Sample records for ageing human fibroblasts

  1. 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.

  2. Age-related changes in cyclic phosphatidic acid-induced hyaluronic acid synthesis in human fibroblasts.

    PubMed

    Sano, Katsura; Gotoh, Mari; Dodo, Kyoko; Tajima, Noriaki; Shimizu, Yoshibumi; Murakami-Murofushi, Kimiko

    2018-01-01

    Hyaluronic acid is a major component of the extracellular matrix, which is important for skin hydration. As aging brings skin dehydration, we aimed to clarify the mRNA expression of hyaluronic acid-related proteins in human skin fibroblasts from donors of various ages (range 0.7-69 years). Previously, we reported that cyclic phosphatidic acid (cPA), a unique phospholipid mediator, stimulated the expression of HAS2 and increased hyaluronic acid synthesis in human skin fibroblasts (donor age: 3 days). In this study, we measured the mRNA expression of hyaluronic acid-related proteins: hyaluronan synthase (HAS) 1-3, hyaluronidase-1, -2, and hyaluronic acid-binding protein (versican). In addition, we tested whether cPA could increase hyaluronic acid synthesis in skin fibroblasts derived from donors of various ages. The expression of HAS1, 3, hyaluronidase-1, and -2 did not change with aging. However, the mRNA expression of versican decreased with aging. Although it is thought that the amount of hyaluronic acid in the dermis decreases with aging, the mRNA expression of HAS2 was increased. But the amount of hyaluronic acid secreted by fibroblasts did not increase with aging. This suggests that the activity and/or protein expression of HAS2 decrease with aging. Furthermore, we observed that cPA caused the increase of hyaluronic acid synthesis at any age, and this effect was increased with aging. These results suggest that aging made the fibroblasts more sensitive to cPA treatment. Therefore, cPA represents a suitable candidate for the health maintenance and improvement of the skin by increasing the level of hyaluronic acid in the dermis.

  3. AGEs trigger autophagy in diabetic skin tissues and fibroblasts.

    PubMed

    Sun, Kan; Wang, Wei; Wang, Chuan; Lao, Guojuan; Liu, Dan; Mai, Lifang; Yan, Li; Yang, Chuan; Ren, Meng

    2016-03-11

    Accumulation of advanced glycation end products (AGEs) contributes to the development of diabetic ulcers. Recent evidence indicates that AGEs administration enhanced autophagy in many cell types. As a positive trigger of autophagy, the effect of AGEs on autophagy in skin tissues and fibroblasts remains unknown. Skin tissues were isolated from Spreqne-Dawley rats and immunohistochemical staining was performed to analyze the location of LC3 and FOXO1 in skin tissues. Then primary cultured foreskin fibroblast cells with treated with AGEs and the effect of AGEs on autophagy was investigated. Protein level expressions of LC3, Beclin-1 and FOXO1 in fibroblasts were analyzed by Western blotting. Autophagic flux is detected with autophagy inhibitor chloroquine and mRFP-GFP-LC3 tandem construct. Compared with skin from normal rats, immunohistochemical staining shows a predominant LC3 localization in fibroblasts cytoplasm in diabetic rats. Elevated expression of FOXO1 also existed in diabetic rats dermis fibroblasts when compared with normal rats in immunohistochemical analysis. In human skin fibroblasts cells, AGEs administration stimulated the autophagy related LC3-II/LC3-I and Beclin-1 expressions and increased autophagy flux. In mRFP-GFP-LC3 puncta formation assays, both autolysosome and autophagosome were increased in human fibroblasts after treatment with AGEs. Fibroblasts exposed to AGEs also have increased FOXO1 expression compared with control group. AGEs could induce autophagy at least in part via regulating the FOXO1 activity in diabetic skin tissues and fibroblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. 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...

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tashiro, Kanae; Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka; Shishido, Mayumi

    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. Westernmore » 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.« less

  6. 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.

  7. Aging Alters Functionally Human Dermal Papillary Fibroblasts but Not Reticular Fibroblasts: A New View of Skin Morphogenesis and Aging

    PubMed Central

    Mine, Solène; Fortunel, Nicolas O.; Pageon, Hervé; Asselineau, Daniel

    2008-01-01

    Understanding the contribution of the dermis in skin aging is a key question, since this tissue is particularly important for skin integrity, and because its properties can affect the epidermis. Characteristics of matched pairs of dermal papillary and reticular fibroblasts (Fp and Fr) were investigated throughout aging, comparing morphology, secretion of cytokines, MMPs/TIMPs, growth potential, and interaction with epidermal keratinocytes. We observed that Fp populations were characterized by a higher proportion of small cells with low granularity and a higher growth potential than Fr populations. However, these differences became less marked with increasing age of donors. Aging was also associated with changes in the secretion activity of both Fp and Fr. Using a reconstructed skin model, we evidenced that Fp and Fr cells do not possess equivalent capacities to sustain keratinopoiesis. Comparing Fp and Fr from young donors, we noticed that dermal equivalents containing Fp were more potent to promote epidermal morphogenesis than those containing Fr. These data emphasize the complexity of dermal fibroblast biology and document the specific functional properties of Fp and Fr. Our results suggest a new model of skin aging in which marked alterations of Fp may affect the histological characteristics of skin. PMID:19115004

  8. Human serum reduces mitomycin-C cytotoxicity in human tenon's fibroblasts.

    PubMed

    Crowston, Jonathan G; Wang, Xiao Y; Khaw, Peng T; Zoellner, Hans; Healey, Paul R

    2006-03-01

    To determine the effect of human serum factors on mitomycin-C (MMC) cytotoxicity in cultured human subconjunctival Tenon's capsule fibroblasts. Fibroblast monolayers were treated with 5-minute applications of mitomycin-C (0.4 mg/mL) and incubated in culture medium with or without additional human serum. Fibroblast apoptosis was quantified by direct cell counts based on nuclear morphology, flow cytometry with annexin-V/propidium iodide, and a lactate dehydrogenase release assay. The number of viable fibroblasts and fibroblast proliferation were measured with a colorimetric MTT assay and by bromodeoxyuridine (BrdU) labeling. Mitomycin-C induced significant levels of fibroblast apoptosis. The addition of human serum resulted in a 40% reduction in MMC-induced fibroblast apoptosis (range, 31.3%-55.3%; P = 0.021) as determined by nuclear morphology and a 32.4% reduction measured by annexin-V/PI. There was a corresponding dose-dependent increase in the number of viable fibroblasts. Serum did not restore proliferation in MMC-treated fibroblasts. Factors present in human serum reduce MMC cytotoxicity in cultured human Tenon's fibroblasts. Human serum increased the number of viable fibroblasts by inhibiting MMC-induced fibroblast apoptosis. Serum factors access aqueous humor after trabeculectomy and may therefore influence the clinical outcome of MMC treatment.

  9. 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

  10. Lidocaine impairs proliferative and biosynthetic functions of aged human dermal fibroblasts

    PubMed Central

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

    2016-01-01

    Background The aged are at increased risk of postoperative wound healing complications. Since local anesthetics are commonly infiltrated into the dermis of surgical wounds, we sought to determine if local anesthetics adversely affect proliferative and biosynthetic functions of dermal fibroblasts. We also evaluated the effect of local anesthetics on IGF-1 and TGF-ß1, growth factors that are important regulators of wound healing. Methods Human dermal fibroblasts from aged and young donors (HFB) 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 the 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-ß1. Lastly, concurrent exposure to lidocaine and IGF-1 or TGF-ß1 were evaluated for their effects on proliferation and expression of dermal collagens, respectively. Results Lidocaine and mepivacaine inhibited proliferation in aged HFB (for lidocaine 88% of control, 95%CI 80%-98%, p=0.009 and for mepivacaine 90% of control, 95%CI 81%-99%, p=0.032), but not in young HFB. Ropivicaine and bupivicaine did not inhibit proliferation. Due to the clinical utility of lidocaine relative to mepivicaine, we focused on lidocaine. Lidocaine decreased proliferation in aged HFB, which was abrogated by IGF-1. Lidocaine inhibited transcripts for IGF-1 and IGF1R in fibroblasts from aged donors (IGF-1, log2 fold-change −1.25 {42% of control, 95%CI 19%-92%, p=0.035} and IGF1R, log2 fold-change of −1.00 {50% of control, 95%CI 31%-81%, p=0.014}). In contrast, lidocaine did not effect 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 fold-change −1.28 {41% of control, 95%CI 20%-83%, p=0.022} in aged HFB and log2 fold-change −1.60 {33% of

  11. AGE AND MULTIPLICATION OF FIBROBLASTS.

    PubMed

    Carrel, A; Ebeling, A H

    1921-11-30

    Pure cultures of fibroblasts displayed marked differences in their activity in the plasma of young, middle aged, and old chickens. The rate of cell multiplication varied in inverse ratio to the age of the animal from which the plasma was taken. There was a definite relation between the age of the animal and the amount of new tissue produced in its plasma in a given time (Text-figs. 1 to 10). The chart obtained by plotting the rate of cell proliferation in ordinates, and the age of the animal in abscissae, showed that the rate of growth decreased more quickly than the age increased (Text-fig. 12). The decrease in the rate of growth was 50 per cent during the first 3 years of life, while in the following 6 years it was only 30 per cent. When the duration of the life of the cultures in the four plasmas was compared, a curve was obtained which showed about the same characteristics (Text-fig. 11). The duration of life of the fibroblasts in vitro varied in inverse ratio to the age of the animal, and decreased more quickly than the age increased. As the differences in the amount of new tissue produced in the plasma of young, middle aged, and old chickens were large, the growth of a pure culture of fibroblasts could be employed as a reagent for detecting certain changes occurring in the plasma under the influence of age. But the method possesses the necessary accuracy only when it is used as has already been described, and by technicians thoroughly trained in the details of its application. A comparative study of the growth of fibroblasts in media containing no serum, and serum under low and high concentrations, was made in order to ascertain whether the decreasing rate of cell multiplication was due to the loss of an accelerating factor, or to the increase See PDF for Structure of an inhibiting one. In high and low concentrations of the serum of young animals, no difference in the rate of multiplication of fibroblasts was observed. This showed that the serum of an

  12. 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.

  13. Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors.

    PubMed

    Figueroa, R; Lindenmaier, H; Hergenhahn, M; Nielsen, K V; Boukamp, P

    2000-06-01

    The life span of normal fibroblasts in vitro (Hayflick limit) depends on donor age, and telomere shortening has been proposed as a potential mechanism. By quantitative fluorescence in situ hybridization and Southern blot analysis, we show progressive telomere loss to about 5 kb mean telomere restriction fragment length in fibroblasts from two adult donors within 40 population doublings, whereas in fibroblasts from two infant donors, telomere erosion is reduced, leaving a mean telomere restriction fragment length of approximately 7 kb at senescence (after approximately 60 population doublings). Aging of fibroblasts from both infant and adult donors was not accompanied by chromosomal abnormalities but was correlated with increased telomere repeat-binding factor 2 expression at both the protein and transcriptional level.

  14. Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture.

    PubMed

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

    2013-01-01

    The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high-passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high-passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti-skin-ageing strategies. © 2013 John Wiley & Sons A/S.

  15. Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

    PubMed

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

    2014-12-01

    The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley

  16. The relationship between in vitro cellular aging and in vivo human age.

    PubMed Central

    Schneider, E L; Mitsui, Y

    1976-01-01

    Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging. PMID:1068470

  17. Partial uncoupling of oxidative phosphorylation induces premature senescence in human fibroblasts and yeast mother cells.

    PubMed

    Stöckl, Petra; Zankl, Christina; Hütter, Eveline; Unterluggauer, Hermann; Laun, Peter; Heeren, Gino; Bogengruber, Edith; Herndler-Brandstetter, Dietmar; Breitenbach, Michael; Jansen-Dürr, Pidder

    2007-09-15

    The mitochondrial theory of aging predicts that functional alterations in mitochondria leading to reactive oxygen species (ROS) production contribute to the aging process in most if not all species. Using cellular senescence as a model for human aging, we have recently reported partial uncoupling of the respiratory chain in senescent human fibroblasts. In the present communication, we address a potential cause-effect relationship between impaired mitochondrial coupling and premature senescence. Chronic exposure of human fibroblasts to the chemical uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) led to a temporary, reversible uncoupling of oxidative phosphorylation. FCCP inhibited cell proliferation in a dose-dependent manner, and a significant proportion of the cells entered premature senescence within 12 days. Unexpectedly, chronic exposure of cells to FCCP led to a significant increase in ROS production, and the inhibitory effect of FCCP on cell proliferation was eliminated by the antioxidant N-acetyl-cysteine. However, antioxidant treatment did not prevent premature senescence, suggesting that a reduction in the level of oxidative phosphorylation contributes to phenotypical changes characteristic of senescent human fibroblasts. To assess whether this mechanism might be conserved in evolution, the influence of mitochondrial uncoupling on replicative life span of yeast cells was also addressed. Similar to our findings in human fibroblasts, partial uncoupling of oxidative phsophorylation in yeast cells led to a substantial decrease in the mother-cell-specific life span and a concomitant incrase in ROS, indicating that life span shortening by mild mitochondrial uncoupling may represent a "public" mechanism of aging.

  18. Fibroblast heterogeneity: implications for human disease.

    PubMed

    Lynch, Magnus D; Watt, Fiona M

    2018-01-02

    Fibroblasts synthesize the extracellular matrix of connective tissue and play an essential role in maintaining the structural integrity of most tissues. Researchers have long suspected that fibroblasts exhibit functional specialization according to their organ of origin, body site, and spatial location. In recent years, a number of approaches have revealed the existence of fibroblast subtypes in mice. Here, we discuss fibroblast heterogeneity with a focus on the mammalian dermis, which has proven an accessible and tractable system for the dissection of these relationships. We begin by considering differences in fibroblast identity according to anatomical site of origin. Subsequently, we discuss new results relating to the existence of multiple fibroblast subtypes within the mouse dermis. We consider the developmental origin of fibroblasts and how this influences heterogeneity and lineage restriction. We discuss the mechanisms by which fibroblast heterogeneity arises, including intrinsic specification by transcriptional regulatory networks and epigenetic factors in combination with extrinsic effects of the spatial context within tissue. Finally, we discuss how fibroblast heterogeneity may provide insights into pathological states including wound healing, fibrotic diseases, and aging. Our evolving understanding suggests that ex vivo expansion or in vivo inhibition of specific fibroblast subtypes may have important therapeutic applications.

  19. 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

  20. A comparison of human and porcine acellularized dermis: interactions with human fibroblasts in vitro.

    PubMed

    Armour, Alexis D; Fish, Joel S; Woodhouse, Kimberly A; Semple, John L

    2006-03-01

    Dermal substitutes derived from xenograft materials require elaborate processing at a considerable cost. Acellularized porcine dermis is a readily available material associated with minimal immunogenicity. The objective of this study was to evaluate acellularized pig dermis as a scaffold for human fibroblasts. In vitro methods were used to evaluate fibroblast adherence, proliferation, and migration on pig acellularized dermal matrix. Acellular human dermis was used as a control. Pig acellularized dermal matrix was found to be inferior to human acellularized dermal matrix as a scaffold for human fibroblasts. Significantly more samples of human acellularized dermal matrix (83 percent, n = 24; p < 0.05) demonstrated fibroblast infiltration below the cell-seeded surface than pig acellularized dermal matrix (31 percent, n = 49). Significantly more (p < 0.05) fibroblasts infiltrated below the surface of human acellularized dermal matrix (mean, 1072 +/- 80 cells per section; n = 16 samples) than pig acellularized dermal matrix (mean, 301 +/- 48 cells per section; n = 16 samples). Fibroblasts migrated significantly less (p < 0.05) distance from the cell-seeded pig acellularized dermal matrix surface than in the human acellularized dermal matrix (78.8 percent versus 38.3 percent cells within 150 mum from the surface, respectively; n = 5). Fibroblasts proliferated more rapidly (p < 0.05) on pig acellularized dermal matrix (n = 9) than on the human acellularized dermal matrix (7.4-fold increase in cell number versus 1.8-fold increase, respectively; n = 9 for human acellularized dermal matrix). There was no difference between the two materials with respect to fibroblast adherence (8120 versus 7436 average adherent cells per section, for pig and human acellularized dermal matrix, respectively; n = 20 in each group; p > 0.05). Preliminary findings suggest that substantial differences may exist between human fibroblast behavior in cell-matrix interactions of porcine and human

  1. Lung Fibroblasts, Aging, and Idiopathic Pulmonary Fibrosis.

    PubMed

    Pardo, Annie; Selman, Moisés

    2016-12-01

    Idiopathic pulmonary fibrosis (IPF) is an aging-associated, progressive, and irreversible lung disease of unknown etiology, elusive pathogenesis, and very limited therapeutic options. The hallmarks of IPF are aberrant activation of alveolar epithelial cells and accumulation of fibroblasts and myofibroblasts along with excessive production of extracellular matrix. The linkage of aging with this disorder is uncertain, but a number of changes associated with aging, including telomere attrition, cell senescence, and mitochondrial dysfunction, have been revealed in IPF lungs. Also, aging seems to confer a profibrotic phenotype upon fibroblasts and to increase the severity of the fibrogenic response in non-IPF fibrotic lung disorders. Better knowledge of the pathophysiological mechanisms linking aging to IPF will advance understanding of its pathogenesis and may provide new therapeutic windows to treatment of this devastating disease.

  2. CXCL12 overexpression and secretion by aging fibroblasts enhance human prostate epithelial proliferation in vitro.

    PubMed

    Begley, Lesa; Monteleon, Christine; Shah, Rajal B; Macdonald, James W; Macoska, Jill A

    2005-12-01

    The direct relationship between the aging process and the incidence and prevalence of both benign prostatic hyperplasia (BPH) and prostate cancer (PCa) implies that certain risk factors associated with the development of both diseases increase with the aging process. In particular, both diseases share an overly proliferative phenotype, suggesting that mechanisms that normally act to suppress cellular proliferation are disrupted or rendered dysfunctional as a consequence of the aging process. We propose that one such mechanism involves changes in the prostate microenvironment, which 'evolves' during the aging process and disrupts paracrine interactions between epithelial and associated stromal fibroblasts. We show that stromal fibroblasts isolated from the prostates of men 63-81 years of age at the time of surgery express and secrete higher levels of the CXCL12 chemokine compared with those isolated from younger men, and stimulate CXCR4-mediated signaling pathways that induce cellular proliferation. These studies represent an important first step towards a mechanistic elucidation of the role of aging in the etiology of benign and malignant prostatic diseases.

  3. Heat shock protein 47 expression in aged normal human fibroblasts: modulation by Salix alba extract.

    PubMed

    Nizard, Carine; Noblesse, Emmanuelle; Boisdé, Cécille; Moreau, Marielle; Faussat, Anne-Marie; Schnebert, Sylvianne; Mahé, Christian

    2004-06-01

    Heat shock protein (HSP) 47 is a specific chaperone of procollagen. This heat shock protein is responsible for the correct three-dimensional organization of procollagen and its control-quality prior secretion. The aim of the study is to evaluate the level of HSP 47 in aged, photoaged, and senescent fibroblasts and its modulation by a plant extract (Salix alba). The level of HSP 47 and/or procollagen expression in fibroblasts was measured by real-time RT-PCR (mRNA transcripts) and by flow cytometry (immunochemistry technique for measurement of arbitrary fluorescence intensity). Immunochemistry techniques and confocal microscopy were used to visualize the cellular localization of HSP 47 and procollagen. These parameters were compared with different age donors, nonsenescent, and senescent fibroblasts. Fibroblasts were irradiated by a noncytotoxic dose of UVA (6 J/cm(2)), and HSP 47 level was evaluated. S. alba extract was tested for its capacity to modulate HSP 47 expression. Colocalization of HSP 47 and procollagen was shown by confocal microscopy, indicating that HSP 47 could play a role of procollagen molecular chaperone in the cellular model. It was also shown that the HSP 47 level is decreased in old-donor cells, senescent, and irradiated cells. This decrease can be modulated by a S. alba extract (polyphenols rich) in a dose-dependent manner. The evaluation of HSP 47 expression in the experimental conditions can lead to a new approach of aging and photoaging, pointing out the implication of this chaperone in these pathophysiologic phenomena. Modulation of HSP 47 expression by this family of molecules could be of cosmetic and/or dermatologic interest.

  4. 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

  5. Effects of Aging and Cyclosporin A on Collagen Turnover in Human Gingiva

    PubMed Central

    Gagliano, N; Costa, F; Tartaglia, G.M; Pettinari, L; Grizzi, F; Sforza, C; Portinaro, N; Gioia, M; Annoni, G

    2009-01-01

    Background: We aimed at characterizing the aging gingiva analyzing: i) collagen content and turnover in human gingival tissues and fibroblasts obtained from healthy young and aging subjects. ii) the effect of cyclosporin A administration in human cultured gingival fibroblasts obtained from aging compared to young subjects. Methods: Morphological analysis was performed on haematoxylin-eosin and Sirius red stained paraffin-embedded gingival biopsies from young and aging healthy subjects. The expression of the main genes and proteins involved in collagen turnover were determined by real time PCR, dot blot and SDS-zymography on cultured young and aging gingival fibroblasts, and after cyclosporin A administration. Results: Our results suggest that in healthy aged people, gingival connective tissue is characterized by a similar collagen content and turnover. Collagen turnover pathways are similarly affected by cyclosporin A treatment in young and aging gingival fibroblasts. Conclusions: Cyclosporin A administration affects gingival collagen turnover pathways in young and aging fibroblasts at the same extent, suggesting that during aging cyclosporin A administration is not related to relevant collagen turnover modifications. PMID:20148173

  6. 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.

  7. 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.

  8. Intracrine sex steroid synthesis and signaling in human epidermal keratinocytes and dermal fibroblasts.

    PubMed

    Pomari, Elena; Dalla Valle, Luisa; Pertile, Paolo; Colombo, Lorenzo; Thornton, M Julie

    2015-02-01

    Peripheral intracrine sex steroid synthesis from adrenal precursors dehydroepiandrosterone (DHEA) and DHEA-sulfate has evolved in humans. We sought to establish if there are differences in intracrine, paracrine, and endocrine regulation of sex steroids by primary cultures of human skin epidermal keratinocytes and dermal fibroblasts. Microarray analysis identified multifunctional genes modulated by steroids, quantitative RT-PCR (qRT-PCR) mRNA expression, enzymatic assay aromatase activity, scratch assay cell migration, immunocytochemistry α-smooth muscle actin (α-SMA), and collagen gel fibroblast contraction. All steroidogenic components were present, although only keratinocytes expressed the organic anion organic anion transporter protein (OATP) 2B1 transporter. Both expressed the G-protein-coupled estrogen receptor (GPER1). Steroids modulated multifunctional genes, up-regulating genes important in repair and aging [angiopoietin-like 4 (ANGPTL4), chemokine (C-X-C motif) ligand 1 (CXCL1), lamin B1 (LMNB1), and thioredoxin interacting protein (TXNIP)]. DHEA-sulfate (DHEA-S), DHEA, and 17β-estradiol stimulated keratinocyte and fibroblast migration at early (4 h) and late (24-48 h) time points, suggesting involvement of genomic and nongenomic signaling. Migration was blocked by aromatase and steroid sulfatase (STS) inhibitors confirming intracrine synthesis to estrogen. Testosterone had little effect, implying it is not an intermediate. Steroids stimulated fibroblast contraction but not α-SMA expression. Mechanical wounding reduced fibroblast aromatase activity but increased keratinocyte activity, amplifying the bioavailability of intracellular estrogen. Cultured fibroblasts and keratinocytes provide a biologically relevant model system to investigate the complex pathways of sex steroid intracrinology in human skin. © FASEB.

  9. Dimethylaminoethanol affects the viability of human cultured fibroblasts.

    PubMed

    Gragnani, Alfredo; Giannoccaro, Fabiana Bocci; Sobral, Christiane S; Moraes, A A F; França, Jeronimo P; Ferreira, A T; Ferreira, Lydia Masako

    2007-01-01

    In clinical practice, dimethylaminoethanol (DMAE) has been used in the fight against wrinkles and flaccidity in the cervicofacial region. The firming action of DMAE is explained by the fact that its molecule, considered to be a precursor of acetylcholine, alters muscle contraction. However, no experimental studies have confirmed this theory. Because the actual mechanism of DMAE action was not defined and there were no references in the literature regarding its direct action on fibroblasts, this study was performed to evaluate the direct action of DMAE on cultured human fibroblasts. Human fibroblasts obtained from discarded fragments of total skin from patients undergoing plastic or reconstructive surgical procedures performed within the Plastic Surgery Division at the Federal University of São Paulo were used for this study. The explant technique was used. The culture medium was supplemented with different concentrations of DMAE on the fourth cell passage, and the cell proliferation rate, cytosolic calcium levels, and cell cycle were evaluated. Statistical analysis was performed using analysis of variance (ANOVA) followed by a Newman-Keuls test for multiple comparisons. A decrease in fibroblast proliferation was associated with an increase in DMAE concentration. A longer treatment time with trypsin was required for the groups treated with DMAE in a dose-dependent manner. In the presence of DMAE, cytosolic calcium increased in a dose-dependent manner. Apoptosis also increased in groups treated with DMAE. Dimethylaminoethanol reduced the proliferation of fibroblasts, increased cytosolic calcium, and changed the cell cycle, causing an increase in apoptosis in cultured human fibroblasts.

  10. Effects of neuropeptides on human lung fibroblast proliferation and chemotaxis.

    PubMed

    Harrison, N K; Dawes, K E; Kwon, O J; Barnes, P J; Laurent, G J; Chung, K F

    1995-02-01

    An increase in subepithelial mesenchymal cells and associated connective tissue is a feature of bronchial asthma. We determined whether neuropeptides could modulate fibroblast activity, particularly with respect to proliferation and chemotaxis. Human lung fibroblasts were cultured with neurokinin A (NKA), substance P (SP), vasoactive intestinal peptide (VIP), and calcitonin-gene-related peptide (CGRP). After 48 h, fibroblast proliferation was measured by a colorimetric assay based on the uptake and subsequent release of methylene blue. The chemotactic response to neuropeptides was determined with the use of a modified Boyden chamber. Both NKA and SP (10(-7)-10(-4) M) stimulated human lung fibroblast proliferation in HFL1 and IMR-90 fibroblasts. VIP and CGRP had no effect on fibroblast proliferation. NKA alone stimulated fibroblast chemotaxis maximally at 10(-10) M. Neutral endopeptidase (NEP) activity of 0.52 and 5.2 pmol/10(6) cells was assayed in IMR-90 and Hs68 fibroblasts, respectively. Phosphoramidon (5 x 10(-6)-10(-5) M), an NEP inhibitor, enhanced fibroblast proliferation in a dose-dependent manner. Thus neuropeptides have the potential to cause activation of mesenchymal cells, and neuropeptide release may contribute to the structural abnormalities observed in asthmatic airways.

  11. Peptide Regulation of Skin Fibroblast Functions during Their Aging In Vitro.

    PubMed

    Lin'kova, N S; Drobintseva, A O; Orlova, O A; Kuznetsova, E P; Polyakova, V O; Kvetnoy, I M; Khavinson, V Kh

    2016-05-01

    The effect peptides KE, KED, AED and AEDG on proliferation (Ki-67), regeneration and aging (CD98hc), apoptosis (caspase-3), and extracellular matrix remodeling (MMP-9) in skin fibroblasts during their aging in culture were studied by immunofluorescent confocal microscopy. All studied peptides inhibited MMP-9 synthesis that increases during aging of skin fibroblasts and enhanced the expression of Ki-67 and CD98hc that are less intensively synthesized during cell aging. Peptides AED and AEDG suppressed caspase-dependent apoptosis that increases during aging of cell cultures.

  12. 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.

  13. Mitochondrial Aging Defects Emerge in Directly Reprogrammed Human Neurons due to Their Metabolic Profile.

    PubMed

    Kim, Yongsung; Zheng, Xinde; Ansari, Zoya; Bunnell, Mark C; Herdy, Joseph R; Traxler, Larissa; Lee, Hyungjun; Paquola, Apua C M; Blithikioti, Chrysanthi; Ku, Manching; Schlachetzki, Johannes C M; Winkler, Jürgen; Edenhofer, Frank; Glass, Christopher K; Paucar, Andres A; Jaeger, Baptiste N; Pham, Son; Boyer, Leah; Campbell, Benjamin C; Hunter, Tony; Mertens, Jerome; Gage, Fred H

    2018-05-29

    Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain, but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging, in contrast to iPSC differentiation. Here, we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression, impaired axonal mitochondrial morphologies, lower mitochondrial membrane potentials, reduced energy production, and increased oxidized proteins levels. In contrast, the fibroblasts from which iNs were generated show only mild age-dependent changes, consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed, OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. Effects of polyhexamethylene guanidine phosphate on human gingival fibroblasts.

    PubMed

    Vitt, Anton; Slizen, Veronica; Boström, Elisabeth A; Yucel-Lindberg, Tülay; Kats, Anna; Sugars, Rachael V; Gustafsson, Anders; Buhlin, Kåre

    2017-10-01

    Polyhexamethylene guanidine phosphate (PHMG-P) was compared to chlorhexidine (CHX) in order to determine potential cytotoxic and immune-modulatory effects on human gingival fibroblasts. Cytotoxic effects of PHMG-P and CHX on human gingival fibroblasts were assessed using cell viability assay at various time points and concentrations. The effects of PHMG-P and CHX on the secretion of prostaglandin (PG) E 2 , interleukin (IL)-6, IL-8 and matrix metalloproteinase (MMP)-1 by non-stimulated or IL-1β stimulated fibroblasts were evaluated by enzyme-linked immunosorbent assays. PHMG-P concentration 0.00009% led to the total loss of fibroblast viability within 24 h, whereas inhibition of fibroblast viability by CHX occurred at significantly higher concentrations of 0.0009% (p < .001). Short-term exposure to 0.005% PHMG-P led to loss of fibroblast viability after 5 min, whilst cells exposed to 0.005% CHX survived 30 min of treatment (p < .001). IL-1β stimulation induced an inflammatory response with a significant increase in the secretion of PGE 2 , IL-6, IL-8 and MMP-1. Treatment of IL-1β stimulated fibroblasts in combination with PHMG-P or CHX at concentrations of 0.000045 or 0.0.00009% resulted in significantly decreased PGE 2 , IL-6, IL-8 and MMP-1 levels. PHMG-P or CHX alone did not affect the baseline secretion of PGE 2 , IL-6, IL-8 or MMP-1 by gingival fibroblasts. Cytotoxic effects on gingival fibroblasts were triggered by both PHMG-P and CHX at concentrations below those used in clinical practice. The tested antiseptics did not cause inflammation and reduced IL-1β-induced secretion of inflammatory mediators and collagenase by gingival fibroblasts, which suggests anti-inflammatory properties.

  15. Diffuse colonies of human skin fibroblasts in relation to cellular senescence and proliferation.

    PubMed

    Zorin, Vadim; Zorina, Alla; Smetanina, Nadezhda; Kopnin, Pavel; Ozerov, Ivan V; Leonov, Sergey; Isaev, Artur; Klokov, Dmitry; Osipov, Andreyan N

    2017-05-16

    Development of personalized skin treatment in medicine and skin care may benefit from simple and accurate evaluation of the fraction of senescent skin fibroblasts that lost their proliferative capacity. We examined whether enriched analysis of colonies formed by primary human skin fibroblasts, a simple and widely available cellular assay, could reveal correlations with the fraction of senescent cells in heterogenic cell population. We measured fractions of senescence associated β-galactosidase (SA-βgal) positive cells in either mass cultures or colonies of various morphological types (dense, mixed and diffuse) formed by skin fibroblasts from 10 human donors. Although the donors were chosen to be within the same age group (33-54 years), the colony forming efficiency of their fibroblasts (ECO-f) and the percentage of dense, mixed and diffuse colonies varied greatly among the donors. We showed, for the first time, that the SA-βgal positive fraction was the largest in diffuse colonies, confirming that they originated from cells with the least proliferative capacity. The percentage of diffuse colonies was also found to correlate with the SA-βgal positive cells in mass culture. Using Ki67 as a cell proliferation marker, we further demonstrated a strong inverse correlation (r=-0.85, p=0.02) between the percentage of diffuse colonies and the fraction of Ki67+ cells. Moreover, a significant inverse correlation (r=-0.94, p=0.0001) between the percentage of diffuse colonies and ECO-f was found. Our data indicate that quantification of a fraction of diffuse colonies may provide a simple and useful method to evaluate the extent of cellular senescence in human skin fibroblasts.

  16. Effect of procaine on cultivated human WI-38 fibroblasts.

    PubMed

    Pigeolet, E; Raes, M; Houbion, A; Remacle, J

    1988-01-01

    Procaine is a local anesthetic, also used in experimental gerontology and has been tested in cultivated human WI-38 fibroblasts. This molecule was found to enhance growth rate and cell densities in actively dividing cultures. As the cells aged, however, this stimulatory effect diminished and finally vanished. In a long term experiment the enhancement of growth of procaine treated cultures was finally replaced by a toxic effect even at low concentration. The amount of the thermolabile enzyme found in phase III cells did not change when procaine was added to the culture medium. In this cellular aging model, procaine behaved like a metabolic stimulator of actively dividing cells but not as an "antiaging" molecule as it is sometimes assumed.

  17. CHOLESTEROL REQUIREMENT OF PRIMARY DIPLOID HUMAN FIBROBLASTS

    PubMed Central

    Holmes, Richard; Helms, Judy; Mercer, Gretchen

    1969-01-01

    Primary cultures of fibroblast-like cells were obtained from skin and articular cartilage of human donors in the age bracket of 1 to 15 years. For growth these cultures required 1 mg/liter of cholesterol added to Medium A2 plus acetyl choline, Na pyruvate, and D-galactosamine HCl (APG) containing 10% lipoprotein-free human serum. Established cell lines did not require cholesterol for growth. Eagle's medium could be used in place of Medium A2 plus APG with the same results. Desmosterol could replace cholesterol but lansterol or 7 dehydrocholesterol could not. Other cholesterol precursors were tested and found to be inactive. With the proviso that cholesterol precursors entered the cell and had to be converted to cholesterol to function, it was concluded that the particular primaries studied lacked a functional enzyme system to reduce the double bond at carbon 7. PMID:5786984

  18. Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

    PubMed

    Sitte, N; Merker, K; von Zglinicki, T; Grune, T

    2000-03-01

    One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

  19. 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

  20. SV40-transformed human fibroblasts: evidence for cellular aging in pre-crisis cells.

    PubMed

    Stein, G H

    1985-10-01

    Pre-crisis SV40-transformed human diploid fibroblast (HDF) cultures have a finite proliferative lifespan, but they do not enter a viable senescent state at end of lifespan. Little is known about either the mechanism for this finite lifespan in SV40-transformed HDF or its relationship to finite lifespan in normal HDF. Recently we proposed that in normal HDF the phenomena of finite lifespan and arrest in a viable senescent state depend on two separate processes: 1) an age-related decrease in the ability of the cells to recognize or respond to serum and/or other mitogens such that the cells become functionally mitogen-deprived at the end of lifespan; and 2) the ability of the cells to enter a viable, G1-arrested state whenever they experience mitogen deprivation. In this paper, data are presented that suggest that pre-crisis SV40-transformed HDF retain the first process described above, but lack the second process. It is shown that SV40-transformed HDF have a progressively decreasing ability to respond to serum as they age, but they continue to traverse the cell cycle at the end of lifespan. Concomitantly, the rate of cell death increases steadily toward the end of lifespan, thereby causing the total population to cease growing and ultimately to decline. Previous studies have shown that when SV40-transformed HDF are environmentally serum deprived, they likewise exhibit continued cell cycle traverse coupled with increased cell death. Thus, these results support the hypothesis that pre-crisis SV40-transformed HDF still undergo the same aging process as do normal HDF, but they end their lifespan in crisis rather than in the normal G1-arrested senescent state because they have lost their ability to enter a viable, G1-arrested state in response to mitogen deprivation.

  1. Molecular basis of retinol anti-ageing properties in naturally aged human skin in vivo.

    PubMed

    Shao, Y; He, T; Fisher, G J; Voorhees, J J; Quan, T

    2017-02-01

    Retinoic acid has been shown to improve the aged-appearing skin. However, less is known about the anti-ageing effects of retinol (ROL, vitamin A), a precursor of retinoic acid, in aged human skin in vivo. This study aimed to investigate the molecular basis of ROL anti-ageing properties in naturally aged human skin in vivo. Sun-protected buttock skin (76 ± 6 years old, n = 12) was topically treated with 0.4% ROL and its vehicle for 7 days. The effects of topical ROL on skin epidermis and dermis were evaluated by immunohistochemistry, in situ hybridization, Northern analysis, real-time RT-PCR and Western analysis. Collagen fibrils nanoscale structure and surface topology were analysed by atomic force microscopy. Topical ROL shows remarkable anti-ageing effects through three major types of skin cells: epidermal keratinocytes, dermal endothelial cells and fibroblasts. Topical ROL significantly increased epidermal thickness by stimulating keratinocytes proliferation and upregulation of c-Jun transcription factor. In addition to epidermal changes, topical ROL significantly improved dermal extracellular matrix (ECM) microenvironment; increasing dermal vascularity by stimulating endothelial cells proliferation and ECM production (type I collagen, fibronectin and elastin) by activating dermal fibroblasts. Topical ROL also stimulates TGF-β/CTGF pathway, the major regulator of ECM homeostasis, and thus enriched the deposition of ECM in aged human skin in vivo. 0.4% topical ROL achieved similar results as seen with topical retinoic acid, the biologically active form of ROL, without causing noticeable signs of retinoid side effects. 0.4% topical ROL shows remarkable anti-ageing effects through improvement of the homeostasis of epidermis and dermis by stimulating the proliferation of keratinocytes and endothelial cells, and activating dermal fibroblasts. These data provide evidence that 0.4% topical ROL is a promising and safe treatment to improve the naturally aged human skin

  2. Senescence of immortal human fibroblasts by the introduction of normal human chromosome 6

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sandhu, A.K.; Hubbard, K.; Kaur, G.P.

    1994-06-07

    In these studies the authors show that introduction of a normal human chromosome 6 or 6q can suppress the immortal phenotype of simian virus 40-transformed human fibroblasts (SV/HF). Normal human fibroblasts have a limited life span in culture. Immortal clones of SV/HF displayed nonrandom rearrangements in chromosome 6. Single human chromosomes present in mouse/human monochromosomal hybrids were introduced into SV/HF via microcell fusion and maintained by selection for a dominant selectable marker gpt, previously integrated into the human chromosome. Clones of SV/HF cells bearing chromosome 6 displayed limited potential for cell division and morphological characteristics of senescent cells. The lossmore » of chromosome 6 from the suppressed clones correlated with the reappearance of immortal clones. Introduced chromosome 6 in the senescing cells was distinguished from those of parental cells by analysis for DNA sequences specific for the donor chromosome. The results further show that suppression of immortal phenotype in SV/HF is specific to chromosome 6. Introduction of individual human chromosomes 2, 8, or 19 did not impart cellular senescence in SV/HF. In addition, introduction of chromosome 6 into human glioblastoma cells did not lead to senescence. Based upon these results the authors propose that at least one of the genes (SEN6) for cellular senescence in human fibroblasts is present on the long arm of chromosome 6.« less

  3. Human Lung Fibroblasts Present Bacterial Antigens to Autologous Lung Th Cells.

    PubMed

    Hutton, Andrew J; Polak, Marta E; Spalluto, C Mirella; Wallington, Joshua C; Pickard, Chris; Staples, Karl J; Warner, Jane A; Wilkinson, Tom M A

    2017-01-01

    Lung fibroblasts are key structural cells that reside in the submucosa where they are in contact with large numbers of CD4 + Th cells. During severe viral infection and chronic inflammation, the submucosa is susceptible to bacterial invasion by lung microbiota such as nontypeable Haemophilus influenzae (NTHi). Given their proximity in tissue, we hypothesized that human lung fibroblasts play an important role in modulating Th cell responses to NTHi. We demonstrate that fibroblasts express the critical CD4 + T cell Ag-presentation molecule HLA-DR within the human lung, and that this expression can be recapitulated in vitro in response to IFN-γ. Furthermore, we observed that cultured lung fibroblasts could internalize live NTHi. Although unable to express CD80 and CD86 in response to stimulation, fibroblasts expressed the costimulatory molecules 4-1BBL, OX-40L, and CD70, all of which are related to memory T cell activation and maintenance. CD4 + T cells isolated from the lung were predominantly (mean 97.5%) CD45RO + memory cells. Finally, cultured fibroblasts activated IFN-γ and IL-17A cytokine production by autologous, NTHi-specific lung CD4 + T cells, and cytokine production was inhibited by a HLA-DR blocking Ab. These results indicate a novel role for human lung fibroblasts in contributing to responses against bacterial infection through activation of bacteria-specific CD4 + T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

  4. Role of Stromal Paracrine Signals in Proliferative Diseases of the Aging Human Prostate

    PubMed Central

    Takahashi, Sanai; Sugimura, Yoshiki

    2018-01-01

    Androgens are essential for the development, differentiation, growth, and function of the prostate through epithelial–stromal interactions. However, androgen concentrations in the hypertrophic human prostate decrease significantly with age, suggesting an inverse correlation between androgen levels and proliferative diseases of the aging prostate. In elderly males, age- and/or androgen-related stromal remodeling is spontaneously induced, i.e., increased fibroblast and myofibroblast numbers, but decreased smooth muscle cell numbers in the prostatic stroma. These fibroblasts produce not only growth factors, cytokines, and extracellular matrix proteins, but also microRNAs as stromal paracrine signals that stimulate prostate epithelial cell proliferation. Surgical or chemical castration is the standard systemic therapy for patients with advanced prostate cancer. Androgen deprivation therapy induces temporary remission, but the majority of patients eventually progress to castration-resistant prostate cancer, which is associated with a high mortality rate. Androgen deprivation therapy-induced stromal remodeling may be involved in the development and progression of castration-resistant prostate cancer. In the tumor microenvironment, activated fibroblasts stimulating prostate cancer cell proliferation are called carcinoma-associated fibroblasts. In this review, we summarize the role of stromal paracrine signals in proliferative diseases of the aging human prostate and discuss the potential clinical applications of carcinoma-associated fibroblast-derived exosomal microRNAs as promising biomarkers. PMID:29614830

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

    PubMed

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

    2016-01-01

    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. 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. We conclude that leptin selectively enhances the expression and secretion of certain matrix metalloproteinases in human gingival fibroblasts, and

  6. 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

  7. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    PubMed

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts. Copyright © 2016 John Wiley & Sons, Inc.

  8. Direct Reprogramming of Human Fibroblasts toward a Cardiomyocyte-like State

    PubMed Central

    Fu, Ji-Dong; Stone, Nicole R.; Liu, Lei; Spencer, C. Ian; Qian, Li; Hayashi, Yohei; Delgado-Olguin, Paul; Ding, Sheng; Bruneau, Benoit G.; Srivastava, Deepak

    2013-01-01

    Summary Direct reprogramming of adult somatic cells into alternative cell types has been shown for several lineages. We previously showed that GATA4, MEF2C, and TBX5 (GMT) directly reprogrammed nonmyocyte mouse heart cells into induced cardiomyocyte-like cells (iCMs) in vitro and in vivo. However, GMT alone appears insufficient in human fibroblasts, at least in vitro. Here, we show that GMT plus ESRRG and MESP1 induced global cardiac gene-expression and phenotypic shifts in human fibroblasts derived from embryonic stem cells, fetal heart, and neonatal skin. Adding Myocardin and ZFPM2 enhanced reprogramming, including sarcomere formation, calcium transients, and action potentials, although the efficiency remained low. Human iCM reprogramming was epigenetically stable. Furthermore, we found that transforming growth factor β signaling was important for, and improved the efficiency of, human iCM reprogramming. These findings demonstrate that human fibroblasts can be directly reprogrammed toward the cardiac lineage, and lay the foundation for future refinements in vitro and in vivo. PMID:24319660

  9. 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.

  10. 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

  11. Normal Human Fibroblasts Are Resistant to RAS-Induced Senescence

    PubMed Central

    Benanti, Jennifer A.; Galloway, Denise A.

    2004-01-01

    Oncogenic stimuli are thought to induce senescence in normal cells in order to protect against transformation and to induce proliferation in cells with altered p53 and/or retinoblastoma (Rb) pathways. In human fibroblasts, RAS initiates senescence through upregulation of the cyclin-dependent kinase inhibitor p16INK4A. We show here that in contrast to cultured fibroblast strains, freshly isolated normal fibroblasts are resistant to RAS-induced senescence and instead show some characteristics of transformation. RAS did not induce growth arrest or expression of senescence-associated β-galactosidase, and Rb remained hyperphosphorylated despite elevated levels of p16. Instead, RAS promoted anchorage-independent growth of normal fibroblasts, although expression of hTert with RAS increased colony formation and allowed normal fibroblasts to bypass contact inhibition. To test the hypothesis that p16 levels determine how cells respond to RAS, we expressed RAS in freshly isolated fibroblasts that expressed very low levels of p16, in hTert-immortalized fibroblasts that had accumulated intermediate levels of p16, and in IMR90 fibroblasts with high levels of p16. RAS induced growth arrest in cells with higher p16 levels, and this effect was reversed by p16 knockdown in the hTert-immortalized fibroblasts. These findings indicate that culture-imposed stress sensitizes cells to RAS-induced arrest, whereas early passage cells do not arrest in response to RAS. PMID:15024073

  12. Purification and Refolding of Overexpressed Human Basic Fibroblast Growth Factor in Escherichia coli

    PubMed Central

    Alibolandi, Mona; Mirzahoseini, Hasan

    2011-01-01

    This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8 M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. PMID:21837279

  13. Induction of human cardiomyocyte-like cells from fibroblasts by defined factors.

    PubMed

    Wada, Rie; Muraoka, Naoto; Inagawa, Kohei; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Kaneda, Ruri; Suzuki, Tomoyuki; Kamiya, Kaichiro; Tohyama, Shugo; Yuasa, Shinsuke; Kokaji, Kiyokazu; Aeba, Ryo; Yozu, Ryohei; Yamagishi, Hiroyuki; Kitamura, Toshio; Fukuda, Keiichi; Ieda, Masaki

    2013-07-30

    Heart disease remains a leading cause of death worldwide. Owing to the limited regenerative capacity of heart tissue, cardiac regenerative therapy has emerged as an attractive approach. Direct reprogramming of human cardiac fibroblasts (HCFs) into cardiomyocytes may hold great potential for this purpose. We reported previously that induced cardiomyocyte-like cells (iCMs) can be directly generated from mouse cardiac fibroblasts in vitro and vivo by transduction of three transcription factors: Gata4, Mef2c, and Tbx5, collectively termed GMT. In the present study, we sought to determine whether human fibroblasts also could be converted to iCMs by defined factors. Our initial finding that GMT was not sufficient for cardiac induction in HCFs prompted us to screen for additional factors to promote cardiac reprogramming by analyzing multiple cardiac-specific gene induction with quantitative RT-PCR. The addition of Mesp1 and Myocd to GMT up-regulated a broader spectrum of cardiac genes in HCFs more efficiently compared with GMT alone. The HCFs and human dermal fibroblasts transduced with GMT, Mesp1, and Myocd (GMTMM) changed the cell morphology from a spindle shape to a rod-like or polygonal shape, expressed multiple cardiac-specific proteins, increased a broad range of cardiac genes and concomitantly suppressed fibroblast genes, and exhibited spontaneous Ca(2+) oscillations. Moreover, the cells matured to exhibit action potentials and contract synchronously in coculture with murine cardiomyocytes. A 5-ethynyl-2'-deoxyuridine assay revealed that the iCMs thus generated do not pass through a mitotic cell state. These findings demonstrate that human fibroblasts can be directly converted to iCMs by defined factors, which may facilitate future applications in regenerative medicine.

  14. 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.

  15. Lipid profiling of parkin-mutant human skin fibroblasts.

    PubMed

    Lobasso, Simona; Tanzarella, Paola; Vergara, Daniele; Maffia, Michele; Cocco, Tiziana; Corcelli, Angela

    2017-12-01

    Parkin mutations are a major cause of early-onset Parkinson's disease (PD). The impairment of protein quality control system together with defects in mitochondria and autophagy process are consequences of the lack of parkin, which leads to neurodegeneration. Little is known about the role of lipids in these alterations of cell functions. In the present study, parkin-mutant human skin primary fibroblasts have been considered as cellular model of PD to investigate on possible lipid alterations associated with the lack of parkin protein. Dermal fibroblasts were obtained from two unrelated PD patients with different parkin mutations and their lipid compositions were compared with that of two control fibroblasts. The lipid extracts of fibroblasts have been analyzed by combined matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) and thin-layer chromatography (TLC). In parallel, we have performed direct MALDI-TOF/MS lipid analyses of intact fibroblasts by skipping lipid extraction steps. Results show that the proportions of some phospholipids and glycosphingolipids were altered in the lipid profiles of parkin-mutant fibroblasts. The detected higher level of gangliosides, phosphatidylinositol, and phosphatidylserine could be linked to dysfunction of autophagy and mitochondrial turnover; in addition, the lysophosphatidylcholine increase could represent the marker of neuroinflammatory state, a well-known component of PD. © 2017 Wiley Periodicals, Inc.

  16. Production of colony-stimulating factor in human dental pulp fibroblasts.

    PubMed

    Sawa, Y; Horie, Y; Yamaoka, Y; Ebata, N; Kim, T; Yoshida, S

    2003-02-01

    Class II major histocompatilibity complex (MHC)-expressing cells are usually distributed in dental pulp, and it was postulated that the colony-stimulating factor (CSF) derived from dental pulp fibroblasts contributes to the migration of class II MHC-expressing cells into pulp tissue. This study aimed to investigate the CSF production of human dental pulp fibroblasts. In pulp tissue sections, granulocyte (G)-CSF was detected from normal teeth, while G-CSF, macrophage (M)-CSF, and granulocyte-macrophage (GM)-CSF were detected from teeth with dentinal caries. In cultured dental pulp fibroblasts, G-CSF was detected by immunostaining, immunoprecipitation, and ELISA, and mRNAs of G-CSF, M-CSF, and GM-CSF were detected by RT-PCR. The dental pulp fibroblasts cultured with TNF-alpha were found to increase the G-CSF expression and to produce M-CSF and GM-CSF. These findings suggest that dental pulp fibroblasts usually produce G-CSF. In the presence of TNF-alpha, dental pulp fibroblast express M-CSF and GM-CSF.

  17. 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.

  18. Cytotoxicity of four denture adhesives on human gingival fibroblast cells.

    PubMed

    Lee, Yoon; Ahn, Jin-Soo; Yi, Young-Ah; Chung, Shin-Hye; Yoo, Yeon-Jee; Ju, Sung-Won; Hwang, Ji-Yun; Seo, Deog-Gyu

    2015-02-01

    The purpose of this study was to compare the cytotoxicity of four denture adhesives on human gingival fibroblast cells. Immortalized human gingival fibroblasts were cultured with one of four different denture adhesives, Polident, Protefix, Staydent or Denfix-A, which was placed in insert dishes (10% w/v concentration) for 48 h. The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometric apoptosis assay were used to evaluate cell viability and apoptosis rates. The fibroblasts were also examined under a scanning electron microscope. The MTT assay showed that all denture adhesives resulted in a significantly lower cell viability compared to the control cells propagated in normal culture medium (p < 0.05), with Staydent demonstrating the lowest cell viability. According to the flow cytometric apoptosis assay, Staydent and Protefix showed significantly higher apoptosis rates than the control group (p < 0.05), whereas Polident and Denfix-A did not demonstrate any significant differences (p > 0.05). Staydent showed the highest apoptosis rate. Scanning electron microscopy showed that the cells of the Staydent group underwent cytoplasmic membrane shrinkage, with cell free areas containing residual fragments of the membrane of dead cells. The four denture adhesives evaluated in this study imparted cytotoxic effects on human gingival fibroblast cells. Staydent showed the highest toxicity.

  19. Cell autonomous expression of inflammatory genes in biologically aged fibroblasts associated with elevated NF-kappaB activity.

    PubMed

    Kriete, Andres; Mayo, Kelli L; Yalamanchili, Nirupama; Beggs, William; Bender, Patrick; Kari, Csaba; Rodeck, Ulrich

    2008-07-16

    Chronic inflammation is a well-known corollary of the aging process and is believed to significantly contribute to morbidity and mortality of many age-associated chronic diseases. However, the mechanisms that cause age-associated inflammatory changes are not well understood. Particularly, the contribution of cell stress responses to age-associated inflammation in 'non-inflammatory' cells remains poorly defined. The present cross-sectional study focused on differences in molecular signatures indicative of inflammatory states associated with biological aging of human fibroblasts from donors aged 22 to 92 years. Gene expression profiling revealed elevated steady-state transcript levels consistent with a chronic inflammatory state in fibroblast cell-strains obtained from older donors. We also observed enhanced NF-kappaB DNA binding activity in a subset of strains, and the NF-kappaB profile correlated with mRNA expression levels characteristic of inflammatory processes, which include transcripts coding for cytokines, chemokines, components of the complement cascade and MHC molecules. This intrinsic low-grade inflammatory state, as it relates to aging, occurs in cultured cells irrespective of the presence of other cell types or the in vivo context. Our results are consistent with the view that constitutive activation of inflammatory pathways is a phenomenon prevalent in aged fibroblasts. It is possibly part of a cellular survival process in response to compromised mitochondrial function. Importantly, the inflammatory gene expression signature described here is cell autonomous, i.e. occurs in the absence of prototypical immune or pro-inflammatory cells, growth factors, or other inflammatory mediators.

  20. Blocking negative effects of senescence in human skin fibroblasts with a plant extract.

    PubMed

    Lämmermann, Ingo; Terlecki-Zaniewicz, Lucia; Weinmüllner, Regina; Schosserer, Markus; Dellago, Hanna; de Matos Branco, André Dargen; Autheried, Dominik; Sevcnikar, Benjamin; Kleissl, Lisa; Berlin, Irina; Morizot, Frédérique; Lejeune, Francois; Fuzzati, Nicola; Forestier, Sandra; Toribio, Alix; Tromeur, Anaïs; Weinberg, Lionel; Higareda Almaraz, Juan Carlos; Scheideler, Marcel; Rietveld, Marion; El Ghalbzouri, Abdoel; Tschachler, Erwin; Gruber, Florian; Grillari, Johannes

    2018-01-01

    There is increasing evidence that senescent cells are a driving force behind many age-related pathologies and that their selective elimination increases the life- and healthspan of mice. Senescent cells negatively affect their surrounding tissue by losing their cell specific functionality and by secreting a pro-tumorigenic and pro-inflammatory mixture of growth hormones, chemokines, cytokines and proteases, termed the senescence-associated secretory phenotype (SASP). Here we identified an extract from the plant Solidago virgaurea subsp. alpestris , which exhibited weak senolytic activity, delayed the acquisition of a senescent phenotype and induced a papillary phenotype with improved functionality in human dermal fibroblasts. When administered to stress-induced premature senescent fibroblasts, this extract changed their global mRNA expression profile and particularly reduced the expression of various SASP components, thereby ameliorating the negative influence on nearby cells. Thus, the investigated plant extract represents a promising possibility to block age-related loss of tissue functionality.

  1. Human Gingival Fibroblasts Display a Non-Fibrotic Phenotype Distinct from Skin Fibroblasts in Three-Dimensional Cultures

    PubMed Central

    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. PMID

  2. Blue light-irradiated human keloid fibroblasts: an in vitro study

    NASA Astrophysics Data System (ADS)

    Magni, Giada; Rossi, Francesca; Tatini, Francesca; Pini, Roberto; Coppi, Elisabetta; Cherchi, Federica; Fusco, Irene; Pugliese, Anna Maria; Pedata, Felicita; Fraccalvieri, Marco; Gasperini, Stefano; Pavone, Francesco S.; Tripodi, Cristina; Alfieri, Domenico; Targetti, Lorenzo

    2018-02-01

    Blue LED light irradiation is currently under investigation because of its effect in wound healing improvement. In this context, several mechanisms of action are likely to occur at the same time, consistently with the presence of different light absorbers within the skin. In our previous studies we observed the wound healing in superficial abrasions in an in vivo murine model. The results evidenced that both inflammatory infiltrate and myofibroblasts activity increase after irradiation. In this study we focused on evaluating the consequences of light absorption in fibroblasts from human cells culture: they play a key role in wound healing, both in physiological conditions and in pathological ones, such as keloid scarring. In particular we used keloids fibroblasts as a new target in order to investigate a possible metabolic or cellular mechanism correlation. Human keloid tissues were excised during standard surgery and immediately underwent primary cell culture extraction. Fibroblasts were allowed to grow in the appropriate conditions and then exposed to blue light. A metabolic colorimetric test (WST-8) was then performed. The tests evidenced an effect in mitochondrial activity, which could be modulated by the duration of the treatment. Electrophysiology pointed out a different behavior of irradiated fibroblasts. In conclusion, the Blue LED light affects the metabolic activity of fibroblasts and thus the cellular proliferation rate. No specific effect was found on keloid fibroblasts, thus indicating a very basic intracellular component, such as cytochromes, being the target of the treatment.

  3. 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

    2018-02-01

    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.

  4. 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

  5. Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes.

    PubMed

    Limat, A; Hunziker, T; Boillat, C; Bayreuther, K; Noser, F

    1989-05-01

    For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.

  6. 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.

  7. 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

  8. 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. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Rapamycin Inhibits Human Laryngotracheal Stenosis–derived Fibroblast Proliferation, Metabolism, and Function in Vitro

    PubMed Central

    Namba, Daryan R.; Ma, Garret; Samad, Idris; Ding, Dacheng; Pandian, Vinciya; Powell, Jonathan D.; Horton, Maureen R.; Hillel, Alexander T.

    2015-01-01

    Objective To determine if rapamycin inhibits the growth, function, and metabolism of human laryngotracheal stenosis (LTS)–derived fibroblasts. Study Design Controlled in vitro study. Setting Tertiary care hospital in a research university. Subjects and Methods Fibroblasts isolated from biopsies of 5 patients with laryngotracheal stenosis were cultured. Cell proliferation, histology, gene expression, and cellular metabolism of LTS-derived fibroblasts were assessed in 4 conditions: (1) fibroblast growth medium, (2) fibroblast growth medium with dimethylsulfoxide (DMSO), (3) fibroblast growth medium with 10−10 M (low-dose) rapamycin dissolved in DMSO, and (4) fibroblast growth medium with 10−9 M (high-dose) rapamycin dissolved in DMSO. Results The LTS fibroblast count and DNA concentration were reduced after treatment with high-dose rapamycin compared to DMSO (P = .0007) and normal (P = .0007) controls. Collagen I expression decreased after treatment with high-dose rapamycin versus control (P = .0051) and DMSO (P = .0093) controls. Maximal respiration decreased to 68.6 pMoles of oxygen/min/10 mg/protein from 96.9 for DMSO (P = .0002) and 97.0 for normal (P = .0022) controls. Adenosine triphosphate (ATP) production decreased to 66.8 pMoles from 88.1 for DMSO (P = .0006) and 83.3 for normal (P = .0003) controls. Basal respiration decreased to 78.6 pMoles from 108 for DMSO (P = .0002) and 101 for normal (P = .0014) controls. Conclusions Rapamycin demonstrated an anti-fibroblast effect by significantly reducing the proliferation, metabolism, and collagen deposition of human LTS fibroblast in vitro. Rapamycin significantly decreased oxidative phosphorylation of LTS fibroblasts, suggesting at a potential mechanism for the reduced proliferation and differentiation. Furthermore, rapamycin’s anti-fibroblast effects indicate a promising adjuvant therapy for the treatment of laryngotracheal stenosis. PMID:25754184

  10. 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

  11. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vectormore » containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.« less

  12. The growth of human fibroblasts and A431 epidermoid carcinoma cells on gamma-irradiated human amnion collagen substrata.

    PubMed

    Liu, B; Harrell, R; Lamb, D J; Dresden, M H; Spira, M

    1989-10-15

    Human fibroblasts and A431 human epidermoid carcinoma cells were cultured on gamma-irradiated human amnion collagen as well as on plastic dishes and non-irradiated collagen coated dishes. The morphology, attachment, growth and short-term cytotoxicity of these culture conditions have been determined. Both irradiated and non-irradiated amnion collagen enhanced the attachment and proliferation of fibroblasts as compared to the plastic dishes. No differences in these properties were observed for A431 cells cultured on irradiated collagen when compared with culture on non-irradiated collagen substrates. Cytotoxicity assays showed that irradiated and non-irradiated collagens were not cytotoxic for either fibroblasts or A431 cells. The results demonstrated that amnion collagen irradiated at doses of 0.25-2.0 Mrads is optimal for cell growth.

  13. Comparison of human umbilical cord blood-derived mesenchymal stem cells with healthy fibroblasts on wound-healing activity of diabetic fibroblasts.

    PubMed

    Jung, Jae-A; Yoon, Young-Don; Lee, Hyup-Woo; Kang, So-Ra; Han, Seung-Kyu

    2018-02-01

    Various types of skin substitutes composed of fibroblasts and/or keratinocytes have been used for the treatment of diabetic ulcers. However, the effects have generally not been very dramatic. Recently, human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have been commercialised for cartilage repair as a first cell therapy product using allogeneic stem cells. In a previous pilot study, we reported that hUCB-MSCs have a superior wound-healing capability compared with fibroblasts. The present study was designed to compare the treatment effect of hUCB-MSCs with that of fibroblasts on the diabetic wound healing in vitro. Diabetic fibroblasts were cocultured with healthy fibroblasts or hUCB-MSCs. Five groups were evaluated: group I, diabetic fibroblasts without coculture; groups II and III, diabetic fibroblasts cocultured with healthy fibroblasts or hUCB-MSCs; and groups IV and V, no cell cocultured with healthy fibroblasts or hUCB-MSCs. After a 3-day incubation, cell proliferation, collagen synthesis levels and glycosaminoglycan levels, which are the major contributing factors in wound healing, were measured. As a result, a hUCB-MSC-treated group showed higher cell proliferation, collagen synthesis and glycosaminoglycan level than a fibroblast-treated group. In particular, there were significant statistical differences in collagen synthesis and glycosaminoglycan levels (P = 0·029 and P = 0·019, respectively). In conclusion, these results demonstrate that hUCB-MSCs may have a superior effect to fibroblasts in stimulating diabetic wound healing. © 2017 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  14. Establishment of human induced pluripotent stem cell lines from normal fibroblast TIG-1.

    PubMed

    Kumazaki, Tsutomu; Kurata, Sayaka; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

    2011-06-01

    Normal human cells have a replicative life span and therefore senesce. Usually, normal human cell strains are differentiated cells and reach a terminally differentiated state after a number of cell divisions. At present, definitive differences are not known between replicative senescence and terminal differentiation. TIG-1 is a human fibroblast strain established from fetal lung and has been used extensively in studies of cellular senescence, and numerous data were accumulated at the molecular level. Recently, a method for generating induced pluripotent stem cells (iPSCs) was developed. Using the method, we introduced four reprogramming genes to TIG-1 fibroblasts and succeeded in isolating colonies that had embryonic stem cell (ESC)-like morphologies. They showed alkaline phosphatase activity and expressed ESC markers, as shown by immunostaining of OCT4, SOX2, SSEA4, and TRA-1-81 as well as reverse-transcription polymerase chain reaction (RT-PCR) for OCT4 and NANOG transcripts. Thus, we succeeded in establishing iPSC clones from TIG-1. The iPSC clones could differentiate to cells originated from all three germ-cell layers, as shown by RT-PCR, for messenger RNA (mRNA) expression of α-fetoprotein (endoderm), MSX1 (mesoderm) and microtubule-associated protein 2 (ectoderm), and by immunostaining for α-fetoprotein (endoderm), α-smooth muscle actin (mesoderm), and β-III-tubulin (ectoderm). The iPSCs formed teratoma containing the structures developed from all three germ-cell layers in severe combined immune-deficiency mice. Thus, by comparing the aging process of parental TIG-1 cells and the differentiation process of iPSC-derived fibrocytes to fibroblasts, we can reveal the exact differences in processes between senescence and terminal differentiation.

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

    PubMed

    Della Mina, Erika; 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

    2017-01-24

    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 (PAM 2 CSK 4 , LTA, FSL-1), TLR1/2 (PAM 3 CSK 4 ), 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.

  16. 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.

  17. 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.

  18. 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

  19. Effects of titanium surface topography on morphology and in vitro activity of human gingival fibroblasts.

    PubMed

    Ramaglia, L; Capece, G; Di Spigna, G; Bruno, M P; Buonocore, N; Postiglione, L

    2013-01-01

    The aim of the present study was to evaluate in vitro the biological behavior of human gingival fibroblasts cultured on two different titanium surfaces. Titanium test disks were prepared with a machined, relatively smooth (S) surface or a rough surface (O) obtained by a double acid etching procedure. Primary cultures of human gingival fibroblasts were plated on the experimental titanium disks and cultured up to 14 days. Titanium disk surfaces were analysed by scanning electron microscopy (SEM). Cell proliferation and a quantitative analysis by ELISA in situ of ECM components as CoI, FN and TN were performed. Results have shown different effects of titanium surface microtopography on cell expression and differentiation. At 96 hours of culture on experimental surfaces human gingival fibroblasts displayed a favourable cell attachment and proliferation on both surfaces although showing some differences. Both the relatively smooth and the etched surfaces interacted actively with in vitro cultures of human gingival fibroblasts, promoting cell proliferation and differentiation. Results suggested that the microtopography of a double acid-etched rough surface may induce a greater Co I and FN production, thus conditioning in vivo the biological behaviour of human gingival fibroblasts during the process of peri-implant soft tissue healing.

  20. Mitomycin C-induced apoptosis in cultured human Tenon's capsule fibroblasts.

    PubMed

    Kim, J W; Kim, S K; Song, I H; Kim, I T

    1999-06-01

    To investigate the mitomycin C-induced apoptotic cell death of fibroblasts, the primarily cultured human Tenon's capsule fibroblasts were exposed to a clinically used dosage of 0.4 mg/ml of mitomycin C for 5 minutes. TUNEL (TdT-mediated dUTP-biotin nick end labeling) assay and electron microscopic studies were performed to determine the extent of mitomycin C-induced apoptosis. A flow cytometric study was performed to quantify the apoptotic cell population over time. The TUNEL stains were positive and electron microscopy showed features of apoptotic cell death in some fibroblasts 3 and 5 days after treatment. Flow cytometric analysis using Annexin V-propidium iodide double staining detected apoptotic cells 3 days after treatment. These apoptotic cell populations increased at 4 days and were sustained for one week. This study revealed that the clinical effects of mitomycin C on fibroblasts may be mediated not only by antiproliferative but also apoptotic cell death to some degree. Therefore, the apoptotic cell death of fibroblasts induced by mitomycin C should be considered to properly understand the mechanism of wound healing after trabeculectomy with adjunctive mitomycin C.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo

    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,more » 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

  2. Establishment of proliferative tetraploid cells from telomerase-immortalized normal human fibroblasts.

    PubMed

    Ohshima, Susumu; Seyama, Atsushi

    2016-06-01

    Aneuploidy is observed in the majority of human cancers and is considered to be causally related to carcinogenesis. Although malignant aneuploid cells are suggested to develop from polyploid cells formed in precancerous lesions, the mechanisms of this process remain elusive. This is partly because no experimental model is available where nontransformed polyploid human cells propagate in vitro. We previously showed that proliferative tetraploid cells can be established from normal human fibroblasts by treatment with the spindle poison demecolcine (DC). However, the limited lifespan of these cells hampered detailed analysis of a link between chromosomal instability and the oncogenic transformation of polyploid cells. Here, we report the establishment of proliferative tetraploid cells from the telomerase-immortalized normal human fibroblast cell line TIG-1. Treatment of immortalized diploid cells with DC for 4 days resulted in proliferation of cells with tetraploid DNA content and near-tetraploid/tetraploid chromosome counts. Established tetraploid cells had functional TP53 despite growing at almost the same rate as diploid cells. The frequency of clonal and sporadic chromosome aberrations in tetraploid cells was higher than in diploid cells and in one experiment, gradually increased with repeated subculture. This study suggests that tetraploid cells established from telomerase-immortalized normal human fibroblasts can be a valuable model for studying chromosomal instability and the oncogenic potential of polyploid cells. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  3. 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.

  4. Effect of brief exposure to mitomycin C on cultured human nasal mucosa fibroblasts.

    PubMed

    Hu, D; Sires, B S; Tong, D C; Royack, G A; Oda, D

    2000-03-01

    To observe the effect of mitomycin C (MMC) on cultured human nasal mucosa fibroblasts. Cultured human nasal mucosa fibroblasts were exposed to MMC (0.1-0.4 mg/ml) for 1 to 5 minutes. The viability of the fibroblasts was determined by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay; DNA fragmentation (apoptosis) by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL); apoptotic percentage by flow cytometry; and morphology by light microscopy. A portion of the fibroblasts survived the mitomycin treatment and showed evidence of regrowth within 2 to 3 days. These cells reached confluence in 5 to 7 days. The inhibition rates by MTT assay of 0.4 mg/ml MMC for 5-minute exposures was 31.3%. Dose-response effect was noted with the lower concentrations and shorter exposure times where the inhibition rates were lower (but not significantly so). DNA fragmentation was observed in fibroblasts 24 hours after MMC exposure (0.4 mg/ml) for 5 minutes compared with normal control. The apoptotic rate of fibroblasts treated by 0.4 mg/ml MMC was significantly higher than the control (p < 0.05). Short MMC exposure times have a variable cytotoxic effect and inhibit proliferation of cultured nasal mucosa fibroblasts. MMC also can induce apoptosis with a 5-minute exposure time. Therefore, it is possible that MMC has a complex effect in dacryocystorhinostomy.

  5. Non-human Primate and Rat Cardiac Fibroblasts show similar Extracellular Matrix-related and Cellular Adhesion Gene Responses to Substance P

    PubMed Central

    Meléndez, Giselle C.; Manteufel, Edward J.; Dehlin, Heather M.; Register, Thomas C.; Levick, Scott P.

    2015-01-01

    Background The sensory nerve neuropeptide substance P (SP) regulates cardiac fibrosis in rodents under pressure overload conditions. Interestingly, SP induces transient increase expression of specific genes in isolated rat cardiac fibroblasts, without resultant changes in cell function. This suggests that SP ‘primes’ fibroblasts, but does not directly activate them. We investigated whether these unusual findings are specific to rodent fibroblasts or are translatable to a larger animal model more closely related to humans. Methods We compared the effects of SP on genes associated with extracellular matrix (ECM) regulation, cell-cell adhesion, cell-matrix adhesion and ECM in cardiac fibroblasts isolated from a non-human primate and Sprague-Dawley rats. Results We found that rodent and non-human primate cardiac fibroblasts showed similar ECM regulation and cell adhesion gene expression responses to SP. There were, however, large discrepancies in ECM genes which did not result in collagen or laminin synthesis in rat or non-human primate fibroblasts in response to SP. Conclusions This study further supports the notion that SP serves as a ‘primer’ for fibroblasts rather than initiating direct effects and suggests that rodent fibroblasts are a suitable model for studying gene and functional responses to SP in the absence of human or non-human primate fibroblasts. PMID:25550118

  6. 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

  7. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol.

    PubMed

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol.

  8. Biological effects of plasma rich in growth factors (PRGF) on human endometrial fibroblasts.

    PubMed

    Anitua, Eduardo; de la Fuente, María; Ferrando, Marcos; Quintana, Fernando; Larreategui, Zaloa; Matorras, Roberto; Orive, Gorka

    2016-11-01

    To evaluate the biological outcomes of plasma rich in growth factors (PRGF) on human endometrial fibroblasts in culture. PRGF was obtained from three healthy donors and human endometrial fibroblasts (HEF) were isolated from endometrial specimens from five healthy women. The effects of PRGF on cell proliferation and migration, secretion of vascular endothelial growth factor (VEGF), procollagen type I and hyaluronic acid (HA) and contractility of isolated and cultured human endometrial fibroblasts (HEF) were analyzed. Statistical analysis was performed in order to compare the effects of PRGF with respect to control situation (T-test or Mann-Whitney U-test). We report a significantly elevated human endometrial fibroblast proliferation and migration after treatment with PRGF. In addition, stimulation of HEF with PRGF induced an increased expression of the angiogenic factor VEGF and favored the endometrial matrix remodeling by the secretion of procollagen type I and HA and endometrial regeneration by elevating the contractility of HEF. These results were obtained for all PRGF donors and each endometrial cell line. The myriad of growth factors contained in PRGF promoted HEF proliferation, migration and synthesis of paracrine molecules apart from increasing their contractility potential. These preliminary results suggest that PRGF improves the biological activity of HEF in vitro, enhancing the regulation of several cellular processes implied in endometrial regeneration. This innovative treatment deserves further investigation for its potential in "in vivo" endometrial development and especially in human embryo implantation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. 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

  10. ATM-dependent DNA damage checkpoint functions regulate gene expression in human fibroblasts

    PubMed Central

    Zhou, Tong; Chou, Jeff; Zhou, Yingchun; Simpson, Dennis A.; Cao, Feng; Bushel, Pierre R.; Paules, Richard S.; Kaufmann, William K.

    2013-01-01

    The relationships between profiles of global gene expression and DNA damage checkpoint functions were studied in cells from patients with ataxia telangiectasia (AT). Three telomerase-expressing AT fibroblast lines displayed the expected hypersensitivity to ionizing radiation (IR) and defects in DNA damage checkpoints. Profiles of global gene expression in AT cells were determined at 2, 6 and 24 h after treatment with 1.5 Gy IR or sham-treatment, and were compared to those previously recognized in normal human fibroblasts. Under basal conditions 160 genes or ESTs were differentially expressed in AT and normal fibroblasts, and these were associated by gene ontology with insulin-like growth factor binding and regulation of cell growth. Upon DNA damage, 1091 gene mRNAs were changed in at least two of the three AT cell lines. When compared with the 1811 genes changed in normal human fibroblasts after the same treatment, 715 were found in both AT and normal fibroblasts, including most genes categorized by gene ontology into cell cycle, cell growth and DNA damage response pathways. However, the IR-induced changes in these 715 genes in AT cells usually were delayed or attenuated in comparison to normal cells. The reduced change in DNA-damage-response genes and the attenuated repression of cell-cycle-regulated genes may account for the defects in cell cycle checkpoint function in AT cells. PMID:17699107

  11. In vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold.

    PubMed

    Pajoum Shariati, Seyed Ramin; Shokrgozar, Mohammad Ali; Vossoughi, Manouchehr; Eslamifar, Ali

    2009-07-01

    Extensive full-thickness burns require replacement of both epidermis and dermis. In designing skin replacements, the goal has been to re-create this model and make a product which has both essential components. In the present study, we developed procedures for establishing confluent, stratified layers of cultured human keratinocytes on the surface of modified collagen-chitosan scaffold that contains fibroblasts. The culture methods for propagation of keratinocytes and fibroblasts isolated from human neonatal foreskin were developed. The growth and proliferation of normal human keratinocytes were evaluated in serum-free (keratinocyte growth medium) and our modified medium. Characterization of human keratinocytes was determined by using pan-keratin and anti-involucrin monoclonal antibodies. For fabrication of relevant biodegradable and biocompatible collagen-chitosan porous scaffold with improved biostability, modified method of freeze-gelation was used. In generating organotypic co-cultures, epidermal keratinocytes were plated onto the upper surface of scaffold containing embedded fibroblasts. The results showed that the growth of isolated human skin fibroblasts and keratinocytes in our modified medium was more than that in the serum-free medium. The different evaluations of collagen-chitosan scaffold showed that it is relevant to growth of cells (fibroblast and keratinocyte) and has a good flexibility in manipulation of the living skin equivalents. These findings indicate that the integration of collagen-chitosan scaffold with co-cultured keratinocyte and fibroblast in vitro provides a potential source of living skin for grafting in vivo.

  12. Altered chromosome 6 in immortal human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hubbard-Smith, K.; Pardinas, J.R.; Jha, K.K.

    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 tomore » 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. 66 refs., 6 figs., 2 tabs.« less

  13. 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...

  14. 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.

  15. 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

  16. The CC chemokine eotaxin/CCL11 has a selective profibrogenic effect on human lung fibroblasts.

    PubMed

    Puxeddu, Ilaria; Bader, Reem; Piliponsky, Adrian Martin; Reich, Reuven; Levi-Schaffer, Francesca; Berkman, Neville

    2006-01-01

    Eotaxin/CCL11 plays an important role in asthma. It acts through the chemokine receptor CCR3 expressed on hematopoietic and nonhematopoietic cells in the lung. To determine whether eotaxin/CCL11 modulates lung and bronchial fibroblast properties and thereby might contribute to airway remodeling. CCR3 expression was characterized on a lung fibroblast line (MRC-5; flow cytometry, fluorescent microscopy, RT-PCR, and Northern blotting), on primary bronchial fibroblasts (flow cytometry), and on fibroblasts in human lung tissue (confocal laser microscopy). The effects of eotaxin/CCL11 on lung fibroblast migration (Boyden chamber), proliferation (tritiated thymidine incorporation), alpha-smooth muscle actin expression (ELISA), 3-dimensional collagen gel contraction (floating gel), pro-alpha1(I) collagen mRNA (Northern blotting), total collagen synthesis (tritiated proline incorporation), matrix metalloproteinase activity (gelatin zymography), and TGF-beta(1) release (ELISA) were evaluated. The contribution of eotaxin/CCL11/CCR3 binding on lung fibroblasts was also investigated by neutralizing experiments. CCR3 is constitutively expressed in cultured lung and primary bronchial fibroblasts and colocalizes with specific surface markers for human fibroblasts in lung tissue. Eotaxin/CCL11 selectively modulates fibroblast activities by increasing their proliferation, matrix metalloproteinase 2 activity, and collagen synthesis but not their differentiation into myofibroblasts, contractility in collagen gel, or TGF-beta(1) release. Eotaxin/CCL11 enhances migration of lung fibroblasts in response to nonspecific chemoattractants, and this effect is completely inhibited by anti-CCR3-neutralizing antibodies. These data demonstrate that eotaxin/CCL11 has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin/CCL11 can participate in airway remodeling in asthma.

  17. 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

  18. 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.

  19. [A method for the primary culture of fibroblasts isolated from human airway granulation tissues].

    PubMed

    Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu-ling; Pei, Ying-hua

    2013-04-01

    To establish a feasible method to culture primary fibroblasts isolated from human airway granulation tissues, and therefore to provide experimental data for the investigation of the pathogenesis of benign airway stenosis. The granulation tissues were collected from 6 patients during routine bronchoscopy at our department of Beijing Tiantan Hospital from April to June 2011. Primary fibroblasts were obtained by culturing the explanted tissues. Cell growth was observed under inverted microscope. All of these 6 primary cultures were successful. Fibroblast-like cells were observed to migrate from the tissue pieces 3 d after inoculation. After 9-11 d of culture, cells reached to 90% confluence and could be sub-cultured. After passage, the cells were still in a typical elongated spindle-shape and grew well. The cells could be sub-cultured further when they formed a monolayer. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissues.

  20. Vegetable peptones increase production of type I collagen in human fibroblasts by inducing the RSK-CCAAT/enhancer binding protein-β phosphorylation pathway.

    PubMed

    Jung, Eunsun; Cho, Jae Youl; Park, Deokhoon; Kim, Min Hee; Park, Beomseok; Lee, Sang Yeol; Lee, Jongsung

    2015-02-01

    Skin aging appears to be principally attributed to a decrease in type I collagen level and the regeneration ability of dermal fibroblasts. We hypothesized that vegetable peptones promote cell proliferation and production of type I collagen in human dermal fibroblasts. Therefore, we investigated the effects of vegetable peptones on cell proliferation and type I collagen production and their possible mechanisms in human dermal fibroblasts. Vegetable peptones significantly promoted cell proliferation in a concentration-dependent manner. In addition, the human luciferase type I collagen α2 promoter and type I procollagen synthesis assays showed that the vegetable peptones induced type I procollagen production by activating the type I collagen α2 promoter. Moreover, the vegetable peptones activated p90 ribosomal s6 kinase, which was mediated by activating the Raf-p44/42 mitogen-activated protein kinase signaling pathway. Furthermore, the vegetable peptone-induced increase in cell proliferation and type I collagen production decreased upon treatment with the ERK inhibitor PD98059. Taken together, these findings suggest that increased proliferation of human dermal fibroblasts and enhanced production of type I collagen by vegetable peptones occur primarily by inducing the p90 ribosomal s6 kinase-CCAAT/enhancer binding protein β phosphorylation pathway, which is mediated by activating Raf-ERK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Eber, R.M.; Shuler, C.F.; Buchanan, W.

    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 measuredmore » 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.« less

  2. Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer.

    PubMed

    Zammit, C; Coope, R; Gomm, J J; Shousha, S; Johnston, C L; Coombes, R C

    2002-04-08

    Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.

  3. PAL-12, a new anti-aging hexa-peptoid, inhibits UVB-induced photoaging in human dermal fibroblasts and 3D reconstructed human full skin model, Keraskin-FT™.

    PubMed

    Song, Daeun; Park, Hyeonji; Lee, Su-Hyon; Kim, Mi Jung; Kim, Eun-Joo; Lim, Kyung-Min

    2017-11-01

    Peptoids are a class of peptidomimetics whose pharmacological activities are widely investigated owing to their remarkable biological stability. However, the utilities of peptoids as cosmetic functional ingredients have not been fully explored. Here, we investigated anti-aging effects of PAL-12, a new hexa-peptoid, on UVB-induced photoaging in human dermal fibroblasts (HDFs) and a 3D reconstituted human full skin model, Keraskin-FT™. PAL-12 suppressed matrix metalloproteinase-1 (MMP-1) expression induced by UVB irradiation along with the attenuation of MMP-1 secretion as determined by ELISA assay. Interestingly PAL-12 slightly enhanced the expression levels of collagen-1 and fibronectin-1 in HDFs or Keraskin-FT™. In addition, PAL-12 prevented the decrease of cell viability following UVB irradiation. However, PAL-12 failed to affect ROS generation, cell necrosis and apoptosis significantly. Instead, PAL-12 suppressed UVB-induced activation of epidermal growth factor receptors (EGFR), extracellular signal-regulated kinase (ERK) and c-Jun, which may resulted in the attenuation of AP-1-promoted MMP-1 expression. Collectively, these results suggest that PAL-12 might be a novel cosmetic ingredient effective against UVB-induced skin photoaging.

  4. Effects of cranberry components on human aggressive periodontitis gingival fibroblasts.

    PubMed

    Tipton, D A; Babu, J P; Dabbous, M Kh

    2013-08-01

    Aggressive periodontitis (AgP) causes rapid periodontal breakdown involving AgP gingival fibroblast production of cytokines [i.e. interleukin (IL)-6, a bone metabolism regulator], and matrix metalloproteinase (MMP)-3. Lipopolysaccharide upregulates fibroblast IL-6 and MMP-3, via transcription factors (i.e. NF-κB). Cranberry (Vaccinium macrocarpon) inhibits lipopolysaccharide-stimulated macrophage and normal gingival fibroblast activities, but little is known of its effects on AgP fibroblasts. Objectives of this study are to use AgP fibroblasts, to determine cytotoxicity of cranberry components or periodontopathogen (Fusobacterium nucleatum, Porphyromonas gingivalis) lipopolysaccharide ± cranberry components, and effects of cranberry components on lipopolysaccharide-stimulated NF-κB activation and IL-6 and MMP-3 production. AgP fibroblasts were incubated ≤ 6 d with high molecular weight non-dialyzable material (NDM) (derived from cranberry juice (1-500 μg/mL) or lipopolysaccharide (1 μg/mL) ± NDM. Membrane damage and viability were assessed by enzyme activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Secreted IL-6 and MMP-3 were measured by ELISA. NF-κB p65 was measured via binding to an oligonucleotide containing the NF-κB consensus site. Data were analyzed using analysis of variance and Scheffe's F procedure for post hoc comparisons. Short-term exposure to NDM, or lipopolysaccharide ± NDM caused no membrane damage. NDM (≤ 100 μg/mL) or lipopolysaccharide ± NDM had no effect on viability ≤ 7 d exposure. NDM (50 μg/mL) inhibited lipopolysaccharide-stimulated p65 (P ≤ 0.003) and constitutive or lipopolysaccharide-stimulated MMP-3 (P ≤ 0.02). NDM increased AgP fibroblast constitutive or lipopolysaccharide-stimulated IL-6 (P ≤ 0.0001), but inhibited normal human gingival fibroblast IL-6 (P ≤ 0.01). Lack of toxicity of low NDM concentrations, and its inhibition of NF-κB and MMP-3, suggest that

  5. 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.

  6. Viability of human fibroblasts in coconut water as a storage medium.

    PubMed

    Moreira-Neto, J J S; Gondim, J O; Raddi, M S G; Pansani, C A

    2009-09-01

    To evaluate the effectiveness of a new storage medium for avulsed teeth, coconut water, in maintaining the viability of human fibroblasts. Cell viability after different time periods was evaluated in the following storage media: coconut water, coconut water with sodium bicarbonate, milk, saline and still mineral water. Human fibroblasts were seeded in Eagle's minimal essential medium (EMEM) supplemented with 7.5% foetal calf serum. After trypsinisation, 100 microL of culture medium containing approximately 10(4) cells mL(-1) were collected and pipetted into the wells of 96-well plates, which were incubated overnight in 5% CO(2) and 95% air mixture at 37 degrees C. EMEM was then replaced by the storage media and the plates were incubated at 37 degrees C for 1, 2 and 4 h. Cell viability was determined using the neutral red assay. The proportions of viable cells after exposure to the storage media were analysed statistically by anova and the least significant difference (LSD) test (alpha = 5%). Milk had the greatest capacity to maintain cell viability (P < 0.05), followed by coconut water with sodium bicarbonate and saline. Coconut water was significantly worse at maintaining cell viability compared to milk, coconut water with sodium bicarbonate and saline. The smallest number of viable cells was observed for mineral water (P < 0.05). Coconut water was worse than milk in maintaining human fibroblast cell viability.

  7. Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts.

    PubMed

    Benderdour, M; Hess, K; Dzondo-Gadet, M; Nabet, P; Belleville, F; Dousset, B

    1998-05-29

    Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.

  8. Inhibition of hydrogen peroxide induced injuring on human skin fibroblast by Ulva prolifera polysaccharide.

    PubMed

    Cai, Chuner; Guo, Ziye; Yang, Yayun; Geng, Zhonglei; Tang, Langlang; Zhao, Minglin; Qiu, Yuyan; Chen, Yifan; He, Peimin

    2016-10-01

    Ulva prolifera can protect human skin fibroblast from being injured by hydrogen peroxide. This work studied the composition of Ulva prolifera polysaccharide and identified its physicochemical properties. The results showed that the cell proliferation of 0.5mg/mL crude polysaccharide was 154.4% of that in negative control group. Moreover, ROS detection indices, including DCFH-DA, GSH-PX, MDA and CAT, indicated that crude polysaccharide could improve cellular ability to scavenge free radical and decrease the injury on human skin fibroblast by hydrogen peroxide. In purified polysaccharide, the activity of fraction P1-1 was the highest, with 174.6% of that in negative control group. The average molecular weight of P1-1 was 137kD with 18.0% of sulfate content. This work showed the inhibition of hydrogen peroxide induced injuries on human skin fibroblast by Ulva prolifera polysaccharide, which may further evaluate the application of U. prolifera on cosmetics. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Expression of the FAP gene in non-fibroblast human cell lines. Development of cancer-associated fibroblast models.

    PubMed

    Tyulkina, D V; Pleshkan, V V; Alekseenko, I V; Kopantseva, M R; Sverdlov, E D

    2016-09-01

    The fibroblast activation protein (FAP) is selectively expressed in cancer-associated fibroblasts (CAFs) and facilitates tumor progression, which makes this protein an attractive therapeutic target. There are difficulties in obtaining CAFs for studying the function and suppression of FAP. In this work, the expression level of FAP was determined by PCR assay in 25 human cell lines and 8 surgical samples of tumor stroma. The expression of FAP was observed in all tumor stroma samples and in four cell lines: NGP-127, SJCRH30, SJSA-1, and A375. The level of FAP expression in NGP-127, SJCRH30, and SJSA-1 lines as well as in CAFs of patients was comparable, which makes these cell lines a possible model for studying FAP.

  10. Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 inmore » 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.« less

  11. Ganoderma lucidum polysaccharides protect fibroblasts against UVB-induced photoaging

    PubMed Central

    Zeng, Qinghai; Zhou, Fang; Lei, Li; Chen, Jing; Lu, Jianyun; Zhou, Jianda; Cao, Ke; Gao, Lihua; Xia, Fang; Ding, Shu; Huang, Lihua; Xiang, Hong; Wang, Jingjing; Xiao, Yangfan; Xiao, Rong; Huang, Jinhua

    2017-01-01

    Ganoderma lucidum has featured in traditional Chinese medicine for >1,000 years. Ganoderma polysaccharides (GL-PS), a major active ingredient in Ganoderma, confer immune regulation, antitumor effects and significant antioxidant effects. The aim of the present study was to investigate the efficacy and mechanism of GL-PS-associated inhibition of ultraviolet B (UVB)-induced photoaging in human fibroblasts in vitro. Primary human skin fibroblasts were cultured, and a fibroblast photoaging model was built through exposure to UVB. Cell viability was measured by MTT assay. Aged cells were stained using a senescence-associated β-galactosidase staining (SA-β-gal) kit. ELISA kits were used to analyze matrix metalloproteinase (MMP) −1 and C-telopeptides of Type I collagen (CICP) protein levels in cellular supernatant. ROS levels were quantified by flow cytometry. Cells exposed to UVB had decreased cell viability, increased aged cells, decreased CICP protein expression, increased MMP-1 protein expression, and increased cellular ROS levels compared with non-exposed cells. However, cells exposed to UVB and treated with 10, 20 and 40 µg/ml GL-PS demonstrated increased cell viability, decreased aged cells, increased CICP protein expression, decreased MMP-1 protein expression, and decreased cellular ROS levels compared with UVB exposed/GL-PS untreated cells. These results demonstrate that GL-PS protects fibroblasts against photoaging by eliminating UVB-induced ROS. This finding indicates GL-PS treatment may serve as a novel strategy for antiphotoaging. PMID:27959406

  12. Ganoderma lucidum polysaccharides protect fibroblasts against UVB-induced photoaging.

    PubMed

    Zeng, Qinghai; Zhou, Fang; Lei, Li; Chen, Jing; Lu, Jianyun; Zhou, Jianda; Cao, Ke; Gao, Lihua; Xia, Fang; Ding, Shu; Huang, Lihua; Xiang, Hong; Wang, Jingjing; Xiao, Yangfan; Xiao, Rong; Huang, Jinhua

    2017-01-01

    Ganoderma lucidum has featured in traditional Chinese medicine for >1,000 years. Ganoderma polysaccharides (GL-PS), a major active ingredient in Ganoderma, confer immune regulation, antitumor effects and significant antioxidant effects. The aim of the present study was to investigate the efficacy and mechanism of GL‑PS‑associated inhibition of ultraviolet B (UVB)‑induced photoaging in human fibroblasts in vitro. Primary human skin fibroblasts were cultured, and a fibroblast photoaging model was built through exposure to UVB. Cell viability was measured by MTT assay. Aged cells were stained using a senescence‑associated β-galactosidase staining (SA‑β‑gal) kit. ELISA kits were used to analyze matrix metalloproteinase (MMP) ‑1 and C‑telopeptides of Type I collagen (CICP) protein levels in cellular supernatant. ROS levels were quantified by flow cytometry. Cells exposed to UVB had decreased cell viability, increased aged cells, decreased CICP protein expression, increased MMP‑1 protein expression, and increased cellular ROS levels compared with non‑exposed cells. However, cells exposed to UVB and treated with 10, 20 and 40 µg/ml GL‑PS demonstrated increased cell viability, decreased aged cells, increased CICP protein expression, decreased MMP‑1 protein expression, and decreased cellular ROS levels compared with UVB exposed/GL‑PS untreated cells. These results demonstrate that GL‑PS protects fibroblasts against photoaging by eliminating UVB‑induced ROS. This finding indicates GL‑PS treatment may serve as a novel strategy for antiphotoaging.

  13. Expression and Function of Connexin 43 in Human Gingival Wound Healing and Fibroblasts

    PubMed Central

    Tarzemany, Rana; Jiang, Guoqiao; Larjava, Hannu; Häkkinen, Lari

    2015-01-01

    Connexins (C×s) are a family of transmembrane proteins that form hemichannels and gap junctions (GJs) on the cell membranes, and transfer small signaling molecules between the cytoplasm and extracellular space and between connecting cells, respectively. Among C×s, suppressing C×43 expression or function promotes skin wound closure and granulation tissue formation, and may alleviate scarring, but the mechanisms are not well understood. Oral mucosal gingiva is characterized by faster wound closure and scarless wound healing outcome as compared to skin wounds. Therefore, we hypothesized that C×43 function is down regulated during human gingival wound healing, which in fibroblasts promotes expression of genes conducive for fast and scarless wound healing. Cultured gingival fibroblasts expressed C×43 as their major connexin. Immunostaining of unwounded human gingiva showed that C×43 was abundantly present in the epithelium, and in connective tissue formed large C×43 plaques in fibroblasts. At the early stages of wound healing, C×43 was strongly down regulated in wound epithelial cells and fibroblasts, returning to the level of normal tissue by day 60 post-wounding. Blocking of C×43 function by C×43 mimetic peptide Gap27 suppressed GJ-mediated dye transfer, promoted migration, and caused significant changes in the expression of wound healing-associated genes in gingival fibroblasts. In particular, out of 54 genes analyzed, several MMPs and TGF-β1, involved in regulation of inflammation and extracellular matrix (ECM) turnover, and VEGF-A, involved in angiogenesis, were significantly upregulated while pro-fibrotic ECM molecules, including Collagen type I, and cell contractility-related molecules were significantly down regulated. These responses involved MAPK, GSK3α/β and TGF-β signaling pathways, and AP1 and SP1 transcription factors. Thus, suppressed function of C×43 in fibroblasts promotes their migration, and regulates expression of wound healing

  14. Induction of vascular endothelial growth factor expression in human pulp fibroblasts stimulated with black-pigmented Bacteroides.

    PubMed

    Yang, L-C; Tsai, C-H; Huang, F-M; Su, Y-F; Lai, C-C; Liu, C-M; Chang, Y-C

    2004-09-01

    To investigate the effect of black-pigmented Bacteroides on the expression of vascular endothelial growth factor (VEGF) gene in human pulp fibroblasts. The supernatants of Porphyromonas endodontalis, Porphyromonas gingivalis and Prevotella intermedia were used to evaluate VEGF gene expression in human pulp fibroblasts. The levels of mRNAs were measured by the quantitative reverse-transcriptase polymerase chain reaction analysis. Black-pigmented Bacteroides induced significantly high levels of VEGF mRNA gene expression in human pulp fibroblasts (P < 0.05). In addition, the expression of VEGF depended on the bacteria tested. Black-pigmented Bacteroides may be involved in developing pulpal disease through the stimulation of VEGF production that would lead to the expansion of the vascular network coincident to progression of the inflammation.

  15. The polypeptide in Chlamys farreri can protect human dermal fibroblasts from ultraviolet B damage

    NASA Astrophysics Data System (ADS)

    Zhang, Yujiang; Zhan, Songmei; Cao, Pengli; Liu, Ning; Chen, Xuehong; Wang, Yuejun; Wang, Chunbo

    2005-09-01

    To investigate the effect of polypeptide from Chlamys farreri (PCF) on NHDF in vitro, we modeled oxidative damage on normal human dermal fibroblasts (NHDF) exposed to ultraviolet B (UVB). In this study, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) were tested to measure cell viability. Enzymes including superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and xanthine oxidase (XOD) were determined biochemically. Total antioxidative capacity (T-AOC) and anti-superoxide anion capacity (A-SAC) were also determined. Ultrastructure of fibroblasts was observed under transmission electron microscope. The results showed that: UVB (1.176×10-4 J/cm2) suppressed the growth of fibroblasts and the introduction of PCF (0.25% 1%) before UVB reduced the suppression in a concentration-dependent manner. PCF could enhance the activities of SOD, GSH-PX and T-AOC as well as A-SAC. Also PCF could inhibit XOD activity, while it did not affect CAT activity. Ultrastructure of fibroblasts were damaged after UVB irradiation, concentration-dependent PCF reduced the destructive effect of UVB on cells. These results indicated that PCF can protect human dermal fibroblasts from being harmed by UVB irradiation via its antioxidant proerty.

  16. The effect of ICG on mitomycin C cytotoxicity in human tenon fibroblasts.

    PubMed

    Reeves, Graham; Wallis, Richard; Crowston, Jonathan G; Small, Keith M; Wells, Anthony P

    2007-08-01

    To examine the effects of indocyanine green (ICG) with and without mitomycin C (MMC) on proliferation of cultured human Tenon fibroblasts. Fibroblast monolayers were exposed to either MMC [0.4 mg/mL in phosphate buffered saline (PBS)] or PBS containing ICG (0.0625%, 0.125%, 0.25%, and 0.5% in 200 microL PBS) or a combination of MMC (0.4 mg/mL in PBS) and ICG (0.25% and 0.5%) for 5 minutes. Controls were exposed for 5 minutes to MMC, PBS, or culture medium containing no ICG. After treatment, the monolayers were washed and incubated in culture medium for 24, 48, 72 hours, and 1 week periods after which the number of viable cells was quantified. The presence of ICG alone, at concentrations ranging from 0.0625% to 0.5%, had no effect on the rate of fibroblast proliferation measured at any of the incubation periods. As expected, MMC treatment resulted in a significant reduction in viable fibroblast number (8.4+/-0.13x10(3)). ICG in combination with MMC did not significantly alter fibroblast numbers (8.5+/-0.05x10(3)) up to 1 week compared with MMC alone (8.4+/-0.12x10(3)). ICG at concentrations of 0.5% and below do not reduce proliferation of Tenon capsule fibroblasts. ICG did not potentiate or diminish the effect of MMC on Tenon capsule fibroblast proliferation.

  17. Effects of Aloe vera on gap junctional intercellular communication and proliferation of human diabetic and nondiabetic skin fibroblasts.

    PubMed

    Abdullah, Kay M; Abdullah, Ahmed; Johnson, Mary Lynn; Bilski, Jerzy J; Petry, Kimberly; Redmer, Dale A; Reynolds, Lawrence P; Grazul-Bilska, Anna T

    2003-10-01

    To evaluate the effects of Aloe vera on gap junctional intercellular communication (GJIC) and proliferation of human skin fibroblasts in the presence or absence of basic fibroblast growth factor (FGF-2). In vitro study using human type II diabetic and nondiabetic skin fibroblast cell lines. Diabetic (n = 4) and nondiabetic (n = 4) human skin fibroblast cell lines were purchased from Coriell Institute for Medical Research (Camden, NJ). The cells were cultured with or without Aloe vera extract in increasing concentrations (0%, 0.625%, 1.25%, 2.5%, 5%, 10%, and 20%; v/v) in culture medium and with or without FGF-2 (30 ng/mL). GJIC was evaluated after 48-hour incubation with treatments by laser cytometry. Cells were counted after 72-hour incubation with treatments by using a Coulter counter. The rate of GJIC was greater (p < 0.01) for diabetic than for nondiabetic fibroblasts (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute during the first 4 minutes after photobleaching). GJIC was increased ( p < 0.05) for diabetic fibroblasts in the presence of 2.5% and 5% of Aloe vera extract (4.2 +/- 0.1 and 4.0 +/- 0.2 versus 3.5 +/- 0.1% per minute for control, respectively). FGF-2 stimulated (p < 0.01) GJIC for diabetic (4.0 +/- 0.1 versus 3.5 +/- 0.1% per minute for control) and nondiabetic (3.5 +/- 0.1 versus 3.0 +/- 0.1% per minute for control) fibroblasts. Aloe vera extract did not affect GJIC of nondiabetic fibroblast cultured without FGF-2. However, Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on GJIC of diabetic and nondiabetic fibroblasts. Proliferation of diabetic fibroblasts was increased (p < 0.05) by 1.25% and 2.5% Aloe vera extract in medium. Proliferation of nondiabetic fibroblasts was not affected by Aloe vera extract. FGF-2 increased (p < 0.05) proliferation of nondiabetic fibroblasts and FGF-2 did not affect proliferation of diabetic fibroblasts. Aloe vera extract decreased (p < 0.05) FGF-2 stimulatory effects on proliferation of nondiabetic

  18. Early superoxide dismutase alterations during SV40-transformation of human fibroblasts.

    PubMed

    Bravard, A; Hoffschir, F; Sabatier, L; Ricoul, M; Pinton, A; Cassingena, R; Estrade, S; Luccioni, C; Dutrillaux, B

    1992-11-11

    The expression of superoxide dismutases (SOD) 1 and 2 was studied in 4 clones of human fibroblasts after their infection by simian virus 40 (SV40), in parallel with the alterations of chromosomes 21 and chromosome 6q arms, carrying the genes that encode for SOD1 and SOD2 respectively. For all clones, a similar scheme with 2 main phases was observed for both chromosome and SOD variations. The first phase, defined as the pre-crisis phase, was characterized by chromosomal instability, but maintenance of normal numbers of chromosome 6q arms and chromosomes 21. The level of SOD2 mRNA was high, while SOD2 activity and immunoreactive protein were low. SOD1 protein and activity were decreased. In the second phase, defined as the post-crisis phase, the accumulation of clonal chromosomal rearrangements led to the loss of 6q arms, while the number of chromosomes 21 remained normal. SOD2 mRNA level was decreased and SOD2 immunoreactive protein and activity remained low. SOD1 protein and activity increased with passages, reaching values similar to those of control cells at late passages. As in established SV40-transformed human fibroblast cell lines, good correlation was found between SOD2 activity and the relative number of 6q arms. These results allow us to reconstruct the sequence of events leading to the decrease of SOD2, a possible tumor-suppressor gene, during the process of SV40-transformation of human fibroblasts.

  19. Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation

    PubMed Central

    Latif, Najma; Quillon, Alfred; Sarathchandra, Padmini; McCormack, Ann; Lozanoski, Alec; Yacoub, Magdi H.; Chester, Adrian H.

    2015-01-01

    Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the

  20. Effect of novobiocin on the viability of human gingival fibroblasts (HGF-1)

    PubMed Central

    2014-01-01

    Background Novobiocin is a coumarin antibiotic, which affects also eukaryotic cells inhibiting activity of Heat shock protein 90 (Hsp90). The Hsp90 represents a molecular chaperone critical for stabilization and activation of many proteins, particularly oncoproteins that drive cancer progression. Currently, Hsp90 inhibitors focus a significant attention since they form a potentially new class of drugs in therapy of cancer. However, in the process of tumorigenesis a significant role is played also by the microenvironment of the tumour, and, in particular, by cancer-associated fibroblasts (CAFs). This study aimed at examination of the effect played by novobiocin on viability of human gingival fibroblasts (HGF-1). Methods The studies were conducted using 24 h cultures of human gingival fibroblasts – HGF-1 (CRL-2014) in Chamber Slides, in presence of 0.1, 0.5, 1.0, 2.5 or 5.0 mM novobiocin. Cell viability was evaluated using fluorescence test, ATP assay and LDH release. Results Viability of HGF-1 was drastically reduced after 5 hour treatment with novobiocin in concentrations of 1 mM or higher. In turn, the percentage of LDH-releasing cells after 5 h did not differ from control value although it significantly increased after 10 h incubation with 1 mM and continued to increase till the 20th hour. Conclusions The obtained data indicate that novobiocin may induce death of human gingival fibroblasts. Therefore, application of the Hsp90 inhibitor in neoplastic therapy seems controversial: on one hand novobiocin reduces tumour-associated CAFs but, on the other, it may induce a significant destruction of periodontium. PMID:24887242

  1. Differential proteomics reveals S100-A11 as a key factor in aldosterone-induced collagen expression in human cardiac fibroblasts.

    PubMed

    Martínez-Martínez, Ernesto; Ibarrola, Jaime; Lachén-Montes, Mercedes; Fernández-Celis, Amaya; Jaisser, Frederic; Santamaría, Enrique; Fernández-Irigoyen, Joaquín; López-Andrés, Natalia

    2017-08-23

    Aldosterone (Aldo) could induce cardiac fibrosis, a hallmark of heart disease. Aldo direct effects on collagen production in cardiac fibroblasts remain controversial. Our aim is to characterize changes in the proteome of adult human cardiac fibroblasts treated with Aldo to identify new proteins altered that might be new therapeutic targets in cardiovascular diseases. Aldo increased collagens expressions in human cardiac fibroblasts. Complementary, using a quantitative proteomic approach, 30 proteins were found differentially expressed between control and Aldo-treated cardiac fibroblasts. Among these proteins, 7 were up-regulated and 23 were down-regulated by Aldo. From the up-regulated proteins, collagen type I, collagen type III, collagen type VI and S100-A11 were verified by Western blot. Moreover, protein interaction networks revealed a functional link between a third of Aldo-modulated proteome and specific survival routes. S100-A11 was identified as a possible link between Aldo and collagen. Interestingly, CRISPR/Cas9-mediated knock-down of S100-A11 blocked Aldo-induced collagen production in human cardiac fibroblasts. In adult human cardiac fibroblasts treated with Aldo, proteomic analyses revealed an increase in collagen production. S100-A11 was identified as a new regulator of Aldo-induced collagen production in human cardiac fibroblasts. These data could identify new candidate proteins for the treatment of cardiac fibrosis in cardiovascular diseases. S100-A11 is identified by a proteomic approach as a novel regulator of Aldosterone-induced collagen production in human cardiac fibroblasts. Our data could identify new candidate proteins of interest for the treatment of cardiac fibrosis in cardiovascular diseases. Copyright © 2017. Published by Elsevier B.V.

  2. 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

    2018-01-01

    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.

  3. Telocytes and putative stem cells in ageing human heart

    PubMed Central

    Popescu, Laurentiu M; Curici, Antoanela; Wang, Enshi; Zhang, Hao; Hu, Shengshou; Gherghiceanu, Mihaela

    2015-01-01

    Tradition considers that mammalian heart consists of about 70% non-myocytes (interstitial cells) and 30% cardiomyocytes (CMs). Anyway, the presence of telocytes (TCs) has been overlooked, since they were described in 2010 (visit http://www.telocytes.com). Also, the number of cardiac stem cells (CSCs) has not accurately estimated in humans during ageing. We used electron microscopy to identify and estimate the number of cells in human atrial myocardium (appendages). Three age-related groups were studied: newborns (17 days–1 year), children (6–17 years) and adults (34–60 years). Morphometry was performed on low-magnification electron microscope images using computer-assisted technology. We found that interstitial area gradually increases with age from 31.3 ± 4.9% in newborns to 41 ± 5.2% in adults. Also, the number of blood capillaries (per mm2) increased with several hundreds in children and adults versus newborns. CMs are the most numerous cells, representing 76% in newborns, 88% in children and 86% in adults. Images of CMs mitoses were seen in the 17-day newborns. Interestingly, no lipofuscin granules were found in CMs of human newborns and children. The percentage of cells that occupy interstitium were (depending on age): endothelial cells 52–62%; vascular smooth muscle cells and pericytes 22–28%, Schwann cells with nerve endings 6–7%, fibroblasts 3–10%, macrophages 1–8%, TCs about 1% and stem cells less than 1%. We cannot confirm the popular belief that cardiac fibroblasts are the most prevalent cell type in the heart and account for about 20% of myocardial volume. Numerically, TCs represent a small fraction of human cardiac interstitial cells, but because of their extensive telopodes, they achieve a 3D network that, for instance, supports CSCs. The myocardial (very) low capability to regenerate may be explained by the number of CSCs, which decreases fivefold by age (from 0.5% to 0.1% in newborns versus adults). PMID:25545142

  4. 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.

  5. Restoring Effects of Natural Anti-Oxidant Quercetin on Cellular Senescent Human Dermal Fibroblasts.

    PubMed

    Sohn, Eun-Ju; Kim, Jung Min; Kang, Se-Hui; Kwon, Joseph; An, Hyun Joo; Sung, Jung-Suk; Cho, Kyung A; Jang, Ik-Soon; Choi, Jong-Soon

    2018-05-08

    The oxidative damage initiated by reactive oxygen species (ROS) is a major contributor to the functional decline and disability that characterizes aging. The anti-oxidant flavonoid, quercetin, is a plant polyphenol that may be beneficial for retarding the aging process. We examined the restoring properties of quercetin on human dermal fibroblasts (HDFs). Quercetin directly reduced either intracellular or extracellular ROS levels in aged HDFs. To find the aging-related target genes by quercetin, microarray analysis was performed and two up-regulated genes LPL and KCNE2 were identified. Silencing LPL increased the expression levels of senescence proteins such as p16 INK4A and p53 and silencing KCNE2 reversed gene expressions of EGR1 and p-ERK in quercetin-treated aged HDFs. Silencing of LPL and KCNE2 decreased the expression levels of antioxidant enzymes such as superoxide dismutase and catalase. Also, the mitochondrial dysfunction in aged HDFs was ameliorated by quercetin treatment. Taken together, these results suggest that quercetin has restoring effect on the cellular senescence by down-regulation of senescence activities and up-regulation of the gene expressions of anti-oxidant enzymes in aged HDFs.

  6. Hedgehog signaling is synergistically enhanced by nutritional deprivation and ligand stimulation in human fibroblasts of Gorlin syndrome.

    PubMed

    Mizuochi, Hiromi; Fujii, Katsunori; Shiohama, Tadashi; Uchikawa, Hideki; Shimojo, Naoki

    2015-02-13

    Hedgehog signaling is a pivotal developmental pathway that comprises hedgehog, PTCH1, SMO, and GLI proteins. Mutations in PTCH1 are responsible for Gorlin syndrome, which is characterized by developmental defects and tumorigenicity. Although the hedgehog pathway has been investigated extensively in Drosophila and mice, its functional roles have not yet been determined in human cells. In order to elucidate the mechanism by which transduction of the hedgehog signal is regulated in human tissues, we employed human fibroblasts derived from three Gorlin syndrome patients and normal controls. We investigated GLI1 transcription, downstream of hedgehog signaling, to assess native signal transduction, and then treated fibroblasts with a recombinant human hedgehog protein with or without serum deprivation. We also examined the transcriptional levels of hedgehog-related genes under these conditions. The expression of GLI1 mRNA was significantly higher in Gorlin syndrome-derived fibroblasts than in control cells. Hedgehog stimulation and nutritional deprivation synergistically enhanced GLI1 transcription levels, and this was blocked more efficiently by vismodegib, a SMO inhibitor, than by the natural compound, cyclopamine. Messenger RNA profiling revealed the increased expression of Wnt signaling and morphogenetic molecules in these fibroblasts. These results indicated that the hedgehog stimulation and nutritional deprivation synergistically activated the hedgehog signaling pathway in Gorlin syndrome fibroblasts, and this was associated with increments in the transcription levels of hedgehog-related genes such as those involved in Wnt signaling. These fibroblasts may become a significant tool for predicting the efficacies of hedgehog molecular-targeted therapies such as vismodegib. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Cell reprogramming by 3D bioprinting of human fibroblasts in polyurethane hydrogel for fabrication of neural-like constructs.

    PubMed

    Ho, Lin; Hsu, Shan-Hui

    2018-04-01

    3D bioprinting is a technique which enables the direct printing of biodegradable materials with cells into 3D tissue. So far there is no cell reprogramming in situ performed with the 3D bioprinting process. Forkhead box D3 (FoxD3) is a transcription factor and neural crest marker, which was reported to reprogram human fibroblasts into neural crest stem-like cells. In this study, we synthesized a new biodegradable thermo-responsive waterborne polyurethane (PU) gel as a bioink. FoxD3 plasmids and human fibroblasts were co-extruded with the PU hydrogel through the syringe needle tip for cell reprogramming. The rheological properties of the PU hydrogel including the modulus, gelation time, and shear thinning were optimized for the transfection effect of FoxD3 in situ. The corresponding shear rate and shear stress were examined. Results showed that human fibroblasts could be reprogrammed into neural crest stem-like cells with high cell viability during the extrusion process under an average shear stress ∼190 Pa. We further translated the method to the extrusion-based 3D bioprinting, and demonstrated that human fibroblasts co-printed with FoxD3 in the thermo-responsive PU hydrogel could be reprogrammed and differentiated into a neural-tissue like construct at 14 days after induction. The neural-like tissue construct produced by 3D bioprinting from human fibroblasts may be applied to personalized drug screening or neuroregeneration. There is no study so far on cell reprogramming in situ with 3D bioprinting. In this manuscript, a new thermoresponsive polyurethane bioink was developed and employed to deliver FoxD3 plasmid into human fibroblasts by the extrusion-based bioprinting. When the polyurethane gel was extruded through the syringe tip, the shear stress generated may have caused the transient membrane permeability for transfection. The shear stress was optimized for transfection in situ by 3D bioprinting. We demonstrated that human fibroblasts could be

  8. Suitability of human Tenon's fibroblasts as feeder cells for culturing human limbal epithelial stem cells.

    PubMed

    Scafetta, Gaia; Tricoli, Eleonora; Siciliano, Camilla; Napoletano, Chiara; Puca, Rosa; Vingolo, Enzo Maria; Cavallaro, Giuseppe; Polistena, Andrea; Frati, Giacomo; De Falco, Elena

    2013-12-01

    Corneal epithelial regeneration through ex vivo expansion of limbal stem cells (LSCs) on 3T3-J2 fibroblasts has revealed some limitations mainly due to the corneal microenvironment not being properly replicated, thus affecting long term results. Insights into the feeder cells that are used to expand LSCs and the mechanisms underlying the effects of human feeder cells have yet to be fully elucidated. We recently developed a standardized methodology to expand human Tenon's fibroblasts (TFs). Here we aimed to investigate whether TFs can be employed as feeder cells for LSCs, characterizing the phenotype of the co-cultures and assessing what human soluble factors are secreted. The hypothesis that TFs could be employed as alternative human feeder layer has not been explored yet. LSCs were isolated from superior limbus biopsies, co-cultured on TFs, 3T3-J2 or dermal fibroblasts (DFs), then analyzed by immunofluorescence (p63α), colony-forming efficiency (CFE) assay and qPCR for a panel of putative stem cell and epithelial corneal differentiation markers (KRT3). Co-cultures supernatants were screened for a set of soluble factors. Results showed that the percentage of p63α(+)LSCs co-cultured onto TFs was significantly higher than those on DFs (p = 0.032) and 3T3-J2 (p = 0.047). Interestingly, LSCs co-cultures on TFs exhibited both significantly higher CFE and mRNA expression levels of ΔNp63α than on 3T3-J2 and DFs (p < 0.0001), showing also significantly greater levels of soluble factors (IL-6, HGF, b-FGF, G-CSF, TGF-β3) than LSCs on DFs. Therefore, TFs could represent an alternative feeder layer to both 3T3-J2 and DFs, potentially providing a suitable microenvironment for LSCs culture.

  9. Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease.

    PubMed

    Pérez, María J; Ponce, Daniela P; Osorio-Fuentealba, Cesar; Behrens, Maria I; Quintanilla, Rodrigo A

    2017-01-01

    The identification of an early biomarker to diagnose Alzheimer's disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients.

  10. Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease

    PubMed Central

    Pérez, María J.; Ponce, Daniela P.; Osorio-Fuentealba, Cesar; Behrens, Maria I.; Quintanilla, Rodrigo A.

    2017-01-01

    The identification of an early biomarker to diagnose Alzheimer's disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients. PMID:29056898

  11. 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. Copyright © 2015 the American Physiological

  12. Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

    PubMed

    Harris, David M; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

    2011-01-01

    Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

  13. Transformation of Human Mesenchymal Cells and Skin Fibroblasts into Hematopoietic Cells

    PubMed Central

    Harris, David M.; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

    2011-01-01

    Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy. PMID:21731684

  14. Lipopolysaccharide and hypoxia significantly alters interleukin-8 and macrophage chemoattractant protein-1 production by human fibroblasts but not fibrosis related factors.

    PubMed

    Eleftheriadis, T; Liakopoulos, V; Lawson, B; Antoniadi, G; Stefanidis, I; Galaktidou, G

    2011-07-01

    Besides extracellular matrix production, fibroblasts are able to produce various cytokines. Their ubiquitous position makes fibroblasts appropriate cells for sensing various noxious stimuli and for attracting immune cells in the affected area. In the present study the effect of lipopolysaccharide (LPS) and cobalt chloride (CoCl(2)) on the above fibroblasts functions were evaluated in primary human skin fibroblasts cultures. Collagen, matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1, transforming growth factor-β1, interleukin-8 (IL-8) and macrophage chemoattractant protein-1 (MCP-1) were measured in fibroblasts culture supernatants. Fibroblasts proliferation and viability were assessed as well. Hypoxia inducible factor-1α and the phosphorylated p65 portion of NF-κB were assessed in fibroblasts protein extracts. LPS and CoCl(2) had a minor effect on fibrosis related factors in human primary fibroblasts, possibly due to the absence of interplay with other cell types in the used experimental system. On the contrary both LPS and CoCl(2) increased significantly IL-8. LPS also increased considerably MCP-1, but CoCl(2) decreased it. Thus LPS and CoCl(2) induce a sentinel, nevertheless not identical, phenotype in primary human fibroblasts. The last disparity could result in different body response to infectious or hypoxic noxious stimuli.

  15. Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age.

    PubMed

    Wen, Yan; Wani, Prachi; Zhou, Lu; Baer, Tom; Phadnis, Smruti Madan; Reijo Pera, Renee A; Chen, Bertha

    2013-02-01

    We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated β-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.

  16. Antifibrotic effects of tocotrienols on human Tenon's fibroblasts.

    PubMed

    Tappeiner, Christoph; Meyenberg, Alexander; Goldblum, David; Mojon, Daniel; Zingg, Jean-Marc; Nesaretnam, Kalanithi; Kilchenmann, Monika; Frueh, Beatrice E

    2010-01-01

    To compare the antifibrotic effect of vitamin E isoforms alpha-, gamma-, and delta-tocotrienol on human Tenon's fibroblasts (hTf) to the antimetabolite mitomycin C. Antifibrotic effects of alpha- (40, 60, 80, 100, and 120 microM), gamma- (10, 20, 30, and 40 microM) and delta-tocotrienol (10, 20, 30, and 40 microM) on hTf cultures were evaluated by performing proliferation, migration and collagen synthesis assays. Whereas for vitamin E the exposure time was set to 7 days to mimic subconjunctival application, cultures were exposed only 5 min to mitomycin C 100 microg/ml to mimic intraoperative administration. Cell morphology (phase contrast microscopy) as an assessment for cytotoxicity and cell density by measuring DNA content in a fluorometric assay to determine proliferation inhibition was performed on day 0, 4, and 7. Migration ability and collagen synthesis of fibroblasts were measured. All tested tocotrienol isoforms were able to significantly inhibit hTf proliferation in a dose-dependent manner (maximal inhibitory effect without relevant morphological changes at day 4 for alpha-tocotrienol 80 microM with 36.7% and at day 7 for alpha-tocotrienol 80 microM with 42.6% compared to control). Degenerative cell changes were observed in cultures with concentrations above 80 microM for alpha- and above 30 microM for gamma- and delta-tocotrienol. The highest collagen synthesis inhibition has been found with 80 microM alpha-tocotrienol (62.4%) and no significant inhibition for mitomycin C (2.5%). Migration ability was significantly reduced in cultures exposed to 80 microM alpha- and 30 microM gamma-tocotrienol (inhibition of 82.2% and 79.5%, respectively, compared to control) and also after mitomycin C treatment (60.0%). Complete growth inhibition without significant degenerative cell changes could only be achieved with mitomycin C. In vitro, all tested tocotrienol isoforms were able to inhibit proliferation, migration and collagen synthesis of human Tenon's fibroblasts

  17. 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

  18. Multistep carcinogenesis of normal human fibroblasts. Human fibroblasts immortalized by repeated treatment with Co-60 gamma rays were transformed into tumorigenic cells with Ha-ras oncogenes.

    PubMed

    Namba, M; Nishitani, K; Fukushima, F; Kimoto, T

    1988-01-01

    Two normal mortal human fibroblast cell strains were transformed into immortal cell lines, SUSM-1 and KMST-6, by treatment with 4-nitroquinoline 1-oxide (4NQO) and Co-60 gamma rays, respectively. These immortalized cell lines showed morphological changes of cells and remarkable chromosome aberrations, but neither of them grew in soft agar or formed tumors in nude mice. The immortal cell line, KMST-6, was then converted into neoplastic cells by treatment with Harvey murine sarcoma virus (Ha-MSV) or the c-Ha-ras oncogene derived from a human lung carcinoma. These neoplastically transformed cells acquired anchorage-independent growth potential and developed tumors when transplanted into nude mice. All the tumors grew progressively without regression until the animals died of tumors. In addition, the tumors were transplantable into other nude mice. Normal human fibroblasts, on the other hand, were not transformed into either immortal or tumorigenic cells by treatment with Ha-MSV or c-Ha-ras alone. Our present data indicate that (1) the chemical carcinogen, 4NQO, or gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to immortality, and (2) the ras gene played a role in the progression of the immortally transformed cells to more malignant cells showing anchorage-independent growth and tumorigenicity. In other words, the immortalization process of human cells seems to be a pivotal or rate-limiting step in the carcinogenesis of human cells.

  19. Interferon-alpha and interferon-gamma modulate Fas-mediated apoptosis in mitomycin-C-resistant human Tenon's fibroblasts.

    PubMed

    Wang, Xiao Yang; Crowston, Jonathan G; White, Andrew J R; Zoellner, Hans; Healey, Paul R

    2014-08-01

    The aim of the study was to investigate, using a native mitomycin-C-resistant human Tenon's fibroblast cell line, the possibility that interferon-alpha and gamma could be used with Fas agonists as an alternative anti-fibrotic strategy to mitomycin-C in trabeculectomy. A clinically resistant and in vitro verified mitomycin-C-resistant human Tenon's fibroblast cell line was pretreated with interferon-alpha and interferon-gamma for 48 h before stimulation with an agonistic Fas antibody (CH11) for 2 days to induce cell death. Cell death assays were undertaken. Changes in apoptosis-related proteins were determined by flow cytometry and Western blot. Pretreatment with interferon-alpha or interferon-gamma for 48 h increased Fas, Fas-associated protein with death domain and caspase-8 expression. Protein expression was further increased by combined exposure to interferon-alpha and gamma. Pretreatment with cytokines had no effect on Fas-L and Bcl-2. Interferon-alpha alone did not change the rate of induced cell death. A combination of interferon-alpha and gamma synergistically increased the sensitivity of mitomycin-C-resistant human Tenon's fibroblast cell line to induced cell death. An antagonistic anti-Fas antibody (ZB4) completely blocked induced cell death. Broad caspase inhibitors specific for caspases-8 and -3 reduced induced deaths in interferon pretreated mitomycin-C-resistant human Tenon's fibroblast cell line in a dose-dependent manner. Interferon-alpha and interferon-gamma render mitomycin-C-resistant human Tenon's fibroblast cell line sensitive to Fas-mediated apoptosis. The mechanism involves increased death-inducing signalling complex formation by upregulation of Fas, Fas-associated protein with death domain and caspase-8 expression. © 2013 Royal Australian and New Zealand College of Ophthalmologists.

  20. IFN-Dependent and -Independent Reduction in West Nile Virus Infectivity in Human Dermal Fibroblasts

    PubMed Central

    Hoover, Lisa I.; Fredericksen, Brenda L.

    2014-01-01

    Although dermal fibroblasts are one of the first cell types exposed to West Nile virus (WNV) during a blood meal by an infected mosquito, little is known about WNV replication within this cell type. Here, we demonstrate that neuroinvasive, WNV-New York (WNV-NY), and nonneuroinvasive, WNV-Australia (WNV-AUS60) strains are able to infect and replicate in primary human dermal fibroblasts (HDFs). However, WNV-AUS60 replication and spread within HDFs was reduced compared to that of WNV-NY due to an interferon (IFN)-independent reduction in viral infectivity early in infection. Additionally, replication of both strains was constrained late in infection by an IFN-β-dependent reduction in particle infectivity. Overall, our data indicates that human dermal fibroblasts are capable of supporting WNV replication; however, the low infectivity of particles produced from HDFs late in infection suggests that this cell type likely plays a limited role as a viral reservoir in vivo. PMID:24662674

  1. Mitochondrial damage and cytoskeleton reorganization in human dermal fibroblasts exposed to artificial visible light similar to screen-emitted light.

    PubMed

    Rascalou, Adeline; Lamartine, Jérôme; Poydenot, Pauline; Demarne, Frédéric; Bechetoille, Nicolas

    2018-05-05

    Artificial visible light is everywhere in modern life. Social communication confronts us with screens of all kinds, and their use is on the rise. We are therefore increasingly exposed to artificial visible light, the effects of which on skin are poorly known. The purpose of this study was to model the artificial visible light emitted by electronic devices and assess its effect on normal human fibroblasts. The spectral irradiance emitted by electronic devices was optically measured and equipment was developed to accurately reproduce such artificial visible light. Effects on normal human fibroblasts were analyzed on human genome microarray-based gene expression analysis. At cellular level, visualization and image analysis were performed on the mitochondrial network and F-actin cytoskeleton. Cell proliferation, ATP release and type I procollagen secretion were also measured. We developed a device consisting of 36 LEDs simultaneously emitting blue, green and red light at distinct wavelengths (450 nm, 525 nm and 625 nm) with narrow spectra and equivalent radiant power for the three colors. A dose of 99 J/cm 2 artificial visible light was selected so as not to induce cell mortality following exposure. Microarray analysis revealed 2984 light-modulated transcripts. Functional annotation of light-responsive genes revealed several enriched functions including, amongst others, the "mitochondria" and "integrin signaling" categories. Selected results were confirmed by real-time quantitative PCR, analyzing 24 genes representing these two categories. Analysis of micro-patterned culture plates showed marked fragmentation of the mitochondrial network and disorganization of the F-actin cytoskeleton following exposure. Functionally, there was considerable impairment of cell growth and spread, ATP release and type I procollagen secretion in exposed fibroblasts. Artificial visible light induces drastic molecular and cellular changes in normal human fibroblasts. This may impede

  2. Polyphenol-rich strawberry extract protects human dermal fibroblasts against hydrogen peroxide oxidative damage and improves mitochondrial functionality.

    PubMed

    Giampieri, Francesca; Alvarez-Suarez, José M; Mazzoni, Luca; Forbes-Hernandez, Tamara Y; Gasparrini, Massimiliano; Gonzàlez-Paramàs, Ana M; Santos-Buelga, Celestino; Quiles, José L; Bompadre, Stefano; Mezzetti, Bruno; Battino, Maurizio

    2014-06-11

    Strawberry bioactive compounds are widely known to be powerful antioxidants. In this study, the antioxidant and anti-aging activities of a polyphenol-rich strawberry extract were evaluated using human dermal fibroblasts exposed to H2O2. Firstly, the phenol and flavonoid contents of strawberry extract were studied, as well as the antioxidant capacity. HPLC-DAD analysis was performed to determine the vitamin C and β-carotene concentration, while HPLC-DAD/ESI-MS analysis was used for anthocyanin identification. Strawberry extract presented a high antioxidant capacity, and a relevant concentration of vitamins and phenolics. Pelargonidin- and cyanidin-glycosides were the most representative anthocyanin components of the fruits. Fibroblasts incubated with strawberry extract and stressed with H2O2 showed an increase in cell viability, a smaller intracellular amount of ROS, and a reduction of membrane lipid peroxidation and DNA damage. Strawberry extract was also able to improve mitochondrial functionality, increasing the basal respiration of mitochondria and to promote a regenerative capacity of cells after exposure to pro-oxidant stimuli. These findings confirm that strawberries possess antioxidant properties and provide new insights into the beneficial role of strawberry bioactive compounds on protecting skin from oxidative stress and aging.

  3. Human gingival fibroblast response to electropolished NiTi surfaces.

    PubMed

    Es-Souni, Martha; Fischer-Brandies, Helge; Es-Souni, Mohammed

    2007-01-01

    In the present study the in vitro biocompatibility of electropolished NiTi sheets is investigated. The assessment of cytotoxic effects due to potential Ni leaching from metal sheets was performed in direct contact with primary human fibroblast cultures using the 5-bromo-2'-deoxyuridine cell proliferation assay and morphologic studies via light microscopy and scanning electron microscopy. To assess toxic effects related to Ni-ions release, cells cultured in the presence of increasing concentrations of Ni(2+) (NiSO(4).6H(2)O) served as positive controls. It is shown that while the addition of NiSO(4) caused severe proliferation decrease (approximately 80%) and morphologic damage at a concentration of 50 mg/L Ni(2+) no negative effects were observed in fibroblasts cultured in the presence of electropolished NiTi sheets. The results are discussed in terms of surface topography effects on the biocompatibility of NiTi shape memory alloys. (c) 2006 Wiley Periodicals, Inc.

  4. [Possibilities and limitations of fibroblast cultures in the study of animal aging].

    PubMed

    Van Gansen, P; Van Lerberghe, N

    1987-01-01

    INTRODUCTION. Aging--the effect of time--occurs in every living organism. Senescence is the last period of the lifespan, leading to death. It happens in all animals, with the exception of a few didermic species (Hydras) having a stock of embryonic cells and being immortal. The causes of animal senescence are badly known. They depend both on genetic characters (maximal lifespan of a species) and on medium factors (mean expectation of life of the animals of a species). Animal senescence could depend on cell aging: 1) by senescence and death of the differentiated cells, 2) by modified proliferation and differentiation of the stem cells of differentiated tissues, 3) by alterations in the extracellular matrices, 4) by interactions between factors 1) 2) and 3) in each tissue, 5) by interactions between the several tissues of an organism. This complexity badly impedes the experimental study of animal senescence. Normal mammal cells are aging when they are cultivated (in vitro ageing): their phenotype varies and depends on the cell generation (in vitro differentiation); the last cell-generation doesn't divide anymore and declines until death of the culture (in vitro senescence). Analysis of these artificial but well controlled systems allows an experimental approach of the proliferation, differentiation, senescence and death of the cells and of the extracellular matrix functions. Present literature upon in vitro aging of cultivated human cells is essentially made of papers where proliferation and differentiation characteristics are compared between early ("young") and late ("old") cell-generations of the cultures. FIBROBLASTIC CELLS OF THE MOUSE SKIN. This cell type has been studied in our laboratory, using different systems: 1) Primary cultures isolated from peeled skins of 19 day old mouse embryos, 2) Mouse dermis analyzed in the animals, 3) Cultivated explants of skins, 4) Serial sub-cultures of fibroblasts isolated from these explants, 5) Cells cultivated comparably on

  5. Poly(I:C) induces expressions of MMP-1, -2, and -3 through various signaling pathways including IRF3 in human skin fibroblasts.

    PubMed

    Yao, Cheng; Lee, Dong Hun; Oh, Jang-Hee; Kim, Min-Kyoung; Kim, Kyu Han; Park, Chi-Hyun; Chung, Jin Ho

    2015-10-01

    Ultraviolet (UV) irradiation can result in premature skin aging (photoaging) which is characterized by decreased expression of collagen and increased expression of matrix metalloproteinases (MMPs). Double-stranded RNAs (dsRNAs) can be generated at various conditions including virally infected cells or UV-damaged skin cells. Recent studies have shown that a synthetic dsRNA, polyinosinic-polycytidylic acid (poly(I:C)), can reduce procollagen expression in human skin fibroblasts. However, little is known about the effect of poly(I:C) on the expression of MMPs in skin fibroblasts and its underlying mechanisms. We examined the effect of poly(I:C) on MMP-1, -2, and -3 expressions in human skin fibroblasts. Then, we further explored the underlying signaling pathways involved in the processes. Human skin fibroblasts were treated with poly(I:C) for the indicated times in the presence or the absence of various chemical inhibitors or small interfering RNAs (siRNAs) at the indicated concentrations. Protein and mRNA levels of various target molecules were examined by Western blotting and quantitative real-time PCR, respectively. Poly(I:C) induced MMP-1, -2, and -3 expressions, which were dependent on TLR3. Poly(I:C) also induced activations of the mitogen-activated protein kinases (MAPKs), the nuclear factor-kappaB (NF-κB) and the interferon regulatory factor 3 (IRF3) pathways. By using specific inhibitors, we found that poly(I:C)-induced expressions of MMP-1, -2, and -3 were differentially regulated by these signaling pathways. In particular, we found that the inhibition of IRF3 signaling pathways attenuated poly(I:C)-induced expressions of all the three MMPs. Our data show that the expressions of MMP-1, -2, and -3 are induced by poly(I:C) through various signaling pathways in human skin fibroblasts and suggest that TLR3 and/or IRF3 may be good targets for regulating the expressions of MMP-1, -2, and -3 induced by dsRNAs. Copyright © 2015 Elsevier Ireland Ltd. All rights

  6. Different dose rate-dependent responses of human melanoma cells and fibroblasts to low dose fast neutrons.

    PubMed

    Dionet, Claude; Müller-Barthélémy, Melanie; Marceau, Geoffroy; Denis, Jean-Marc; Averbeck, Dietrich; Gueulette, John; Sapin, Vincent; Pereira, Bruno; Tchirkov, Andrei; Chautard, Emmanuel; Verrelle, Pierre

    2016-09-01

    To analyze the dose rate influence in hyper-radiosensitivity (HRS) of human melanoma cells to very low doses of fast neutrons and to compare to the behaviour of normal human skin fibroblasts. We explored different neutron dose rates as well as possible implication of DNA double-strand breaks (DSB), apoptosis, and energy-provider adenosine-triphosphate (ATP) levels during HRS. HRS in melanoma cells appears only at a very low dose rate (VLDR), while a high dose rate (HDR) induces an initial cell-radioresistance (ICRR). HRS does not seem to be due either to DSB or to apoptosis. Both phenomena (HRS and ICRR) appear to be related to ATP availability for triggering cell repair. Fibroblast survival after neutron irradiation is also dose rate-dependent but without HRS. Melanoma cells or fibroblasts exert their own survival behaviour at very low doses of neutrons, suggesting that in some cases there is a differential between cancer and normal cells radiation responses. Only the survival of fibroblasts at HDR fits the linear no-threshold model. This new insight into human cell responses to very low doses of neutrons, concerns natural radiations, surroundings of accelerators, proton-therapy devices, flights at high altitude. Furthermore, ATP inhibitors could increase HRS during high-linear energy transfer (high-LET) irradiation.

  7. 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.

  8. MiR-590 Promotes Transdifferentiation of Porcine and Human Fibroblasts Toward a Cardiomyocyte-Like Fate by Directly Repressing Specificity Protein 1.

    PubMed

    Singh, Vivek P; Mathison, Megumi; Patel, Vivekkumar; Sanagasetti, Deepthi; Gibson, Brian W; Yang, Jianchang; Rosengart, Todd K

    2016-11-10

    Reprogramming of cardiac fibroblasts into induced cardiomyocyte-like cells represents a promising potential new therapy for treating heart disease, inducing significant improvements in postinfarct ventricular function in rodent models. Because reprogramming factors effective in transdifferentiating rodent cells are not sufficient to reprogram human cells, we sought to identify reprogramming factors potentially applicable to human studies. Lentivirus vectors expressing Gata4, Mef2c, and Tbx5 (GMT); Hand2 (H), Myocardin (My), or microRNA (miR)-590 were administered to rat, porcine, and human cardiac fibroblasts in vitro. induced cardiomyocyte-like cell production was then evaluated by assessing expression of the cardiomyocyte marker, cardiac troponin T (cTnT), whereas signaling pathway studies were performed to identify reprogramming factor targets. GMT administration induced cTnT expression in ≈6% of rat fibroblasts, but failed to induce cTnT expression in porcine or human cardiac fibroblasts. Addition of H/My and/or miR-590 to GMT administration resulted in cTNT expression in ≈5% of porcine and human fibroblasts and also upregulated the expression of the cardiac genes, MYH6 and TNNT2. When cocultured with murine cardiomyocytes, cTnT-expressing porcine cardiac fibroblasts exhibited spontaneous contractions. Administration of GMT plus either H/My or miR-590 alone also downregulated fibroblast genes COL1A1 and COL3A1. miR-590 was shown to directly suppress the zinc finger protein, specificity protein 1 (Sp1), which was able to substitute for miR-590 in inducing cellular reprogramming. These data support porcine studies as a surrogate for testing human cardiac reprogramming, and suggest that miR-590-mediated repression of Sp1 represents an alternative pathway for enhancing human cardiac cellular reprogramming. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  9. Accelerated telomere shortening and replicative senescence in human fibroblasts overexpressing mutant and wild-type lamin A

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang Shurong; Risques, Rosa Ana; Martin, George M.

    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. Tomore » 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 detectable 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.« less

  10. Antiproliferative effect of methanolic extraction of tualang honey on human keloid fibroblasts

    PubMed Central

    2011-01-01

    Background Keloid is a type of scar which extends beyond the boundaries of the original wound. It can spread to the surrounding skin by invasion. The use of Tualang honey is a possible approach for keloid treatment. The objective of this study was to determine the antiproliferative effect of methanolic extraction of Tualang honey to primary human keloid fibroblasts and to identify the volatile compounds in methanol extraction of Tualang honey. Methods Crude Tualang honey was extracted with methanol and then dried using rota vapor to remove remaining methanol from honey. Normal and keloid fibroblasts were verified and treated with the extracted honey. Cell proliferation was tested with [3-(4,5-dimethylthiazol-2-yi)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay. Extraction of Tualang honey using methanol was carried out and the extracted samples were analysed using gas chromatography-mass spectrometry (GC-MS). The result was analysed using SPSS and tested with Kruskal-Wallis and Mann-Whitney tests. Results Methanolic extraction of honey has positive anti proliferative effect on keloid fibroblasts in a dose-dependent manner. The presence of fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid and octadecanoic acid may contribute to the anti-proliferative effect in keloid fibroblasts. Conclusions The methanolic honey extraction has an antiproliferative effect on keloid fibroblasts and a range of volatile compounds has been identified from Tualang honey. The antiproliferative effect of keloid fibroblasts towards Tualang honey may involve cell signaling pathway. Identifying other volatile compounds from different organic solvents should be carried out in future. PMID:21943200

  11. Effect of Fish Collagen Hydrolysates on Type I Collagen mRNA Levels of Human Dermal Fibroblast Culture

    PubMed Central

    Sanchez, Ana; Blanco, Maria; Correa, Begoña

    2018-01-01

    Fish discards and subproducts may represent an important source of raw material, not only for the food industry, but for other different kind of industries, such as the nutraceutical and cosmetic industries. Collagen, which is mainly obtained from animal skins, is an important structural protein in the animal kingdom having many different applications. It is well known that fish skins constitute a significant subproduct in the fishery industry, especially in the case of some species, where fish skins may represent up to 20% of the total body weight of fish. Peptides from collagen hydrolysates have been described to be useful for preventing skin aging and osteoarthritis, however, the mechanism for these biological activities is not well known. Fibroblasts are the main cell types involved in the collagen synthesis, and in the present work, human dermal fibroblasts have been exposed to the treatment of collagen peptides of two different molecular weight ranges. Results show that higher molecular weight collagen peptides produce higher synthesis of collagen type I mRNA and, therefore, it may suggest that prior molecular weight selection may be an important step to maximize the effect of collagen hydrolysates on collagen type I synthesis by dermal fibroblasts. PMID:29701725

  12. Comparison of gene expression profiles in primary and immortalized human pterygium fibroblast cells.

    PubMed

    Hou, Aihua; Voorhoeve, P Mathijs; Lan, Wanwen; Tin, Minqi; Tong, Louis

    2013-11-01

    Pterygium is a fibrovascular growth on the ocular surface with corneal tissue destruction, matrix degradation and varying extents of chronic inflammation. To facilitate investigation of pterygium etiology, we immortalized pterygium fibroblast cells and profiled their global transcript levels compared to primary cultured cells. Fibroblast cells were cultured from surgically excised pterygium tissue using the explant method and propagated to passage number 2-4. We hypothesized that intervention with 3 critical molecular intermediates may be necessary to propage these cells. Primary fibroblast cells were immortalized sequentially by a retroviral construct containing the human telomerase reverse transcriptase gene and another retroviral expression vector expressing p53/p16 shRNAs. Primary and immortalized fibroblast cells were evaluated for differences in global gene transcript levels using an Agilent Genechip microarray. Light microscopic morphology of immortalized cells was similar to primary pterygium fibroblast at passage 2-4. Telomerase reverse transcriptase was expressed, and p53 and p16 levels were reduced in immortalized pterygium fibroblast cells. There were 3308 significantly dysregulated genes showing at least 2 fold changes in transcript levels between immortalized and primary cultured cells (2005 genes were up-regulated and 1303 genes were down-regulated). Overall, 13.58% (95% CI: 13.08-14.10) of transcripts in immortalized cells were differentially expressed by at least 2 folds compared to primary cells. Pterygium primary fibroblast cells were successfully immortalized to at least passage 11. Although a variety of genes are differentially expressed between immortalized and primary cells, only genes related to cell cycle are significantly changed, suggesting that the immortalized cells may be used as an in vitro model for pterygium pathology. © 2013 Elsevier Inc. All rights reserved.

  13. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization

    PubMed Central

    Deglesne, Pierre-Antoine; Arroyo, Rodrigo; Ranneva, Evgeniya; Deprez, Philippe

    2016-01-01

    Mesotherapy/biorevitalization with hyaluronic acid (HA) is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates) HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15%) and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts. PMID:26966384

  14. Sodium arsenite induces chromosome endoreduplication and inhibits protein phosphatase activity in human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Rong-Nan Huang; I-Ching Ho; Ling-Hui Yih

    Arsenic, strongly associated with increased risks of human cancers, is a potent clastogen in a variety of mammalian cell systems. The effect of sodium arsenite (a trivalent arsenic compound) on chromatid separation was studied in human skin fibroblasts (HFW). Human fibroblasts were arrested in S phase by the aid of serum starvation and aphidicolin blocking and then these cells were allowed to synchronously progress into G2 phase. Treatment of the G2-enriched HFW cells with sodium arsenite (0-200 {mu}M) resulted in arrest of cells in the G2 phase, interference with mitotic division, inhibition of spindle assembly, and induction of chromosome endoreduplicationmore » in their second mitosis. Sodium arsenite treatment also inhibited the activities of serine/threonine protein phosphatases and enhanced phosphorylation levels of a small heat shock protein (HSP27). These results suggest that sodium arsenite may mimic okadaic acid to induce chromosome endoreduplication through its inhibitory effect on protein phosphatase activity. 61 refs., 6 figs., 2 tabs.« less

  15. Role of human pulmonary fibroblast-derived MCP-1 in cell activation and migration in experimental silicosis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Xueting; Fang, Shencun; Liu, Haijun

    Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO{sub 2}). Phagocytosis of SiO{sub 2} in the lung initiates an inflammatory cascade that results in fibroblast proliferation and migration and subsequent fibrosis. Clinical evidence indicates that the activation of alveolar macrophages by SiO{sub 2} produces rapid and sustained inflammation that is characterized by the generation of monocyte chemotactic protein 1 (MCP-1), which induces fibrosis. Pulmonary fibroblast-derived MCP-1 may play a critical role in fibroblast proliferation and migration. Methods and results: Experiments using primary cultured adult human pulmonary fibroblasts (HPF-a) demonstrated the following results: 1) SiO{sub 2} treatment resultedmore » in the rapid and sustained induction of MCP-1 as well as the elevation of the CC chemokine receptor type 2 (CCR2) protein levels; 2) pretreatment of HPF-a with RS-102895, a specific CCR2 inhibitor, abolished the SiO{sub 2}-induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO{sub 2}-induced increase in cell migration. Conclusion: These data demonstrated that the up-regulation of pulmonary fibroblast-derived MCP-1 is involved in pulmonary fibroblast migration induced by SiO{sub 2}. CCR2 was also up-regulated in response to SiO{sub 2}, and this up-regulation facilitated the effect of MCP-1 on fibroblasts. Our study deciphered the link between fibroblast-derived MCP-1 and SiO{sub 2}-induced cell migration. This finding provides novel insight into the potential of MCP-1 in the development of novel therapeutic strategies for silicosis. - Highlights: • Role of pulmonary fibroblast-derived MCP-1 in experimental silicosis was studied. • SiO{sub 2} induced MCP-1 release from cultured human pulmonary fibroblast (HPF-a). • SiO{sub 2} directly activated HPF-a via the MCP-1/CCR2 pathway. • SiO{sub 2} increased HPF-a migration in both 2D

  16. 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

  17. 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

  18. 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.

  19. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Murata, Naohiko; Ito, Satoru, E-mail: itori@med.nagoya-u.ac.jp; Furuya, Kishio

    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 intracellularmore » 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.« less

  20. 4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

    PubMed Central

    1984-01-01

    The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts. PMID:6538202

  1. Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts.

    PubMed

    Crowston, Jonathan G; Chang, Lydia H; Constable, Peter H; Daniels, Julie T; Akbar, Arne N; Khaw, Peng T

    2002-03-01

    To examine the effect of mitomycin-C on the expression of apoptosis genes in human Tenon capsule fibroblasts and to evaluate whether death receptor signaling modulates mitomycin-C cytotoxicity. Bcl-2, Bax, Bcl-x, Fas (CD95) and tumor necrosis factor (TNF) receptor expression was determined by flow cytometry in control and mitomycin-C-treated Tenon fibroblasts. Fibroblast death was quantified using a lactate dehydrogenase release assay. The effect of Fas and TNF-receptor signaling was evaluated using Fas-specific antibodies and soluble TNF-alpha. Tenon fibroblasts constitutively express Bcl-2, Bax, and Bcl-x in culture. Mitomycin-C (0.4 mg/mL) induced a small but consistent increase in the expression of all three proteins. Tenon fibroblasts express low levels of Fas but are resistant to the effects of Fas-receptor ligation. Mitomycin-C (0.01-1.0 mg/mL) led to a significant increase in Fas expression at all concentrations tested (P < 0.01). Pretreatment with mitomycin-C (0.4 mg/mL) rendered fibroblasts susceptible to agonistic anti-Fas monoclonal IgM antibodies (50-500 ng/mL) and led to a further 50% reduction in viable fibroblasts at 48 hours, compared with mitomycin-C alone (P < 0.05). Antibodies that block the Fas receptor did not inhibit mitomycin-C-induced apoptosis. Mitomycin-C alters apoptosis gene expression and primes fibroblasts to the effects of Fas receptor ligation. Factors other than the level of Fas receptor expression modulate the response to Fas receptor signaling. Determining the signals that regulate fibroblast apoptosis may help to refine therapeutic strategies for switching off the subconjunctival healing response and maintaining intraocular pressure control.

  2. Anti-inflammatory activity of clove (Eugenia caryophyllata) essential oil in human dermal fibroblasts.

    PubMed

    Han, Xuesheng; Parker, Tory L

    2017-12-01

    Clove (Eugenia caryophyllata Thunb. [Myrtaceae]) essential oil (CEO) has been shown to possess antimicrobial, antifungal, antiviral, antioxidant, anti-inflammatory and anticancer properties. However, few studies have focused on its topical use. We investigated the biological activity of a commercially available CEO in a human skin disease model. We evaluated the effect of CEO on 17 protein biomarkers that play critical roles in inflammation and tissue remodelling in a validated human dermal fibroblast system, which was designed to model chronic inflammation and fibrosis. Four concentrations of CEO (0.011, 0.0037, 0.0012, and 0.00041%, v/v) were studied. The effect of 0.011% CEO on genome-wide gene expression was also evaluated. CEO at a concentration of 0.011% showed robust antiproliferative effects on human dermal fibroblasts. It significantly inhibited the increased production of several proinflammatory biomarkers such as vascular cell adhesion molecule-1 (VCAM-1), interferon γ-induced protein 10 (IP-10), interferon-inducible T-cell α chemoattractant (I-TAC), and monokine induced by γ interferon (MIG). CEO also significantly inhibited tissue remodelling protein molecules, namely, collagen-I, collagen-III, macrophage colony-stimulating factor (M-CSF), and tissue inhibitor of metalloproteinase 2 (TIMP-2). Furthermore, it significantly modulated global gene expression and altered signalling pathways critical for inflammation, tissue remodelling, and cancer signalling processes. CEO significantly inhibited VCAM-1 and collagen III at both protein and gene expression levels. This study provides important evidence of CEO-induced anti-inflammatory and tissue remodelling activity in human dermal fibroblasts. This study also supports the anticancer properties of CEO and its major active component eugenol.

  3. Stochastic variation in telomere shortening rate causes heterogeneity of human fibroblast replicative life span.

    PubMed

    Martin-Ruiz, Carmen; Saretzki, Gabriele; Petrie, Joanne; Ladhoff, Juliane; Jeyapalan, Jessie; Wei, Wenyi; Sedivy, John; von Zglinicki, Thomas

    2004-04-23

    The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.

  4. Oxidants produced by methylglyoxal-modified collagen trigger ER stress and apoptosis in skin fibroblasts.

    PubMed

    Nowotny, Kerstin; Castro, José Pedro; Hugo, Martín; Braune, Sabine; Weber, Daniela; Pignitter, Marc; Somoza, Veronika; Bornhorst, Julia; Schwerdtle, Tanja; Grune, Tilman

    2018-05-20

    Methylglyoxal (MG), a highly reactive dicarbonyl, interacts with proteins to form advanced glycation end products (AGEs). AGEs include a variety of compounds which were shown to have damaging potential and to accumulate in the course of different conditions such as diabetes mellitus and aging. After confirming collagen as a main target for MG modifications in vivo within the extracellular matrix, we show here that MG-collagen disrupts fibroblast redox homeostasis and induces endoplasmic reticulum (ER) stress and apoptosis. In particular, MG-collagen-induced apoptosis is associated with the activation of the PERK-eIF2α pathway and caspase-12. MG-collagen contributes to altered redox homeostasis by directly generating hydrogen peroxide and oxygen-derived free radicals. The induction of ER stress in human fibroblasts was confirmed using collagen extracts isolated from old mice in which MG-derived AGEs were enriched. In conclusion, MG-derived AGEs represent one factor contributing to diminished fibroblast function during aging. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  5. circHIPK2-mediated σ-1R promotes endoplasmic reticulum stress in human pulmonary fibroblasts exposed to silica.

    PubMed

    Cao, Zhouli; Xiao, Qingling; Dai, Xiaoniu; Zhou, Zewei; Jiang, Rong; Cheng, Yusi; Yang, Xiyue; Guo, Huifang; Wang, Jing; Xi, Zhaoqing; Yao, Honghong; Chao, Jie

    2017-12-13

    Silicosis is characterized by fibroblast accumulation and excessive deposition of extracellular matrix. Although the roles of SiO 2 -induced chemokines and cytokines released from alveolar macrophages have received significant attention, the direct effects of SiO 2 on protein production and functional changes in pulmonary fibroblasts have been less extensively studied. Sigma-1 receptor, which has been associated with cell proliferation and migration in the central nervous system, is expressed in the lung, but its role in silicosis remains unknown. To elucidate the role of sigma-1 receptor in fibrosis induced by silica, both the upstream molecular mechanisms and the functional effects on cell proliferation and migration were investigated. Both molecular biological assays and pharmacological techniques, combined with functional experiments, such as migration and proliferation, were applied in human pulmonary fibroblasts from adults to analyze the molecular and functional changes induced by SiO 2 . SiO 2 induced endoplasmic reticulum stress in association with enhanced expression of sigma-1 receptor. Endoplasmic reticulum stress promoted migration and proliferation of human pulmonary fibroblasts-adult exposed to SiO 2 , inducing the development of silicosis. Inhibition of sigma-1 receptor ameliorated endoplasmic reticulum stress and fibroblast functional changes induced by SiO 2 . circHIPK2 is involved in the regulation of sigma-1 receptor in human pulmonary fibroblasts-adult exposed to SiO 2 . Our study elucidated a link between SiO 2 -induced fibrosis and sigma-1 receptor signaling, thereby providing novel insight into the potential use of sigma-1 receptor/endoplasmic reticulum stress in the development of novel therapeutic strategies for silicosis treatment.

  6. The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts.

    PubMed

    Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

    2017-01-01

    Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6-78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts.

  7. The effects of collagen-rich extracellular matrix on the intracellular delivery of glycol chitosan nanoparticles in human lung fibroblasts

    PubMed Central

    Yhee, Ji Young; Yoon, Hong Yeol; Kim, Hyunjoon; Jeon, Sangmin; Hergert, Polla; Im, Jintaek; Panyam, Jayanth; Kim, Kwangmeyung; Nho, Richard Seonghun

    2017-01-01

    Recent progress in nanomedicine has shown a strong possibility of targeted therapy for obstinate chronic lung diseases including idiopathic pulmonary fibrosis (IPF). IPF is a fatal lung disease characterized by persistent fibrotic fibroblasts in response to type I collagen-rich extracellular matrix. As a pathological microenvironment is important in understanding the biological behavior of nanoparticles, in vitro cellular uptake of glycol chitosan nanoparticles (CNPs) in human lung fibroblasts was comparatively studied in the presence or absence of type I collagen matrix. Primary human lung fibroblasts from non-IPF and IPF patients (n=6/group) showed significantly increased cellular uptake of CNPs (>33.6–78.1 times) when they were cultured on collagen matrix. To elucidate the underlying mechanism of enhanced cellular delivery of CNPs in lung fibroblasts on collagen, cells were pretreated with chlorpromazine, genistein, and amiloride to inhibit clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis, respectively. Amiloride pretreatment remarkably reduced the cellular uptake of CNPs, suggesting that lung fibroblasts mainly utilize the macropinocytosis-dependent mechanism when interacted with collagen. In addition, the internalization of CNPs was predominantly suppressed by a phosphoinositide 3-kinase (PI3K) inhibitor in IPF fibroblasts, indicating that enhanced PI3K activity associated with late-stage macropinocytosis can be particularly important for the enhanced cellular delivery of CNPs in IPF fibroblasts. Our study strongly supports the concept that a pathological microenvironment which surrounds lung fibroblasts has a significant impact on the intracellular delivery of nanoparticles. Based on the property of enhanced intracellular delivery of CNPs when fibroblasts are made to interact with a collagen-rich matrix, we suggest that CNPs may have great potential as a drug-carrier system for targeting fibrotic lung fibroblasts. PMID

  8. 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

  9. MiR-191 Regulates Primary Human Fibroblast Proliferation and Directly Targets Multiple Oncogenes

    PubMed Central

    Polioudakis, Damon; Abell, Nathan S.; Iyer, Vishwanath R.

    2015-01-01

    miRNAs play a central role in numerous pathologies including multiple cancer types. miR-191 has predominantly been studied as an oncogene, but the role of miR-191 in the proliferation of primary cells is not well characterized, and the miR-191 targetome has not been experimentally profiled. Here we utilized RNA induced silencing complex immunoprecipitations as well as gene expression profiling to construct a genome wide miR-191 target profile. We show that miR-191 represses proliferation in primary human fibroblasts, identify multiple proto-oncogenes as novel miR-191 targets, including CDK9, NOTCH2, and RPS6KA3, and present evidence that miR-191 extensively mediates target expression through coding sequence (CDS) pairing. Our results provide a comprehensive genome wide miR-191 target profile, and demonstrate miR-191’s regulation of primary human fibroblast proliferation. PMID:25992613

  10. Inhibitory effect of mitomycin C on proliferation of primary cultured fibroblasts from human airway granulation tissues.

    PubMed

    Chen, Nan; Zhang, Jie; Xu, Min; Wang, Yu Ling; Pei, Ying Hua

    2013-01-01

    Airway granulation tissue and scar formation pose a challenge because of the high incidence of recurrence after treatment. As an emerging treatment modality, topical application of mitomycin C has potential value in delaying the recurrence of airway obstruction. Several animal and clinical studies have already proven its feasibility and efficacy. However, the ideal dosage has still not been determined. To establish a novel method for culturing primary fibroblasts isolated from human airway granulation tissue, and to investigate the dose-effect of mitomycin C on the fibroblast proliferation in vitro, so as to provide an experimental reference for clinical practitioners. Granulation tissues were collected during the routine bronchoscopy at our department. The primary fibroblasts were obtained by culturing the explanted tissues. The cells were treated with different concentrations of mitomycin C (0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml) for 5 min followed by additional 48-hour culture before an MTT assay was performed to measure cell viability. MTT assay showed that mitomycin C reduced cell viability at all tested concentrations. The inhibitory ratios were 10.26, 26.77, 32.88, 64.91 and 80.45% for cells treated with mitomycin C at 0.1, 0.2, 0.4, 0.8 and 1.6 mg/ml, respectively. Explant culture is a reliable method for culturing primary fibroblasts from human airway granulation tissue, and mitomycin C can inhibit proliferation of the fibroblasts in vitro. Copyright © 2013 S. Karger AG, Basel.

  11. Novel aspects of intrinsic and extrinsic aging of human skin: beneficial effects of soy extract.

    PubMed

    Südel, Kirstin M; Venzke, Kirsten; Mielke, Heiko; Breitenbach, Ute; Mundt, Claudia; Jaspers, Sören; Koop, Urte; Sauermann, Kirsten; Knussman-Hartig, Elke; Moll, Ingrid; Gercken, Günther; Young, Anthony R; Stäb, Franz; Wenck, Horst; Gallinat, Stefan

    2005-01-01

    Biochemical and structural changes of the dermal connective tissue substantially contribute to the phenotype of aging skin. To study connective tissue metabolism with respect to ultraviolet (UV) exposure, we performed an in vitro (human dermal fibroblasts) and an in vivo complementary DNA array study in combination with protein analysis in young and old volunteers. Several genes of the collagen metabolism such as Collagen I, III and VI as well as heat shock protein 47 and matrix metalloproteinase-1 are expressed differentially, indicating UV-mediated effects on collagen expression, processing and degradation. In particular, Collagen I is time and age dependently reduced after a single UV exposure in human skin in vivo. Moreover, older subjects display a lower baseline level and a shorter UV-mediated increase in hyaluronan (HA) levels. To counteract these age-dependent changes, cultured fibroblasts were treated with a specific soy extract. This treatment resulted in increased collagen and HA synthesis. In a placebo-controlled in vivo study, topical application of an isoflavone-containing emulsion significantly enhanced the number of dermal papillae per area after 2 weeks. Because the flattening of the dermal-epidermal junction is the most reproducible structural change in aged skin, this soy extract appears to rejuvenate the structure of mature skin.

  12. Possible involvement of loss of imprinting in immortalization of human fibroblasts.

    PubMed

    Okamura, Kotaro; Ohno, Maki; Tsutsui, Takeki

    2011-04-01

    Disruption of the normal pattern of parental origin-specific gene expression is referred to as loss of imprinting (LOI), which is common in various cancers. To investigate a possible role of LOI in the early stage of human cell transformation, we studied LOI in 18 human fibroblast cell lines immortalized spontaneously, by viral oncogenes, by chemical or physical carcinogens, or by infection with a retrovirus vector encoding the human telomerase catalytic subunit, hTERT cDNA. LOI was observed in all the 18 immortal cell lines. The gene most commonly exhibiting LOI was NDN which displayed LOI in 15 of the 18 cell lines (83%). The other genes exhibiting LOI at high frequencies were PEG3 (50%), MAGE-L2 (61%) and ZNF 127 (50%). Expression of NDN that was lost in the immortal cell lines was restored by treatment with 5-aza-2'-deoxycytidine. The ratio of histone H3 lysine 9 methylation to histone H3 lysine 4 methylation of the chromatin containing the NDN promoter in the immortal WI-38VA13 cells was greater than that in the parental cells, suggesting chromatin structure-mediated regulation of NDN expression. We previously demonstrated that inactivation of the p16INK4a/pRb pathway is necessary for immortalization of human cells. Human fibroblasts in the pre-crisis phase and cells with an extended lifespan that eventually senesce, both of which have the normal p16INK4a/pRb pathway, did not show LOI at any imprinted gene examined. Although it is not clear if LOI plays a causal role in immortalization of human cells or is merely coincidental, these findings indicate a possible involvement of LOI in immortalization of human cells or a common mechanism involved in both processes.

  13. Keratinocyte growth factor expression in human gingival fibroblasts and stimulation of in vitro gene expression by retinoic acid.

    PubMed

    Mackenzie, I C; Gao, Z

    2001-04-01

    Keratinocyte growth factor (KGF) is a stromally derived growth factor of the fibroblast growth factor (FGF) family with paracrine effects targeted to influence the growth and differentiation of epithelia. Regional and temporal changes in KGF expression play important roles in the development and maintenance of epithelial structures and in epithelial wound healing. Differing patterns of expression of KGF by fibroblasts in the gingival region could therefore be related to the observed regional variation in the differentiation and behavior of gingival epithelia. The in vitro and in vivo patterns of expression of KGF mRNA in human gingival and periodontal fibroblasts were examined using reverse transcription polymerase chain reactions (RT-PCR) and in situ hybridization with digoxigenin-labeled riboprobes. The patterns observed for human gingiva were compared with those for human skin and for murine tissues. Gingival and periodontal fibroblasts showed expression of KGF transcripts in vitro, and the degree of expression was markedly influenced by the presence of retinoic acid, an agent known to influence patterns of epithelial differentiation. Sections of human and murine gingiva and skin showed regionally variable expression of transcripts with the cells expressing KGF in the subepithelial, rather than the deeper, connective tissues and periodontium. The results point to a role of KGF in the maintenance of normal growth and differentiation of gingival epithelia. A lack of KGF expression by periodontal fibroblasts in vivo is expected to hinder apical epithelial migration and thus stabilize the epithelial attachment. The effects of retinoic acid (RA) on KGF expression in vitro provide an indirect mechanism by which RA may regulate the growth and differentiation of gingival epithelia.

  14. Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women.

    PubMed

    Tanaka, Miyuki; Misawa, Eriko; Yamauchi, Koji; Abe, Fumiaki; Ishizaki, Chiaki

    2015-01-01

    Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. First, we investigated the capability of Aloe sterols (cycloartenol and lophenol) to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP) containing 40 μg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake period. The present study confirms that daily oral Aloe sterol-containing AVGP significantly reduced facial wrinkles in women aged ≥40 years, and Aloe sterols stimulate collagen and hyaluronic acid production by human dermal fibroblasts.

  15. Quercetin inhibits hexose transport in a human diploid fibroblast

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Salter, D.W.; Custead-Jones, S.; Cook, J.S.

    1978-01-01

    The flavonol quercetin, a phloretin analog, inhibits transport of 2-deoxyglucose and 3-O-methylglucose in a cultured human diploid fibroblast. This inhibition is related to transport itself and not to the reported effects of flavonoids on membrane-bound ATPases. From concentration-inhibition curves at several pH's we conclude that uncharged (acid) quercetin (pH = 7.65) is the inhibitory form of the molecule (K/sub I/ = 10 ..mu..m). Quercetin, unlike phloretin, is rapidly degraded in 0.1 N NaOH; the degradation products are weakly inhibitory to hexose transport.

  16. ANO1 contributes to angiotensin-II-activated Ca2+-dependent Cl- current in human atrial fibroblasts.

    PubMed

    El Chemaly, Antoun; Norez, Caroline; Magaud, Christophe; Bescond, Jocelyn; Chatelier, Aurelien; Fares, Nassim; Findlay, Ian; Jayle, Christophe; Becq, Frederic; Faivre, Jean-François; Bois, Patrick

    2014-03-01

    Cardiac fibroblasts are an integral part of the myocardial tissue and contribute to its remodelling. This study characterises for the first time the calcium-dependent chloride channels (CaCC) in the plasma membrane of primary human atrial cardiac fibroblasts by means of the iodide efflux and the patch clamp methods. The calcium ionophore A23187 and Angiotensin II (Ang II) activate a chloride conductance in cardiac fibroblasts that shares pharmacological similarities with calcium-dependent chloride channels. This chloride conductance is depressed by RNAi-mediated selective Anoctamine 1 (ANO1) but not by Anoctamine 2 (ANO2) which has been revealed as CaCC and is inhibited by the selective ANO1 inhibitor, T16inh-A01. The effect of Ang II on anion efflux is mediated through AT1 receptors (with an EC50 = 13.8 ± 1.3 nM). The decrease of anion efflux by calphostin C and bisindolylmaleimide I (BIM I) suggests that chloride conductance activation is dependent on PKC. We conclude that ANO1 contributes to CaCC current in human cardiac fibroblasts and that this is regulated by Ang II acting via the AT1 receptor pathway. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Pharmacological NAD-Boosting Strategies Improve Mitochondrial Homeostasis in Human Complex I-Mutant Fibroblasts.

    PubMed

    Felici, Roberta; Lapucci, Andrea; Cavone, Leonardo; Pratesi, Sara; Berlinguer-Palmini, Rolando; Chiarugi, Alberto

    2015-06-01

    Mitochondrial disorders are devastating genetic diseases for which efficacious therapies are still an unmet need. Recent studies report that increased availability of intracellular NAD obtained by inhibition of the NAD-consuming enzyme poly(ADP-ribose) polymerase (PARP)-1 or supplementation with the NAD-precursor nicotinamide riboside (NR) ameliorates energetic derangement and symptoms in mouse models of mitochondrial disorders. Whether these pharmacological approaches also improve bioenergetics of human cells harboring mitochondrial defects is unknown. It is also unclear whether the same signaling cascade is prompted by PARP-1 inhibitors and NR supplementation to improve mitochondrial homeostasis. Here, we show that human fibroblasts mutant for the NADH dehydrogenase (ubiquinone) Fe-S protein 1 (NDUFS1) subunit of respiratory complex I have similar ATP, NAD, and mitochondrial content compared with control cells, but show reduced mitochondrial membrane potential. Interestingly, mutant cells also show increased transcript levels of mitochondrial DNA but not nuclear DNA respiratory complex subunits, suggesting activation of a compensatory response. At variance with prior work in mice, however, NR supplementation, but not PARP-1 inhibition, increased intracellular NAD content in NDUFS1 mutant human fibroblasts. Conversely, PARP-1 inhibitors, but not NR supplementation, increased transcription of mitochondrial transcription factor A and mitochondrial DNA-encoded respiratory complexes constitutively induced in mutant cells. Still, both NR and PARP-1 inhibitors restored mitochondrial membrane potential and increased organelle content as well as oxidative activity of NDUFS1-deficient fibroblasts. Overall, data provide the first evidence that in human cells harboring a mitochondrial respiratory defect exposure to NR or PARP-1, inhibitors activate different signaling pathways that are not invariantly prompted by NAD increases, but equally able to improve energetic

  18. Human fibroblast matrices bio-assembled under macromolecular crowding support stable propagation of human embryonic stem cells.

    PubMed

    Peng, Yanxian; Bocker, Michael Thomas; Holm, Jennifer; Toh, Wei Seong; Hughes, Christopher Stephen; Kidwai, Fahad; Lajoie, Gilles Andre; Cao, Tong; Lyko, Frank; Raghunath, Michael

    2012-11-01

    Stable pluripotent feeder-free propagation of human embryonic stem cells (hESCs) prior to their therapeutic applications remains a major challenge. Matrigel™ (BD Singapore) is a murine sarcoma-derived extracellular matrix (ECM) widely used as a cell-free support combined with conditioned or chemically defined media; however, inherent xenogenic and immunological threats invalidate it for clinical applications. Using human fibrogenic cells to generate ECM is promising but currently suffers from inefficient and time-consuming deposition in vitro. We recently showed that macromolecular crowding (MMC) accelerated ECM deposition substantially in vitro. In the current study, we used dextran sulfate 500 kDa as a macromolecular crowder to induce WI-38 fetal human lung fibroblasts at 0.5% serum condition to deposit human ECM in three days. After decellularization, the generated ECMs allowed stable propagation of H9 hESCs over 20 passages in chemically-defined medium (mTEsR1) with an overall improved outcome compared to Matrigel in terms of population doubling while retaining teratoma formation and differentiation capacity. Of significance, only ECMs generated by MMC allowed the successful propagation of hESCs. ECMs were highly complex and in contrast to Matrigel, contained no vitronectin but did contain collagen XII, ig-h3 and novel for hESC-supporting human matrices, substantial amounts of transglutaminase 2. Genome-wide analysis of promoter DNA methylation states revealed high overall similarity between human ECM- and Matrigel-cultured hESCs; however, distinct differences were observed with 49 genes associated with a variety of cellular functions. Thus, human ECMs deposited by MMC by selected fibroblast lines are a suitable human microenvironment for stable hESC propagation and clinically translational settings. Copyright © 2012 John Wiley & Sons, Ltd.

  19. Effects of Panax ginseng extract on human dermal fibroblast proliferation and collagen synthesis.

    PubMed

    Lee, Geum-Young; Park, Kang-Gyun; Namgoong, Sik; Han, Seung-Kyu; Jeong, Seong-Ho; Dhong, Eun-Sang; Kim, Woo-Kyung

    2016-03-01

    Current studies of Panax ginseng (or Korean ginseng) have demonstrated that it has various biological effects, including angiogenesis, immunostimulation, antimicrobial and anti-inflammatory effects. Therefore, we hypothesised that P. ginseng may also play an important role in wound healing. However, few studies have been conducted on the wound-healing effects of P. ginseng. Thus, the purpose of this in vitro pilot study was to determine the effects of P. ginseng on the activities of fibroblasts, which are key wound-healing cells. Cultured human dermal fibroblasts were treated with one of six concentrations of P. ginseng: 0, 1, 10 and 100 ng/ml and 1 and 10 µg/ml. Cell proliferation was determined 3 days post-treatment using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay, and collagen synthesis was evaluated by the collagen type I carboxy-terminal propeptide method. Cell proliferation levels and collagen synthesis were compared among the groups. The 10 ng/ml to 1 µg/ml P. ginseng treatments significantly increased cell proliferation, and the 1 ng/ml to 1 µg/ml concentrations significantly increased collagen synthesis. The maximum effects for both parameters were observed at 10 ng/ml. P. ginseng stimulated human dermal fibroblast proliferation and collagen synthesis at an optimal concentration of 10 ng/ml. © 2015 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

  20. Receptors and aging: structural selectivity of the rhamnose-receptor on fibroblasts as shown by Ca(2+)-mobilization and gene-expression profiles.

    PubMed

    Faury, G; Molinari, J; Rusova, E; Mariko, B; Raveaud, S; Huber, P; Velebny, V; Robert, A M; Robert, L

    2011-01-01

    Qualitative and quantitative modifications of receptors were shown to play a key role in cell and tissue aging. We recently described the properties of a rhamnose-recognizing receptor on fibroblasts involved in the mediation of age-dependent functions of these cells. Using Ca(2+)-mobilization and DNA-microarrays we could show in the presence of rhamnose-rich oligo- and polysaccharides (RROPs) Ca(2+)-mobilization and changes in gene regulation. Here, we compared the effects of several RROPs, differing in their carbohydrate sequence and molecular weights, in normal human dermal fibroblasts (NHDFs). It appeared that different structural features were required for maximal effects on Ca(2+)-mobilization and gene-expression profiles. Maximal effect on Ca(2+) influx and intracellular free calcium regulation was exhibited by RROP-1, a 50 kDa average molecular weight polysaccharide, and RROP-3, a 5 kDa average molecular weight oligosaccharide with a different carbohydrate sequence. Maximal effect on gene-expression profiles was obtained with RROP-3. These results suggest the possibility of several different transmission pathways from the rhamnose-receptor to intracellular targets, differentially affecting these two intracellular functions, with potential consequences on aging. Although of only relative specificity, this receptor site exhibits a high affinity for rhamnose, absent from vertebrate glycoconjugates. The rhamnose-receptor might well represent an evolutionary conserved conformation of a prokaryote lectin. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  1. Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

    PubMed

    Reisner, P D; Brandt, P C; Vanaman, T C

    1997-01-01

    It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

  2. Protective effect of maghemite nanoparticles on ultraviolet-induced photo-damage in human skin fibroblasts

    NASA Astrophysics Data System (ADS)

    Lee, Kwon-Jai; An, Jeung-Hee; Shin, Jae-Soo; Kim, Dong-Hee; Kim, Changman; Ozaki, Hajime; Koh, Jae-Gui

    2007-11-01

    This study examined the optical properties of an oxidized form of maghemite (γ-Fe2O3) nanoparticles and their protective effects against the photoaging of human skin fibroblasts irradiated with ultraviolet (UV) light. Nanoparticles with diameters ranging from 8.7 to 12 nm were prepared using a chemical co-precipitation method. The nanoparticles were coated with two surfactants to obtain a water-based product. The onset of the absorption of the γ-Fe2O3 nanoparticles in the UV-visible absorption spectra increased with increasing particle size. The γ-Fe2O3 nanoparticles significantly inhibited the production of matrix metalloproteinase-1 in human skin fibroblast HS 68 cells by 60% compared with the UV-irradiated control. These results suggest that γ-Fe2O3 nanoparticles have photoprotective properties, and have potential use as an agent against photoaging.

  3. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts.

    PubMed

    Questa, María; Romorini, Leonardo; Blüguermann, Carolina; Solari, Claudia María; Neiman, Gabriel; Luzzani, Carlos; Scassa, María Élida; Sevlever, Gustavo Emilio; Guberman, Alejandra Sonia; Miriuka, Santiago Gabriel

    2016-03-01

    Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC) line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls. Copyright © 2015 University of Texas at Austin Dell Medical School. Published by Elsevier B.V. All rights reserved.

  4. The Inhibitory Effects of Anti-Oxidants on Ultraviolet-Induced Up-Regulation of the Wrinkling-Inducing Enzyme Neutral Endopeptidase in Human Fibroblasts

    PubMed Central

    Nakajima, Hiroaki; Terazawa, Shuko; Niwano, Takao; Yamamoto, Yorihiro; Imokawa, Genji

    2016-01-01

    We recently reported that the over-expression of skin fibroblast-derived neutral endopeptidase (NEP) plays a pivotal role in impairing the three-dimensional architecture of dermal elastic fibers during the biological mechanism of ultraviolet (UV)-induced skin wrinkling. In that process, a UVB-associated epithelial-mesenchymal cytokine interaction as well as a direct UVA-induced cellular stimulation are associated with the up-regulation of NEP in human fibroblasts. In this study, we characterized the mode of action of ubiquinol10 which may abrogate the up-regulation of NEP by dermal fibroblasts, resulting in a reported in vivo anti-wrinkling action, and compared that with 3 other anti-oxidants, astaxanthin (AX), riboflavin (RF) and flavin mononucleotide (FMN). Post-irradiation treatment with all 4 of those anti-oxidants elicited an interrupting effect on the UVB-associated epithelial-mesenchymal cytokine interaction leading to the up-regulation of NEP in human fibroblasts but with different modes of action. While AX mainly served as an inhibitor of the secretion of wrinkle-inducing cytokines, such as interleukin-1α (IL-1α) and granulocyte macrophage colony stimulatory factor (GM-CSF) in UVB-exposed epidermal keratinocytes, ubiquinol10, RF and FMN predominantly interrupted the IL-1α and GM-CSF-stimulated expression of NEP in dermal fibroblasts. On the other hand, as for the UVA-associated mechanism, similar to the abrogating effects reported for AX and FMN, ubiquinol10 but not RF had the potential to abrogate the increased expression of NEP and matrix-metalloproteinase-1 in UVA-exposed human fibroblasts. Our findings strongly support the in vivo anti-wrinkling effects of ubiquinol10 and AX on human and animal skin and provide convincing proof of the UV-induced wrinkling mechanism that essentially focuses on the over-expression of NEP by dermal fibroblasts as an intrinsic causative factor. PMID:27648570

  5. 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.

  6. Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Zhong Xin; Sun, Cong Cong; Wenzhou People's Hospital, Wenzhou, Zhejiang

    Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Westernmore » blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-β-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of β-catenin, suggesting that β-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy. - Highlights: • bFGF regulates Hedgehog (Hh) signaling in fibroblasts. • The Smo and Gli two master regulators of Hh signaling positively regulate fibroblast migration. • Smo facilitates β-catenin nuclear translocation via activation PI3K/JNK/GSK3β. • β-catenin positively regulates fibroblast cell migration and the expression of Hh signaling genes including Smo and Gli.« less

  7. ATF6α regulates morphological changes associated with senescence in human fibroblasts

    PubMed Central

    Martin, Nathalie; Saas, Laure; Cormenier, Johanna; Malaquin, Nicolas; Huot, Ludovic; Slomianny, Christian; Bouali, Fatima; Vercamer, Chantal; Hot, David; Pourtier, Albin; Chevet, Eric; Abbadie, Corinne; Pluquet, Olivier

    2016-01-01

    Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated β-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6α signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6α in senescent cells. Moreover, ATF6α drives the increase of senescence associated-β-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6α in the establishment of morphological features of senescence in normal human primary fibroblasts. PMID:27563820

  8. ATF6α regulates morphological changes associated with senescence in human fibroblasts.

    PubMed

    Druelle, Clémentine; Drullion, Claire; Deslé, Julie; Martin, Nathalie; Saas, Laure; Cormenier, Johanna; Malaquin, Nicolas; Huot, Ludovic; Slomianny, Christian; Bouali, Fatima; Vercamer, Chantal; Hot, David; Pourtier, Albin; Chevet, Eric; Abbadie, Corinne; Pluquet, Olivier

    2016-10-18

    Cellular senescence is known as an anti-tumor barrier and is characterized by a number of determinants including cell cycle arrest, senescence associated β-galactosidase activity and secretion of pro-inflammatory mediators. Senescent cells are also subjected to enlargement, cytoskeleton-mediated shape changes and organelle alterations. However, the underlying molecular mechanisms responsible for these last changes remain still uncharacterized. Herein, we have identified the Unfolded Protein Response (UPR) as a player controlling some morphological aspects of the senescent phenotype. We show that senescent fibroblasts exhibit ER expansion and mild UPR activation, but conserve an ER stress adaptive capacity similar to that of exponentially growing cells. By genetically invalidating the three UPR sensors in senescent fibroblasts, we demonstrated that ATF6α signaling dictates senescence-associated cell shape modifications. We also show that ER expansion and increased secretion of the pro-inflammatory mediator IL6 were partly reversed by silencing ATF6α in senescent cells. Moreover, ATF6α drives the increase of senescence associated-β-galactosidase activity. Collectively, these findings unveil a novel and central role for ATF6α in the establishment of morphological features of senescence in normal human primary fibroblasts.

  9. Matrix Stiffness Corresponding to Strictured Bowel Induces a Fibrogenic Response in Human Colonic Fibroblasts

    PubMed Central

    Johnson, Laura A.; Rodansky, Eva S.; Sauder, Kay L.; Horowitz, Jeffrey C.; Mih, Justin D.; Tschumperlin, Daniel J.; Higgins, Peter D.

    2013-01-01

    Background Crohn’s disease is characterized by repeated cycles of inflammation and mucosal healing which ultimately progress to intestinal fibrosis. This inexorable progression towards fibrosis suggests that fibrosis becomes inflammation-independent and auto-propagative. We hypothesized that matrix stiffness regulates this auto-propagation of intestinal fibrosis. Methods The stiffness of fresh ex vivo samples from normal human small intestine, Crohn’s disease strictures, and the unaffected margin were measured with a microelastometer. Normal human colonic fibroblasts were cultured on physiologically normal or pathologically stiff matrices corresponding to the physiological stiffness of normal or fibrotic bowel. Cellular response was assayed for changes in cell morphology, α-smooth muscle actin (αSMA) staining, and gene expression. Results Microelastometer measurements revealed a significant increase in colonic tissue stiffness between normal human colon and Crohn’s strictures as well as between the stricture and adjacent tissue margin. In Ccd-18co cells grown on stiff matrices corresponding to Crohn’s strictures, cellular proliferation increased. Pathologic stiffness induced a marked change in cell morphology and increased αSMA protein expression. Growth on a stiff matrix induced fibrogenic gene expression, decreased matrix metalloproteinase and pro-inflammatory gene expression, and was associated with nuclear localization of the transcriptional cofactor MRTF-A. Conclusions Matrix stiffness, representative of the pathological stiffness of Crohn’s strictures, activates human colonic fibroblasts to a fibrogenic phenotype. Matrix stiffness affects multiple pathways suggesting the mechanical properties of the cellular environment are critical to fibroblast function and may contribute to autopropagation of intestinal fibrosis in the absence of inflammation, thereby contributing to the intractable intestinal fibrosis characteristic of Crohn’s disease. PMID

  10. Differential Gene Expression in Primary Human Skin Keratinocytes and Fibroblasts in Response to Ionizing Radiation

    PubMed Central

    Warters, Raymond L.; Packard, Ann T.; Kramer, Gwen F.; Gaffney, David K.; Moos, Philip J.

    2009-01-01

    Although skin is usually exposed during human exposures to ionizing radiation, there have been no thorough examinations of the transcriptional response of skin fibroblasts and keratinocytes to radiation. The transcriptional response of quiescent primary fibroblasts and keratinocytes exposed to from 10 cGy to 5 Gy and collected 4 h after treatment was examined. RNA was isolated and examined by microarray analysis for changes in the levels of gene expression. Exposure to ionizing radiation altered the expression of 279 genes across both cell types. Changes in RNA expression could be arranged into three main categories: (1) changes in keratinocytes but not in fibroblasts, (2) changes in fibroblasts but not in keratinocytes, and (3) changes in both. All of these changes were primarily of p53 target genes. Similar radiation-induced changes were induced in immortalized fibroblasts or keratinocytes. In separate experiments, protein was collected and analyzed by Western blotting for expression of proteins observed in microarray experiments to be overexpressed at the mRNA level. Both Q-PCR and Western blot analysis experiments validated these transcription changes. Our results are consistent with changes in the expression of p53 target genes as indicating the magnitude of cell responses to ionizing radiation. PMID:19580510

  11. Alterations in TNF- and IL-related gene expression in space-flown WI38 human fibroblasts

    NASA Technical Reports Server (NTRS)

    Semov, Alexandre; Semova, Nathalia; Lacelle, Chantale; Marcotte, Richard; Petroulakis, Emmanuel; Proestou, Gregory; Wang, Eugenia

    2002-01-01

    Spaceflight, just like aging, causes profound changes in musculoskeletal parameters, which result in decreased bone density and muscular weakness. As these conditions decrease our ability to conduct long-term manned space missions, and increase bone frailty in the elderly, the identification of genes responsible for the apparition of these physiological changes will be of great benefit. Thus, we developed and implemented a new microarray approach to investigate the changes in normal WI38 human fibroblast gene expression that arise as a consequence of space flight. Using our microarray, we identified changes in the level of expression of 10 genes, belonging to either the tumor necrosis factor- (TNF) or interleukin- (IL) related gene families in fibroblasts when WI38 cells exposed to microgravity during the STS-93 Space Shuttle mission were compared with ground controls. The genes included two ligands from the TNF superfamily, TWEAK and TNFSF15; two TNF receptor-associated proteins, NSMAF and PTPN13; three TNF-inducible genes, ABC50, PTX3, and SCYA13; TNF-alpha converting enzyme, IL-1 receptor antagonist, and IL-15 receptor alpha chain. Most of these are involved in either the regulation of bone density, and as such the development of spaceflight osteopenia, or in the development of proinflammatory status.

  12. Anethole prevents hydrogen peroxide-induced apoptosis and collagen metabolism alterations in human skin fibroblasts.

    PubMed

    Galicka, Anna; Krętowski, Rafał; Nazaruk, Jolanta; Cechowska-Pasko, Marzanna

    2014-09-01

    The collagen metabolism alterations triggered by reactive oxygen species are involved in the development of various connective tissue diseases and skin aging. This study was designed to examine whether (E)-anethole possesses a protective effect on H2O2-induced alterations in collagen metabolism as well as whether it can prevent apoptosis in human skin fibroblasts. In cells treated with 300 µM H₂O₂, a decrease in collagen biosynthesis of 54% was observed. Pretreatment of cells with 0.5 µM anethole for 1 h completely prevented this alteration. Changes at the protein level positively correlated with alterations of type I collagen mRNA expression. We have shown that H2O2 caused increase in the activity of MMP-2 and MMP-9 as well as that an increase in MMP-2 activity can contribute to the 8% decrease in the amount of collagen secreted into the medium. The most efficient suppression of these changes was observed in the presence of 0.5 µM of anethole. At 10 µM, in addition to suppression, an inhibitory effect of anethole on MMP-9 activity was documented. Additionally, the 60% H₂O₂-induced decrease in cell viability was suppressed by 1 µM of anethole and a 4-fold increase in cell apoptosis was suppressed by 0.5 µM of anethole. Our results suggest that anethole, which is a small lipophilic and non-toxic molecule with the ability to prevent H₂O₂-induced collagen metabolism alterations and apoptosis in human skin fibroblasts, would prove useful in the development of effective agents in pharmacotherapy of oxidative stress-related skin diseases.

  13. Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts.

    PubMed

    Gothai, Sivapragasam; Arulselvan, Palanisamy; Tan, Woan Sean; Fakurazi, Sharida

    2016-01-01

    Wounds are the outcome of injuries to the skin that interrupt the soft tissue. Healing of a wound is a complex and long-drawn-out process of tissue repair and remodeling in response to injury. A large number of plants are used by folklore traditions for the treatment of cuts, wounds and burns. Moringa oleifera (MO) is an herb used as a traditional folk medicine for the treatment of various skin wounds and associated diseases. The underlying mechanisms of wound healing activity of ethyl acetate fraction of MO leaves extract are completely unknown. In the current study, ethyl acetate fraction of MO leaves was investigated for its efficacy on cell viability, proliferation and migration (wound closure rate) in human normal dermal fibroblast cells. Results revealed that lower concentration (12.5 µg/ml, 25 µg/ml, and 50 µg/ml) of ethyl acetate fraction of MO leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. This study suggested that ethyl acetate fraction of MO leaves might be a potential therapeutic agent for skin wound healing by promoting fibroblast proliferation and migration through increasing the wound closure rate corroborating its traditional use.

  14. Effects of conditioned medium from LL-37 treated adipose stem cells on human fibroblast migration.

    PubMed

    Yang, Eun-Jung; Bang, Sa-Ik

    2017-07-01

    Adipose stem cell-conditioned medium may promote human dermal fibroblast (HDF) proliferation and migration by activating paracrine peptides during the re-epithelization phase of wound healing. Human antimicrobial peptide LL-37 is upregulated in the skin epithelium as part of the normal response to injury. The effects of conditioned medium (CM) from LL-37 treated adipose stem cells (ASCs) on cutaneous wound healing, including the mediation of fibroblast migration, remain to be elucidated, therefore the aim of the present study was to determine how ASCs would react to an LL-37-rich microenvironment and if CM from LL-37 treated ASCs may influence the migration of HDFs. The present study conducted migration assays with HDFs treated with CM from LL-37 treated ASCs. Expression of CXC chemokine receptor 4 (CXCR4), which controls the recruitment of HDFs, was analyzed at the mRNA and protein levels. To further characterize the stimulatory effects of LL-37 on ASCs, the expression of stromal cell-derived factor-1α (SDF-1α), a CXC chemokine, was investigated. CM from LL-37-treated ASCs induced migration of HDFs in a time- and dose-dependent manner, with a maximum difference in migration observed 24 h following stimulation with LL-37 at a concentration of 10 µg/ml. The HDF migration and the expression of CXCR4 in fibroblasts was markedly increased upon treatment with CM from LL-37-treated ASCs compared with CM from untreated ASCs. SDF-1α expression was markedly increased in CM from LL-37 treated ASCs. It was additionally observed that SDF-1α blockade significantly reduced HDF migration. These findings suggest the feasibility of CM from LL-37-treated ASCs as a potential therapeutic for human dermal fibroblast migration.

  15. The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts.

    PubMed

    Schultz, Francisca; Hasan, Alveera; Alvarez-Laviada, Anita; Miragoli, Michele; Bhogal, Navneet; Wells, Sarah; Poulet, Claire; Chambers, Jenny; Williamson, Catherine; Gorelik, Julia

    2016-01-01

    Bile acids are elevated in the blood of women with intrahepatic cholestasis of pregnancy (ICP) and this may lead to fetal arrhythmia, fetal hypoxia and potentially fetal death in utero. The bile acid taurocholic acid (TC) causes abnormal calcium dynamics and contraction in neonatal rat cardiomyocytes. Ursodeoxycholic acid (UDCA), a drug clinically used to treat ICP, prevents adverse effects of TC. During development, the fetus is in a state of relative hypoxia. Although this is essential for the development of the heart and vasculature, resident fibroblasts can transiently differentiate into myofibroblasts and form gap junctions with cardiomyocytes in vitro, resulting in cardiomyocyte depolarization. We expanded on previously published work using an in vitro hypoxia model to investigate the differentiation of human fetal fibroblasts into myofibroblasts. Recent evidence shows that potassium channels are involved in maintaining the membrane potential of ventricular fibroblasts and that ATP-dependent potassium (KATP) channel subunits are expressed in cultured fibroblasts. KATP channels are a valuable target as they are thought to have a cardioprotective role during ischaemic and hypoxic conditions. We investigated whether UDCA could modulate fibroblast membrane potential. We established the isolation and culture of human fetal cardiomyocytes and fibroblasts to investigate the effect of hypoxia, TC and UDCA on human fetal cardiac cells. UDCA hyperpolarized myofibroblasts and prevented TC-induced depolarisation, possibly through the activation of KATP channels that are expressed in cultured fibroblasts. Also, similar to the rat model, UDCA can counteract TC-induced calcium abnormalities in human fetal cultures of cardiomyocytes and myofibroblasts. Under normoxic conditions, we found a higher number of myofibroblasts in cultures derived from human fetal hearts compared to cells isolated from neonatal rat hearts, indicating a possible increased number of myofibroblasts

  16. Role of fibroblast-derived factors in the pathogenesis of melasma.

    PubMed

    Byun, J W; Park, I S; Choi, G S; Shin, J

    2016-08-01

    The hyperactive melanocytes present in melasma skin are confined to the epidermis, but epidermal ablation to treat melasma pigmentation may lead to disease recurrence and aggravation. Melanocyte function is regulated by interactions between melanocytes and neighbouring cells such as keratinocytes and fibroblasts. Because melasma skin usually shows dermal changes after exposure to sunlight, we hypothesized that sun-damaged fibroblasts might play a crucial role in the pathogenesis of melasma. In this study, the melanogenic role of primary cultured fibroblasts from human melasma skin was investigated. We explored whether primary cultured fibroblasts from melasma tissue have a melanogenic function on cultured human epidermal melanocytes and artificial skin. The cytokine profile derived from fibroblasts and their effect on the pigmented epidermal equivalents were investigated. Fibroblasts from the melasma lesion and perilesional skin increased melanogenesis in cultured human epidermal melanocytes and in artificial skin. Fibroblasts from the melasma lesion and perilesional skin secreted more nerve growth factor (NGF)-β than those in normal buttock skin, and also increased melanogenesis and the expression level of NGF-β in cultured human epidermal melanocytes and artificial skin. These results suggest that fibroblasts may play a role in melanogenesis and the pathogenesis of melasma. © 2016 British Association of Dermatologists.

  17. Immortalization of normal human fibroblasts by treatment with 4-nitroquinoline 1-oxide.

    PubMed

    Bai, L; Mihara, K; Kondo, Y; Honma, M; Namba, M

    1993-02-01

    Normal human fibroblasts (the OUMS-24 strain), derived from a 6-week-old human embryo, were transformed (into the OUMS-24F line) and immortalized by repeated treatments (59 times) with 4-nitroquinoline 1-oxide (4NQO). Treatment began during primary culture and ended at the 51st population doubling level (PDL). At the 57th PDL (146 days after the last treatment), morphologically altered, epithelial-type cells appeared, began to grow and became immortal (now past the 100th PDL). However, the control fibroblasts, which were not treated with 4NQO, senesced at the 62nd PDL. The finding that extensive, repeated treatments with 4NQO are required for the immortalization of normal human cells, indicates that multiple mutational events are involved in the immortalization of human cells in general. In other words, immortalization itself seems to be a multi-step process. Karyotypic analysis showed that many cells were hypodiploid before immortalization, but that afterwards chromosomes were distributed broadly in the diploid to tetraploid regions. The immortalized cells showed amplification and enhanced expression of c-myc. Two-dimensional electrophoretic analysis showed that the number of disappearing cellular proteins was greater than the number of the newly appearing ones after the cells became immortalized. Since the immortalized cells showed neither anchorage-independent growth nor tumorigenicity, they are useful for studying factors that can contribute to multi-step carcinogenesis in human cells. In addition, genetically matched normal (OUMS-24) and immortalized (OUMS-24F) cells will be useful for analyzing the genes related to cellular mortality and immortalization.

  18. Elevated CD26 Expression by Skin Fibroblasts Distinguishes a Profibrotic Phenotype Involved in Scar Formation Compared to Gingival Fibroblasts.

    PubMed

    Mah, Wesley; Jiang, Guoqiao; Olver, Dylan; Gallant-Behm, Corrie; Wiebe, Colin; Hart, David A; Koivisto, Leeni; Larjava, Hannu; Häkkinen, Lari

    2017-08-01

    Compared to skin, wound healing in oral mucosa is faster and produces less scarring, but the mechanisms involved are incompletely understood. Studies in mice have linked high expression of CD26 to a profibrotic fibroblast phenotype, but this has not been tested in models more relevant for humans. We hypothesized that CD26 is highly expressed by human skin fibroblasts (SFBLs), and this associates with a profibrotic phenotype distinct from gingival fibroblasts (GFBLs). We compared CD26 expression in human gingiva and skin and in gingival and hypertrophic-like scar-forming skin wound healing in a pig model, and used three-dimensional cultures of human GFBLs and SFBLs. In both humans and pigs, nonwounded skin contained abundantly CD26-positive fibroblasts, whereas in gingiva they were rare. During skin wound healing, CD26-positive cells accumulated over time and persisted in forming hypertrophic-like scars, whereas few CD26-positive cells were present in the regenerated gingival wounds. Cultured human SFBLs displayed significantly higher levels of CD26 than GFBLs. This was associated with an increased expression of profibrotic genes and transforming growth factor-β signaling in SFBLs. The profibrotic phenotype of SFBLs partially depended on expression of CD26, but was independent of its catalytic activity. Thus, a CD26-positive fibroblast population that is abundant in human skin but not in gingiva may drive the profibrotic response leading to excessive scarring. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  19. Effect of static magnetic fields and phloretin on antioxidant defense system of human fibroblasts.

    PubMed

    Pawłowska-Góral, Katarzyna; Kimsa-Dudek, Magdalena; Synowiec-Wojtarowicz, Agnieszka; Orchel, Joanna; Glinka, Marek; Gawron, Stanisław

    2016-08-01

    The available evidence from in vitro and in vivo studies is deemed not sufficient to draw conclusions about the potential health effects of static magnetic field (SMF) exposure. Therefore, the aim of the present study was to determine the influence of static magnetic fields and phloretin on the redox homeostasis of human dermal fibroblasts. Control fibroblasts and fibroblasts treated with phloretin were subjected to the influence of static magnetic fields. Three chambers with static magnetic fields of different intensities (0.4, 0.55, and 0.7 T) were used in the study. Quantification of superoxide dismutase 1 (SOD1), superoxide dismutase 2 (SOD2), glutathione peroxidase 1 (GPX1), microsomal glutathione S-transferase 1 (MGST1), glutathione reductase (GSR), and catalase (CAT) messenger RNAs (mRNAs) was performed by means of real-time reverse transcription PCR (QRT-PCR) technique. Superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were measured using a commercially available kit. No significant differences were found in SOD1, SOD2, GPX1, MGST1, GSR, and CAT mRNA levels among the studied groups in comparison to the control culture without phloretin and without the magnet. There were also no changes in SOD, GPx, and CAT activities. In conclusion, our study indicated that static magnetic fields generated by permanent magnets do not exert a negative influence on the oxidative status of human dermal fibroblasts. Based on these studies, it may also be concluded that phloretin does not increase its antioxidant properties under the influence of static magnetic fields. However, SMF-induced modifications at the cellular and molecular level require further clarification.

  20. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst, E-mail: e.wolvetang@uq.edu.au

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatmentsmore » we observed marked differences in mitochondrial structure, membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans. -- Highlights: ► Cells respond to certain environmental toxins by increasing mitochondrial biogenesis. ► We investigated the effect of Chloramphenicol and EtBr in primary human fibroblasts. ► Inhibiting mitochondrial protein synthesis or DNA replication elicit different effects. ► We provide novel insights into the cellular responses toxins and antibiotics.« less

  1. Lysophosphatidic acid stimulates epidermal growth factor-family ectodomain shedding and paracrine signaling from human lung fibroblasts.

    PubMed

    Shiomi, Tetsuya; Boudreault, Francis; Padem, Nurcicek; Higashiyama, Shigeki; Drazen, Jeffrey M; Tschumperlin, Daniel J

    2011-01-01

    Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase-tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme-linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase-tagged heparin-binding epidermal growth factor (HB-EGF), amphiregulin, and transforming growth factor-a; non-transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB-EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal-regulated kinase 1/2, enhanced expression of mRNA for c-fos, HB-EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA-stimulated cells, and found enhanced EGFR and extracellular signal-regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF-family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells. © 2011 by the Wound Healing Society.

  2. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

    PubMed

    Buskermolen, Jeroen K; Reijnders, Christianne M A; Spiekstra, Sander W; Steinberg, Thorsten; Kleverlaan, Cornelis J; Feilzer, Albert J; Bakker, Astrid D; Gibbs, Susan

    2016-08-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  3. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts

    PubMed Central

    Buskermolen, Jeroen K.; Reijnders, Christianne M.A.; Spiekstra, Sander W.; Steinberg, Thorsten; Kleverlaan, Cornelis J.; Feilzer, Albert J.; Bakker, Astrid D.

    2016-01-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  4. Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

    PubMed

    Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

    2010-09-01

    Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

  5. The use of a biostatic fascia lata thigh allograft as a scaffold for autologous human culture of fibroblasts--An in vitro study.

    PubMed

    Żurek, Jarek; Dominiak, Marzena; Botzenhart, Ute; Bednarz, Wojciech

    2015-05-01

    The method for covering gingival recession defects and augmenting keratinized gingiva involves the use of autogenuous connective tissue grafts obtained from palatal mucosa in combination with various techniques of flap repositioning or tunnel techniques. In the case of multiple gingival recession defects the amount of connective tissue available for grafting is insufficient. Therefore, the use of substitutes is necessary. The most widely used material in recent years has been the acellular dermal matrix allograft. The disadvantage of its application lies in the absence of cells and blood vessels, which increases incorporation time. Primary cultured human autologic fibroblasts are commonly used to optimize the healing process. The aim of this study was to examine the in vitro biocompatibility of human fascia lata allograft as a new scaffold for primary cultured human autologic fibroblasts. For that, a fibroblast culture obtained from a fragment of gingival tissue taken from the hard palate mucosa of a subject was used. After 14 days the colony cells were inoculated on a fragment of human fascia lata allograft. After a further 7 days of incubation the material was frozen, cut and prepared for histochemical examination. After two weeks of incubation, and 7 days after inoculation on a fragment of fascia lata allograft numerous accumulations of the cultured fibroblast were found that had a typical structure and produced collagen fibres. A human fascia lata allograft can be used as a scaffold for primary cultured human autologic fibroblasts. Further studies should confirm the clinical efficacy of this solution. Copyright © 2014 Elsevier GmbH. All rights reserved.

  6. 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.

  7. 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

  8. Anti-fibrotic effects of pirfenidone and rapamycin in primary IPF fibroblasts and human alveolar epithelial cells.

    PubMed

    Molina-Molina, M; Machahua-Huamani, C; Vicens-Zygmunt, V; Llatjós, R; Escobar, I; Sala-Llinas, E; Luburich-Hernaiz, P; Dorca, J; Montes-Worboys, A

    2018-04-27

    Pirfenidone, a pleiotropic anti-fibrotic treatment, has been shown to slow down disease progression of idiopathic pulmonary fibrosis (IPF), a fatal and devastating lung disease. Rapamycin, an inhibitor of fibroblast proliferation could be a potential anti-fibrotic drug to improve the effects of pirfenidone. Primary lung fibroblasts from IPF patients and human alveolar epithelial cells (A549) were treated in vitro with pirfenidone and rapamycin in the presence or absence of transforming growth factor β1 (TGF-β). Extracellular matrix protein and gene expression of markers involved in lung fibrosis (tenascin-c, fibronectin, collagen I [COL1A1], collagen III [COL3A1] and α-smooth muscle actin [α-SMA]) were analyzed. A cell migration assay in pirfenidone, rapamycin and TGF-β-containing media was performed. Gene and protein expression of tenascin-c and fibronectin of fibrotic fibroblasts were reduced by pirfenidone or rapamycin treatment. Pirfenidone-rapamycin treatment did not revert the epithelial to mesenchymal transition pathway activated by TGF-β. However, the drug combination significantly abrogated fibroblast to myofibroblast transition. The inhibitory effect of pirfenidone on fibroblast migration in the scratch-wound assay was potentiated by rapamycin combination. These findings indicate that the combination of pirfenidone and rapamycin widen the inhibition range of fibrogenic markers and prevents fibroblast migration. These results would open a new line of research for an anti-fibrotic combination therapeutic approach.

  9. Establishment of ultra long-lived cell lines by transfection of TERT into normal human fibroblast TIG-1 and their characterization.

    PubMed

    Kamada, Mizuna; Kumazaki, Tsutomu; Matsuo, Taira; Mitsui, Youji; Takahashi, Tomoko

    2012-06-01

    To establish useful human normal cell lines, TERT (telomerase reverse transcriptase) cDNA was transfected into normal female lung fibroblast, TIG-1. After long-term-sub-cultivation of 74 individual clones selected for resistance to G418, we obtained 55 cultures with normal range of life span [75 PDL (population doubling level)], 16 cultures with extended life span (75-140 PDL). In addition, 3 immortal cell strains and unexpectedly, one ultra long-lived cell line (ULT-1) with life span of 166 PDL were established. IMT-1, one of the immortal cell strains was confirmed to maintain long telomere length, high telomerase activity and an extremely low level of p16INK4A. They also showed moderate p53 and p21CIP1 expression, keeping vigorous growth rate even at 450 PDL. High level of fibronectin and collagen 1α expression confirmed IMT-1 as normal fibroblasts, although one X chromosome had been lost. ULT-1, however, kept a near normal karyotypes and had shortening of telomere length, high expression of p16INK4A, moderate levels of senescence associated-β-galactosidase positive cells and decreased growth rate only after 150 PDs (population doublings), and finally reached senescence at 166 PDL with morphology of normal senescent fibroblasts. As resources of standard normal human cell, abundant vials of early and middle passages of ULT-1 have been stocked. The use of the cell line is discussed, focusing on isograft of artificial skin and screening of anti-aging or safe chemical agents.

  10. Immortalisation of a human diploid fibroblast cell strain: a DT-diaphorase paradox.

    PubMed Central

    Kuehl, B. L.; Brezden, C. B.; Traver, R. D.; Siegel, D.; Ross, D.; Renzing, J.; Rauth, A. M.

    1996-01-01

    Transfection of a normal human diploid fibroblast cell strain, GM38, with a simian virus 40 (SV40) large T antigen containing plasmid, yielded an immortal cell line, G38-8X, which had a similar sensitivity as the parental cell strain to the quinone-containing chemotherapeutic agent mitomycin C (MMC), under both aerobic and hypoxic exposure conditions. The activity level of DT-diaphorase was similar in both the parental GM38 and G38-8X cells. Although DT-diaphorase could be detected by Western blot analysis, using two mouse anti-human monoclonal antibodies, in GM38 cells, it was not detected in the G38-8X cells. G38-8X cells have a slightly increased P450R activity (2-fold), and have elevated P-glycoprotein levels compared with the parental GM38 cell strain. The immortal G38-8X cell line is 2-fold more resistant to ionising radiation than the parental GM38 cell strain (D10 approximately 5 Gy). Although these SV40 large T antigen immortalised human diploid fibroblasts behaved similarly to their parental cell strain in terms of MMC sensitivity and DT-diaphorase activity, careful characterisation revealed that these cells had enhanced P-glycoprotein activity and had a decreased sensitivity to ionising radiation. Images Figure 3 PMID:8763839

  11. Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus.

    PubMed

    Chen, Daxiang; Long, Mingjian; Xiao, Bin; Xiong, Yufeng; Chen, Huiqin; Chen, Yu; Kuang, Zhenzhan; Li, Ming; Wu, Yingsong; Rock, Daniel L; Gong, Daoyuan; Wang, Yong; He, Haijian; Liu, Fang; Luo, Shuhong; Hao, Wenbo

    2017-08-29

    Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

  12. Chondrogenic potential of physically treated bovine cartilage matrix derived porous scaffolds on human dermal fibroblast cells.

    PubMed

    Moradi, Ali; Ataollahi, Forough; Sayar, Katayoun; Pramanik, Sumit; Chong, Pan-Pan; Khalil, Alizan Abdul; Kamarul, Tunku; Pingguan-Murphy, Belinda

    2016-01-01

    Extracellular matrices have drawn attention in tissue engineering as potential biomaterials for scaffold fabrication because of their bioactive components. Noninvasive techniques of scaffold fabrication and cross-linking treatments are believed to maintain the integrity of bioactive molecules while providing proper architectural and mechanical properties. Cartilage matrix derived scaffolds are designed to support the maintenance of chondrocytes and provide proper signals for differentiation of chondroinducible cells. Chondroinductive potential of bovine articular cartilage matrix derived porous scaffolds on human dermal fibroblasts and the effect of scaffold shrinkage on chondrogenesis were investigated. An increase in sulfated glycosaminoglycans production along with upregulation of chondrogenic genes confirmed that physically treated cartilage matrix derived scaffolds have chondrogenic potential on human dermal fibroblasts. © 2015 Wiley Periodicals, Inc.

  13. Aluminum is More Cytotoxic than Lunar Dust in Human Skin and Lung Fibroblasts

    NASA Technical Reports Server (NTRS)

    Hammond, D.; Shehata, T.; Hammond, D.; Shehata, T.; Wise, J.P.; Martino, J; Wise, J.P.; Wise, J.P.

    2009-01-01

    NASA plans to build a permanent space station on the moon to explore its surface. The surface of the moon is covered in lunar dust, which consists of fine particles that contain silicon, aluminum and titanium, among others. Because this will be a manned base, the potential toxicity of this dust has to be studied. Also, toxicity standards for potential exposure have to be set. To properly address the potential toxicity of lunar dust we need to understand the toxicity of its individual components, as well as their combined effects. In order to study this we compared NASA simulant JSC-1AVF (volcanic ash particles), that simulates the dust found on the moon, to aluminum, the 3rd most abundant component in lunar dust. We tested the cytotoxicity of both compounds on human lung and skin fibroblasts (WTHBF-6 and BJhTERT cell lines, respectively). Aluminum oxide was more cytotoxic than lunar dust to both cell lines. In human lung fibroblasts 5, 10 and 50 g/sq cm of aluminum oxide induced 85%, 61% and 30% relative survival, respectively. For human skin fibroblasts the same concentrations induced 58%, 41% and 58% relative survival. Lunar dust was also cytotoxic to both cell lines, but its effects were seen at higher concentrations: 50, 100, 200 and 400 g/sq cm of lunar dust induced a 69%, 46%, 35% and 30% relative survival in the skin cells and 53%, 16%, 8% and 2% on the lung cells. Overall, for both compounds, lung cells were more sensitive than skin cells. This work was supported by a NASA EPSCoR grant through the Maine Space Grant Consortium (JPW), the Maine Center for Toxicology and Environmental Health., a Fulbright Grant (JM) and a Delta Kappa Gamma Society International World Fellowship (JM).

  14. Rejoining of isochromatid breaks induced by heavy ions in G2-phase normal human fibroblasts

    NASA Technical Reports Server (NTRS)

    Kawata, T.; Durante, M.; Furusawa, Y.; George, K.; Ito, H.; Wu, H.; Cucinotta, F. A.

    2001-01-01

    We reported previously that exposure of normal human fibroblasts in G2 phase of the cell cycle to high-LET radiation produces a much higher frequency of isochromatid breaks than exposure to gamma rays. We concluded that an increase in the production of isochromatid breaks is a signature of initial high-LET radiation-induced G2-phase damage. In this paper, we report the repair kinetics of isochromatid breaks induced by high-LET radiation in normal G2-phase human fibroblasts. Exponentially growing human fibroblasts (AG1522) were irradiated with gamma rays or energetic carbon (290 MeV/nucleon), silicon (490 MeV/nucleon), or iron (200 MeV/nucleon) ions. Prematurely condensed chromosomes were induced by calyculin A after different postirradiation incubation times ranging from 0 to 600 min. Chromosomes were stained with Giemsa, and aberrations were scored in cells at G2 phase. G2-phase fragments, the result of the induction of isochromatid breaks, decreased quickly with incubation time. The curve for the kinetics of the rejoining of chromatid-type breaks showed a slight upward curvature with time after exposure to 440 keV/microm iron particles, probably due to isochromatid-isochromatid break rejoining. The formation of chromatid exchanges after exposure to high-LET radiation therefore appears to be underestimated, because isochromatid-isochromatid exchanges cannot be detected. Increased induction of isochromatid breaks and rejoining of isochromatid breaks affect the overall kinetics of chromatid-type break rejoining after exposure to high-LET radiation.

  15. p53/PUMA expression in human pulmonary fibroblasts mediates cell activation and migration in silicosis.

    PubMed

    Wang, Wei; Liu, Haijun; Dai, Xiaoniu; Fang, Shencun; Wang, Xingang; Zhang, Yingming; Yao, Honghong; Zhang, Xilong; Chao, Jie

    2015-11-18

    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.

  16. Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts

    PubMed Central

    Baroni, S; Romero-Cordoba, S; Plantamura, I; Dugo, M; D'Ippolito, E; Cataldo, A; Cosentino, G; Angeloni, V; Rossini, A; Daidone, M G; Iorio, M V

    2016-01-01

    It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma. PMID:27468688

  17. Exosome-mediated delivery of miR-9 induces cancer-associated fibroblast-like properties in human breast fibroblasts.

    PubMed

    Baroni, S; Romero-Cordoba, S; Plantamura, I; Dugo, M; D'Ippolito, E; Cataldo, A; Cosentino, G; Angeloni, V; Rossini, A; Daidone, M G; Iorio, M V

    2016-07-28

    It is established that the interaction between microenvironment and cancer cells has a critical role in tumor development, given the dependence of neoplastic cells on stromal support. However, how this communication promotes the activation of normal (NFs) into cancer-associated fibroblasts (CAFs) is still not well understood. Most microRNA (miRNA) studies focused on tumor cell, but there is increasing evidence of their involvement in reprogramming NFs into CAFs. Here we show that miR-9, upregulated in various breast cancer cell lines and identified as pro-metastatic miRNA, affects the properties of human breast fibroblasts, enhancing the switch to CAF phenotype, thus contributing to tumor growth. Expressed at higher levels in primary triple-negative breast CAFs versus NFs isolated from patients, miR-9 improves indeed migration and invasion capabilities when transfected in immortalized NFs; viceversa, these properties are strongly impaired in CAFs upon miR-9 inhibition. We also demonstrate that tumor-secreted miR-9 can be transferred via exosomes to recipient NFs and this uptake results in enhanced cell motility. Moreover, we observed that this miRNA is also secreted by fibroblasts and in turn able to alter tumor cell behavior, by modulating its direct target E-cadherin, and NFs themselves. Consistently with the biological effects observed, gene expression profiles of NFs upon transient transfection with miR-9 show the modulation of genes mainly involved in cell motility and extracellular matrix remodeling pathways. Finally, we were able to confirm the capability of NFs transiently transfected with miR-9 to promote in vivo tumor growth. Taken together, these data provide new insights into the role of miR-9 as an important player in the cross-talk between cancer cells and stroma.

  18. Alterations in cell migration and cell viability of wounded human skin fibroblasts following visible red light exposure

    NASA Astrophysics Data System (ADS)

    Prabhu, Vijendra; Rao, Bola Sadashiva S.; Mahato, Krishna Kishore

    2014-02-01

    The present study intended to examine the effect of visible red light on structural and cellular parameters on wounded skin fibroblast cells. To achieve the stated objective, uniform scratch was created on confluent monolayered human skin fibroblast cells, and were exposed to single dose of He-Ne laser (15 mm spot, 6.6808 mWcm-2) at 1, 2, 3, 4, 5, 6 and 7 Jcm-2 in the presence and absence of 10% fetal bovine serum (FBS). Beam profile measurements of the expanded laser beam were conducted to ensure the beam uniformity. The influence of laser dose on the change in temperature was recorded using sensitive temperature probe. Additionally, following laser exposure cell migration and cell survival were documented at different time intervals on wounded human skin fibroblast cells grown in vitro. Beam profile measurements indicated more or less uniform power distribution over the whole beam area. Temperature monitoring of sham irradiated control and laser treatment groups displayed negligible temperature change indicating the absence of thermal effect at the tested laser doses. In the absence of 10% FBS, single exposure of different laser doses failed to produce any significant effects on cell migration or cell survival. However, in the presence of serum single exposure of 5 J/cm2 on wounded skin fibroblasts significantly enhanced the cell migration (P<0.05) compared to the other tested doses (1, 2, 3, 4, 6 and 7 J/cm2) and sham irradiated controls. In conclusion, the LLLT acts by improving cell migration and cell proliferation to produce measurable changes in wounded fibroblast cells.

  19. Fibroblast growth factor receptors in breast cancer.

    PubMed

    Wang, Shuwei; Ding, Zhongyang

    2017-05-01

    Fibroblast growth factor receptors are growth factor receptor tyrosine kinases, exerting their roles in embryogenesis, tissue homeostasis, and development of breast cancer. Recent genetic studies have identified some subtypes of fibroblast growth factor receptors as strong genetic loci associated with breast cancer. In this article, we review the recent epidemiological findings and experiment results of fibroblast growth factor receptors in breast cancer. First, we summarized the structure and physiological function of fibroblast growth factor receptors in humans. Then, we discussed the common genetic variations in fibroblast growth factor receptors that affect breast cancer risk. In addition, we also introduced the potential roles of each fibroblast growth factor receptors isoform in breast cancer. Finally, we explored the potential therapeutics targeting fibroblast growth factor receptors for breast cancer. Based on the biological mechanisms of fibroblast growth factor receptors leading to the pathogenesis in breast cancer, targeting fibroblast growth factor receptors may provide new opportunities for breast cancer therapeutic strategies.

  20. Diaminopurine-Resistant Mutants of Cultured, Diploid Human Fibroblasts

    PubMed Central

    Rappaport, Harriet; DeMars, Robert

    1973-01-01

    Clones of cells resistant to 2,6-diaminopurine were detected in skin fibroblast cultures derived from 13 of 21 normal humans of both sexes from 17 unrelated families. Almost all of the cultures that yielded mutants were chosen for further study from among a total of 83 surveyed because they displayed a slight resistance to low concentrations of diaminopurine. The incidences of mutant colonies ranged between about 10-5 and 10-4 per cell surviving prior mutagenic treatment with MNNG. The incidences of spontaneous mutants were about 10-7 to 10-5 in three unrelated cultures. Most independent mutants had distinctly reduced activity of adenine phosphoribosyltransferase but some had apparently normal amounts of activity. Two mutants from unrelated boys had little or no detectable enzyme activity and were unable to effectively use exogenous adenine for growth when purine biosynthesis was blocked with azaserine. Most mutants could utilize exogenous adenine, just as most azaguanine-resistant fibroblast mutants can utilize exogenous hypoxanthine, even when their hypoxanthine-guanine phosphoribosyltransferase activity is reduced. Diverse genetic changes conferred diaminopurine resistance but their specific natures are still undefined. Gross numerical or structural chromosome abnormalities were not observed in the mutants examined so far. Since at least one gene responsible for adenine phosphoribosyltransferase activity is on autosome No. 16 our results suggest that at least some of the cultures yielding mutants were heterozygous and that alleles conferring diaminopurine resistance may be frequent enough to comprise a polymorphism. PMID:4358687

  1. Generation of Footprint-Free Induced Pluripotent Stem Cells from Human Fibroblasts Using Episomal Plasmid Vectors.

    PubMed

    Ovchinnikov, Dmitry A; Sun, Jane; Wolvetang, Ernst J

    2015-01-01

    Human induced pluripotent stem cells (hiPSCs) have provided novel insights into the etiology of disease and are set to transform regenerative medicine and drug screening over the next decade. The generation of human iPSCs free of a genetic footprint of the reprogramming process is crucial for the realization of these potential uses. Here we describe in detail the generation of human iPSC from control and disease-carrying individuals' fibroblasts using episomal plasmids.

  2. Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts

    PubMed Central

    Gothai, Sivapragasam; Arulselvan, Palanisamy; Tan, Woan Sean; Fakurazi, Sharida

    2016-01-01

    Background/Aim: Wounds are the outcome of injuries to the skin that interrupt the soft tissue. Healing of a wound is a complex and long-drawn-out process of tissue repair and remodeling in response to injury. A large number of plants are used by folklore traditions for the treatment of cuts, wounds and burns. Moringa oleifera (MO) is an herb used as a traditional folk medicine for the treatment of various skin wounds and associated diseases. The underlying mechanisms of wound healing activity of ethyl acetate fraction of MO leaves extract are completely unknown. Materials and Methods: In the current study, ethyl acetate fraction of MO leaves was investigated for its efficacy on cell viability, proliferation and migration (wound closure rate) in human normal dermal fibroblast cells. Results: Results revealed that lower concentration (12.5 µg/ml, 25 µg/ml, and 50 µg/ml) of ethyl acetate fraction of MO leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. Conclusion: This study suggested that ethyl acetate fraction of MO leaves might be a potential therapeutic agent for skin wound healing by promoting fibroblast proliferation and migration through increasing the wound closure rate corroborating its traditional use. PMID:27069722

  3. Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women

    PubMed Central

    Tanaka, Miyuki; Misawa, Eriko; Yamauchi, Koji; Abe, Fumiaki; Ishizaki, Chiaki

    2015-01-01

    Background Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. Methods First, we investigated the capability of Aloe sterols (cycloartenol and lophenol) to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP) containing 40 μg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. Results After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake period. Conclusion The present study confirms that daily oral Aloe sterol-containing AVGP significantly reduced facial wrinkles in women aged ≥40 years, and Aloe sterols stimulate collagen and hyaluronic acid production by human dermal fibroblasts. PMID:25759593

  4. Mitomycin C induces apoptosis in human epidural scar fibroblasts after surgical decompression for spinal cord injury.

    PubMed

    Sui, Tao; Ge, Da-Wei; Yang, Lei; Tang, Jian; Cao, Xiao-Jian; Ge, Ying-Bin

    2017-04-01

    Numerous studies have shown that topical application of mitomycin C after surgical decompression effectively reduces scar adhesion. However, the underlying mechanisms remain unclear. In this study, we investigated the effect of mitomycin C on the proliferation and apoptosis of human epidural scar fibroblasts. Human epidural scar fibroblasts were treated with various concentrations of mitomycin C (1, 5, 10, 20, 40 μg/mL) for 12, 24 and 48 hours. Mitomycin C suppressed the growth of these cells in a dose- and time-dependent manner. Mitomycin C upregulated the expression levels of Fas, DR4, DR5, cleaved caspase-8/9, Bax, Bim and cleaved caspase-3 proteins, and it downregulated Bcl-2 and Bcl-xL expression. In addition, inhibitors of caspase-8 and caspase-9 (Z-IETD-FMK and Z-LEHD-FMK, respectively) did not fully inhibit mitomycin C-induced apoptosis. Furthermore, mitomycin C induced endoplasmic reticulum stress by increasing the expression of glucose-regulated protein 78, CAAT/enhancer-binding protein homologous protein (CHOP) and caspase-4 in a dose-dependent manner. Salubrinal significantly inhibited the mitomycin C-induced cell viability loss and apoptosis, and these effects were accompanied by a reduction in CHOP expression. Our results support the hypothesis that mitomycin C induces human epidural scar fibroblast apoptosis, at least in part, via the endoplasmic reticulum stress pathway.

  5. Evidence of two distinct functionally specialized fibroblast lineages in breast stroma.

    PubMed

    Morsing, Mikkel; Klitgaard, Marie Christine; Jafari, Abbas; Villadsen, René; Kassem, Moustapha; Petersen, Ole William; Rønnov-Jessen, Lone

    2016-11-03

    The terminal duct lobular unit (TDLU) is the most dynamic structure in the human breast and the putative site of origin of human breast cancer. Although stromal cells contribute to a specialized microenvironment in many organs, this component remains largely understudied in the human breast. We here demonstrate the impact on epithelium of two lineages of breast stromal fibroblasts, one of which accumulates in the TDLU while the other resides outside the TDLU in the interlobular stroma. The two lineages are prospectively isolated by fluorescence activated cell sorting (FACS) based on different expression levels of CD105 and CD26. The characteristics of the two fibroblast lineages are assessed by immunocytochemical staining and gene expression analysis. The differentiation capacity of the two fibroblast populations is determined by exposure to specific differentiating conditions followed by analysis of adipogenic and osteogenic differentiation. To test whether the two fibroblast lineages are functionally imprinted by their site of origin, single cell sorted CD271 low /MUC1 high normal breast luminal epithelial cells are plated on fibroblast feeders for the observation of morphological development. Epithelial structure formation and polarization is shown by immunofluorescence and digitalized quantification of immunoperoxidase-stained cultures. Lobular fibroblasts are CD105 high /CD26 low while interlobular fibroblasts are CD105 low /CD26 high . Once isolated the two lineages remain phenotypically stable and functionally distinct in culture. Lobular fibroblasts have properties in common with bone marrow derived mesenchymal stem cells and they specifically convey growth and branching morphogenesis of epithelial progenitors. Two distinct functionally specialized fibroblast lineages exist in the normal human breast, of which the lobular fibroblasts have properties in common with mesenchymal stem cells and support epithelial growth and morphogenesis. We propose that

  6. PD-1 ligand expression by human colonic myofibroblasts/fibroblasts regulates CD4+ T-cell activity.

    PubMed

    Pinchuk, Irina V; Saada, Jamal I; Beswick, Ellen J; Boya, Gushyalatha; Qiu, Sumin M; Mifflin, Randy C; Raju, Gottumukkala S; Reyes, Victor E; Powell, Don W

    2008-10-01

    A prominent role for inhibitory molecules PD-L1 and PD-L2 in peripheral tolerance has been proposed. However, the phenotype and function of PD-L-expressing cells in human gut remains unclear. Recent studies suggest that colonic myofibroblasts (CMFs) and fibroblasts are important in the switch from acute inflammation to adaptive immunity. In the normal human colon, CMFs represent a distinct population of major histocompatibility complex class II(+) cells involved in the regulation of mucosal CD4(+) T-cell responses. PD-L1 and PD-L2 expression on human CMFs was determined using Western blot, fluorescence-activated cell sorter analysis and confocal microscopy. Lymphoproliferation assays and cytokine enzyme-linked immunosorbent assays were used to evaluate the role of B7 costimulators expressed by CMFs with regard to the regulation of preactivated T-helper cell responses. We demonstrate here the expression of PD-L1/2 molecules by normal human CMF and fibroblasts in situ and in culture. Both molecules support suppressive functions of CMFs in the regulation of activated CD4(+) T-helper cell proliferative responses; blocking this interaction reverses the suppressive effect of CMFs on T-cell proliferation and leads to increased production of the major T-cell growth factor, interleukin (IL)-2. PD-L1/2-mediated CMF suppressive functions are mainly due to the inhibition of IL-2 production, because supplementation of the coculture media with exogenous IL-2 led to partial recovery of activated T-cell proliferation. Our data suggest that stromal myofibroblasts and fibroblasts may limit T-helper cell proliferative activity in the gut and, thus, might play a prominent role in mucosal intestinal tolerance.

  7. Cyclic tension promotes fibroblastic differentiation of human MSCs cultured on collagen-fibre scaffolds.

    PubMed

    Qiu, Yongzhi; Lei, Jennifer; Koob, Thomas J; Temenoff, Johnna S

    2016-12-01

    Mesenchymal stem cells (MSCs) have been suggested as a potential cell source for tendon/ligament tissue engineering. Extrinsic cues, such as the chemical and physical properties of scaffolds, as well as external forces, play an important role in fibroblastic differentiation of these cells. In this study, we employed a collagen-fibre scaffold that mimics the chemical and fibrous structure and mechanical properties of tendon/ligament, and studied how imparting cyclic tension to these fibrous collagen scaffolds affects tendon/ligament fibroblastic differentiation of MSCs. Human MSCs attached and spread on the surface of the scaffolds, and appeared aligned along the fibres 24 h after seeding. Cyclic tension was then applied to cell-laden scaffolds over a period of 14 days (10% strain, 1 Hz, 3 h on/3 h off). Real time RT-PCR analysis indicated that scleraxis, a transcription factor associated with the tendon fibroblast phenotype, was found to be significantly upregulated only under cyclic tension. Immunohistochemical staining demonstrated that MSCs cultured under cyclic tension after 14 days secreted more extracellular matrix, including collagen I, collagen III and tenascin-C, compared to constructs in static culture, after 14 days in vitro. Our data indicate that cyclic tension can promote fibroblastic differentiation of MSCs in these fibrous collagen-based scaffolds, which may have significant applications in the development of tissue-engineered graft alternatives for tendon and ligament injuries. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

  8. Cytotoxicity Evaluation of Two Bis-Acryl Composite Resins Using Human Gingival Fibroblasts.

    PubMed

    Gonçalves, Fabiano Palmeira; Alves, Gutemberg; Guimarães, Vladi Oliveira; Gallito, Marco Antônio; Oliveira, Felipe; Scelza, Míriam Zaccaro

    2016-01-01

    Bis-acryl resins are used for temporary dental restorations and have shown advantages over other materials. The aim of this work was to evaluate the in vitro cytotoxicity of two bis-acryl composite resins (Protemp 4 and Luxatemp Star), obtained at 1, 7 and 40 days after mixing the resin components, using a standardized assay employing human primary cells closely related to oral tissues. Human gingival fibroblast cell cultures were exposed for 24 h to either bis-acryl composite resins, polystyrene beads (negative control) and latex (positive control) extracts obtained after incubation by the different periods, at 37 °C under 5% CO2. Cell viability was evaluated using a multiparametric procedure involving sequential assessment (using the same cells) of mitochondrial activity (XTT assay), membrane integrity (neutral red test) and total cell density (crystal violet dye exclusion test). The cells exposed to the resin extracts showed cell viability indexes exceeding 75% after 24 h. Even when cells were exposed to extracts prepared with longer conditioning times, the bis-acryl composite resins showed no significant cytotoxic effects (p>0.05), compared to the control group or in relation to the first 24 h of contact with the products. There were no differences among the results obtained for the bis-acryl composite resins evaluated 24 h, 7 days and 40 days after mixing. It may be concluded that the bis-acryl resins Protemp 4 and Luxatemp Star were cytocompatible with human gingival fibroblasts, suggesting that both materials are suitable for use in contact with human tissues.

  9. Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes.

    PubMed

    El Ghalbzouri, Abdoelwaheb; Jonkman, Marcel F; Dijkman, Remco; Ponec, Maria

    2005-01-01

    This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.

  10. Possible role of ginsenoside Rb1 in skin wound healing via regulating senescent skin dermal fibroblast.

    PubMed

    Hou, Jingang; Kim, Sunchang

    2018-05-05

    Cellular senescence suppresses cancer by inducing irreversible cell growth arrest. Nevertheless, senescent cells is proposed as causal link with aging and aging-related pathologies. The physiological beneficial functions of senescent cells are still of paucity. Here we show that senescent human dermal fibroblast accelerates keratinocytes scratch wound healing and stimulates differentiation of fibroblast. Using oxidative stress (100 μM H 2 O 2 exposure for 1 h) induction, we successfully triggered fibroblast senescence and developed senescence associated secretory phenotype (SASP). The induction of SASP was regulated by p38MAPK/MSK2/NF-κB pathway. Interestingly, inhibition of p38MAPK activation only partially suppressed SASP. However, SASP was significantly inhibited by SB747651A, a specific MSK inhibitor. Additionally, we demonstrate that SASP stimulates migration of keratinocytes and myofibroblast transition of fibroblast, through fold-increased secretion of growth factors, platelet-derived growth factor AA (PDGF-AA) and AB (PDGF-AB), transforming growth factor beta 1 (TGF-β1) and beta 2 (TGF-β2), vascular endothelial growth factor A (VEGF-A) and D (VEGF-D), vascular endothelial growth factor receptor 2 (VEGFR2) and 3 (VEGFR3). Importantly, we also confirmed ginsenoside Rb1 promoted SASP-mediated healing process via p38MAPK/MSK2/NF-κB pathway. The results pointed to senescent fibroblast as a potential mechanism of wound healing control in human skin. Further, it provided a candidate targeted for wound therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Comparative study of human-induced pluripotent stem cells derived from bone marrow cells, hair keratinocytes, and skin fibroblasts.

    PubMed

    Streckfuss-Bömeke, Katrin; Wolf, Frieder; Azizian, Azadeh; Stauske, Michael; Tiburcy, Malte; Wagner, Stefan; Hübscher, Daniela; Dressel, Ralf; Chen, Simin; Jende, Jörg; Wulf, Gerald; Lorenz, Verena; Schön, Michael P; Maier, Lars S; Zimmermann, Wolfram H; Hasenfuss, Gerd; Guan, Kaomei

    2013-09-01

    Induced pluripotent stem cells (iPSCs) provide a unique opportunity for the generation of patient-specific cells for use in disease modelling, drug screening, and regenerative medicine. The aim of this study was to compare human-induced pluripotent stem cells (hiPSCs) derived from different somatic cell sources regarding their generation efficiency and cardiac differentiation potential, and functionalities of cardiomyocytes. We generated hiPSCs from hair keratinocytes, bone marrow mesenchymal stem cells (MSCs), and skin fibroblasts by using two different virus systems. We show that MSCs and fibroblasts are more easily reprogrammed than keratinocytes. This corresponds to higher methylation levels of minimal promoter regions of the OCT4 and NANOG genes in keratinocytes than in MSCs and fibroblasts. The success rate and reprogramming efficiency was significantly higher by using the STEMCCA system than the OSNL system. All analysed hiPSCs are pluripotent and show phenotypical characteristics similar to human embryonic stem cells. We studied the cardiac differentiation efficiency of generated hiPSC lines (n = 24) and found that MSC-derived hiPSCs exhibited a significantly higher efficiency to spontaneously differentiate into beating cardiomyocytes when compared with keratinocyte-, and fibroblast-derived hiPSCs. There was no significant difference in the functionalities of the cardiomyocytes derived from hiPSCs with different origins, showing the presence of pacemaker-, atrial-, ventricular- and Purkinje-like cardiomyocytes, and exhibiting rhythmic Ca2+ transients and Ca2+ sparks in hiPSC-derived cardiomyocytes. Furthermore, spontaneously and synchronously beating and force-developing engineered heart tissues were generated. Human-induced pluripotent stem cells can be reprogrammed from all three somatic cell types, but with different efficiency. All analysed iPSCs can differentiate into cardiomyocytes, and the functionalities of cardiomyocytes derived from different cell

  12. Human fibroblast-derived extracellular matrix constructs for bone tissue engineering applications.

    PubMed

    Tour, Gregory; Wendel, Mikael; Tcacencu, Ion

    2013-10-01

    We exploited the biomimetic approach to generate constructs composed of synthetic biphasic calcium phosphate ceramic and extracellular matrix (SBC-ECM) derived from adult human dermal fibroblasts in complete xeno-free culture conditions. The construct morphology and composition were assessed by scanning electron microscopy, histology, immunohistochemistry, Western blot, glycosaminoglycan, and hydroxyproline assays. Residual DNA quantification, endotoxin testing, and local inflammatory response after implantation in a rat critical-sized calvarial defect were used to access the construct biocompatibility. Moreover, in vitro interaction of human mesenchymal stem cells (hMSCs) with the constructs was studied. The bone marrow- and adipose tissue-derived mesenchymal stem cells were characterized by flow cytometry and tested for osteogenic differentiation capacity prior seeding onto SBC-ECM, followed by alkaline phosphatase, 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and real-time quantitative polymerase chain reaction to assess the osteogenic differentiation of hMSCs after seeding onto the constructs at different time intervals. The SBC-ECM constructs enhanced osteogenic differentiation of hMSCs in vitro and exhibited excellent handling properties and high biocompatibility in vivo. Our results highlight the ability to generate in vitro fibroblast-derived ECM constructs in complete xeno-free conditions as a step toward clinical translation, and the potential use of SBC-ECM in craniofacial bone tissue engineering applications. Copyright © 2013 Wiley Periodicals, Inc., a Wiley Company.

  13. Cytotoxicity evaluation of sodium alendronate on cultured human periodontal ligament fibroblasts.

    PubMed

    Correia, Vera de Fátima Padrão; Caldeira, Celso L; Marques, Márcia Martins

    2006-12-01

    External root resorption processes are usually associated with dental trauma, mainly avulsion and intrusion. In such cases endodontic therapy aims to prevent this process by using medications that can inhibit osteoclastic activity, such as bisphosphonates. However, these drugs must be biocompatible to the periapical tissues. The aim of this study was to analyze the cytotoxicity of a bisphosphonate (sodium alendronate) on human periodontal ligament fibroblasts (PDL cells). Cells were plated in a density of 1 x 10(3) cells per dish. The experimental groups were GI (control) no sodium alendronate, and GII, GIII, and GIV with sodium alendronate at the concentrations of 10(-5), 10(-6), and 10(-7) M, respectively. The experimental times were 1, 6, 12, and 24 h (short-term) for viability and 2, 4, 6, and 8 days (long-term) for cell survival. Data in triplicate were statistically analyzed. Cultures treated with the highest alendronate concentration (GII) showed cell viability percentages significantly lower (P < 0.01) than those of the other groups (GI, GIII, and GIV), at 12 and 24 h. Cell growth on GII and GIII groups was similar. GII presented smaller growth than the other groups (P < 0.05). We concluded that sodium alendronate, on direct contact with human periodontal ligament fibroblasts, is cytotoxic in concentrations higher than of 10(-6) M.

  14. Evaluation of the cytotoxicity of selected conventional glass ionomer cements on human gingival fibroblasts.

    PubMed

    Marczuk-Kolada, Grażyna; Łuczaj-Cepowicz, Elżbieta; Pawińska, Małgorzata; Hołownia, Adam

    2017-10-01

    Dentistry materials are the most frequently used substitutes of human tissues. Therefore, an assessment of dental filling materials should cover not only their chemical, physical, and mechanical characteristics, but also their cytotoxicity. To compare the cytotoxic effects of 13 conventional glass ionomer cements on human gingival fibroblasts. The assessment was conducted using the MTT test. Six samples were prepared for each material. Culture plates with cells and inserts with the materials were incubated at 37°C, 5% CO2, and 95% humidity for 24 h. Then the inserts were removed, 1 mL of MTT was added in the amount of 0.5 mg/1 mL of the medium, and the samples were incubated in the described conditions without light for 2 h. The optical density was measured with an absorption spectrophotometer at a wavelength of 560 nm. The cytotoxic effects of the Argion Molar was significantly stronger than the Fuji Triage (p = 0.007), Chemfil Molar (p < 0.0001), and Ionofil Molar AC Quick (p < 0.001). The Fuji IX GP and Fuji IX Extra had a significantly stronger adverse effect than the Chemfil Molar (p = 0.014, p = 0.029, respectively) and Ionofil Molar AC Quick (p = 0.017, p = 0.034, respectively). The cements from the low cytotoxicity group were significantly more toxic vs materials whose presence resulted in fibroblast growth (p < 0.001). The research conducted indicates that, although the materials studied may belong to the same group, they are characterized by low, yet not uniform, cytotoxicity on human gingival fibroblasts. The toxic effects should not be assigned to a relevant group of materials, but each dentistry product should be evaluated individually.

  15. Angiotensin II Type 1 Receptor Knockdown Impairs Interleukin-1β-Induced Cytokines in Human Periodontal Fibroblasts.

    PubMed

    Gabriele, Lilian Gobbo; Morandini, Ana Carolina; Dionísio, Thiago José; Santos, Carlos Ferreira

    2017-01-01

    The renin-angiotensin (Ang) system (RAS) has been reported as an important modulator of inflammatory and immune responses. Evidence suggests an alternative Ang 1-7/Mas receptor axis as counter-regulatory to the classic RAS Ang II/Ang II Type 1 (AT1) receptor axis. It is known that periodontal pathogens elicit host-derived immune response due to release of cytokines such as interleukin (IL)-1β, and fibroblasts are among the most numerous sentinel cells that contribute to this production. The aim of this study is to determine whether AT1 receptor (AT1R) contributes to production of inflammatory cytokines that are important for periodontal pathogenesis using primary human gingival fibroblasts (HGFs) and human periodontal ligament fibroblasts (HPLFs) stimulated with IL-1β. Through RNA interference or pharmacologic inhibition using AT1R antagonist losartan, HGF and HPLF were stimulated by IL-1β for 3 (messenger RNA [mRNA]) or 24 (protein) hours. IL-1β upregulated mRNA expression of AT1R, IL-1β, IL-6, IL-8, tumor necrosis factor-alpha, and osteoprotegerin (OPG) in HGF and HPLF. AT1R knockdown impaired IL-1β-induced IL-6 and IL-8 secretion in cultured HGF and HPLF. AT1R silencing also increased OPG gene expression in HGF only. Pharmacologic inhibition of AT1R through losartan modulated mRNA transcription of IL-6 and IL-8 in HPLF but not in HGF. In contrast, IL-1β-induced secretion of IL-6 and IL-8 was not influenced by losartan in HGF or HPLF. These results suggest that AT1R knockdown and AT1R pharmacologic blockade by losartan may differently control balance of inflammatory cytokines, such as IL-6 and IL-8, in primary human periodontal fibroblasts.

  16. TNF-alpha and endotoxin increase hypoxia-induced VEGF production by cultured human nasal fibroblasts in synergistic fashion.

    PubMed

    Sun, Dong; Matsune, Shoji; Ohori, Junichiro; Fukuiwa, Tatsuya; Ushikai, Masato; Kurono, Yuichi

    2005-09-01

    Vascular endothelial growth factor (VEGF) promotes angiogenesis and is associated with the invasion and metastasis of malignant tumors. It enhances vascular permeability and is expressed in inflammatory nasal as well as middle-ear mucosa. As the mechanism of VEGF induction during chronic inflammation, such as chronic paranasal sinusitis (CPS) remains to be clarified, we studied the factors regulating the production of VEGF in cultured human nasal fibroblasts and discussed the role of VEGF in the pathogenesis of CPS. We used ELISA to quantify VEGF levels in paranasal sinus effusions, nasal secretions, and serum from patients with CPS. In addition, we cultured human nasal fibroblasts isolated from nasal polyps of CPS patients and studied the effects of hypoxia, TNF-alpha, and endotoxin on their production of VEGF using ELISA and PCR. The VEGF concentration was significantly higher in paranasal sinus effusions than in nasal secretions and serum. Nasal fibroblasts produced high levels of VEGF, when cultured under hypoxic condition and this production was remarkably enhanced in the presence of TNF-alpha or endotoxin. VEGF is locally produced in paranasal sinuses as well as nasal mucosa and its production is increased in patients with CPS. Hypoxia is associated with the production of VEGF by nasal fibroblasts and TNF-alpha and endotoxin may act synergistically to enhance VEGF production in paranasal sinuses under hypoxic condition.

  17. Computational modeling for cardiac safety pharmacology analysis: Contribution of fibroblasts.

    PubMed

    Gao, Xin; Engel, Tyler; Carlson, Brian E; Wakatsuki, Tetsuro

    2017-09-01

    Drug-induced proarrhythmic potential is an important regulatory criterion in safety pharmacology. The application of in silico approaches to predict proarrhythmic potential of new compounds is under consideration as part of future guidelines. Current approaches simulate the electrophysiology of a single human adult ventricular cardiomyocyte. However, drug-induced proarrhythmic potential can be different when cardiomyocytes are surrounded by non-muscle cells. Incorporating fibroblasts in models of myocardium is important particularly for predicting a drugs cardiac liability in the aging population - a growing population who take more medications and exhibit increased cardiac fibrosis. In this study, we used computational models to investigate the effects of fibroblast coupling on the electrophysiology and response to drugs of cardiomyocytes. A computational model of cardiomyocyte electrophysiology and ion handling (O'Hara, Virag, Varro, & Rudy, 2011) is coupled to a passive model of fibroblast electrophysiology to test the effects of three compounds that block cardiomyocyte ion channels. Results are compared to model results without fibroblast coupling to see how fibroblasts affect cardiomyocyte action potential duration at 90% repolarization (APD 90 ) and propensity for early after depolarization (EAD). Simulation results show changes in cardiomyocyte APD 90 with increasing concentration of three drugs that affect cardiac function (dofetilide, vardenafil and nebivolol) when no fibroblasts are coupled to the cardiomyocyte. Coupling fibroblasts to cardiomyocytes markedly shortens APD 90 . Moreover, increasing the number of fibroblasts can augment the shortening effect. Coupling cardiomyocytes and fibroblasts are predicted to decrease proarrhythmic susceptibility under dofetilide, vardenafil and nebivolol block. However, this result is sensitive to parameters which define the electrophysiological function of the fibroblast. Fibroblast membrane capacitance and

  18. The effect of isosaponarin isolated from wasabi leaf on collagen synthesis in human fibroblasts and its underlying mechanism.

    PubMed

    Nagai, Masashi; Akita, Keiko; Yamada, Kazuno; Okunishi, Isao

    2010-07-01

    Wasabi has been used as an important spice in Japanese foods. The wasabi leaves were used as a cosmetic material, but its biological activities have not yet been examined. We investigated the effect of isosaponarin derived from wasabi leaf on collagen synthesis in human fibroblasts. The production of type I collagen in human fibroblasts was increased with treatment of wasabi leaf extract. Isosaponarin isolated from wasabi leaves belonged to the group of flavone glycoside, and was the key compound in collagen synthesis from the wasabi leaf ingredients. Isosaponarin increased the type I collagen production at the mRNA gene level. The treatment of isosaponarin did not influence the production of transforming growth factor-beta (TGF-beta) protein, but increased the production of TGF-beta type II receptor (TbetaR-II) protein and TbetaR-II mRNA. Prolyl 4-hydroxylase (P4H) protein and P4H mRNA were increased by treatment with isosaponarin. Heat shock protein 47 (HSP47) was not increased by treatment with isosaponarin. These results suggested that isosaponarin increased collagen synthesis in human fibroblasts, caused by up-regulated TbetaR-II and P4H production.

  19. Electron spin resonance detection of oxygen radicals released by UVA-irradiated human fibroblasts

    NASA Astrophysics Data System (ADS)

    Souchard, J. P.; Pierlot, G.; Barbacanne, M. A.; Charveron, M.; Bonafé, J.-L.; Nepveu, F.

    1999-01-01

    This work reports the electron spin resonance (ESR) detection of oxygenated radicals (OR) released by cultured human fibroblasts after UVA (365 nm) exposure. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as spin trap. After a UVA irradiation of one hour, followed by a latent period of at least 45 min., and an incubation time of 30 min. in a trapping medium containing DMPO, glucose, Na^+, K+ and Ca2+ an ESR signal was recorded. By contrast, an ESR signal was produced after only 15 min. incubation when calcium ionophore A23187 was used. Although the ESR signal was characteristic of the hydroxyl adduct DMPO-OH, the use of catalase and superoxide dismutase (SOD) revealed that UVA stimulated fibroblasts released the superoxide anion O2- in the medium. SOD, vitamin C and (+)-catechin inhibited the release of superoxide generated by human fibroblasts stimulated with A23187 calcium ionophore at 5 units/ml, 10-5 M and 2× 10-4 M, respectively. Dans ce travail nous présentons la détection par résonance de spin électronique (RSE) de radicaux oxygénés (RO) libérés par des fibroblastes humains en culture après irradiation aux UVA (365 nm). Le 5,5-diméthyl-1-pyrroline-N-oxyde (DMPO) a été utilisé comme piégeur de spin. Après une irradiation aux UVA d'une heure, suivie d'une période de latence d'au moins 45 min. et d'une incubation de 30 min. dans un milieu de piégeage composé de DMPO, glucose, Na^+, K+ et Ca2+, un signal RPE est enregistré. L'ionophore calcique A23187 entraîne l'apparition d'un signal RPE après seulement 15 min. d'incubation. Bien que le signal RPE obtenu corresponde à l'adduit DMPO-OH du radical hydroxyle, l'utilisation de catalase et de superoxyde dismutase (SOD) a révélé que les fibroblastes libéraient l'anion superoxyde dans le milieu de culture. Sur ce modèle cellulaire la SOD, la vitamine C et la (+) catéchine inhibent la production du radical superoxyde aux concentrations respectivement de 5 unités/ml, 10-5 M et 2× 10-4M.

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

    PubMed

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

    2017-12-01

    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, α 2 β 1 integrin (VLA-2) and the laminin receptor very late antigen 6, α 6 β 1 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.

  1. 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.

  2. Feeder & basic fibroblast growth factor-free culture of human embryonic stem cells: Role of conditioned medium from immortalized human feeders.

    PubMed

    Teotia, Pooja; Sharma, Shilpa; Airan, Balram; Mohanty, Sujata

    2016-12-01

    Human embryonic stem cell (hESC) lines are commonly maintained on inactivated feeder cells, in the medium supplemented with basic fibroblast growth factor (bFGF). However, limited availability of feeder cells in culture, and the high cost of growth factors limit their use in scalable expansion of hESC cultures for clinical application. Here, we describe an efficient and cost-effective feeder and bFGF-free culture of hESCs using conditioned medium (CM) from immortalized feeder cells. KIND-1 hESC cell line was cultured in CM, collected from primary mouse embryonic fibroblast, human foreskin fibroblast (HFF) and immortalized HFF (I-HFF). Pluripotency of KIND-1 hESC cell line was confirmed by expression of genes, proteins and cell surface markers. In culture, these cells retained normal morphology, expressed all cell surface markers, could differentiate to embryoid bodies upon culture in vitro. Furthermore, I-HFF feeder cells without supplementation of bFGF released ample amount of endogenous bFGF to maintain stemness of hESC cells. The study results described the use of CM from immortalized feeder cells as a consistent source and an efficient, inexpensive feeder-free culture system for the maintenance of hESCs. Moreover, it was possible to maintain hESCs without exogenous supplementation of bFGF. Thus, the study could be extended to scalable expansion of hESC cultures for therapeutic purposes.

  3. Comparison of the effects of agalsidase alfa and agalsidase beta on cultured human Fabry fibroblasts and Fabry mice.

    PubMed

    Sakuraba, Hitoshi; Murata-Ohsawa, Mai; Kawashima, Ikuo; Tajima, Youichi; Kotani, Masaharu; Ohshima, Toshio; Chiba, Yasunori; Takashiba, Minako; Jigami, Yoshifumi; Fukushige, Tomoko; Kanzaki, Tamotsu; Itoh, Kohji

    2006-01-01

    We compared two recombinant alpha-galactosidases developed for enzyme replacement therapy for Fabry disease, agalsidase alfa and agalsidase beta, as to specific alpha-galactosidase activity, stability in plasma, mannose 6-phosphate (M6P) residue content, and effects on cultured human Fabry fibroblasts and Fabry mice. The specific enzyme activities of agalsidase alfa and agalsidase beta were 1.70 and 3.24 mmol h(-1) mg protein(-1), respectively, and there was no difference in stability in plasma between them. The M6P content of agalsidase beta (3.6 mol/mol protein) was higher than that of agalsidase alfa (1.3 mol/mol protein). The administration of both enzymes resulted in marked increases in alpha-galactosidase activity in cultured human Fabry fibroblasts, and Fabry mouse kidneys, heart, spleen and liver. However, the increase in enzyme activity in cultured fibroblasts, kidneys, heart and spleen was higher when agalsidase beta was used. An immunocytochemical analysis revealed that the incorporated recombinant enzyme degraded the globotriaosyl ceramide accumulated in cultured Fabry fibroblasts in a dose-dependent manner, with the effect being maintained for at least 7 days. Repeated administration of agalsidase beta apparently decreased the number of accumulated lamellar inclusion bodies in renal tubular cells of Fabry mice.

  4. Novel signatures of cancer-associated fibroblasts.

    PubMed

    Bozóky, Benedek; Savchenko, Andrii; Csermely, Péter; Korcsmáros, Tamás; Dúl, Zoltán; Pontén, Fredrik; Székely, László; Klein, George

    2013-07-15

    Increasing evidence indicates the importance of the tumor microenvironment, in particular cancer-associated fibroblasts, in cancer development and progression. In our study, we developed a novel, visually based method to identify new immunohistochemical signatures of these fibroblasts. The method employed a protein list based on 759 protein products of genes identified by RNA profiling from our previous study, comparing fibroblasts with differential growth-modulating effect on human cancers cells, and their first neighbors in the human protein interactome. These 2,654 proteins were analyzed in the Human Protein Atlas online database by comparing their immunohistochemical expression patterns in normal versus tumor-associated fibroblasts. Twelve new proteins differentially expressed in cancer-associated fibroblasts were identified (DLG1, BHLHE40, ROCK2, RAB31, AZI2, PKM2, ARHGAP31, ARHGAP26, ITCH, EGLN1, RNF19A and PLOD2), four of them can be connected to the Rho kinase signaling pathway. They were further analyzed in several additional tumor stromata and revealed that the majority showed congruence among the different tumors. Many of them were also positive in normal myofibroblast-like cells. The new signatures can be useful in immunohistochemical analysis of different tumor stromata and may also give us an insight into the pathways activated in them in their true in vivo context. The method itself could be used for other similar analysis to identify proteins expressed in other cell types in tumors and their surrounding microenvironment. Copyright © 2013 UICC.

  5. Molecular mechanisms of UVB-induced senescence of dermal fibroblasts and its relevance for photoaging of the human skin.

    PubMed

    Cavinato, Maria; Jansen-Dürr, Pidder

    2017-08-01

    Due to its ability to cross the epidermis and reach the upper dermis where it causes cumulative DNA damage and increased oxidative stress, UVB is considered the most harmful component of sunlight to the skin. The consequences of chronic exposition to UVB are related to photoaging and photocarcinogenesis. There are limitations to the study of human skin aging and for this reason the use of models is required. Human dermal fibroblasts submitted to mild and repeated doses of UVB are considered a versatile model to study UVB effects in the process of skin photoaging, which depends on the accumulation of senescent cells, in particular in the dermis. Here we provide updated information about the current model of UVB-induced senescence with special emphasis on the process of protein quality control. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Pirfenidone Induces G1 Arrest in Human Tenon's Fibroblasts In Vitro Involving AKT and MAPK Signaling Pathways.

    PubMed

    Guo, Xiujuan; Yang, Yangfan; Liu, Liling; Liu, Xiaoan; Xu, Jiangang; Wu, Kaili; Yu, Minbin

    2017-06-01

    To investigate the underlying mechanism by which pirfenidone blocks the transition from the G1 to S phase in primary human Tenon's fibroblasts. Primary human Tenon's fibroblasts were characterized by immunocytofluorescence staining with vimentin, fibroblast surface protein, and cytokeratin. After treating Tenon's fibroblasts with pirfenidone under proliferation conditions (10% fetal bovine serum), cell proliferation was measured using a WST-1 assay. Progression through the cell cycle was analyzed by flow cytometry. The expression of CDK2, CDK6, cyclinD1, cyclinD3, and cyclinE and the phosphorylation of AKT, ERK1/2/MAPK, JNK/MAPK, and p38 MAPK were estimated using western blot analysis. Under proliferative conditions, pirfenidone inhibited Tenon's fibroblasts proliferation and arrested the cell cycle at the G1 phase; decreased the phosphorylation of AKT, GSK3β, ERK1/2/MAPK, and JNK/MAPK; increased the phosphorylation of p38 MAPK; and inhibited CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E in a dose-dependent manner. Inhibitors of AKT (LY294002), ERK1/2 (U0126), and JNK (SP600125) arrested the G1/S transition, similar to the effect of pirfenidone. The p38 inhibitor (SB202190) decreased the G1-blocking effect of pirfenidone. The expression of CDK2, CDK6, cyclin D1, and cyclin D3 were inhibited by LY294002, U0126, and SP600125. SB202190 attenuated the pirfenidone-induced reduction of CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E. Pirfenidone inhibited HTFs proliferation and induced G1 arrest by downregulating CDKs and cyclins involving the AKT/GSK3β and MAPK signaling pathways.

  7. Dose-dependent effect of mitomycin C on human vocal fold fibroblasts.

    PubMed

    Li, Nicole Y K; Chen, Fei; Dikkers, Frederik G; Thibeault, Susan L

    2014-03-01

    The purpose of this study was to evaluate in vitro cytotoxicity and antifibrotic effects of mitomycin C on normal and scarred human vocal fold fibroblasts. Fibroblasts were subjected to mitomycin C treatment at 0.2, 0.5, or 1 mg/mL, or serum control. Cytotoxicity, immunocytochemistry, and Western blot for collagen I/III were performed at days 0, 1, 3, and 5. Significant decreases in live cells were measured for mitomycin C-treated cells on days 3 and 5 for all doses. Extracellular staining of collagen I/III was observed in mitomycin C-treated cells across all doses and times. Extracellular staining suggests apoptosis with necrosis, compromising the integrity of cell membranes and release of cytosolic proteins into the extracellular environment. Western blot indicates inhibition of collagen at all doses except 0.2 mg/mL at day 1. A total of 0.2 mg/mL mitomycin C may provide initial and transient stimulation of collagen for necessary repair to damaged tissue without the long-term risk of fibrosis. Copyright © 2013 Wiley Periodicals, Inc., A Wiley Company.

  8. Dose-dependent effect of mitomycin C on human vocal fold fibroblasts

    PubMed Central

    Li, Nicole Y. K.; Chen, Fei; Dikkers, Frederik G.; Thibeault, Susan L.

    2014-01-01

    Background The purpose of this study was to evaluate in vitro cytotoxicity and antifibrotic effects of mitomycin C on normal and scarred human vocal fold fibroblasts. Methods Fibroblasts were subjected to mitomycin C treatment at 0.2, 0.5, or 1 mg/mL, or serum control. Cytotoxicity, immunocytochemistry, and Western blot for collagen I/III were performed at days 0, 1, 3, and 5. Results Significant decreases in live cells were measured for mitomycin C-treated cells on days 3 and 5 for all doses. Extracellular staining of collagen I/III was observed in mitomycin C-treated cells across all doses and times. Extracellular staining suggests apoptosis with necrosis, compromising the integrity of cell membranes and release of cytosolic proteins into the extracellular environment. Western blot indicates inhibition of collagen at all doses except 0.2 mg/mL at day 1. Conclusion A total of 0.2 mg/mL mitomycin C may provide initial and transient stimulation of collagen for necessary repair to damaged tissue without the long-term risk of fibrosis. PMID:23765508

  9. Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

    PubMed

    Berning, Manuel; Prätzel-Wunder, Silke; Bickenbach, Jackie R; Boukamp, Petra

    2015-09-01

    Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

  10. Protective effect of chromene isolated from Sargassum horneri against UV-A-induced damage in skin dermal fibroblasts.

    PubMed

    Kim, Jung-Ae; Ahn, Byul-Nim; Kong, Chang-Suk; Kim, Se-Kwon

    2012-08-01

    Skin homoeostasis is interrupted during UV-A irradiation. How the UV-A-altered skin components influences photoageing of skin should be investigated using human in vitro models that are important for understanding skin ageing. In this study, chromene compound, sargachromenol, was isolated from Sargassum horneri, and its potency on inhibition of photoageing was investigated in UV-A-irradiated dermal fibroblasts. Effects of sargachromenol on the prevention of photoageing were evaluated by measuring ROS production, membrane protein oxidation, lipid peroxidation and ageing-related gene expression in UV-A-irradiated human skin dermal fibroblasts. The results indicated that treatment with sargachromenol suppressed the collagenase matrix metalloproteinases (MMPs), MMP-1, MMP-2 and MMP-9 expression without any cytotoxicity and phototoxicity. It was further found that these inhibitions were because of increase in the expression of TIMP-1 and TIMP-2 genes. Furthermore, we confirmed that the UV-A-induced transcriptions of AP-1 signalling pathway were regulated by sargachromenol treatment in UV-A-irradiated dermal fibroblasts. © 2012 John Wiley & Sons A/S.

  11. EMMPRIN Is Secreted by Human Uterine Epithelial Cells in Microvesicles and Stimulates Metalloproteinase Production by Human Uterine Fibroblast Cells

    PubMed Central

    Dayger, C. A.; Mehrotra, P.; Belton, R. J.; Nowak, R. A.

    2012-01-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C. PMID:22729071

  12. EMMPRIN is secreted by human uterine epithelial cells in microvesicles and stimulates metalloproteinase production by human uterine fibroblast cells.

    PubMed

    Braundmeier, A G; Dayger, C A; Mehrotra, P; Belton, R J; Nowak, R A

    2012-12-01

    Endometrial remodeling is a physiological process involved in the gynecological disease, endometriosis. Tissue remodeling is directed by uterine fibroblast production of matrix metalloproteinases (MMPs). Several MMPs are regulated directly by the protein extracellular matrix metalloproteinase inducer (EMMPRIN) and also by proinflammatory cytokines such as interleukin (IL)1-α/β. We hypothesized that human uterine epithelial cells (HESs) secrete intact EMMPRIN to stimulate MMPs. Microvesicles from HES cell-conditioned medium (CM) expressed intact EMMPRIN protein. Treatment of HES cells with estradiol or phorbyl 12-myristate-13-acetate increased the release of EMMPRIN-containing microvesicles. The HES CM stimulated MMP-1, -2, and -3 messenger RNA levels in human uterine fibroblasts (HUFs) and EMMPRIN immunodepletion from HES-cell concentrated CM reduced MMP stimulation (P < .05). Treatment of HUF cells with low concentrations of IL-1β/α stimulated MMP production (P < .05). These results indicate that HES cells regulate MMP production by HUF cells by secretion of EMMPRIN, in response to ovarian hormones, proinflammatory cytokines as well as activation of protein kinase C.

  13. Genome-wide co-localization of Polycomb orthologs and their effects on gene expression in human fibroblasts

    PubMed Central

    2014-01-01

    Background Polycomb group proteins form multicomponent complexes that are important for establishing lineage-specific patterns of gene expression. Mammalian cells encode multiple permutations of the prototypic Polycomb repressive complex 1 (PRC1) with little evidence for functional specialization. An aim of this study is to determine whether the multiple orthologs that are co-expressed in human fibroblasts act on different target genes and whether their genomic location changes during cellular senescence. Results Deep sequencing of chromatin immunoprecipitated with antibodies against CBX6, CBX7, CBX8, RING1 and RING2 reveals that the orthologs co-localize at multiple sites. PCR-based validation at representative loci suggests that a further six PRC1 proteins have similar binding patterns. Importantly, sequential chromatin immunoprecipitation with antibodies against different orthologs implies that multiple variants of PRC1 associate with the same DNA. At many loci, the binding profiles have a distinctive architecture that is preserved in two different types of fibroblast. Conversely, there are several hundred loci at which PRC1 binding is cell type-specific and, contrary to expectations, the presence of PRC1 does not necessarily equate with transcriptional silencing. Interestingly, the PRC1 binding profiles are preserved in senescent cells despite changes in gene expression. Conclusions The multiple permutations of PRC1 in human fibroblasts congregate at common rather than specific sites in the genome and with overlapping but distinctive binding profiles in different fibroblasts. The data imply that the effects of PRC1 complexes on gene expression are more subtle than simply repressing the loci at which they bind. PMID:24485159

  14. Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

    PubMed Central

    Paxson, Julia A.; Gruntman, Alisha; Parkin, Christopher D.; Mazan, Melissa R.; Davis, Airiel; Ingenito, Edward P.; Hoffman, Andrew M.

    2011-01-01

    While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration. PMID:21912590

  15. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Malpass, Gloria E., E-mail: gloria.malpass@gmail.com; Arimilli, Subhashini, E-mail: sarimill@wakehealth.edu; Prasad, G.L., E-mail: prasadg@rjrt.com

    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 5more » 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.« less

  16. Simulation of Atrial Fibrosis Using Coupled Myocyte-Fibroblast Cellular and Human Atrial Models

    PubMed Central

    Gao, Yuan

    2017-01-01

    Atrial fibrosis is characterized by expansion of extracellular matrix and increase in the number of fibroblasts which has been associated with the development and maintenance of atrial arrhythmias. However, the mechanisms how the fibrosis contributes to atrial arrhythmia remain incompletely understood. In this study, we used a proposed fibroblast model coupled with the human atrial myocyte to investigate the effects of fibrosis on atrial excitability and repolarization at both cellular and macroscopic levels. The 12-lead electrocardiogram (ECG) was also simulated to explore the index of clinical diagnosis for fibrosis. The simulation results showed that the fibrosis can modify action potential morphology of human atrial myocyte, slow down wave propagation, and have rate adaptation, thus causing the atrial electrical heterogeneity. The fibrosis alone was sufficient to cause arrhythmia, induce reentry wave, and result in low amplitude and wide P waves at normal heart rate and significant prolonged and inverse P waves at high heart rate. All these symptoms aggravated when the level of fibrosis increased. Our simulations demonstrated that fibrosis is the substrate of atrial arrhythmia and thereby may be a potential target in the treatment of atrial arrhythmias. PMID:29441121

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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,more » 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.« less

  18. Resveratrol Prevents High Fluence Red Light-Emitting Diode Reactive Oxygen Species-Mediated Photoinhibition of Human Skin Fibroblast Migration.

    PubMed

    Mamalis, Andrew; Koo, Eugene; Isseroff, R Rivkah; Murphy, William; Jagdeo, Jared

    2015-01-01

    Skin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL) is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease. The goal of our study was to investigate the how reactive oxygen species (ROS) free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed. High fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR). For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2) on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2. High fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158.4% relative to matched controls

  19. 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

  20. Inhibition of interleukin-17-stimulated interleukin-6 and -8 production by cranberry components in human gingival fibroblasts and epithelial cells.

    PubMed

    Tipton, D A; Cho, S; Zacharia, N; Dabbous, M K

    2013-10-01

    Gingival epithelial cells and fibroblasts participate in periodontal inflammation and destruction, producing interleukin (IL)-6, a regulator of osteoclastic bone resorption, and the neutrophil chemoattractant IL-8. IL-17, a product of T-helper 17 cells, may play a role in periodontitis by stimulating cytokine production by gingival cells. The cranberry (Vaccinium macrocarpon) is rich in polyphenols, particularly proanthocyanidins, which have antioxidant and other beneficial properties. Cranberry components inhibit pro-inflammatory activities of lipopolysaccharide-stimulated human macrophages, gingival fibroblasts, and epithelial cells, but little is known of its effects on IL-17-stimulated cytokine production. The objectives were to determine the effects of IL-17 ± cranberry components on IL-6 and IL-8 production by human gingival epithelial cells and fibroblasts. Cranberry high molecular weight non-dialyzable material (NDM), which is rich in proanthocyanidins, was derived from cranberry juice. Human gingival epithelial cells and normal human gingival fibroblasts were incubated with NDM (5-50 μg/mL), IL-17 (0.5-100 ng/mL), or NDM + IL-17 in serum-free medium for 6 d. IL-6 and IL-8 in culture supernatants were measured by ELISA. Membrane damage and viability were assessed by lactate dehydrogenase activity released into cell supernatants and activity of a mitochondrial enzyme, respectively. Data were analyzed using ANOVA and Scheffe's F procedure for post hoc comparisons. In both cell lines, IL-17 (≥ ~5-10 ng/mL) significantly stimulated production of IL-6 (p < 0.005) and IL-8 (p < 0.03). Non-toxic levels of NDM inhibited constitutive IL-6 and IL-8 production by epithelial cells (p ≤ 0.01) and fibroblasts (p ≤ 0.03) as well as IL-17-stimulated cytokine production by epithelial cells [IL-6 (maximum ~80% inhibition; p ≤ 0.0001); IL-8 (maximum ~70% inhibition; p ≤ 0.03)] and fibroblasts [IL-6 (maximum ~90% inhibition; p ≤ 0.0001); IL

  1. Transforming growth factor-β1 induces expression of human coagulation factor XII via Smad3 and JNK signaling pathways in human lung fibroblasts.

    PubMed

    Jablonska, Ewa; Markart, Philipp; Zakrzewicz, Dariusz; Preissner, Klaus T; Wygrecka, Malgorzata

    2010-04-09

    Coagulation factor XII (FXII) is a liver-derived serine protease involved in fibrinolysis, coagulation, and inflammation. The regulation of FXII expression is largely unknown. Transforming growth factor-beta1 (TGF-beta1) is a multifunctional cytokine that has been linked to several pathological processes, including tissue fibrosis by modulating procoagulant and fibrinolytic activities. This study investigated whether TGF-beta1 may regulate FXII expression in human lung fibroblasts. Treatment of human lung fibroblasts with TGF-beta1 resulted in a time-dependent increase in FXII production, activation of p44/42, p38, JNK, and Akt, and phosphorylation and translocation into the nucleus of Smad3. However, TGF-beta1-induced FXII expression was repressed only by the JNK inhibitor and JNK and Smad3 antisense oligonucleotides but not by MEK, p38, or phosphoinositide 3-kinase blockers. JNK inhibition had no effect on TGF-beta1-induced Smad3 phosphorylation, association with Smad4, and its translocation into the nucleus but strongly suppressed Smad3-DNA complex formation. FXII promoter analysis revealed that the -299/+1 region was sufficient for TGF-beta1 to induce FXII expression. Sequence analysis of this region detected a potential Smad-binding element at position -272/-269 (SBE-(-272/-269)). Chromatin immunoprecipitation and streptavidin pulldown assays demonstrated TGF-beta1-dependent Smad3 binding to SBE-(-272/-269). Mutation or deletion of SBE-(-272/-269) substantially reduced TGF-beta1-mediated activation of the FXII promoter. Clinical relevance was demonstrated by elevated FXII levels and its co-localization with fibroblasts in the lungs of patients with acute respiratory distress syndrome. Our results show that JNK/Smad3 pathway plays a critical role in TGF-beta1-induced FXII expression in human lung fibroblasts and implicate its possible involvement in pathological conditions characterized by elevated TGF-beta1 levels.

  2. From Hayflick to Walford: the role of T cell replicative senescence in human aging.

    PubMed

    Effros, Rita B

    2004-06-01

    The immunologic theory of aging, proposed more than 40 years ago by Roy Walford, suggests that the normal process of aging in man and in animals is pathogenetically related to faulty immunological processes. Since that time, research on immunological aging has undergone extraordinary expansion, leading to new information in areas spanning from molecular biology and cell signaling to large-scale clinical studies. Investigation in this area has also provided unexpected insights into HIV disease, many aspects of which represent accelerated immunological aging. This article describes the initial insights and vision of Roy Walford into one particular facet of human immunological aging, namely, the potential relevance of the well-studied human fibroblast replicative senescence model, initially developed by Leonard Hayflick, to cells of the immune system. Extensive research on T cell senescence in cell culture has now documented changes in vitro that closely mirror alterations occurring during in vivo aging in humans, underscoring the biological significance of T cell replicative senescence. Moreover, the inclusion of high proportions of putatively senescent T cells in the 'immune risk phenotype' that is associated with early mortality in octogenarians provides initial clinical confirmation of both the immunologic theory of aging and the role of the T cell Hayflick Limit in human aging, two areas of gerontological research pioneered by Roy Walford.

  3. Chemical Carcinogen (Hydrazine et al.) Induced Carcinogenesis of Human Diploid Fibroblasts in vitro.

    DTIC Science & Technology

    1985-06-12

    compound then is converted to a carbonium ion and the radical interacts /, with the purine bases in DNA. Methylazoxymethanol acetate, ( MAMA ) in the...interacts with the purine bases in DNA. Methylazoxymethanol acetate, ( MAMA ) in the presence of colon, secum and liver homogenates reduced NAD+ to NADH...3. Human foreskin fibroblast populations blocked in G1 , released and treated with methylazoxy methanol acetate ( MAMA ) from the time of

  4. Biotin deficiency inhibits heme synthesis and impairs mitochondria in human lung fibroblasts.

    PubMed

    Atamna, Hani; Newberry, Justin; Erlitzki, Ronit; Schultz, Carla S; Ames, Bruce N

    2007-01-01

    Four of the 5 biotin-dependent carboxylases (BDC) are in the mitochondria. BDC replace intermediates in the Krebs [tricarboxylic acid (TCA)] cycle that are regularly removed for the synthesis of key metabolites such as heme or amino acids. Heme, unlike amino acids, is not recycled to regenerate these intermediates, is not utilized from the diet, and must be synthesized in situ. We studied whether biotin deficiency (BD) lowers heme synthesis and whether mitochondria would be disrupted. Biotin-deficient medium was prepared by using bovine serum stripped of biotin with charcoal/dextran or avidin. Biotin-deficient primary human lung fibroblasts (IMR90) lost their BDC and senesced before biotin-sufficient cells. BD caused heme deficiency; there was a decrease in heme content and heme synthesis, and biotin-deficient cells selectively lost mitochondrial complex IV, which contains heme-a. Loss of complex IV, which is part of the electron transport chain, triggered oxidant release and oxidative damage, hallmarks of heme deficiency. Restoring biotin to the biotin-deficient medium prevented the above changes. Old cells were more susceptible to biotin shortage than young cells. These findings highlight the biochemical connection among biotin, heme, and iron metabolism, and the mitochondria, due to the role of biotin in maintaining the biochemical integrity of the TCA cycle. The findings are discussed in relation to aging and birth defects in humans.

  5. Regulation of gene expression by tobacco product preparations in cultured human dermal fibroblasts

    PubMed Central

    Malpass, Gloria E.; Arimilli, Subhashini; Prasad, G. L.; Howlett, Allyn C.

    2015-01-01

    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. PMID:24927667

  6. The fibroblast-derived paracrine factor neuregulin-1 has a novel role in regulating the constitutive color and melanocyte function in human skin

    PubMed Central

    Choi, Wonseon; Wolber, Rainer; Gerwat, Wolfram; Mann, Tobias; Batzer, Jan; Smuda, Christoph; Liu, Hongfang; Kolbe, Ludger; Hearing, Vincent J.

    2010-01-01

    Interactions between melanocytes and neighboring cells in the skin are important in regulating skin color in humans. We recently demonstrated that the less pigmented and thicker skin on the palms and soles is regulated by underlying fibroblasts in those areas, specifically via a secreted factor (DKK1) that modulates Wnt signaling. In this study, we tested the hypothesis that dermal fibroblasts regulate the constitutive skin color of individuals ranging from very light to very dark. We used microarray analysis to compare gene expression patterns in fibroblasts derived from lighter skin types compared to darker skin types, with a focus on secreted proteins. We identified a number of genes that differ dramatically in expression and, among the expressed proteins, neuregulin-1, which is secreted by fibroblasts derived from dark skin, effectively increases the pigmentation of melanocytes in tissue culture and in an artificial skin model and regulates their growth, suggesting that it is one of the major factors determining human skin color. PMID:20736300

  7. Neuregulin-1β induces proliferation, survival and paracrine signaling in normal human cardiac ventricular fibroblasts.

    PubMed

    Kirabo, Annet; Ryzhov, Sergey; Gupte, Manisha; Sengsayadeth, Seng; Gumina, Richard J; Sawyer, Douglas B; Galindo, Cristi L

    2017-04-01

    Neuregulin-1β (NRG-1β) is critical for cardiac development and repair, and recombinant forms are currently being assessed as possible therapeutics for systolic heart failure. We previously demonstrated that recombinant NRG-1β reduces cardiac fibrosis in an animal model of cardiac remodeling and heart failure, suggesting that there may be direct effects on cardiac fibroblasts. Here we show that NRG-1β receptors (ErbB2, ErbB3, and ErbB4) are expressed in normal human cardiac ventricular (NHCV) fibroblast cell lines. Treatment of NHCV fibroblasts with recombinant NRG-1β induced activation of the AKT pathway, which was phosphoinositide 3-kinase (PI3K)-dependent. Moreover, the NRG-1β-induced PI3K/AKT signaling in these cells required phosphorylation of both ErbB2 and ErbB3 receptors at tyrosine (Tyr)1248 and Tyr1289 respectively. RNASeq analysis of NRG-1β-treated cardiac fibroblasts obtained from three different individuals revealed a global gene expression signature consistent with cell growth and survival. We confirmed enhanced cellular proliferation and viability in NHCV fibroblasts in response to NRG-1β, which was abrogated by PI3K, ErbB2, and ErbB3 inhibitors. NRG-1β also induced production and secretion of cytokines (interleukin-1α and interferon-γ) and pro-reparative factors (angiopoietin-2, brain-derived neurotrophic factor, and crypto-1), suggesting a role in cardiac repair through the activation of paracrine signaling. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

    PubMed Central

    Reijnders, Christianne M.A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin–eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely

  9. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts.

    PubMed

    Reijnders, Christianne M A; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J; Gibbs, Susan

    2015-09-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve these limitations. The aim was to develop a fully differentiated HSE constructed entirely from human skin cell lines, which could be applied for in vitro wound-healing assays. Skin equivalents were constructed from human TERT-immortalized keratinocytes and fibroblasts (TERT-HSE) and compared with native skin and primary HSEs. HSEs were characterized by hematoxylin-eosin and immunohistochemical stainings with markers for epidermal proliferation and differentiation, basement membrane (BM), fibroblasts, and the extracellular matrix (ECM). Ultrastructure was determined with electron microscopy. To test the functionality of the TERT-HSE, burn and cold injuries were applied, followed by immunohistochemical stainings, measurement of reepithelialization, and determination of secreted wound-healing mediators. The TERT-HSE was composed of a fully differentiated epidermis and a fibroblast-populated dermis comparable to native skin and primary HSE. The epidermis consisted of proliferating keratinocytes within the basal layer, followed by multiple spinous layers, a granular layer, and cornified layers. Within the TERT-HSE, the membrane junctions such as corneosomes, desmosomes, and hemidesmosomes were well developed as shown by ultrastructure pictures. Furthermore, the BM consisted of a lamina lucida and lamina densa comparable to native skin. The dermal matrix of the TERT-HSE was more similar to native skin than the primary construct, since collagen III, an ECM marker, was present in TERT-HSEs and absent in primary HSEs. After wounding, the TERT-HSE was able to reepithelialize and secrete inflammatory wound-healing mediators. In conclusion, the novel TERT-HSE, constructed entirely

  10. bFGF Promotes the Migration of Human Dermal Fibroblasts under Diabetic Conditions through Reactive Oxygen Species Production via the PI3K/Akt-Rac1- JNK Pathways

    PubMed Central

    Shi, Hongxue; Cheng, Yi; Ye, Jingjing; Cai, Pingtao; Zhang, Jinjing; Li, Rui; Yang, Ying; Wang, Zhouguang; Zhang, Hongyu; Lin, Cai; Lu, Xianghong; Jiang, Liping; Hu, Aiping; Zhu, Xinbo; Zeng, Qiqiang; Fu, Xiaobing; Li, Xiaokun; Xiao, Jian

    2015-01-01

    Fibroblasts play a pivotal role in the process of cutaneous wound repair, whereas their migratory ability under diabetic conditions is markedly reduced. In this study, we investigated the effect of basic fibroblast growth factor (bFGF) on human dermal fibroblast migration in a high-glucose environment. bFGF significantly increased dermal fibroblast migration by increasing the percentage of fibroblasts with a high polarity index and reorganizing F-actin. A significant increase in intracellular reactive oxygen species (ROS) was observed in dermal fibroblasts under diabetic conditions following bFGF treatment. The blockage of bFGF-induced ROS production by either the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI) almost completely neutralized the increased migration rate of dermal fibroblasts promoted by bFGF. Akt, Rac1 and JNK were rapidly activated by bFGF in dermal fibroblasts, and bFGF-induced ROS production and promoted dermal fibroblast migration were significantly attenuated when suppressed respectively. In addition, bFGF-induced increase in ROS production was indispensable for the activation of focal adhesion kinase (FAK) and paxillin. Therefore, our data suggested that bFGF promotes the migration of human dermal fibroblasts under diabetic conditions through increased ROS production via the PI3K/Akt-Rac1-JNK pathways. PMID:26078726

  11. Functional roles of fibroblast growth factor receptors (FGFRs) signaling in human cancers.

    PubMed

    Tiong, Kai Hung; Mah, Li Yen; Leong, Chee-Onn

    2013-12-01

    The fibroblast growth factor receptors (FGFRs) regulate important biological processes including cell proliferation and differentiation during development and tissue repair. Over the past decades, numerous pathological conditions and developmental syndromes have emerged as a consequence of deregulation in the FGFRs signaling network. This review aims to provide an overview of FGFR family, their complex signaling pathways in tumorigenesis, and the current development and application of therapeutics targeting the FGFRs signaling for treatment of refractory human cancers.

  12. 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.

  13. Interplay between Selenium Levels and Replicative Senescence in WI-38 Human Fibroblasts: A Proteomic Approach.

    PubMed

    Hammad, Ghania; Legrain, Yona; Touat-Hamici, Zahia; Duhieu, Stéphane; Cornu, David; Bulteau, Anne-Laure; Chavatte, Laurent

    2018-01-20

    Selenoproteins are essential components of antioxidant defense, redox homeostasis, and cell signaling in mammals, where selenium is found in the form of a rare amino acid, selenocysteine. Selenium, which is often limited both in food intake and cell culture media, is a strong regulator of selenoprotein expression and selenoenzyme activity. Aging is a slow, complex, and multifactorial process, resulting in a gradual and irreversible decline of various functions of the body. Several cellular aspects of organismal aging are recapitulated in the replicative senescence of cultured human diploid fibroblasts, such as embryonic lung fibroblast WI-38 cells. We previously reported that the long-term growth of young WI-38 cells with high (supplemented), moderate (control), or low (depleted) concentrations of selenium in the culture medium impacts their replicative lifespan, due to rapid changes in replicative senescence-associated markers and signaling pathways. In order to gain insight into the molecular link between selenium levels and replicative senescence, in the present work, we have applied a quantitative proteomic approach based on 2-Dimensional Differential in-Gel Electrophoresis (2D-DIGE) to the study of young and presenescent cells grown in selenium-supplemented, control, or depleted media. Applying a restrictive cut-off (spot intensity ±50% and a p value < 0.05) to the 2D-DIGE analyses revealed 81 differentially expressed protein spots, from which 123 proteins of interest were identified by mass spectrometry. We compared the changes in protein abundance for three different conditions: (i) spots varying between young and presenescent cells, (ii) spots varying in response to selenium concentration in young cells, and (iii) spots varying in response to selenium concentration in presenescent cells. Interestingly, a 72% overlap between the impact of senescence and selenium was observed in our proteomic results, demonstrating a strong interplay between selenium

  14. Defective Wound-healing in Aging Gingival Tissue.

    PubMed

    Cáceres, M; Oyarzun, A; Smith, P C

    2014-07-01

    Aging may negatively affect gingival wound-healing. However, little is known about the mechanisms underlying this phenomenon. The present study examined the cellular responses associated with gingival wound-healing in aging. Primary cultures of human gingival fibroblasts were obtained from healthy young and aged donors for the analysis of cell proliferation, cell invasion, myofibroblastic differentiation, and collagen gel remodeling. Serum from young and old rats was used to stimulate cell migration. Gingival repair was evaluated in Sprague-Dawley rats of different ages. Data were analyzed by the Mann-Whitney and Kruskal-Wallis tests, with a p value of .05. Fibroblasts from aged donors showed a significant decrease in cell proliferation, migration, Rac activation, and collagen remodeling when compared with young fibroblasts. Serum from young rats induced higher cell migration when compared with serum from old rats. After TGF-beta1 stimulation, both young and old fibroblasts demonstrated increased levels of alpha-SMA. However, alpha-SMA was incorporated into actin stress fibers in young but not in old fibroblasts. After 7 days of repair, a significant delay in gingival wound-healing was observed in old rats. The present study suggests that cell migration, myofibroblastic differentiation, collagen gel remodeling, and proliferation are decreased in aged fibroblasts. In addition, altered cell migration in wound-healing may be attributable not only to cellular defects but also to changes in serum factors associated with the senescence process. © International & American Associations for Dental Research.

  15. Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

    PubMed Central

    Pierce, Elizabeth M.; Carpenter, Kristin; Jakubzick, Claudia; Kunkel, Steven L.; Flaherty, Kevin R.; Martinez, Fernando J.; Hogaboam, Cory M.

    2007-01-01

    Idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 × 106 primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7. PMID:17392156

  16. MicroRNA Expression Profiles in Cultured Human Fibroblasts in Space

    NASA Technical Reports Server (NTRS)

    Wu, Honglu; Lu, Tao; Jeevarajan, John; Rohde, Larry; Zhang, Ye

    2014-01-01

    Microgravity, or an altered gravity environment from the static 1g, has been shown to influence global gene expression patterns and protein levels in living organisms. However, it is unclear how these changes in gene and protein expressions are related to each other or are related to other factors regulating such changes. A different class of RNA, the small non-coding microRNA (miRNA), can have a broad effect on gene expression networks by mainly inhibiting the translation process. Previously, we investigated changes in the expression of miRNA and related genes under simulated microgravity conditions on the ground using the NASA invented bioreactor. In comparison to static 1 g, simulated microgravity altered a number of miRNAs in human lymphoblastoid cells. Pathway analysis with the altered miRNAs and RNA expressions revealed differential involvement of cell communication and catalytic activity, as well as immune response signaling and NGF activation of NF-kB pathways under simulated microgravity condition. The network analysis also identified several projected networks with c- Rel, ETS1 and Ubiquitin C as key factors. In a flight experiment on the International Space Station (ISS), we will investigate the effects of actual spaceflight on miRNA expressions in nondividing human fibroblast cells in mostly G1 phase of the cell cycle. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, the structural framework for tissues, and plays a critical role in wound healing and other functions. In addition to miRNA expressions, we will investigate the effects of spaceflight on the cellular response to DNA damages from bleomycin treatment.

  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. Maternal nutrient restriction in baboon programs later-life cellular growth and respiration of cultured skin fibroblasts: a potential model for the study of aging-programming interactions.

    PubMed

    Salmon, Adam B; Dorigatti, Jonathan; Huber, Hillary F; Li, Cun; Nathanielsz, Peter W

    2018-05-25

    Compelling data exist for programming of chronic later-life diseases and longevity by perinatal developmental programming challenges. Understanding mechanisms by which life course health trajectory and longevity are set is fundamental to understanding aging. Appropriate approaches are needed to determine programming effects on cellular function. We have developed a baboon model in which control mothers eat ad libitum while a second group eat 70% of the global diet fed controls, leading to male and female offspring intrauterine growth restriction (IUGR). We have shown that IUGR suffer from acceleration of several age-related physiological declines. Here, we report on a skin-derived fibroblast model with potential relevance for mechanistic studies on how IUGR impacts aging. Fibroblasts were cultured from the skin biopsies taken from adult baboons from control and IUGR cohorts. IUGR-derived fibroblasts grew in culture less well than controls and those derived from male, but not female, IUGR baboons had a significant reduction in maximum respiration rate compared to control-derived fibroblasts. We also show that relative levels of several mitochondrial protein subunits, including NDUFB8 and cytochrome c oxidase subunit IV, were reduced in IUGR-derived fibroblasts even after serial passaging in culture. The lower levels of electron transport system components provide potential mechanisms for accelerated life course aging in the setting of programmed IUGR. This observation fits with the greater sensitivity of males compared with females to many, but not all, outcomes in response to programming challenges. These approaches will be powerful in the determination of programming-aging interactions.

  19. Hypoxia induces pulmonary fibroblast proliferation through NFAT signaling.

    PubMed

    Senavirathna, Lakmini Kumari; Huang, Chaoqun; Yang, Xiaoyun; Munteanu, Maria Cristina; Sathiaseelan, Roshini; Xu, Dao; Henke, Craig A; Liu, Lin

    2018-02-09

    Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

  20. The fibroblast surface markers FAP, anti-fibroblast, and FSP are expressed by cells of epithelial origin and may be altered during epithelial-to-mesenchymal transition.

    PubMed

    Kahounová, Zuzana; Kurfürstová, Daniela; Bouchal, Jan; Kharaishvili, Gvantsa; Navrátil, Jiří; Remšík, Ján; Šimečková, Šárka; Študent, Vladimír; Kozubík, Alois; Souček, Karel

    2017-04-06

    The identification of fibroblasts and cancer-associated fibroblasts from human cancer tissue using surface markers is difficult, especially because the markers used currently are usually not expressed solely by fibroblasts, and the identification of fibroblast-specific surface molecules is still under investigation. It was aimed to compare three commercially available antibodies in the detection of different surface epitopes of fibroblasts (anti-fibroblast, fibroblast activation protein α, and fibroblast surface protein). The specificity of their expression, employing fibroblast cell lines and tumor-derived fibroblasts from breast and prostate tissues was investigated. Both the established fibroblast cell line HFF-1 and ex vivo primary fibroblasts isolated from breast and prostate cancer tissues expressed the tested surface markers to different degrees. Surprisingly, those markers were expressed also by permanent cell lines of epithelial origin, both benign and cancer-derived (breast-cell lines MCF 10A, HMLE and prostate-cell lines BPH-1, DU 145, and PC-3). The expression of fibroblast activation protein α increased on the surface of previously described models of epithelial cells undergoing epithelial-to-mesenchymal transition in response to treatment with TGF-β1. To prove the co-expression of the fibroblast markers on cells of epithelial origin, we used freshly dissociated human prostate and breast cancer tissues. The results confirmed the co-expression of anti-fibroblast and fibroblast surface protein on CD31/CD45-negative/EpCAM-positive epithelial cells. In summary, our data support the findings that the tested fibroblast markers are not fibroblast specific and may be expressed also by cells of epithelial origin (e.g., cells undergoing EMT). Therefore, the expression of these markers should be interpreted with caution, and the combination of several epitopes for both positive (anti-fibroblast or fibroblast activation protein α) and negative (Ep

  1. Extracellular Collagen Promotes Interleukin-1β-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts.

    PubMed

    Sugioka, Koji; Kodama-Takahashi, Aya; Yoshida, Koji; Aomatsu, Keiichi; Okada, Kiyotaka; Nishida, Teruo; Shimomura, Yoshikazu

    2017-03-01

    Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1β (IL-1β). Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. Collagen and IL-1β synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1β, were all abolished by a selective uPA inhibitor. Collagen and IL-1β cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1β-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.

  2. 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.

  3. Human beta defensin-4 and keratinocyte growth factor gene expression in cultured keratinocyte and fibroblasts of burned patients.

    PubMed

    Noronha, Silvana Aparecida Alves Corrêa de; Noronha, Samuel Marcos Ribeiro de; Lanziani, Larissa Elias; Ipolito, Michele Zampieri; Ferreira, Lydia Masako; Gragnani, Alfredo

    2014-01-01

    To evaluate KGF and human beta defensin-4 (HBD-4) levels produced by dermic fibroblasts and keratinocytes cultivated from burned patients' skin samples. Keratinocytes and fibroblasts of 10 patients (four major burns, four minor burns and two controls) were primarily cultivated according to standard methods. HBD-4 and KGF genes were analyzed by quantitative PCR. In fibroblasts, KGF gene expression was 220±80 and 33.33±6.67 (M±SD; N=4), respectively for major and minor burn groups. In keratinocytes, KGF gene expression was 11.2±1.9 and 3.45±0.37 (M±SD; N=4), respectively for major and minor burn groups. In fibroblasts, HBD-4 gene expression was 15.0±4.0 and 11.5±0.5 (M±SD; N=4), respectively for major and minor burn. In keratinocyte, HBD-4 gene expression was 0.0±0.0 and 13.4±4.8 (M±SD; N=4), respectively for major and minor burn. KGF expression was increased in burn patient fibroblasts compared to control group. In keratinocytes culture, KGF suppression is inversely proportional to burn extension; it is active and increased in major burn but decreased in minor burn. HBD-4 expression was increased in fibroblasts and decreased in keratinocytes from all burned patients.

  4. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts - A comparative study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meeuwen, J.A. van; Nijmeijer, S.; Mutarapat, T.

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissuemore » in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts.« less

  5. WISP1 mediates IL-6-dependent proliferation in primary human lung fibroblasts.

    PubMed

    Klee, S; Lehmann, M; Wagner, D E; Baarsma, H A; Königshoff, M

    2016-02-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. IPF is characterized by epithelial cell injury and reprogramming, increases in (myo)fibroblasts, and altered deposition of extracellular matrix. The Wnt1-inducible signaling protein 1 (WISP1) is involved in impaired epithelial-mesenchymal crosstalk in pulmonary fibrosis. Here, we aimed to further investigate WISP1 regulation and function in primary human lung fibroblasts (phLFs). We demonstrate that WISP1 is directly upregulated by Transforming growth factor β1 (TGFβ1) and Tumor necrosis factor α (TNFα) in phLFs, using a luciferase-based reporter system. WISP1 mRNA and protein secretion increased in a time- and concentration-dependent manner by TGFβ1 and TNFα in phLFs, as analysed by qPCR and ELISA, respectively. Notably, WISP1 is required for TGFβ1- and TNFα-dependent induction of interleukin 6 (IL-6), a mechanism that is conserved in IPF phLFs. The siRNA-mediated WISP1 knockdown led to a significant IL-6 reduction after TGFβ1 or TNFα stimulation. Furthermore, siRNA-mediated downregulation or antibody-mediated neutralization of WISP1 reduced phLFs proliferation, a process that was in part rescued by IL-6. Taken together, these results strongly indicate that WISP1-induced IL-6 expression contributes to the pro-proliferative effect on fibroblasts, which is likely orchestrated by a variety of profibrotic mediators, including Wnts, TGFβ1 and TNFα.

  6. MicroRNA-26a modulates transforming growth factor beta-1-induced proliferation in human fetal lung fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Xiaoou; Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan; Liu, Lian

    2014-11-28

    Highlights: • Endogenous miR-26a inhibits TGF-beta 1 induced proliferation of lung fibroblasts. • miR-26a induces G1 arrest through directly targeting 3′-UTR of CCND2. • TGF indispensable receptor, TGF-beta R I, is regulated by miR-26a. • miR-26a acts through inhibiting TGF-beta 2 feedback loop to reduce TGF-beta 1. • Collagen type I and connective tissue growth factor are suppressed by miR-26a. - Abstract: MicroRNA-26a is a newly discovered microRNA that has a strong anti-tumorigenic capacity and is capable of suppressing cell proliferation and activating tumor-specific apoptosis. However, whether miR-26a can inhibit the over-growth of lung fibroblasts remains unclear. The relationship betweenmore » miR-26a and lung fibrosis was explored in the current study. We first investigated the effect of miR-26a on the proliferative activity of human lung fibroblasts with or without TGF-beta1 treatment. We found that the inhibition of endogenous miR-26a promoted proliferation and restoration of mature miR-26a inhibited the proliferation of human lung fibroblasts. We also examined that miR-26a can block the G1/S phase transition via directly targeting 3′-UTR of CCND2, degrading mRNA and decreasing protein expression of Cyclin D2. Furthermore, we showed that miR-26a mediated a TGF-beta 2-TGF-beta 1 feedback loop and inhibited TGF-beta R I activation. In addition, the overexpression of miR-26a also significantly suppressed the TGF-beta 1-interacting-CTGF–collagen fibrotic pathway. In summary, our studies indicated an essential role of miR-26a in the anti-fibrotic mechanism in TGF-beta1-induced proliferation in human lung fibroblasts, by directly targeting Cyclin D2, regulating TGF-beta R I as well as TGF-beta 2, and suggested the therapeutic potential of miR-26a in ameliorating lung fibrosis.« less

  7. 18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts.

    PubMed

    Hung, Chi-Feng; Hsiao, Chien-Yu; Hsieh, Wen-Hao; Li, Hsin-Ju; Tsai, Yi-Ju; Lin, Chun-Nan; Chang, Hsun-Hsien; Wu, Nan-Lin

    2017-01-01

    Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18β-glycyrrhetinic acid (18β-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18β-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18β-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18β-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18β-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18β-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18β-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects.

  8. Telomerase-mediated life-span extension of human primary fibroblasts by human artificial chromosome (HAC) vector

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shitara, Shingo; Kakeda, Minoru; Nagata, Keiko

    2008-05-09

    Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-{beta}-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120 daysmore » after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70 days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.« less

  9. Effect of Microalgal Extracts of Tetraselmis suecica against UVB-Induced Photoaging in Human Skin Fibroblasts.

    PubMed

    Jo, Wol Soon; Yang, Kwang Mo; Park, Hee Sung; Kim, Gi Yong; Nam, Byung Hyouk; Jeong, Min Ho; Choi, Yoo Jin

    2012-12-01

    Exposure of cells to ultraviolet B (UVB) radiation can induce production of free radicals and reactive oxygen species (ROS), which damage cellular components. In addition, these agents can stimulate the expression of matrix metalloproteinase (MMP) and decrease collagen synthesis in human skin cells. In this study, we examined the anti-photoaging effects of extracts of Tetraselmis suecica (W-TS). W-TS showed the strongest scavenging activity against 2,2-difenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals, followed by superoxide anions from the xanthine/xanthine oxidase system. We observed that the levels of both intracellular ROS and lipid peroxidation significantly increased in UVB-irradiated human skin fibroblast cells. Furthermore, the activities of enzymatic antioxidants (e.g., superoxide dismutase) and the levels of non-enzymatic antioxidants (e.g., glutathione) significantly decreased in cells. However, W-TS pretreatment, at the maximum tested concentration, significantly decreased intracellular ROS and malondialdehyde (MDA) levels, and increased superoxide dismutase and glutathione levels in the cells. At this same concentration, W-TS did not show cytotoxicity. Type 1 procollagen and MMP-1 released were quantified using RT-PCR techniques. The results showed that W-TS protected type 1 procollagen against UVBinduced depletion in fibroblast cells in a dose-dependent manner via inhibition of UVB-induced MMP-1. Taken together, the results of the study suggest that W-TS effectively inhibits UVB-induced photoaging in skin fibroblasts by its strong anti-oxidant ability.

  10. Effect of Microalgal Extracts of Tetraselmis suecica against UVB-Induced Photoaging in Human Skin Fibroblasts

    PubMed Central

    Jo, Wol Soon; Yang, Kwang Mo; Park, Hee Sung; Kim, Gi Yong; Nam, Byung Hyouk

    2012-01-01

    Exposure of cells to ultraviolet B (UVB) radiation can induce production of free radicals and reactive oxygen species (ROS), which damage cellular components. In addition, these agents can stimulate the expression of matrix metalloproteinase (MMP) and decrease collagen synthesis in human skin cells. In this study, we examined the anti-photoaging effects of extracts of Tetraselmis suecica (W-TS). W-TS showed the strongest scavenging activity against 2,2-difenyl-1-picrylhydrazyl (DPPH) and peroxyl radicals, followed by superoxide anions from the xanthine/xanthine oxidase system. We observed that the levels of both intracellular ROS and lipid peroxidation significantly increased in UVB-irradiated human skin fibroblast cells. Furthermore, the activities of enzymatic antioxidants (e.g., superoxide dismutase) and the levels of non-enzymatic antioxidants (e.g., glutathione) significantly decreased in cells. However, W-TS pretreatment, at the maximum tested concentration, significantly decreased intracellular ROS and malondialdehyde (MDA) levels, and increased superoxide dismutase and glutathione levels in the cells. At this same concentration, W-TS did not show cytotoxicity. Type 1 procollagen and MMP-1 released were quantified using RT-PCR techniques. The results showed that W-TS protected type 1 procollagen against UVBinduced depletion in fibroblast cells in a dose-dependent manner via inhibition of UVB-induced MMP-1. Taken together, the results of the study suggest that W-TS effectively inhibits UVB-induced photoaging in skin fibroblasts by its strong anti-oxidant ability. PMID:24278616

  11. Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Usuki, S.; Lyu, S.C.; Sweeley, C.C.

    1988-05-15

    Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pHmore » 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ((1-14C)N-acetylmannosamine) and a radioactive precursor of ceramide ((3,3-3H2)serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.« less

  12. Increased fibroblast proliferation and activity after applying intense pulsed light 800-1200 nm.

    PubMed

    Cuerda-Galindo, E; Díaz-Gil, G; Palomar-Gallego, M A; Linares-GarcíaValdecasas, R

    2015-03-01

    Light devices emitting near infrared have been shown to be highly effective for the skin rejuvenation but biochemical and molecular mechanism or optimum dose treatment are not well-known. In our study we try to elucidate why systems emitting near infrared produce skin improvement such as fibroblasts proliferation, increase in gene expression or extracellular matrix (ECM) protein production. 1BR3G human skin fibroblasts were used to test the effects of an intense pulsed light device emitting with an 800-1200 nm filter (MiniSilk FT manufactured by Deka(®)). In our protocol, fibroblasts were irradiated twice successively with a 10 Hz frequency, with a total fluence up to 60 J/cm(2) for 15s each pass. After incubating for 48 h, fibroblasts were harvested from the culture plates to test cell proliferation by flow cytometer. To determine changes in gene expression (mRNA levels for collagen types I and III and metalloproteinase 1 (MMP-1)) and protein production (hyaluronic acid, versican and decorin) tests were performed after irradiation. After 48 h irradiation, 1BR3G human skin fibroblasts were observed to proliferate at a fast rate. The study of ECM macromolecules production using ELISA showed an increase of hyaluronic acid and versican production but no changes were observed for decorin. With RT-PCR assays, an increase in mRNA for collagen type I, type III and MMP-1 were observed. Intense pulsed light emitting near infrared applied in vitro cultured cells increases fibroblasts proliferation and activity, which can be a possible mechanism of action for these devices in aging skin treatment. Copyright © 2014 Elsevier GmbH. All rights reserved.

  13. TβRIII Expression in Human Breast Cancer Stroma and the Role of Soluble TβRIII in Breast Cancer Associated Fibroblasts.

    PubMed

    Jovanović, Bojana; Pickup, Michael W; Chytil, Anna; Gorska, Agnieszka E; Johnson, Kimberly C; Moses, Harold L; Owens, Philip

    2016-11-04

    The TGF-β pathway plays a major role in tumor progression through regulation of epithelial and stromal cell signaling. Dysfunction of the pathway can lead to carcinoma progression and metastasis. To gain insight into the stromal role of the TGF-β pathway in breast cancer, we performed laser capture microdissection (LCM) from breast cancer patients and reduction mammoplasty patients. Microdissected tumor stroma and normal breast stroma were examined for gene expression. Expression of the TGF-β type III receptor ( TGFBR3 ) was greatly decreased in the tumor stroma compared to control healthy breast tissue. These results demonstrated a 44-fold decrease in TGFBR3 mRNA in tumor stroma in comparison to control tissue. We investigated publicly available databases, and have identified that TGFBR3 mRNA levels are decreased in tumor stroma. We next investigated fibroblast cell lines derived from cancerous and normal breast tissue and found that in addition to mRNA levels, TβRIII protein levels were significantly reduced. Having previously identified that cancer-associated fibroblasts secrete greater levels of tumor promoting cytokines, we investigated the consequences of soluble-TβRIII (sTβRIII) on fibroblasts. Fibroblast conditioned medium was analyzed for 102 human secreted cytokines and distinct changes in response to sTβRIII were observed. Next, we used the fibroblast-conditioned medium to stimulate human monocyte cell line THP-1. These results indicate a distinct transcriptional response depending on sTβRIII treatment and whether it was derived from normal or cancerous breast tissue. We conclude that the effect of TβRIII has distinct roles not only in cancer-associated fibroblasts but that sTβRIII has distinct paracrine functions in the tumor microenvironment.

  14. Silencing of the pentose phosphate pathway genes influences DNA replication in human fibroblasts.

    PubMed

    Fornalewicz, Karolina; Wieczorek, Aneta; Węgrzyn, Grzegorz; Łyżeń, Robert

    2017-11-30

    Previous reports and our recently published data indicated that some enzymes of glycolysis and the tricarboxylic acid cycle can affect the genome replication process by changing either the efficiency or timing of DNA synthesis in human normal cells. Both these pathways are connected with the pentose phosphate pathway (PPP pathway). The PPP pathway supports cell growth by generating energy and precursors for nucleotides and amino acids. Therefore, we asked if silencing of genes coding for enzymes involved in the pentose phosphate pathway may also affect the control of DNA replication in human fibroblasts. Particular genes coding for PPP pathway enzymes were partially silenced with specific siRNAs. Such cells remained viable. We found that silencing of the H6PD, PRPS1, RPE genes caused less efficient enterance to the S phase and decrease in efficiency of DNA synthesis. On the other hand, in cells treated with siRNA against G6PD, RBKS and TALDO genes, the fraction of cells entering the S phase was increased. However, only in the case of G6PD and TALDO, the ratio of BrdU incorporation to DNA was significantly changed. The presented results together with our previously published studies illustrate the complexity of the influence of genes coding for central carbon metabolism on the control of DNA replication in human fibroblasts, and indicate which of them are especially important in this process. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Influence of human fibroblasts on development and quality of multilayered composite grafts in athymic nude mice.

    PubMed

    Cedidi, C Can; Wilkens, L; Berger, A; Ingianni, G

    2007-11-05

    In patients after extensive burn injury the lack of split thickness skin graft donor sites, and consecutive delay in wound closure are critical factors of morbidity and mortality. In addition limited functional and aesthetic results after transplantation of split thickness skin grafts present a socioeconomic problem. For improved wound closure the aim of this study was the development of a one stage technique for the establishment of a multi layer composite graft, existing of a collagen-GAG-matrix with silicon layer of a two layer synthetic dermal equivalent (DE) with integrated fibroblasts, and ceratinocytes. - In 64 athymic nude mice the evaluation of the multi layer skin grafts potential to re-establish a human epidermis, and high quality dermal structure was performed. In addition to clinical investigations we measured wound contraction, and analyzed histomorphologic, immunohistologic, "in situ hybridisation", and electro microscopic data. - Our results show, that the seeding of DE with human fibroblasts and ceratinocytes as a composite skin graft reproducible enabled a wound healing with an organised human dermis and epidermis within 10 - 15 days. The histological studies of the grafted composite skin grafts in this model showed morphologically a characteristic dermal-epidermal skin structure with a cornifying epithelium, being of human origin ("in situ hybridisation"). Through the co-cultivation of fibroblasts and ceratinocytes in the DE the generation and structural morphology of collagen fibres, and inflammatory reaction in the neodermis is positively influenced, and as a consequence wound contraction significantly reduced. In regard to the early preparation of composite grafts, and the minimal requirements for donor sites - with dependable stable reconstruction of the integument - this technique may present a step forward in the treatment of patients with extensive burns.

  16. Protein kinase-A-dependent osteoprotegerin production on interleukin-1 stimulation in human gingival fibroblasts is distinct from periodontal ligament fibroblasts

    PubMed Central

    Hormdee, D; Nagasawa, T; Kiji, M; Yashiro, R; Kobayashi, H; Koshy, G; Noguchi, K; Nitta, H; Ishikawa, I

    2005-01-01

    Periodontitis, a chronic inflammatory disease, is characterized by increased expression of interleukin (IL)-1 and other inflammatory mediators resulting in extensive osteoclast formation and bone loss. Expression of receptor activator of nuclear factor kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), by osteoblasts is important to regulate osteoclast differentiation. The aim of the present study was to investigate the regulatory effects of IL-1 on RANKL and OPG production by mesenchymal fibroblasts in periodontal tissue. Human gingival fibroblasts (HGF) and periodontal ligament fibroblasts (PDL) were stimulated with IL-1α with or without protein synthesis inhibitor cycloheximide (CHX), protein kinase A (PKA) inhibitors, protein kinase C (PKC) inhibitors and prostaglandin E2 (PGE2) inhibitor. In some experiments, the cultured cells were directly stimulated with either PKA or PKC activators. In HGF, IL-1α-stimulated OPG mRNA expression was high and could be reduced by CHX. PKA inhibitor completely abrogated IL-1α-induced OPG mRNA expression and OPG production. Endogenous PGE2 further enhanced IL-1α-induced OPG production in HGF. In PDL, RANKL mRNA expression was greatly augmented by IL-1α. IL-1α induced OPG mRNA expression and protein production. PKC inhibitor partially reduced IL-1α-induced OPG production and PKC activator enhanced OPG production in PDL. The IL-1α-stimulated OPG mRNA expression in HGF was greater than PDL. These results provide new evidence for the possible osteoclastogenesis-inhibitory function of HGF through PKA activity pathway. PDL utilized PKC for OPG production. Thus, we emphasize that HGF and PDL have different characteristics of host defence mechanism against inflammatory process. PMID:16297161

  17. Effect of diode low-level lasers on fibroblasts derived from human periodontal tissue: a systematic review of in vitro studies.

    PubMed

    Ren, Chong; McGrath, Colman; Jin, Lijian; Zhang, Chengfei; Yang, Yanqi

    2016-09-01

    This study aimed to systematically assess the parameter-specific effects of the diode low-level laser on human gingival fibroblasts (HGFs) and human periodontal ligament fibroblasts (HPDLFs). An extensive search was performed in major electronic databases including PubMed (1997), EMBASE (1947) and Web of Science (1956) and supplemented by hand search of reference lists and relevant laser journals for cell culture studies investigating the effect of diode low-level lasers on HGFs and HPDLFs published from January 1995 to December 2015. A total of 21 studies were included after screening 324 independent records, amongst which eight targeted HPDLFs and 13 focussed on HGFs. The diode low-level laser showed positive effects on promoting fibroblast proliferation and osteogenic differentiation and modulating cellular inflammation via changes in gene expression and the release of growth factors, bone-remodelling markers or inflammatory mediators in a parameter-dependent manner. Repeated irradiations with wavelengths in the red and near-infrared range and at an energy density below 16 J/cm(2) elicited favourable responses. However, considerable variations and weaknesses in the study designs and laser protocols limited the interstudy comparison and clinical transition. Current evidence showed that diode low-level lasers with adequate parameters stimulated the proliferation and modulated the inflammation of fibroblasts derived from human periodontal tissue. However, further in vitro studies with better designs and more appropriate study models and laser parameters are anticipated to provide sound evidence for clinical studies and practice.

  18. Synthetic ligands of the elastin receptor induce elastogenesis in human dermal fibroblasts via activation of their IGF-1 receptors.

    PubMed

    Qa'aty, Nour; Vincent, Matthew; Wang, Yanting; Wang, Andrew; Mitts, Thomas F; Hinek, Aleksander

    2015-12-01

    We have previously reported that a mixture of peptides obtained after chemical or enzymatic degradation of bovine elastin, induced new elastogenesis in human skin. Now, we investigated the elastogenic potential of synthetic peptides mimicking the elastin-derived, VGVAPG sequence, IGVAPG sequence that we found in the rice bran, and a similar peptide, VGVTAG that we identified in the IGF-1-binding protein-1 (IGFBP-1). We now demonstrate that treatment with each of these xGVxxG peptides (recognizable by the anti-elastin antibody), up-regulated the levels of elastin-encoding mRNA, tropoelastin protein, and the deposition of new elastic fibers in cultures of human dermal fibroblasts and in cultured explants of human skin. Importantly, we found that such induction of new elastogenesis may involve two parallel signaling pathways triggered after activation of IGF-1 receptor. In the first one, the xGVxxG peptides interact with the cell surface elastin receptor, thereby causing the downstream activation of the c-Src kinase and a consequent cross-activation of the adjacent IGF-1R, even in the absence of its principal ligand. In the second pathway their hydrophobic association with the N-terminal domain (VGVTAG) of the serum-derived IGFBP-1 induces conformational changes of this IGF-1 chaperone allowing for the release of its cargo and a consequent ligand-specific phosphorylation of IGF-1R. We present a novel, clinically relevant mechanism in which products of partial degradation of dermal elastin may stimulate production of new elastic fibers by dermal fibroblasts. Our findings particularly encourage the use of biologically safe synthetic xGVxxG peptides for regeneration of the injured or aged human skin. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. [Energy corrective and antioxidative actions of cytoflavin during postischemic period of human dermal fibroblasts in vitro].

    PubMed

    Tiuriaeva, I I; Kuranova, M L; Gonchar, I V; Rozanov, Iu M

    2012-01-01

    The influence of metabolic drug Cytoflavin (CF) with antihypoxic and antioxidative properties on human dermal fibroblasts in a model of ischemia-reoxygenation in vitro was studied. It was revealed that the restoration of ATP synthesis in fibroblasts in the postischemic period was considerably accelerated (in 2.1 times) by the addition of CF to the culture medium. The drug had a cell protective effect of reducing cell mortality during the reoxygenation after ischemia by 2-2.7 times. CF effectively reduced the level of reactive oxygen species (ROS) in fibroblasts after H2O2 treatment which allowed maintaining their survival at the level of control cells. Pretreatment of the cells with CF for one day ensured the maintenance of normal levels of ROS during the investigated time period in the fibroblasts subjected to H2O2 treatment, and reduced H2O2-induced cell death by almost a third compared to control cells. The introduction of CF in culture medium after ischemia showed no influence on Hsp70 synthesis, but led to decrease in GRP78 synthesis, raised after ischemia, to the control level, indicating a resolve of the endoplasmic reticulum (ER) stress and functional normalization of ER.

  20. Preparation of individual human diploid fibroblasts and study of ion transport.

    PubMed

    Abraham, E H; Breslow, J L; Epstein, J; Chang-Sing, P; Lechene, C

    1985-01-01

    A method for analyzing individual mammalian cells with electron probe microanalysis has been developed using human diploid fibroblasts. Cells were grown on the same support that is used for experimental manipulations and analysis. Steady-state cation and anion concentrations and kinetic processes during experimental perturbations could be measured on populations of less than 1,000 cells. Human diploid fibroblasts in normal tissue culture medium had the following intracellular concentrations (in mM): K, 168; Na, 25.0; Cl, 51.2; P, 84.1; S, 16.5; Ca, 6.04; and Mg, 10.0. The ratios of K to Na were equivalent when measured in the nuclear or cytoplasmic area of the cells. Serum in the incubation medium was found to increase the cellular effective permeability to Na by a factor of 2.5, while leaving the effective permeability to K unchanged. When returned to control medium after 7 h of incubation in K-free medium, the cells recovered normal K/Na in less than 1 h. In some experiments the coupling ratio of the ouabain-inhibitable cellular transport of Na to K was 3:2 and the ratio of Cl to K was 1:2. The sum of intracellular content (Na + K) (an estimate of cellular volume) did not change when the cells were placed in K-free medium and increased by less than 30% after ouabain treatment. After 5-7 h of ouabain treatment or of incubation in K-free medium, long after the intracellular K had been replaced by Na, the cellular chloride content had not changed significantly.

  1. The anti-oxidant effects of melatonin derivatives on human gingival fibroblasts.

    PubMed

    Phiphatwatcharaded, Chawapon; Puthongking, Ploenthip; Chaiyarit, Ponlatham; Johns, Nutjaree Pratheepawanit; Sakolchai, Sumon; Mahakunakorn, Pramote

    2017-07-01

    Aim of this in vitro study was to evaluate the anti-oxidant activity of indole ring modified melatonin derivatives as compared with melatonin in primary human gingival fibroblast (HGF) cells. Anti-oxidant activity of melatonin (MLT), acetyl-melatonin (AMLT) and benzoyl-melatonin (BMLT) was evaluated by5 standard methods as follows: 2, 2-diphenyl-1-picrylhydrazyl (DPPH); ferric ion reducing antioxidant power (FRAP); superoxide anion scavenging; nitric oxide (NO) scavenging; and thiobarbituric acid reactive substances (TBARs).Evaluation of cellular antioxidant activity (CAA) and protectivity against H 2 O 2 induced cellular damage was performed via MTT assay in HGF cells. According to the standard anti-oxidant assays, the antioxidant power of AMLT and BMLT were slightly less than MLT in FRAP and superoxide scavenging assays. In the NO scavenging and TBARs assays, BMLT and AMLT were more potent than MLT, whereas DPPH assays demonstrated that MLT was more potent than others. BMLT and AMLT had more potent anti-oxidant and protective activities against H 2 O 2 in HGF cells as compared with MLT. MLT derivatives demonstrated different anti-oxidant activities as compared with MLT, depending upon assays. These findings imply that N-indole substitution of MLT may help to improve hydrogen atom transfer to free radicals but electron transfer property is slightly decreased. Anti-oxidant and protective effects of melatonin derivatives (AMLT and BMLT) on human gingival fibroblasts imply the potential use of these molecules as alternative therapeutics for chronic inflammatory oral diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. The Possible Pre- and Post-UVA Radiation Protective Effect of Amaranth Oil on Human Skin Fibroblast Cells.

    PubMed

    Wolosik, Katarzyna; Zareba, Ilona; Surazynski, Arkadiusz; Markowska, Agnieszka

    2017-07-01

    The health effects of Amaranth Oil (AO) are attributed to its specific chemical composition. That makes it an outstanding natural product for the prevention and treatment of ultraviolet (UV) irradiation-related pathologies such as sunburn, photoaging, photoimmunosuppression, and photocarcinogenesis. Most of the studies are taken on animal model, and there is a lack of research on the endogenous effect of AO on fibroblast level, where UVA takes it harmful place. The aim of this study was evaluation if AO can protect or abolish UVA exposure effect on human skin fibroblast. The 0.1% AO, 0.25% AO, and 0.5% AO concentration and irradiation for 15 min under UVA-emitting lamp were studied in various condition. In all experiments, the mean values for six assays ± standard deviations were calculated. Pretreatment with various concentrations of AO was tested. The highest concentration of AO where cell survival was observed was 0.5%. Cytotoxicity assays provided evidence for pre- and post-UVA protective effect of 0.1% AO among three tested concentrations. The results also provide evidence that UVA has inhibitory effect on collagen biosynthesis in confluent skin fibroblast, but presence of 0.1% AO abolishes pre- and post-UVA effect comparing to other used AO concentration. The assessment results on DNA biosynthesis show the significant abolished post-UVA effect when 0.1% and 0.5% of AO were added. AO gives pre- and post-UVA protection in low concentration. This provides the evidence for using it not as a main protective factor against UV but as one of the combined components in cosmetic formulation. The recommended Amaranth Oil (AO) concentration in cosmetic formulation is between 0.1 and 5%Pretreatment with various concentrations of AO suggests to use the highest 0.5% concentration of AO in human skin fibroblast culturesThe 0.1% of AO in fibroblast cultures, protects and abolishes effect of ultraviolet A (UVA) exposureUVA has inhibitory effect on collagen biosynthesis in

  3. Radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in culture human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chigorno, V.; Cardace, G.; Pitto, M.

    1986-03-01

    A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GD/sub 1a/ isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated (/sup 3/H)GM/sub 1/ was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM/sub 1/, using as low as 10 ..mu..g of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that,more » however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm/sup 2/ plastic flasks was 5.8 -/+ 2.5 (SD) nmol liberated GM/sub 1/ h/sup -1/ mg protein/sup -1/. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate.« less

  4. Basic Fibroblast Growth Factor Influences Epidermal Homeostasis of Living Skin Equivalents through Affecting Fibroblast Phenotypes and Functions.

    PubMed

    Yang, Lujun; Zhang, Dangui; Wu, Hongjuan; Xie, Sitian; Zhang, Mingjun; Zhang, Bingna; Tang, Shijie

    2018-05-30

    To elucidate the possible mechanisms of how basic fibroblast growth factor (bFGF) influences epidermal homeostasis in a living skin equivalent (LSE) model. Several wound healing-related growth factors were analyzed at protein and mRNA levels for dermal fibroblasts of induced alpha-smooth muscle actin (α-SMA)-positive or α-SMA-negative phenotypes. During culturing an LSE model by seeding normal human keratinocytes on a fibroblast-populated type I collagen gel, bFGF or neutralizing antibody for keratinocyte growth factor (KGF) was added to investigate its effects on fibroblast phenotypes and, subsequently, epidermal homeostasis by histology and immunohistochemistry. The α-SMA-positive phenotype of fibroblasts induced by transforming growth factor beta-1 (TGF-β1) markedly suppressed the expression of KGF and hepatocyte growth factor (HGF), and slightly upregulated vascular endothelial growth factor (VEGF) and TGF-β1 at mRNA and protein levels, compared with α-SMA-negative fibroblasts treated with bFGF. α-SMA expression of fibroblasts at the epidermal-mesenchymal junction of the LSEs was suppressed by the addition of bFGF, and a better-differentiated epidermis was presented. The abrogation of KGF from fibroblasts by the addition of the KGF neutralizing antibody disenabled the LSE culturing system to develop an epidermis. bFGF, through affecting the phenotypes and functions of fibroblasts, especially KGF expression, influenced epidermal homeostasis in an LSE model. © 2018 S. Karger AG, Basel.

  5. Transduction of Oct6 or Oct9 gene concomitant with Myc family gene induced osteoblast-like phenotypic conversion in normal human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mizoshiri, N.; Department of Orthopaedics, Kyoto Prefectural University of Medicine, Kyoto; Kishida, T.

    Introduction: Osteoblasts play essential roles in bone formation and regeneration, while they have low proliferation potential. Recently we established a procedure to directly convert human fibroblasts into osteoblasts (dOBs). Transduction of Runx2 (R), Osterix (X), Oct3/4 (O) and L-myc (L) genes followed by culturing under osteogenic conditions induced normal human fibroblasts to express osteoblast-specific genes and produce calcified bone matrix both in vitro and in vivo Intriguingly, a combination of only two factors, Oct3/4 and L-myc, significantly induced osteoblast-like phenotype in fibroblasts, but the mechanisms underlying the direct conversion remains to be unveiled. Materials and Methods: We examined which Oct family genesmore » and Myc family genes are capable of inducing osteoblast-like phenotypic conversion. Results: As result Oct3/4, Oct6 and Oct9, among other Oct family members, had the capability, while N-myc was the most effective Myc family gene. The Oct9 plus N-myc was the best combination to induce direct conversion of human fibroblasts into osteoblast-like cells. Discussion: The present findings may greatly contribute to the elucidation of the roles of the Oct and Myc proteins in osteoblast direct reprogramming. The results may also lead to establishment of novel regenerative therapy for various bone resorption diseases. - Highlights: • Introducing L-myc in a combination with either Oct3/4, Oct6 or Oct9 enables the conversion of fibroblasts to osteoblasts. • A combination of L-myc with Oct3/4 or Oct9 can induce the cells to a phenotype closer to normal osteoblasts. • N-myc was considered the most appropriate Myc family gene for induction of osteoblast-like phenotype in fibroblasts. • The combination of Oct9 plus N-myc has the strongest capability of inducing osteoblast-like phenotype.« less

  6. PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts.

    PubMed

    Sun, Qingzhu; Liu, Li; Mandal, Jyotshna; Molino, Antonio; Stolz, Daiana; Tamm, Michael; Lu, Shemin; Roth, Michael

    2016-04-01

    Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

    PubMed

    Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

    2016-09-01

    To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

  8. The effect of mitomycin C after long-term storage on human Tenon's fibroblast proliferation.

    PubMed

    Hu, D; Chen, P P; Oda, D

    1999-10-01

    To investigate the effect of mitomycin C (MMC) after long-term storage on proliferation of human Tenon's fibroblasts in vitro. Human Tenon's fibroblasts in tissue culture were exposed for 5 minutes to MMC (0.4 mg/mL) that was either freshly prepared or had been stored for as long as 18 months at either 4 degrees C or -20 degrees C. The MTT colorimetric assay was used to determine the inhibition of proliferation as measured indirectly by mitochondrial activity. The inhibition rate was 88% using fresh MMC, and declined to a mean of 73% when using MMC that had been stored for as long as 18 months at 4 degrees C; this decrease was not statistically significant. The mean inhibition for MMC stored at -20 degrees C was 68%, and this was significantly less than inhibition with fresh MMC. Inhibition did not vary significantly with MMC after different storage times. Mitomycin C continues to have strong in vitro antiproliferative effects when stored for as long as 18 months at 4 degrees C or -20 degrees C. A significant decline in potency compared with fresh MMC occurs when MMC is stored at -20 degrees C.

  9. An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts.

    PubMed

    Jaquemar, D; Schenker, T; Trueb, B

    1999-03-12

    We have identified a novel transformation-sensitive mRNA, which is present in cultured fibroblasts but is lacking in SV40 transformed cells as well as in many mesenchymal tumor cell lines. The corresponding gene is located on human chromosome 8 in band 8q13. The open reading frame of the mRNA encodes a protein of 1119 amino acids forming two distinct domains. The N-terminal domain consists of 18 repeats that are related to the cytoskeletal protein ankyrin. The C-terminal domain contains six putative transmembrane segments that resemble many ion channels. This overall structure is reminiscent of TRP-like proteins that function as store-operated calcium channels. The novel protein with an Mr of 130 kDa is expressed at a very low level in human fibroblasts and at a moderate level in liposarcoma cells. Overexpression in eukaryotic cells appears to interfere with normal growth, suggesting that it might play a direct or indirect role in signal transduction and growth control.

  10. Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF.

    PubMed

    Meng, G L; Zur Nieden, N I; Liu, S Y; Cormier, J T; Kallos, M S; Rancourt, D E

    2008-04-01

    In embryonic stem (ES) cells, leukemia inhibitory factor (LIF)/STAT3, wnt and nodal/activin signaling are mainly active to control pluripotency during expansion. To maintain pluripotency, ES cells are typically cultured on feeder cells of varying origins. Murine ES cells are commonly cultured on murine embryonic fibroblasts (MEFs), which senesce early and must be frequently prepared. This process is laborious and leads to batch variation presenting a challenge for high-throughput ES cell expansion. Although some cell lines can be sustained by exogenous LIF, this method is costly. We present here a novel and inexpensive culture method for expanding murine ES cells on human foreskin fibroblast (HFF) feeders. After 20 passages on HFFs without LIF, ES cell lines showed normal expression levels of pluripotency markers, maintained a normal karyotype and retained the ability to contribute to the germline. As HFFs do not senesce for at least 62 passages, they present a vast supply of feeders. Copyright 2007 Wiley-Liss, Inc.

  11. Cellular Dysfunction in the Diabetic Fibroblast

    PubMed Central

    Lerman, Oren Z.; Galiano, Robert D.; Armour, Mary; Levine, Jamie P.; Gurtner, Geoffrey C.

    2003-01-01

    selective impairments in discrete cellular processes critical for tissue repair including cellular migration, VEGF production, and the response to hypoxia. The VEGF abnormalities developed concurrently with the onset of hyperglycemia and were not seen in normoglycemic, leptin receptor-deficient db/db mice. These observations support a role for fibroblast dysfunction in the impaired wound healing observed in human diabetics, and also suggest a mechanism for the poor clinical outcomes that occur after ischemic injury in diabetic patients. PMID:12507913

  12. 18ß-glycyrrhetinic acid derivative promotes proliferation, migration and aquaporin-3 expression in human dermal fibroblasts

    PubMed Central

    Hung, Chi-Feng; Hsiao, Chien-Yu; Hsieh, Wen-Hao; Li, Hsin-Ju; Tsai, Yi-Ju; Lin, Chun-Nan; Chang, Hsun-Hsien; Wu, Nan-Lin

    2017-01-01

    Licorice (Glycyrrhiza) species have been widely used as a traditional medicine and a natural sweetener in foods. The 18β-glycyrrhetinic acid (18β-GA) is a bioactive compound in licorice that exhibits potential anti-cancer, anti-inflammatory, and anti-microbial activities. Many synthesized derivatives of 18β-GA have been reported to be cytotoxic and suggested for the treatment of malignant diseases. In this study, we explored the possible pharmacological roles of an 18β-GA derivative in skin biology using primary human dermal fibroblasts and HaCaT keratinocytes as cell models. We found that this 18β-GA derivative did not cause cell death, but significantly enhanced the proliferation of dermal fibroblasts and HaCaT keratinocytes. A scratch wound healing assay revealed that the 18β-GA derivative promoted the migration of fibroblasts. Due to the important role of aquaporin-3 in cell migration and proliferation, we also investigated the expression of aquaporin-3 and found this compound up-regulated the expression of aquaporin-3 in dermal fibroblasts and HaCaT keratinocytes. In dermal fibroblasts, the 18β-GA derivative induced the phosphorylation of Akt, ERK, and p38. The inhibitor of Akt predominantly suppressed the 18β-GA derivative-induced expression of aquaporin-3. Collectively, this compound had a positive effect on the proliferation, migration, and aquaporin-3 expression of skin cells, implying its potential role in the treatment of skin diseases characterized by impaired wound healing or dermal defects. PMID:28813533

  13. Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity.

    PubMed

    Kooistra, T; Lloyd, J B

    1985-01-01

    The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.

  14. Hydrostatic pressure suppresses fibrotic changes via Akt/GSK-3 signaling in human cardiac fibroblasts.

    PubMed

    Tanaka, Ryo; Umemura, Masanari; Narikawa, Masatoshi; Fujita, Takayuki; Yokoyama, Utako; Ishigami, Tomoaki; Kimura, Kazuo; Tamura, Kouichi; Ishikawa, Yoshihiro

    2018-05-01

    Mechanical stresses play important roles in the process of constructing and modifying heart structure. It has been well established that stretch force acting on cardiac fibroblasts induces fibrosis. However, the effects of compressive force, that is, hydrostatic pressure (HP), have not been well elucidated. We thus evaluated the effects of HP using a pressure-loading apparatus in human cardiac fibroblasts (HCFs) in vitro. In this study, high HP (200 mmHg) resulted in significant phosphorylation of Akt in HCFs. HP then greatly inhibited glycogen synthase kinase 3 (GSK-3)α, which acts downstream of the PI3K/Akt pathway. Similarly, HP suppressed mRNA transcription of inflammatory cytokine-6, collagen I and III, and matrix metalloproteinase 1, compared with an atmospheric pressure condition. Furthermore, HP inhibited collagen matrix production in a three-dimensional HCF culture. Taken together, high HP suppressed the differentiation of fibroblasts into the myofibroblast phenotype. HP under certain conditions suppressed cardiac fibrosis via Akt/GSK-3 signaling in HCFs. These results might help to elucidate the pathology of some types of heart disease. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  15. A three-dimensional skin equivalent reflecting some aspects of in vivo aged skin.

    PubMed

    Diekmann, Johanna; Alili, Lirija; Scholz, Okka; Giesen, Melanie; Holtkötter, Olaf; Brenneisen, Peter

    2016-01-01

    Human skin undergoes morphological, biochemical and functional modifications during the ageing process. This study was designed to produce a 3-dimensional (3D) skin equivalent in vitro reflecting some aspects of in vivo aged skin. Reconstructed skin was generated by co-culturing skin fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan scaffold, and ageing was induced by the exposition of fibroblasts to Mitomycin-C (MMC). Recently published data showed that MMC treatment resulted in a drug-induced accelerated senescence (DIAS) in human dermal fibroblast cultures. Next to established ageing markers, histological changes were analysed in comparison with in vivo aged skin. In aged epidermis, the filaggrin expression is reduced in vivo and in vitro. Furthermore, in dermal tissue, the amount of elastin and collagen is lowered in aged skin in vivo as well as after the treatment of 3D skin equivalents with MMC in vitro. Our results show histological signs and some aspects of ageing in a 3D skin equivalent in vitro, which mimics aged skin in vivo. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines.

    PubMed

    Kono, Kiyomi; Maeda, Hidefumi; Fujii, Shinsuke; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Monnouchi, Satoshi; Teramatsu, Yoko; Hamano, Sayuri; Koori, Katsuaki; Akamine, Akifumi

    2013-05-01

    Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

  17. A stochastic step model of replicative senescence explains ROS production rate in ageing cell populations.

    PubMed

    Lawless, Conor; Jurk, Diana; Gillespie, Colin S; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F

    2012-01-01

    Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells.One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence.

  18. A Stochastic Step Model of Replicative Senescence Explains ROS Production Rate in Ageing Cell Populations

    PubMed Central

    Lawless, Conor; Jurk, Diana; Gillespie, Colin S.; Shanley, Daryl; Saretzki, Gabriele; von Zglinicki, Thomas; Passos, João F.

    2012-01-01

    Increases in cellular Reactive Oxygen Species (ROS) concentration with age have been observed repeatedly in mammalian tissues. Concomitant increases in the proportion of replicatively senescent cells in ageing mammalian tissues have also been observed. Populations of mitotic human fibroblasts cultured in vitro, undergoing transition from proliferation competence to replicative senescence are useful models of ageing human tissues. Similar exponential increases in ROS with age have been observed in this model system. Tracking individual cells in dividing populations is difficult, and so the vast majority of observations have been cross-sectional, at the population level, rather than longitudinal observations of individual cells. One possible explanation for these observations is an exponential increase in ROS in individual fibroblasts with time (e.g. resulting from a vicious cycle between cellular ROS and damage). However, we demonstrate an alternative, simple hypothesis, equally consistent with these observations which does not depend on any gradual increase in ROS concentration: the Stochastic Step Model of Replicative Senescence (SSMRS). We also demonstrate that, consistent with the SSMRS, neither proliferation-competent human fibroblasts of any age, nor populations of hTERT overexpressing human fibroblasts passaged beyond the Hayflick limit, display high ROS concentrations. We conclude that longitudinal studies of single cells and their lineages are now required for testing hypotheses about roles and mechanisms of ROS increase during replicative senescence. PMID:22359661

  19. High-efficiency generation of induced pluripotent mesenchymal stem cells from human dermal fibroblasts using recombinant proteins.

    PubMed

    Chen, Fanfan; Zhang, Guoqiang; Yu, Ling; Feng, Yanye; Li, Xianghui; Zhang, Zhijun; Wang, Yongting; Sun, Dapeng; Pradhan, Sriharsa

    2016-07-30

    Induced pluripotent mesenchymal stem cells (iPMSCs) are novel candidates for drug screening, regenerative medicine, and cell therapy. However, introduction of transcription factor encoding genes for induced pluripotent stem cell (iPSC) generation which could be used to generate mesenchymal stem cells is accompanied by the risk of insertional mutations in the target cell genome. We demonstrate a novel method using an inactivated viral particle to package and deliver four purified recombinant Yamanaka transcription factors (Sox2, Oct4, Klf4, and c-Myc) resulting in reprogramming of human primary fibroblasts. Whole genome bisulfite sequencing was used to analyze genome-wide CpG methylation of human iPMSCs. Western blot, quantitative PCR, immunofluorescence, and in-vitro differentiation were used to assess the pluripotency of iPMSCs. The resulting reprogrammed fibroblasts show high-level expression of stem cell markers. The human fibroblast-derived iPMSC genome showed gains in DNA methylation in low to medium methylated regions and concurrent loss of methylation in previously hypermethylated regions. Most of the differentially methylated regions are close to transcription start sites and many of these genes are pluripotent pathway associated. We found that DNA methylation of these genes is regulated by the four iPSC transcription factors, which functions as an epigenetic switch during somatic reprogramming as reported previously. These iPMSCs successfully differentiate into three embryonic germ layer cells, both in vitro and in vivo. Following multipotency induction in our study, the delivered transcription factors were degraded, leading to an improved efficiency of subsequent programmed differentiation. Recombinant transcription factor based reprogramming and derivatization of iPMSC offers a novel high-efficiency approach for regenerative medicine from patient-derived cells.

  20. 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.

  1. Structure of a highly stable mutant of human fibroblast growth factor 1.

    PubMed

    Szlachcic, Anna; Zakrzewska, Małgorzata; Krowarsch, Daniel; Os, Vibeke; Helland, Ronny; Smalås, Arne O; Otlewski, Jacek

    2009-01-01

    Fibroblast growth factors (FGFs) are involved in diverse cellular processes such as cell migration, angiogenesis, osteogenesis, wound healing and embryonic and foetal development. Human acidic fibroblast growth factor (FGF-1) is the only member of the FGF family that binds with high affinity to all four FGF receptors and thus is considered to be the human mitogen with the broadest specificity. However, pharmacological applications of FGF-1 are limited owing to its low stability. It has previously been reported that the introduction of single mutations can significantly improve the stability of FGF-1 and its resistance to proteolytic degradation. Here, the structure of the Q40P/S47I/H93G triple mutant of FGF-1, which exhibits much higher stability, a prolonged half-life and enhanced mitogenic activity, is presented. Compared with the wild-type structure, three localized conformational changes in the stable triple mutant were observed, which is in agreement with the perfect energetic additivity of the single mutations described in a previous study. The huge change in FGF-1 stability (the denaturation temperature increased by 21.5 K, equivalent to DeltaDeltaG(den) = 24.3 kJ mol(-1)) seems to result from the formation of a short 3(10)-helix (position 40), an improvement in the propensity of amino acids to form beta-sheets (position 47) and the rearrangement of a local hydrogen-bond network (positions 47 and 93).

  2. Transcriptional and Cell Cycle Alterations Mark Aging of Primary Human Adipose-Derived Stem Cells.

    PubMed

    Shan, Xiaoyin; Roberts, Cleresa; Kim, Eun Ji; Brenner, Ariana; Grant, Gregory; Percec, Ivona

    2017-05-01

    Adult stem cells play a critical role in the maintenance of tissue homeostasis and prevention of aging. While the regenerative potential of stem cells with low cellular turnover, such as adipose-derived stem cells (ASCs), is increasingly recognized, the study of chronological aging in ASCs is technically difficult and remains poorly understood. Here, we use our model of chronological aging in primary human ASCs to examine genome-wide transcriptional networks. We demonstrate first that the transcriptome of aging ASCs is distinctly more stable than that of age-matched fibroblasts, and further, that age-dependent modifications in cell cycle progression and translation initiation specifically characterize aging ASCs in conjunction with increased nascent protein synthesis and a distinctly shortened G1 phase. Our results reveal novel chronological aging mechanisms in ASCs that are inherently different from differentiated cells and that may reflect an organismal attempt to meet the increased demands of tissue and organ homeostasis during aging. Stem Cells 2017;35:1392-1401. © 2017 AlphaMed Press.

  3. Stimulation of plasmin activity in cultured human fibroblast cells by Porphyromonas endodontalis.

    PubMed

    Oikawa, T; Ogura, N; Akiba, M; Abiko, Y; Takiguchi, H; Izumi, H

    1993-09-01

    1. Plasmin activity in the conditioned medium of Gin-1 cells, a human gingival fibroblast cell line, was stimulated by Porphyromonas endodontalis, a putative pathogen of oral submucous abscesses, in a time- and dose-dependent manner. 2. P. endodontalis stimulated the activity of plasminogen activator in both the conditioned medium and the cell lysate. The plasminogen activator in Gin-1 cells was approx. 50 kDa by zymography. 3. The conditioned medium of Gin-1 cells exposed to P. endodontalis stimulated the conversion of human serum prekallikrein to kallikrein. 4. These results suggested that P. endodontalis stimulates the plasminogen activator-plasmin system in Gin-1 cells, and that activated plasmin plays a role in the progress of periodontal tissue inflammation.

  4. 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

  5. Antifibrotic effect of pirfenidone on human pterygium fibroblasts.

    PubMed

    Lee, Kihwang; Young Lee, Sun; Park, So Yean; Yang, Hongseok

    2014-07-01

    The effects of pirfenidone were investigated on cultured human pterygium fibroblasts (HPFs). HPFs were obtained from pterygium surgery and subjected to primary culture. After treatment with 0.5, 1.0 or 1.5 mg/mL pirfenidone, MTT and cell migration assays were performed, and procollagen secretion and TGF-β expression were measured by Western blotting and immunofluorescence analysis. Pirfenidone had a significant inhibitory effect on HPF proliferation, migration and collagen synthesis. There were no differences between the cells treated with 0.5, 1.0 and 1.5 mg/mL pirfenidone and the controls in the MTT assay. After 48 h of treatment with 1.0 or 1.5 mg/mL pirfenidone, TGF-β expression was significantly decreased. These findings demonstrate that pirfenidone inhibits the proliferation, migration and procollagen secretion of HPFs at nontoxic concentrations by decreasing TGF-β expression. Thus, pirfenidone may be considered as a safe adjuvant for pterygium surgery to prevent recurrence.

  6. A Prospective, Randomized, Double-blind, Split-face Clinical Trial Comparing the Efficacy of Two Topical Human Growth Factors for the Rejuvenation of the Aging Face

    PubMed Central

    Goldman, Mitchel P.

    2017-01-01

    Background: Cosmeceutical products represent an increasingly important therapeutic option for anti-aging and rejuvenation, either used alone or in combination with dermatologic surgical procedures. Among this group of products, topical growth factors have demonstrated efficacy in randomized, controlled clinical trials. However, comparisons between different products remain uncommon. Objective: The objective of this randomized, double-blind, split-face clinical trial was to compare two different topical growth factor formulations derived from either human fibroblasts or human adipose tissue derived mesenchymal stem cells. Methods: This was an institutional review board-approved, randomized, double-blind, split-face clinical trial involving 20 healthy subjects with moderate-to-severe facial wrinkling secondary to photodamage. One half of the face was randomized to receive topical human fibroblast growth factors and the other topical human mesenchymal stem cell growth factors. Treatment was continued for three months, and evaluations were performed in a double-blind fashion. Results: Both growth factor formulations achieved significant improvement in facial wrinkling. Blinded investigator and subject evaluations did not detect any significant differences between the two formulations in terms of efficacy, safety, or tolerability. Conclusion: Both human fibroblast growth factors and human mesenchymal stem cell growth factors are effective at facial rejuvenation. Topical growth factors represent a useful therapeutic modality. PMID:28670356

  7. A Prospective, Randomized, Double-blind, Split-face Clinical Trial Comparing the Efficacy of Two Topical Human Growth Factors for the Rejuvenation of the Aging Face.

    PubMed

    Wu, Douglas C; Goldman, Mitchel P

    2017-05-01

    Background: Cosmeceutical products represent an increasingly important therapeutic option for anti-aging and rejuvenation, either used alone or in combination with dermatologic surgical procedures. Among this group of products, topical growth factors have demonstrated efficacy in randomized, controlled clinical trials. However, comparisons between different products remain uncommon. Objective: The objective of this randomized, double-blind, split-face clinical trial was to compare two different topical growth factor formulations derived from either human fibroblasts or human adipose tissue derived mesenchymal stem cells. Methods: This was an institutional review board-approved, randomized, double-blind, split-face clinical trial involving 20 healthy subjects with moderate-to-severe facial wrinkling secondary to photodamage. One half of the face was randomized to receive topical human fibroblast growth factors and the other topical human mesenchymal stem cell growth factors. Treatment was continued for three months, and evaluations were performed in a double-blind fashion. Results: Both growth factor formulations achieved significant improvement in facial wrinkling. Blinded investigator and subject evaluations did not detect any significant differences between the two formulations in terms of efficacy, safety, or tolerability. Conclusion: Both human fibroblast growth factors and human mesenchymal stem cell growth factors are effective at facial rejuvenation. Topical growth factors represent a useful therapeutic modality.

  8. Pericellular Versican Regulates the Fibroblast-Myofibroblast Transition

    PubMed Central

    Hattori, Noriko; Carrino, David A.; Lauer, Mark E.; Vasanji, Amit; Wylie, James D.; Nelson, Courtney M.; Apte, Suneel S.

    2011-01-01

    The cell and its glycosaminoglycan-rich pericellular matrix (PCM) comprise a functional unit. Because modification of PCM influences cell behavior, we investigated molecular mechanisms that regulate PCM volume and composition. In fibroblasts and other cells, aggregates of hyaluronan and versican are found in the PCM. Dermal fibroblasts from Adamts5−/− mice, which lack a versican-degrading protease, ADAMTS5, had reduced versican proteolysis, increased PCM, altered cell shape, enhanced α-smooth muscle actin (SMA) expression and increased contractility within three-dimensional collagen gels. The myofibroblast-like phenotype was associated with activation of TGFβ signaling. We tested the hypothesis that fibroblast-myofibroblast transition in Adamts5−/− cells resulted from versican accumulation in PCM. First, we noted that versican overexpression in human dermal fibroblasts led to increased SMA expression, enhanced contractility, and increased Smad2 phosphorylation. In contrast, dermal fibroblasts from Vcan haploinsufficient (Vcanhdf/+) mice had reduced contractility relative to wild type fibroblasts. Using a genetic approach to directly test if myofibroblast transition in Adamts5−/− cells resulted from increased PCM versican content, we generated Adamts5−/−;Vcanhdf/+ mice and isolated their dermal fibroblasts for comparison with dermal fibroblasts from Adamts5−/− mice. In Adamts5−/− fibroblasts, Vcan haploinsufficiency or exogenous ADAMTS5 restored normal fibroblast contractility. These findings demonstrate that altering PCM versican content through proteolytic activity of ADAMTS5 profoundly influenced the dermal fibroblast phenotype and may regulate a phenotypic continuum between the fibroblast and its alter ego, the myofibroblast. We propose that a physiological function of ADAMTS5 in dermal fibroblasts is to maintain optimal versican content and PCM volume by continually trimming versican in hyaluronan-versican aggregates. PMID:21828051

  9. GSK126 (EZH2 inhibitor) interferes with ultraviolet A radiation-induced photoaging of human skin fibroblast cells

    PubMed Central

    Qin, Haiyan; Zhang, Guang; Zhang, Lianbo

    2018-01-01

    Polycomb group genes (PcG) encode chromatin modification proteins that are involved in the epigenetic regulation of cell differentiation, proliferation and the aging processes. The key subunit of the PcG complex, enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), has a central role in a variety of mechanisms, such as the formation of chromatin structure, gene expression regulation and DNA damage. In the present study, ultraviolet A (UVA) was used to radiate human dermal fibroblasts in order to construct a photo-aged cell model. Subsequently, the cell viability assay, Hoechst staining, apoptosis detection using flow cytometry, senescence-associated β-galactosidase (SA-β-gal) staining and erythrocyte exclusion experiments were performed. GSK126, a histone methylation enzyme inhibitor of EZH2, was used as an experimental factor. Results suggested that GSK126 downregulated the mRNA expression levels of EZH2 and upregulated the mRNA expression levels of BMI-1. Notably, GSK126 affected the transcription of various photoaging-related genes and thus protected against photoaging induced by UVA radiation. PMID:29545866

  10. SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

    PubMed

    Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

    2018-05-01

    SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

  11. Determine the yield of micronucleated cells in primary human fibroblasts exposed to focused soft X-rays.

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kevin M. Prise

    This project was a small part of a larger collaborative study headed by Dr Aloke Chatterjee, (Lawrence Berkeley National Laboratory) and including Drs Les Braby, John Ford (Texas A&M) and Kathy Held (MGH Boston), which was developing an integrated theoretical and experimental model of the radiation-induced bystander response. Our part of the study has been to determine the effectiveness of soft X-rays at inducing chromosomal damage under conditions of direct and bystander exposure. The aim was to compare this with the effectiveness of the low energy 60 kV electron microbeam available at Texas A&M. Previous studies have been performed withmore » primary human fibroblasts measuring micronuclei formation to determine the relative yields of direct versus bystander mediated micronuclei formation after cells were individually irradiated utilizing our novel focused soft X-ray microprobe, which is capable of producing localized submicron beams of carbon-K (278 eV) X-rays. Only a brief overview is given here as the study has been published in several papers. Our original hypothesis was to study yields of bystander-induced micronucleated cells in both wild-type and mutant fibroblast from mouse embryo fibroblasts. Difficulties with the level of background micronuclei in the MEFs prevented systematic studies of bystander responses in the laboratories involved in the collaboration. We then performed these studies with AG1522 primary human fibroblast cells using a siRNA approach developed by John Ford at Texas A&M to knock down DNA PKcs in the first instance. Our soft X-ray source has been in routine use for carbon-K X-rays and is now available with Aluminium-K (1.49 keV) and titanium-K (4.5 keV), although the dose-rate from titanium is still too low at present for most experiments, where large numbers of cells need to be exposed. A separately funded project developed a new soft X-ray microprobe which will give much greater flexibility for changing energies and giving high

  12. Comparison of primary human fibroblasts and keratinocytes with immortalized cell lines regarding their sensitivity to sodium dodecyl sulfate in a neutral red uptake cytotoxicity assay.

    PubMed

    Olschläger, Veronika; Schrader, Andreas; Hockertz, Stefan

    2009-01-01

    Cell lines present a valuable tool for in vitro assessment of skin damage caused by application of cosmeticals or pharmaceuticals. They form a reproducible test system under controllable test conditions and, in many cases, can be used as alternatives to animal testing in order to assess the compatibility of drugs or cosmetics and human skin. Yet, it can not necessarily be assumed that the behavior of cultured cells, when treated with different substances, is exactly consistent with the behavior of cells being part of a live organism. Becoming immortal, cells exhibit changes in genotype and/or phenotype, possibly resulting in modified reactions to external influences. Therefore, to obtain results close to in vivo studies, it seems apparent to use primary cells for testing that have not yet undergone any modifications. To compare the properties of primary fibroblasts (Normal Human Dermal Fibroblasts, NHDF) and primary keratinocytes (Normal Human Epidermal Keratinocytes, NHEK) with those of immortal cell lines (3T3 (ACC 173) Swiss albino mouse fibroblasts and HaCaT (human, adult, low calcium, high temperature, human adult skin keratinocytes) cells), their sensitivities in cytotoxicity assays have been assessed. While both fibroblast cell cultures showed similar sensitivities towards sodium dodecyl sulfate (SDS), primary keratinocytes died at SDS concentrations about three times lower than the immortal HaCaT cells.

  13. Distinct Fibroblasts in the Papillary and Reticular Dermis: Implications for Wound Healing.

    PubMed

    Woodley, David T

    2017-01-01

    Human skin wounds heal largely by reparative wound healing rather than regenerative wound healing. Human skin wounds heal with scarring and without pilosebaceous units or other appendages. Dermal fibroblasts come from 2 distinct lineages of cells that have distinct cell markers and, more importantly, distinct functional abilities. Human skin wound healing largely involves the dermal fibroblast lineage from the reticular dermis and not the papillary dermis. If scientists could find a way to stimulate the dermal fibroblast lineages from the papillary dermis in early wound healing, perhaps human skin wounds could heal without scarring and with skin appendages. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Homogeneous Inflammatory Gene Profiles Induced in Human Dermal Fibroblasts in Response to the Three Main Species of Borrelia burgdorferi sensu lato

    PubMed Central

    Meddeb, Mariam; Carpentier, Wassila; Cagnard, Nicolas; Nadaud, Sophie; Grillon, Antoine; Barthel, Cathy; De Martino, Sylvie Josiane; Jaulhac, Benoît; Boulanger, Nathalie

    2016-01-01

    In Lyme borreliosis, the skin is the key site for bacterial inoculation by the infected tick and for cutaneous manifestations. We previously showed that different strains of Borrelia burgdorferi sensu stricto isolated from tick and from different clinical stages of the Lyme borreliosis (erythema migrans, and acrodermatitis chronica atrophicans) elicited a very similar transcriptional response in normal human dermal fibroblasts. In this study, using whole transcriptome microarray chips, we aimed to compare the transcriptional response of normal human dermal fibroblasts stimulated by 3 Borrelia burgdorferi sensu lato strains belonging to 3 main pathogenic species (B. afzelii, B. garinii and B. burgdorferi sensu stricto) in order to determine whether “species-related” inflammatory pathways could be identified. The three Borrelia strains tested exhibited similar transcriptional profiles, and no species-specific fingerprint of transcriptional changes in fibroblasts was observed. Conversely, a common core of chemokines/cytokines (CCL2, CXCL1, CXCL2, CXCL6, CXCL10, IL-6, IL-8) and interferon-related genes was stimulated by all the 3 strains. Dermal fibroblasts appear to play a key role in the cutaneous infection with Borrelia, inducing a homogeneous inflammatory response, whichever Borrelia species was involved. PMID:27706261

  15. Modulation of hepatocyte growth factor secretion in human female reproductive tract stromal fibroblasts by poly (I:C) and estradiol.

    PubMed

    Coleman, Kimberly D; Ghosh, Mimi; Crist, Sarah G; Wright, Jacqueline A; Rossoll, Richard M; Wira, Charles R; Fahey, John V

    2012-01-01

    Hepatocyte Growth Factor (HGF) secretion facilitates epithelial cell growth and development in the female reproductive tract (FRT) and may contribute to pathological conditions such as cancer and endometriosis. We hypothesized that estradiol and poly (I:C), a synthetic RNA mimic, may have a regulatory effect on HGF secretion by stromal fibroblasts from FRT tissues. Following hysterectomies, normal tissue from the uterus, endocervix, and ectocervix were dispersed into stromal cell fractions by enzymatic digestion and differential filtering. Stromal fibroblasts were cultured and treated with estradiol and/or poly (I:C), and conditioned media were analyzed for HGF via enzyme-linked immunosorbent assay. Treating uterine fibroblasts with estradiol or poly (I:C) significantly increased HGF secretion. When uterine fibroblasts were co-treated with estradiol and poly (I:C), the effect on HGF secretion was additive. In contrast, stromal fibroblasts from endo- and ecto-cervix were unresponsive to estradiol, but were stimulated to secrete HGF by poly (I:C). HGF secretion is uniquely regulated in the uterus, but not in ecto- and endo-cervix, by estradiol. Moreover, potential viral pathogens further induce HGF. These findings have potential applications in understanding both hormonal regulation of normal tissue as well as the role of HGF in tumorogenesis, endometriosis, and human immunodeficiency virus infection. © 2011 John Wiley & Sons A/S.

  16. AhR-dependent secretion of PDGF-BB by human classically activated macrophages exposed to DEP extracts stimulates lung fibroblast proliferation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jaguin, Marie; Fardel, Olivier; Pôle Biologie, Centre Hospitalier Universitaire

    2015-06-15

    Lung diseases are aggravated by exposure to diesel exhaust particles (DEPs) found in air pollution. Macrophages are thought to play a crucial role in lung immune response to these pollutants, even if the mechanisms involved remain incompletely characterized. In the present study, we demonstrated that classically and alternative human macrophages (MΦ) exhibited increased secretion of PDGF-B in response to DEP extract (DEPe). This occurred via aryl hydrocarbon receptor (AhR)-activation because DEPe-induced PDGF-B overexpression was abrogated after AhR expression knock-down by RNA interference, in both M1 and M2 polarizing MΦ. In addition, TCDD and benzo(a)pyrene, two potent AhR ligands, also significantlymore » increased mRNA expression of PDGF-B in M1 MΦ, whereas some weak ligands of AhR did not. We next evaluated the impact of conditioned media (CM) from MΦ culture exposed to DEPe or of recombinant PDGF-B onto lung fibroblast proliferation. The tyrosine kinase inhibitor, AG-1295, prevents phosphorylations of PDGF-Rβ, AKT and ERK1/2 and the proliferation of MRC-5 fibroblasts induced by recombinant PDGF-B and by CM from M1 polarizing MΦ, strongly suggesting that the PDGF-BB secreted by DEPe-exposed MΦ is sufficient to activate the PDGF-Rβ pathway of human lung fibroblasts. In conclusion, we demonstrated that human MΦ, whatever their polarization status, secrete PDGF-B in response to DEPe and that PDGF-B is a target gene of AhR. Therefore, induction of PDGF-B by DEP may participate in the deleterious effects towards human health triggered by such environmental urban contaminants. - Highlights: • PDGF-B expression and secretion are increased by DEPe exposure in human M1 and M2 MΦ. • DEPe-induced PDGF-B expression is aryl-hydrocarbon-dependent. • DEPe-exposed M1 MΦ secrete sufficient PDGF-B to increase lung fibroblast proliferation.« less

  17. [Subretinal transplantation of human fetal lung fibroblasts expressed ciliary neurotrophic factor gene prevent photoreceptor degeneration in RCS rats].

    PubMed

    Huang, Qian; Xu, Ping; Xia, Xin; Hu, Hong-hui; Wang, Feng; Li, Hui-ming

    2006-02-01

    To investigate the efficacy of subretinal transplantation of CNTF gene transfected fibroblasts for preventing photoreceptor degeneration in RCS. The human fetal lung fibroblasts with high level expression of CNTF were established by liposome mediated gene transfer and MTX selection. A 5 microl of cell suspension, containing 1 x 10(5) cells, was injected through pars plana of ciliary body into the subretinal space of the right eye at postnatal 4-5 weeks, the left eye was left without injection or injected with PBS as controls. The both eyes were enucleated for histopathological examinations at 2, 4, 6, 8, 10, 12 and 15 weeks following transplantation. The level of CNTF protein (91,046.15 pg/ml) expressed in the transfected cells was determined by sandwich enzyme-linked immunosorbent assay (ELISA). The four of seven eyes examined by light microscopy and the ten of 14 eyes examined by electro microscopy showed rescue effect. The prolonged photoreceptor survival, reduction of apoptotic cells and debris were observed in transplanted eyes in comparison with untreated or sham-injected eyes. This study provides the first indication that transplanted human fibroblasts with high level expression of CNTF are able to rescue photoreceptor degeneration in RCS dystrophic rat retina.

  18. Transforming growth factor (TGF. beta. ) decreases the proliferation of human bone marrow fibroblasts by inhibiting the platelet-derived growth factor (PDGF) binding

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bryckaert, M.C.; Tobelem, G.; Lindroth, M.

    1988-12-01

    Human bone marrow fibroblasts were cultivated and characterized by immunofluorescent staining and electron microscopy. Their interactions with PDGF and TGF{beta} were studied. While a positive intracellular antifibronectin staining was observed, the cultured cells were not labeled with specific antibodies toward factor VIII von Willebrand factor (F VIII/vWF), desmin, and macrophage antigen. The binding of pure human PDGF to the cultured bone marrow fibroblasts was investigated. Addition of an excess of unlabeled PDGF decreased the binding to 75 and 80%, which means that the nonspecific binding represented 20-25% of total binding, whereas epidermal growth factor (EGF) had no effect. Two classesmore » of sites were detected by Scatchard analysis. The stimulation of DNA synthesis of PDGF was quantified by ({sup 3}H)thymidine incorporation. The results suggested that PDGF and TGF{beta} could modulate the growth of bone marrow fibroblasts.« less

  19. Ex-vivo transduced autologous skin fibroblasts expressing human Lim Mineralization Protein-3 efficiently form new bone in animal models

    PubMed Central

    Lattanzi, Wanda; Parrilla, Claudio; Fetoni, Annarita; Logroscino, Giandomenico; Straface, Giuseppe; Pecorini, Giovanni; Stigliano, Egidio; Tampieri, Anna; Bedini, Rossella; Pecci, Raffaella; Michetti, Fabrizio; Gambotto, Andrea; Robbins, Paul D.; Pola, Enrico

    2012-01-01

    Local gene transfer of the human LIM Mineralization Protein (LMP), a novel intracellular positive regulator of the osteoblast differentiation program, can induce efficient bone formation in rodents. In order to develop a clinically relevant gene therapy approach to facilitate bone healing, we have used primary dermal fibroblasts transduced ex vivo with Ad.LMP3 and seeded on an hydroxyapatite/collagen matrix prior to autologous implantation. Here we demonstrate that genetically modified autologous dermal fibroblasts expressing Ad.LMP-3 are able to induce ectopic bone formation following implantation of the matrix into the mouse triceps and paravertebral muscles. Moreover, implantation of the Ad.LMP-3-modified dermal fibroblasts into a rat mandibular bone critical size defect model results in efficient healing as determined by X-ray, histology and three dimensional micro computed tomography (3DμCT). These results demonstrate the effectiveness of the non-secreted intracellular osteogenic factor LMP-3, in inducing bone formation in vivo. Moreover, the utilization of autologous dermal fibroblasts implanted on a biomaterial represents a promising approach for possible future clinical applications aimed at inducing new bone formation. PMID:18633445

  20. Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells

    PubMed Central

    Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

    2017-01-01

    Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn’t differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication. PMID:28886142

  1. Effects of ozone exposure on human epithelial adenocarcinoma and normal fibroblasts cells.

    PubMed

    Poma, Anna; Colafarina, Sabrina; Aruffo, Eleonora; Zarivi, Osvaldo; Bonfigli, Antonella; Di Bucchianico, Sebastiano; Di Carlo, Piero

    2017-01-01

    Previous studies show variable ozone cytotoxicity and genotoxicity in cell cultures, laboratory animals and humans directly exposed to tropospheric ozone. The aim of this study was therefore to investigate and compare the cyto and genotoxic effects of ozone using adenocarcinoma human alveolar basal epithelial cells A549 and normal human fibroblasts Hs27. A cell culture chamber with controlled atmosphere (a simulation reactor) was built to inject a flow of 120 ppb of ozone, which is two times the threshold value for the protection of human health, fixed by the EU legislation. Cell proliferation was evaluated by a luminescent cell viability assay while we assessed the genotoxic potential of ozone by the induction of micronuclei as well as evaluating DNA strand breaks by the induction of micronuclei evaluated by means of the cytokinesis-block micronucleus (CBMN) assay as well as evaluating DNA strand breaks by Alkaline Comet Assay (CA) or Comet Assay. A549 cells viability decreases significantly at 24 hours treatment with 120 ppb of O3 while at 48 hours and 72 hours O3 treated cells viability doesn't differ in respect to the control. However a significative decrease of A549 viability is shown at 72 hours vs. 48 hours in both treated and not-treated cells. The viability trend in the Hs27 cells did not show any significant changes in treated samples compared to the control in all conditions. The two genotoxicity biomarkers, the micronucleus and the comet tests, showed in both the cell types exposed to ozone, a significant increase in the number of micronuclei and in the tail DNA % in respect to the control even if at different times/cell type. Moreover, we found that O3 provokes genotoxic effects more evident in A549 cancer cells than in normal fibroblasts Hs27 ones. We applied a cell growth simulation model referred to ozone treated or not cell lines to confirm that the ozone exposure causes a slackening in the cells replication.

  2. Evaluating the potential of poly(beta-amino ester) nanoparticles for reprogramming human fibroblasts to become induced pluripotent stem cells.

    PubMed

    Bhise, Nupura S; Wahlin, Karl J; Zack, Donald J; Green, Jordan J

    2013-01-01

    Gene delivery can potentially be used as a therapeutic for treating genetic diseases, including neurodegenerative diseases, as well as an enabling technology for regenerative medicine. A central challenge in many gene delivery applications is having a safe and effective delivery method. We evaluated the use of a biodegradable poly(beta-amino ester) nanoparticle-based nonviral protocol and compared this with an electroporation-based approach to deliver episomal plasmids encoding reprogramming factors for generation of human induced pluripotent stem cells (hiPSCs) from human fibroblasts. A polymer library was screened to identify the polymers most promising for gene delivery to human fibroblasts. Feeder-independent culturing protocols were developed for nanoparticle-based and electroporation-based reprogramming. The cells reprogrammed by both polymeric nanoparticle-based and electroporation-based nonviral methods were characterized by analysis of pluripotency markers and karyotypic stability. The hiPSC-like cells were further differentiated toward the neural lineage to test their potential for neurodegenerative retinal disease modeling. 1-(3-aminopropyl)-4-methylpiperazine end-terminated poly(1,4-butanediol diacry-late-co-4-amino-1-butanol) polymer (B4S4E7) self-assembled with plasmid DNA to form nanoparticles that were more effective than leading commercially available reagents, including Lipofectamine® 2000, FuGENE® HD, and 25 kDa branched polyethylenimine, for nonviral gene transfer. B4S4E7 nanoparticles showed effective gene delivery to IMR-90 human primary fibroblasts and to dermal fibroblasts derived from a patient with retinitis pigmentosa, and enabled coexpression of exogenously delivered genes, as is needed for reprogramming. The karyotypically normal hiPSC-like cells generated by conventional electroporation, but not by poly(beta-amino ester) reprogramming, could be differentiated toward the neuronal lineage, specifically pseudostratified optic cups. This

  3. Effects of human umbilical cord blood-derived mesenchymal stromal cells and dermal fibroblasts on diabetic wound healing.

    PubMed

    Moon, Kyung-Chul; Lee, Jong-Seok; Han, Seung-Kyu; Lee, Hyup-Woo; Dhong, Eun-Sang

    2017-07-01

    A previous study demonstrated that human umbilical cord blood-derived mesenchymal stromal cells (hUCB-MSCs) have superior wound-healing activity compared with fibroblasts in vitro. However, wound healing in vivo is a complex process that involves multiple factors. The purpose of this study was to compare the effects of hUCB-MSCs and fibroblasts on diabetic wound healing in vivo. This study especially focused on collagen synthesis and angiogenesis, which are considered to be the important factors affecting diabetic wound healing. Porous polyethylene discs were loaded with either fibroblasts or hUCB-MSCs, and a third group, which served as a control, was not loaded with cells. The discs were then implanted in the back of diabetic mice. During the first and the second week after implantation, the discs were harvested, and collagen level and microvascular density were compared. In terms of collagen synthesis, the hUCB-MSC group showed the highest collagen level (117.7 ± 8.9 ng/mL), followed by the fibroblast group (83.2 ± 5.2 ng/mL) and the no-cell group (60.0 ± 4.7 ng/mL) in the second week after implantation. In terms of angiogenesis, the microvascular density in the hUCB-MSC group was 56.8 ± 16.4, which was much higher than that in the fibroblast group (14.3 ± 4.0) and the no-cell group (5.7 ± 2.1) in the second week after implantation. These results demonstrate that hUCB-MSCs are superior to fibroblasts in terms of their effect on diabetic wound healing in vivo. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

  4. Response of human corneal fibroblasts on silk film surface patterns.

    PubMed

    Gil, Eun Seok; Park, Sang-Hyug; Marchant, Jeff; Omenetto, Fiorenzo; Kaplan, David L

    2010-06-11

    Transparent, biodegradable, mechanically robust, and surface-patterned silk films were evaluated for the effect of surface morphology on human corneal fibroblast (hCF) cell proliferation, orientation, and ECM deposition and alignment. A series of dimensionally different surface groove patterns were prepared from optically graded glass substrates followed by casting poly(dimethylsiloxane) (PDMS) replica molds. The features on the patterned silk films showed an array of asymmetric triangles and displayed 37-342 nm depths and 445-3 582 nm widths. hCF DNA content on all patterned films were not significantly different from that on flat silk films after 4 d in culture. However, the depth and width of the grooves influenced cell alignment, while the depth differences affected cell orientation; overall, deeper and narrower grooves induced more hCF orientation. Over 14 d in culture, cell layers and actin filament organization demonstrated that confluent hCFs and their cytoskeletal filaments were oriented along the direction of the silk film patterned groove axis. Collagen type V and proteoglycans (decorin and biglycan), important markers of corneal stromal tissue, were highly expressed with alignment. Understanding corneal stromal fibroblast responses to surface features on a protein-based biomaterial applicable in vivo for corneal repair potential suggests options to improve corneal tissue mimics. Further, the approaches provide fundamental biomaterial designs useful for bioengineering oriented tissue layers, an endemic feature in most biological tissue structures that lead to critical tissue functions.

  5. The levels and kinetics of oxygen tension detectable at the surface of human dermal fibroblast cultures.

    PubMed

    Tokuda, Y; Crane, S; Yamaguchi, Y; Zhou, L; Falanga, V

    2000-03-01

    Low oxygen tension has recently been shown to stimulate cell growth and clonal expansion, as well as synthesis and transcription of certain growth factors and extracellular matrix components. These results have been obtained by exposing cell cultures to a hypoxic environment. Using an oxygen probe, we have now studied how experimental conditions affect the oxygen tension detectable at the cell surface. Dissolved oxygen tension was directly related to the height of the medium above the cell surface (r = 0.8793, P = 0.021), but was constant when no cells were present in the flask (r = -0. 9732, P = 0.001). In both human dermal fibroblasts and NIH/3T3 cultures, oxygen tension decreased linearly as cell density increased (r = -0.835, P < 0.0001; r = -0.916, P < 0.0001, respectively). When human dermal fibroblasts were exposed to 2% O(2), maximum hypoxic levels (0 mmHg) were achieved within approximately 15 min, and the recovery time was within a similar time frame. The addition of rotenone, an inhibitor of cellular respiration, blocked this decrease in oxygen tension at the cell surface, suggesting that cellular consumption of oxygen is responsible for the decline. Finally, we examined the cell-surface oxygen tension in control and acutely wounded human skin equivalents (HSE), consisting of a keratinocyte layer over a type I collagen matrix containing fibroblasts. We found that oxygen tension dropped significantly (P < 0.0001) in acutely wounded areas of HSE as compared to unwounded areas of HSE and that this drop was prevented by the addition of mitomycin C. These results indicate that cell-surface oxygen tension is indirectly related to cell density, and that the amount of detectable oxygen at the cell surface is a function of cell density, the oxygen tension in the incubator, and increased cellular activity, as occurs after injury. Copyright 2000 Wiley-Liss, Inc.

  6. Detection of DNA Damage by Space Radiation in Human Fibroblasts Flown on the International Space Station

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; hide

    2017-01-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37 degrees Centigrade in space for 14 days before being fixed for analysis of DNA damages with the gamma-H2AX assay. The 3-dimensional gamma-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate gamma rays at 37 degrees Centigrade. Cells exposed to chronic gamma rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET (Linear Energy Transfer) protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

  7. Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station.

    PubMed

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2017-02-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37°C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37°C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space. Published by Elsevier Ltd.

  8. Detection of DNA damage by space radiation in human fibroblasts flown on the International Space Station

    NASA Astrophysics Data System (ADS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2017-02-01

    Although charged particles in space have been detected with radiation detectors on board spacecraft since the discovery of the Van Allen Belts, reports on the effects of direct exposure to space radiation in biological systems have been limited. Measurement of biological effects of space radiation is challenging due to the low dose and low dose rate nature of the radiation environment, and due to the difficulty in distinguishing the radiation effects from microgravity and other space environmental factors. In astronauts, only a few changes, such as increased chromosome aberrations in their lymphocytes and early onset of cataracts, are attributed primarily to their exposure to space radiation. In this study, cultured human fibroblasts were flown on the International Space Station (ISS). Cells were kept at 37 °C in space for 14 days before being fixed for analysis of DNA damage with the γ-H2AX assay. The 3-dimensional γ-H2AX foci were captured with a laser confocal microscope. Quantitative analysis revealed several foci that were larger and displayed a track pattern only in the Day 14 flight samples. To confirm that the foci data from the flight study was actually induced from space radiation exposure, cultured human fibroblasts were exposed to low dose rate γ rays at 37 °C. Cells exposed to chronic γ rays showed similar foci size distribution in comparison to the non-exposed controls. The cells were also exposed to low- and high-LET protons, and high-LET Fe ions on the ground. Our results suggest that in G1 human fibroblasts under the normal culture condition, only a small fraction of large size foci can be attributed to high-LET radiation in space.

  9. Skin fibroblasts from individuals hemizygous for the familial adenopolyposis susceptibility gene show delayed crisis in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, S.; Kazim, D.; Kraveka, J.

    Normal human fibroblast cells have not been reported to escape crisis--that is they die after about 24 doublings in culture. The authors have been studying the growth properties of skin fibroblast cells from persons in families with familial adenopolyposis of the colon (FAP). An individual hemizygous at the FAP locus will develop hyperplasia of the colonic epithelium followed by colonic polyps, both at an early age. Polyps themselves still retain a single functional FAP allele. A mutation or deletion in this allele in a polyp is hypothesized to lead to further loss of growth control; thus, a tumor is formed.more » They found that the in vitro life-span of skin fibroblast cells from FAP individuals and from some asymptomatic children were markedly extended when compared with normal individuals.« less

  10. Investigation of the phototoxic effect of ZnO nanorods on fibroblasts and melanoma human cells

    NASA Astrophysics Data System (ADS)

    Kishwar, S.; Siddique, M.; Israr-Qadir, M.; Nur, O.; Willander, M.; Öllinger, K.

    2014-11-01

    Photocytotoxic effects of as-grown and zinc oxide (ZnO) nanorods coated with 5-aminolevulinic acid (ALA) have been studied on human cells, i.e. melanoma and foreskin fibroblast, under dark and ultraviolet light exposures. Zinc oxide nanorods have been grown on the very sharp tip (diameter = 700 nm) of borosilicate glass pipettes and then were coated by the photosensitizer for targeted investigations inside human cells. The coated glass pipette’s tip with photosensitizer has been inserted inside the cells with the help of a micro-manipulator and irradiated through ultraviolet light (UVA), which reduces the membrane potential of the mitochondria leading to cell death. Cell viability loss has been detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay when exposed to the dissolved ZnO nanorods and the production of the reactive oxygen species (ROS) has been detected along with the enhanced cytotoxic effect under UVA irradiation. Additionally, the influence of the lipid soluble antioxidant vitamin E and water-soluble N-acetyl-cysteine toward the enhancement or reduction of the toxicity has been investigated. A comparative analysis of the toxic nature of ZnO nanorods has been drawn between normal human fibroblast and melanoma cells, which can be favorable for understanding the clinical setting for killing tumor cells.

  11. Calmodulin and calmodulin-binding proteins in cystic fibrosis and normal human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tallant, E.A.; Wallace, R.W.

    1986-05-01

    The authors have investigated the possibility that a lesion in a calmodulin (CaM)-dependent regulatory mechanism may be involved in cystic fibrosis (CF). The level of CaM, CaM-binding proteins (CaM-BP) and a CaM-dependent phosphatase (CaM-Ptase) have been compared in cultured fibroblasts from CF patients versus age- and sex-matched control subjects. The CaM concentration, measured by radioimmunoassay, ranged from 0.20 to 0.76 ..mu..g/mg protein (n=8); there was no significant difference in the average CaM concentration from CF patients vs controls. Using Western blotting techniques with /sup 125/I-CaM, they detected at least ten distinct CaM-BPs in fibroblasts with molecular weights ranging from 230Kmore » to 37K; the only consistent difference between control and CF cell lines was in a 46.5K CaM-BP, which was depressed in all three CF samples. The 46.5 K CaM-BP was found only in the particulate fraction. A 59K CaM-BP was identified as a CaM-Ptase by its crossreactivity with an antibody against a brain CaM-Ptase. There was no significant difference in CaM-Ptase activity or in the amount of the phosphatase as determined by radioimmunoassay in CF vs. normal samples (n=8). Thus, the level of CaM as well as its various enzymes and proteins do not appear to be altered in CF fibroblasts except for a CaM-BP of 46.5K, the identity of which is currently being investigated.« less

  12. 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

  13. Cytokinesis-block micronucleus assay in primary human liver fibroblasts exposed to griseofulvin and mitomycin C.

    PubMed

    Nesti, C; Trippi, F; Scarpato, R; Migliore, L; Turchi, G

    2000-03-01

    Primary liver fibroblasts were applied in a cytokinesis-block micronucleus assay in combination with fluorescence in situ hybridization (FISH) using two protocols. In protocol A (Prot. A), cytochalasin B (Cyt B) was added at the end of the treatment time directly to the medium containing the standard compounds, whereas in protocol B (Prot. B) the chemical-containing medium was removed and fresh medium with Cyt B was added. The study was performed using the aneugen griseofulvin (GF) and the clastogen mitomycin C (MMC) as standard compounds. With both protocols GF induced a significant increase in MN frequency over controls in a dose-related manner at the lower concentrations tested (7.5 and 15 microg/ml). At the highest dose (30 microg/ml) the aneugen effect was substantially reduced. MN induction obtained with Prot. A was significantly higher ( approximately 3-fold) than with Prot. B at the most effective concentration. The aneugen effect induced by GF did not change when different cell densities were used, but again with Prot. A we obtained the highest effect. MN induced by MMC showed a dose- and time-dependent increase in both protocols. In contrast to GF, the greater clastogenic response induced by MMC in human liver fibroblasts was obtained with Prot. B, approximately 3-fold higher than Prot. A at the most effective concentration and approximately 2-fold with 24 h treatment at 0.17 microg/ml MMC. With GF, the FISH data in human liver fibroblasts (80% C+MN) were fairly consistent with those obtained in the rodent cell lines. In human whole blood cultures, the same dose used in our experiment produced a relatively higher percentage of C+MN. FISH analysis showed that MMC induced mainly MN containing acentric fragments rather than whole chromosomes. In conclusion we have demostrated that chemically induced genetic effects are strongly dependent on the cell culture employed, treatment schedule and intra- and post-treatment experimental conditions.

  14. Epidermal regulation of dermal fibroblast activity.

    PubMed

    Garner, W L

    1998-07-01

    Although the association between delayed burn wound healing and subsequent hypertrophic scar formation is well-established, the mechanism for this relationship is unknown. Unhealed burn wounds lack an epidermis, suggesting a possible regulatory role for the epidermis in controlling dermal fibroblast matrix synthesis. Therefore, we examined the effect of epidermal cells and media conditioned by epidermal cells on fibroblast collagen synthesis and replication. Purified fibroblast and keratinocyte cell strains were developed from discarded normal adult human skin. Conditioned media were created by incubation of cytokine-free and serum-free medium with either confluent fibroblast or keratinocyte cultures for 18 hours (n = 3). Nearly confluent fibroblast cultures were exposed for 48 hours to graded concentrations of either unconditioned medium (control), conditioned medium, or varying numbers of keratinocytes. Replication was quantified by the incorporation of 3H-thymidine. Collagen synthesis was measured by the incorporation of 3H-proline into collagenase-sensitive protein. Data were compared using analysis of variance (ANOVA) and linear regression. Keratinocyte conditioned medium induced a significant increase in replication (n = 3) (p = 0.004) and a decrease in collagen synthesis (n = 6) (p < 0.001). In contrast, neither fibroblast conditioned medium nor control medium had an effect on fibroblast replication or collagen synthesis. Co-culture of fibroblast with a graded number of keratinocytes similarly decreased collagen synthesis (n = 6) (p < 0.001). Dermal fibroblast collagen synthesis appears to be regulated by a soluble keratinocyte product. This result suggests a mechanism for the clinical observation that unhealed burn wounds, which lack the epidermis, demonstrate excess collagen production and scar. Clinical strategies to decrease hypertrophic scar should include an attempt at early wound closure with skin grafting or the application of cultured epithelial

  15. Modulation of the Senescence-Associated Inflammatory Phenotype in Human Fibroblasts by Olive Phenols

    PubMed Central

    Menicacci, Beatrice; Cipriani, Caterina; Margheri, Francesca

    2017-01-01

    Senescent cells display an increase in the secretion of growth factors, inflammatory cytokines and proteolytic enzymes, termed the “senescence-associated-secretory-phenotype” (SASP), playing a major role in many age-related diseases. The phenolic compounds present in extra-virgin olive oil are inhibitors of oxidative damage and have been reported to play a protective role in inflammation-related diseases. Particularly, hydroxytyrosol and oleuropein are the most abundant and more extensively studied. Pre-senescent human lung (MRC5) and neonatal human dermal (NHDF) fibroblasts were used as cellular model to evaluate the effect of chronic (4–6 weeks) treatment with 1 μM hydroxytyrosol (HT) or 10 μM oleuropein aglycone (OLE) on senescence/inflammation markers. Both phenols were effective in reducing β-galactosidase-positive cell number and p16 protein expression. In addition, senescence/inflammation markers such as IL-6 and metalloprotease secretion, and Ciclooxigenase type 2 (COX-2) and α-smooth-actin levels were reduced by phenol treatments. In NHDF, COX-2 expression, Nuclear Factor κ-light-chain-enhancer of activated B cells (NFκB) protein level and nuclear localization were augmented with culture senescence and decreased by OLE and HT treatment. Furthermore, the inflammatory effect of Tumor Necrosis Factor α (TNFα) exposure was almost completely abolished in OLE- and HT-pre-treated NHDF. Thus, the modulation of the senescence-associated inflammatory phenotype might be an important mechanism underlying the beneficial effects of olive oil phenols. PMID:29084133

  16. Focal Adhesion Kinase Regulates Fibroblast Migration via Integrin beta-1 and Plays a Central Role in Fibrosis

    PubMed Central

    Zhao, Xue-Ke; Cheng, Yiju; Liang Cheng, Ming; Yu, Lei; Mu, Mao; Li, Hong; Liu, Yang; Zhang, Baofang; Yao, Yumei; Guo, Hui; Wang, Rong; Zhang, Quan

    2016-01-01

    Lung fibrosis is a major medical problem for the aging population worldwide. Fibroblast migration plays an important role in fibrosis. Focal Adhesion Kinase (FAK) senses the extracellular stimuli and initiates signaling cascades that promote cell migration. This study first examined the dose and time responses of FAK activation in human lung fibroblasts treated with platelet derived growth factor BB (PDGF-BB). The data indicate that FAK is directly recruited by integrin β1 and the subsequent FAK activation is required for fibroblast migration on fibronectin. In addition, the study has identified that α5β1 and α4β1 are the major integrins for FAK-mediated fibroblast migration on fibronect. In contrast, integrins αvβ3, αvβ6, and αvβ8 play a minor but distinct role in fibroblast migration on fibronectin. FAK inhibitor significantly reduces PDGF-BB stimulated fibroblast migration. Importantly, FAK inhibitor protects bleomycin-induced lung fibrosis in mice. FAK inhibitor blocks FAK activation and significantly reduces signaling cascade of fibroblast migration in bleomycin-challenged mice. Furthermore, FAK inhibitor decreases lung fibrotic score, collagen accumulation, fibronectin production, and myofibroblast differentiation in in bleomycin-challenged mice. These data demonstrate that FAK mediates fibroblast migration mainly via integrin β1. Furthermore, the findings suggest that targeting FAK signaling is an effective therapeutic strategy against fibrosis. PMID:26763945

  17. Fibroblastic connective tissue nevus.

    PubMed

    Velez, Moises J; Billings, Steven D; Weaver, Joshua A

    2016-01-01

    Fibroblastic connective tissue nevus (FCTN) is a newly recognized, benign cutaneous mesenchymal lesion of fibroblasts/myofibroblastic lineage, which expands the classification of connective tissue nevi. We present three cases of FCTN and discuss significant clinical, morphologic and immunophenotypic overlap with dermatomyofibroma. Our cases were from young women, aged 32, 24 and 10, and presented as 1.2 and 1 cm nodules on the posterior neck and right upper flank, respectively while presenting as a linear plaque of the right posterior thigh in the latter case. The lesions showed a poorly circumscribed proliferation of hypercellular spindle cells arranged in short to longer intersecting fascicles entrapping adnexal structures. Superficial adipose tissue was also entrapped in one case. The spindle cells had fibroblastic features with pale eosinophilic cytoplasmic extensions and inconspicuous nucleoli. The spindle cells were positive for CD34 in two cases. One case was negative for CD34, smooth muscle actin (SMA), desmin and S100. The overall features were consistent with a diagnosis of FCTN. In two cases, we further elucidated the fibroblastic differentiation of the spindle cells in FCTN with electron microscopy, which has not been previously described. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  19. In vitro cytotoxicity of white MTA, MTA Fillapex® and Portland cement on human periodontal ligament fibroblasts.

    PubMed

    Yoshino, Patrícia; Nishiyama, Celso Kenji; Modena, Karin Cristina da Silva; Santos, Carlos Ferreira; Sipert, Carla Renata

    2013-01-01

    The aim of this study was to compare the in vitro cytotoxicity of white mineral trioxide aggregate (MTA), MTA Fillapex® and Portland cement (PC) on human cultured periodontal ligament fibroblasts. Periodontal ligament fibroblast culture was established and the cells were used for cytotoxic tests after the fourth passage. Cell density was set at 1.25 X10 4 cells/well in 96-well plates. Endodontic material extracts were prepared by placing sealer/cement specimens (5x3mm) in 1mL of culture medium for 72 h. The extracts were then serially two-fold diluted and inserted into the cell-seeded wells for 24, 48 and 72 h. MTT assay was employed for analysis of cell viability. Cell supernatants were tested for nitric oxide using the Griess reagent system. MTA presented cytotoxic effect in undiluted extracts at 24 and 72 h. MTA Fillapex® presented the highest cytotoxic levels with important cell viability reduction for pure extracts and at ½ and ¼ dilutions. In this study, PC did not induce alterations in fibroblast viability. Nitric oxide was detected in extract-treated cell supernatants and also in the extracts only, suggesting presence of nitrite in the soluble content of the tested materials. In the present study, MTA Fillapex displayed the highest cytotoxic effect on periodontal ligament fibroblasts followed by white MTA and PC.

  20. p63 Silencing induces reprogramming of cardiac fibroblasts into cardiomyocyte-like cells.

    PubMed

    Patel, Vivekkumar; Singh, Vivek P; Pinnamaneni, Jaya Pratap; Sanagasetti, Deepthi; Olive, Jacqueline; Mathison, Megumi; Cooney, Austin; Flores, Elsa R; Crystal, Ronald G; Yang, Jianchang; Rosengart, Todd K

    2018-04-13

    Reprogramming of fibroblasts into induced cardiomyocytes represents a potential new therapy for heart failure. We hypothesized that inactivation of p63, a p53 gene family member, may help overcome human cell resistance to reprogramming. p63 Knockout ( -/- ) and knockdown murine embryonic fibroblasts (MEFs), p63 -/- adult murine cardiac fibroblasts, and human cardiac fibroblasts were assessed for cardiomyocyte-specific feature changes, with or without treatment by the cardiac transcription factors Hand2-Myocardin (HM). Flow cytometry revealed that a significantly greater number of p63 -/- MEFs expressed the cardiac-specific marker cardiac troponin T (cTnT) in culture compared with wild-type (WT) cells (38% ± 11% vs 0.9% ± 0.9%, P < .05). HM treatment of p63 -/- MEFs increased cTnT expression to 74% ± 3% of cells but did not induce cTnT expression in wild-type murine embryonic fibroblasts. shRNA-mediated p63 knockdown likewise yielded a 20-fold increase in cTnT microRNA expression compared with untreated MEFs. Adult murine cardiac fibroblasts demonstrated a 200-fold increase in cTnT gene expression after inducible p63 knockout and expressed sarcomeric α-actinin as well as cTnT. These p63 -/- adult cardiac fibroblasts exhibited calcium transients and electrically stimulated contractions when co-cultured with neonatal rat cardiomyocytes and treated with HM. Increased expression of cTnT and other marker genes was also observed in p63 knockdown human cardiac fibroblasts procured from patients undergoing procedures for heart failure. Downregulation of p63 facilitates direct cardiac cellular reprogramming and may help overcome the resistance of human cells to reprogramming. Copyright © 2018 The American Association for Thoracic Surgery. Published by Elsevier Inc. All rights reserved.

  1. Expression of cholesteryl glucoside by heat shock in human fibroblasts

    PubMed Central

    Kunimoto, Shohko; Kobayashi, Tetsuyuki; Kobayashi, Susumu; Murakami-Murofushi, Kimiko

    2000-01-01

    ABSTRACT We investigated the heat-induced alteration of glycolipids in human cultured cells, TIG-3 fibroblasts, to show the expression of steryl glucoside by heat shock. A glycolipid band was detected on a thin-layer chromatography plate in lipid extracts from TIG-3 cells exposed to high temperature (42°C) for 15 and 30 minutes, while it was hardly detectable without heat shock. Both cholesterol and glucose were almost exclusively detected by gas liquid chromatography as degradation products of the lipid. The structure of the lipid molecule was elucidated by electrospray mass spectrometry to be a cholesteryl glucoside. This is the first report to show the occurrence of a steryl glucoside in mammalian cells, and this substance is considered to have a significant role in heat shock responses in mammalian cells. PMID:10701833

  2. Evaluation of human embryonic stem cells and their differentiated fibroblastic progenies as cellular models for in vitro genotoxicity screening.

    PubMed

    Vinoth, Kumar Jayaseelan; Manikandan, Jayapal; Sethu, Swaminathan; Balakrishnan, Lakshmidevi; Heng, Alexis; Lu, Kai; Hande, Manoor Prakash; Cao, Tong

    2014-08-20

    This study evaluated human embryonic stem cells (hESC) and their differentiated fibroblastic progenies as cellular models for genotoxicity screening. The DNA damage response of hESCs and their differentiated fibroblastic progenies were compared to a fibroblastic cell line (HEPM, CRL1486) and primary cultures of peripheral blood lymphocytes (PBL), upon exposure to Mitomycin C, gamma irradiation and H2O2. It was demonstrated that hESC-derived fibroblastic progenies (H1F) displayed significantly higher chromosomal aberrations, micronuclei formation and double strand break (DSB) formation, as compared to undifferentiated hESC upon exposure to genotoxic stress. Nevertheless, H1F cell types displayed comparable sensitivities to genotoxic challenge as HEPM and PBL, both of which are representative of somatic cell types commonly used for genotoxicity screening. Subsequently, transcriptomic and pathways analysis identified differential expression of critical genes involved in cell death and DNA damage response upon exposure to gamma irradiation. The results thus demonstrate that hESC-derived fibroblastic progenies are as sensitive as commonly-used somatic cell types for genotoxicity screening. Moreover, hESCs have additional advantages, such as their genetic normality compared to immortalized cell lines, as well as their amenability to scale-up for producing large, standardized quantities of cells for genotoxicity screening on an industrial scale, something which can never be achieved with primary cell cultures. Copyright © 2014. Published by Elsevier B.V.

  3. Estrogens Induce Rapid Cytoskeleton Re-Organization in Human Dermal Fibroblasts via the Non-Classical Receptor GPR30

    PubMed Central

    Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

    2015-01-01

    The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation. PMID:25781607

  4. Estrogens induce rapid cytoskeleton re-organization in human dermal fibroblasts via the non-classical receptor GPR30.

    PubMed

    Carnesecchi, Julie; Malbouyres, Marilyne; de Mets, Richard; Balland, Martial; Beauchef, Gallic; Vié, Katell; Chamot, Christophe; Lionnet, Claire; Ruggiero, Florence; Vanacker, Jean-Marc

    2015-01-01

    The post-menopausal decrease in estrogen circulating levels results in rapid skin deterioration pointing out to a protective effect exerted by these hormones. The identity of the skin cell type responding to estrogens is unclear as are the cellular and molecular processes they elicit. Here, we reported that lack of estrogens induces rapid re-organization of the human dermal fibroblast cytoskeleton resulting in striking cell shape change. This morphological change was accompanied by a spatial re-organization of focal adhesion and a substantial reduction of their number as evidenced by vinculin and actin co-staining. Cell morphology and cytoskeleton organization was fully restored upon 17β-estradiol (E2) addition. Treatment with specific ER antagonists and cycloheximide respectively showed that the E2 acts independently of the classical Estrogen Receptors and that cell shape change is mediated by non-genomic mechanisms. E2 treatment resulted in a rapid and transient activation of ERK1/2 but not Src or PI3K. We show that human fibroblasts express the non-classical E2 receptor GPR30 and that its agonist G-1 phenocopies the effect of E2. Inhibiting GPR30 through treatment with the G-15 antagonist or specific shRNA impaired E2 effects. Altogether, our data reveal a novel mechanism by which estrogens act on skin fibroblast by regulating cell shape through the non-classical G protein-coupled receptor GPR30 and ERK1/2 activation.

  5. Induction and repair of radiation-induced DNA double-strand breaks in human fibroblasts are not affected by terminal differentiation.

    PubMed

    Brammer, Ingo; Herskind, Carsten; Haase, Oliver; Rodemann, H Peter; Dikomey, Ekkehard

    2004-02-03

    It was studied for human skin fibroblasts, whether the induction or repair of DNA double-strand breaks (dsb) depend on the differentiation status. These studies were performed (a) with a fibroblast strain (HSF1) kept in progenitor state (mitotic fibroblasts, MF) or triggered to premature terminal differentiation (postmitotic fibrocytes, PMF) by exposure to mitomycin C or (b) with 20 fibroblast strains differing intrinsically in their differentiation status. The differentiation status was quantified by determining the fraction of postmitotic fibrocytes by light microscopy. DNA dsb were measured by constant-field gel electrophoresis, and the fraction of apoptotic cells by comet assay. MF and PMF cultures of HSF1 cells were irradiated with X-ray doses up to 160 Gy, and dsb were measured either immediately after irradiation or after a repair incubation of 4 or 24 h. There were a difference neither in the number of initial nor residual dsb. PMF cultures, however, showed a slightly higher number of dsb already present in non-irradiated cells, which was measured to result from a small fraction of 5% apoptotic cells. The 20 analysed fibroblast strains showed a substantial variation in the fraction of postmitotic fibrocytes (9-51%) as well as in the number of dsb remaining at 24 h after irradiation (1.9-4.9%), but there was no correlation between these two parameters. These data demonstrate that for fibroblasts the terminal differentiation has an effect neither on the induction nor the repair of radiation-induced dsb. This result indicates that the variation in dsb-repair capacity previously observed for fibroblast strains and which was considered to be the main cause for the variation in the cellular radiosensitivity, cannot be ascribed to differences in the differentiation status.

  6. Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

    PubMed

    Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

    1990-01-01

    We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

  7. Inhibition by rebamipide of cytokine-induced or lipopolysaccharide-induced chemokine synthesis in human corneal fibroblasts.

    PubMed

    Fukuda, Ken; Ishida, Waka; Tanaka, Hiroshi; Harada, Yosuke; Fukushima, Atsuki

    2014-12-01

    The dry-eye drug rebamipide has mucin secretagogue activity in and anti-inflammatory effects on corneal epithelial cells. Corneal stromal fibroblasts (transdifferentiated keratocytes) function as immune modulators in the pathogenesis of chronic ocular allergic inflammation and in innate immune responses at the ocular surface. The possible anti-inflammatory effects of rebamipide on human corneal stromal fibroblasts were examined. Serum-deprived cells were incubated for 1 h with rebamipide and then for various times in the additional absence or presence of cytokines or bacterial lipopolysaccharide (LPS). The release of chemokines into culture supernatants was determined with ELISAs. The intracellular abundance of chemokine mRNAs was quantitated by reverse transcription and real-time PCR analysis. Degradation of the nuclear factor κB (NFκB) inhibitor IκBα was detected by immunoblot analysis. Rebamipide suppressed the release of interleukin (IL)-8 and the upregulation of IL-8 mRNA induced by tumour necrosis factor α (TNF-α) or LPS in corneal fibroblasts. It also inhibited eotaxin-1 (CCL-11) expression at the protein and mRNA levels induced by the combination of TNF-α and IL-4. In addition, rebamipide attenuated the degradation of IκBα induced by TNF-α or LPS. Rebamipide inhibited the synthesis of chemokines by corneal fibroblasts in association with suppression of NFκB signalling. Rebamipide may therefore prove effective for the treatment of corneal stromal inflammation associated with allergy or bacterial infection. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

  8. Halomethane-induced cytotoxicity and cell proliferation in human lung MRC-5 fibroblasts and NL20-TA epithelial cells.

    PubMed

    Nájera-Martínez, Minerva; García-Latorre, Ethel A; Reyes-Maldonado, Elba; Domínguez-López, M Lilia; Vega-López, Armando

    2012-09-01

    Halomethanes (HMs) can be formed during the chlorination process to obtain drinking water. In liver cells, HMs had been shown to be mutagenic and carcinogenic; however, their bioactivation by CYP 2E1 and GSTT1 is required. Although inhalation is the most common pathway of exposure, reports on the toxic effects induced by HMs in human lung are contradictory. The aim of this study was therefore to evaluate in vitro cytotoxicity and cell proliferation induced by CH(2)Cl(2), CHCl(3) and BrCHCl(2) in human lung NL20-TA epithelial cells and MRC-5 fibroblasts, and their relationship with CYP 2E1 and GSTT1 activity. High concentrations of these HMs induced cytotoxicity, particularly in cells treated with BrCHCl(2). Low concentrations of BrCHCl(2) stimulated hyperproliferation of fibroblasts, the most probable consequence of which is regenerative proliferation related to collagen induction. Fibroblasts exposed to BrCHCl(2) exhibited low levels of CYP 2E1 activity suggesting that released bromine is able to alter this activity by affecting the active site or auto regulating the activity itself. GSTT1 was up to ten times more active than CYP 2E1 in both cell lines, indicating that potential lung damage is due to formation of pro-carcinogens such as formaldehyde.

  9. Rapid fibroblast removal from high density human embryonic stem cell cultures.

    PubMed

    Turner, William S; McCloskey, Kara E

    2012-10-28

    Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation(1). This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity(4). Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types(5-8)). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

  10. 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.

  11. 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

  12. Micronuclei Induction in Human Fibroblasts Exposed In Vitro to Los Alamos High-Energy Neutrons

    NASA Technical Reports Server (NTRS)

    Gersey, Brad; Sodolak, John; Hada, Megumi; Saganti, Prem; Wilkins, Richard; Cucinotta, Francis; Wu, Honglu

    2006-01-01

    High-energy secondary neutrons, produced by the interaction of galactic cosmic rays with the atmosphere, spacecraft structure and planetary surfaces, contribute to a significant fraction to the dose equivalent in crew members and passengers during commercial aviation travel, and astronauts in space missions. The Los Alamos Nuclear Science Center (LANSCE) neutron facility#s ICE House 30L beamline is known to generate neutrons that simulate the secondary neutron spectra of earth#s atmosphere. The neutron spectrum is also similar to that measured onboard spacecraft like the MIR and International Space Station (ISS). To evaluate the biological damage, we exposed human fibroblasts in vitro to the LANSCE neutron beams without degrader at an entrance dose rate of 25 mGy/hr and analyzed the micronuclei (MN) induction. The cells were also placed behind a 9.9 cm water column to study effect of shielding in the protection of neutron induced damages. It was found that the dose response in the MN frequency was linear for the samples with and without shielding and the slope of the MN yield behind the shielding was reduced by a factor of 3.5. Compared to the MN induction in human fibroblasts exposed to a gamma source at a low dose rate, the RBE was found to be 16.7 and 10.0 for the neutrons without and with 9.9 cm water shielding, respectively.

  13. Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

    PubMed

    Kitakaze, Keisuke; Tasaki, Chikako; Tajima, Youichi; Hirokawa, Takatsugu; Tsuji, Daisuke; Sakuraba, Hitoshi; Itoh, Kohji

    2016-09-01

    GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA , HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.

  14. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Han, Yanfu; Chai, Jiake, E-mail: cjk304@126.com; Sun, Tianjun

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. Inmore » this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  15. Anti-inflammatory effects of zinc in PMA-treated human gingival fibroblast cells

    PubMed Central

    Kim, Sangwoo; Jeon, Sangmi; Hui, Zheng; Kim, Young; Im, Yeonggwan; Lim, Wonbong; Kim, Changsu; Choi, Hongran; Kim, Okjoon

    2015-01-01

    Objectives: Abnormal cellular immune response has been considered to be responsible for oral lesions in recurrent aphthous stomatitis. Zinc has been known to be an essential nutrient metal that is necessary for a broad range of biological activities including antioxidant, immune mediator, and anti-inflammatory drugs in oral mucosal disease. The objective of this study was to investigate the effects of zinc in a phorbol-12-myristate-13-acetate (PMA)-treated inflammatory model on human gingival fibroblast cells (hGFs). Study Design: Cells were pre-treated with zinc chloride, followed by PMA in hGFs. The effects were assessed on cell viability, cyclooxygenease-1,2(COX-1/2) protein expression, PGE2 release, ROS production and cytokine release, Results: The effects were assessed on cell viability, COX1/2 protein expression, PGE2 release, ROS production, cytokine release. The results showed that, in the presence of PMA, zinc treatment leads to reduce the production of ROS, which results in decrease of COX-2 expression and PGE2 release. Conclusions: Thus, we suggest that zinc treatment leads to the mitigation of oral inflammation and may prove to be an alternative treatment for recurrent aphthous stomatitis. Key words:Zinc, inflammatory response, cytokines, phorbol-12-myristate-13-acetate, gingival fibroblasts cells. PMID:25662537

  16. 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.

  17. Age-dependent oxidation of extracellular cysteine/cystine redox state (Eh(Cys/CySS)) in mouse lung fibroblasts is mediated by a decline in Slc7a11 expression.

    PubMed

    Zheng, Yuxuan; Ritzenthaler, Jeffrey D; Burke, Tom J; Otero, Javier; Roman, Jesse; Watson, Walter H

    2018-04-01

    Aging is associated with progressive oxidation of the extracellular environment. The redox state of human plasma, defined by the concentrations of cysteine (Cys) and cystine (CySS), becomes more oxidized as we age. Recently, we showed that fibroblasts isolated from the lungs of young and old mice retain this differential phenotype; old cells produce and maintain a more oxidizing extracellular redox potential (E h (Cys/CySS)) than young cells. Microarray analysis identified down-regulation of Slc7a11, the light subunit of the CySS/glutamate transporter, as a potential mediator of age-related oxidation in these cells. The purpose of the present study was to investigate the mechanistic link between Slc7a11 expression and extracellular E h (Cys/CySS). Sulforaphane treatment or overexpression of Slc7a11 was used to increase Slc7a11 in lung fibroblasts from old mice, and sulfasalazine treatment or siRNA-mediated knock down was used to decrease Slc7a11 in young fibroblasts. Slc7a11 mRNA levels were measured by real-time PCR, Slc7a11 activity was determined by measuring the rate of glutamate release, Cys, CySS, glutathione (GSH) and its disulfide (GSSG) were measured by HPLC, and E h (Cys/CySS) was calculated from the Nernst equation. The results showed that both E h (Cys/CySS) and E h (GSH/GSSG) were more oxidized in the conditioned media of old cells than in young cells. Up-regulation of Slc7a11 via overexpression or sulforaphane treatment restored extracellular E h (Cys/CySS) in cultures of old cells, whereas down-regulation reproduced the oxidizing E h (Cys/CySS) in young cells. Only sulforaphane treatment was able to increase total GSH and restore E h (GSH/GSSG), whereas overexpression, knock down and sulfasalazine had no effect on these parameters. In addition, inhibition of GSH synthesis with buthionine sulfoximine had no effect on the ability of cells to restore their extracellular redox potential in response to an oxidative challenge. In conclusion, our study

  18. Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei and Prometaphase Rosettes

    PubMed Central

    Bolzer, Andreas; Kreth, Gregor; Solovei, Irina; Koehler, Daniela; Saracoglu, Kaan; Fauth, Christine; Müller, Stefan; Eils, Roland; Cremer, Christoph; Speicher, Michael R

    2005-01-01

    Studies of higher-order chromatin arrangements are an essential part of ongoing attempts to explore changes in epigenome structure and their functional implications during development and cell differentiation. However, the extent and cell-type-specificity of three-dimensional (3D) chromosome arrangements has remained controversial. In order to overcome technical limitations of previous studies, we have developed tools that allow the quantitative 3D positional mapping of all chromosomes simultaneously. We present unequivocal evidence for a probabilistic 3D order of prometaphase chromosomes, as well as of chromosome territories (CTs) in nuclei of quiescent (G0) and cycling (early S-phase) human diploid fibroblasts (46, XY). Radial distance measurements showed a probabilistic, highly nonrandom correlation with chromosome size: small chromosomes—independently of their gene density—were distributed significantly closer to the center of the nucleus or prometaphase rosette, while large chromosomes were located closer to the nuclear or rosette rim. This arrangement was independently confirmed in both human fibroblast and amniotic fluid cell nuclei. Notably, these cell types exhibit flat-ellipsoidal cell nuclei, in contrast to the spherical nuclei of lymphocytes and several other human cell types, for which we and others previously demonstrated gene-density-correlated radial 3D CT arrangements. Modeling of 3D CT arrangements suggests that cell-type-specific differences in radial CT arrangements are not solely due to geometrical constraints that result from nuclear shape differences. We also found gene-density-correlated arrangements of higher-order chromatin shared by all human cell types studied so far. Chromatin domains, which are gene-poor, form a layer beneath the nuclear envelope, while gene-dense chromatin is enriched in the nuclear interior. We discuss the possible functional implications of this finding. PMID:15839726

  19. Senescent fibroblasts enhance early skin carcinogenic events via a paracrine MMP-PAR-1 axis.

    PubMed

    Malaquin, Nicolas; Vercamer, Chantal; Bouali, Fatima; Martien, Sébastien; Deruy, Emeric; Wernert, Nicolas; Chwastyniak, Maggy; Pinet, Florence; Abbadie, Corinne; Pourtier, Albin

    2013-01-01

    The incidence of carcinoma increases greatly with aging, but the cellular and molecular mechanisms underlying this correlation are only partly known. It is established that senescent fibroblasts promote the malignant progression of already-transformed cells through secretion of inflammatory mediators. We investigated here whether the senescent fibroblast secretome might have an impact on the very first stages of carcinogenesis. We chose the cultured normal primary human epidermal keratinocyte model, because after these cells reach the senescence plateau, cells with transformed and tumorigenic properties systematically and spontaneously emerge from the plateau. In the presence of medium conditioned by autologous senescent dermal fibroblasts, a higher frequency of post-senescence emergence was observed and the post-senescence emergent cells showed enhanced migratory properties and a more marked epithelial-mesenchymal transition. Using pharmacological inhibitors, siRNAs, and blocking antibodies, we demonstrated that the MMP-1 and MMP-2 matrix metalloproteinases, known to participate in late stages of cancer invasion and metastasis, are responsible for this enhancement of early migratory capacity. We present evidence that MMPs act by activating the protease-activated receptor 1 (PAR-1), whose expression is specifically increased in post-senescence emergent keratinocytes. The physiopathological relevance of these results was tested by analyzing MMP activity and PAR-1 expression in skin sections. Both were higher in skin sections from aged subjects than in ones from young subjects. Altogether, our results suggest that during aging, the dermal and epidermal skin compartments might be activated coordinately for initiation of skin carcinoma, via a paracrine axis in which MMPs secreted by senescent fibroblasts promote very early epithelial-mesenchymal transition of keratinocytes undergoing transformation and oversynthesizing the MMP-activatable receptor PAR-1.

  20. Perilla frutescens leaves extract ameliorates ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin.

    PubMed

    Bae, Jung-Soo; Han, Mira; Shin, Hee Soon; Kim, Min-Kyoung; Shin, Chang-Yup; Lee, Dong Hun; Chung, Jin Ho

    2017-01-04

    Perilla frutescens (L.) Britt. (Lamiaceae) is a traditional herb that is consumed in East Asian countries as a traditional medicine. This traditional herb has been documented for centuries to treat various diseases such as depression, allergies, inflammation and asthma. However, the effect of Perilla frutescens on skin has not been characterized well. The present study aimed to investigate the effect of Perilla frutescens leaves extract (PLE) on ultraviolet radiation-induced extracellular matrix damage in human dermal fibroblasts and hairless mice skin. Human dermal fibroblasts and Skh-1 hairless mice were irradiated with UV and treated with PLE. Protein and mRNA levels of various target molecules were analyzed by western blotting and quantitative RT-PCR, respectively. Histological changes of mouse skin were analyzed by H&E staining. To elucidate underlying mechanism of PLE, activator protein-1 (AP-1) DNA binding assay and the measurement of reactive oxygen species (ROS) were performed. PLE significantly inhibited basal and UV-induced MMP-1 and MMP-3 expression dose-dependently, and also decreased UV-induced phosphorylation of extracellular signal-regulated kinases and c-Jun N-terminal kinases. This inhibitory effects of PLE on MMP-1 and MMP-3 were mediated by reduction of ROS generation and AP-1 DNA binding activity induced by UV. Furthermore, PLE promoted type I procollagen production irrespective of UV irradiation. In the UV-irradiated animal model, PLE significantly reduced epidermal skin thickness and MMP-13 expression induced by UV. Our results demonstrate that PLE has the protective effect against UV-induced dermal matrix damage. Therefore, we suggest that PLE can be a potential agent for prevention of skin aging. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Knock-in fibroblasts and transgenic blastocysts for expression of human FGF2 in the bovine β-casein gene locus using CRISPR/Cas9 nuclease-mediated homologous recombination.

    PubMed

    Jeong, Young-Hee; Kim, Yeong Ji; Kim, Eun Young; Kim, Se Eun; Kim, Jiwoo; Park, Min Jee; Lee, Hong-Gu; Park, Se Pill; Kang, Man-Jong

    2016-06-01

    Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland, and this approach is one of the most important methods for agricultural and biomedical applications. However, expression and secretion of a protein varies because transgenes are integrated at random sites in the genome. In addition, distal enhancers are very important for transcriptional gene regulation and tissue-specific gene expression. Development of a vector system regulated accurately in the genome is needed to improve production of therapeutic proteins. The objective of this study was to develop a knock-in system for expression of human fibroblast growth factor 2 (FGF2) in the bovine β-casein gene locus. The F2A sequence was fused to the human FGF2 gene and inserted into exon 3 of the β-casein gene. We detected expression of human FGF2 mRNA in the HC11 mouse mammary epithelial cells by RT-PCR and human FGF2 protein in the culture media using western blot analysis when the knock-in vector was introduced. We transfected the knock-in vector into bovine ear fibroblasts and produced knock-in fibroblasts using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Moreover, the CRISPR/Cas9 system was more efficient than conventional methods. In addition, we produced knock-in blastocysts by somatic cell nuclear transfer using the knock-in fibroblasts. Our knock-in fibroblasts may help to create cloned embryos for development of transgenic dairy cattle expressing human FGF2 protein in the mammary gland via the expression system of the bovine β-casein gene.

  2. Merkel Cell Polyomavirus Infection of Animal Dermal Fibroblasts.

    PubMed

    Liu, Wei; Krump, Nathan A; MacDonald, Margo; You, Jianxin

    2018-02-15

    Merkel cell polyomavirus (MCPyV) is the first polyomavirus to be associated with human cancer. Mechanistic studies attempting to fully elucidate MCPyV's oncogenic mechanisms have been hampered by the lack of animal models for MCPyV infection. In this study, we examined the ability of MCPyV-GFP pseudovirus (containing a green fluorescent protein [GFP] reporter construct), MCPyV recombinant virions, and several MCPyV chimeric viruses to infect dermal fibroblasts isolated from various model animals, including mouse ( Mus musculus ), rabbit ( Oryctolagus cuniculus ), rat ( Rattus norvegicus ), chimpanzee ( Pan troglodytes ), rhesus macaque ( Macaca mulatta ), patas monkey ( Erythrocebus patas ), common woolly monkey ( Lagothrix lagotricha ), red-chested mustached tamarin ( Saguinus labiatus ), and tree shrew ( Tupaia belangeri ). We found that MCPyV-GFP pseudovirus was able to enter the dermal fibroblasts of all species tested. Chimpanzee dermal fibroblasts were the only type that supported vigorous MCPyV gene expression and viral replication, and they did so to a level beyond that of human dermal fibroblasts. We further demonstrated that both human and chimpanzee dermal fibroblasts produce infectious MCPyV virions that can successfully infect new cells. In addition, rat dermal fibroblasts supported robust MCPyV large T antigen expression after infection with an MCPyV chimeric virus in which the entire enhancer region of the MCPyV early promoter has been replaced with the simian virus 40 (SV40) analog. Our results suggest that viral transcription and/or replication events represent the major hurdle for MCPyV cross-species transmission. The capacity of rat dermal fibroblasts to support MCPyV early gene expression suggests that the rat is a candidate model organism for studying viral oncogene function during Merkel cell carcinoma (MCC) oncogenic progression. IMPORTANCE MCPyV plays an important role in the development of a highly aggressive form of skin cancer, Merkel

  3. Methylparaben-induced decrease in collagen production and viability of cultured human dermal fibroblasts.

    PubMed

    Majewska, Natalia; Zaręba, Ilona; Surażyński, Arkadiusz; Galicka, Anna

    2017-09-01

    Parabens owing to their many advantageous properties are widely applied in cosmetics, food products and pharmaceuticals. However, recent research results have shown that they possess the ability to accumulate in the human body and exert many adverse effects. In this study, the impact of methylparaben (MP) as the most frequently used preservative in cosmetics, on human dermal fibroblasts and collagen production was evaluated. In cells treated with 0.01, 0.03 and 0.05% MP a dose-dependent decrease in collagen biosynthesis was revealed, which was positively correlated with the activity of prolidase responsible for the recovery of proline. Consequently, the concentration of total collagen secreted into the medium was markedly diminished. A similar reduction in expression of the major skin collagen type I at both the protein and mRNA level as well as collagen type III and VI at the mRNA level was also detected. The decrease in the collagen level may result not only from the reduced synthesis but also increased degradation owing to MP-induced activation of pro-MMP-2 (72 kDa). The increase in activity of MMP-2 (66 kDa) was accompanied by a reduction in the inhibitory activity of TIMP-2. In addition, an inhibitory effect of MP on cell survival and proliferation was revealed in this study. The increased expression and nuclear translocation of caspase-3 as well as increased Bax and decreased Bcl-2 expression may suggest MP-induced cell apoptosis. In summary, we have provided new data on the adverse effects of methylparaben on human dermal fibroblasts and the main structural protein of the skin. Further studies on the mechanisms responsible for its action are in progress. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Nonmuscle myosin IIA and IIB differentially contribute to intrinsic and directed migration of human embryonic lung fibroblasts.

    PubMed

    Kuragano, Masahiro; Murakami, Yota; Takahashi, Masayuki

    2018-03-25

    Nonmuscle myosin II (NMII) plays an essential role in directional cell migration. In this study, we investigated the roles of NMII isoforms (NMIIA and NMIIB) in the migration of human embryonic lung fibroblasts, which exhibit directionally persistent migration in an intrinsic manner. NMIIA-knockdown (KD) cells migrated unsteadily, but their direction of migration was approximately maintained. By contrast, NMIIB-KD cells occasionally reversed their direction of migration. Lamellipodium-like protrusions formed in the posterior region of NMIIB-KD cells prior to reversal of the migration direction. Moreover, NMIIB KD led to elongation of the posterior region in migrating cells, probably due to the lack of load-bearing stress fibers in this area. These results suggest that NMIIA plays a role in steering migration by maintaining stable protrusions in the anterior region, whereas NMIIB plays a role in maintenance of front-rear polarity by preventing aberrant protrusion formation in the posterior region. These distinct functions of NMIIA and NMIIB might promote intrinsic and directed migration of normal human fibroblasts. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts.

    PubMed

    Newman, Donna R; Sills, W Shane; Hanrahan, Katherine; Ziegler, Amanda; Tidd, Kathleen McGinnis; Cook, Elizabeth; Sannes, Philip L

    2016-02-01

    The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF. © 2016 The Histochemical Society.

  6. Photobiomodulation of distinct lineages of human dermal fibroblasts: a rational approach towards the selection of effective light parameters for skin rejuvenation and wound healing

    NASA Astrophysics Data System (ADS)

    Mignon, Charles; Uzunbajakava, Natallia E.; Raafs, Bianca; Moolenaar, Mitchel; Botchkareva, Natalia V.; Tobin, Desmond J.

    2016-03-01

    Distinct lineages of human dermal fibroblasts play complementary roles in skin rejuvenation and wound healing, which makes them a target of phototherapy. However, knowledge about differential responses of specific cell lineages to different light parameters and moreover the actual molecular targets remain to be unravelled. The goal of this study was to investigate the impact of a range of parameters of light on the metabolic activity, collagen production, and cell migration of distinct lineages of human dermal fibroblasts. A rational approach was used to identify parameters with high therapeutic potential. Fibroblasts exhibited both inhibitory and cytotoxic change when exposed to a high dose of blue and cyan light in tissue culture medium containing photo-reactive species, but were stimulated by high dose red and near infrared light. Cytotoxic effects were eliminated by refreshing the medium after light exposure by removing potential ROS formed by extracellular photo-reactive species. Importantly, distinct lineages of fibroblasts demonstrated opposite responses to low dose blue light treatment when refreshing the medium after exposure. Low dose blue light treatment also significantly increased collagen production by papillary fibroblasts; high dose significantly retarded closure of the scratch wound without signs of cytotoxicity, and this is likely to have involved effects on both cell migration and proliferation. We recommend careful selection of fibroblast subpopulations and their culture conditions, a systematic approach in choosing and translating treatment parameters, and pursuit of fundamental research on identification of photoreceptors and triggered molecular pathways, while seeking effective parameters to address different stages of skin rejuvenation and wound healing.

  7. Postmitotic human dermal fibroblasts preserve intact feeder properties for epithelial cell growth after long-term cryopreservation.

    PubMed

    Limat, A; Hunziker, T; Boillat, C; Noser, F; Wiesmann, U

    1990-07-01

    In vitro, human dermal fibroblasts (HDF) differentiate through morphologically and biochemically identified compartments. In the course of this spontaneous differentiation through mitotic and postmitotic states, a tremendous increase in cellular and nuclear size occurs. Induction of postmitotic states can be accelerated by chemical (e.g., mitomycin C) or physical (e.g., x-ray) treatments. Such experimentally induced postmitotic HDF cells support very efficiently the growth of cutaneous epithelial cells, i.e. interfollicular keratinocytes and follicular outer root sheath cells, especially in primary cultures starting from very low cell seeding densities. The HDF feeder system provides more fundamental and also practical advantages, i.e. use of initially diploid human fibroblasts from known anatomic locations, easy handling and excellent reproducibility, and the possibility of long-term storage by incubation at 37 degrees C. Conditions for the cryogenic storage of postmitotic HDF cells in liquid nitrogen are presented and related to the feeder capacity for epithelial cell growth. Because postmitotic HDF cells preserve intact feeder properties after long-term storage, the immediate availability of feeder cells and the possibility to repeat experiments with identical materials further substantiate the usefulness of this feeder system.

  8. Royal jelly protects against ultraviolet B-induced photoaging in human skin fibroblasts via enhancing collagen production.

    PubMed

    Park, Hye Min; Hwang, Eunson; Lee, Kwang Gill; Han, Sang-Mi; Cho, Yunhi; Kim, Sun Yeou

    2011-09-01

    Royal jelly (RJ) is a honeybee product containing proteins, carbohydrates, fats, free amino acids, vitamins, and minerals. As its principal unsaturated fatty acid, RJ contains 10-hydroxy-2-decenoic acid (10-HDA), which may have antitumor and antibacterial activity and a capacity to stimulate collagen production. RJ has attracted interest in various parts of the world for its pharmacological properties. However, the effects of RJ on ultraviolet (UV)-induced photoaging of the skin have not been reported. In this study we measured the 10-HDA content of RJ by high-performance liquid chromatography and tested the effects of RJ on UVB-induced skin photoaging in normal human dermal fibroblasts. The effects of RJ and 10-HDA on UVB-induced photoaging were tested by measuring procollagen type I, transforming growth factor (TGF)-β1, and matrix metalloproteinase (MMP)-1 after UVB irradiation. The RJ contained about 0.211% 10-HDA. The UVB-irradiated human skin fibroblasts treated with RJ and 10-HDA had increased procollagen type I and TGF-β1 productions, but the level of MMP-1 was not changed. Thus RJ may potentially protect the skin from UVB-induced photoaging by enhancing collagen production.

  9. Protective effect of crocin on ultraviolet B‑induced dermal fibroblast photoaging.

    PubMed

    Deng, Mingwu; Li, Dong; Zhang, Yichen; Zhou, Guangdong; Liu, Wei; Cao, Yilin; Zhang, Wenjie

    2018-06-11

    Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS), resulting in the aging of dermal fibroblasts. Crocin, a bioactive constituent of Crocus sativus, possesses anti‑oxidation effects. The purpose of the present study was to evaluate the protective effect of crocin on UVB‑induced dermal fibroblast photoaging. Human dermal fibroblasts were isolated and cultured with different concentrations of crocin prior to and following exposure to UVB irradiation. The senescent phenotypes of cells were evaluated, including cell proliferation, cell cycle, senescence‑associated β‑galactosidase (SA‑β‑gal) expression, intracellular ROS, expression of antioxidant protein glutathione peroxidase 1 (GPX‑1) and extracellular matrix protein collagen type 1 (Col‑1). Crocin rescued the cell proliferation inhibited by UVB irradiation, prevented cell cycle arrest and markedly decreased the number of SA‑β‑gal‑positive cells. In addition, crocin reduced UVB‑induced ROS by increasing GPX‑1 expression and other direct neutralization effects. Furthermore, crocin promoted the expression of the extracellular matrix protein Col‑1. Crocin could effectively prevent UVB‑induced cell damage via the reduction of intracellular ROS; thus, it could potentially be used in the prevention of skin photoaging.

  10. Random integration of SV40 in SV40-transformed, immortalized human fibroblasts.

    PubMed

    Hara, H; Kaji, H

    1987-02-01

    We have studied the relationship between immortalization of SV40-transformed human embryonic fibroblasts and their SV40 integration sites. From several independently transformed cell pools, we have isolated clones which do not harbor unintegrated SV40 DNA. We have analysed whole-cell DNA from these clones, using the Southern blot method. Our results suggest that no specific integration sites in the cellular genome exist which are a prerequisite for the immortalization process. Although some integration sites were found to be predominant in pre-crisis clones, they could not be detected in the post-crisis clones. This suggests that none of these predominating sites is selected for during the crisis period.

  11. Resident fibroblasts in the kidney: a major driver of fibrosis and inflammation.

    PubMed

    Sato, Yuki; Yanagita, Motoko

    2017-01-01

    Chronic kidney disease (CKD) is a leading cause of end stage renal disease (ESRD) and cardiovascular morbidity and mortality worldwide, resulting in a growing social and economic burden. The prevalence and burden of CKD is anticipated to further increase over the next decades as a result of aging. In the pathogenesis of CKD, irrespective of the etiology, resident fibroblasts are key players and have been demonstrated to play crucial roles for disease initiation and progression. In response to injury, resident fibroblasts transdifferentiate into myofibroblasts that express alpha smooth muscle actin (αSMA) and have an increased capacity to produce large amounts of extracellular matrix (ECM) proteins, leading to renal fibrosis. In addition to this fundamental role of fibroblasts as drivers for renal fibrosis, growing amounts of evidence have shown that resident fibroblasts are also actively involved in initiating and promoting inflammation during kidney injury. During the myofibroblastic transition described above, resident fibroblasts activate NF-κB signaling and produce pro-inflammatory cytokines and chemokines, promoting inflammation. Furthermore, under aging milieu, resident fibroblasts transdifferentiate into several distinct phenotypic fibroblasts, including CXCL13/CCL19-producing fibroblasts, retinoic acid-producing fibroblasts, and follicular dendritic cells, in response to injury and orchestrate tertiary lymphoid tissue (TLT) formation, which results in uncontrolled aberrant inflammation and retards tissue repair. Anti-inflammatory agents can improve myofibroblastic transdifferentiation and abolish TLT formation, suggesting that targeting these inflammatory fibroblasts can potentially ameliorate kidney disease. Beyond its conventional role as an executor of fibrosis, resident fibroblasts display more pro-inflammatory phenotypes and contribute actively to driving inflammation during kidney injury.

  12. Short and prolonged exposure to hyperglycaemia in human fibroblasts and endothelial cells: metabolic and osmotic effects.

    PubMed

    Moruzzi, Noah; Del Sole, Marianna; Fato, Romana; Gerdes, Jantje M; Berggren, Per-Olof; Bergamini, Christian; Brismar, Kerstin

    2014-08-01

    High blood glucose levels are the main feature of diabetes. However, the underlying mechanism linking high glucose concentration to diabetic complications is still not fully elucidated, particularly with regard to human physiology. Excess of glucose is likely to trigger a metabolic response depending on the cell features, activating deleterious pathways involved in the complications of diabetes. In this study, we aim to elucidate how acute and prolonged hyperglycaemia alters the biology and metabolism in human fibroblasts and endothelial cells. We found that hyperglycaemia triggers a metabolic switch from oxidative phosphorylation to glycolysis that is maintained over prolonged time. Moreover, osmotic pressure is a major factor in the early metabolic response, decreasing both mitochondrial transmembrane potential and cellular proliferation. After prolonged exposure to hyperglycaemia we observed decreased mitochondrial steady-state and uncoupled respiration, together with a reduced ATP/ADP ratio. At the same time, we could not detect major changes in mitochondrial transmembrane potential and reactive oxygen species. We suggest that the physiological and metabolic alterations observed in healthy human primary fibroblasts and endothelial cells are an adaptive response to hyperglycaemia. The severity of metabolic and bioenergetics impairment associated with diabetic complications may occur after longer glucose exposure or due to interactions with cell types more sensitive to hyperglycaemia. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. 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 (IC 50 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. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Transgene-free human induced pluripotent stem cell line (HS5-SV.hiPS) generated from cesarean scar-derived fibroblasts.

    PubMed

    Rungsiwiwut, Ruttachuk; Pavarajarn, Wipawee; Numchaisrika, Pranee; Virutamasen, Pramuan; Pruksananonda, Kamthorn

    2016-01-01

    Transgene-free human HS5-SV.hiPS line was generated from human cesarean scar-derived fibroblasts using temperature-sensitive Sendai virus vectors carrying Oct4, Sox2, cMyc and Klf4 exogenous transcriptional factors. The viral constructs were eliminated from HS5-SV.hiPS line through heat treatment. Transgene-free HS5-SV.hiPS cells expressed pluripotent associated transcription factors Oct4, Nanog, Sox2, Rex1 and surface markers SSEA-4, TRA-1-60 and OCT4. HS5-SV.hiPS cells formed embryoid bodies and differentiated into three embryonic germ layers in vivo. HS5-SV.hiPS cells maintained their normal karyotype (46, XX) after culture for extended period. HS5-SV.hiPS displayed the similar pattern of DNA fingerprinting to the parenteral scar-derived fibroblasts. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  15. From the Hayflick mosaic to the mosaics of ageing. Role of stress-induced premature senescence in human ageing.

    PubMed

    Toussaint, Olivier; Remacle, Jose; Dierick, Jean-François; Pascal, Thierry; Frippiat, Christophe; Zdanov, Stéphanie; Magalhaes, Joao Pedro; Royer, Véronique; Chainiaux, Florence

    2002-11-01

    The Hayflick limit-senescence of proliferative cell types-is a fundamental feature of proliferative cells in vitro. Various human proliferative cell types exposed in vitro to many types of subcytotoxic stresses undergo stress-induced premature senescence (SIPS) (also called stress-induced premature senescence-like phenotype, according to the definition of senescence). The known mechanisms of appearance the main features of SIPS are reviewed: senescent-like morphology, growth arrest, senescence-related changes in gene expression, telomere shortening. Long before telomere-shortening induces senescence, other factors such as culture conditions or lack of 'feeder cells' can trigger either SIPS or prolonged reversible G(0) phase of the cell cycle. In vivo, 'proliferative' cell types of aged individuals are likely to compose a mosaic made of cells irreversibly growth arrested or not. The higher level of stress to which these cells have been exposed throughout their life span, the higher proportion of the cells of this mosaic will be in SIPS rather than in telomere-shortening dependent senescence. All cell types undergoing SIPS in vivo, most notably the ones in stressful conditions, are likely to participate in the tissular changes observed along ageing. For instance, human diploid fibroblasts (HDFs) exposed in vivo and in vitro to pro-inflammatory cytokines display biomarkers of senescence and might participate in the degradation of the extracellular matrix observed in ageing.

  16. Matrix metalloproteinase-9 activates TGF-β and stimulates fibroblast contraction of collagen gels.

    PubMed

    Kobayashi, Tetsu; Kim, HuiJung; Liu, Xiangde; Sugiura, Hisatoshi; Kohyama, Tadashi; Fang, Qiuhong; Wen, Fu-Qiang; Abe, Shinji; Wang, Xingqi; Atkinson, Jeffrey J; Shipley, James M; Senior, Robert M; Rennard, Stephen I

    2014-06-01

    Matrix metalloproteinase-9 (MMP-9) is a matrix-degrading enzyme implicated in many biological processes, including inflammation. It is produced by many cells, including fibroblasts. When cultured in three-dimensional (3D) collagen gels, fibroblasts contract the surrounding matrix, a function that is thought to model the contraction that characterizes both normal wound repair and fibrosis. The current study was designed to evaluate the role of endogenously produced MMP-9 in fibroblast contraction of 3D collagen gels. Fibroblasts from mice lacking expression of MMP-9 and human lung fibroblasts (HFL-1) transfected with MMP-9 small-interfering RNA (siRNA) were used. Fibroblasts were cast into type I collagen gels and floated in culture medium with or without transforming growth factor (TGF)-β1 for 5 days. Gel size was determined daily using an image analysis system. Gels made from MMP-9 siRNA-treated human fibroblasts contracted less than control fibroblasts, as did fibroblasts incubated with a nonspecific MMP inhibitor. Similarly, fibroblasts cultured from MMP-9-deficient mice contracted gels less than did fibroblasts from control mice. Transfection of the MMP-9-deficient murine fibroblasts with a vector expressing murine MMP-9 restored contractile activity to MMP-9-deficient fibroblasts. Inhibition of MMP-9 reduced active TGF-β1 and reduced several TGF-β1-driven responses, including activity of a Smad3 reporter gene and production of fibronectin. Because TGF-β1 also drives fibroblast gel contraction, this suggests the mechanism for MMP-9 regulation of contraction is through the generation of active TGF-β1. This study provides direct evidence that endogenously produced MMP-9 has a role in regulation of tissue contraction of 3D collagen gels mediated by fibroblasts. Copyright © 2014 the American Physiological Society.

  17. HSP27 regulates TGF-β mediated lung fibroblast differentiation through the Smad3 and ERK pathways.

    PubMed

    Wang, Gang; Jiao, Hao; Zheng, Jun-Nian; Sun, Xia

    2017-01-01

    Idiopathic pulmonary fibrosis (IPF) is a chronic lethal interstitial lung disease with unknown etiology. Recent studies have indicated that heat-shock protein 27 (HSP27) contributes to the pathogenesis of IPF through the regulation of epithelial-mesenchymal transition (EMT). However, the expression and role of HSP27 in fibroblasts during pulmonary fibrogenesis has not been investigated to date, at least to the best of our knowledge. In this study, we examined the expression of HSP27 in fibrotic lung tissue and fibroblasts from bleomycin (BLM)-challenged mice and human lung fibroblasts treated with transforming growth factor-β (TGF-β). The results revealed that the expression of HSP27 was significantly increased in fibrotic lung tissue and fibroblasts from BLM-challenged mice. In vitro, TGF-β stimulated HSP27 expression in and the differentiation of human lung fibroblasts. The knockdown of Smad3 expression or nuclear factor-κB p65 subunit attenuated the TGF-β-induced increase in HSP27 expression and the differentiation of human lung fibroblasts. In addition, the knockdown of HSP27 expression attenuated the TGF-β-induced activation of ERK and Smad3, and inhibited the differentiation of human lung fibroblasts. On the whole, the findings of our study demonstrate that HSP27 expression is upregulated in lung fibroblasts during pulmonary fibrosis, and subsequently, HSP27 modulates lung fibroblast differentiation through the Smad3 and ERK pathways.

  18. v-myb transformation of Xeroderma pigmentosum human fibroblasts: Overexpression of the c-Ha-ras oncogene in the transformed cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Michelin, S.; Varlet, I.; Sarasin, A.

    1991-10-01

    Human Xeroderma pigmentosum normal' fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental normal' AS16 cells and a revertant clone (ASKXAmore » Cl 1.1 G). The results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.« less

  19. RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells

    PubMed Central

    Serban, Andreea Iren; Stanca, Loredana; Geicu, Ovidiu Ionut; Munteanu, Maria Cristina; Dinischiotu, Anca

    2016-01-01

    AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE’s proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications. PMID:27015414

  20. RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells.

    PubMed

    Serban, Andreea Iren; Stanca, Loredana; Geicu, Ovidiu Ionut; Munteanu, Maria Cristina; Dinischiotu, Anca

    2016-01-01

    AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE's proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.

  1. Cell cycle perturbations and genotoxic effects in human primary fibroblasts induced by low-energy protons and X/gamma-rays.

    PubMed

    Antoccia, Antonio; Sgura, Antonella; Berardinelli, Francesco; Cavinato, Maria; Cherubini, Roberto; Gerardi, Silvia; Tanzarella, Caterina

    2009-09-01

    The effect of graded doses of high-linear energy transfer (LET) low-energy protons to induce cycle perturbations and genotoxic damage was investigated in normal human fibroblasts. Furthermore, such effects were compared with those produced by low-LET radiations. HFFF2, human primary fibroblasts were exposed to either protons (LET = 28.5 keV/microm) or X/gamma-rays, and endpoints related to cell cycle kinetics and DNA damage analysed. Following both type of irradiations, unsynchronized cells suffered an inhibition to entry into S-phase for doses of 1-4 Gy and remained arrested in the G(1)-phase for several days. The levels of induction of regulator proteins, such as TP53 and CDKN1A showed a clear LET-dependence. DSB induction and repair as measured by scoring for gamma-H2AX foci indicated that protons, with respect to X-rays, yielded a lower number of DSBs per Gy, which showed a slower kinetics of disappearance. Such result was in agreement with the extent of MN induction in binucleated cells after X-irradiation. No significant differences between the two types of radiations were observed with the clonogenic assay, resulting anyway the slope of gamma-ray curve higher than that the proton one. In conclusion, in normal human primary fibroblasts cell cycle arrest at the G(1)/S transition can be triggered shortly after irradiation and maintained for several hours post-irradiation of both protons and X-rays. DNA damage produced by protons appears less amenable to be repaired and could be transformed in cytogenetic damage in the form of MN.

  2. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    PubMed

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  3. Autoradiographic detection of diphtheria toxin resistant mutants in human diploid fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gupta, R.S.; Singh, B.

    1985-01-01

    An autoradiographic procedure for the detection of diphtheria toxin (DT) resistant (Dip/sub R/) mutants in human diploid fibroblast (HDF) cells has been developed. The assay is based on the observation that when HDFs from confluent cultures are seeded in medium containing 0.01 flocculating units/ml or higher concentration of DT, protein synthesis in sensitive cells is severely inhibited by 4-6 hr. If at this or later time, a radiolabeled protein precursor (eg, /sup 3/H-leucine) is added to the culture, it is almost exclusively incorporated into the resistant cells, which are then readily identified by autoradiography. These studies provide strong evidence thatmore » the labeled cells identified by autoradiography are bona fide Dip/sub R/ mutants. The detection of Dip/sub R/ cells by autoradiography is apparently not affected by the presence of the sensitive cells in the mixtures. The spontaneous frequency of Dip/sub R/ cells in HDFs has been found to be in the range of 1-5 x 10/sup -6/, and this increases in a dose dependent manner upon treatment with the mutagen ethyl methanesulfonate. These results indicate that the autoradiographic assay could be used for quantitative mutagenesis. Since the autoradiographic assay does not depend on cell division, it may prove useful in estimating the incidence of pre-existing mutations in cell populations that either do not divide or have very limited growth potential (eg, lymphocytes, muscle cells, neurons, senescent fibroblasts, etc.).« less

  4. P-glycoprotein Blockers Augment the Effect of Mitomycin C on Human Tenon's Fibroblasts.

    PubMed

    White, Andrew J R; Kelly, Elizabeth; Healey, Paul R; Crowston, Jonathan G; Mitchell, Paul; Zoellner, Hans

    2013-08-01

    Mitomycin C (MMC), which induces apoptosis in human Tenon's fibroblasts (HTF), is frequently used to retard wound healing after glaucoma surgery. The aim of this in vitro study was to examine whether adjunctive Verapamil and Cyclosporine could augment the cytotoxic effect of MMC on HTF. Fibroblast cell lines were established by explant culture from human tissue biopsy samples obtained during trabeculectomy procedures. Cells were exposed to MMC at varying concentrations (0.01-0.4 mg/ml) for 3 minutes, prior to washing in the presence or absence of the following drugs: Staurosporine (0.003mg/ml), Verapamil (2.5-0.25 mg/ml), or Cyclosporine (50-0.5 mg/ml). Following exposure, cells were cultured for 6 hours and surviving cells quantitated by haemocytometer counts. Both Verapamil and Staurosporine exhibited mild toxic effects on their own, but greatly enhanced the apoptotic effect of MMC. Staurosporine is too toxic to be considered clinically, so its augmentive effect on the activity of MMC was not studied further here. Doses as low as 0.25 mg/ml of Verapamil continued to show significant augmentation of the apoptotic effect of MMC Cyclosporine at a clinically used concentration (5 mg/ml) exhibited modest augmentation of the effect of MMC. Verapamil and Cyclosporine in clinically acceptable concentrations potentiate the effect of MMC and may obviate the need for high dose antimetabolites in trabeculectomy; however, further preclinical study is required. Adjunctive Verapamil or Cyclosporine may allow lower dose MMC to be used in glaucoma filtration surgery while maintaining the same antifibrotic effects.

  5. 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.

  6. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    PubMed

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  7. Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

    PubMed

    Epstein Shochet, Gali; Wollin, Lutz; Shitrit, David

    2018-03-12

    Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. Activated fibroblasts are the key effector cells in fibrosis, producing excessive amounts of collagen and extracellular matrix (ECM) proteins. Whether the ECM conditioned by IPF fibroblasts determines the phenotype of naïve fibroblasts is difficult to explore. IPF-derived primary fibroblasts were cultured on Matrigel and then cleared using ammonium hydroxide, creating an IPF-conditioned matrix (CM). Normal fibroblast CM served as control. Normal fibroblasts were cultured on both types of CM, and cell count, cell distribution and markers of myofibroblast differentiation; transforming growth factor beta (TGFβ) signalling; and ECM expression were assessed. The effects of the anti-fibrotic drugs nintedanib and pirfenidone at physiologically relevant concentrations were also explored. Normal fibroblasts cultured on IPF-CM arranged in large aggregates as a result of increased proliferation and migration. Moreover, increased levels of pSmad3, pSTAT3 (phospho signal transducer and activator of transcription 3), alpha smooth muscle actin (αSMA) and Collagen1a were found, suggesting a differentiation towards a myofibroblast-like phenotype. SB505124 (10 μmol/L) partially reversed these alterations, suggesting a TGFβ contribution. Furthermore, nintedanib at 100 nmol/L and, to a lesser extent, pirfenidone at 100 μmol/L prevented the IPF-CM-induced fibroblast phenotype alterations, suggesting an attenuation of the ECM-fibroblast interplay. IPF fibroblasts alter the ECM, thus creating a CM that further propagates an IPF-like phenotype in normal fibroblasts. This assay demonstrated differences in drug activities for approved IPF drugs at clinically relevant concentrations. Thus, the matrix-fibroblast phenotype interplay might be a relevant assay to explore drug candidates for IPF treatment. © 2018 Asian Pacific Society of Respirology.

  8. Uroporphyrin I Stimulation of Collagen Biosynthesis in Human Skin Fibroblasts

    PubMed Central

    Varigos, George; Schiltz, John R.; Bickers, David R.

    1982-01-01

    Porphyria cutanea tarda and erythropoietic porphyria are disorders of heme synthesis that originate in the liver and bone marrow, respectively. Each is characterized by increased accumulation of uroporphyrin, I, by cutaneous photosensitivity, and in some patients by indurated plaques and scarring that resemble scleroderma. These scleroderma-like lesions occur in light-exposed and light-protected body areas. In these studies we evaluated the role of uroporphyrin I and of light in evoking the scleroderma-like cutaneous changes. Normal human skin fibroblasts were exposed to uroporphyrin I and to 400 nm radiation and the effect of these agents on collagen accumulation by the cells was determined. Radioactive tracer studies showed that uroporphyrin I caused a specific increase in the accumulation of newly synthesized collagen by fibroblast monolayer cultures, as verified by [3H]hydroxyproline and collagenase digestion assays. Collagen accumulation was stimulated 1.5- to 2.7-fold by uroporphyrin I, whereas noncollagenous protein accumulation was unchanged. The increased collagen accumulation was time and uroporphyrin I-concentration-dependent, and occurred both in the presence or absence of ultraviolet light exposure. Further studies demonstrated that the increased accumulation was not the result of decreased rates of collagen degradation nor was it due to changes in cell population growth parameters (generation times and saturation densities). No changes in morphology of the treated cells occurred. These studies indicate that porphyrins possess previously undemonstrated biological effects that are independent of their photosensitizing properties. This novel dark effect of uroporphyrin I may account for the sclerodermatous lesions seen in the skin of patients with porphyria cutanea tarda and erythropoietic porphyria. PMID:7054234

  9. Hereditary orotic aciduria, Lesch-Nyhan syndrome, and xeroderma pigmentosum probed by herpes simplex virus: /sup 125/I-iododeoxycytidine incorporation as an assay for viral growth. [Human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Campisi, J.; Hafner, J.; Boorstein, R.

    /sup 125/I-Iododeoxycytidine (/sup 125/IdC) incorporation into acid-insoluble material was a sensitive, rapid, and quantitative assay for the growth of herpes simplex virus type 1 (HSV-1) in human fibroblasts. Cellular utilization of the isotope was 10 to 25% of the incorporation by infected cells and could be 80% inhibited by tetrahydrouridine (THU). Viral utilization was inhibited by acycloguanosine, thioguanine (TG), and cytosine arabinoside. Isotope was incorporated equally well by growing or quiescent infected cells. HSV-1 was used to probe the metabolic capabilities of three mutant human fibroblast strains. /sup 125/IdC incorporation quantitatively measured the ability of the virus to grow inmore » these cells. Viral /sup 125/IdC incorporation was sensitive to TG in normal fibroblasts but showed a 8- to 10-fold greater resistance to TG in fibroblasts derived from patients with Lesch-Nyhan syndrome (LN). Similarly, the growth of ultraviolet irradiated HSV-1 in normal fibroblasts was 5-fold greater than in fibroblasts derived from patients with xeroderma pigmentosum. In fibroblasts derived from patients with hereditary orotic aciduria, viral /sup 125/IdC incorporation was sensitive to adenosine (AD) at concentrations which were slightly stimulatory in normal fibroblasts. This was a 2-fold difference in AD sensitivity, which the radioassay reliably and quantitatively documented. HSV-1 infected cells could be individually identified by their incorporated /sup 125/IdC; such cells had blackened nuclei in autoradiograms prepared 12 hr after infection. Normal cells infected in the presence of TG had many fewer labeled nuclei than LN cells similarly infected in the presence of the drug. (JMT)« less

  10. Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts.

    PubMed

    Heo, Jung Sun; Lee, Seung-Youp; Lee, Jeong-Chae

    2010-11-01

    Wnt/β-catenin signaling has been known to influence bone formation and homeostasis. In this study, we investigated the canonical Wnt signaling regulation of osteogenic differentiation from periodontal ligament (PDL) fibroblasts. Stimulating PDL fibroblasts with lithium chloride (LiCl), a canonical Wnt activator, significantly increased mineralized nodule and alkaline phosphatase (ALP) activity in a time- and dose-dependent manner. LiCl up-regulated protein expression of osteogenic transcription factors, including the runt-related gene 2, Msx2, and Osterix 2, in the PDL fibroblasts. Treatment of these cells with LiCl also increased the mRNA levels of ALP, FosB, and Fra1 in a dose-dependent manner. Blockage of canonical Wnt signaling by treating the cells with DKK1 inhibited Wnt1-stimulated mRNA expression of these osteogenic factors. Furthermore, pretreatment with DKK1 reduced the ALP activity and matrix mineralization stimulated by Wnt1. Collectively, these results suggest that canonical Wnt signaling leads to the differentiation of PDL fibroblasts into osteogenic lineage with the attendant stimulation of osteogenic transcription factors.

  11. Endoplasmic reticulum stress signaling is involved in mitomycin C (MMC)-induced apoptosis in human fibroblasts via PERK pathway.

    PubMed

    Shi, Kun; Wang, Daode; Cao, Xiaojian; Ge, Yingbin

    2013-01-01

    Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation.

  12. Endoplasmic Reticulum Stress Signaling Is Involved in Mitomycin C(MMC)-Induced Apoptosis in Human Fibroblasts via PERK Pathway

    PubMed Central

    Cao, Xiaojian; Ge, Yingbin

    2013-01-01

    Endoplasmic reticulum (ER) stress-mediated cell apoptosis has been implicated in various cell types, including fibroblasts. Previous studies have shown that mitomycin C (MMC)-induced apoptosis occurs in fibroblasts, but the effects of MMC on ER stress-mediated apoptosis in fibroblasts have not been examined. Here, MMC-induced apoptosis in human primary fibroblasts was investigated by exposing cells to a single dose of MMC for 5 minutes. Significant inhibition of cell proliferation and increased apoptosis were observed using a cell viability assay, Annexin V/propidium iodide double staining, cell cycle analysis, and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) staining. Upregulation of proapoptotic factors, including cleaved caspase-3 and poly ADP-ribose polymerase (PARP), was detected by Western blotting. MMC-induced apoptosis was correlated with elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP homologous protein (CHOP), which are hallmarks of ER stress. Three unfolded protein response (UPR) sensors (inositol-requiring enzyme 1, IRE1; activating transcription factor 6, ATF6; and PKR-like ER kinase, PERK) and their downstream signaling pathways were also activated. Knockdown of CHOP attenuated MMC-induced apoptosis by increasing the ratio of BCL-2/BAX and decreasing BIM expression, suggesting that ER stress is involved in MMC-induced fibroblast apoptosis. Interestingly, knockdown of PERK significantly decreased ER stress-mediated apoptosis by reducing the expression of CHOP, BIM and cleaved caspase-3. Reactive oxygen species (ROS) scavenging also decreased the expression of GRP78, phospho-PERK, CHOP, and BIM. These results demonstrate that MMC-induced apoptosis is triggered by ROS generation and PERK activation. PMID:23533616

  13. Small molecular antioxidants effectively protect from PUVA-induced oxidative stress responses underlying fibroblast senescence and photoaging.

    PubMed

    Briganti, Stefania; Wlaschek, Meinhard; Hinrichs, Christina; Bellei, Barbara; Flori, Enrica; Treiber, Nicolai; Iben, Sebastian; Picardo, Mauro; Scharffetter-Kochanek, Karin

    2008-09-01

    Exposure of human fibroblasts to 8-methoxypsoralen plus ultraviolet-A irradiation (PUVA) results in stress-induced cellular senescence in fibroblasts. We here studied the role of the antioxidant defense system in the accumulation of reactive oxygen species (ROS) and the effect of the antioxidants alpha-tocopherol, N-acetylcysteine, and alpha-lipoic acid on PUVA-induced cellular senescence. PUVA treatment induced an immediate and increasing generation of intracellular ROS. Supplementation of PUVA-treated fibroblasts with alpha-tocopherol (alpha-Toc), N-acetylcysteine (NAC), or alpha-lipoic acid (alpha-LA) abrogated the increased ROS generation and rescued fibroblasts from the ROS-dependent changes into the cellular senescence phenotype, such as cytoplasmic enlargement, enhanced expression of senescence-associated-beta-galactosidase and matrix-metalloproteinase-1, hallmarks of photoaging and intrinsic aging. PUVA treatment disrupted the integrity of cellular membranes and impaired homeostasis and function of the cellular antioxidant system with a significant decrease in glutathione and hydrogen peroxide-detoxifying enzymes activities. Supplementation with NAC, alpha-LA, and alpha-Toc counteracted these changes. Our data provide causal evidence that (i) oxidative stress due to an imbalance in the overall cellular antioxidant capacity contributes to the induction and maintenance of the PUVA-induced fibroblast senescence and that (ii) low molecular antioxidants protect effectively against these deleterious alterations.

  14. Gadolinium-induced fibrosis is counter-regulated by CCN3 in human dermal fibroblasts: a model for potential treatment of nephrogenic systemic fibrosis.

    PubMed

    Riser, Bruce L; Bhagavathula, Narasimharao; Perone, Patricia; Garchow, Kendra; Xu, Yiru; Fisher, Gary J; Najmabadi, Feridoon; Attili, Durga; Varani, James

    2012-06-01

    We recently show that CCN3 is a counter-regulatory molecule for the pro-fibrotic protein CCN2, and a potentially novel fibrosis therapy. The goal of this study was to assess the role of CCN3 in fibroproliferative/fibrotic responses in human dermal fibroblasts exposed to Omniscan, one of the gadolinium-based contrast agents associated with development of nephrogenic systemic fibrosis (NSF) a rare but life-threatening disease thought to be complication of NMR diagnostics in renal impaired patients. Human dermal fibroblasts were exposed to Omniscan; or to platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β) as controls. Proliferation was assessed along with matrix metalloproteinase-1, tissue inhibitor of metalloproteinases-1 and type 1 procollagen in the absence and presence of CCN3. In parallel, CCN3 production was assessed in control and Omniscan-treated cells. The results showed that PDGF stimulated fibroblast proliferation, production of Timp-1 and MMP-1 whereas exogenous CCN3 inhibited, in a dose response manner, cell proliferation (approx. 50 % max.) and production of MMP-1 (approx 35 % max.) but had little effect on TIMP-1. TGF-β stimulated type 1 procollagen production but not proliferation, Timp-1 or MMP-1 compared to non-TGF-ß treated control cells, and CCN3 treatment blocked (approx. 80 % max.) this up-regulation. Interestingly, untreated, control fibroblasts produced high constitutive levels of CCN3 and concentrations of Omniscan that induced fibroproliferative/fibrogenic changes in dermal fibroblasts correspondingly suppressed CCN3 production. The use of PDGF and TGF-β as positive controls, and the study of differential responses, including that to Omniscan itself, provide the first evidence for a role of fibroblast-derived CCN3 as an endogenous regulator of pro-fibrotic changes, elucidating possible mechanism(s). In conclusion, these data support our hypothesis of a role for fibroblast-derived CCN3 as an endogenous

  15. Cell Toxicity in Fibroblasts, Tenocytes, and Human Mesenchymal Stem Cells-A Comparison of Necrosis and Apoptosis-Inducing Ability in Ropivacaine, Bupivacaine, and Triamcinolone.

    PubMed

    Zhang, Anja Z; Ficklscherer, Andreas; Gülecyüz, Mehmet F; Paulus, Alexander C; Niethammer, Thomas R; Jansson, Volkmar; Müller, Peter E

    2017-04-01

    To analyze the ability of ropivacaine, bupivacaine, and triamcinolone to induce apoptosis and necrosis in fibroblasts, tenocytes, and human mesenchymal stem cells. Human dermal fibroblasts, adipose-derived human mesenchymal stem cells (hMSCs), and tenocytes gained from the rotator cuff tendon were seeded with a cell density of 0.5 × 10 4 /cm 2 . One specimen of ropivacaine, bupivacaine, and triamcinolone was tested separately on the cells with separate concentrations of 0.5%, 0.25%, and 0.125% for each specimen. The negative control received no agent, only a change of medium. The incubation period for each agent was 30 minutes. After a change of medium and 1 hour, 24 hours, and 7 days of incubation, 10 4  cells were harvested and analyzed via fluorescence-activated cell sorting with double-staining with annexin V and propidium iodide. Statistical analysis to determine significant difference (P < .05) between the groups with SPSS statistics 23 through one-way analysis of variance with a univariate general linear model was performed. Bupivacaine showed necrosis-inducing effects on fibroblasts and tenocytes, with the necrotic effect peaking at 0.5% and 0.25%. Ropivacaine and triamcinolone caused no significant necrosis. Compared with fibroblasts and tenocytes, hMSCs did not show significant necrotic or apoptotic effects after exposure to bupivacaine. Overall, no significant differences in apoptosis were detected between different cell lines, varying concentrations, or time measurements. Bupivacaine 0.5% and 0.25% have the most necrosis-inducing effects on fibroblasts and tenocytes. Ropivacaine caused less necrosis than bupivaine. Compared with fibroblasts and tenocytes, hMSCs were not affected by necrosis using any of the tested agents. A significant apoptosis-inducing effect could not be detected for the different cell lines. Possible cell toxicity raises questions of concern for intra-articular injections using local anesthetics and corticosteroids. The

  16. Gene expression analysis upon lncRNA DDSR1 knockdown in human fibroblasts

    PubMed Central

    Jia, Li; Sun, Zhonghe; Wu, Xiaolin; Misteli, Tom; Sharma, Vivek

    2015-01-01

    Long non-coding RNAs (lncRNAs) play important roles in regulating diverse biological processes including DNA damage and repair. We have recently reported that the DNA damage inducible lncRNA DNA damage-sensitive RNA1 (DDSR1) regulates DNA repair by homologous recombination (HR). Since lncRNAs also modulate gene expression, we identified gene expression changes upon DDSR1 knockdown in human fibroblast cells. Gene expression analysis after RNAi treatment targeted against DDSR1 revealed 119 genes that show differential expression. Here we provide a detailed description of the microarray data (NCBI GEO accession number GSE67048) and the data analysis procedure associated with the publication by Sharma et al., 2015 in EMBO Reports [1]. PMID:26697398

  17. A Novel Role of Peripheral Corticotropin-Releasing Hormone (CRH) on Dermal Fibroblasts

    PubMed Central

    Rassouli, Olga; Liapakis, George; Lazaridis, Iakovos; Sakellaris, George; Gkountelias, Kostas; Gravanis, Achille; Margioris, Andrew N.

    2011-01-01

    Corticotropin-releasing hormone, or factor, (CRH or CRF) exerts important biological effects in multiple peripheral tissues via paracrine/autocrine actions. The aim of our study was to assess the effects of endogenous CRH in the biology of mouse and human skin fibroblasts, the primary cell type involved in wound healing. We show expression of CRH and its receptors in primary fibroblasts, and we demonstrate the functionality of fibroblast CRH receptors by induction of cAMP. Fibroblasts genetically deficient in Crh (Crh−/−) had higher proliferation and migration rates and compromised production of IL-6 and TGF-β1 compared to the wildtype (Crh+/+) cells. Human primary cultures of foreskin fibroblasts exposed to the CRF1 antagonist antalarmin recapitulated the findings in the Crh−/− cells, exhibiting altered proliferative and migratory behavior and suppressed production of IL-6. In conclusion, our findings show an important role of fibroblast-expressed CRH in the proliferation, migration, and cytokine production of these cells, processes associated with the skin response to injury. Our data suggest that the immunomodulatory effects of CRH may include an important, albeit not explored yet, role in epidermal tissue remodeling and regeneration and maintenance of tissue homeostasis. PMID:21765902

  18. Quiescent Fibroblasts Exhibit High Metabolic Activity

    PubMed Central

    Lemons, Johanna M. S.; Feng, Xiao-Jiang; Bennett, Bryson D.; Legesse-Miller, Aster; Johnson, Elizabeth L.; Raitman, Irene; Pollina, Elizabeth A.; Rabitz, Herschel A.; Rabinowitz, Joshua D.; Coller, Hilary A.

    2010-01-01

    Many cells in mammals exist in the state of quiescence, which is characterized by reversible exit from the cell cycle. Quiescent cells are widely reported to exhibit reduced size, nucleotide synthesis, and metabolic activity. Much lower glycolytic rates have been reported in quiescent compared with proliferating lymphocytes. In contrast, we show here that primary human fibroblasts continue to exhibit high metabolic rates when induced into quiescence via contact inhibition. By monitoring isotope labeling through metabolic pathways and quantitatively identifying fluxes from the data, we show that contact-inhibited fibroblasts utilize glucose in all branches of central carbon metabolism at rates similar to those of proliferating cells, with greater overflow flux from the pentose phosphate pathway back to glycolysis. Inhibition of the pentose phosphate pathway resulted in apoptosis preferentially in quiescent fibroblasts. By feeding the cells labeled glutamine, we also detected a “backwards” flux in the tricarboxylic acid cycle from α-ketoglutarate to citrate that was enhanced in contact-inhibited fibroblasts; this flux likely contributes to shuttling of NADPH from the mitochondrion to cytosol for redox defense or fatty acid synthesis. The high metabolic activity of the fibroblasts was directed in part toward breakdown and resynthesis of protein and lipid, and in part toward excretion of extracellular matrix proteins. Thus, reduced metabolic activity is not a hallmark of the quiescent state. Quiescent fibroblasts, relieved of the biosynthetic requirements associated with generating progeny, direct their metabolic activity to preservation of self integrity and alternative functions beneficial to the organism as a whole. PMID:21049082

  19. [In vitro tendon engineering using human dermal fibroblasts].

    PubMed

    Deng, Dan; Liu, Wei; Xu, Feng; Wu, Xiao-Li; Wei, Xian; Zhong, Bin; Cui, Lei; Cao, Yi-Lin

    2008-04-01

    To examine the feasibility of using human dermal fibroblasts (DFbs) and polyglycolic acids (PGA) to engineer tendon in vitro. Human dermal fibroblasts (DFbs) were isolated from the foreskin tissues of children obtained during operation with collagenase and cultured in vitro. Human tendon was obtained from a patient undergoing amputation during operation to isolate tenocytes. The DFbs of second passage were seeded on PGA fibers to form cell-scaffold constructs in shape of tendons. Those constructs were divided into 4 groups: experimental group (n = 15) with the DFbs inoculated on PGA scaffold under constant tension generated by a U-shaped spring, control group 1 (n = 15) with the DFbs inoculated on PGA scaffold without tension, control group 2 (n = 3), i. e., cell-free pure PGA scaffolds under tension, and control group 3 (n = 5), i. e., tenocyte-scaffold constructs under tension that was harvested only at the ninth week. Samples were harvested 2, 5, 9, 14, and 18 weeks later to undergo histological examination and biomechanical test. Two weeks later histological examination showed that the constructs were mainly composed of PGA fibers in both the experimental group and the group without tension. Transmission electron microscopy showed fine cell attachment and stretching on the scaffold. By the 5th week, a neo-tendon was formed in all groups except for the cell-free group, and histology revealed the formation of collagen fibers. At the 9th week, the PGA fibers of the cell-free group were broken and partially degraded, the neo-tendon's diameter of the experimental group was (1.18 +/- 0.25) mm, significantly thinner than that of the group without tension[ (2.43 +/- 0.49) mm, P = 0.017]. The gross morphology of tendons of the experimental group and tenocyte group were similar to each other except for more cells in the experimental group. In experimental group, immunohistochemistry revealed the production of fibers of collagen type I & III that were aligned

  20. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bruzzone, Santina, E-mail: santina.bruzzone@unige.it; Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova; Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova

    Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated themore » effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.« less

  1. Cd²⁺-induced alteration of the global proteome of human skin fibroblast cells.

    PubMed

    Prins, John M; Fu, Lijuan; Guo, Lei; Wang, Yinsheng

    2014-03-07

    Cadmium (Cd(2+)) is a toxic heavy metal and a well-known human carcinogen. The toxic effects of Cd(2+) on biological systems are diverse and thought to be exerted through a complex array of mechanisms. Despite the large number of studies aimed to elucidate the toxic mechanisms of action of Cd(2+), few have been targeted toward investigating the ability of Cd(2+) to disrupt multiple cellular pathways simultaneously and the overall cellular responses toward Cd(2+) exposure. In this study, we employed a quantitative proteomic method, relying on stable isotope labeling by amino acids in cell culture (SILAC) and LC-MS/MS, to assess the Cd(2+)-induced simultaneous alterations of multiple cellular pathways in cultured human skin fibroblast cells. By using this approach, we were able to quantify 2931 proteins, and 400 of them displayed significantly changed expression following Cd(2+) exposure. Our results unveiled that Cd(2+) treatment led to the marked upregulation of several antioxidant enzymes (e.g., metallothionein-1G, superoxide dismutase, pyridoxal kinase, etc.), enzymes associated with glutathione biosynthesis and homeostasis (e.g., glutathione S-transferases, glutathione synthetase, glutathione peroxidase, etc.), and proteins involved in cellular energy metabolism (e.g., glycolysis, pentose phosphate pathway, and the citric acid cycle). Additionally, we found that Cd(2+) treatment resulted in the elevated expression of two isoforms of dimethylarginine dimethylaminohydrolase (DDAH I and II), enzymes known to play a key role in regulating nitric oxide biosynthesis. Consistent with these findings, we observed elevated formation of nitric oxide in human skin (GM00637) and lung (IMR-90) fibroblast cells following Cd(2+) exposure. The upregulation of DDAH I and II suggests a role of nitric oxide synthesis in Cd(2+)-induced toxicity in human cells.

  2. The influence of retraction agents on cytoskeleton reorganization and oxidative stress in primary human gingival fibroblasts (HGFs).

    PubMed

    Nowakowska, Danuta; Saczko, Jolanta; Bieżuńska-Kusiak, Katarzyna; Choromańska, Anna; Dubińska-Magiera, Magda; Ziętek, Marek; Kulbacka, Julita

    2014-03-01

    Contemporary gingival retraction chemicals are not without disagreeable side-effects; there appears to be no best gingival retraction agent. The aim of this research was to select the most biocompatible retraction agents based on examination of the parameters of oxidative stress in fibroblasts derived from human primary cell culture. In this in vitro study we evaluated parameters of oxidative stress after treatment with retraction agents. Visine, Afrin, Neosynephrin, Strazolin and Adrenaline were the commercial products studied as gingival retraction agents. Additionally we examined three experimental agents. We determined lipid peroxidation and protein damage and monitored changes in cellular cytoskeleton proteins. Proliferative and survival efficiency were also evaluated. Oxidative changes included by evaluated retraction agents were at the lowest level in the case of the experimental gels. Also cytoskeleton observations suggest that the experimental agents did not degrade the cellular structure of human gingival fibroblasts (HGFs). The current study was performed because of a need to project new nontoxic and save retraction agents for peridontological therapeutic usage. We suggest that the new investigational gels are most biocompatible with periodontal tissues and can be applied as new vasoconstrictor chemical retraction agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Basic Fibroblast Growth Factor Accelerates Matrix Degradation Via a Neuro-Endocrine Pathway in Human Adult Articular Chondrocytes

    PubMed Central

    IM, HEE-JEONG; LI, XIN; MUDDASANI, PRASUNA; KIM, GUN-HEE; DAVIS, FRANCESCA; RANGAN, JAYANTHI; FORSYTH, CHRISTOPHER B.; ELLMAN, MICHAEL; THONAR, EUGENE JMA

    2010-01-01

    Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK1-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1β accelerate matrix degradation via a neural pathway upregulation of substance P and NK1-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK1-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK1-R is, in part, through an IL-1β-dependent pathway. PMID:17960584

  4. Basic fibroblast growth factor accelerates matrix degradation via a neuro-endocrine pathway in human adult articular chondrocytes.

    PubMed

    Im, Hee-Jeong; Li, Xin; Muddasani, Prasuna; Kim, Gun-Hee; Davis, Francesca; Rangan, Jayanthi; Forsyth, Christopher B; Ellman, Michael; Thonar, Eugene J M A

    2008-05-01

    Pain-related neuropeptides released from synovial fibroblasts, such as substance P, have been implicated in joint destruction. Substance P-induced inflammatory processes are mediated via signaling through a G-protein-coupled receptor, that is, neurokinin-1 tachykinin receptor (NK(1)-R). We determined the pathophysiological link between substance P and its receptor in human adult articular cartilage homeostasis. We further examined if catabolic growth factors such as basic fibroblast growth factor (bFGF or FGF-2) or IL-1beta accelerate matrix degradation via a neural pathway upregulation of substance P and NK(1)-R. We show here that substance P stimulates the production of cartilage-degrading enzymes, such as matrix metalloproteinase-13 (MMP-13), and suppresses proteoglycan deposition in human adult articular chondrocytes via NK(1)-R. Furthermore, we have demonstrated that substance P negates proteoglycan stimulation promoted by bone morphogenetic protein-7, suggesting the dual role of substance P as both a pro-catabolic and anti-anabolic mediator of cartilage homeostasis. We report that bFGF-mediated stimulation of substance P and its receptor NK(1)-R is, in part, through an IL-1beta-dependent pathway. (c) 2007 Wiley-Liss, Inc.

  5. RAGE-dependent activation of gene expression of superoxide dismutase and vanins by AGE-rich extracts in mice cardiac tissue and murine cardiac fibroblasts.

    PubMed

    Leuner, Beatrice; Ruhs, Stefanie; Brömme, Hans-Jürgen; Bierhaus, Angelika; Sel, Saadettin; Silber, Rolf-Edgar; Somoza, Veronika; Simm, Andreas; Nass, Norbert

    2012-10-01

    Advanced glycation end products (AGEs) are stable compounds formed from initial Maillard reaction products. They are considered as markers for ageing and often associated with age-related, degenerative diseases. Bread crust represents an established model for nutritional compounds rich in AGEs and is able to induce antioxidative defense genes such as superoxide dismutases and vanins in cardiac cells. The aim of this study was to investigate to what extend the receptor for AGEs (RAGE) contributes to this response. Signal transduction in response to bread crust extract was analysed in cardiac fibroblasts derived from C57/B6-NCrl (RAGE +/+) and the corresponding RAGE-knock out C57/B6-NCrl mouse strain (RAGE -/-). Activation of superoxide dismutases in animals was then analysed upon bread crust feeding in these two mice strains. Cardiac fibroblasts from RAGE -/- mice did not express RAGE, but the expression of AGER-1 and AGER-3 was up-regulated, whereas the expression of SR-B1 was down-regulated. RAGE -/- cells were less sensitive to BCE in terms of MAP-kinase phosphorylation and NF-κB reporter gene activation. Bread crust extract induced mRNA levels of MnSOD and Vnn-1 were also reduced in RAGE -/- cells, whereas Vnn-3 mRNA accumulation seemed to be RAGE receptor independent. In bread crust feeding experiments, RAGE -/- mice did not exhibit an activation of MnSOD-mRNA and -protein accumulation as observed for the RAGE +/+ animals. In conclusion, RAGE was clearly a major factor for the induction of antioxidant defense signals derived from bread crust in cardiac fibroblast and mice. Nevertheless higher doses of bread crust extract could overcome the RAGE dependency in cell cultures, indicating that additional mechanisms are involved in BCE-mediated activation of SOD and vanin expression.

  6. FGF9 and FGF18 in idiopathic pulmonary fibrosis promote survival and migration and inhibit myofibroblast differentiation of human lung fibroblasts in vitro.

    PubMed

    Joannes, Audrey; Brayer, Stéphanie; Besnard, Valérie; Marchal-Sommé, Joëlle; Jaillet, Madeleine; Mordant, Pierre; Mal, Hervé; Borie, Raphael; Crestani, Bruno; Mailleux, Arnaud A

    2016-04-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by an accumulation of extracellular matrix proteins and fibroblasts in the distal airways. Key developmental lung signaling pathways are reactivated in IPF. For instance, fibroblast growth factor 9 (FGF9) and FGF18, involved in epithelial-mesenchymal interactions, are critical for lung development. We evaluated the expression of FGF9, FGF18, and FGF receptors (FGFRs) in lung tissue from controls and IPF patients and assessed their effect on proliferation, survival, migration, and differentiation of control and IPF human lung fibroblasts (HLFs). FGF9, FGF18, and all FGFRs were present in the remodeled alveolar epithelium close to the fibroblast foci in IPF lungs. FGFR3 was generally detected in fibroblast foci by immunohistochemistry. In vitro, HLFs mainly expressed mesenchyme-associated FGFR isoforms (FGFR1c and FGFR3c) and FGFR4. FGF9 did not affect fibroblast proliferation, whereas FGF18 inhibited cell growth in control fibroblasts. FGF9 and FGF18 decreased Fas-ligand-induced apoptosis in control but not in IPF fibroblasts. FGF9 prevented transforming growth factor β1-induced myofibroblast differentiation. FGF9 and FGF18 increased the migratory capacities of HLF, and FGF9 actively modulated matrix metalloproteinase activity. In addition, FGFR3 inhibition by small interfering RNA impacted p-ERK activation by FGF9 and FGF18 and their effects on differentiation and migration. These results identify FGF9 as an antiapoptotic and promigratory growth factor on HLF, maintaining fibroblasts in an undifferentiated state. The biological effects of FGF9 and FGF18 were partially driven by FGFR3. FGF18 was a less potent molecule. Both growth factors likely contribute to the fibrotic process in vivo. Copyright © 2016 the American Physiological Society.

  7. Suppression of TGF-β pathway by pirfenidone decreases extracellular matrix deposition in ocular fibroblasts in vitro.

    PubMed

    Stahnke, Thomas; Kowtharapu, Bhavani S; Stachs, Oliver; Schmitz, Klaus-Peter; Wurm, Johannes; Wree, Andreas; Guthoff, Rudolf Friedrich; Hovakimyan, Marina

    2017-01-01

    In glaucoma surgery, fibrotic processes occur, leading to impairment of liquid outflow. Activated fibroblasts are responsible for postoperative scarring. The transforming growth factor-β (TGF-β) pathway plays a key role in fibroblast function, differentiation and proliferation. The aim of this study was the characterization of the fibrotic potential of two subtypes of primary human ocular fibroblasts and the attempt to inhibit fibrotic processes specifically, without impairing cell viability. For fibrosis inhibition we focused on the small molecule pirfenidone, which has been shown to prevent pulmonary fibrosis by the decrease of the expression of TGF-β1, TGF-β2 and TGF-β3 cytokines. For in vitro examinations, isolated human primary fibroblasts from Tenon capsule and human intraconal orbital fat tissues were used. These fibroblast subpopulations were analyzed in terms of the expression of matrix components responsible for postoperative scarring. We concentrated on the expression of collagen I, III, VI and fibronectin. Additionally, we analyzed the expression of α-smooth muscle actin, which serves as a marker for fibrosis and indicates transformation of fibroblasts into myofibroblasts. Gene expression was analyzed by rtPCR and synthesized proteins were examined by immunofluorescence and Western blot methods. Proliferation of fibroblasts under different culture conditions was assessed using BrdU assay. TGF-β1 induced a significant increase of cell proliferation in both cell types. Also the expression of some fibrotic markers was elevated. In contrast, pirfenidone decreased cell proliferation and matrix synthesis in both fibroblast subpopulations. Pirfenidone slightly attenuated TGF-β1 induced expression of fibronectin and α-smooth muscle actin in fibroblast cultures, without impairing cell viability. To summarize, manipulation of the TGF-β signaling pathway by pirfenidone represents a specific antifibrotic approach with no toxic side effects in two human

  8. Fibroblast extracellular matrix gene expression in response to keratinocyte-releasable stratifin.

    PubMed

    Ghaffari, Abdi; Li, Yunyaun; Karami, Ali; Ghaffari, Mazyar; Tredget, Edward E; Ghahary, Aziz

    2006-05-15

    Termination of wound-healing process requires a fine balance between connective tissue deposition and its hydrolysis. Previously, we have demonstrated that keratinocyte-releasable stratifin, also known as 14-3-3 sigma protein, stimulates collagenase (MMP-1) expression in dermal fibroblasts. However, role of extracellular stratifin in regulation of extracellular matrix (ECM) factors and other matrix metalloproteinases (MMPs) in dermal fibroblast remains unexplored. To address this question, large-scale ECM gene expression profile were analyzed in human dermal fibroblasts co-cultured with keratinocytes or treated with recombinant stratifin. Superarray pathway-specific microarrays were utilized to identify upregulation or downregulation of 96 human ECM and adhesion molecule genes. RT-PCR and Western blot were used to validate microarray expression profiles of selected genes. Comparison of gene profiles with the appropriate controls showed a significant (more than twofold) increase in expression of collagenase-1, stromelysin-1 and -2, neutrophil collagenase, and membrane type 5 MMP in dermal fibroblasts treated with stratifin or co-cultured with keratinocytes. Expression of type I collagen and fibronectin genes decreased in the same fibroblasts. The results of a dose-response experiment showed that stratifin stimulates the expression of stromelysin-1 (MMP-3) mRNA by dermal fibroblasts in a concentration-dependent fashion. Furthermore, Western blot analysis of fibroblast-conditioned medium showed a peak in MMP-3 protein levels 48 h following treatment with recombinant stratifin. In a lasting-effect study, MMP-3 protein was detected in fibroblast-condition medium for up to 72 h post removal of stratifin. In conclusion, our results suggest that keratinocyte-releasable stratifin plays a major role in induction of ECM degradation by dermal fibroblasts through stimulation of key MMPs, such as MMP-1 and MMP-3. Therefore, stratifin protein may prove to be a useful target for

  9. Feeder-free reprogramming of human fibroblasts with messenger RNA.

    PubMed

    Warren, Luigi; Wang, Jiwu

    2013-11-13

    This unit describes a feeder-free protocol for deriving induced pluripotent stem cells (iPSCs) from human fibroblasts by transfection of synthetic mRNA. The reprogramming of somatic cells requires transient expression of a set of transcription factors that collectively activate an endogenous gene regulatory network specifying the pluripotent phenotype. The necessary ectopic factor expression was first effected using retroviruses; however, as viral integration into the genome is problematic for cell therapy applications, the use of footprint-free vectors such as mRNA is increasingly preferred. Strong points of the mRNA approach include high efficiency, rapid kinetics, and obviation of a clean-up phase to purge the vector. Still, the method is relatively laborious and has, up to now, involved the use of feeder cells, which brings drawbacks including poor applicability to clinically oriented iPSC derivation. Using the methods described here, mRNA reprogramming can be performed without feeders at much-reduced labor and material costs relative to established protocols. Copyright © 2013 John Wiley & Sons, Inc.

  10. Human gingival fibroblasts express functional chemokine receptor CXCR6.

    PubMed

    Hosokawa, Y; Hosokawa, I; Ozaki, K; Nakae, H; Matsuo, T

    2009-06-01

    We have reported that CXCL16, a recently discovered transmembrane chemokine, is expressed in human gingival fibroblasts (HGF). However, it is not known whether HGF express CXCR6, the receptor for CXCL16, or CXCL16 affects HGF biology. We have shown that HGF expressed CXCR6 by reverse transcription-polymerase chain reaction and flow cytometric analysis. Moreover, we elucidated that tumour necrosis factor (TNF)-alpha and cytosine-guanine dinucleotide (CpG) DNA (Toll-like receptor-9 ligand) treatment enhanced CXCR6 expression by HGF. Interleukin (IL)-4, IL-13 and CpG DNA up-regulated CXCR6 expression by TNF-alpha-stimulated HGF. On the other hand, IL-1beta and interferon-gamma inhibited CXCR6 expression on TNF-alpha-treated HGF. CXCL16 treatment induced HGF proliferation and phosphorylation of extracellular regulated kinase (ERK) and protein kinase B (AKT) in HGF. In conclusion, HGF expressed CXCR6 functionally, because CXCL16 induced HGF proliferation and ERK and AKT phosphorylation in HGF. These results indicate that CXCL16 may play an important role in the pathogenesis and remodelling in periodontally diseased tissues.

  11. Lung Beractant Increases Free Cytosolic Levels of Ca2+ in Human Lung Fibroblasts

    PubMed Central

    Guzmán-Silva, Alejandro; Vázquez de Lara, Luis G.; Torres-Jácome, Julián; Vargaz-Guadarrama, Ajelet; Flores-Flores, Marycruz; Pezzat Said, Elias; Lagunas-Martínez, Alfredo; Mendoza-Milla, Criselda; Tanzi, Franco; Moccia, Francesco; Berra-Romani, Roberto

    2015-01-01

    Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca2+ signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca2+ concentration ([Ca2+]i) in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 μM) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 μg/ml. The application of beractant, at a concentration of 500 μg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a) a single Ca2+ spike which could be followed by b) Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cβ (PLCβ), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs) and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF. PMID:26230503

  12. Retinoic acid stimulation of human dermal fibroblast proliferation is dependent on suboptimal extracellular Ca2+ concentration.

    PubMed Central

    Varani, J.; Shayevitz, J.; Perry, D.; Mitra, R. S.; Nickoloff, B. J.; Voorhees, J. J.

    1990-01-01

    Human dermal fibroblasts failed to proliferate when cultured in medium containing 0.15 mmol/l (millimolar) Ca2+ (keratinocyte growth medium [KGM]) but did when the external Ca2+ concentration was raised to 1.4 mmol/l. All-trans retinoic acid (retinoic acid) stimulated proliferation in KGM but did not further stimulate growth in Ca2(+)-supplemented KGM. The ability of retinoic acid to stimulate proliferation was inhibited in KGM prepared without Ca2+ or prepared with 0.03 mmol/l Ca2+ and in KGM treated with 1 mmol/l ethylene-glycol-bis-(beta-aminoethyl ether)N,N'-tetra acetic acid. Using 45Ca2+ to measure Ca2+ influx and efflux, it was found that retinoic acid minimally increased Ca2+ uptake into fibroblasts. In contrast, retinoic acid treatment of fibroblasts that had been pre-equilibrated for 1 day with 45Ca2+ inhibited release of intracellular Ca2+ into the extracellular fluid. Retinoic acid also stimulated 35S-methionine incorporation into trichloroacetic acid-precipitable material but in contrast to its effect on proliferation, stimulation of 35S-methionine incorporation occurred in both high-Ca2+ and low-Ca2+ medium. These data indicate that retinoic acid stimulation of proliferation, but not protein synthesis, is dependent on the concentration of Ca2+ in the extracellular environment. PMID:2356860

  13. A co-culture system with three different primary human cell populations reveals that biomaterials and MSC modulate macrophage-driven fibroblast recruitment.

    PubMed

    Caires, Hugo R; Barros da Silva, Patrícia; Barbosa, Mário A; Almeida, Catarina R

    2018-03-01

    The biological response to implanted biomaterials is a complex and highly coordinated phenomenon involving many different cell types that interact within 3D microenvironments. Here, we increased the complexity of a 3D platform to include at least 3 cell types that play a role in the host response upon scaffold implantation. With this system, it was possible to address how immune responses triggered by 3D biomaterials mediate recruitment of stromal cells that promote tissue regeneration, mesenchymal stromal/stem cells (MSC), or a foreign body response, fibroblasts. Primary human macrophages yielded the highest fibroblast recruitment when interacting with chitosan scaffolds but not polylactic acid. Interestingly, when there were MSC and fibroblasts in the same environment, macrophages in chitosan scaffolds again promoted a significant increase on fibroblast recruitment, but not of MSC. However, macrophages that were firstly allowed to interact with MSC within the scaffolds were no longer able to recruit fibroblasts. This study illustrates the potential to use different scaffolds to regulate the dynamics of recruitment of proregenerative or fibrotic cell types through immunomodulation. Overall, this work strengths the idea that ex vivo predictive systems need to consider the different players involved in the biological response to biomaterials and that timing of arrival of specific cell types will affect the outcome. Copyright © 2017 John Wiley & Sons, Ltd.

  14. Trivalent chromium activates Rac-1 and Src and induces switch in the cell death mode in human dermal fibroblasts.

    PubMed

    Rudolf, Emil; Cervinka, Miroslav

    2009-08-10

    In this study we examined interactions between human dermal fibroblasts and chromium acetate hydroxide originating from environmental waste sediments. We show that initially exposure of fibroblasts to Cr (III) induced membrane-dependent signaling including activation of Rac1 GTPase, Src and apoptosis signal-regulating kinase 1 (ASK-1) kinases leading to increased activities of p38 and particularly Jun N-terminal kinase (JNK) and subsequent activation of caspase-3. At later treatment intervals (48-96 h), caspase-3 activity became suppressed and markedly increased lactate dehydrogenase (LDH) release was observed. Further experiments demonstrated that LDH release occurred in the presence of increased oxidative stress, extensive DNA damage, overactivation of poly(ADP-ribose)polymerase-1 (PARP-1) and depletion of ATP. Using specific inhibitors it was demonstrated that oxidative stress along with PARP-1 activity are responsible for cell death mode switch and upon their inhibition caspase-3 activity could be restored. In conclusion, Cr (III) seems to induce a biphasic response in dermal fibroblasts, with initial apoptosis switched to necrosis via increased DNA damage and resulting PARP-1 activity.

  15. Fibroblast Activation Protein Cleaves and Inactivates Fibroblast Growth Factor 21*

    PubMed Central

    Dunshee, Diana Ronai; Bainbridge, Travis W.; Kljavin, Noelyn M.; Zavala-Solorio, Jose; Schroeder, Amy C.; Chan, Ruby; Corpuz, Racquel; Wong, Manda; Zhou, Wei; Deshmukh, Gauri; Ly, Justin; Sutherlin, Daniel P.; Ernst, James A.; Sonoda, Junichiro

    2016-01-01

    FGF21 is a stress-induced hormone with potent anti-obesity, insulin-sensitizing, and hepatoprotective properties. Although proteolytic cleavage of recombinant human FGF21 in preclinical species has been observed previously, the regulation of endogenously produced FGF21 is not well understood. Here we identify fibroblast activation protein (FAP) as the enzyme that cleaves and inactivates human FGF21. A selective chemical inhibitor, immunodepletion, or genetic deletion of Fap stabilized recombinant human FGF21 in serum. In addition, administration of a selective FAP inhibitor acutely increased circulating intact FGF21 levels in cynomolgus monkeys. On the basis of our findings, we propose selective FAP inhibition as a potential therapeutic approach to increase endogenous FGF21 activity for the treatment of obesity, type 2 diabetes, non-alcoholic steatohepatitis, and related metabolic disorders. PMID:26797127

  16. Effect of the cytostatic agent idarubicin on fibroblasts of the human Tenon's capsule compared with mitomycin C.

    PubMed

    Heilmann, C; Schönfeld, P; Schlüter, T; Bohnensack, R; Behrens-Baumann, W

    1999-08-01

    To investigate the in vitro effect of a short time exposure to the anthracycline idarubicin on proliferation, protein synthesis, and motility of human Tenon's capsule fibroblasts in comparison with the antitumour antibiotic mitomycin C. After determination of effective concentrations of idarubicin, fibroblasts of the human Tenon's capsule were exposed to idarubicin or mitomycin C at concentrations ranging from 0.1 microg/ml to 1 microg/ml or from 2.5 microg/ml to 250 microg/ml, respectively, for 0.5, 2, or 5 minutes and cultured for 60 days. Cell death by apoptosis caused by idarubicin treatment was confirmed by Hoechst 33258 staining. Further proliferation was explored by cell counting and by (3)H-thymidine uptake. Protein synthesis was measured by (3)H-proline uptake and motility was assessed by agarose droplet motility assay. Idarubicin is able to exert toxicity and to induce apoptosis during a short time exposure of 0.5 minutes at concentrations of 0.3-1 microg/ml resulting in a significant reduction in cell number compared with the control after 60 days. For mitomycin C, higher concentrations and longer expositions were necessary. Even after treatment with 1 microg/ml idarubicin or 250 microg/ml mitomycin C a few cells were able to incorporate (3)H-thymidine. (3)H-proline uptake up to 10 days after exposure to 0.3 microg/ml idarubicin was found not to be decreased. Cell motility was reduced after treatment with 1 microg/ml idarubicin for 5 minutes or with 250 microg/ml mitomycin C for 2 or 5 minutes. For low mitomycin C concentrations, an increase in motility was found during the first 10 days. Idarubicin reduces proliferation of human Tenons's capsule fibroblasts after incubation for 0.5 minutes at concentrations as low as 0.3-1 microg/ml. In comparison, mitomycin C requires longer exposure times and higher doses for equal results. Therefore, idarubicin may be useful in the prevention of glaucoma filtering surgery failure.

  17. Minoxidil specifically decreases the expression of lysine hydroxylase in cultured human skin fibroblasts.

    PubMed Central

    Hautala, T; Heikkinen, J; Kivirikko, K I; Myllylä, R

    1992-01-01

    The levels of lysine hydroxylase protein and the levels of the mRNAs for lysine hydroxylase and the alpha- and beta-subunits of proline 4-hydroxylase were measured in cultured human skin fibroblasts treated with 1 mM-minoxidil. The data demonstrate that minoxidil decreases the amount of lysine hydroxylase protein, this being due to a decrease in the level of lysine hydroxylase mRNA. The effect of minoxidil appears to be highly specific, as no changes were observed in the amounts of mRNAs for the alpha- and beta-subunits of proline 4-hydroxylase. Images Fig. 1. Fig. 2. Fig. 3. PMID:1314568

  18. The influence of SrO and CaO in silicate and phosphate bioactive glasses on human gingival fibroblasts.

    PubMed

    Massera, J; Kokkari, A; Närhi, T; Hupa, L

    2015-06-01

    In this paper, we investigate the effect of substituting SrO for CaO in silicate and phosphate bioactive glasses on the human gingival fibroblast activity. In both materials the presence of SrO led to the formation of a CaP layer with partial Sr substitution for Ca. The layer at the surface of the silicate glass consisted of HAP whereas at the phosphate glasses it was close to the DCPD composition. In silicate glasses, SrO gave a faster initial dissolution and a thinner reaction layer probably allowing for a continuous ion release into the solution. In phosphate glasses, SrO decreased the dissolution process and gave a more strongly bonded reaction layer. Overall, the SrO-containing silicate glass led to a slight enhancement in the activity of the gingival fibroblasts cells when compared to the SrO-free reference glass, S53P4. The cell activity decreased up to 3 days of culturing for all phosphate glasses containing SrO. Whereas culturing together with the SrO-free phosphate glass led to complete cell death at 7 days. The glasses containing SrO showed rapid cell proliferation and growth between 7 and 14 days, reaching similar activity than glass S53P4. The addition of SrO in both silicate and phosphate glasses was assumed beneficial for proliferation and growth of human gingival fibroblasts due to Sr incorporation in the reaction layer at the glass surface and released in the cell culture medium.

  19. C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice.

    PubMed

    Kimura, Toru; Nojiri, Takashi; Hino, Jun; Hosoda, Hiroshi; Miura, Koichi; Shintani, Yasushi; Inoue, Masayoshi; Zenitani, Masahiro; Takabatake, Hiroyuki; Miyazato, Mikiya; Okumura, Meinoshin; Kangawa, Kenji

    2016-02-19

    Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts. C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription. CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22

  20. Curcumin inhibits TGF-β1-induced connective tissue growth factor expression through the interruption of Smad2 signaling in human gingival fibroblasts.

    PubMed

    Chen, Jung-Tsu; Wang, Chen-Ying; Chen, Min-Huey

    2018-01-13

    Many fibrotic processes are associated with an increased level of transforming growth factor-β1 (TGF-β1). TGF-β1 can increase synthesis of matrix proteins and enhance secretion of protease inhibitors, resulting in matrix accumulation. Connective tissue growth factor (CTGF) is a downstream profibrotic effector of TGF-β1 and is associated with the fibrosis in several human organs. Curcumin has been applied to reduce matrix accumulation in fibrotic diseases. This study was aimed to evaluate whether curcumin could suppress TGF-β1-induced CTGF expression and its related signaling pathway involving in this inhibitory action in primary human gingival fibroblasts. The differences in CTGF expression among three types of gingival overgrowth and normal gingival tissues were assessed by immunohistochemistry. Gingival fibroblast viability in cultured media with different concentrations of curcumin was studied by MTT assay. The effect of curcumin on TGF-β1-induced CTGF expression in primary human gingival fibroblasts was examined by immunoblotting. Moreover, the proteins involved in TGF-β1 signaling pathways including TGF-β1 receptors and Smad2 were also analyzed by immunoblotting. CTGF was highly expressed in fibroblasts, epithelial cells and some of endothelial cells, smooth muscle cells, and inflammatory cells in phenytoin-induced gingival overgrowth tissues rather than in those of hereditary and inflammatory gingival overgrowth tissues. Moreover, CTGF expression in the epithelial and connective tissue layers was higher in phenytoin-induced gingival overgrowth tissues than in normal gingival tissues. Curcumin was nontoxic and could reduce TGF-β1-induced CTGF expression by attenuating the phosphorylation and nuclear translocation of Smad2. Curcumin can suppress TGF-β1-induced CTGF expression through the interruption of Smad2 signaling. Copyright © 2018. Published by Elsevier B.V.

  1. Endothelin-1 stimulates colon cancer adjacent fibroblasts.

    PubMed

    Knowles, Jonathan P; Shi-Wen, Xu; Haque, Samer-ul; Bhalla, Ashish; Dashwood, Michael R; Yang, Shiyu; Taylor, Irving; Winslet, Marc C; Abraham, David J; Loizidou, Marilena

    2012-03-15

    Endothelin-1 (ET-1) is produced by and stimulates colorectal cancer cells. Fibroblasts produce tumour stroma required for cancer development. We investigated whether ET-1 stimulated processes involved in tumour stroma production by colonic fibroblasts. Primary human fibroblasts, isolated from normal tissues adjacent to colon cancers, were cultured with or without ET-1 and its antagonists. Cellular proliferation, migration and contraction were measured. Expression of enzymes involved in tumour stroma development and alterations in gene transcription were determined by Western blotting and genome microarrays. ET-1 stimulated proliferation, contraction and migration (p < 0.01 v control) and the expression of matrix degrading enzymes TIMP-1 and MMP-2, but not MMP-3. ET-1 upregulated genes for profibrotic growth factors and receptors, signalling molecules, actin modulators and extracellular matrix components. ET-1 stimulated colonic fibroblast cellular processes in vitro that are involved in developing tumour stroma. Upregulated genes were consistent with these processes. By acting as a strong stimulus for tumour stroma creation, ET-1 is proposed as a target for adjuvant cancer therapy. Copyright © 2011 UICC.

  2. Cryopreservation of dermal fibroblasts and keratinocytes in hydroxyethyl starch-based cryoprotectants.

    PubMed

    Naaldijk, Yahaira; Johnson, Adiv A; Friedrich-Stöckigt, Annett; Stolzing, Alexandra

    2016-12-01

    Preservation of human skin fibroblasts and keratinocytes is essential for the creation of skin tissue banks. For successful cryopreservation of cells, selection of an appropriate cryoprotectant agent (CPA) is imperative. The aim of this study was to identify CPAs that minimize toxic effects and allow for the preservation of human fibroblasts and keratinocytes in suspension and in monolayers. We cryopreserved human fibroblasts and keratinocytes with different CPAs and compared them to fresh, unfrozen cells. Cells were frozen in the presence and absence of hydroxyethyl starch (HES) or dimethyl sulfoxide (DMSO), the latter of which is a commonly used CPA known to exert toxic effects on cells. Cell numbers were counted immediately post-thaw as well as three days after thawing. Cellular structures were analyzed and counted by labeling nuclei, mitochondria, and actin filaments. We found that successful cryopreservation of suspended or adherent keratinocytes can be accomplished with a 10% HES or a 5% HES, 5% DMSO solution. Cell viability of fibroblasts cryopreserved in suspension was maintained with 10% HES or 5% HES, 5% DMSO solutions. Adherent, cryopreserved fibroblasts were successfully maintained with a 5% HES, 5% DMSO solution. We conclude that skin tissue cells can be effectively cryopreserved by substituting all or a portion of DMSO with HES. Given that DMSO is the most commonly used CPA and is believed to be more toxic than HES, these findings are of clinical significance for tissue-based replacement therapies. Therapies that require the use of keratinocyte and fibroblast cells, such as those aimed at treating skin wounds or skin burns, may be optimized by substituting a portion or all of DMSO with HES during cryopreservation protocols.

  3. The observed human sperm mutation frequency cannot explain the achondroplasia paternal age effect

    PubMed Central

    Tiemann-Boege, Irene; Navidi, William; Grewal, Raji; Cohn, Dan; Eskenazi, Brenda; Wyrobek, Andrew J.; Arnheim, Norman

    2002-01-01

    The lifelong spermatogonial stem cell divisions unique to male germ cell production are thought to contribute to a higher mutation frequency in males. The fact that certain de novo human genetic conditions (e.g., achondroplasia) increase in incidence with the age of the father is consistent with this idea. Although it is assumed that the paternal age effect is the result of an increasing frequency of mutant sperm as a man grows older, no direct molecular measurement of the germ-line mutation frequency has been made to confirm this hypothesis. Using sperm DNA from donors of different ages, we determined the frequency of the nucleotide substitution in the fibroblast growth factor receptor 3 (FGFR3) gene that causes achondroplasia. Surprisingly, the magnitude of the increase in mutation frequency with age appears insufficient to explain why older fathers have a greater chance of having a child with this condition. A number of alternatives may explain this discrepancy, including selection for sperm that carry the mutation or an age-dependent increase in premutagenic lesions that remain unrepaired in sperm and are inefficiently detected by the PCR assay. PMID:12397172

  4. Induced pluripotent stem cells from goat fibroblasts.

    PubMed

    Song, Hui; Li, Hui; Huang, Mingrui; Xu, Dan; Gu, Chenghao; Wang, Ziyu; Dong, Fulu; Wang, Feng

    2013-12-01

    Embryonic stem cells (ESCs) are a powerful model for genetic engineering, studying developmental biology, and modeling disease. To date, ESCs have been established from the mouse (Evans and Kaufman, 1981, Nature 292:154-156), non-human primates (Thomson et al., , Proc Nat Acad Sci USA 92:7844-7848), humans (Thomson et al., 1998, Science 282:1145-1147), and rats (Buehr et al., , Cell 135:1287-1298); however, the derivation of ESCs from domesticated ungulates such as goats, sheep, cattle, and pigs have not been successful. Alternatively, induced pluripotent stem cells (iPSCs) can be generated by reprogramming somatic cells with several combinations of genes encoding transcription factors (OCT3/4, SOX2, KLF4, cMYC, LIN28, and NANOG). To date, iPSCs have been isolated from various species, but only limited information is available regarding goat iPSCs (Ren et al., 2011, Cell Res 21:849-853). The objectives of this study were to generate goat iPSCs from fetal goat primary ear fibroblasts using lentiviral transduction of four human transcription factors: OCT4, SOX2, KLF4, and cMYC. The goat iPSCs were successfully generated by co-culture with mitomycin C-treated mouse embryonic fibroblasts using medium supplemented with knockout serum replacement and human basic fibroblast growth factor. The goat iPSCs colonies are flat, compact, and closely resemble human iPSCs. They have a normal karyotype; stain positive for alkaline phosphatase, OCT4, and NANOG; express endogenous pluripotency genes (OCT4, SOX2, cMYC, and NANOG); and can spontaneously differentiate into three germ layers in vitro and in vivo. © 2013 Wiley Periodicals, Inc.

  5. Effects of mitomycin-C on normal dermal fibroblasts.

    PubMed

    Chen, Theodore; Kunnavatana, Shaun S; Koch, R James

    2006-04-01

    To evaluate the effects of mitomycin-C on the growth and autocrine growth factor production of human dermal fibroblasts from the face. In vitro study using normal adult dermal fibroblast cell lines in a serum-free model. Cell cultures were exposed to 4 mg/mL, 0.4 mg/mL, 0.04 mg/mL, 0.004 mg/mL, and 0.0004 mg/mL concentrations of mitomycin-C solution. Cell counts were performed, and the cell-free supernatants were collected at 0, 1, 3, and 5 days after the initial exposure. Population doubling times were calculated and supernatants were quantitatively assayed for basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-beta1. Continuous exposure to mitomycin-C caused fibroblast cell death by day 7 at all tested concentrations. A 4 minute exposure to mitomycin-C at 4 mg/mL caused rapid fibroblast cell death. A 4-minute exposure to mitomycin-C at either 0.4 mg/mL or 0.04 mg/mL resulted in decreased fibroblast proliferation. A 4 minute exposure to mitomycin-C at 0.4 mg/mL resulted in a marked increase in the production of both bFGF and TGF-beta1. A clinically ideal concentration of mitomycin-C would slow fibroblast proliferation yet not cause cell death to allow for a wound healing response. Mitomycin-C 0.4 mg/mL for 4 minutes satisfies the above criteria in vitro.

  6. Chromosome Conformation of Human Fibroblasts Grown in 3-Dimensional Spheroids

    PubMed Central

    Chen, Haiming; Comment, Nicholas; Chen, Jie; Ronquist, Scott; Hero, Alfred; Ried, Thomas; Rajapakse, Indika

    2015-01-01

    In the study of interphase chromosome organization, genome-wide chromosome conformation capture (Hi-C) maps are often generated using 2-dimensional (2D) monolayer cultures. These 2D cells have morphological deviations from cells that exist in 3-dimensional (3D) tissues in vivo, and may not maintain the same chromosome conformation. We used Hi-C maps to test the extent of differences in chromosome conformation between human fibroblasts grown in 2D cultures and those grown in 3D spheroids. Significant differences in chromosome conformation were found between 2D cells and those grown in spheroids. Intra-chromosomal interactions were generally increased in spheroid cells, with a few exceptions, while inter-chromosomal interactions were generally decreased. Overall, chromosomes located closer to the nuclear periphery had increased intra-chromosomal contacts in spheroid cells, while those located more centrally had decreased interactions. This study highlights the necessity to conduct studies on the topography of the interphase nucleus under conditions that mimic an in vivo environment. PMID:25738643

  7. Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing.

    PubMed

    Micera, Alessandra; Lambiase, Alessandro; Puxeddu, Ilaria; Aloe, Luigi; Stampachiacchiere, Barbara; Levi-Schaffer, Francesca; Bonini, Sergio; Bonini, Stefano

    2006-10-01

    In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing.

  8. Effect of a feeder layer composed of mouse embryonic and human foreskin fibroblasts on the proliferation of human embryonic stem cells.

    PubMed

    Yang, Hua; Qiu, Ying; Zeng, Xianghui; Ding, Yan; Zeng, Jianye; Lu, Kehuan; Li, Dongsheng

    2016-06-01

    The aim of the present study was to investigate the effects of feeder layers composed of various ratios of mouse embryonic fibroblasts (MEFs) and human foreskin fibroblasts (hFFs) on the growth of human embryonic stem cells (hESCs). In addition, the secretion levels of basic fibroblast growth factor (bFGF) by the feeder layers was detected. MEFs and hFFs were treated with mitomycin C and seeded onto gelatin-coated plates at a density of 1×10 8 cells/l. The hFFs and MEFs were combined and plated at the following ratios: 0:1, 1:2, 1:1, 2:1 and 1:0. The secretion of bFGF by the various hFF/MEF ratio feeder layers was detected using an enzyme-linked immunosorbent assay. Subsequently, hESCs were cultured on top of the various feeder layers. The differences in the cellular morphology of the hESCs were observed using microscopy, and the expression levels alkaline phosphatase (AKP) and octamer-binding transcription factor 4 (OCT-4) were detected using immunohistochemical analysis as indicators of differentiation status. The results showed that the hFFs secreted substantial quantities of bFGF, while no bFGF was secreted by the MEFs. The clones of hESC growing on the feeder layer containing MEF or hFF alone were flat. By contrast, hESC clones grown on a mixed feeder layer containing hFFs + MEFs at a ratio of 1:1 exhibited an accumulated growth with a clear edge, as compared with the other ratios. In addition, hESCs growing on the feeder layer were positive for the expression of AKP and OCT-4. In summary, feeder layer hFFs secreted bFGF, while MEFs did not, indicating that bFGF is not the only factor that supports the growth and differentiation of hESCs. The optimal growth of hESCs was achieved using a mixed feeder layer composed of hFFs + MEFs at a ratio of 1:1.

  9. Short form FLICE-inhibitory protein promotes TNFα-induced necroptosis in fibroblasts derived from CFLARs transgenic mice.

    PubMed

    Shindo, Ryodai; Yamazaki, Soh; Ohmuraya, Masaki; Araki, Kimi; Nakano, Hiroyasu

    2016-11-04

    Cellular FLICE-inhibitory protein (cFLIP) is a catalytically inactive homolog of the initiator caspase, caspase 8 and blocks apoptosis through binding to caspase 8. Human CFLAR gene encodes two proteins, a long form cFLIP (cFLIP L ) and a short form cFLIP (cFLIPs) due to an alternative splicing. Recent studies have shown that expression of cFLIPs, but not cFLIP L promotes programmed necrosis (also referred to as necroptosis) in an immortalized human keratinocyte cell line, HaCaT. Here, we found that expression of cFLIPs similarly promoted necroptosis in immortalized fibroblasts. To further expand this observation and exclude the possibility that immortalization process of keratinocytes or fibroblasts might affect the phenotype induced by cFLIPs expression, we generated human CFLARs transgenic (Tg) mice. Primary fibroblasts derived from CFLARs Tg mice were increased in susceptibility to TNFα-induced necroptosis, but not apoptosis compared to wild-type (WT) fibroblasts. Moreover, hallmarks of necroptosis, such as phosphorylation of receptor-interacting protein kinase (RIPK)1 and RIPK3, and oligomer formation of mixed lineage kinase domain-like (MLKL) were robustly induced in CFLARs Tg fibroblasts compared to wild-type fibroblasts following TNFα stimulation. Thus, cFLIPs-dependent promotion of necroptosis is not unique to immortalized keratinocytes or fibroblasts, but also to generalized to primary fibroblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Immortalization of normal human embryonic fibroblasts by introduction of either the human papillomavirus type 16 E6 or E7 gene alone.

    PubMed

    Yamamoto, Akito; Kumakura, Shin-ichi; Uchida, Minoru; Barrett, J Carl; Tsutsui, Takeki

    2003-09-01

    The ability of the human papillomavirus type 16 (HPV-16) E6 or E7 gene to induce immortalization of normal human embryonic fibroblast WHE-7 cells was examined. WHE-7 cells at 9 population doublings (PD) were infected with retrovirus vectors encoding either HPV-16 E6 or E7 alone or both E6 and E7 (E6/E7). One of 4 isolated clones carrying E6 alone became immortal and is currently at >445 PD. Four of 4 isolated clones carrying E7 alone escaped from crisis and are currently at >330 PD. Three of 5 isolated clones carrying E6/E7 were also immortalized and are currently at >268 PD. The immortal clone carrying E6 only and 2 of the 3 immortal clones carrying E6/E7 expressed a high level of E6 protein, and all the immortal clones carrying E7 alone and the other immortal clone carrying E6/E7 expressed a high level of E7 protein when compared to their mortal or precrisis clones. The immortal clones expressing a high level of E6 or E7 protein were positive for telomerase activity or an alternative mechanism of telomere maintenance, respectively, known as ALT (alternative lengthening of telomeres). All the mortal or precrisis clones were negative for both phenotypes. All the immortal clones exhibited abrogation of G1 arrest after DNA damage by X-ray irradiation. The expression of INK4a protein (p16(INK4a)) was undetectable in the E6-infected mortal and immortal clones, whereas Rb protein (pRb) was hyperphosphorylated only in the immortal clone. The p16(INK4a) protein was overexpressed in all the E7-infected immortal clones and their clones in the pre-crisis period as well as all the E6/E7-infected mortal and immortal clones, but the pRb expression was downregulated in all of these clones. These results demonstrate for the first time to our knowledge that HPV-16 E6 or E7 alone can induce immortalization of normal human embryonic fibroblasts. Inactivation of p16(INK4a)/pRb pathways in combination with activation of a telomere maintenance mechanism is suggested to be necessary for

  11. Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

    PubMed Central

    Wang, Yao-wen; Ren, Ji-hao; Xia, Kun; Wang, Shu-hui; Yin, Tuan-fang; Xie, Ding-hua; Li, Li-hua

    2012-01-01

    Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell), particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast. Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro. Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution, and serum-free culture medium was used as control. The cellular morphology change, growth characteristics, cell proliferation, and apoptosis were observed at different intervals. For the fibroblasts, the mRNA expression changes of transforming growth factor (TGF)-β1, basic fibroblast growth factor (bFGF), procollagen I, and III were detected by reverse transcription polymerase chain reaction (RT-PCR). Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially. A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells. Cell morphology changed, cell density decreased, and the growth curves were without an exponential phase. The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml. Meanwhile, 5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis. The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05). A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1, procollagen I and III, and a marked increase in the mRNA production of bFGF. Conclusions: Mitomycin can inhibit fibroblast proliferation, induce fibroblast apoptosis, and regulate intracellular protein expression on mRNA levels. In additon, mitomycin can inhibit HaCat cell proliferation, so epithelial cell needs more protecting to avoid mitomycin’s side effect when it is applied clinically. PMID

  12. The attachment of V79 and human periodontal ligament fibroblasts on periodontally involved root surfaces following treatment with EDTA, citric acid, or tetracycline HCL: an SEM in vitro study.

    PubMed

    Chandra, R Viswa; Jagetia, Ganesh Chandra; Bhat, K Mahalinga

    2006-02-15

    The present in vitro study has been designed to establish and compare the effects of citric acid, EDTA, and tetracycline HCl on human periodontally diseased roots on the structure, attachment, and orientation of V79 (primary Chinese hamster lung fibroblasts) cells and human periodontal ligament fibroblasts (HPDL). Commercially available V79 cells and HPDL derived from healthy human third molars were used in this study. These fibroblasts were left in solution for seven days in order to attain confluence. Forty single-rooted teeth were obtained from patients diagnosed with periodontitis. The crown part was removed under constant irrigation and the root was split vertically into two equal halves, thus, yielding 80 specimens. Following scaling and root planing, the specimens were washed with phosphate buffered saline (PBS) and kept in 50 microg/ml gentamycin sulphate solution for 24 hours. The root pieces were then treated as follows: citric acid at pH 1, 24% EDTA, or with a 10% solution of tetracycline HCl and were then placed in V79 fibroblast cultures and HPDL cultures. The specimens were harvested after four weeks and were fixed in 2.5% glutaraldehyde in PBS before preparation for scanning electron microscopy (SEM). The behavior of V79 cells was similar to that of human periodontal ligament cells on root conditioned surfaces. V79 and HPDL showed a healthy morphology on root surfaces treated with citric acid and EDTA and a relatively unhealthy appearance on root surfaces treated with tetracycline HCl and distilled water (control group). The results suggest the use of citric acid and EDTA as root conditioning agents favorably affects the migration, attachment, and morphology of fibroblasts on human root surfaces, which may play a significant role in periodontal healing and regeneration.

  13. Effect of mitomycin C on keloid fibroblasts: an in vitro study.

    PubMed

    Simman, Richard; Alani, Hashim; Williams, Frances

    2003-01-01

    Keloids are the result of aberrant wound healing of human skin after dermal injury. Therapeutic options include excision followed by radiation therapy, steroid injection, and compression with silicone sheets among others. Local invasion and recurrence after excision has provoked interest in treating keloids as neoplasms. The purpose of this study was to determine the effect of mitomycin C (MMC) on keloid fibroblasts. Keloid fibroblasts obtained from five different patients were exposed to MMC. A control group of normal and keloid cells was treated with phosphate buffered saline only. Contrast microscopy showed a decrease of fibroblast density during the 3 weeks after exposure for normal and keloid fibroblasts relative to untreated fibroblasts. This was confirmed by total cell counts ( = 0.1) and measurement of DNA synthesis. By the third week, there was a recovery in DNA synthesis and increased cell count for some of the treated fibroblasts. We concluded that at an appropriate concentration, MMC shows proliferation of keloid fibroblasts in vitro for a period of 3 weeks. This agent may be considered in clinical trials after surgical excision of keloids.

  14. Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro.

    PubMed

    Park, Hyun-Jin; Salem, Mabrouka; Semlali, Abdelhabib; Leung, Kai P; Rouabhia, Mahmoud

    2017-07-01

    We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Pirfenidone induces intercellular adhesion molecule-1 (ICAM-1) down-regulation on cultured human synovial fibroblasts

    PubMed Central

    Kaneko, M; Inoue, H; Nakazawa, R; Azuma, N; Suzuki, M; Yamauchi, S; Margolin, S B; Tsubota, K; Saito, I

    1998-01-01

    Pirfenidone has been shown to modify some cytokine regulatory actions and inhibit fibroblast biochemical reactions resulting in inhibition of proliferation and collagen matrix synthesis by fibroblast. We have investigated the effect of pirfenidone on the expression of cell adhesion molecules. The synovial fibroblasts were treated with IL-1α in the presence or absence of pirfenidone (range 0–1000 μm), and assayed for the expression of adhesion molecules such as ICAM-1 and endothelial-leucocyte adhesion molecule-1 (E-selectin) by cell ELISA. Pirfenidone significantly down-regulated the expression of ICAM-1 on cultured synovial fibroblasts in a dose-dependent manner. In contrast, expression of E-selectin was not affected. Furthermore, we examined whether pirfenidone affects the cellular binding between cultured lymphocytes and IL-1α-stimulated synovial fibroblasts by in vitro binding assay and found their mutual binding was significantly suppressed in a dose-dependent manner by pirfenidone. It is speculated that down-regulation of ICAM-1 might be one of the novel mechanisms of action of pirfenidone. These data indicate a novel mechanism of action for pirfenidone to reduce the activation of synovial fibroblasts. PMID:9697986

  16. Tissue factor-dependent vascular endothelial growth factor production by human fibroblasts in response to activated factor VII.

    PubMed

    Ollivier, V; Bentolila, S; Chabbat, J; Hakim, J; de Prost, D

    1998-04-15

    The transmembrane protein tissue factor (TF) is the cell surface receptor for coagulation factor VII (FVII) and activated factor VII (FVIIa). Recently, TF has been identified as a regulator of angiogenesis, tumor growth, and metastasis. This study was designed to link the binding of FVII(a) to its receptor, TF, with the subsequent triggering of angiogenesis through vascular endothelial growth factor (VEGF) production by human lung fibroblasts. We report that incubation of fibroblasts, which express constitutive surface TF, with FVII(a) induces VEGF synthesis. FVII(a)-induced VEGF secretion, assessed by a specific enzyme-linked immunosorbent assay, was time- and concentration-dependent. VEGF secretion was maximal after 24 hours of incubation of the cells with 100 nmol/L FVII(a) and represented a threefold induction of the basal VEGF level. Reverse transcriptase-polymerase chain reaction analysis of VEGF detected three mRNA species of 180, 312, and 384 bp corresponding, respectively, to VEGF121, VEGF165, and VEGF189. A 2.5- to 3.5-fold increase was observed for the 180- and 312-bp transcripts at 12 and 24 hours, respectively. FVII(a)-dependent VEGF production was inhibited by a pool of antibodies against TF, pointing to the involvement of this receptor. On specific active-site inhibition with dansyl-glutamyl-glycinyl-arginyl chloromethyl ketone, FVIIa lost 70% of its capacity to elicit VEGF production. Consistent with this, the native form (zymogen) of FVII only had a 1.8-fold stimulating effect. Protein tyrosine kinase and protein kinase C are involved in signal transduction leading to VEGF production, as shown by the inhibitory effects of genistein and GF 109203X. The results of this study indicate that TF is essential for VIIa-induced VEGF production by human fibroblasts and that its role is mainly linked to the proteolytic activity of the TF-VIIa complex.

  17. Adiponectin Attenuates Lung Fibroblasts Activation and Pulmonary Fibrosis Induced by Paraquat

    PubMed Central

    He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose

  18. Adiponectin attenuates lung fibroblasts activation and pulmonary fibrosis induced by paraquat.

    PubMed

    Yao, Rong; Cao, Yu; He, Ya-rong; Lau, Wayne Bond; Zeng, Zhi; Liang, Zong-an

    2015-01-01

    Pulmonary fibrosis is one of the most common complications of paraquat (PQ) poisoning, which demands for more effective therapies. Accumulating evidence suggests adiponectin (APN) may be a promising therapy against fibrotic diseases. In the current study, we determine whether the exogenous globular APN isoform protects against pulmonary fibrosis in PQ-treated mice and human lung fibroblasts, and dissect the responsible underlying mechanisms. BALB/C mice were divided into control group, PQ group, PQ + low-dose APN group, and PQ + high-dose APN group. Mice were sacrificed 3, 7, 14, and 21 days after PQ treatment. We compared pulmonary histopathological changes among different groups on the basis of fibrosis scores, TGF-β1, CTGF and α-SMA pulmonary content via Western blot and real-time quantitative fluorescence-PCR (RT-PCR). Blood levels of MMP-9 and TIMP-1 were determined by ELISA. Human lung fibroblasts WI-38 were divided into control group, PQ group, APN group, and APN receptor (AdipoR) 1 small-interfering RNA (siRNA) group. Fibroblasts were collected 24, 48, and 72 hours after PQ exposure for assay. Cell viability and apoptosis were determined via Kit-8 (CCK-8) and fluorescein Annexin V-FITC/PI double labeling. The protein and mRNA expression level of collagen type III, AdipoR1, and AdipoR2 were measured by Western blot and RT-PCR. APN treatment significantly decreased the lung fibrosis scores, protein and mRNA expression of pulmonary TGF-β1, CTGF and α-SMA content, and blood MMP-9 and TIMP-1 in a dose-dependent manner (p<0.05). Pretreatment with APN significantly attenuated the reduced cell viability and up-regulated collagen type III expression induced by PQ in lung fibroblasts, (p<0.05). APN pretreatment up-regulated AdipoR1, but not AdipoR2, expression in WI-38 fibroblasts. AdipoR1 siRNA abrogated APN-mediated protective effects in PQ-exposed fibroblasts. Taken together, our data suggests APN protects against PQ-induced pulmonary fibrosis in a dose

  19. Soy milk as a storage medium to preserve human fibroblast cell viability: an in vitro study.

    PubMed

    Moura, Camilla Christian Gomes; Soares, Priscilla Barbosa Ferreira; Reis, Manuella Verdinelli de Paula; Fernandes Neto, Alfredo Júlio; Soares, Carlos José

    2012-01-01

    Soy milk (SM) is widely consumed worldwide as a substitute for cow milk. It is a source of vitamins, carbohydrates and sugars, but its capacity to preserve cell viability has not been evaluated. The purpose of the present study was to investigate the efficacy of SM to maintain the viability of human fibroblasts at short periods compared with different cow milks. Human mouth fibroblasts were cultured and stored in the following media at room temperature: 10% Dulbecco's Modified Eagle Medium (DMEM) (positive control group); long shelf-life ultra-high temperature whole cow milk (WM); long shelf-life ultra-high temperature skim cow milk (SKM); powdered cow milk (PM); and soy milk (SM). After 5, 15, 30 and 45 min, cell viability was analyzed using the MTT assay. Data were analyzed statistically by the Kruskal-Wallis test with post-analysis using the Dunn's method (α=0.05). SKM showed the lowest capacity to maintain cell viability in all analyzed times (p<0.05). At 30 and 45 min, the absorbance levels in control group (DMEM) and SM were significantly higher than in SKM (p<0.05). Cell viability decreased along the time (5-45 min). The results indicate that SM can be used as a more adequate storage medium for avulsed teeth. SKM was not as effective in preserving cell viability as the cell culture medium and SM.

  20. Combined CSL and p53 downregulation promotes cancer-associated fibroblast activation

    PubMed Central

    Procopio, Maria-Giuseppina; Laszlo, Csaba; Labban, Dania Al; Kim, Dong Eun; Bordignon, Pino; Jo, Seunghee; Goruppi, Sandro; Menietti, Elena; Ostano, Paola; Ala, Ugo; Provero, Paolo; Hoetzenecker, Wolfram; Neel, Victor; Kilarski, Witek; Swartz, Melody A.; Brisken, Cathrin; Lefort, Karine; Dotto, G. Paolo

    2015-01-01

    Stromal fibroblast senescence has been linked to aging-associated cancer risk. However, density and proliferation of cancer-associated fibroblasts (CAF) are frequently increased. Loss or down-modulation of the Notch effector CSL/RBP-Jκ in dermal fibroblasts is sufficient for CAF activation and ensuing keratinocyte-derived tumors. We report that CSL silencing induces senescence of primary fibroblasts from dermis, oral mucosa, breast and lung. CSL functions in these cells as direct repressor of multiple senescence- and CAF-effector genes. It also physically interacts with p53, repressing its activity. CSL is down-modulated in stromal fibroblasts of premalignant skin actinic keratosis lesions and squamous cell carcinomas (SCC), while p53 expression and function is down-modulated only in the latter, with paracrine FGF signaling as likely culprit. Concomitant loss of CSL and p53 overcomes fibroblast senescence, enhances expression of CAF effectors and promotes stromal and cancer cell expansion. The findings support a CAF activation/stromal co-evolution model under convergent CSL/p53 control. PMID:26302407

  1. Cytotoxic effects of denture adhesives on primary human oral keratinocytes, fibroblasts and permanent L929 cell lines.

    PubMed

    Chen, Fengying; Wu, Tianfu; Cheng, Xiangrong

    2014-03-01

    To date, there have been very little data on the cytotoxic responses of different cell lines to denture adhesives. To determine the cytotoxicity of three denture adhesives on primary human oral keratinocytes (HOKs), fibroblasts (HOFs) and permanent mouse fibroblasts cell lines (L929). Three commercial denture adhesives (two creams and one powder) were prepared for indirect contact using the agar diffusion test, as well as extracts in MTT assay. The results of the MTT assay were statistically analysed by one-way anova and Tukey's test (p < 0.05). All of the tested denture adhesives showed mild to moderate cytotoxicity to primary HOKs (p < 0.001), whereas none of three was toxic to L929 cells (p > 0.05) in both assays. For primary HOFs cultures, slight cytotoxicity was observed for one of the products from the agar diffusion test and undiluted eluates of all tested adhesives with MTT assay (p < 0.01). Denture adhesives are toxic to the primary HOKs and HOFs cultures, whereas non-toxic to L929 cells. The results suggest that primary human oral mucosal cells may provide more valuable information in toxicity screening of denture adhesives. © 2012 John Wiley & Sons A/S and The Gerodontology Association. Published by John Wiley & Sons Ltd.

  2. Photocytotoxicity in human dermal fibroblasts elicited by permanent makeup inks containing titanium dioxide.

    PubMed

    Wamer, Wayne G; Yin, Jun-Jie

    2011-01-01

    Titanium dioxide (TiO2) is a pigment widely used in decorative tattoo and permanent makeup inks. However, little is known about the risks associated with its presence in these products. We have developed an in vitro assay to identify inks containing TiO2 that are cytotoxic and/or photocytotoxic. The presence of TiO2 in ten permanent makeup inks was established by X-ray fluorescence. Using X-ray diffraction, we found that seven inks contained predominately TiO2 (anatase), the more photocatalytically active crystalline form of TiO2. The remaining inks contained predominately TiO2 (rutile). To identify cytotoxic and/or photocytotoxic inks, human dermal fibroblasts were incubated for 18 h in media containing inks or pigments isolated from inks. Fibroblasts were then irradiated with 10 J/cm2 UVA radiation combined with 45 J/cm2 visible light for determining photocytotoxicity, or kept in the dark for determining cytotoxicity. Toxicity was assessed as inhibition of colony formation. No inks were cytotoxic. However eight inks, and the pigments isolated from these inks, were photocytotoxic. Using ESR, we found that most pigments from photocytotoxic inks generated hydroxyl radicals when photoexcited with UV radiation. Therefore, the possibility of photocytotoxicity should be considered when evaluating the safety of permanent makeup inks containing TiO2.

  3. 8,12;8,20-diepoxy-8,14-secopregnane glycosides from roots of Asclepias tuberosa and their effect on proliferation of human skin fibroblasts.

    PubMed

    Warashina, Tsutomu; Umehara, Kaoru; Miyase, Toshio; Noro, Tadataka

    2011-10-01

    A pregnane glycoside fraction from the roots of Asclepias tuberosa L. caused normal human skin fibroblasts to proliferate. This fraction contained 21 pregnane glycosides whose structures were established using NMR spectroscopic analysis and chemical evidence. The aglycones of most of these compounds were identified as 8,12;8,20-diepoxy-8,14-secopregnanes, such as tuberogenin or 5,6-didehydrotuberogenin, the same aglycones as constituents of the aerial parts of this plant. Some of these compounds also caused proliferation of skin fibroblasts. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. Spiral-Wave Dynamics in a Mathematical Model of Human Ventricular Tissue with Myocytes and Fibroblasts

    PubMed Central

    Nayak, Alok Ranjan; Shajahan, T. K.; Panfilov, A. V.; Pandit, Rahul

    2013-01-01

    Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as , the fibroblast resting-membrane potential, the fibroblast conductance , and the MF gap-junctional coupling . Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as , and , and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity decreases as a function of , for zero-sided and one-sided couplings; however, for two-sided coupling, decreases initially and then increases as a function of , and, eventually, we observe that conduction failure occurs for low values of . In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling or . Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac tissue, in our MF model both with and without heterogeneities. PMID:24023798

  5. Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53

    PubMed Central

    Chen, Bo; Simpson, Dennis A.; Zhou, Yingchun; Mitra, Amritava; Mitchell, David L.; Cordeiro-Stone, Marila; Kaufmann, William K.

    2015-01-01

    After treatment with ultraviolet radiation (UV), human fibroblasts that express the HPV type 16 E6 oncoprotein display defects in repair of cyclobutane pyrimidine dimers, hypersensitivity to inactivation of clonogenic survival and an inability to sustain DNA replication. To determine whether these effects are specific to depletion of p53 or inactivation of its function, fibroblast lines were constructed with ectopic expression of a dominant-negative p53 allele (p53-H179Q) to inactivate function or a short-hairpin RNA (p53-RNAi) to deplete expression of p53. Only the expression of HPV16E6 sensitized fibroblasts to UV or the chemical carcinogen, benzo[a]pyrene diolepoxide I (BPDE). Carcinogen-treated cells expressing p53-H179Q or p53-RNAi were resistant to inactivation of colony formation and did not suffer replication arrest. CHK1 is a key checkpoint kinase in the response to carcinogen-induced DNA damage. Control and p53-RNAi-expressing fibroblasts displayed phosphorylation of Ser345 on CHK1 45–120 min after carcinogen treatment with a return to near baseline phosphorylation by 6 h after treatment. HPV16E6-expressing fibroblasts displayed enhanced and sustained phosphorylation of CHK1. This was associated with enhanced phosphorylation of Thr68 on CHK2 and Ser139 on H2AX, both markers of severe replication stress and DNA double strand breaks. Incubation with the phosphatase inhibitor okadaic acid produced more phosphorylation of CHK1 in UV-treated HPV16E6-expressing cells than in p53-H179Q-expressing cells suggesting that HPV16E6 may interfere with the recovery of coupled DNA replication at replication forks that are stalled at [6-4]pyrimidine-pyrimidone photoproducts and BPDE-DNA adducts. The results indicate that HPV16E6 targets a protein or proteins other than p53 to deregulate the activity of CHK1 in carcinogen-damaged cells. PMID:19411857

  6. Comparison of the cidal activity of tea tree oil and terpinen-4-ol against clinical bacterial skin isolates and human fibroblast cells.

    PubMed

    Loughlin, R; Gilmore, B F; McCarron, P A; Tunney, M M

    2008-04-01

    The aim of this study was to compare both the antimicrobial activity of terpinen-4-ol and tea tree oil (TTO) against clinical skin isolates of meticillin-resistant Staphylococcus aureus (MRSA) and coagulase-negative staphylococci (CoNS) and their toxicity against human fibroblast cells. Antimicrobial activity was compared by using broth microdilution and quantitative in vitro time-kill test methods. Terpinen-4-ol exhibited significantly greater bacteriostatic and bactericidal activity, as measured by minimum inhibitory and bactericidal concentrations, respectively, than TTO against both MRSA and CoNS isolates. Although not statistically significant, time-kill studies also clearly showed that terpinen-4-ol exhibited greater antimicrobial activity than TTO. Comparison of the toxicity of terpinen-4-ol and TTO against human fibroblasts revealed that neither agent, at the concentrations tested, were toxic over the 24-h test period. Terpinen-4-ol is a more potent antibacterial agent against MRSA and CoNS isolates than TTO with neither agent exhibiting toxicity to fibroblast cells at the concentrations tested. Terpinen-4-ol should be considered for inclusion as a single agent in products formulated for topical treatment of MRSA infection. However, further work would initially be required to ensure that resistance would not develop with the use of terpinen-4-ol as a single agent.

  7. Trehalose Improves Human Fibroblast Deficits in a New CHIP-Mutation Related Ataxia

    PubMed Central

    Casarejos, Maria Jose; Perucho, Juan; López-Sendón, Jose Luis; García de Yébenes, Justo; Bettencourt, Conceição; Gómez, Ana; Ruiz, Carolina; Heutink, Peter; Rizzu, Patrizia; Mena, Maria Angeles

    2014-01-01

    In this work we investigate the role of CHIP in a new CHIP-mutation related ataxia and the therapeutic potential of trehalose. The patient's fibroblasts with a new form of hereditary ataxia, related to STUB1 gene (CHIP) mutations, and three age and sex-matched controls were treated with epoxomicin and trehalose. The effects on cell death, protein misfolding and proteostasis were evaluated. Recent studies have revealed that mutations in STUB-1 gene lead to a growing list of molecular defects as deregulation of protein quality, inhibition of proteasome, cell death, decreased autophagy and alteration in CHIP and HSP70 levels. In this CHIP-mutant patient fibroblasts the inhibition of proteasome with epoxomicin induced severe pathophysiological age-associated changes, cell death and protein ubiquitination. Additionally, treatment with epoxomicin produced a dose-dependent increase in the number of cleaved caspase-3 positive cells. However, co-treatment with trehalose, a disaccharide of glucose present in a wide variety of organisms and known as a autophagy enhancer, reduced these pathological events. Trehalose application also increased CHIP and HSP70 expression and GSH free radical levels. Furthermore, trehalose augmented macro and chaperone mediated autophagy (CMA), rising the levels of LC3, LAMP2, CD63 and increasing the expression of Beclin-1 and Atg5-Atg12. Trehalose treatment in addition increased the percentage of immunoreactive cells to HSC70 and LAMP2 and reduced the autophagic substrate, p62. Although this is an individual case based on only one patient and the statistical comparisons are not valid between controls and patient, the low variability among controls and the obvious differences with this patient allow us to conclude that trehalose, through its autophagy activation capacity, anti-aggregation properties, anti-oxidative effects and lack of toxicity, could be very promising for the treatment of CHIP-mutation related ataxia, and possibly a wide spectrum

  8. Differential effects of eicosapentaenoic acid and docosahexaenoic acid on human skin fibroblasts.

    PubMed

    Brown, E R; Subbaiah, P V

    1994-12-01

    To better understand the mode of action of omega 3 fatty acids in cell membranes, human foreskin fibroblasts were grown in serum-free medium supplemented with 50 microM oleic acid linoleic acid, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), and the effects on membrane composition, fluorescence polarization and enzyme activities were followed. The cells were enriched with EPA and DHA up to 7 and 13% of total lipids, respectively, of which > 95% was associated with phospholipids. In addition, the concentration of 22:5n-3 increased with both EPA and DHA to 7.5, and 2.1% of the total fatty acids, respectively. When compared to controls (oleic acid), cells treated with DHA showed a decrease in cholesterol, phospholipids, arachidonic acid (AA) and free cholesterol/phospholipid ratio (P < 0.05). In the presence of EPA, only decreases in AA and cholesterol were significant (P < 0.05). Membrane fluidity, assessed by fluorescence anisotropy, was increased 16% in cells enriched with DHA (P < 0.05), but showed no change with EPA or linoleic acid. There was an increase in membrane-associated 5'-nucleotidase (+27%) and adenylate cyclase (+19%) activities (P < 0.05), in DHA-enriched, but not in EPA-enriched cells, when compared with oleate controls. The studies show that incorporation of DHA, but not EPA, into cell membranes of fibroblasts alters membrane biophysical characteristics and function. We suggest that these two major n-3 fatty acids of fish oils have differential effects on cell membranes, and this may be related to the known differences in their physiological effects.

  9. Silibinin prevents prostate cancer cell-mediated differentiation of naïve fibroblasts into cancer-associated fibroblast phenotype by targeting TGF β2.

    PubMed

    Ting, Harold J; Deep, Gagan; Jain, Anil K; Cimic, Adela; Sirintrapun, Joseph; Romero, Lina M; Cramer, Scott D; Agarwal, Chapla; Agarwal, Rajesh

    2015-09-01

    Tumor microenvironment (TM) is an essential element in prostate cancer (PCA), offering unique opportunities for its prevention. TM includes naïve fibroblasts that are recruited by nascent neoplastic lesion and altered into 'cancer-associated fibroblasts' (CAFs) that promote PCA. A better understanding and targeting of interaction between PCA cells and fibroblasts and inhibiting CAF phenotype through non-toxic agents are novel approaches to prevent PCA progression. One well-studied cancer chemopreventive agent is silibinin, and thus, we examined its efficacy against PCA cells-mediated differentiation of naïve fibroblasts into a myofibroblastic-phenotype similar to that found in CAFs. Silibinin's direct inhibitory effect on the phenotype of CAFs derived directly from PCA patients was also assessed. Human prostate stromal cells (PrSCs) exposed to control conditioned media (CCM) from human PCA PC3 cells showed more invasiveness, with increased alpha-smooth muscle actin (α-SMA) and vimentin expression, and differentiation into a phenotype we identified in CAFs. Importantly, silibinin (at physiologically achievable concentrations) inhibited α-SMA expression and invasiveness in differentiated fibroblasts and prostate CAFs directly, as well as indirectly by targeting PCA cells. The observed increase in α-SMA and CAF-like phenotype was transforming growth factor (TGF) β2 dependent, which was strongly inhibited by silibinin. Furthermore, induction of α-SMA and CAF phenotype by CCM were also strongly inhibited by a TGFβ2-neutralizing antibody. The inhibitory effect of silibinin on TGFβ2 expression and CAF-like biomarkers was also observed in PC3 tumors. Together, these findings highlight the potential usefulness of silibinin in PCA prevention through targeting the CAF phenotype in the prostate TM. © 2014 Wiley Periodicals, Inc.

  10. Pretreatment of Ferulic Acid Protects Human Dermal Fibroblasts against Ultraviolet A Irradiation

    PubMed Central

    Hahn, Hyung Jin; Kim, Ki Bbeum; Bae, Seunghee; Choi, Byung Gon; An, Sungkwan

    2016-01-01

    Background Approximately 90%~99% of ultraviolet A (UVA) ray reaches the Earth's surface. The deeply penetrating UVA rays induce the formation of reactive oxygen species (ROS), which results in oxidative stress such as photoproducts, senescence, and cell death. Thus, UVA is considered a primary factor that promotes skin aging. Objective Researchers investigated whether pretreatment with ferulic acid protects human dermal fibroblasts (HDFs) against UVA-induced cell damages. Methods HDF proliferation was analyzed using the water-soluble tetrazolium salt assay. Cell cycle distribution and intracellular ROS levels were assessed by flow cytometric analysis. Senescence was evaluated using a senescence-associated β-galactosidase assay, while Gadd45α promoter activity was analyzed through a luciferase assay. The expression levels of superoxide dismutase 1 (SOD1), catalase (CAT), xeroderma pigmentosum complementation group A and C, matrix metalloproteinase 1 and 3, as well as p21 and p16 were measured using quantitative real-time polymerase chain reaction. Results Inhibition of proliferation and cell cycle arrest were detected in cells that were irradiated with UVA only. Pretreatment with ferulic acid significantly increased the proliferation and cell cycle progression in HDFs. Moreover, ferulic acid pretreatment produced antioxidant effects such as reduced DCF intensity, and affected SOD1 and CAT mRNA expression. These effects were also demonstrated in the analysis of cell senescence, promoter activity, expression of senescent markers, and DNA repair. Conclusion These results demonstrate that ferulic acid exerts protective effects on UVA-induced cell damages via anti-oxidant and stress-inducible cellular mechanisms in HDFs. PMID:27904274

  11. Effect of the cytostatic agent idarubicin on fibroblasts of the human Tenon's capsule compared with mitomycin C

    PubMed Central

    Heilmann, C.; Schonfeld, P.; Schluter, T.; Bohnensack, R.; Behrens-Baumann, W.

    1999-01-01

    BACKGROUND/AIMS—To investigate the in vitro effect of a short time exposure to the anthracycline idarubicin on proliferation, protein synthesis, and motility of human Tenon's capsule fibroblasts in comparison with the antitumour antibiotic mitomycin C.
METHODS—After determination of effective concentrations of idarubicin, fibroblasts of the human Tenon's capsule were exposed to idarubicin or mitomycin C at concentrations ranging from 0.1 µg/ml to 1 µg/ml or from 2.5 µg/ml to 250 µg/ml, respectively, for 0.5, 2, or 5 minutes and cultured for 60 days. Cell death by apoptosis caused by idarubicin treatment was confirmed by Hoechst 33258 staining. Further proliferation was explored by cell counting and by 3H-thymidine uptake. Protein synthesis was measured by 3H-proline uptake and motility was assessed by agarose droplet motility assay.
RESULTS—Idarubicin is able to exert toxicity and to induce apoptosis during a short time exposure of 0.5 minutes at concentrations of 0.3-1 µg/ml resulting in a significant reduction in cell number compared with the control after 60 days. For mitomycin C, higher concentrations and longer expositions were necessary. Even after treatment with 1 µg/ml idarubicin or 250 µg/ml mitomycin C a few cells were able to incorporate 3H-thymidine. 3H-proline uptake up to 10 days after exposure to 0.3 µg/ml idarubicin was found not to be decreased. Cell motility was reduced after treatment with 1 µg/ml idarubicin for 5 minutes or with 250 µg/ml mitomycin C for 2 or 5 minutes. For low mitomycin C concentrations, an increase in motility was found during the first 10 days.
CONCLUSION—Idarubicin reduces proliferation of human Tenons's capsule fibroblasts after incubation for 0.5 minutes at concentrations as low as 0.3-1 µg/ml. In comparison, mitomycin C requires longer exposure times and higher doses for equal results. Therefore, idarubicin may be useful in the prevention of glaucoma filtering surgery failure

  12. Effect of exposure to mitomycin C on cultured tympanic membrane fibroblasts.

    PubMed

    Jang, Chul Ho; Song, Chang-Hun; Pak, Sok Cheon

    2003-02-01

    Recently, attempts have been made to prolong the patency of myringotomy site with topical use of mitomycin C (MMC). It has been shown that MMC inhibits mitosis and proliferation of ocular fibroblasts, however, there are no studies of MMC's effect on tympanic membrane fibroblasts. To investigate the effects of MMC on cultured human tympanic membrane fibroblasts and understand the cellular basis of MMC for maintain myringotomy patency, cultured fibroblasts were exposed to various concentrations of MMC for periods of 5-10 min. Effect of MMC on cultured fibroblasts was assessed by microscopic observation and cell viability test. Dose-, time- dependent relationship of MMC on cultured fibroblasts was revealed. There was a significant difference between the inhibition effects of MMC at concentrations of 0.4 mg/ml and control following 5 and 10 min exposure intervals. Phase-contrast microscopy showed consistency with the antiproliferative effect of MMC at higher concentration. Therefore, it would appear that intraoperative use of MMC could be effective in delaying the healing of the myringotomy site and extending the period of time for myringotomy patency.

  13. Spiral-wave dynamics in a mathematical model of human ventricular tissue with myocytes and fibroblasts.

    PubMed

    Nayak, Alok Ranjan; Shajahan, T K; Panfilov, A V; Pandit, Rahul

    2013-01-01

    Cardiac fibroblasts, when coupled functionally with myocytes, can modulate the electrophysiological properties of cardiac tissue. We present systematic numerical studies of such modulation of electrophysiological properties in mathematical models for (a) single myocyte-fibroblast (MF) units and (b) two-dimensional (2D) arrays of such units; our models build on earlier ones and allow for zero-, one-, and two-sided MF couplings. Our studies of MF units elucidate the dependence of the action-potential (AP) morphology on parameters such as [Formula: see text], the fibroblast resting-membrane potential, the fibroblast conductance [Formula: see text], and the MF gap-junctional coupling [Formula: see text]. Furthermore, we find that our MF composite can show autorhythmic and oscillatory behaviors in addition to an excitable response. Our 2D studies use (a) both homogeneous and inhomogeneous distributions of fibroblasts, (b) various ranges for parameters such as [Formula: see text], and [Formula: see text], and (c) intercellular couplings that can be zero-sided, one-sided, and two-sided connections of fibroblasts with myocytes. We show, in particular, that the plane-wave conduction velocity [Formula: see text] decreases as a function of [Formula: see text], for zero-sided and one-sided couplings; however, for two-sided coupling, [Formula: see text] decreases initially and then increases as a function of [Formula: see text], and, eventually, we observe that conduction failure occurs for low values of [Formula: see text]. In our homogeneous studies, we find that the rotation speed and stability of a spiral wave can be controlled either by controlling [Formula: see text] or [Formula: see text]. Our studies with fibroblast inhomogeneities show that a spiral wave can get anchored to a local fibroblast inhomogeneity. We also study the efficacy of a low-amplitude control scheme, which has been suggested for the control of spiral-wave turbulence in mathematical models for cardiac

  14. A Study of Parameters Affecting Fibroblast Morphology in Response to an Applied Mechanical Force

    NASA Technical Reports Server (NTRS)

    Grymes, Rosalind A.; Sawyer, Christine

    1994-01-01

    A precisely controlled stretch/relaxation regimen (20% elongation at 6.6 cycles/min) was applied to normal human fetal, neonatal and aged dermal fibroblasts cultured on flexible membranes. Culture conditions included poly (NH2) or collagen type I coated substrate membranes; control cultures were grown on the same pliable material in the absence of applied stretch. Direct observation and immunofluorescence analyses revealed a progressive change in cell body orientation limited to the stretched dermal fibroblast cultures. Monolayers gradually (over 4 days) acquired a symmetric, radial distribution equivalent to the biaxial array of the applied force. At high seeding density, alignment was inhibited in the fetal cell cultures. This cell strain required collagen type I coating for optimal attachment to the flexible membrane, preferring growth in three-dimensional cell 'balls' on the poly(NH2) coated substrate. Neonatal cells also required the collagen type I coating, but both neonatal and aged dermal fibroblasts aligned efficiently at all seeding densities examined. The randomly oriented neonatal cells on the unstretched control membranes spontaneously detached at confluence, as a single cell sheet. Their aligned counterparts did not detach until the applied stretch stimulus was removed. Low concentrations of cytochalasin D (62.5 ng/ml) disrupted the stretch-related alignment response. Rhodamine phalloidin staining visualized fewer actin stress fibers in stretched, aligned cells than in controls. Both intercellular interactions and cytoskeletal integrity mediate the response to mechanical strain. Normal rabbit corneal stroma fibroblasts (NRC) were also analyzed, and failed to orient under these conditions. This cell type may require a different regimen, or a longer time period, to demonstrate alignment behavior. Supported by NASA Space Biology RTOP 199-40-22 and the NASA-ARC Director's Discretionary Fund.

  15. Effects of a vegetable extract from Lupinus albus (LU105) on the production of matrix metalloproteinases (MMP1, MMP2, MMP9) and tissue inhibitor of metalloproteinases (TIMP1, TIMP2) by human gingival fibroblasts in culture.

    PubMed

    Gaultier, F; Foucault-Bertaud, A; Lamy, E; Ejeil, A L; Dridi, S M; Piccardi, N; Piccirilli, A; Msika, P; Godeau, G; Gogly, B

    2003-12-01

    This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture. LU105 was extracted from seeds of L. albus and is freely soluble in water. Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48 h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected. Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B). LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9. Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue. TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts. Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva.

  16. Effect of Fibroblast-Like Cells of Mesenchymal Origin of Cytotoxic Activity of Lymphocytes against NK-Sensitive Target Cells.

    PubMed

    Lupatov, A Yu; Kim, Ya S; Bystrykh, O A; Vakhrushev, I V; Pavlovich, S V; Yarygin, K N; Sukhikh, G T

    2017-02-01

    We studied immunosuppressive properties of skin fibroblasts and mesenchymal stromal cells against NK cells. In vitro experiments showed that mesenchymal stromal cells isolated from human umbilical cord and human skin fibroblasts can considerably attenuate cytotoxic activity of NK cells against Jurkat cells sensitive to NK-mediated lysis. NK cells cultured in lymphocyte population exhibited higher cytotoxic activity than isolated NK cells. Mesenchymal stromal cells or fibroblasts added 1:1 to lymphocyte culture almost completely suppressed NK cell cytotoxicity. This suggests that fibroblast-like cells can suppress not only isolated NK cells, but also NK cells in natural cell microenvironment.

  17. Synthesis and biological activity of M6-P and M6-P analogs on fibroblast and keratinocyte proliferation.

    PubMed

    Clavel, Caroline; Barragan-Montero, Véronique; Garric, Xavier; Molès, Jean-Pierre; Montero, Jean-Louis

    2005-09-01

    A new synthetic route to obtain the carboxylate analog of mannose 6-phosphate (M6-P) is presented. The effects of the M6-P, the carboxylate and two other analogs (the phosphonate and the alpha,beta ethylenic carboxylate) on the proliferation of human keratinocytes and dermal fibroblasts as well as on the proliferation of a murine fibroblast cell line, 3T3-J2 are tested. We observed that M6-P is a potent inhibitor of proliferation of both fibroblasts and keratinocytes. Among its analogs, the phosphonate showed a similar effect on human dermal fibroblasts but not on keratinocytes.

  18. Redox-active cerium oxide nanoparticles protect human dermal fibroblasts from PQ-induced damage.

    PubMed

    von Montfort, Claudia; Alili, Lirija; Teuber-Hanselmann, Sarah; Brenneisen, Peter

    2015-01-01

    Recently, it has been published that cerium (Ce) oxide nanoparticles (CNP; nanoceria) are able to downregulate tumor invasion in cancer cell lines. Redox-active CNP exhibit both selective pro-oxidative and antioxidative properties, the first being responsible for impairment of tumor growth and invasion. A non-toxic and even protective effect of CNP in human dermal fibroblasts (HDF) has already been observed. However, the effect on important parameters such as cell death, proliferation and redox state of the cells needs further clarification. Here, we present that nanoceria prevent HDF from reactive oxygen species (ROS)-induced cell death and stimulate proliferation due to the antioxidative property of these particles. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

  19. Detection of advanced glycation end products (AGEs) on human skin by in vivo confocal Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Martin, A. A.; Pereira, L.; Ali, S. M.; Pizzol, C. D.; Tellez, C. A.; Favero, P. P.; Santos, L.; da Silva, V. V.; Praes, C. E. O.

    2016-03-01

    The aging process involves the reduction in the production of the major components of skin tissue. During intrinsic aging and photoaging processes, in dermis of human skin, fibroblasts become senescent and have decreased activity, which produce low levels of collagen. Moreover, there is accumulation of advanced glycation end products (AGEs). AGEs have incidence in the progression of age-related diseases, principally in diabetes mellitus and in Alzheimer's diseases. AGEs causes intracellular damage and/or apoptosis leading to an increase of the free radicals, generating a crosslink with skin proteins and oxidative stress. The aim of this study is to detect AGEs markers on human skin by in vivo Confocal Raman spectroscopy. Spectra were obtained by using a Rivers Diagnostic System, 785 nm laser excitation and a CCD detector from the skin surface down to 120 μm depth. We analyzed the confocal Raman spectra of the skin dermis of 30 women volunteers divided into 3 groups: 10 volunteers with diabetes mellitus type II, 65-80 years old (DEW); 10 young healthy women, 20-33 years old (HYW); and 10 elderly healthy women, 65-80 years old (HEW). Pentosidine and glucosepane were the principally identified AGEs in the hydroxyproline and proline Raman spectral region (1000-800 cm-1), in the 1.260-1.320 cm-1 region assignable to alpha-helical amide III modes, and in the Amide I region. Pentosidine and glucosepane calculated vibrational spectra were performed through Density Functional Theory using the B3LYP functional with 3-21G basis set. Difference between the Raman spectra of diabetic elderly women and healthy young women, and between healthy elderly women and healthy young women were also obtained with the purpose of identifying AGEs Raman bands markers. AGEs peaks and collagen changes have been identified and used to quantify the glycation process in human skin.

  20. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures.

    PubMed

    Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

    2014-07-17

    Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. © 2014 The Authors.

  1. MiR-133 promotes cardiac reprogramming by directly repressing Snai1 and silencing fibroblast signatures

    PubMed Central

    Muraoka, Naoto; Yamakawa, Hiroyuki; Miyamoto, Kazutaka; Sadahiro, Taketaro; Umei, Tomohiko; Isomi, Mari; Nakashima, Hanae; Akiyama, Mizuha; Wada, Rie; Inagawa, Kohei; Nishiyama, Takahiko; Kaneda, Ruri; Fukuda, Toru; Takeda, Shu; Tohyama, Shugo; Hashimoto, Hisayuki; Kawamura, Yoshifumi; Goshima, Naoki; Aeba, Ryo; Yamagishi, Hiroyuki; Fukuda, Keiichi; Ieda, Masaki

    2014-01-01

    Fibroblasts can be directly reprogrammed into cardiomyocyte-like cells (iCMs) by overexpression of cardiac transcription factors or microRNAs. However, induction of functional cardiomyocytes is inefficient, and molecular mechanisms of direct reprogramming remain undefined. Here, we demonstrate that addition of miR-133a (miR-133) to Gata4, Mef2c, and Tbx5 (GMT) or GMT plus Mesp1 and Myocd improved cardiac reprogramming from mouse or human fibroblasts by directly repressing Snai1, a master regulator of epithelial-to-mesenchymal transition. MiR-133 overexpression with GMT generated sevenfold more beating iCMs from mouse embryonic fibroblasts and shortened the duration to induce beating cells from 30 to 10 days, compared to GMT alone. Snai1 knockdown suppressed fibroblast genes, upregulated cardiac gene expression, and induced more contracting iCMs with GMT transduction, recapitulating the effects of miR-133 overexpression. In contrast, overexpression of Snai1 in GMT/miR-133-transduced cells maintained fibroblast signatures and inhibited generation of beating iCMs. MiR-133-mediated Snai1 repression was also critical for cardiac reprogramming in adult mouse and human cardiac fibroblasts. Thus, silencing fibroblast signatures, mediated by miR-133/Snai1, is a key molecular roadblock during cardiac reprogramming. PMID:24920580

  2. Human dermal and gingival fibroblasts in a three-dimensional culture: a comparative study on matrix remodeling.

    PubMed

    Chaussain Miller, C; Septier, D; Bonnefoix, M; Lecolle, S; Lebreton-Decoster, C; Coulomb, B; Pellat, B; Godeau, G

    2002-03-01

    Free-floating collagen lattice is considered a useful tool for assessing wound healing in vitro. This work compared extracellular matrix remodeling in collagen lattices populated by gingival or dermal fibroblasts. For 21 days we followed gel contraction and changes in cell number of collagen lattices seeded with l.5 x 10(5) fibroblasts of each tissue. We also used indirect immunodetection to study extracellular matrix components, metalloproteinases (MMPs), and their tissues inhibitors (TIMPs). In addition, the presence of MMPs and TIMPs in the culture media was analyzed by zymography and western blotting. No significant difference was found concerning gel contraction and changes in cell number. We observed the early expression of fibrillin I and collagen type III, apparently codistributed and at the end of the gel contraction their disappearance. Concomitantly we demonstrated the expression of MMPs and TIMPs, initially localized in cellular cytoplasm, then spreading in the extracellular compartment, and even found in the culture medium. This remodeling was more rapid and intense with gingival fibroblasts than dermal fibroblasts. In conclusion, gingival fibroblasts seem more efficient at remodeling the connective tissue than dermal fibroblasts and could lead to the better wound healing observed in vivo.

  3. Effects of mechanical strain on human mesenchymal stem cells and ligament fibroblasts in a textured poly(L-lactide) scaffold for ligament tissue engineering.

    PubMed

    Kreja, Ludwika; Liedert, Astrid; Schlenker, Heiter; Brenner, Rolf E; Fiedler, Jörg; Friemert, Benedikt; Dürselen, Lutz; Ignatius, Anita

    2012-10-01

    The purpose of this study was to prove the effect of cyclic uniaxial intermittent strain on the mRNA expression of ligament-specific marker genes in human mesenchymal stem cells (MSC) and anterior cruciate ligament-derived fibroblasts (ACL-fibroblasts) seeded onto a novel textured poly(L-lactide) scaffold (PLA scaffold). Cell-seeded scaffolds were mechanically stimulated by cyclic uniaxial stretching. The expression of ligament matrix gene markers: collagen types I and III, fibronectin, tenascin C and decorin, as well as the proteolytic enzymes matrix metalloproteinase MMP-1 and MMP-2 and their tissue specific inhibitors TIMP-1 and TIMP-2 was investigated by analysing the mRNA expression using reverse transcriptase polymerase chain reaction and related to the static control. In ACL-fibroblasts seeded on PLA, mechanical load induced up-regulation of collagen types I and III, fibronectin and tenascin C. No effect of mechanical stimulation on the expression of ligament marker genes was found in undifferentiated MSC seeded on PLA. The results indicated that the new textured PLA scaffold could transfer the mechanical load to the ACL-fibroblasts and improved their ligament phenotype. This scaffold might be suitable as a cell-carrying component of ACL prostheses.

  4. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    PubMed Central

    Laranjeira, Marta S; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-01-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior. PMID:27877662

  5. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    NASA Astrophysics Data System (ADS)

    Laranjeira, Marta S.; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-04-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol-gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.

  6. Biomass Smoke Exposure Enhances Rhinovirus-Induced Inflammation in Primary Lung Fibroblasts

    PubMed Central

    Capistrano, Sarah J.; Zakarya, Razia; Chen, Hui; Oliver, Brian G.

    2016-01-01

    Biomass smoke is one of the major air pollutants and contributors of household air pollution worldwide. More than 3 billion people use biomass fuels for cooking and heating, while other sources of exposure are from the occurrence of bushfires and occupational conditions. Persistent biomass smoke exposure has been associated with acute lower respiratory infection (ALRI) as a major environmental risk factor. Children under the age of five years are the most susceptible in developing severe ALRI, which accounts for 940,000 deaths globally. Around 90% of cases are attributed to viral infections, such as influenza, adenovirus, and rhinovirus. Although several epidemiological studies have generated substantial evidence of the association of biomass smoke and respiratory infections, the underlying mechanism is still unknown. Using an in vitro model, primary human lung fibroblasts were stimulated with biomass smoke extract (BME), specifically investigating hardwood and softwood types, and human rhinovirus-16 for 24 h. Production of pro-inflammatory mediators, such as IL-6 and IL-8, were measured via ELISA. Firstly, we found that hardwood and softwood smoke extract (1%) up-regulate IL-6 and IL-8 release (p ≤ 0.05). In addition, human rhinovirus-16 further increased biomass smoke-induced IL-8 in fibroblasts, in comparison to the two stimulatory agents alone. We also investigated the effect of biomass smoke on viral susceptibility by measuring viral load, and found no significant changes between BME exposed and non-exposed infected fibroblasts. Activated signaling pathways for IL-6 and IL-8 production by BME stimulation were examined using signaling pathway inhibitors. p38 MAPK inhibitor SB239063 significantly attenuated IL-6 and IL-8 release the most (p ≤ 0.05). This study demonstrated that biomass smoke can modulate rhinovirus-induced inflammation during infection, which can alter the severity of the disease. The mechanism by which biomass smoke exposure increases

  7. Biomass Smoke Exposure Enhances Rhinovirus-Induced Inflammation in Primary Lung Fibroblasts.

    PubMed

    Capistrano, Sarah J; Zakarya, Razia; Chen, Hui; Oliver, Brian G

    2016-08-25

    Biomass smoke is one of the major air pollutants and contributors of household air pollution worldwide. More than 3 billion people use biomass fuels for cooking and heating, while other sources of exposure are from the occurrence of bushfires and occupational conditions. Persistent biomass smoke exposure has been associated with acute lower respiratory infection (ALRI) as a major environmental risk factor. Children under the age of five years are the most susceptible in developing severe ALRI, which accounts for 940,000 deaths globally. Around 90% of cases are attributed to viral infections, such as influenza, adenovirus, and rhinovirus. Although several epidemiological studies have generated substantial evidence of the association of biomass smoke and respiratory infections, the underlying mechanism is still unknown. Using an in vitro model, primary human lung fibroblasts were stimulated with biomass smoke extract (BME), specifically investigating hardwood and softwood types, and human rhinovirus-16 for 24 h. Production of pro-inflammatory mediators, such as IL-6 and IL-8, were measured via ELISA. Firstly, we found that hardwood and softwood smoke extract (1%) up-regulate IL-6 and IL-8 release (p ≤ 0.05). In addition, human rhinovirus-16 further increased biomass smoke-induced IL-8 in fibroblasts, in comparison to the two stimulatory agents alone. We also investigated the effect of biomass smoke on viral susceptibility by measuring viral load, and found no significant changes between BME exposed and non-exposed infected fibroblasts. Activated signaling pathways for IL-6 and IL-8 production by BME stimulation were examined using signaling pathway inhibitors. p38 MAPK inhibitor SB239063 significantly attenuated IL-6 and IL-8 release the most (p ≤ 0.05). This study demonstrated that biomass smoke can modulate rhinovirus-induced inflammation during infection, which can alter the severity of the disease. The mechanism by which biomass smoke exposure increases

  8. Production and in vitro evaluation of soy protein-based biofilms as a support for human keratinocyte and fibroblast culture.

    PubMed

    Curt, Sèverine; Subirade, Muriel; Rouabhia, Mahmoud

    2009-06-01

    This study presents results on soy protein isolate (SPI) biofilm production and the corresponding effect on the stability and toxicity of the derived films. SPI biofilms were prepared from SPI chemically treated with formaldehyde at various concentrations (0%, 1%, 2%, and 3%) as cross-linking agents. In vitro SPI biofilm degradation was evaluated as a function of water absorption leading to weight and size modifications. SPI biofilm toxicity was determined as a function of human keratinocyte and fibroblast adhesion, viability, and proliferation. Cytokine gene expression supported this using reverse transcriptase polymerase chain reaction techniques. Our results confirm that SPI can be used to produce biofilms. The resulting SPI biofilms without formaldehyde swell significantly, which leads to their physical instability. Formaldehyde treatment enhanced the mechanical properties of these biofilms by covalently cross-linking polypeptide chains. The decreased water absorption was dependent on the amount of formaldehyde present. SPI biofilms with 2% and 3% formaldehyde were highly stable and easier to manipulate than those with 0% and 1% formaldehyde. Tissue culture analyses revealed that the SPI biofilms without formaldehyde were non-toxic to human cells (keratinocytes and fibroblasts). The presence of formaldehyde in biofilms did not have any effects on cell viability, adhesion, or proliferation. This was supported by the high level of messenger RNA expression of interleukin-1 beta (IL-1beta) and tumor necrosis factor alpha by the keratinocytes and of IL-6 and IL-8 by the fibroblasts. Overall, we produced a stable, non-toxic soy protein support, which may be of potential interest in medical applications such as cell culture matrices and damaged tissue replacement.

  9. A drug-induced accelerated senescence (DIAS) is a possibility to study aging in time lapse.

    PubMed

    Alili, Lirija; Diekmann, Johanna; Giesen, Melanie; Holtkötter, Olaf; Brenneisen, Peter

    2014-06-01

    Currently, the oxidative stress (or free radical) theory of aging is the most popular explanation of how aging occurs at the molecular level. Accordingly, a stress-induced senescence-like phenotype of human dermal fibroblasts can be induced in vitro by the exposure of human diploid fibroblasts to subcytotoxic concentrations of hydrogen peroxide. However, several biomarkers of replicative senescence e.g. cell cycle arrest and enlarged morphology are abrogated 14 days after treatment, indicating that reactive oxygen species (ROS) rather acts as a trigger for short-term senescence (1-3 days) than being responsible for the maintenance of the senescence-like phenotype. Further, DNA-damaging factors are discussed resulting in a permanent senescent cell type. To induce long-term premature senescence and to understand the molecular alterations occurring during the aging process, we analyzed mitomycin C (MMC) as an alkylating DNA-damaging agent and ROS producer. Human dermal fibroblasts (HDF), used as model for skin aging, were exposed to non-cytotoxic concentrations of MMC and analyzed for potential markers of cellular aging, for example enlarged morphology, activity of senescence-associated-ß-galactosidase, cell cycle arrest, increased ROS production and MMP1-activity, which are well-documented for HDF in replicative senescence. Our data show that mitomycin C treatment results in a drug-induced accelerated senescence (DIAS) with long-term expression of senescence markers, demonstrating that a combination of different susceptibility factors, here ROS and DNA alkylation, are necessary to induce a permanent senescent cell type.

  10. Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

    PubMed

    Abdian, Narges; Ghasemi-Dehkordi, Payam; Hashemzadeh-Chaleshtori, Morteza; Ganji-Arjenaki, Mahbobe; Doosti, Abbas; Amiri, Beheshteh

    2015-12-01

    Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p < 0.05). The findings of the present study demonstrate that appropriate supplementation of culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast

  11. Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway.

    PubMed

    Wang, Jun; Si, Yanfang; Wu, Chen; Sun, Lu; Ma, Yudong; Ge, Aili; Li, Baomin

    2012-10-17

    Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of

  12. Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway

    PubMed Central

    2012-01-01

    Background Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. Results In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Conclusions Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to

  13. Chronic Exposure to Rhodobacter Sphaeroides Extract Lycogen™ Prevents UVA-Induced Malondialdehyde Accumulation and Procollagen I Down-Regulation in Human Dermal Fibroblasts

    PubMed Central

    Yang, Tsai-Hsiu; Lai, Ying-Hsiu; Lin, Tsuey-Pin; Liu, Wen-Sheng; Kuan, Li-Chun; Liu, Chia-Chyuan

    2014-01-01

    UVA contributes to the pathogenesis of skin aging by downregulation of procollagen I content and induction of matrix metalloproteinase (MMP)-associated responses. Application of antioxidants such as lycopene has been demonstrated as a convenient way to achieve protection against skin aging. Lycogen™, derived from the extracts of Rhodobacter sphaeroides, exerts several biological effects similar to that of lycopene whereas most of its anti-aging efficacy remains uncertain. In this study, we attempted to examine whether Lycogen™ could suppress malondialdehyde (MDA) accumulation and restore downregulated procollagen I expression induced by UVA exposure. In human dermal fibroblasts Hs68 cells, UVA repressed cell viability and decreased procollagen I protein content accompanied with the induction of MMP-1 and MDA accumulation. Remarkably, incubation with 50 μM Lycogen™ for 24 h ameliorated UVA-induced cell death and restored UVA-induced downregulation of procollagen in a dose-related manner. Lycogen™ treatment also prevented the UVA-induced MMP-1 upregulation and intracellular MDA generation in Hs68 cells. Activation of NFκB levels, one of the downstream events induced by UVA irradiation and MMP-1 induction, were also prevented by Lycogen™ administration. Taken together, our findings demonstrate that Lycogen™ may be an alternative agent that prevents UVA-induced skin aging and could be used in cosmetic and pharmaceutical applications. PMID:24463291

  14. Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibroatheromata

    PubMed Central

    Brokopp, Chad E.; Schoenauer, Roman; Richards, Peter; Bauer, Stefan; Lohmann, Christine; Emmert, Maximilian Y.; Weber, Benedikt; Winnik, Stephan; Aikawa, Elena; Graves, Kirk; Genoni, Michele; Vogt, Peter; Lüscher, Thomas F.; Renner, Christoph; Hoerstrup, Simon P.; Matter, Christian M.

    2011-01-01

    Aims Collagen degradation in atherosclerotic plaques with thin fibrous caps renders them more prone to rupture. Fibroblast activation protein (FAP) plays a role in arthritis and tumour formation through its collagenase activity. However, the significance of FAP in thin-cap human fibroatheromata remains unknown. Methods and results We detected enhanced FAP expression in type IV–V human aortic atheromata (n = 12), compared with type II–III lesions (n = 9; P < 0.01) and healthy aortae (n = 8; P < 0.01) by immunostaining and western blot analyses. Fibroblast activation protein was also increased in thin-cap (<65 µm) vs. thick-cap (≥65 µm) human coronary fibroatheromata (n = 12; P < 0.01). Fibroblast activation protein was expressed by human aortic smooth muscle cells (HASMC) as shown by colocalization on immunofluorescent aortic plaque stainings (n = 10; P < 0.01) and by flow cytometry in cell culture. Although macrophages did not express FAP, macrophage burden in human aortic plaques correlated with FAP expression (n = 12; R2= 0.763; P < 0.05). Enzyme-linked immunosorbent assays showed a time- and dose-dependent up-regulation of FAP in response to human tumour necrosis factor α (TNFα) in HASMC (n = 6; P < 0.01). Moreover, supernatants from peripheral blood-derived macrophages induced FAP expression in cultured HASMC (n = 6; P < 0.01), an effect abolished by blocking TNFα (n = 6; P < 0.01). Fibroblast activation protein associated with collagen-poor regions in human coronary fibrous caps and digested type I collagen and gelatin in vitro (n = 6; P < 0.01). Zymography revealed that FAP-mediated collagenase activity was neutralized by an antibody directed against the FAP catalytic domain both in HASMC (n = 6; P < 0.01) and in fibrous caps of atherosclerotic plaques (n = 10; P < 0.01). Conclusion Fibroblast activation protein expression in HASMC is induced by macrophage-derived TNFα. Fibroblast activation protein associates with thin-cap human coronary

  15. Comparative human cellular radiosensitivity: I. The effect of SV40 transformation and immortalisation on the gamma-irradiation survival of skin derived fibroblasts from normal individuals and from ataxia-telangiectasia patients and heterozygotes.

    PubMed

    Arlett, C F; Green, M H; Priestley, A; Harcourt, S A; Mayne, L V

    1988-12-01

    We have compared cell killing following 60Co gamma irradiation in 22 primary human fibroblast strains, nine SV40-immortalized human fibroblast lines and seven SV40-transformed pre-crisis human fibroblast cultures. We have examined material from normal individuals, from ataxia-telangiectasia (A-T) patients and from A-T heterozygotes. We have confirmed the greater sensitivity of A-T derived cells to gamma radiation. The distinction between A-T and normal cells is maintained in cells immortalized by SV40 virus but the immortal cells are more gamma radiation resistant than the corresponding primary fibroblasts. Cells transformed by plasmids (pSV3gpt and pSV3neo) expressing SV40 T-antigen, both pre- and post-crisis, show this increased resistance, indicating that it is expression of SV40 T-antigen, rather than immortalization per se which is responsible for the change. We use D0, obtained from a straight line fit, and D, estimated from a multitarget curve, as parameters to compare radiosensitivity. We suggest that both have their advantages; D0 is perhaps more reproducible, but D is more realistic when comparing shouldered and non-shouldered data.

  16. Alleviation of Ultraviolet B-Induced Photodamage by Coffea arabica Extract in Human Skin Fibroblasts and Hairless Mouse Skin

    PubMed Central

    Wu, Po-Yuan; Huang, Chi-Chang; Chu, Yin; Huang, Ya-Han; Lin, Ping; Liu, Yu-Han; Wen, Kuo-Ching; Lin, Chien-Yih; Hsu, Mei-Chich; Chiang, Hsiu-Mei

    2017-01-01

    Coffea arabica extract (CAE) containing 48.3 ± 0.4 mg/g of chlorogenic acid and a trace amount of caffeic acid was found to alleviate photoaging activity in human skin fibroblasts. In this study, polyphenol-rich CAE was investigated for its antioxidant and antiinflammatory properties, as well as for its capability to alleviate ultraviolet B (UVB)-induced photodamage in BALB/c hairless mice. The results indicated that 500 μg/mL of CAE exhibited a reducing power of 94.7%, ferrous ion chelating activity of 46.4%, and hydroxyl radical scavenging activity of 20.3%. The CAE dose dependently reduced UVB-induced reactive oxygen species (ROS) generation in fibroblasts. Furthermore, CAE inhibited the UVB-induced expression of cyclooxygenase-2 and p-inhibitor κB, and the translocation of nuclear factor-kappa B (NF-κB) to the nucleus of fibroblasts. In addition, CAE alleviated UVB-induced photoaging and photodamage in BALB/c hairless mice by restoring the collagen content and reduced UVB-induced epidermal hyperplasia. CAE also inhibited UVB-induced NF-κB, interleukin-6, and matrix metalloproteinase-1 expression in the hairless mouse skin. The results indicated that CAE exhibits antiphotodamage activity by inhibiting UV-induced oxidative stress and inflammation. Therefore, CAE is a candidate for use in antioxidant, antiinflammatory, and antiphotodamage products. PMID:28387707

  17. SORBS2 and TLR3 induce premature senescence in primary human fibroblasts and keratinocytes

    PubMed Central

    2013-01-01

    Background Genetic aberrations are required for the progression of HPV-induced cervical precancers. A prerequisite for clonal expansion of cancer cells is unlimited proliferative capacity. In a cell culture model for cervical carcinogenesis loss of genes located on chromosome 4q35→qter and chromosome 10p14-p15 were found to be associated with escape from senescence. Moreover, by LOH and I-FISH analyses a higher frequency of allele loss of these regions was also observed in cervical carcinomas as compared to CIN3. The aim of this study was to identify candidate senescence-related genes located on chromosome 4q35→qter and chromosome 10p14-p15 which may contribute to clonal expansion at the transition of CIN3 to cancer. Methods Microarray expression analyses were used to identify candidate genes down-regulated in cervical carcinomas as compared to CIN3. In order to relate these genes with the process of senescence their respective cDNAs were overexpressed in HPV16-immortalized keratinocytes as well as in primary human fibroblasts and keratinocytes using lentivirus mediated gene transduction. Results Overall fifteen genes located on chromosome 4q35→qter and chromosome 10p14-p15 were identified. Ten of these genes could be validated in biopsies by RT-PCR. Of interest is the novel finding that SORBS2 and TLR3 can induce senescence in primary human fibroblasts and keratinocytes but not in HPV-immortalized cell lines. Intriguingly, the endogenous expression of both genes increases during finite passaging of primary keratinocytes in vitro. Conclusions The relevance of the genes SORBS2 and TLR3 in the process of cellular senescence warrants further investigation. In ongoing experiments we are investigating whether this increase in gene expression is also characteristic of replicative senescence. PMID:24165198

  18. Profiles of Global Gene Expression in Ionizing-Radiation–Damaged Human Diploid Fibroblasts Reveal Synchronization behind the G1 Checkpoint in a G0-like State of Quiescence

    PubMed Central

    Zhou, Tong; Chou, Jeff W.; Simpson, Dennis A.; Zhou, Yingchun; Mullen, Thomas E.; Medeiros, Margarida; Bushel, Pierre R.; Paules, Richard S.; Yang, Xuebin; Hurban, Patrick; Lobenhofer, Edward K.; Kaufmann, William K.

    2006-01-01

    Cell cycle arrest and stereotypic transcriptional responses to DNA damage induced by ionizing radiation (IR) were quantified in telomerase-expressing human diploid fibroblasts. Analysis of cytotoxicity demonstrated that 1.5 Gy IR inactivated colony formation by 40–45% in three fibroblast lines; this dose was used in all subsequent analyses. Fibroblasts exhibited > 90% arrest of progression from G2 to M at 2 hr post-IR and a similarly severe arrest of progression from G1 to S at 6 and 12 hr post-IR. Normal rates of DNA synthesis and mitosis 6 and 12 hr post-IR caused the S and M compartments to empty by > 70% at 24 hr. Global gene expression was analyzed in IR-treated cells. A microarray analysis algorithm, EPIG, identified nine IR-responsive patterns of gene expression that were common to the three fibroblast lines, including a dominant p53-dependent G1 checkpoint response. Many p53 target genes, such as CDKN1A, GADD45, BTG2, and PLK3, were significantly up-regulated at 2 hr post-IR. Many genes whose expression is regulated by E2F family transcription factors, including CDK2, CCNE1, CDC6, CDC2, MCM2, were significantly down-regulated at 24 hr post-IR. Numerous genes that participate in DNA metabolism were also markedly repressed in arrested fibroblasts apparently as a result of cell synchronization behind the G1 checkpoint. However, cluster and principal component analyses of gene expression revealed a profile 24 hr post-IR with similarity to that of G0 growth quiescence. The results reveal a highly stereotypic pattern of response to IR in human diploid fibroblasts that reflects primarily synchronization behind the G1 checkpoint but with prominent induction of additional markers of G0 quiescence such as GAS1. PMID:16581545

  19. Substance P and neurokinin A metabolism by cultured human skeletal muscle myocytes and fibroblasts.

    PubMed

    Russell, J S; Chi, H; Lantry, L E; Stephens, R E; Ward, P E

    1996-01-01

    A recent study determined that cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts degraded angiotensins and kinins via neutral endopeptidase-24.11 (NEP-24.11: EC 3.4.24.11) and aminopeptidase N (APN: EC 3.4.11.2). Due to the possible importance of other peptides to skeletal muscle blood flow and function, the present study looked specifically at the metabolism of the neurokinins substance P (SP) and neurokinin A (NKA) by skeletal muscle peptidases. The results show that SP is degraded not only by NEP-24.11, but also sequentially by dipeptidyl(amino)peptidase IV (DAP IV: EC 3.4.14.5)/APN. NKA is unaffected by DAP IV but is metabolized by NEP-24.11 and APN. NEP-24.11 was inhibited by phosphoramidon (IC50 = 80 nM), thiorphan and ZINCOV, DAP IV by diprotin A (IC50 = 8 microM), and APN by amastatin (IC50 = 50 nM) and bestatin (IC50 = 100 microM). Skeletal muscle myocyte and fibroblast metabolism of SP and NKA may regulate local skeletal muscle vascular and extravascular functions including SP- and NKA-mediated nerve-induced vasodilation. Inhibition of both NEP-24.11 and DAP IV/APN may increase skeletal muscle blood flow and decrease peripheral vascular resistance via potentiation of local neurokinin levels.

  20. Effect of pirfenidone on proliferation, TGF-β-induced myofibroblast differentiation and fibrogenic activity of primary human lung fibroblasts.

    PubMed

    Conte, Enrico; Gili, Elisa; Fagone, Evelina; Fruciano, Mary; Iemmolo, Maria; Vancheri, Carlo

    2014-07-16

    Pirfenidone is an orally active small molecule that has been shown to inhibit the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis. Although pirfenidone exhibits well documented antifibrotic and antiinflammatory activities, in vitro and in vivo, its molecular targets and mechanisms of action have not been elucidated. In this study, we investigated the effects of pirfenidone on proliferation, TGF-β-induced differentiation and fibrogenic activity of primary human lung fibroblasts (HLFs). Pirfenidone reduced fibroblast proliferation and attenuated TGF-β-induced α-smooth muscle actin (SMA) and pro-collagen (Col)-I mRNA and protein levels. Importantly, pirfenidone inhibited TGF-β-induced phosphorylation of Smad3, p38, and Akt, key factors in the TGF-β pathway. Together, these results demonstrate that pirfenidone modulates HLF proliferation and TGF-β-mediated differentiation into myofibroblasts by attenuating key TGF-β-induced signaling pathways. Copyright © 2014 Elsevier B.V. All rights reserved.