These altered genes were distributed into 20 different pathways. In addition, gene ontology (GO) analysis indicated that 31 GO terms were enriched. Among the pathways identified, nuclear factor κB (NF‑κB) pathway genes were highly expressed, and the addition of Bay11‑7082, a typical NF‑κB signaling inhibitor, blocked the previously observed alterations in plasminogen activator inhibitor 1 (PAI1), an inflammation marker and frizzled class receptor 8 (FZD8), a Wnt signaling gene, expression that resulted from HG stress. Furthermore, an inhibitor of Wnt signaling diminished the role of Bay11‑7082 in the regulation of PAI1 expression under HG conditions, suggesting that Wnt signaling may function downstream of the NF‑κB pathway to protect fibroblast cells from HG stress. To the best of our knowledge, the current study is the first analysis of transcriptomic responses under HG stress in human fibroblasts. The data provided here may aid the understanding of the molecular mechanisms by which fibroblast cells are damaged in the skin of patients with DM.

  13. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts.

    PubMed

    Carmona-Rodríguez, Bruno; Alvarez-Pérez, Marco Antonio; Narayanan, A Sampath; Zeichner-David, Margarita; Reyes-Gasga, José; Molina-Guarneros, Juan; García-Hernández, Ana Lilia; Suárez-Franco, José Luis; Chavarría, Ivet Gil; Villarreal-Ramírez, Eduardo; Arzate, Higinio

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  14. Human Cementum Protein 1 induces expression of bone and cementum proteins by human gingival fibroblasts

    SciTech Connect

    Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath; Zeichner-David, Margarita; Reyes-Gasga, Jose; Molina-Guarneros, Juan; Garcia-Hernandez, Ana Lilia; Suarez-Franco, Jose Luis; Chavarria, Ivet Gil; Villarreal-Ramirez, Eduardo; Arzate, Higinio . E-mail: harzate@servidor.unam.mx

    2007-07-06

    We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment protein (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.

  15. Ameliorative effects of Eriobotrya japonica seed extract on cellular aging in cultured rat fibroblasts.

    PubMed

    Muramoto, Kazuyo; Quan, Rong-Dan; Namba, Toshiharu; Kyotani, Shojiro; Miyamura, Mitsuhiko; Nishioka, Yutaka; Tonosaki, Keiichi; Doi, Yoshinori L; Kaba, Hideto

    2011-04-01

    To investigate the effects of Eriobotrya japonica seed extract (ESE) on cellular aging, intracellular calcium homeostasis in young and senescent cells was analyzed using a rat fibroblast culture as an in vitro model system and a calcium imaging technique. The application of bradykinin (BK) transiently elicited intracellular calcium ion (Ca(2+)) increased in most of the young fibroblasts, whereas these responses were scarcely observed or were significantly attenuated in senescent cells. However, the long-term treatment of senescent cells with ESE (for 7 days) dose-dependently increased the amplitude of BK-induced responses and the percentage of BK-responding cells. In particular, most senescent cells could respond to BK with long-term treatment with ESE (1.0% or 2.0%), an effect that reinstated the percentage of BK-responding cells to the same level as that in young cells. The effects of ESE on amplitude or percentage of responding cells were not observed in young cells. Moreover, the time to half decay, which was significantly longer in senescent cells than that in young cells, was shortened in senescent cells with long-term treatment with ESE. These results suggest that treatment with an adequate concentration of ESE renders BK-induced Ca(2+) dynamics in senescent cells similar to those in young cells. Therefore, ESE can retard and/or protect against cellular aging and may be useful for elucidating the antiaging processes.

  16. Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence.

    PubMed

    Carvalho, Ana C; Gomes, Andreia C; Pereira-Wilson, Cristina; Lima, Cristovao F

    2015-06-01

    Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

  17. Assessment of human gingival fibroblast interaction with dental implant abutment materials.

    PubMed

    Rutkunas, Vygandas; Bukelskiene, Virginija; Sabaliauskas, Vaidotas; Balciunas, Evaldas; Malinauskas, Mangirdas; Baltriukiene, Daiva

    2015-04-01

    The biocompatibility of dental implant abutment materials depends on numerous factors including the nature of the material, its chemical composition, roughness, texture, hydrophilicity and surface charge. The aim of the present study was to compare the viability and adhesion strength of human gingival fibroblasts (HGFs) grown on several dental materials used in implant prosthodontics. Surfaces of the tested materials were assessed using an optical imaging profiler. For material toxicity and cellular adhesion evaluation, primary human gingival fibroblast cells were used. To evaluate the strength of cellular adhesion, gingival fibroblasts were cultured on the tested materials and subjected to lateral shear forces by applying 300 and 500 rpm shaking intensities. Focal adhesion kinase (FAK) expression and phosphorylation in cells grown on the specimens were registered by cell-based ELISA. There was a tendency of fibroblast adhesion strength to decrease in the following order: sandblasted titanium, polished titanium, sandblasted zirconium oxide, polished zirconium oxide, gold-alloy, chrome-cobalt alloy. Higher levels of total as well as phospho-FAK protein were registered in HGFs grown on roughened titanium. Material type and surface processing technique have an impact on gingival fibroblast interaction with dental implant abutment materials.

  18. Towards Scarless Wound Healing: A Comparison of Protein Expression between Human, Adult and Foetal Fibroblasts

    PubMed Central

    Ho, Sonia; Marçal, Helder; Foster, Leslie John Ray

    2014-01-01

    Proteins from human adult and foetal fibroblast cell lines were compared, focusing on those involved in wound healing. Proteins were separated through two-dimensional gel electrophoresis (2DE). Differences in protein spot intensity between the lineages were quantified through 3D gel scanning densitometry. Selected protein spots were excised, subjected to tryptic digests, prior to separation using HPLC with a linear ion trap mass spectrometer, and identified. Protein maps representing the proteomes from adult and foetal fibroblasts showed similar distributions but revealed differences in expression levels. Heat shock cognate 71 kDA protein, Tubulin alpha-1A chain, actin cytoplasmic-1, and neuron cytoplasmic protein were all expressed in significantly higher concentrations by foetal fibroblasts, nearly double those observed for their adult counterparts. Fructose bisphosphate aldolase A, Cofilin-1, Peroxiredoxin-1, Lactotransferrin Galectin-1, Profilin-1, and Calreticulin were expressed at comparatively higher concentrations by the adult fibroblasts. Significant differences in the expression levels of some proteins in human adult and foetal fibroblasts correlated with known differences in wound healing behaviour. This data may assist in the development of technologies to promote scarless wound healing and better functional tissue repair and regeneration. PMID:24605334

  19. Towards scarless wound healing: a comparison of protein expression between human, adult and foetal fibroblasts.

    PubMed

    Ho, Sonia; Marçal, Helder; Foster, Leslie John Ray

    2014-01-01

    Proteins from human adult and foetal fibroblast cell lines were compared, focusing on those involved in wound healing. Proteins were separated through two-dimensional gel electrophoresis (2DE). Differences in protein spot intensity between the lineages were quantified through 3D gel scanning densitometry. Selected protein spots were excised, subjected to tryptic digests, prior to separation using HPLC with a linear ion trap mass spectrometer, and identified. Protein maps representing the proteomes from adult and foetal fibroblasts showed similar distributions but revealed differences in expression levels. Heat shock cognate 71 kDA protein, Tubulin alpha-1A chain, actin cytoplasmic-1, and neuron cytoplasmic protein were all expressed in significantly higher concentrations by foetal fibroblasts, nearly double those observed for their adult counterparts. Fructose bisphosphate aldolase A, Cofilin-1, Peroxiredoxin-1, Lactotransferrin Galectin-1, Profilin-1, and Calreticulin were expressed at comparatively higher concentrations by the adult fibroblasts. Significant differences in the expression levels of some proteins in human adult and foetal fibroblasts correlated with known differences in wound healing behaviour. This data may assist in the development of technologies to promote scarless wound healing and better functional tissue repair and regeneration.

  20. Prostaglandin E₂ inhibits human lung fibroblast chemotaxis through disparate actions on different E-prostanoid receptors.

    PubMed

    Li, Ying-Ji; Wang, Xing-Qi; Sato, Tadashi; Kanaji, Nobuhiro; Nakanishi, Masanori; Kim, Miok; Michalski, Joel; Nelson, Amy J; Sun, Jian-Hong; Farid, Maha; Basma, Hesham; Patil, Amol; Toews, Myron L; Liu, Xiangde; Rennard, Stephen I

    2011-01-01

    The migration of fibroblasts is believed to play a key role in both normal wound repair and abnormal tissue remodeling. Prostaglandin E (PGE)(2), a mediator that can inhibit many fibroblast functions including chemotaxis, was reported to be mediated by the E-prostanoid (EP) receptor EP2. PGE(2), however, can act on four receptors. This study was designed to determine if EP receptors, in addition to EP2, can modulate fibroblast chemotaxis. Using human fetal lung fibroblasts, the expression of all four EP receptors was demonstrated by Western blotting. EP2-selective and EP4-selective agonists inhibited both chemotaxis toward fibronectin in the blindwell assay and migration in a wound-closure assay. In contrast, EP1-selective and EP3-selective agonists stimulated cell migration in both assay systems. These results were confirmed using EP-selective antagonists. The role of both EP2 and EP4 receptors in mediating the PGE(2) inhibition of chemotaxis was also confirmed by small interfering RNA suppression. Furthermore, the role of EP receptors was confirmed by blocking the expected signaling pathways. Taken together, these results demonstrate that PGE(2) can act on multiple EP receptors in human lung fibroblasts, to exert disparate effects. Alterations in EP receptor expression may have the potential to alter PGE(2) action. Targeting specific EP receptors may offer therapeutic opportunities in conditions characterized by abnormal tissue repair and remodeling.

  1. Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation.

    PubMed

    Agley, Chibeza C; Rowlerson, Anthea M; Velloso, Cristiana P; Lazarus, Norman R; Harridge, Stephen D R

    2013-12-15

    We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells. The CD56(-) fraction obtained from the first sort was highly enriched in TE-7(+) fibroblasts. Using quantitative analysis of immunofluorescent staining for lipid content, lineage markers and transcription factors, we tested if the purified cell populations could differentiate into adipocytes in response to treatment with either fatty acids or adipocyte-inducing medium. Both treatments caused the fibroblasts to differentiate into adipocytes, as shown by loss of intracellular TE-7, upregulation of the adipogenic transcription factors PPARγ and C/EBPα, and adoption of a lipid-laden adipocyte morphology. By contrast, myogenic cells did not undergo adipogenesis and showed differential regulation of PPARγ and C/EBPα in response to these adipogenic treatments. Our results show that human skeletal muscle fibroblasts are at least bipotent progenitors that can remain as extracellular-matrix-producing cells or differentiate into adipocytes.

  2. Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through αVβ3 integrin in human skin fibroblasts.

    PubMed

    Qin, Zhaoping; Fisher, Gary J; Quan, Taihao

    2013-04-26

    Human skin largely comprises collagenous extracellular matrix. The hallmark of skin aging is fragmentation of collagen fibrils. Matrix metalloproteinases (MMPs) are largely responsible for collagen degradation. MMP-1, principally derived from dermal fibroblasts, is the major protease capable of initiating degradation of native fibrillar collagens. Presently, we report that CCN1, a secreted and extracellular matrix-associated protein, is elevated in aged human skin dermal fibroblasts in vivo and stimulates MMP-1 expression through functional interaction with αVβ3 integrin in human dermal fibroblasts. CCN1 contains four conserved structural domains. Our results indicate that the three N-terminal domains (IGFBP, VWC, and TSP1), but not the C-terminal CT domain, are required for CCN1 to stimulate MMP-1 expression. This stimulation is dependent on interaction between the active structural domains and αVβ3 integrin. The interaction of VWC domain with integrin αVβ3 is necessary and requires functional cooperation with adjacent IGFBP and TSP1 domains to stimulate MMP-1 expression. Finally, induction of MMP-1 expression in dermal fibroblasts by CCN1 N-terminal domains resulted in fragmentation of type I collagen fibrils in a three-dimensional collagen lattice model. These data suggest that domain-specific interactions of CCN1 with αVβ3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

  3. Malignant transformation of diploid human fibroblasts by transfection of oncogenes

    SciTech Connect

    McCormick, J.J.

    1992-01-01

    This document consist of brief reports prepared by postdoctoral students supported by the project, each describing his accomplishments under the grant. Topics include (1) Malignant Transformation of MSU-1. 1 Cells by Gamma Radiation, (2) Correlation between Levels of ras Expression and Presence of Transformed Phenotypes Including Tumorigenicity, Using a Modulatable Promoter, (3) Relation between Specific rad Oncogene Expression, (4) Correlation of Genetic Changes in Fibroblastic Tumors with Malignancies, (5)Transformation of MSU-1.1 Cells by sis Oncogene, (6) Malignant Transformation of MSU-1.0 Cells, (7) Correlation of Urokinase Plasminogen Activation (mu-PA) with Malignant Phenotype, (8)Two Dimensional Gel Electrophoresis Studies of the Proteins of the Major Cell Strains of the MSU-1 Family of Cells, and (9) Correlation between Proteinase Activity Levels and Malignancy.

  4. Pathomechanisms in Coenzyme Q10-Deficient Human Fibroblasts

    PubMed Central

    López, Luis C.; Luna-Sánchez, Marta; García-Corzo, Laura; Quinzii, Catarina M.; Hirano, Michio

    2014-01-01

    Primary coenzyme Q10 (CoQ10) deficiency is a rare mitochondrial disorder associated with 5 major clinical phenotypes: (1) encephalomyopathy, (2) severe infantile multisystemic disease, (3) cerebellar ataxia, (4) isolated myopathy, and (5) steroid-resistant nephrotic syndrome. Growth retardation, deafness and hearing loss have also been described in CoQ10-deficient patients. This heterogeneity in the clinical presentations suggests that multiple pathomechanisms may exist. To investigate the biochemical and molecular consequences of CoQ10 deficiency, different laboratories have studied cultures of skin fibroblasts from patients with CoQ10 deficiency. In this review, we summarize the results obtained in these studies over the last decade. PMID:25126049

  5. Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

    PubMed Central

    Ballou, L R; Barker, S C; Postlethwaite, A E; Kang, A H

    1991-01-01

    IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol. PMID:1845871

  6. Concentration- and time-dependent response of human gingival fibroblasts to fibroblast growth factor 2 immobilized on titanium dental implants

    PubMed Central

    Ma, Qianli; Wang, Wei; Chu, Paul K; Mei, Shenglin; Ji, Kun; Jin, Lei; Zhang, Yumei

    2012-01-01

    Background Titanium (Ti) implants are widely used clinically, but peri-implantitis remains one of the most common and serious complications. Healthy integration between gingival tissue and the implant surface is critical to long-term success in dental implant therapy. The objective of this study was to investigate how different concentrations of immobilized fibroblast growth factor 2 (FGF2) on the titania nanotubular surface influence the response of human gingival fibroblasts (HGFs). Methods Pure Ti metal was anodized at 20 V to form a vertically organized titanium dioxide nanotube array on which three concentrations of FGF2 (250 ng/mL, 500 ng/mL, or 1000 ng/mL) were immobilized by repeated lyophilization. Surface topography was observed and FGF2 elution was detected using enzyme-linked immunosorbent assay. The bioactivity changes of dissolvable immobilized FGF2 were measured by methyl-thiazolyl-tetrazolium assay. Behavior of HGFs was evaluated using adhesion and methyl-thiazolyl-tetrazolium bromide assays. Results The FGF2 remained for several days on the modified surface on which HGFs were cultured. Over 90% of the dissolvable immobilized FGF2 had been eluted by Day 9, whereas the FGF2 activity was found to diminish gradually from Day 1 to Day 9. The titania nanotubular surface with an optimal preparing concentration (500 ng/mL) of FGF2 immobilization exhibited improved HGF functions such as cellular attachment, proliferation, and extracellular matrix-related gene expression. Moreover, significant bidirectional as well as concentration- and time-dependent bioactivity was observed. Conclusion Synergism of the FGF2-impregnated titanium dioxide nanotubular surface revealed good gingival-implant integration, indicating that these materials might have promising applications in dentistry and other biomedical devices. PMID:22619534

  7. Melatonin and human skin aging

    PubMed Central

    Kleszczynski, Konrad; Fischer, Tobias W.

    2012-01-01

    Like the whole organism, skin follows the process of aging during life-time. Additional to internal factors, several environmental factors, such as solar radiation, considerably contribute to this process. While fundamental mechanisms regarding skin aging are known, new aspects of anti-aging agents such as melatonin are introduced. Melatonin is a hormone produced in the glandula pinealis that follows a circadian light-dependent rhythm of secretion. It has been experimentally implicated in skin functions such as hair cycling and fur pigmentation, and melatonin receptors are expressed in many skin cell types including normal and malignant keratinocytes, melanocytes and fibroblasts. It possesses a wide range of endocrine properties as well as strong antioxidative activity. Regarding UV-induced solar damage, melatonin distinctly counteracts massive generation of reactive oxygen species, mitochondrial and DNA damage. Thus, there is considerable evidence for melatonin to be an effective anti-skin aging compound, and its various properties in this context are described in this review. PMID:23467217

  8. Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

    SciTech Connect

    Clingen, Peter H. . E-mail: p.clingen@ucl.ac.uk; Arlett, Colin F.; Hartley, John A.; Parris, Christopher N.

    2007-02-15

    Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.

  9. Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization.

    PubMed

    Gutiérrez-Venegas, Gloria; Kawasaki-Cárdenas, Perla; Garcés, Carla Portillo; Román-Alvárez, Patricia; Barajas-Torres, Carolina; Contreras-Marmolejo, Luis Arturo

    2007-09-01

    Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.

  10. Monoclonal antibody specific for human colon fibroblast-derived T-PA

    SciTech Connect

    Schaumann, J.P.; Olander, J.V.; Harakas, N.K.; Feder, J

    1989-05-23

    This patent describes a murine-derived hybridoma cell line capable of producing monoclonal antibody against human colon fibroblast-derived tissue plasminogen activator and the cell line selected from the group consisting of cell lines 63-4 (ATCC HB 9155), 54-2 (ATCC HB 9157) or 79-7 (ATCC HB 9156).

  11. In Vitro Comparison of Cytotoxicity of Four Root Canal Sealers on Human Gingival Fibroblasts

    PubMed Central

    Konjhodzic-Prcic, Alma; Gorduysus, Omer; Kucukkaya, Selen; Atila, Burcu; Muftuoglu, Sevda; Zeybek, Dilara

    2015-01-01

    The goal of this in vitro study was to evaluate the relative biocompatibility of four endodontic sealers on the cell culture of the human fibroblast through cytotoxicity. Materials and Methods: In this study four endodontics sealers was used GuttaFlow (Roeko)silicone based sealer, AH plus (De Tray-DENTSPLY) epoxy resin based, Apexit (Vivadent) calcium hydroxide based and Endorez (Ultradent) methacrylate based sealer. Sealers were tested on primary cell lines of human gingival fibroblasts. Experiments were preformed in laboratories of Hacettepe University of Ankara, Turkey and Faculty of Dentistry, University of Sarajevo, Bosnia and Herzegovina Cytotoxicity was determinate using WST-1 assay. Results: Results were analyzed by SPSS 19 program. Kolgomorov-Smirnov test, Shapiro-Wilk and descriptive statistics also were used, as well as Kriskall-Wallis, ANOVA test and T- test. According to our results all four sealers showed different cytotoxicity effects on human gingival fibroblast cell culture, but all of them are slightly cytotoxic. Conclusions: According to results of this study it can be concluded: all four sealers showed different cytotoxicity effects on primary cell lines of human gingival fibroblasts, but all of them are slightly cytotoxicity. PMID:25870472

  12. Analysis of Gene Expression in Human Dermal Fibroblasts Treated with Senescence-Modulating COX Inhibitors

    PubMed Central

    Han, Jeong A.; Kim, Jong-Il

    2017-01-01

    We have previously reported that NS-398, a cyclooxygenase-2 (COX-2)–selective inhibitor, inhibited replicative cellular senescence in human dermal fibroblasts and skin aging in hairless mice. In contrast, celecoxib, another COX-2–selective inhibitor, and aspirin, a non-selective COX inhibitor, accelerated the senescence and aging. To figure out causal factors for the senescence-modulating effect of the inhibitors, we here performed cDNA microarray experiment and subsequent Gene Set Enrichment Analysis. The data showed that several senescence-related gene sets were regulated by the inhibitor treatment. NS-398 up-regulated gene sets involved in the tumor necrosis factor β receptor pathway and the fructose and mannose metabolism, whereas it down-regulated a gene set involved in protein secretion. Celecoxib up-regulated gene sets involved in G2M checkpoint and E2F targets. Aspirin up-regulated the gene set involved in protein secretion, and down-regulated gene sets involved in RNA transcription. These results suggest that COX inhibitors modulate cellular senescence by different mechanisms and will provide useful information to understand senescence-modulating mechanisms of COX inhibitors. PMID:28638310

  13. Interplay between Selenium Levels, Selenoprotein Expression, and Replicative Senescence in WI-38 Human Fibroblasts*

    PubMed Central

    Legrain, Yona; Touat-Hamici, Zahia; Chavatte, Laurent

    2014-01-01

    Selenium is an essential trace element, which is incorporated as selenocysteine into at least 25 selenoproteins using a unique translational UGA-recoding mechanism. Selenoproteins are important enzymes involved in antioxidant defense, redox homeostasis, and redox signaling pathways. Selenium levels decline during aging, and its deficiency is associated with a marked increase in mortality for people over 60 years of age. Here, we investigate the relationship between selenium levels in the culture medium, selenoprotein expression, and replicative life span of human embryonic lung fibroblast WI-38 cells. Selenium levels regulate the entry into replicative senescence and modify the cellular markers characteristic for senescent cells. Whereas selenium supplementation extends the number of population doublings, its deficiency impairs the proliferative capacity of WI-38 cells. We observe that the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence. Their expression is selectively controlled by the modulation of mRNA levels and translational recoding efficiencies. Our data provide novel mechanistic insights into how selenium impacts the replicative life span of mammalian cells by identifying several selenoproteins as new targets of senescence. PMID:24425862

  14. Analysis of Gene Expression in Human Dermal Fibroblasts Treated with Senescence-Modulating COX Inhibitors.

    PubMed

    Han, Jeong A; Kim, Jong-Il

    2017-06-01

    We have previously reported that NS-398, a cyclooxygenase-2 (COX-2)-selective inhibitor, inhibited replicative cellular senescence in human dermal fibroblasts and skin aging in hairless mice. In contrast, celecoxib, another COX-2-selective inhibitor, and aspirin, a non-selective COX inhibitor, accelerated the senescence and aging. To figure out causal factors for the senescence-modulating effect of the inhibitors, we here performed cDNA microarray experiment and subsequent Gene Set Enrichment Analysis. The data showed that several senescence-related gene sets were regulated by the inhibitor treatment. NS-398 up-regulated gene sets involved in the tumor necrosis factor β receptor pathway and the fructose and mannose metabolism, whereas it down-regulated a gene set involved in protein secretion. Celecoxib up-regulated gene sets involved in G2M checkpoint and E2F targets. Aspirin up-regulated the gene set involved in protein secretion, and down-regulated gene sets involved in RNA transcription. These results suggest that COX inhibitors modulate cellular senescence by different mechanisms and will provide useful information to understand senescence-modulating mechanisms of COX inhibitors.

  15. Interplay between selenium levels, selenoprotein expression, and replicative senescence in WI-38 human fibroblasts.

    PubMed

    Legrain, Yona; Touat-Hamici, Zahia; Chavatte, Laurent

    2014-02-28

    Selenium is an essential trace element, which is incorporated as selenocysteine into at least 25 selenoproteins using a unique translational UGA-recoding mechanism. Selenoproteins are important enzymes involved in antioxidant defense, redox homeostasis, and redox signaling pathways. Selenium levels decline during aging, and its deficiency is associated with a marked increase in mortality for people over 60 years of age. Here, we investigate the relationship between selenium levels in the culture medium, selenoprotein expression, and replicative life span of human embryonic lung fibroblast WI-38 cells. Selenium levels regulate the entry into replicative senescence and modify the cellular markers characteristic for senescent cells. Whereas selenium supplementation extends the number of population doublings, its deficiency impairs the proliferative capacity of WI-38 cells. We observe that the expression of several selenoproteins involved in antioxidant defense is specifically affected in response to cellular senescence. Their expression is selectively controlled by the modulation of mRNA levels and translational recoding efficiencies. Our data provide novel mechanistic insights into how selenium impacts the replicative life span of mammalian cells by identifying several selenoproteins as new targets of senescence.

  16. Derivation and characterization of goat fetal fibroblast cells induced with human telomerase reverse transcriptase.

    PubMed

    Xie, Ying; Zhao, Xiaoe; Jia, Hongxiang; Ma, Baohua

    2013-01-01

    Fetal fibroblast cells (FFCs) are often used as donor cells for somatic cell nuclear transfer (SCNT) because they are easy to culture and suitable for genetic manipulation. However, through genetic modification process, which required FFCs to be cultured in vitro for several passages, cells tended to age very rapidly and became inappropriate for SCNT. Human telomerase reverse transcriptase (hTERT) possessed the activity of human telomerase and maintains telomere in dividing cells; therefore, hTERT can be transfected into somatic cells to extend their lifespan. In this study, we transfected a Xinong Saanen Dairy Goat FFC line with hTERT. Then, we tested several characteristics of transfected cells, including growth curve, expression and activity of hTERT, tumorigenicity, and expression of oct4 and nanog. The result showed that hTERT could significantly extend the lifespan of transfected cells in vitro. hTERT mRNA was expressed in hTERT-transfected cells. Moreover, hTERT-transfected cells presented enhanced telomerase activity and longer telomere than untransfected cells at the same passage. On the other hand, hTERT-transfected cells can maintain normal karyotype even after several times of subculture in vitro. After inoculation of hTERT-transfected cells in nude mouse, none of them developed tumors on the vaccination site. Interestingly, transfection of hTERT can improve expression of nanog and oct4 in Xinong Saanen Dairy Goat FFCs, especially in low generation after transfection, but with increasing subculture, this effect gradually weakened.

  17. Deceleration of senescence in normal human fibroblasts by withanone extracted from ashwagandha leaves.

    PubMed

    Widodo, Nashi; Shah, Navjot; Priyandoko, Didik; Ishii, Tetsuro; Kaul, Sunil C; Wadhwa, Renu

    2009-10-01

    Ashwagandha is an Ayurvedic shrub that forms a common ingredient of health supplements, tonics, and Indian home remedies designed to promote health and quality of life. Though sustained through experience and history, there are only a limited laboratory studies and experimental evidence to its effects. In our efforts to characterize Ashwagandha activities and their molecular mechanisms, we initially prepared leaf extract of Ashwagandha (i-Extract) that showed tumor-inhibitory activity. In the present study, we demonstrate that a major component of i-Extract and withanone (i-Factor) protected the normal human fibroblasts against the toxicity caused by withaferin A. It increased the in vitro division potential of normal human cells that appeared to be mediated by decreased accumulation of molecular damage, downregulation of the senescence-specific beta-galactosidase activity and the senescence marker protein, p21(WAF-1), protection against oxidative damage, and induction of proteasomal activity. To the best of our knowledge, we provide the first example of phytochemical(s) (i-Extract and withanone) that have both anticancer and antiaging activities and point to the molecular link between aging and cancer.

  18. Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium

    PubMed Central

    Ruvinov, Emil; Sharabani-Yosef, Orna; Nagler, Arnon; Einbinder, Tom; Feinberg, Micha S; Holbova, Radka; Douvdevani, Amos; Leor, Jonathan

    2008-01-01

    Background Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). Methods and results RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. Conclusion In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat. PMID:19014419

  19. Defect of hepatocyte growth factor production by fibroblasts in human pulmonary emphysema.

    PubMed

    Plantier, Laurent; Marchand-Adam, Sylvain; Marchal-Sommé, Joëlle; Lesèche, Guy; Fournier, Michel; Dehoux, Monique; Aubier, Michel; Crestani, Bruno

    2005-04-01

    Pulmonary emphysema results from an excessive degradation of lung parenchyma associated with a failure of alveolar repair. Secretion by pulmonary fibroblasts of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) is crucial to an effective epithelial repair after lung injury. We hypothesized that abnormal HGF or KGF secretion by pulmonary fibroblasts could play a role in the development of emphysema. We measured in vitro production of HGF and KGF by human fibroblasts cultured from emphysematous and normal lung samples. HGF and KGF production was quantified at basal state and after stimulation. Intracellular content of HGF was lower in emphysema (1.52 pg/mug, range of 0.15-7.40 pg/mug) than in control fibroblasts (14.16 pg/mug, range of 2.50-47.62 pg/mug; P = 0.047). HGF production by emphysema fibroblasts (19.3 pg/mug protein, range of 10.4-39.2 pg/mug) was lower than that of controls at baseline (57.5 pg/mug, range of 20.4-116 pg/mug; P = 0.019) and after stimulation with interleukin-1beta or prostaglandin E(2). Neither retinoic acids (all-trans and 9-cis) nor N-acetylcysteine could reverse this abnormality. KGF production by emphysema fibroblasts (5.3 pg/mug, range of 2.2-9.3 pg/mug) was similar to that of controls at baseline (2.6 pg/mug, range of 1-6.1 pg/mug; P = 0.14) but could not be stimulated with interleukin-1beta. A decreased secretion of HGF by pulmonary fibroblasts could contribute to the insufficient alveolar repair in pulmonary emphysema.

  20. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    SciTech Connect

    Tao, Jun; Xiang, Jun-Jian; Li, Dan; Deng, Ning; Wang, Hong; Gong, Yi-Ping

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  1. Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

    PubMed

    Nash, Claire; Boufaied, Nadia; Mills, Ian G; Franco, Omar E; Hayward, Simon W; Thomson, Axel A

    2017-05-05

    The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue

  2. Vitamin E inhibits proliferation of human Tenon's capsule fibroblasts in vitro.

    PubMed

    Haas, A L; Boscoboinik, D; Mojon, D S; Böhnke, M; Azzi, A

    1996-01-01

    Failure of glaucoma surgery is mostly due to fibrocellular scar formation, derived from Tenon's capsule fibroblasts. In high-risk cases, postoperative Tenon's capsule fibroblast proliferation is inhibited by mitomycin C or 5-fluorouracil. Toxicity to other ocular cell types and the risk of ocular hypotony limits the use of these agents. We have found that d-alpha-tocopherol (vitamin E) was able to inhibit proliferation of in vitro human Tenon's capsule fibroblasts obtained from seven different donors. At 48 h, inhibition of cell proliferation was 30-78% (mean 60%) for 50 microM d-alpha-tocopherol and 46-97% (mean 77%) for 100 microM d-alpha-tocopherol. This inhibition was statistically significant. No cytotoxic effects were observed.

  3. Proliferation of human fibroblasts in vitro after exposure to orbital implants.

    PubMed

    Mawn, L A; Jordan, D R; Gilberg, S

    2001-08-01

    Porous orbital implants allow fibrovascular ingrowth and integration with the extraocular muscles. The available implants have different structural characteristics, which may influence orbital response. We studied the proliferation of orbital fibroblasts in vitro after exposure to four different orbital implants. Four orbital implant biomaterials were studied: hydroxyapatite (Bio-Eye), synthetic hydroxyapatite, porous polyethylene (Medpor) (pore sizes 150 microm and 400 microm) and aluminium oxide (Bioceramic implant). Human fibroblasts obtained from orbital fat at the time of elective blepharoplasty were cultured and then exposed to the individual implants. Cell growth was assessed with immunocytochemical analysis using bromodeoxyuridine, a thymidine analogue. After DNA denaturation, the cells were washed, incubated with secondary antibody and visualized. The fibroblasts growing on the Bio-Eye, synthetic hydroxyapatite, and 150-microm and 400-microm Medpor implants all had debris associated with them. The Bioceramic implant was free of this debris. The Bioceramic implant and the 150-microm Medpor implant had the greatest number of fibroblasts on the coverslips. The proliferation of fibroblasts, as determined by visualization of actively dividing cells with bromodeoxyuridine, differed on the various implants studied. The lack of debris associated with the Bioceramic implant may be related to the crystalline structure of the implant.

  4. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis

    PubMed Central

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-01-01

    Phagocytosis of SiO2 into the lung causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Clinical evidence has indicated that the activation of alveolar macrophages by SiO2 produces rapid and sustained inflammation characterized by the generation of monocyte chemotactic protein 1, which, in turn, induces fibrosis. However, the details of events downstream of monocyte chemotactic protein 1 activity in pulmonary fibroblasts remain unclear. Here, to elucidate the role of p53 in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Experiments using primary cultured adult human pulmonary fibroblasts led to the following results: 1) SiO2 treatment resulted in a rapid and sustained increase in p53 and PUMA protein levels; 2) the MAPK and PI3K pathways were involved in the SiO2-induced alteration of p53 and PUMA expression; and 3) RNA interference targeting p53 and PUMA prevented the SiO2-induced increases in fibroblast activation and migration. Our study elucidated a link between SiO2-induced p53/PUMA expression in fibroblasts and cell migration, thereby providing novel insight into the potential use of p53/PUMA in the development of novel therapeutic strategies for silicosis treatment. PMID:26576741

  5. Effects of whole cigarette smoke on human gingival fibroblast adhesion, growth, and migration.

    PubMed

    Semlali, Abdelhabib; Chakir, Jamila; Rouabhia, Mahmoud

    2011-01-01

    The aim of this study was to investigate the effects of a single exposure to whole cigarette smoke on human gingival fibroblast behavior. Normal oral mucosa fibroblasts were exposed once to whole cigarette smoke for 5, 15, or 30 min, and then were used to analyze cell adhesion, β1-integrin expression, cell growth and viability, cell capacity to contract collagen gel, and cell migration following wound infliction. Our findings showed that when gingival fibroblasts were exposed once to whole cigarette smoke, this resulted in a significant inhibition of cell adhesion, a decrease in the number of β1-integrin-positive cells, increased LDH activity in the target cells, and reduced growth. The smoke-exposed fibroblasts were also not able to contract collagen gel matrix and migrate following insult. Overall results demonstrate that a single exposure to whole cigarette smoke produced significant morphological and functional deregulation in gingival fibroblasts. This may explain the higher predisposition of tobacco users to oral infections and diseases such as cancer.

  6. Cytotoxicity evaluation of root repair materials in human-cultured periodontal ligament fibroblasts

    PubMed Central

    Samyuktha, Voruganti; Ravikumar, Pabbati; Nagesh, Bolla; Ranganathan, K.; Jayaprakash, Thumu; Sayesh, Vemuri

    2014-01-01

    Aim: To evaluate the cytotoxicity of three root repair materials, mineral trioxide aggregate (MTA), Endosequence Root Repair Material and Biodentine in human periodontal ligament fibroblasts. Materials and Methods: Periodontal ligament fibroblasts were cultured from healthy premolar extracted for orthodontic purpose. Cells in the third passage were used in the study. The cultured fibroblast cells were placed in contact with root repair materials: (a) Biodentine, (b) MTA, (c) Endosequence, (d) control. The effects of these three materials on the viability of Periodontal ligament (PDL) fibroblasts were determined by trypan blue dye assay after 24 hours and 48-hour time period. Cell viability was determined using inverted phase contrast microscope. Statistical Analysis: Cell viability was compared for all the experimental groups with Wilcoxons matched pair test. Results: At the 24-hour examination period, all the materials showed increased cell viability. At 48-hour time period, there is slight decrease in cell viability. Mineral trioxide aggregate showed statistically significant increase in the cell viability when compared to other root repair materials. Conclusion: Mineral trioxide aggregate was shown to be less toxic to periodontal ligament fibroblasts than Endosequence Root Repair Material and Biodentine. PMID:25298650

  7. Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

    NASA Astrophysics Data System (ADS)

    Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

    1990-09-01

    The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

  8. Mitochondrial trifunctional protein deficiency in human cultured fibroblasts: effects of bezafibrate.

    PubMed

    Djouadi, Fatima; Habarou, Florence; Le Bachelier, Carole; Ferdinandusse, Sacha; Schlemmer, Dimitri; Benoist, Jean François; Boutron, Audrey; Andresen, Brage S; Visser, Gepke; de Lonlay, Pascale; Olpin, Simon; Fukao, Toshiyuki; Yamaguchi, Seiji; Strauss, Arnold W; Wanders, Ronald J A; Bastin, Jean

    2016-01-01

    Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 μM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes.

  9. Cytotoxic evaluation of biomechanically improved crosslinked ovine collagen on human dermal fibroblasts.

    PubMed

    Awang, M A; Firdaus, M A B; Busra, M B; Chowdhury, S R; Fadilah, N R; Wan Hamirul, W K; Reusmaazran, M Y; Aminuddin, M Y; Ruszymah, B H I

    2014-01-01

    Earlier studies in our laboratory demonstrated that collagen extracted from ovine tendon is biocompatible towards human dermal fibroblast. To be able to use this collagen as a scaffold in skin tissue engineering, a mechanically stronger scaffold is required that can withstand manipulation before transplantation. This study was conducted to improve the mechanical strength of this collagen sponge using chemical crosslinkers, and evaluate their effect on physical, chemical and biocompatible properties. Collagen sponge was crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and glutaraldehyde (GA). Tensile test, FTIR study and mercury porosimetry were used to evaluate mechanical properties, chemical property and porosity, respectively. MTT assay was performed to evaluate the cytotoxic effect of crosslinked collagen sponge on human dermal fibroblasts. The FTIR study confirmed the successful crosslinking of collagen sponge. Crosslinking with EDC and GA significantly increased the mechanical strength of collagen sponge, with GA being more superior. Crosslinking of collagen sponge significantly reduced the porosity and the effect was predominant in GA-crosslinked collagen sponge. The GA-crosslinked collagen showed significantly lower, 60% cell viability towards human dermal fibroblasts compared to that of EDC-crosslinked collagen, 80% and non-crosslinked collagen, 100%. Although the mechanical strength was better when using GA but the more toxic effect on dermal fibroblast makes EDC a more suitable crosslinker for future skin tissue engineering.

  10. Induction of interleukin-1 and -6 in human gingival fibroblast cultures stimulated with Bacteroides lipopolysaccharides.

    PubMed Central

    Takada, H; Mihara, J; Morisaki, I; Hamada, S

    1991-01-01

    Normal human gingival fibroblasts stimulated in vitro by lipopolysaccharides (LPS) from oral Bacteroides species produced cell-free and cell-associated thymocyte-activating factors (TAF). Neutralization assays using antisera to human interleukin-1 alpha (HuIL-1 alpha), HuIL-1 beta, and HuIL-6 revealed that cell-free TAF was attributable mainly to IL-1 beta and that IL-6 augmented the TAF activity of IL-1 beta in the culture supernatant. Another factor(s), however, may also be involved in cell-free TAF. By contrast, the active entity of cell-associated TAF was ascribed to IL-1 alpha alone. Furthermore, IL-6 was detected mainly in the supernatant of fibroblast cultures stimulated with Bacteroides LPS. Fibroblasts pretreated with natural human beta or gamma interferon, but not those pretreated with alpha interferon, synthesized higher levels of cell-associated IL-1 alpha in response to stimulation by Bacteroides LPS; however, no interferons exhibited direct IL-1-inducing activity or synergistic IL-1-inducing activity with LPS. Endogenously induced beta interferon was suggested to be necessary for fibroblasts to produce cell-associated IL-1 alpha in response to Bacteroides LPS. PMID:1702762

  11. Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts.

    PubMed

    Greussing, Ruth; Hackl, Matthias; Charoentong, Pornpimol; Pauck, Alexander; Monteforte, Rossella; Cavinato, Maria; Hofer, Edith; Scheideler, Marcel; Neuhaus, Michael; Micutkova, Lucia; Mueck, Christoph; Trajanoski, Zlatko; Grillari, Johannes; Jansen-Dürr, Pidder

    2013-04-04

    Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts. We observed a parallel activation of the p53/p21(WAF1) and p16(INK4a)/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101. We performed a comprehensive screen for UVB-regulated microRNAs in human diploid fibroblasts, and identified a

  12. Identification of microRNA-mRNA functional interactions in UVB-induced senescence of human diploid fibroblasts

    PubMed Central

    2013-01-01

    Background Cellular senescence can be induced by a variety of extrinsic stimuli, and sustained exposure to sunlight is a key factor in photoaging of the skin. Accordingly, irradiation of skin fibroblasts by UVB light triggers cellular senescence, which is thought to contribute to extrinsic skin aging, although molecular mechanisms are incompletely understood. Here, we addressed molecular mechanisms underlying UVB induced senescence of human diploid fibroblasts. Results We observed a parallel activation of the p53/p21WAF1 and p16INK4a/pRb pathways. Using genome-wide transcriptome analysis, we identified a transcriptional signature of UVB-induced senescence that was conserved in three independent strains of human diploid fibroblasts (HDF) from skin. In parallel, a comprehensive screen for microRNAs regulated during UVB-induced senescence was performed which identified five microRNAs that are significantly regulated during the process. Bioinformatic analysis of miRNA-mRNA networks was performed to identify new functional mRNA targets with high confidence for miR-15a, miR-20a, miR-20b, miR-93, and miR-101. Already known targets of these miRNAs were identified in each case, validating the approach. Several new targets were identified for all of these miRNAs, with the potential to provide new insight in the process of UVB-induced senescence at a genome-wide level. Subsequent analysis was focused on miR-101 and its putative target gene Ezh2. We confirmed that Ezh2 is regulated by miR-101 in human fibroblasts, and found that both overexpression of miR-101 and downregulation of Ezh2 independently induce senescence in the absence of UVB irradiation. However, the downregulation of miR-101 was not sufficient to block the phenotype of UVB-induced senescence, suggesting that other UVB-induced processes induce the senescence response in a pathway redundant with upregulation of miR-101. Conclusion We performed a comprehensive screen for UVB-regulated microRNAs in human diploid

  13. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    SciTech Connect

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  14. Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

    PubMed

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be considered a

  15. Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

    PubMed

    Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

    2016-01-01

    This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

  16. Helium Generated Cold Plasma Finely Regulates Activation of Human Fibroblast-Like Primary Cells

    PubMed Central

    Brun, Paola; Pathak, Surajit; Castagliuolo, Ignazio; Palù, Giorgio; Brun, Paola; Zuin, Matteo; Cavazzana, Roberto; Martines, Emilio

    2014-01-01

    Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS) in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2′,7′-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR)-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine) cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and can be

  17. Donor age and long-term culture do not negatively influence the stem potential of limbal fibroblast-like stem cells.

    PubMed

    Tomasello, Laura; Musso, Rosa; Cillino, Giovanni; Pitrone, Maria; Pizzolanti, Giuseppe; Coppola, Antonina; Arancio, Walter; Di Cara, Gianluca; Pucci-Minafra, Ida; Cillino, Salvatore; Giordano, Carla

    2016-06-13

    In regenerative medicine the maintenance of stem cell properties is of crucial importance. Ageing is considered a cause of reduced stemness capability. The limbus is a stem niche of easy access and harbors two stem cell populations: epithelial stem cells and fibroblast-like stem cells. Our aim was to investigate whether donor age and/or long-term culture have any influence on stem cell marker expression and the profiles in the fibroblast-like stem cell population. Fibroblast-like stem cells were isolated and digested from 25 limbus samples of normal human corneo-scleral rings and long-term cultures were obtained. SSEA4 expression and sphere-forming capability were evaluated; cytofluorimetric assay was performed to detect the immunophenotypes HLA-DR, CD45, and CD34 and the principle stem cell markers ABCG2, OCT3/4, and NANOG. Molecular expression of the principal mesenchymal stem cell genes was investigated by real-time PCR. Two-dimensional gel electrophoresis and mass spectrometric sequencing were performed and a stable proteomic profile was identified. The proteins detected were explored by gene ontology and STRING analysis. The data were reported as means ± SD, compared by Student's unpaired t test and considering p < 0.05 as statistically significant. The isolated cells did not display any hematopoietic surface marker (CD34 and CD45) and HLA-DR and they maintained these features in long-term culture. The expression of the stemness genes and the multilineage differentiation under in-vitro culture conditions proved to be well maintained. Proteomic analysis revealed a fibroblast-like stem cell profile of 164 proteins with higher expression levels. Eighty of these showed stable expression levels and were involved in maintenance of "the stem gene profile"; 84 were differentially expressed and were involved in structural activity. The fibroblast-like limbal stem cells confirmed that they are a robust source of adult stem cells and that they have good plasticity, good

  18. Development of a high-throughput screening system for identification of novel reagents regulating DNA damage in human dermal fibroblasts.

    PubMed

    Bae, Seunghee; An, In-Sook; An, Sungkwan

    2015-09-01

    Ultraviolet (UV) radiation is a major inducer of skin aging and accumulated exposure to UV radiation increases DNA damage in skin cells, including dermal fibroblasts. In the present study, we developed a novel DNA repair regulating material discovery (DREAM) system for the high-throughput screening and identification of putative materials regulating DNA repair in skin cells. First, we established a modified lentivirus expressing the luciferase and hypoxanthine phosphoribosyl transferase (HPRT) genes. Then, human dermal fibroblast WS-1 cells were infected with the modified lentivirus and selected with puromycin to establish cells that stably expressed luciferase and HPRT (DREAM-F cells). The first step in the DREAM protocol was a 96-well-based screening procedure, involving the analysis of cell viability and luciferase activity after pretreatment of DREAM-F cells with reagents of interest and post-treatment with UVB radiation, and vice versa. In the second step, we validated certain effective reagents identified in the first step by analyzing the cell cycle, evaluating cell death, and performing HPRT-DNA sequencing in DREAM-F cells treated with these reagents and UVB. This DREAM system is scalable and forms a time-saving high-throughput screening system for identifying novel anti-photoaging reagents regulating DNA damage in dermal fibroblasts.

  19. Plasma Rich in Growth Factors Inhibits Ultraviolet B Induced Photoageing of the Skin in Human Dermal Fibroblast Culture.

    PubMed

    Anitua, Eduardo; Pino, Ander; Orive, Gorka

    Ultraviolet irradiation is able to deeply penetrate into the dermis and alter fibroblast structure and function, leading to a degradation of the dermal extracellular matrix. The regenerative effect of plasma rich in growth factors (PRGF) on skin ageing was investigated using UVB photo-stressed human dermal fibroblasts as an in vitro culture model. PRGF was assessed over the main indicative features of ultraviolet B irradiation, including ROS formation, cell viability and death detection, apoptosis/ necrosis analysis and biosynthetic activity measurement. Four different UV irradiation protocols were tested in order to analyze the beneficial effects of PRGF. Ultraviolet irradiation exhibited a dose dependent cytotoxicity and dose of 400mJ/cm2 was selected for subsequent experiments. PRGF increased the cell viability and decreased the cell death comparing to the non-treated group. The apoptosis and necrosis were significantly lower in PRGF treated fibroblasts. ROS production after UV irradiation was significantly reduced in the presence of PRGF. Procollagen type I, hyaluronic acid and TIMP-1 levels were higher in the when treated with PRGF. This preliminary in vitro study suggests that PRGF is able to prevent UVB derived photooxidative stress and to diminish the cell damage caused by ultraviolet irradiation.

  20. Photoprotective Potential of Anthocyanins Isolated from Acanthopanax divaricatus Var. albeofructus Fruits against UV Irradiation in Human Dermal Fibroblast Cells.

    PubMed

    Lyu, Su-Yun; Park, Won-Bong

    2012-03-01

    Ultraviolet (UV) A penetrates deeply into the skin and induces the generation of reactive oxygen species (ROS) causing damage to fibroblasts, which leads to aging of the skin. However, the body has developed an antioxidant defence system against the harmful effects of ROS. Enzymes such as superoxide dismutase (SOD) and catalase (CAT) play critical roles on the removal of excess ROS in living organisms. In this study, the antioxidant activities of anthocyanins (cyanidin 3-galactoside and cyanidin 3-lathyroside) from Acanthopanax divaricatus var. albeofructus (ADA) fruits were investigated by xylenol orange, thiobarbituric acid reactive substances (TBARS), and antioxidant enzyme assay. As a result, generation of H2O2 and lipid peroxide induced by UVA-irradiation in human dermal fibroblast (HDF-N) cells was reduced by treatment of anthocyanins. Also, augmented enzyme (SOD and CAT) activities were observed in UVA-irradiated cells when treated with anthocyanin. In conclusion, the results obtained show that anthocyanins from ADA fruits are potential candidates for the protection of fibroblast against the damaging effects of UVA irradiation. Furthermore, anthocyanin may be a good candidate for antioxidant agent development.

  1. Hydrogen sulfide suppresses transforming growth factor-β1-induced differentiation of human cardiac fibroblasts into myofibroblasts.

    PubMed

    Zhang, YouEn; Wang, JiaNing; Li, Hua; Yuan, LiangJun; Wang, Lei; Wu, Bing; Ge, JunBo

    2015-11-01

    In heart disease, transforming growth factor-β1 (TGF-β1) converts fibroblasts into myofibroblasts, which synthesize and secrete fibrillar type I and III collagens. The purpose of the present study was to investigate how hydrogen sulfide (H2S) suppresses TGF-β1-induced differentiation of human cardiac fibroblasts to myofibroblasts. Human cardiac fibroblasts were serum-starved in fibroblast medium for 16 h before exposure to TGF-β1 (10 ng mL(-1)) for 24 h with or without sodium hydrosulfide (NaHS, 100 µmol L(-1), 30 min pretreatment) treatment. NaHS, an exogenous H2S donor, potently inhibited the proliferation and migration of TGF-β1-induced human cardiac fibroblasts and regulated their cell cycle progression. Furthermore, NaHS treatment led to suppression of fibroblast differentiation into myofibroblasts, and reduced the levels of collagen, TGF-β1, and activated Smad3 in TGF-β1-induced human cardiac fibroblasts in vitro. We therefore conclude that H2S suppresses TGF-β1-stimulated conversion of fibroblasts to myofibroblasts by inhibiting the TGF-β1/Smad3 signaling pathway, as well as by inhibiting the proliferation, migration, and cell cycle progression of human cardiac myofibroblasts. These effects of H2S may play significant roles in cardiac remodeling associated with heart failure.

  2. Episomal-based generation of an iPS cell line from human fetal foreskin fibroblasts.

    PubMed

    Matz, Peggy; Adjaye, James

    2016-01-01

    Human fetal foreskin fibroblasts (HFF1) were used to generate the iPSC line epiHFF1-B1 employing a combination of three episomal-based plasmids expressing OCT4, SOX2, NANOG, LIN28, c-MYC, and KLF4. Pluripotency was confirmed both in vivo and in vitro. The transcriptome profile of epiHFF1-B1 and the human embryonic stem cell line-H1 have a pearson correlation of 0.936.

  3. Expression of integrins by human periodontal ligament and gingival fibroblasts and their involvement in fibroblast adhesion to enamel matrix-derived proteins.

    PubMed

    van der Pauw, M T M; Everts, V; Beertsen, W

    2002-10-01

    We showed recently that human periodontal ligament (PDL) and gingival fibroblasts adhere and spread on enamel matrix protein (EMP) coatings. In the present study, we investigated whether this interaction can be attributed to integrin expression. Human PDL and gingival fibroblasts were cultured for periods up to 24 h on EMP coatings, in the presence of synthetic RGD-containing peptide or an antibody against the beta1 integrin subunit. The cells were first cultured for 24 h under serum-free conditions and then cultured on EMP coatings for 48 h. Integrin expression levels were assessed by flow cytometry analysis. It was found that attachment and spreading on EMP was inhibited by the synthetic RGD-containing peptide, but not by a synthetic RGE-peptide. Both PDL and gingival fibroblasts showed expression of the integrin subunits, alpha2, alpha5, beta1, and the integrin, alphavbeta3. Incubation with an antibody against the beta1 subunit significantly inhibited the attachment and spreading of PDL and gingival fibroblasts on EMP coatings. We conclude that integrins are involved in the interaction of PDL and gingival fibroblasts with EMP.

  4. Activated Human T Lymphocytes Express Cyclooxygenase-2 and Produce Proadipogenic Prostaglandins that Drive Human Orbital Fibroblast Differentiation to Adipocytes

    PubMed Central

    Feldon, Steven E.; O’Loughlin, Charles W.; Ray, Denise M.; Landskroner-Eiger, Shira; Seweryniak, Kathryn E.; Phipps, Richard P.

    2006-01-01

    The differentiation of preadipocyte fibroblasts to adipocytes is a crucial process to many disease states including obesity, cardiovascular, and autoimmune diseases. In Graves’ disease, the orbit of the eye can become severely inflamed and infiltrated with T lymphocytes as part of the autoimmune process. The orbital fibroblasts convert to fat-like cells causing the eye to protrude, which is disfiguring and can lead to blindness. Recently, the transcription factor peroxisome proliferator activated receptor (PPAR)-γ and its natural (15d-PGJ2) and synthetic (thiazolidinedione-type) PPAR-γ agonists have been shown to be crucial to the in vitro differentiation of preadipocyte fibroblasts to adipocytes. We show herein several novel findings. First, that activated T lymphocytes from Graves’ patients drive the differentiation of PPAR-γ-expressing orbital fibroblasts to adipocytes. Second, this adipogenic differentiation is blocked by nonselective small molecule cyclooxygenase (Cox)-1/Cox-2 inhibitors and by Cox-2 selective inhibitors. Third, activated, but not naïve, human T cells highly express Cox-2 and synthesize prostaglandin D2 and related prostaglandins that are PPAR-γ ligands. These provocative new findings provide evidence for how activated T lymphocytes, through production of PPAR-γ ligands, profoundly influence human fibroblast differentiation to adipocytes. They also suggest the possibility that, in addition to the orbit, T lymphocytes influence the deposition of fat in other tissues. PMID:17003477

  5. The effectiveness of potent dental adhesives on the viability of LPS challenged human gingival fibroblasts.

    PubMed

    Garner, Angelia D; Tucci, Michelle A; Benghuzzi, Hamed A

    2014-01-01

    Dental adhesives are necessary for the retention of specific dental restorations utilized to repair the anatomy of the tooth after dental decay is removed. Adhesives come into contact with healthy and diseased periodontal tissues. Porphyromonas gingivalis is a gram negative bacterial pathogen, and lipopolysaccharide (LPS-PG) is an endotoxin found in gingival connective tissues of patients who suffer from periodontal disease. The presence of the endotoxin causes inflammation. This study aims to evaluate the effectiveness of potent dental adhesives when human gingival fibroblasts are challenged with LPS-PG. The fibroblasts were exposed to the dental adhesives polymethly methacrylate (PMMA), OptiBond®, and Prime & Bond® which were purchased from Patterson Dental, a national dental materials supplier. The human gingival fibroblasts (HGF-1, ATCC® CRL-2014™) were purchased from American Type Culture Collection (ATCC). The porphyromonas gingival lipopolysaccharide (LPS-PG) was purchased from Fisher Scientific (Pittsburg, PA). No significant differences in metabolic behavior was detected among the groups (p<0.132). While the glutathione assay determined that there was not any significant increase in oxidative stress levels; the lactate dehydrogenase assay identified significant cellular damage in the group exposed to combinations of the Prime & Bond® adhesives and LPS-PG at 48 hour intervals (p<0.003). No significant changes were noted in cellular morphology at any phases, and all cells demonstrated typical fibroblast spindle shape.

  6. A large-conductance calcium-regulated K+ channel in human dermal fibroblast mitochondria.

    PubMed

    Kicinska, Anna; Augustynek, Bartlomiej; Kulawiak, Bogusz; Jarmuszkiewicz, Wieslawa; Szewczyk, Adam; Bednarczyk, Piotr

    2016-12-01

    Potassium channels have been found in the inner mitochondrial membrane of various cells. These channels regulate the mitochondrial membrane potential, respiration and production of reactive oxygen species. In the present study, we identified the activity of a mitochondrial large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) in mitoplasts isolated from a primary human dermal fibroblast cell line. A potassium selective current was recorded with a mean conductance of 280 ± 2 pS in a symmetrical 150 mM KCl solution. The mitoBKCa channel was activated by the Ca(2+) and by potassium channel opener NS1619. The channel activity was irreversibly inhibited by paxilline, a selective inhibitor of the BKCa channels. In isolated fibroblast mitochondria NS1619 depolarized the mitochondrial membrane potential, stimulated nonphosphorylating respiration and decreased superoxide formation. Additionally, the α- and β-subunits (predominantly the β3-form) of the BKCa channels were identified in fibroblast mitochondria. Our findings indicate, for the first time, the presence of a large-conductance Ca(2+)-regulated potassium channel in the inner mitochondrial membrane of human dermal fibroblasts. © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  7. Direct conversion of human fibroblasts into functional osteoblasts by defined factors.

    PubMed

    Yamamoto, Kenta; Kishida, Tsunao; Sato, Yoshiki; Nishioka, Keisuke; Ejima, Akika; Fujiwara, Hiroyoshi; Kubo, Toshikazu; Yamamoto, Toshiro; Kanamura, Narisato; Mazda, Osam

    2015-05-12

    Osteoblasts produce calcified bone matrix and contribute to bone formation and remodeling. In this study, we established a procedure to directly convert human fibroblasts into osteoblasts by transducing some defined factors and culturing in osteogenic medium. Osteoblast-specific transcription factors, Runt-related transcription factor 2 (Runx2), and Osterix, in combination with Octamer-binding transcription factor 3/4 (Oct4) and L-Myc (RXOL) transduction, converted ∼ 80% of the fibroblasts into osteocalcin-producing cells. The directly converted osteoblasts (dOBs) induced by RXOL displayed a similar gene expression profile as normal human osteoblasts and contributed to bone repair after transplantation into immunodeficient mice at artificial bone defect lesions. The dOBs expressed endogenous Runx2 and Osterix, and did not require continuous expression of the exogenous genes to maintain their phenotype. Another combination, Oct4 plus L-Myc (OL), also induced fibroblasts to produce bone matrix, but the OL-transduced cells did not express Osterix and exhibited a more distant gene expression profile to osteoblasts compared with RXOL-transduced cells. These findings strongly suggest successful direct reprogramming of fibroblasts into functional osteoblasts by RXOL, a technology that may provide bone regeneration therapy against bone disorders.

  8. Colonization by human fibroblasts of polypropylene prosthesis in a composite form for hernia repair.

    PubMed

    Canuto, R A; Saracino, S; Oraldi, M; Festa, V; Festa, F; Muzio, G; Chiaravalloti, A

    2013-04-01

    Abdominal wall hernia is one of the commonest surgical disorders worldwide, and there is no single gold-standard operative technique to repair it. In an effort to improve techniques and technologies to reinforce hernia repair, synthetic meshes are employed. In this study, a new prosthesis (named composite) formed of two polypropylene layers, one macroporous (named mesh) and one transparent (named film), was examined to evaluate its capability to enable cell proliferation without inducing cell death. Inflammatory processes were also examined. Human fibroblasts BJ were seeded on multiwells, on which composite or film had been placed. After 7, 14, and 21 days, cell growth and viability, deposition of collagen, and release of IL-6, IL-1β, and TNF-α were evaluated. The "in vitro" protocol showed the composite to be colonized by human fibroblasts on the polypropylene macroporous mesh side; no cell growth occurred on the film. The slowdown of cell growth observed between 14 and 21 days was accompanied by an increase in type I collagen deposition and marked fibroblast activity. Inflammatory cytokines initially increased, followed by their reduction beginning at 14 days. The new prosthesis comprising two polypropylene layers of differing morphologies can be colonized by fibroblasts on the side facing the abdominal wall, whereas no cell growth occurs on the side facing the viscera. The transient inflammation, observed at early experimental times, is probably important for the healing process.

  9. Biological effects of helium-neon laser irradiation on normal and wounded human skin fibroblasts.

    PubMed

    Hawkins, D; Abrahamse, H

    2005-06-01

    This study aimed to investigate a number of structural, cellular, and molecular responses to heliumneon (632.8 nm) laser irradiation following a single dose of 0.5, 2.5, 5, or 10 J/cm2 on normal and wounded human skin fibroblasts. Low-level laser therapy (LLLT) is a form of phototherapy, involving the application of low-power monochromatic and coherent light to injuries and lesions to stimulate healing. 1 This therapy has been successfully used for pain attenuation and to induce wound healing in nonhealing defects. Changes in normal and wounded fibroblast cell morphology were evaluated by light microscopy. Cellular parameters evaluated cell proliferation, cell viability, and cytotoxicity while molecular parameters assessed the extent of DNA damage. The results clearly demonstrate that LLLT has an effect on normal and wounded(3) human skin fibroblasts. The parameters showed that doses of 0.5, 2.5, 5, and 10 J/cm2 were sufficient to produce measurable changes in fibroblast cells. A dose of 10 J/cm2 appeared to produce a significant amount of cellular and molecular damage, which could be an important consideration for other therapies, such as photodynamic therapy.

  10. Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

    PubMed Central

    Alanazi, Humidah; Semlali, Abdelhabib; Perraud, Laura; Chmielewski, Witold; Zakrzewski, Andrew

    2014-01-01

    The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers. PMID:25302312

  11. Cigarette smoke-exposed Candida albicans increased chitin production and modulated human fibroblast cell responses.

    PubMed

    Alanazi, Humidah; Semlali, Abdelhabib; Perraud, Laura; Chmielewski, Witold; Zakrzewski, Andrew; Rouabhia, Mahmoud

    2014-01-01

    The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC) on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P < 0.01) sensitive to oxidation but significantly (P < 0.01) resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P < 0.01) slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

  12. Fibronectin functional domains coupled to hyaluronan stimulate adult human dermal fibroblast responses critical for wound healing.

    PubMed

    Ghosh, Kaustabh; Ren, Xiang-Dong; Shu, Xiao Zheng; Prestwich, Glenn D; Clark, Richard A F

    2006-03-01

    Fibronectin (FN) facilitates dermal fibroblast migration during normal wound healing. Proteolytic degradation of FN in chronic wounds hampers healing. Previously, three FN functional domains (FNfd) have been shown to be sufficient for optimal adult human dermal fibroblast migration. Here we report the development of an acellular hydrogel matrix comprised of the FNfds coupled to a hyaluronan (HA) backbone to stimulate wound repair. Employing Michael-type addition, the cysteine- tagged FNfds were first coupled to a homobifunctional PEG derivative. Thereafter, these PEG derivative FNfd solutions, containing bifunctional PEG-derivative crosslinker were coupled to thiol-modified HA (HA-DTPH) to obtain a crosslinked hydrogel matrix. When evaluated in vitro, these acellular hydrogels were completely cytocompatible. While spreading and proliferation of adult human dermal fibroblasts plateaued at higher FNfd bulk densities, their rapid and robust migration followed a typical bell-shaped response. When implanted in porcine cutaneous wounds, these acellular matrices, besides being completely biocompatible, induced rapid and en masse recruitment of stromal fibroblasts that was not observed with RGD-tethered or unmodified hydrogels. Such constructs might be of great benefit in clinical settings where rapid formation of new tissue is needed.

  13. Inadequate mito-biogenesis in primary dermal fibroblasts from old humans is associated with impairment of PGC1A-independent stimulation.

    PubMed

    Kalfalah, Faiza; Sobek, Stefan; Bornholz, Beatrice; Götz-Rösch, Christine; Tigges, Julia; Fritsche, Ellen; Krutmann, Jean; Köhrer, Karl; Deenen, René; Ohse, Sebastian; Boerries, Melanie; Busch, Hauke; Boege, Fritz

    2014-08-01

    Extrinsic skin ageing converges on the dermis, a post-mitotic tissue compartment consisting of extracellular matrix and long-lived fibroblasts prone to damage accumulation and maladaptation. Aged human fibroblasts exhibit mitochondrial and nuclear dysfunctions, which may be a cause or consequence of ageing. We report on a systematic study of human dermal fibroblasts retrieved from female donors aged 20-67 years and analysed ex vivo at low population doubling precluding replicative senescence. According to gene set enrichment analysis of genome wide array data, the most prominent age-associated change of the transcriptome was decreased expression of mitochondrial genes. Consistent with that, mitochondrial content and cell proliferation declined with donor age. This was associated with upregulation of AMP-dependent protein kinase (AMPK), increased mRNA levels of PPARγ-coactivator 1α (PGC1A) and decreased levels of NAD(+)-dependent deacetylase sirtuin 1. In the old cells the PGC1A-mediated mito-biogenetic response to direct AMPK-stimulation by AICAR was undiminished, while the PGC1A-independent mito-biogenetic response to starvation was attenuated and accompanied by increased ROS-production. In summary, these observations suggest an age-associated decline in PGC1A-independent mito-biogenesis, which is insufficiently compensated by upregulation of the AMPK/PGC1A-axis leading under baseline conditions to decreased mitochondrial content and reductive overload of residual respiratory capacity. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System

    PubMed Central

    Ueno, Takayuki; Utsumi, Jun; Toi, Masakazu; Shimizu, Kazuharu

    2015-01-01

    The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment. PMID:26171396

  15. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System.

    PubMed

    Ueno, Takayuki; Utsumi, Jun; Toi, Masakazu; Shimizu, Kazuharu

    2015-01-01

    The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment.

  16. [The mechanisms of the antiherpetic action of aqueous propolis extracts. I. The antioxidant action on human fibroblast cultures].

    PubMed

    Dumitrescu, M; Eşanu, V; Crişan, I

    1992-01-01

    A redox state modulation model was worked out in human fibroblast cultures treated with oxidation stress inducing agents and a redox agent, virtually protecting cell against the stress. Quantification of the global redox changes in fibroblasts was done using the hemoglobin electronic spectrum, in the presence and in the absence of H2O2.

  17. MCPIP1 mediates silica-induced cell migration in human pulmonary fibroblasts.

    PubMed

    Liu, Haijun; Dai, Xiaoniu; Cheng, Yusi; Fang, Shencun; Zhang, Yingming; Wang, Xingang; Zhang, Wei; Liao, Hong; Yao, Honghong; Chao, Jie

    2016-01-15

    Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO2). Phagocytosis of SiO2 in the lungs initiates an inflammatory cascade that results in fibroblast proliferation and migration followed by fibrosis. According to previous data from our laboratory, monocyte chemotactic protein-1 (MCP-1) plays a critical role in fibroblast proliferation and migration in conventional two-dimensional (2D) monolayer cultures. The present study aimed to explore the downstream cascade of MCP-1 in both 2D and three-dimensional (3D) cell culture models of silicosis. Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following: 1) SiO2 treatment induces expression of MCP-1-induced protein (MCPIP1) in a time- and dose-dependent manner in both 2D and 3D cultures; 2) the MAPK and phosphatidylinositol-3-kinase (PI3K)/Akt pathways are involved in SiO2-induced MCPIP1 expression; and 3) MCPIP1 induction mediates the SiO2-induced increase in cell migration in both 2D and 3D cultures. The effect of MCP-1 in silicosis occurs mainly through MCPIP1, which, in turn, mediates the observed SiO2-induced increase in pulmonary fibroblast migration. However, the time frame for MCPIP1 induction differed between 2D and 3D cultures, indicating that, compared with conventional 2D cell culture systems, 3D culture may be useful for analyses of fibroblast physiology under conditions that more closely resemble in vivo environments. Our study determined the link between fibroblast-derived MCPIP1 and SiO2-induced cell migration, and this finding provides novel evidence of the potential of MCPIP1 in the development of novel therapeutic strategies for silicosis.

  18. Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

    SciTech Connect

    Espinoza, B.; Wharton, W.

    1986-08-01

    Cholera toxin produced a dose-dependent decrease in the restimulation of G0/G1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition in the growth of sparse human fibrobasts. IBMX produced an inhibition both in the G1 and in the G2 phases of the cell cycle by a mechanism(s) that was not related to the magnitude of the increases in adenosine 3,5-cyclic monophosphate concentrations.

  19. Cu,Zn Superoxide Dismutase is a Peroxisomal Enzyme in Human Fibroblast and Hepatoma Cells

    NASA Astrophysics Data System (ADS)

    Keller, Gilbert-Andre; Warner, Thomas G.; Steimer, Kathelyn S.; Hallewell, Robert A.

    1991-08-01

    The intracellular localization of Cu,Zn superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) has been examined by immunofluorescence using four monoclonal anti-Cu,Zn superoxide dismutase antibodies raised against a recombinant human Cu,Zn superoxide dismutase derivative produced and purified from Escherichia coli. Colocalization with catalase, a peroxisomal matrix enzyme, was used to demonstrate the peroxisomal localization of Cu,Zn superoxide dismutase in human fibroblasts and hepatoma cells. In the fibroblasts of Zellweger syndrome patients, the enzyme is not transported to the peroxisomal ghosts but, like catalase, remains in the cytoplasm. In addition, immunocryoelectron microscopy of yeast cells expressing human Cu,Zn superoxide dismutase showed that the enzyme is translocated to the peroxisomes.

  20. 199 EFFECTS OF REPROGRAMMING-CONDITIONED MEDIUM ON ULTRAVIOLET RAY A-DAMAGED HUMAN DERMAL FIBROBLASTS.

    PubMed

    Kang, J; Lee, S G; Kang, J H; Park, S-M; Heo, S Y; Lee, S Y; Kim, S; Lo, E; Ahn, K S; Shim, H

    2016-01-01

    Ultraviolet ray A (UVA) is an electromagnetic light with a long wavelength from the sun. The penetration of UVA deep into the human dermis causes changes in cells, such as DNA fragmentation, apoptosis, and senescence, eventually leading a decline of proliferation and wound-healing ability. These changes induced by UVA exposure are similar to those seen in the process of stem cell differentiation. We postulated that the condition that reverses cellular differentiation may alleviate the UVA-induced damage in skin cells. Human dermal fibroblasts (HDF) could be reprogrammed to induced pluripotent stem cells (iPSC). Conditioned medium (CM) was prepared during the process of iPSC reprogramming (referred to as Repro-CM). The UVA-irradiated HDF were cultured in Repro-CM for 24h. In comparison with CM prepared from the culture of normal HDF and iPSC (referred to as HDF-CM and iPSC-CM, respectively), effects of Repro-CM on UVA-irradiated cells were investigated. Viability, wound-healing ability, apoptosis, and senescence of HDF were analysed by WST-1 assay, scratch assay, Annexin V assay, and senescence-associated β-galactosidase assay, respectively. Upon recovering from the UVA-induced damage, viability and wound-healing ability of HDF were significantly different (P<0.05) among the treatments in the order of Repro-, HDF-, and iPSC-CM. In the same context, apoptosis and senescence were significantly different (P<0.05) in the order of iPSC-, HDF-, and Repro-CM. Interestingly, iPSC-CM did not substantially ameliorate UVA-induced damage, suggesting that the conditions optimized to pluripotent stem cells may not be suitable for the recovery from damage in terminally differentiated cells, such as fibroblasts. The RNA-seq analysis was performed to assess the genome-wide transcriptional profile in the process of recovery. Repro- and HDF-CM were categorized more closely than iPSC-CM in hierarchical cluster analysis. In comparison with iPSC-CM, the up-regulated genes by Repro

  1. Distinct Cell Stress Responses Induced by ATP Restriction in Quiescent Human Fibroblasts

    PubMed Central

    Yalamanchili, Nirupama; Kriete, Andres; Alfego, David; Danowski, Kelli M.; Kari, Csaba; Rodeck, Ulrich

    2016-01-01

    Quiescence is the prevailing state of many cell types under homeostatic conditions. Yet, surprisingly little is known about how quiescent cells respond to energetic and metabolic challenges. To better understand compensatory responses of quiescent cells to metabolic stress, we established, in human primary dermal fibroblasts, an experimental ‘energy restriction’ model. Quiescence was achieved by short-term culture in serum-deprived media and ATP supply restricted using a combination of glucose transport inhibitors and mitochondrial uncouplers. In aggregate, these measures led to markedly reduced intracellular ATP levels while not compromising cell viability over the observation period of 48 h. Analysis of the transcription factor (TF) landscape induced by this treatment revealed alterations in several signal transduction nodes beyond the expected biosynthetic adaptations. These included increased abundance of NF-κB regulated TFs and altered TF subsets regulated by Akt and p53. The observed changes in gene regulation and corresponding alterations in key signaling nodes are likely to contribute to cell survival at intracellular ATP concentrations substantially below those achieved by growth factor deprivation alone. This experimental model provides a benchmark for the investigation of cell survival pathways and related molecular targets that are associated with restricted energy supply associated with biological aging and metabolic diseases. PMID:27757122

  2. Passiflora tarminiana fruits reduce UVB-induced photoaging in human skin fibroblasts.

    PubMed

    Bravo, Karent; Duque, Luisa; Ferreres, Federico; Moreno, Diego A; Osorio, Edison

    2017-03-01

    Skin aging is a complex process that is strongly affected by UV radiation, which stimulates the production of reactive oxygen species (ROS) in the epidermis and dermis and subsequently causes skin damage. Among the major consequences are increased collagen degradation and reduced collagen synthesis. Previous reports have demonstrated the beneficial effects of polyphenols for healthy skin. Passiflora tarminiana Coppens & V.E. Barney, a species of the Passifloraceae family, is widely distributed in South America and is rich in flavonoids. We show that UVB radiation increases metalloproteinase 1 (MMP-1) and reduces procollagen production in human dermal fibroblast (HDF) cells in a dose- and time-dependent manner. We examined the antioxidant and antiaging effects of the extract and fractions of P. tarminiana fruits. The fractions showed high polyphenol content (620mg EAG/g) and antioxidant activity, as measured by ORAC (4097μmol ET/g) and ABTS (2992μmol ET/g) assays. The aqueous fraction drastically inhibited the collagenase enzyme (IC50 0.43μg/mL). The extract and fractions presented photoprotective effects by reducing UVB-induced MMP-1 production, increasing UVB-inhibited procollagen production, and decreasing ROS production after UVB irradiation in HDF. Finally, the polyphenol contents of the extracts and fractions from P. tarminiana were analyzed by HPLC-DAD-ESI-MS(n), and procyanidins and glycosylated flavonoids were identified.

  3. Cordyceps militaris Extract Protects Human Dermal Fibroblasts against Oxidative Stress-Induced Apoptosis and Premature Senescence

    PubMed Central

    Park, Jun Myoung; Lee, Jong Seok; Lee, Ki Rim; Ha, Suk-Jin; Hong, Eock Kee

    2014-01-01

    Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS. PMID:25230212

  4. Cordyceps militaris extract protects human dermal fibroblasts against oxidative stress-induced apoptosis and premature senescence.

    PubMed

    Park, Jun Myoung; Lee, Jong Seok; Lee, Ki Rim; Ha, Suk-Jin; Hong, Eock Kee

    2014-09-16

    Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-β-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

  5. Human dermal fibroblast proliferation activity of usimine-C from Antarctic lichen Ramalina terebrata.

    PubMed

    Lee, Sung Gu; Koh, Hye Yeon; Oh, Hyuncheol; Han, Se Jong; Kim, Il-Chan; Lee, Hong Kum; Yim, Joung Han

    2010-04-01

    Type I collagen is the major structural protein in dermis and its presence is used to monitor skin cell proliferation and aging. Recently, novel usimine compounds have been found in the Antarctic lichen Ramalina terebrata. In the present study, usimine-C induced cell proliferation of human dermal fibroblast, CCD-986SK, up to 1.6-fold after treating with 90 microg/ml for 48 h. Type I procollagen synthesis was significantly increased 1.3-fold, 3-fold, and 5-fold after treating with 0.14, 0.72, and 3.6 microg usimine-C/ml for 24 h, respectively, whereas no significant increase in type I procollagen was observed after treating with usimine-A or -B. Usimines are usnic acid derivatives. Considering that the difference among the derivatives is a side chain, the proliferation activity may be related to this side chain, triggering an internal signal for type I procollagen expression. Further studies still remain to clarify the signaling pathways for the type I procollagen induction, which is activated by usimine-C.

  6. UVB-Induced Senescence of Human Dermal Fibroblasts Involves Impairment of Proteasome and Enhanced Autophagic Activity.

    PubMed

    Cavinato, Maria; Koziel, Rafal; Romani, Nikolaus; Weinmüllner, Regina; Jenewein, Brigitte; Hermann, Martin; Dubrac, Sandrine; Ratzinger, Gudrun; Grillari, Johannes; Schmuth, Matthias; Jansen-Dürr, Pidder

    2017-05-01

    In the current study, we have extended previous findings aiming at a better understanding of molecular mechanisms underlying UVB-induced senescence of diploid human dermal fibroblasts (HDFs), an experimental model to study the process of photoaging in the skin. We provide evidence that the inhibition of proteasomal degradation of damaged proteins and the activation of autophagosome formation are early events in UVB-induced senescence of HDFs, dependent on UVB-induced accumulation of reactive oxygen species. Our data suggest that autophagy is required for the establishment of the senescent phenotype in UVB-treated HDFs and that inhibition of autophagy is sufficient to change the cell fate from senescence to cell death by apoptosis. Studies in reconstructed skin equivalents revealed that UVB irradiation triggers hallmarks of autophagy induction in the dermal layer. These findings have potential implications for fundamental as well as translational research into skin aging, in particular photoaging. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Comparative proteomic analysis of human lung telocytes with fibroblasts

    PubMed Central

    Zheng, Yonghua; Cretoiu, Dragos; Yan, Guoquan; Cretoiu, Sanda Maria; Popescu, Laurentiu M; Wang, Xiangdong

    2014-01-01

    Telocytes (TCs) were recently described as interstitial cells with very long prolongations named telopodes (Tps; http://www.telocytes.com). Establishing the TC proteome is a priority to show that TCs are a distinct type of cells. Therefore, we examined the molecular aspects of lung TCs by comparison with fibroblasts (FBs). Proteins extracted from primary cultures of these cells were analysed by automated 2-dimensional nano-electrospray ionization liquid chromatography tandem mass spectrometry (2D Nano-ESI LC-MS/MS). Differentially expressed proteins were screened by two-sample t-test (P < 0.05) and fold change (>2), based on the bioinformatics analysis. We identified hundreds of proteins up- or down-regulated, respectively, in TCs as compared with FBs. TC proteins with known identities are localized in the cytoskeleton (87%) and plasma membrane (13%), while FB up-regulated proteins are in the cytoskeleton (75%) and destined to extracellular matrix (25%). These identified proteins were classified into different categories based on their molecular functions and biological processes. While the proteins identified in TCs are mainly involved in catalytic activity (43%) and as structural molecular activity (25%), the proteins in FBs are involved in catalytic activity (24%) and in structural molecular activity, particularly synthesis of collagen and other extracellular matrix components (25%). Anyway, our data show that TCs are completely different from FBs. In conclusion, we report here the first extensive identification of proteins from TCs using a quantitative proteomics approach. Protein expression profile shows many up-regulated proteins e.g. myosin-14, periplakin, suggesting that TCs might play specific roles in mechanical sensing and mechanochemical conversion task, tissue homoeostasis and remodelling/renewal. Furthermore, up-regulated proteins matching those found in extracellular vesicles emphasize TCs roles in intercellular signalling and stem cell niche

  8. Ascorbic acid extends replicative life span of human embryonic fibroblast by reducing DNA and mitochondrial damages.

    PubMed

    Hwang, Won-Sang; Park, Seong-Hoon; Kim, Hyun-Seok; Kang, Hong-Jun; Kim, Min-Ju; Oh, Soo-Jin; Park, Jae-Bong; Kim, Jaebong; Kim, Sung Chan; Lee, Jae-Yong

    2007-01-01

    Ascorbic acid has been reported to extend replicative life span of human embryonic fibroblast (HEF). Since the detailed molecular mechanism of this phenomenon has not been investigated, we attempted to elucidate. Continuous treatment of HEF cells with ascorbic acid (at 200 microM) from 40 population doubling (PD) increased maximum PD numbers by 18% and lowered SA-beta-gal positive staining, an aging marker, by 2.3 folds, indicating that ascorbic acid extends replicative life span of HEF cells. Ascorbic acid treatment lowered DCFH by about 7 folds and Rho123 by about 70%, suggesting that ascorbic acid dramatically decreased ROS formation. Ascorbic acid also increased aconitase activity, a marker of mitochondrial aging, by 41%, indicating that ascorbic acid treatment restores age-related decline of mitochondrial function. Cell cycle analysis by flow cytometry revealed that ascorbic acid treatment decreased G1 population up to 12%. Further western blot analysis showed that ascorbic acid treatment decreased levels of p53, phospho-p53 at ser 15, and p21, indicating that ascorbic acid relieved senescence-related G1 arrest. Analysis of AP (apurinic/apyrimidinic) sites showed that ascorbic acid treatment decreased AP site formation by 35%. We also tested the effect of hydrogen peroxide treatment, as an additional oxidative stress. Continuous treatment of 20 microM of hydrogen peroxide from PD 40 of HEF cells resulted in premature senescence due to increased ROS level, and increased AP sites. Taken together, the results suggest that ascorbic acid extends replicative life span of HEF cells by reducing mitochondrial and DNA damages through lowering cellular ROS.

  9. Extracellular low pH affects circadian rhythm expression in human primary fibroblasts.

    PubMed

    Lee, Sang Kil; Achieng, Elsie; Maddox, Connie; Chen, Suephy C; Iuvone, P Michael; Fukuhara, Chiaki

    2011-12-16

    Circadian rhythm is a fundamental biological system involved in the regulation of various physiological functions. However, little is known about a nature or function of circadian clock in human primary cells. In the present study, we have applied in vitro real time circadian rhythm monitoring to study human clock properties using primary skin fibroblasts. Among factors that affect human physiology, slightly lower extracellular pH was chosen to test its effects on circadian rhythm expression. We established human primary fibroblast cultures obtained from three healthy subjects, stably delivered a circadian reporter gene Bmal1-luciferase, and recorded circadian rhythms in the culture medium at pH 7.2 and 6.7. At pH 7.2, robust and sustained circadian rhythms were observed with average period length 24.47 ± 0.03 h. Such rhythms were also found at pH 6.7; however, period length was significantly shortened to 22.60 ± 0.20, amplitude was increased, and damping rate was decreased. The effect of exposure to low pH on the period length was reversible. The shortened period was unlikely caused by factors affecting cell viability because cell morphology and MTT assay showed no significant difference between the two conditions. In summary, our results showed that the circadian rhythm expression is affected at pH 6.7 in human primary fibroblasts without affecting cell viability. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Cyclobutane-type pyrimidine photodimer formation and induction of ornithine decarboxylase in human skin fibroblasts after UV irradiation

    SciTech Connect

    Niggli, H.J.; Roethlisberger, R.

    1988-12-01

    Cyclobutane-type pyrimidine photodimers as well as the induction of ornithine decarboxylase (ODC) may serve as biochemical markers of the mutagenic and carcinogenic effects of ultraviolet light (UV). For this reason, it is important to compare the formation of pyrimidine dimers with the induction of ODC in human skin fibroblasts after irradiation with UVC (200-290 nm) and UVB (290-320 nm). In our studies we determined cytosine-thymine (C-T) as well as thymine-thymine dimer yields (T-T) by high-pressure liquid chromatography in cultures of neonatal normal human foreskin-derived fibroblasts after irradiation with UVC and UVB light. It was found that the yield of dimerization and the ratio of T-T/C-T decreased from the UVC to the UVB region. Time-course studies of ODC-induction in the same cells indicated that the maximal activity after UVB irradiation was retarded compared to UVC exposure. For the UV-induced ODC-levels, however, no significant difference in maximal induction could be measured after UVC and UVB irradiation at fluences where comparable yields of thymine dimerization are produced. Similar ODC-maxima were obtained with strains from children, while cells from adults showed significantly less pronounced ODC induction, indicating that ODC-response decreases with age and may therefore be used as a marker of aging.

  11. Serum from calorie-restricted animals delays senescence and extends the lifespan of normal human fibroblasts in vitro.

    PubMed

    de Cabo, Rafael; Liu, Lijuan; Ali, Ahmed; Price, Nathan; Zhang, Jing; Wang, Mingyi; Lakatta, Edward; Irusta, Pablo M

    2015-03-01

    The cumulative effects of cellular senescence and cell loss over time in various tissues and organs are considered major contributing factors to the ageing process. In various organisms, caloric restriction (CR) slows ageing and increases lifespan, at least in part, by activating nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases of the sirtuin family. Here, we use an in vitro model of CR to study the effects of this dietary regime on replicative senescence, cellular lifespan and modulation of the SIRT1 signaling pathway in normal human diploid fibroblasts. We found that serum from calorie-restricted animals was able to delay senescence and significantly increase replicative lifespan in these cells, when compared to serum from ad libitum fed animals. These effects correlated with CR-mediated increases in SIRT1 and decreases in p53 expression levels. In addition, we show that manipulation of SIRT1 levels by either over-expression or siRNA-mediated knockdown resulted in delayed and accelerated cellular senescence, respectively. Our results demonstrate that CR can delay senescence and increase replicative lifespan of normal human diploid fibroblasts in vitro and suggest that SIRT1 plays an important role in these processes.

  12. Serum from calorie-restricted animals delays senescence and extends the lifespan of normal human fibroblasts in vitro

    PubMed Central

    Ali, Ahmed; Price, Nathan; Zhang, Jing; Wang, Mingyi; Lakatta, Edward; Irusta, Pablo M.

    2015-01-01

    The cumulative effects of cellular senescence and cell loss over time in various tissues and organs are considered major contributing factors to the ageing process. In various organisms, caloric restriction (CR) slows ageing and increases lifespan, at least in part, by activating nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases of the sirtuin family. Here, we use an in vitro model of CR to study the effects of this dietary regime on replicative senescence, cellular lifespan and modulation of the SIRT1 signaling pathway in normal human diploid fibroblasts. We found that serum from calorie-restricted animals was able to delay senescence and significantly increase replicative lifespan in these cells, when compared to serum from ad libitum fed animals. These effects correlated with CR-mediated increases in SIRT1 and decreases in p53 expression levels. In addition, we show that manipulation of SIRT1 levels by either over-expression or siRNA-mediated knockdown resulted in delayed and accelerated cellular senescence, respectively. Our results demonstrate that CR can delay senescence and increase replicative lifespan of normal human diploid fibroblasts in vitro and suggest that SIRT1 plays an important role in these processes. (185 words). PMID:25855056

  13. Generation of human iPS cell line SKiPSc1 from healthy Human Neonatal Foreskin Fibroblast cells.

    PubMed

    Alawad, Abdullah; Alhazzaa, Othman; Altuwaijri, Saleh; Alkhrayef, Mohammad; Alagrafi, Faisal; Alhamdan, Ziyad; Alenazi, Abdullah; Alharbi, Sultan; Hammad, Mohamed

    2016-06-25

    The SKiPSc1 induced pluripotent stem (iPS) cell line was generated from Human Neonatal Foreskin Fibroblasts (HNFFs) obtained from a healthy donor infant that were reprogrammed using non-integrating Sendai viral vectors expressing Oct3/4, Sox2, c-Myc, and Klf4.

  14. Regulation of PI3K/Akt dependent apoptotic markers during b virus infection of human and macaque fibroblasts.

    PubMed

    Vasireddi, Mugdha; Hilliard, Julia K

    2017-01-01

    B virus (Macacine herpesvirus 1), a simplex virus endemic in macaques, causes encephalitis, encephalomyelitis, and death in 80% of untreated zoonotically infected humans with delayed or no treatment. Here we report a significant difference in PI3K/Akt-dependent apoptosis between B virus infected human and macaque dermal fibroblasts. Our data show that B virus infection in either human or macaque fibroblasts results in activation of Akt via PI3K and this activation does not require viral de novo protein synthesis. Inhibition of PI3K with LY294002 results in a significant reduction of viral titers in B virus infected macaque and human fibroblasts with only a modest difference in the reduction of virus titers between the two cell types. We, therefore, tested the hypothesis that B virus results in the phosphorylation of Akt (S473), which prevents apoptosis, enhancing virus replication in B virus infected macaque dermal fibroblasts. We observed markers of intrinsic apoptosis when PI3K activation of Akt was inhibited in B virus infected macaque cells, while, these apoptotic markers were absent in B virus infected human fibroblasts under the same conditions. From these data we suggest that PI3K activates Akt in B virus infected macaque and human fibroblasts, but this enhances virus replication in macaque fibroblast cells by blocking apoptosis.

  15. Human anterior cruciate ligament fibroblasts from immature patients have a stronger in vitro response to platelet concentrates than those from mature individuals

    PubMed Central

    Magarian, Elise M.; Vavken, Patrick; Murray, Martha M.

    2010-01-01

    A number of recently published studies have established a substantial age dependence of the response of ACL fibroblasts to stimulation by platelet-rich plasma (PRP). Further in-depth research of this age dependence revealed negative effects on both histological and biomechanical results in a large animal model. However, while it has been postulated that this association could affect potential human applications negatively too it remains to be proven that the same effects occur in human cells. Thus it was the objective of this study to search for age dependence in human fibroblasts before further human experiments are done. Human fibroblasts were obtained from 10 immature and adolescent patients, based on a-priori power calculations, and culture in a collagen-PRP composite. Three parameters that are pivotal for defect remodeling and wound healing – cell migration, cell proliferation, and scaffold contraction – were chosen as endpoints. Both migration and proliferation were significantly higher in immature cells, but no differences were seen in wound contraction. The former findings suggest that immature patients respond more favorably to treatment with PRP, which consequently might translate into better results in ACL tissue engineering. PMID:20728363

  16. Lamin A and lamin-associated polypeptide 2 (LAP-2) in human skin in the process of aging.

    PubMed

    Golubtsova, N N; Filippov, F N; Gunin, A G

    2016-01-01

    At present time, relationships between lamins and processes leading to aging are established. Mutations of genes of lamins lead to diseases, one of them is progeria. This disease is caused by violation of splaysing of lamin A gene and accumulation the farnezylated prelamin A (progerin) in the nucleus. LAP-2 is an important factor which regulates and stabilizes the lamin A. However, roles of lamin A and LAP-2 in behavior of population of dermal fibroblasts in relation to age were not examined. The aim of this research was to study A type lamin and LAP-2 in human skin at different ages. Lamin A and LAP-2 were detected in sections of the skin by indirect immunohistochemistry. The number of fibroblasts containing lamin A was gradually decreased from 90,4 to 76,9 % from 20 weeks of gestation to 85 years old. There were 32 % of dermal fibroblasts with positive staining for LAP-2 at the period from 20 weeks of gestation to 20 years old. From 21 to 40 years, 37,8 % of fibroblasts containing lamin A were found in the dermis. In age interval 41-85 years, 49-51 % of dermal fibroblasts had a positive staining for LAP-2. Content of lamin A in the nuclei of fibroblasts was almost constant from 20 weeks of gestation to 85 years old. Expression of LAP-2 in the nuclei of fibroblasts was reduced from birth to 20 years old but increased from 21 years old. Number of fibroblasts and PCNA+ fibroblasts in dermis was diminished with age. The most significant decrease in the number of fibroblasts was observed from 20 weeks of gestation to 20 years old. Results allow to assume the participation of lamin A and LAP-2 in triggering age-dependent decrease in the number of fibroblasts in the dermis in humans.

  17. Regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor, and lymphotoxin.

    PubMed Central

    Elias, J A; Krol, R C; Freundlich, B; Sampson, P M

    1988-01-01

    Mononuclear cells may be important regulators of fibroblast glycosaminoglycan (GAG) biosynthesis. However, the soluble factors mediating these effects, the importance of intercytokine interactions in this regulation and the mechanisms of these alterations remain poorly understood. We analyzed the effect of recombinant (r) tumor necrosis factor (TNF), lymphotoxin (LT), and gamma, alpha, and beta 1 interferons (INF-gamma, -alpha and -beta 1), alone and in combination, on GAG production by normal human lung fibroblasts. rTNF, rLT, and rINF-gamma each stimulated fibroblast GAG production. In addition, rIFN-gamma synergized with rTNF and rLT to further augment GAG biosynthesis. In contrast, IFN-alpha A, -alpha D, and -beta 1 neither stimulated fibroblast GAG production nor interacted with rTNF or rLT to regulate GAG biosynthesis. The effects of the stimulatory cytokines and cytokine combinations were dose dependent and were abrogated by the respective monoclonal antibodies. In addition, these cytokines did not cause an alteration in the distribution of GAG between the fibroblast cell layer and supernatant. However, the stimulation was at least partially specific for particular GAG moieties with hyaluronic acid biosynthesis being markedly augmented without a comparable increase in the production of sulfated GAGs. Fibroblast prostaglandin production did not mediate these alterations since indomethacin did not decrease the stimulatory effects of the cytokines. In contrast, protein and mRNA synthesis appeared to play a role since the stimulatory effects of the cytokines were abrogated by cyclohexamide and actinomycin D, respectively. In addition, the cytokines and cytokine combinations increased cellular hyaluronate synthetase activity in proportion to their effects on hyaluronic acid suggesting that induction of this enzyme(s) is important in this stimulatory process. These studies demonstrate that IFN-gamma, TNF, and LT are important stimulators of fibroblast GAG

  18. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products.

    PubMed

    El Ghalbzouri, Abdoelwaheb; Commandeur, Suzan; Rietveld, Marion H; Mulder, Aat A; Willemze, Rein

    2009-01-01

    Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.

  19. Transcriptional profiling of fibroblasts from patients with mutations in MCT8 and comparative analysis with the human brain transcriptome.

    PubMed

    Visser, W Edward; Swagemakers, Sigrid M A; Ozgur, Zeliha; Schot, Rachel; Verheijen, Frans W; van Ijcken, Wilfred F J; van der Spek, Peter J; Visser, Theo J

    2010-11-01

    Thyroid hormone (TH) is crucial for normal brain development. TH transporters control TH homeostasis in brain as evidenced by the complex endocrine and neurological phenotype of patients with mutations in monocarboxylate transporter 8 (MCT8). We investigated the mechanisms of disease by analyzing gene expression profiles in fibroblasts from patients with MCT8 mutations. Studying MCT8 and its transcriptional context in different comprehensive spatial and temporal human brain transcriptome data sets revealed distinct region-specific MCT8 expression. Furthermore, MCT8 demonstrated a clear age-dependent decrease, suggesting its importance in early brain development. Performing comparative transcriptome analysis, we linked the genes differentially expressed (DE) in patient fibroblasts to the human brain transcriptome. DE genes in patient fibroblasts were strongly over-represented among genes highly correlated with MCT8 expression in brain. Furthermore, using the same approach we identified which genes in the classical TH signaling pathway are affected in patients. Finally, we provide evidence that the TRα2 receptor variant is closely connected to MCT8. The present study provides a molecular basis for understanding which pathways are likely affected in the brains of patients with mutations in MCT8. Our data regarding a functional relationship between MCT8 and TRα2 suggest an unanticipated role for TRα2 in the (patho)physiology of TH signaling in the brain. This study demonstrates how genome-wide expression data from patient-derived non-neuronal tissue related to the human brain transcriptome may be successfully employed to improve our understanding of neurological disease.

  20. Influence of E-smoking liquids on human periodontal ligament fibroblasts

    PubMed Central

    2014-01-01

    Introduction Over the last years, electronic cigarettes (ECs) have become more popular, particularly in individuals who want to give up smoking tobacco. The aim of the present study was to assess the influence of the different e-smoking liquids on the viability and proliferation of human periodontal ligament fibroblasts. Method and materials For this study six test solutions with components from ECs were selected: lime-, hazelnut- and menthol-flavored liquids, nicotine, propylene glycol, and PBS as control group. The fibroblasts were incubated up to 96 h with the different liquids, and cell viability was measured by using the PrestoBlue® reagent, the ATP detection and the migration assay. Fluorescence staining was carried out to visualize cell growth and morphology. Data were statistically analyzed by two-tailed one-way ANOVA. Results The cell viability assay showed that the proliferation rates of the cells incubated with nicotine or the various flavored liquids of the e-cigarettes were reduced in comparison to the controls, though not all reductions were statistically significant. After an incubation of 96 h with the menthol-flavored liquid the fibroblasts were statistically significant reduced (p < 0.001). Similar results were found for the detection of ATP in fibroblasts; the incubation with menthol-flavored liquids (p < 0.001) led to a statistically significant reduction. The cell visualization tests confirmed these findings. Conclusion Within its limits, the present in vitro study demonstrated that menthol additives of e-smoking have a harmful effect on human periodontal ligament fibroblasts. This might indicate that menthol additives should be avoided for e-cigarettes. PMID:25224853

  1. Novel Therapy To Reverse The Cellular Effects of Bisphosphonates on Primary Human Oral Fibroblasts

    PubMed Central

    Cozin, Matthew; Pinker, Bradley M.; Solemani, Kimberley; Zuniga, Jeremy M.; Dadaian, Stephen C.; Cremers, Serge; Landesberg, Regina; Raghavan, Srikala

    2011-01-01

    Purpose Osteonecrosis of the Jaws is a clinical condition that is characterized by a non-healing breach in the oral mucosa resulting in exposure of bone and has been increasingly reported in patients receiving bisphosphonate (BP) therapy. Although the pathogenesis and natural history of ONJ remain ill defined, it appears that the oral soft tissues play a critical role in the development of this condition. In this report we examined the effects of the nitrogen-containing BPs pamidronate and zoledronate on primary human gingival fibroblasts. Materials and Methods Primary gingival fibroblasts were exposed to clinically relevant doses of pamidronate and zoledronate. Cellular proliferation was measured using a MTS/PMS reagent-based kit, scratch wound assays were performed to measure cellular migration and apoptosis was measured by using TUNEL and caspase assays. The BP exposed cells were treated with 10ng/mL recombinant human Platelet-Derived Growth Factor-BB (rhPDGF-BB, GEM21™) and 50μM geranylgeraniol (GGOH). Results Gingival fibroblasts are significantly more sensitive to inhibition of proliferation by zoledronate compared to pamidronate. Exposure of these cells to pamidronate but not zoledronate resulted in an increase in cellular apoptosis. Furthermore, exposure of gingival fibroblasts to pamidronate or zoledronate resulted in a decrease in cellular migration. We show that these defects are due to a loss of cell-substratum adhesion, and a reduction of F-actin bundles. Finally, we show that the addition of rhPDGF-BB, (GEM21™) and GGOH in vitro are able to partially rescue the cell proliferation, migration and adhesion defects. Conclusion The cytotoxic affects BPs on oral fibroblasts and its significant reversal by the addition of GGOH and rhPDGF-BB, (GEM21™) provide both the potential mechanism and treatment options for ONJ. PMID:21807448

  2. Anti-wrinkle effects of a tuna heart H2O fraction on Hs27 human fibroblasts

    PubMed Central

    KIM, YOUNG-MIN; JUNG, HEE-JIN; CHOI, JAE-SUE; NAM, TAEK-JEONG

    2016-01-01

    With the increase in life expectancy, there is also growing interest in anti-aging treatments and technologies. The development of anti-aging functional drugs for the skin, and foods from natural sources, may offer solutions to this global matter. Aging involves structural, functional and biochemical changes that occur throughout cells and bodily tissues; the amount of hormones secreted from of all human organs, including the skin, decreases over time. Matrix metalloproteinase (MMP) genes (MMP-1 and -8) play an important role in the aging of skin fibroblasts. For example, an increased MMP expression causes accelerated aging and the degradation of skin elasticity-related genes. In the present study, we examined the anti-wrinkle effects of tuna heart extract which are mediated through the inhibition of MMPs in skin cells. Generally, tuna contains high concentrations of selenium and antioxidants, which serve to remove free radicals, and is known to delay skin and body aging. In addition, unsaturated fatty acids in tuna help to maintain the natural glossy look of skin, and increase skin elasticity, providing moisture for dry skin. A recent study confirmed the various bio-effects of boiled tuna extract and muscle. However, bioactivity studies using tuna heart are limited. Thus, in the present study, we obtained extracts and fractions of tuna heart, and examined their effects on Hs27 human fibroblast proliferation using an MTS assay. In addition, we measured procollagen type 1 levels and elastase activity, and performed β-galactosidase staining. We then measured the expression levels of phosphatidylinositol 3-kinase/Akt and MMP-related genes by western blot analysis and RT-PCR. Our results revealed that tuna heart extract decreased MMP expression by upregulating tissue inhibitors of metallopro-teinase-1 (TIMP-1) and decreasing elastase activity, thus exerting anti-aging and anti-wrinkle effects by increasing collagen synthesis and promoting skin fibroblast proliferation

  3. Anti-wrinkle effects of a tuna heart H2O fraction on Hs27 human fibroblasts.

    PubMed

    Kim, Young-Min; Jung, Hee-Jin; Choi, Jae-Sue; Nam, Taek-Jeong

    2016-01-01

    With the increase in life expectancy, there is also growing interest in anti-aging treatments and technologies. The development of anti-aging functional drugs for the skin, and foods from natural sources, may offer solutions to this global matter. Aging involves structural, functional and biochemical changes that occur throughout cells and bodily tissues; the amount of hormones secreted from of all human organs, including the skin, decreases over time. Matrix metalloproteinase (MMP) genes (MMP-1 and -8) play an important role in the aging of skin fibroblasts. For example, an increased MMP expression causes accelerated aging and the degradation of skin elasticity-related genes. In the present study, we examined the anti-wrinkle effects of tuna heart extract which are mediated through the inhibition of MMPs in skin cells. Generally, tuna contains high concentrations of selenium and antioxidants, which serve to remove free radicals, and is known to delay skin and body aging. In addition, unsaturated fatty acids in tuna help to maintain the natural glossy look of skin, and increase skin elasticity, providing moisture for dry skin. A recent study confirmed the various bio-effects of boiled tuna extract and muscle. However, bioactivity studies using tuna heart are limited. Thus, in the present study, we obtained extracts and fractions of tuna heart, and examined their effects on Hs27 human fibroblast proliferation using an MTS assay. In addition, we measured procollagen type 1 levels and elastase activity, and performed β-galactosidase staining. We then measured the expression levels of phosphatidylinositol 3-kinase/Akt and MMP-related genes by western blot analysis and RT-PCR. Our results revealed that tuna heart extract decreased MMP expression by upregulating tissue inhibitors of metalloproteinase-1 (TIMP-1) and decreasing elastase activity, thus exerting anti-aging and anti-wrinkle effects by increasing collagen synthesis and promoting skin fibroblast

  4. Structural chromosome abnormalities, increased DNA strand breaks and DNA strand break repair deficiency in dermal fibroblasts from old female human donors

    PubMed Central

    Kalfalah, Faiza; Seggewiß, Sabine; Walter, Regina; Tigges, Julia; Moreno-Villanueva, María; Bürkle, Alexander; Ohse, Sebastian; Busch, Hauke; Boerries, Melanie; Hildebrandt, Barbara; Royer-Pokora, Brigitte; Boege, Fritz

    2015-01-01

    Dermal fibroblasts provide a paradigmatic model of cellular adaptation to long-term exogenous stress and ageing processes driven thereby. Here we addressed whether fibroblast ageing analysed ex vivo entails genome instability. Dermal fibroblasts from human female donors aged 20–67 years were studied in primary culture at low population doubling. Under these conditions, the incidence of replicative senescence and rates of age-correlated telomere shortening were insignificant. Genome-wide gene expression analysis revealed age-related impairment of mitosis, telomere and chromosome maintenance and induction of genes associated with DNA repair and non-homologous end-joining, most notably XRCC4 and ligase 4. We observed an age-correlated drop in proliferative capacity and age-correlated increases in heterochromatin marks, structural chromosome abnormalities (deletions, translocations and chromatid breaks), DNA strand breaks and histone H2AX-phosphorylation. In a third of the cells from old and middle-aged donors repair of X-ray induced DNA strand breaks was impaired despite up-regulation of DNA repair genes. The distinct phenotype of genome instability, increased heterochromatinisation and (in 30% of the cases futile) up-regulation of DNA repair genes was stably maintained over several cell passages indicating that it represents a feature of geroconversion that is distinct from cellular senescence, as it does not encompass a block of proliferation. PMID:25678531

  5. SIRT3 mediates decrease of oxidative damage and prevention of ageing in porcine fetal fibroblasts.

    PubMed

    Xie, Xiaoxian; Wang, Liangliang; Zhao, Binggong; Chen, Yangyang; Li, Jiaqi

    2017-05-15

    Sirtuin 3 (SIRT3) is a mitochondria-specific protein required for the deacetylation of metabolic enzymes and the action of oxidative phosphorylation by acting as a nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase. SIRT3 increases oxidative stress resistance and prevents mitochondrial decay associated with ageing in response to caloric restriction. However, the effects of SIRT3 on oxidative damage and ageing are not well understood. We investigated the physiological functions of porcine SIRT3 on the damage and ageing in porcine fetal fibroblasts (PFFs). Overexpression and knockdown of SIRT3 were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis, respectively. All cells were treated with three different stress reagents 12-o-tetradecanoylphorbol-13-acetate (TPA), methanesulfonic acid methylester (MMS), and tert-butylhydroperoxide (t-BHP), respectively, and then examined by flow cytometry following JC-1 (5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazol-carbocyanine iodide) staining. SIRT3 overexpression enhanced the ability of superoxide dismutase 2 (SOD2) to reduce cellular reactive oxygen species (ROS), which further decreased the damage to the membranes and the organelles of the cells, especially to mitochondria. It inhibited the initial decrease of mitochondrial membrane potential, and prevented the decrease of adenosine triphosphate (ATP) production and activity of Nampt. In contrast, SIRT3 knockdown reduced the ability of SOD2 to increase cellular ROS which was directly correlated with stress-induced oxidative damage and ageing in PFFs. Our findings identify one function of SIRT3 in PFFs was to dampen cytotoxicity, and, therefore, to decrease oxidative damage and attenuate ageing possibly by enhancing the activity of SOD2. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Enhanced Biological Behavior of In Vitro Human Gingival Fibroblasts on Cold Plasma-Treated Zirconia.

    PubMed

    Zheng, Miao; Yang, Yang; Liu, Xiao-Qiang; Liu, Ming-Yue; Zhang, Xiao-Fei; Wang, Xin; Li, He-Ping; Tan, Jian-Guo

    2015-01-01

    To evaluate whether atmospheric-pressure dielectric-barrier-discharge plasma treatment of zirconia enhances its biocompatibility with human gingival fibroblasts. The zirconia disks were divided into four groups and treated using helium atmospheric-pressure dielectric-barrier-discharge plasmas for 30, 60 or 90 s or left untreated. The surface morphology, wettability and chemical elements were analyzed. Fibroblasts density, morphology, morphometry and attachment-related genes expression were measured at different time points from 3 to 72 h. After plasma treatment, the surface morphology and roughness remained the same, while the contact angle decreased from 78.31° to 43.71°, and the surface C/O ratio decreased from 3.17 to 0.89. The surficial areas and perimeters of HGFs were increased two-fold in the treated groups at 3 h. Fibroblasts density increased on treated disks at all time points, especially the ones treated for 60 s. Attachment-related genes in the groups treated for 30 and 60 s were significantly higher at 3 and 24 h. The helium atmospheric-pressure dielectric-barrier-discharge plasma treatment enhances the biological behavior of fibroblasts on zirconia by increasing the expression of attachment-related genes within 24 h and promoting the cell density during longer culture times. Wettability of zirconia, an important physicochemical property, has a vital influence on the cell behaviors.

  7. Ca2+ influx and ATP release mediated by mechanical stretch in human lung fibroblasts.

    PubMed

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-10-10

    One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca(2+) concentration ([Ca(2+)]i) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10-30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca(2+)]i transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca(2+)]i. The stretch-induced [Ca(2+)]i elevation was attenuated in Ca(2+)-free solution. In contrast, the increase of [Ca(2+)]i by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd(3+), ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca(2+)]i elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca(2+) influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  8. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    SciTech Connect

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G.L.; Howlett, Allyn C.

    2014-09-01

    Skin fibroblasts comprise the first barrier of defense against wounds, and tobacco products directly contact the oral cavity. Cultured human dermal fibroblasts were exposed to smokeless tobacco extract (STE), total particulate matter (TPM) from tobacco smoke, or nicotine at concentrations comparable to those found in these extracts for 1 h or 5 h. Differences were identified in pathway-specific genes between treatments and vehicle using qRT-PCR. At 1 h, IL1α was suppressed significantly by TPM and less significantly by STE. Neither FOS nor JUN was suppressed at 1 h by tobacco products. IL8, TNFα, VCAM1, and NFκB1 were suppressed after 5 h with STE, whereas only TNFα and NFκB1 were suppressed by TPM. At 1 h with TPM, secreted levels of IL10 and TNFα were increased. Potentially confounding effects of nicotine were exemplified by genes such as ATF3 (5 h), which was increased by nicotine but suppressed by other components of STE. Within 2 h, TPM stimulated nitric oxide production, and both STE and TPM increased reactive oxygen species. The biological significance of these findings and utilization of the gene expression changes reported herein regarding effects of the tobacco product preparations on dermal fibroblasts will require additional research. - Highlights: • Tobacco product preparations (TPPs) alter gene expression in dermal fibroblasts. • Some immediate early genes critical to the inflammatory process are affected. • Different TPPs produce differential responses in certain pro-inflammatory genes.

  9. Enhanced Biological Behavior of In Vitro Human Gingival Fibroblasts on Cold Plasma-Treated Zirconia

    PubMed Central

    Zheng, Miao; Yang, Yang; Liu, Xiao-Qiang; Liu, Ming-Yue; Zhang, Xiao-Fei; Wang, Xin; Li, He-Ping; Tan, Jian-Guo

    2015-01-01

    Objective To evaluate whether atmospheric-pressure dielectric-barrier-discharge plasma treatment of zirconia enhances its biocompatibility with human gingival fibroblasts. Materials and Methods The zirconia disks were divided into four groups and treated using helium atmospheric-pressure dielectric-barrier-discharge plasmas for 30, 60 or 90 s or left untreated. The surface morphology, wettability and chemical elements were analyzed. Fibroblasts density, morphology, morphometry and attachment-related genes expression were measured at different time points from 3 to 72 h. Results After plasma treatment, the surface morphology and roughness remained the same, while the contact angle decreased from 78.31° to 43.71°, and the surface C/O ratio decreased from 3.17 to 0.89. The surficial areas and perimeters of HGFs were increased two-fold in the treated groups at 3 h. Fibroblasts density increased on treated disks at all time points, especially the ones treated for 60 s. Attachment-related genes in the groups treated for 30 and 60 s were significantly higher at 3 and 24 h. Conclusion The helium atmospheric-pressure dielectric-barrier-discharge plasma treatment enhances the biological behavior of fibroblasts on zirconia by increasing the expression of attachment-related genes within 24 h and promoting the cell density during longer culture times. Wettability of zirconia, an important physicochemical property, has a vital influence on the cell behaviors. PMID:26461253

  10. Bleomycin-induced synthesis of type I procollagen by human lung and skin fibroblasts in culture.

    PubMed

    Clark, J G; Starcher, B C; Uitto, J

    1980-08-13

    Bleomycin is a chemotherapeutic agent sometimes associated with pulmonary fibrosis and skin lesions in patients undergoing treatment. We examined the mechanisms of increased collagen deposition on bleomycin-induced fibrosis by incubating human lung and skin fibroblast cultures with [14C]proline; the synthesis of [14C]hydroxyproline relative to DNA or cell protein was taken as an index of procollagen formation. Procollagen synthesis by lung cells in the presence of 0.1 and 1.0 microgram/ml bleomycin was significantly increased and similar results were obtained with skin fibroblasts. The relative synthesis of genetically distinct types of collagen was measured by isolating the newly synthesized type I and type III procollagens by DEAE-cellulose chromatography. The proportion of type III procollagen of total newly synthesized procollagen in control lung fibroblast cultures was 17.4 +/0 0.6% (mean +/- S.E.) while the corresponding value in cells incubated in 1 microgram/ml bleomycin was 12.5 +/- 0.6% (n = 6, P < 0.01). Similar results were obtained when the ratios of newly synthesized type I and type III collagens were estimated by interrupted polyacrylamide disc gel electrophoresis in sodium dodecyl sulfate after a limited proteolytic digestion with pepsin. The results indicate that the increased procollagen synthesis induced by bleomycin in fibroblast cultures is predominantly directed towards the synthesis of type I procollagen.

  11. Protective role of vitamin E preconditioning of human dermal fibroblasts against thermal stress in vitro.

    PubMed

    Butt, Hira; Mehmood, Azra; Ali, Muhammad; Tasneem, Saba; Anjum, Muhammad Sohail; Tarar, Moazzam N; Khan, Shaheen N; Riazuddin, Sheikh

    2017-09-01

    Oxidative microenvironment of burnt skin restricts the outcome of cell based therapies of thermal skin injuries. The aim of this study was to precondition human dermal fibroblasts with an antioxidant such as vitamin E to improve their survival and therapeutic abilities in heat induced oxidative in vitro environment. Fibroblasts were treated with 100μM vitamin E for 24h at 37°C followed by heat shock for 10min at 51°C in fresh serum free medium. Preconditioning with vitamin E reduced cell injury as demonstrated by decreased expression of annexin-V, cytochrome p450 (CYP450) mediated oxidative reactions, senescence and release of lactate dehydrogenase (LDH) accomplished by down-regulated expression of pro-apoptotic BAX gene. Vitamin E preconditioned cells exhibited remarkable improvement in cell viability, release of paracrine factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), stromal derived factor-1alpha (SDF-1α) and also showed significantly up-regulated levels of PCNA, VEGF, BCL-XL, FGF7, FGF23, FLNβ and Col7α genes presumably through activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. The results suggest that pretreatment of fibroblasts with vitamin E prior to transplantation in burnt skin speeds up the wound healing process by improving the antioxidant scavenging responses in oxidative environment of transplanted burn wounds. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Proteomic identification of cathepsin B and nucleophosmin as novel UVA-targets in human skin fibroblasts.

    PubMed

    Lamore, Sarah D; Qiao, Shuxi; Horn, David; Wondrak, Georg T

    2010-01-01

    Solar UVA exposure plays a causative role in skin photoaging and photocarcinogenesis. Here, we describe the proteomic identification of novel UVA-targets in human dermal fibroblasts following a two-dimensional-difference-gel-electrophoresis (2D-DIGE) approach. Fibroblasts were exposed to noncytotoxic doses of UVA or left untreated, and total protein extracts underwent CyDye-labeling followed by 2D-DIGE/mass-spectrometric identification of differentially expressed proteins, confirmed independently by immunodetection. The protein displaying the most pronounced UVA-induced upregulation was identified as the nucleolar protein nucleophosmin. The protein undergoing the most pronounced UVA-induced downregulation was identified as cathepsin B, a lysosomal cysteine-protease displaying loss of enzymatic activity and altered maturation after cellular UVA exposure. Extensive lysosomal accumulation of lipofuscin-like autofluorescence and osmiophilic material occurred in UVA-exposed fibroblasts as detected by confocal fluorescence microscopy and transmission electron microscopy, respectively. Array analysis indicated UVA-induced upregulation of oxidative stress response gene expression, and UVA-induced loss of cathepsin B enzymatic activity in fibroblasts was suppressed by antioxidant intervention. Pharmacological cathepsin B inhibition using CA074Me mimicked UVA-induced accumulation of lysosomal autofluorescence and deficient cathepsin B maturation. Taken together, these data support the hypothesis that cathepsin B is a crucial target of UVA-induced photo-oxidative stress causatively involved in dermal photodamage through the impairment of lysosomal removal of lipofuscin.

  13. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed

    Speirs, V; Ray, K P; Freshney, R I

    1991-10-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts.

  14. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed Central

    Speirs, V.; Ray, K. P.; Freshney, R. I.

    1991-01-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts. Images Figure 5 PMID:1654985

  15. Conserved genes and pathways in primary human fibroblast strains undergoing replicative and radiation induced senescence.

    PubMed

    Marthandan, Shiva; Menzel, Uwe; Priebe, Steffen; Groth, Marco; Guthke, Reinhard; Platzer, Matthias; Hemmerich, Peter; Kaether, Christoph; Diekmann, Stephan

    2016-07-28

    Cellular senescence is induced either internally, for example by replication exhaustion and cell division, or externally, for example by irradiation. In both cases, cellular damages accumulate which, if not successfully repaired, can result in senescence induction. Recently, we determined the transcriptional changes combined with the transition into replicative senescence in primary human fibroblast strains. Here, by γ-irradiation we induced premature cellular senescence in the fibroblast cell strains (HFF and MRC-5) and determined the corresponding transcriptional changes by high-throughput RNA sequencing. Comparing the transcriptomes, we found a high degree of similarity in differential gene expression in replicative as well as in irradiation induced senescence for both cell strains suggesting, in each cell strain, a common cellular response to error accumulation. On the functional pathway level, "Cell cycle" was the only pathway commonly down-regulated in replicative and irradiation-induced senescence in both fibroblast strains, confirming the tight link between DNA repair and cell cycle regulation. However, "DNA repair" and "replication" pathways were down-regulated more strongly in fibroblasts undergoing replicative exhaustion. We also retrieved genes and pathways in each of the cell strains specific for irradiation induced senescence. We found the pathways associated with "DNA repair" and "replication" less stringently regulated in irradiation induced compared to replicative senescence. The strong regulation of these pathways in replicative senescence highlights the importance of replication errors for its induction.

  16. Protective Effect of Strawberry Extract against Inflammatory Stress Induced in Human Dermal Fibroblasts.

    PubMed

    Gasparrini, Massimiliano; Forbes-Hernandez, Tamara Y; Giampieri, Francesca; Afrin, Sadia; Mezzetti, Bruno; Quiles, Josè L; Bompadre, Stefano; Battino, Maurizio

    2017-01-21

    A protracted pro-inflammatory state is a major contributing factor in the development, progression and complication of the most common chronic pathologies. Fruit and vegetables represent the main sources of dietary antioxidants and their consumption can be considered an efficient tool to counteract inflammatory states. In this context an evaluation of the protective effects of strawberry extracts on inflammatory stress induced by E. coli LPS on human dermal fibroblast cells was performed in terms of viability assays, ROS and nitrite production and biomarkers of oxidative damage of the main biological macromolecules. The results demonstrated that strawberry extracts exerted an anti-inflammatory effect on LPS-treated cells, through an increase in cell viability, and the reduction of ROS and nitrite levels, and lipid, protein and DNA damage. This work showed for the first time the potential health benefits of strawberry extract against inflammatory and oxidative stress in LPS-treated human dermal fibroblast cells.

  17. Immediate induction of heat shock proteins is not protective against cryopreservation in normal human fibroblasts.

    PubMed

    Park, S J; Choi, H R; Nam, K M; Na, J I; Huh, C H; Park, K C

    2013-01-01

    Heat shock proteins (HSPs) were first identified as proteins whose synthesis was enhanced by stresses, such as increased temperature. HSPs can protect cells from various cytotoxic factors by stabilizing proteins. Thus, it could be hypothesized that heat induced HSPs can provide protective effects against cryopreservation-induced cell death. The aim of this study was to determine whether induction of HSPs can increase the cell viability of normal human fibroblasts after cryopreservation. Cytotoxic effects of heat treatment were tested and the induction of HSPs was assessed by examining time-dependent HSP expression. A cell counting method using fluorescence microscopy was used to determine the viability of cells. In addition, the effects of geranylgeranylacetone were evaluated in terms of HSP expression and cytoskeleton changes. The results of this study showed that immediate induction of HSPs does not protect normal human fibroblasts against cryopreservation-induced cell death possibly by inducing cytoskeleton changes.

  18. Hyaluronic acid abrogates ethanol-dependent inhibition of collagen biosynthesis in cultured human fibroblasts

    PubMed Central

    Donejko, Magdalena; Przylipiak, Andrzej; Rysiak, Edyta; Miltyk, Wojciech; Galicka, Elżbieta; Przylipiak, Jerzy; Zaręba, Ilona; Surazynski, Arkadiusz

    2015-01-01

    Introduction The aim of the study was to evaluate the effect of ethanol on collagen biosynthesis in cultured human skin fibroblasts, and the role of hyaluronic acid (HA) in this process. Regarding the mechanism of ethanol action on human skin fibroblasts we investigated: expression of β1 integrin and insulin-like growth factor 1 receptor (IGF-IR), signaling pathway protein expression: mitogen-activated protein kinases (MAPKs), protein kinase B (Akt), nuclear factor kappa B (NF-κB) transcription factor, cytotoxicity assay and apoptosis, metalloproteinase activity, as well as the influence of HA on these processes. Materials and methods Collagen biosynthesis, activity of prolidase, DNA biosynthesis, and cytotoxicity were measured in confluent human skin fibroblast cultures that have been treated with 25, 50, and 100 mM ethanol and with ethanol and 500 µg/mL HA. Western blot analysis and zymography were performed to evaluate expression of collagen type I, β1 integrin receptor, IGF-IR, NF-κB protein, phospho-Akt protein, kinase MAPK, caspase 9 activity, and matrix metalloproteinases (MMP-9 and MMP-2). Results Ethanol in a dose-dependent manner lead to the impairment of collagen biosynthesis in fibroblast cultures through decreasing prolidase activity and expression of β1 integrin and IGF-IR. This was accompanied by an increased cytotoxicity, apoptosis and lowered expression of the signaling pathway proteins induced by β1 integrin and IGF-IR, that is, MAPK (ERK1/2) kinases. The lowered amount of synthesized collagen and prolidase activity disturbance may also be due to the activation of NF-κB transcription factor, which inhibits collagen gene expression. It suggests that the decrease in fibroblast collagen production may be caused by the disturbance in its biosynthesis but not degradation. The application of HA has a protective effect on disturbances caused by the examined substances. It seems that regulatory mechanism of ethanol-induced collagen aberration take

  19. DNA damage in human skin fibroblasts exposed to UVA light used in clinical PUVA treatment

    SciTech Connect

    Bredberg, A.

    1981-06-01

    Human skin fibroblasts were irradiated with a clinically used UVA light source. The doses (1.1 and 3 J/cm2) were similar to those reaching the dermis during clinical PUVA treatment of psoriasis. DNA strand breaks, as determined by alkaline elution, were formed in a dose-dependent way and disappeared within 1 hr of postincubation at 37 degrees C. These findings have clinical implications since UVA-induced DNA damage may be accompanied by mutagenic and tumor promoting effects.

  20. DNA repair in human fibroblasts treated with a combination of chemicals

    SciTech Connect

    Ahmed, F.E.; Setlow, R.B.

    1981-07-01

    Excision repair of DNA damage was measured by the photolysis of bromodeoxyuridine incorporated during repair in normal human and xeroderma pigmentosum group C fibroblasts (XP C) treated with a combination of the carcinogens N-acetoxy-2-acetylamino fluorene (AAAF), and 4-nitroquinoline 1-oxide (4NQO). Repair was additive in normal and XP C cells treated with AAAF plus 4NQO, indicating that there are different rate limiting steps for removal of 4NQO and AAAF lesions.

  1. Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts.

    PubMed

    Chang, Y C; Hu, C C; Tseng, T H; Tai, K W; Lii, C K; Chou, M Y

    2001-09-01

    Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.

  2. LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

    PubMed

    Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

    2015-02-15

    Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

  3. Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

    PubMed

    Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

    2008-11-01

    This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

  4. The Role of Human Adult Peripheral and Umbilical Cord Blood Platelet-Rich Plasma on Proliferation and Migration of Human Skin Fibroblasts.

    PubMed

    Hashemi, Seyedeh-Sara; Mahmoodi, Mahdokht; Rafati, Ali Reza; Manafi, Farzad; Mehrabani, Davood

    2017-05-01

    Wound healing is a complex and dynamic process following damage in tissue structures. Due to extensive skin damage caused by burn injuries, this study determined the role of human adult peripheral and umbilical cord blood platelet-rich plasma on proliferation and migration in human skin fibroblasts. Platelet-rich plasma (5, 10, 15, 20 and 50% PRP) from human umbilical cord blood and adult peripheral blood were provided and added to fibroblasts cultured from a human skin sample. Migration and proliferation of fibroblasts were assessed in comparison to 10% FBS and by the fibroblast responses to a concentration gradient. All components of the umbilical cord blood PRP significantly stim