The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

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Jelena Markovic

2009-07-01

Full Text Available Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM and buthionine sulfoximine (BSO, and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

The small Rho GTPase Rac1 controls normal human dermal fibroblasts proliferation with phosphorylation of the oncoprotein c-myc

International Nuclear Information System (INIS)

Nikolova, Ekaterina; Mitev, Vanio; Zhelev, Nikolai; Deroanne, Christophe F.; Poumay, Yves

2007-01-01

Proliferation of dermal fibroblasts is crucial for the maintenance of skin. The small Rho GTPase, Rac1, has been identified as a key transducer of proliferative signals in various cell types, but in normal human dermal fibroblasts its significance to cell growth control has not been studied. In this study, we applied the method of RNA interference to suppress endogenous Rac1 expression and examined the consequences on human skin fibroblasts. Rac1 knock-down resulted in inhibition of DNA synthesis. This effect was not mediated by inhibition of the central transducer of proliferative stimuli, ERK1/2 or by activation of the pro-apoptotic p38. Rather, as a consequence of the suppressed Rac1 expression we observed a significant decrease in phosphorylation of c-myc, revealing for the first time that in human fibroblasts Rac1 exerts control on proliferation through c-myc phosphorylation. Thus Rac1 activates proliferation of normal fibroblasts through stimulation of c-myc phosphorylation without affecting ERK1/2 activity

Fibroblast growth factor-mediated proliferation of central nervous system precursors depends on endogenous production of insulin-like growth factor I

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Drago, J.; Murphy, M.; Carroll, S.M.; Harvey, R.P.; Bartlett, P.F.

1991-01-01

Fibroblast growth factor stimulates proliferation and subsequent differentiation of precursor cells isolated from the neuroepithelium of embryonic day 10 mice in vitro. Here we show that fibroblast growth factor-induced proliferation is dependent on the presence of insulin-like growth factors (IGFs) and that IGF-I is endogenously produced by the neuroepithelial cells. Blocking of endogenous IGF-I activity with anti-IGF-I antibodies results in complete inhibition of fibroblast growth factor-mediated proliferation and in cell death. IGF-I alone acts as a survival agent. These observations correlate with the detection of transcripts for IGF-I and basic fibroblast growth factor in freshly isolated neuroepithelium and are consistent with an autocrine action of these factors in early brain development in vivo

Induction of growth and proliferation of fibroblast cells in magnetic field

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Naghmeh Ezatti

2015-02-01

Full Text Available Background: Tissue engineering is generally defined as developing and changing the laboratory growth of molecules and cells in tissues or organs to replace and repair the damaged part of body. This study was carried out to stimulate the growth of cultured fibroblast cells by a physical electromagnetic method. Methods: First, an air-core coil was prepared and the cell culture plate was placed comfortably into the mold, then the plate containing the culture medium and human fibroblast cell along with air-core coil were placed in an incubator and then connected to the power supply. Thus, the sample underwent electromagnetic field at different times, and cell proliferation was studied by MTTassay. Results: Microscopic images indicated that the cells undergoing electromagnetic field (0.35 amps had a significant growth compared to the cells in control group in a definit range of stimulation. Conclusion: In conclusion, electromagnetic stimulation in a definite range led to cell proliferation and could be used as a positive factor in tissue engineering.

Inhibition of proliferation and migration of stricture fibroblasts by epithelial cell-conditioned media

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Nilima Nath

2015-01-01

Conclusion: These results demonstrate the ability of ECCM to inhibit the proliferation and migration of stricture fibroblasts and present it as an effective adjunct in urethroplasty, which may influence stricture wound healing and inhibit the recurrence of stricture.

Effects of plant lectins on in vitro fibroblast proliferation

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Ana Maria Sell

2003-06-01

Full Text Available Lectins are carbohydrate-binding proteins that have been isolated from various sources and presented a wide spectrum of biological activities. The effects of four lectins, namely, Phaseolus vulgaris phytohemagglutinin, PHA, wheat germ agglutinin, WGA, Artocarpus integrifolia seed lectins, jacalin and artocarpin, on in vitro fibroblasts proliferation were investigated. The lectins did not influence the initial cell adhesion to the plate. PHA and WGA at 10-20 µg/mL concentrations significantly decreased fibroblasts proliferation. At these concentrations, they caused morphological alterations on cells and over 80 µg/mL, promoted cell death. Neither jacalin nor artocarpin significantly affected cell proliferation.O objetivo deste trabalho foi avaliar a influência das lectinas PHA, WGA, jacalina e artocarpina sobre a proliferação de fibroblastos in vitro. Para tanto, fibroblastos gengivais de voluntários saudáveis foram cultivados, por cinco dias, em DMEM suplementado com soro bovino fetal (10% v/v e na presença das lectinas nas concentrações finais de 0.1 a 300 µg/mL. A adesão, o crescimento e a morfologia celular foram acompanhados por microscopia de inversão e contraste de fase. O índice de proliferação foi avaliado pelo método calorimétrico usando MTT. As lectinas não alteraram a adesão inicial dos fibroblastos à placa de poliestireno. PHA e WGA, nas concentrações de 10 a 20 µg/mL, diminuíram significativamente a proliferação celular. Nestas concentrações a morfologia celular é alterada e acima de 80 µg/mL, houve100% de morte celular. As lectinas jacalina e artocarpina não influenciaram a proliferação celular.

Background Levels of Neomorphic 2-hydroxyglutarate Facilitate Proliferation of Primary Fibroblasts

Czech Academy of Sciences Publication Activity Database

Dvořák, Aleš; Zelenka, Jaroslav; Smolková, Katarína; Vítek, L.; Ježek, Petr

2017-01-01

Roč. 66, č. 2 (2017), s. 293-304 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GPP301/12/P381; GA ČR(CZ) GA16-04788S Institutional support: RVO:67985823 Keywords : 2-hydroxyglutarate * fibroblast proliferation * SH-SY5Y neuroblastoma cells Subject RIV: CE - Biochemistry OBOR OECD: Oncology Impact factor: 1.461, year: 2016

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

International Nuclear Information System (INIS)

Li, Xiaoou; Liu, Lian; Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan; Wen, Fuqiang

2014-01-01

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 between 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

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

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Li, Xiaoou [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Liu, Lian [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Shen, Yongchun; Wang, Tao; Chen, Lei; Xu, Dan [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Wen, Fuqiang, E-mail: wenfuqiang.scu@gmail.com [Division of Pulmonary Diseases, State Key Laboratory of Biotherapy of China, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China); Department of Respiratory Medicine, West China Hospital, West China School of Medicine, Sichuan University, Chengdu, Sichuan (China)

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 between 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.

Effects of recombinant human epidermal growth factor on the proliferation and radiation survival of human fibroblast cell lines in vitro

International Nuclear Information System (INIS)

Kim, Hyun Sook; Kang, Ki Mun; Na, Jae Boem; Chai, Gyu Young; Lee, Sang Wook

2006-01-01

To explore the effect of recombinant human EGF on the proliferation and survival of human fibroblast cell lines following irradiation. Fibroblast was originated human skin and primary cultured. The trypan blue stain assay and MTT assay were used to study the proliferative effects of EGF on human fibroblast cell lines in vitro. An incubation of fibroblasts with rhEGF for 24 hours immediately after irradiation was counted everyday. Cell cycle distributions were analyzed by FACS analysis. Number of fibroblast was significant more increased rhEGF (1.0 nM, 10 nM, 100 nM, 1,000 nM) treated cell than control after 8 Gy irradiation. Most effective dose of rhEGF was at 160 nM. These survival differences were maintained at 1 week later. Proportion of S phase was significantly increased on rhEGF treated cells. rhEGF cause increased fibroblast proliferation following irradiation. We expect that rhEGF was effective for radiation induced wound healing

Proliferation-promoting effect of platelet-rich plasma on human adipose-derived stem cells and human dermal fibroblasts.

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Kakudo, Natsuko; Minakata, Tatsuya; Mitsui, Toshihito; Kushida, Satoshi; Notodihardjo, Frederik Zefanya; Kusumoto, Kenji

2008-11-01

This study evaluated changes in platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 release from platelets by platelet-rich plasma activation, and the proliferation potential of activated platelet-rich plasma and platelet-poor plasma on human adipose-derived stem cells and human dermal fibroblasts. Platelet-rich plasma was prepared using a double-spin method, with the number of platelets counted in each preparation stage. Platelet-rich and platelet-poor plasma were activated with autologous thrombin and calcium chloride, and levels of platelet-released PDGF-AB and TGF-beta1 were determined by enzyme-linked immunosorbent assay. Cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 5% whole blood plasma, nonactivated platelet-rich plasma, nonactivated platelet-poor plasma, activated platelet-rich plasma, or activated platelet-poor plasma. In parallel, these cells were cultured for 1, 4, or 7 days in serum-free Dulbecco's Modified Eagle Medium supplemented with 1%, 5%, 10%, or 20% activated platelet-rich plasma. The cultured human adipose-derived stem cells and human dermal fibroblasts were assayed for proliferation. Platelet-rich plasma contained approximately 7.9 times as many platelets as whole blood, and its activation was associated with the release of large amounts of PDGF-AB and TGF-beta1. Adding activated platelet-rich or platelet-poor plasma significantly promoted the proliferation of human adipose-derived stem cells and human dermal fibroblasts. Adding 5% activated platelet-rich plasma to the medium maximally promoted cell proliferation, but activated platelet-rich plasma at 20% did not promote it. Platelet-rich plasma can enhance the proliferation of human adipose-derived stem cells and human dermal fibroblasts. These results support clinical platelet-rich plasma application for cell-based, soft-tissue engineering and wound healing.

Plasma rich in growth factors promotes dermal fibroblast proliferation, migration and biosynthetic activity.

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Anitua, E; Pino, A; Orive, G

2016-11-02

The use of plasma rich in growth factors (PRGF) has gained importance in many medical fields due to its regenerative potential. The aim of this study is to evaluate the effects of PRGF on primary skin fibroblasts assessing cell proliferation, migration and secretion of growth factors. The age of the patients from who PRGF was prepared was also studied to determine whether it influenced the outcomes. Human dermal fibroblasts were isolated from three healthy volunteers. Using PRGF-Endoret technology, PRGF was prepared from two groups of different ages (18-35 years and 50+ years). The effects of increasing concentration of PRGF (5%, 10% and 20%) on cell proliferation and migration was evaluated. Biosynthetic behaviour of cells was also analysed measuring vascular endothelial growth factor (VEGF), transforming growth factor b1 (TGFb1) and pro-collagen type I secreted levels with or without PRGF treatment. Mean platelet enrichment reached 2.4X and 2X in 18-35 and 50+ groups respectively. A dose-dependent response was observed in proliferation assays achieving the highest levels with 20% PRGF. Migration was also promoted in cells but not in a dose-dependent manner. Cell proliferation and migration outcomes obtained with PRGF (from both groups) were significantly higher compared to non-stimulated groups (pPRGF, however, with the exception of VEGF, no statistical significances were observed between the different age groups. Results from this study concluded that PRGF is safe and effective in stimulating skin regeneration by enhancing proliferation, migration and expression of pivotal bioactive molecules involved in wound healing and haemostasis.

In vitro vitamin K3 effect on conjunctival fibroblast migration and proliferation.

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Pinilla, I; Izaguirre, L B; Gonzalvo, F J; Piazuelo, E; Garcia-Gonzalez, M A; Sanchez-Cano, A I; Sopeña, F

2014-01-01

To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [(3)H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

In Vitro Vitamin K3 Effect on Conjunctival Fibroblast Migration and Proliferation

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I. Pinilla

2014-01-01

Full Text Available Purpose. To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Methods. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [3H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L, group 4/vitamin K3 2 mg/L, group 5/vitamin K3 4 mg/L, and group 6/vitamin K3 6 mg/L. Each experiment was carried out in triplicate and 4 times. Results. There were no differences among groups at the initial time. In vitro wound repair was slower in groups 4, 5, and 6. There were no differences between control and ethanol groups and between control and vitamin K3 1 mg/L groups. Fibroblast mitogenic activity was statistically decreased in all vitamin K groups; statistical differences were found among vitamin K3 1 mg/mL and higher doses too. In groups 5 and 6, cellular toxicity was presented. Conclusions. Vitamin K3 is able to inhibit fibroblast proliferation. Vitamin K3 2 mg/L or higher doses inhibit wound healing repair, exhibiting cellular toxicity at 4 and 6 mg/L.

Proliferating fibroblasts and HeLa cells co-cultured in vitro reciprocally influence growth patterns, protein expression, chromatin features and cell survival.

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Delinasios, John G; Angeli, Flora; Koumakis, George; Kumar, Shant; Kang, Wen-Hui; Sica, Gigliola; Iacopino, Fortunata; Lama, Gina; Lamprecht, Sergio; Sigal-Batikoff, Ina; Tsangaris, George T; Farfarelos, Christos D; Farfarelos, Maria C; Vairaktaris, Eleftherios; Vassiliou, Stavros; Delinasios, George J

2015-04-01

to identify biological interactions between proliferating fibroblasts and HeLa cells in vitro. Fibroblasts were isolated from both normal and tumour human tissues. Coverslip co-cultures of HeLa and fibroblasts in various ratios with medium replacement every 48 h were studied using fixed cell staining with dyes such as Giemsa and silver staining, with immunochemistry for Ki-67 and E-cadherin, with dihydrofolate reductase (DHFR) enzyme reaction, as well as live cell staining for non-specific esterases and lipids. Other techniques included carmine cell labeling, autoradiography and apoptosis assessment. Under conditions of feeding and cell: cell ratios allowing parallel growth of human fibroblasts and HeLa cells, co-cultured for up to 20 days, a series of phenomena occur consecutively: profound affinity between the two cell types and exchange of small molecules; encircling of the HeLa colonies by the fibroblasts and enhanced growth of both cell types at their contact areas; expression of carbonic anhydrase in both cell types and high expression of non-specific esterases and cytoplasmic argyrophilia in the surrounding fibroblasts; intense production and secretion of lipid droplets by the surrounding fibroblasts; development of a complex net of argyrophilic projections of the fibroblasts; E-cadherin expression in the HeLa cells; from the 10th day onwards, an increasing detachment of batches of HeLa cells at the peripheries of colonies and appearance of areas with many multi-nucleated and apoptotic HeLa cells, and small HeLa fragments; from the 17th day, appearance of fibroblasts blocked at the G2-M phase. Co-cultures at approximately 17-20 days display a cell-cell fight with foci of (a) sparse growth of both cell types, (b) overgrowth of the fibroblasts and (c) regrowth of HeLa in small colonies. These results indicate that during their interaction with HeLa cells in vitro, proliferating fibroblasts can be activated against HeLa. This type of activation is not observed

Increased p21ras activity in human fibroblasts transduced with survivin enhances cell proliferation

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Temme, Achim; Diestelkoetter-Bachert, Petra; Schmitz, Marc; Morgenroth, Agnieszka; Weigle, Bernd; Rieger, Michael A.; Kiessling, Andrea; Rieber, E. Peter

2005-01-01

Survivin is critically involved in mitosis and when overexpressed enhances the activity of the Aurora B kinase, a serine-threonine kinase belonging to the family of oncogenic Aurora/IpI1p-related kinases. Both proteins interact with Ras GTPase-activating protein suggesting an impact on the Ras pathway. This study aimed at defining the role of survivin in proliferation and potential transformation of cells. When survivin was overexpressed in normal human lung fibroblasts, the characteristic track lanes of fibroblasts were disturbed and the rate of cell proliferation was increased. An enhanced level of p21 ras mRNA and protein expression and concomitant rise in levels of activated p21 ras were observed. Despite increased proliferation cell survival remained dependent on serum and cells were not able to form colonies in soft agar assays. These data suggest that overexpression of survivin increases cell growth but, despite the increase in active p21 ras , is not sufficient to transform primary cells. Yet, in addition to its anti-apoptotic function it might contribute to the accelerated growth of tumour cells by increasing p21 ras activity

Effects of hydroxysafflor yellow A on proliferation and collagen synthesis of rat vascular adventitial fibroblasts induced by angiotensin II.

Science.gov (United States)

Yuan, Wendan; Yang, Dongxia; Sun, Xuhong; Liu, Wei; Wang, Liang; Li, Xiaoyan; Man, Xuejing; Fu, Qiang

2014-01-01

1) examine the effects of hydroxysafflor yellow A (HSYA) on the proliferation, collagen and cytokine synthesis of vascular adventitial fibroblasts as induced by angiotensin II (Ang II) in normal Sprague-Dawley (SD) rats in vitro, and 2) to assess the effects of HSYA on morphological changes and collagen accumulation of vascular adventitia in spontaneously hypertensive rats (SHR) in vivo. In vitro experiment, vascular adventitial fibroblasts from SD rats were isolated, cultured, and divided into control groups, model groups and HSYA groups. Cell morphology of adventitial fibroblasts was assessed using laser confocal microscopy, while cell proliferation with the MTT assay, and collagen synthesis was determined using hydroxyproline chromatometry. Immunocytochemistry and reverse transcription PCR were used for detecting the expression of TGF-β1, MMP-1, α-SMA and NF-κB in adventitial fibroblasts. In vivo experiment, vascular adventitia proliferation and collagen synthesis were analyzed using hematoxylin-eosin and Sirius staining. Our results showed that: 1) in vitro experiment of SD rats, HSYA inhibited proliferative activity and collagen synthesis of adventitial fibroblasts as induced by Ang II, and the inhibitory effects of HSYA on the increased expression of MMP-1, TGF-β1, α-SMA and NF-κB p65 as induced by Ang II were assessed, and 2) in vivo experiment of SHR, histological analysis displayed fewer pathological changes of vascular adventitia in HSYA treatment groups as compared with no HSYA treatment groups, and MMP-1, TGF-β1, α-SMA and NF-κB p65 expression significantly reduced after HSYA treatment (P adventitia components. This study provides experimental evidence demonstrating that HSYA has the capacity to decrease vascular adventitia proliferation and hyperplasia during vascular remodeling.

Heterogeneity in Fibroblast Proliferation and Survival in Idiopathic Pulmonary Fibrosis

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David Michael Habiel

2014-01-01

Full Text Available Idiopathic pulmonary fibrosis (IPF is the most common form of interstitial lung disease characterized by the persistence of activated myofibroblasts resulting in excessive deposition of extracellular matrix proteins and profound tissue remodeling. Myofibroblasts have been shown to arise from interstitial fibroblasts, epithelial to mesenchymal transition of type II alveolar epithelial cells, and the differentiation of recruited fibrocytes. There are many mechanisms that are utilized by these cells for survival, proliferation and persistent activation including up-regulation of cytokines (i.e. Interlukin 6 (IL-6, cytokine receptors (i.e. Interlukin 6 Receptor 1 (IL-6R1, Glycoprotein 130 (gp130 and C-C Chemokine Receptor type 7 (CCR7 and innate pattern recognition receptors (PRRs; i.e. Toll Like Receptor 9 (TLR9. In this review, we will discuss the role of the cytokines IL-6 and CCL21, their receptors and the pattern recognition receptor (PRR, TLR9, in fibroblast recruitment, activation, survival and differentiation into myofibroblasts in IPF.

The gene expression program of prostate fibroblast senescence modulates neoplastic epithelial cell proliferation through paracrine mechanisms.

Science.gov (United States)

Bavik, Claes; Coleman, Ilsa; Dean, James P; Knudsen, Beatrice; Plymate, Steven; Nelson, Peter S

2006-01-15

The greatest risk factor for developing carcinoma of the prostate is advanced age. Potential molecular and physiologic contributors to the frequency of cancer occurrence in older individuals include the accumulation of somatic mutations through defects in genome maintenance, epigenetic gene silencing, oxidative stress, loss of immune surveillance, telomere dysfunction, chronic inflammation, and alterations in tissue microenvironment. In this context, the process of prostate carcinogenesis can be influenced through interactions between intrinsic cellular alterations and the extrinsic microenvironment and macroenvironment, both of which change substantially as a consequence of aging. In this study, we sought to characterize the molecular alterations that occur during the process of prostate fibroblast senescence to identify factors in the aged tissue microenvironment capable of promoting the proliferation and potentially the neoplastic progression of prostate epithelium. We evaluated three mechanisms leading to cell senescence: oxidative stress, DNA damage, and replicative exhaustion. We identified a consistent program of gene expression that includes a subset of paracrine factors capable of influencing adjacent prostate epithelial growth. Both direct coculture and conditioned medium from senescent prostate fibroblasts stimulated epithelial cell proliferation, 3-fold and 2-fold, respectively. The paracrine-acting proteins fibroblast growth factor 7, hepatocyte growth factor, and amphiregulin (AREG) were elevated in the extracellular environment of senescent prostate fibroblasts. Exogenous AREG alone stimulated prostate epithelial cell growth, and neutralizing antibodies and small interfering RNA targeting AREG attenuated, but did not completely abrogate the growth-promoting effects of senescent fibroblast conditioned medium. These results support the concept that aging-related changes in the prostate microenvironment may contribute to the progression of prostate

Notoginsenoside Ft1 Promotes Fibroblast Proliferation via PI3K/Akt/mTOR Signaling Pathway and Benefits Wound Healing in Genetically Diabetic Mice.

Science.gov (United States)

Zhang, Eryun; Gao, Bo; Yang, Li; Wu, Xiaojun; Wang, Zhengtao

2016-02-01

Wound healing requires the essential participation of fibroblasts, which is impaired in diabetic foot ulcers (DFU). Notoginsenoside Ft1 (Ft1), a saponin from Panax notoginseng, can enhance platelet aggregation by activating signaling network mediated through P2Y12 and induce proliferation, migration, and tube formation in cultured human umbilical vein endothelial cells. However, whether it can accelerate fibroblast proliferation and benefit wound healing, especially DFU, has not been elucidated. In the present study on human dermal fibroblast HDF-a, Ft1 increased cell proliferation and collagen production via PI3K/Akt/mTOR signaling pathway. On the excisional wound splinting model established on db/db diabetic mouse, topical application of Ft1 significantly shortened the wound closure time by 5.1 days in contrast with phosphate-buffered saline (PBS) treatment (15.8 versus 20.9 days). Meanwhile, Ft1 increased the rate of re-epithelialization and the amount of granulation tissue at day 7 and day 14. The molecule also enhanced mRNA expressions of COL1A1, COL3A1, transforming growth factor (TGF)-β1 and TGF-β3 and fibronectin, the genes that contributed to collagen expression, fibroblast proliferation, and consequent scar formation. Moreover, Ft1 facilitated the neovascularization accompanied with elevated vascular endothelial growth factor, platelet-derived growth factor, and fibroblast growth factor at either mRNA or protein levels and alleviated the inflammation of infiltrated monocytes indicated by reduced tumor necrosis factor-α and interleukin-6 mRNA expressions in the diabetic wounds. Altogether, these results indicated that Ft1 might accelerate diabetic wound healing by orchestrating multiple processes, including promoting fibroblast proliferation, enhancing angiogenesis, and attenuating inflammatory response, which provided a great potential application of it in clinics for patients with DFU. Copyright © 2016 by The American Society for Pharmacology and

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

International Nuclear Information System (INIS)

Wang, Xianwei; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

2012-01-01

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22 phox , p47 phox , p67 phox , NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H 2 O 2 . Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

Energy Technology Data Exchange (ETDEWEB)

Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

2012-03-15

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

Effect of macrophage and matrix metalloproteinase-9 on proliferation of pulmonary fibroblast and synthesis of collagen IV

International Nuclear Information System (INIS)

Song Liangwen; Sun Li; Diao Ruiying; Li Yang; Zhang Yong; Yin Jiye

2006-01-01

Objective: To explore pathogenetic mechanism in initiation of radiation-induced pulmonary fibrosis. Methods: Alveolar macrophages in Wistar rats irradiated by 60 Co γ-ray were collected by alveolar lavage; condition medium was prepared for stimulating human lung fibroblast (HLF) proliferation; HLF proliferation activity was determined by MTT method; collagen IV (Col IV) in HLF was determined by Western blot; the activity of matrix metalloproteinase-9 (MMP-9) was determined by zymography. Results: HLF proliferation activity was significantly increased after stimulation of condition medium, and the increase was most evident within 48-72 hs. Col IV synthesis in HLF was increased and reached a peak at 12 h after stimulation and then began to decrease. MMP-9 activity began to increase at 12 h and reached a peak at 48 h and then decreased after 72 h. Conclusions: Cobalt-60 gamma ray irradiation of 20 Gy can stimulate secretion of some cytokines in alveolar macrophage to promote pulmonary interstitial fibroblast proliferation and synthesis of Col IV . Col IV can stimulate MMP-9 increase; MMP-9 can degrade excess Col IV. Such changes are involved in remodeling process of early pulmonary injury. (authors)

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

International Nuclear Information System (INIS)

Hecht, Emelia; Zago, Michela; Sarill, Miles; Rico de Souza, Angela; Gomez, Alvin; Matthews, Jason; Hamid, Qutayba; Eidelman, David H.; Baglole, Carolyn J.

2014-01-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR −/− ) and wild-type (AhR +/+ ) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR −/− cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR −/− compared to AhR +/+ cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR +/+ fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR +/+ lung fibroblasts in response to serum, corresponding to a decrease in p27 KIP1 protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27 KIP1 in AhR −/− fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates the expression of the microRNA miR-196a independent of

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

DEFF Research Database (Denmark)

Anisimov, Sergey V.; Christophersen, Nicolaj S.; Correia, Ana S.

2011-01-01

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both...... the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early...... foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates....

TGF-β1 activates the canonical NF-κB signaling to promote cell survival and proliferation in dystrophic muscle fibroblasts in vitro

Energy Technology Data Exchange (ETDEWEB)

Ma, Zhen-Yu [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Department of Neurology, The Second Affiliated Hospital, Guangzhou Medical University, No.250 Changgang East Road, Guangzhou 510260, Guangdong Province (China); Zhong, Zhi-Gang; Qiu, Meng-Yao; Zhong, Yu-Hua [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China); Zhang, Wei-Xi, E-mail: weixizhang@qq.com [Department of Neurology, The First Affiliated Hospital, Sun Yat-sen University, No. 58 Zhongshan 2nd Road, Guangzhou 510080, Guangdong Province (China)

2016-03-18

Activated fibroblasts continue to proliferate at injury sites, leading to progressive muscular fibrosis in Duchenne muscular dystrophy (DMD). TGF-β1 is a dominant profibrotic mediator thought to play a critical role in muscle fibrosis; however, the implicated mechanisms are not fully understood. Here we showed that TGF-β1 increased the resistance to apoptosis and stimulated cell cycle progression in dystrophic muscle fibroblasts under serum deprivation conditions in vitro. TGF-β1 treatment activated the canonical NF-κB pathway; and we found that pharmacological inhibition of IKKβ with IMD-0354 and RelA gene knockdown with siRNA attenuated these effects of TGF-β1 on dystrophic muscle fibroblasts. Collectively, our data suggest that TGF-β1 prevents apoptosis and cell cycle arrest in dystrophic muscle fibroblasts through the canonical NF-κB signaling pathway. - Highlights: • TGF-β1 promotes survival and proliferation in dystrophic muscle fibroblasts. • TGF-β1 activated the canonical NF-κB pathway in dystrophic muscle fibroblasts. • Canonical NF-κB pathway mediates these effects of TGF-β1.

TGF-β1 activates the canonical NF-κB signaling to promote cell survival and proliferation in dystrophic muscle fibroblasts in vitro

International Nuclear Information System (INIS)

Ma, Zhen-Yu; Zhong, Zhi-Gang; Qiu, Meng-Yao; Zhong, Yu-Hua; Zhang, Wei-Xi

2016-01-01

Activated fibroblasts continue to proliferate at injury sites, leading to progressive muscular fibrosis in Duchenne muscular dystrophy (DMD). TGF-β1 is a dominant profibrotic mediator thought to play a critical role in muscle fibrosis; however, the implicated mechanisms are not fully understood. Here we showed that TGF-β1 increased the resistance to apoptosis and stimulated cell cycle progression in dystrophic muscle fibroblasts under serum deprivation conditions in vitro. TGF-β1 treatment activated the canonical NF-κB pathway; and we found that pharmacological inhibition of IKKβ with IMD-0354 and RelA gene knockdown with siRNA attenuated these effects of TGF-β1 on dystrophic muscle fibroblasts. Collectively, our data suggest that TGF-β1 prevents apoptosis and cell cycle arrest in dystrophic muscle fibroblasts through the canonical NF-κB signaling pathway. - Highlights: • TGF-β1 promotes survival and proliferation in dystrophic muscle fibroblasts. • TGF-β1 activated the canonical NF-κB pathway in dystrophic muscle fibroblasts. • Canonical NF-κB pathway mediates these effects of TGF-β1.

GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

Science.gov (United States)

Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

2016-01-01

Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

Aryl hydrocarbon receptor-dependent regulation of miR-196a expression controls lung fibroblast apoptosis but not proliferation

Energy Technology Data Exchange (ETDEWEB)

Hecht, Emelia [Department of Medicine, McGill University, Montreal, Quebec (Canada); Zago, Michela [Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Sarill, Miles [Department of Medicine, McGill University, Montreal, Quebec (Canada); Rico de Souza, Angela [Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Gomez, Alvin; Matthews, Jason [Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON (Canada); Hamid, Qutayba; Eidelman, David H. [Department of Medicine, McGill University, Montreal, Quebec (Canada); Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada); Baglole, Carolyn J., E-mail: Carolyn.baglole@McGill.ca [Department of Medicine, McGill University, Montreal, Quebec (Canada); Research Institute of the McGill University Health Centre, McGill University, Montreal, Quebec (Canada)

2014-11-01

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor implicated in the regulation of apoptosis and proliferation. Although activation of the AhR by xenobiotics such as dioxin inhibits the cell cycle and control apoptosis, paradoxically, AhR expression also promotes cell proliferation and survival independent of exogenous ligands. The microRNA (miRNA) miR-196a has also emerged as a regulator of proliferation and apoptosis but a relationship between the AhR and miR-196a is not known. Therefore, we hypothesized that AhR-dependent regulation of endogenous miR-196a expression would promote cell survival and proliferation. Utilizing lung fibroblasts from AhR deficient (AhR{sup −/−}) and wild-type (AhR{sup +/+}) mice, we show that there is ligand-independent regulation of miRNA, including low miR-196a in AhR{sup −/−} cells. Validation by qRT-PCR revealed a significant decrease in basal expression of miR-196a in AhR{sup −/−} compared to AhR{sup +/+} cells. Exposure to AhR agonists benzo[a]pyrene (B[a]P) and FICZ as well as AhR antagonist CH-223191 decreased miR-196a expression in AhR{sup +/+} fibroblasts concomitant with decreased AhR protein levels. There was increased proliferation only in AhR{sup +/+} lung fibroblasts in response to serum, corresponding to a decrease in p27{sup KIP1} protein, a cyclin-dependent kinase inhibitor. Increasing the cellular levels of miR-196a had no effect on proliferation or expression of p27{sup KIP1} in AhR{sup −/−} fibroblasts but attenuated cigarette smoke-induced apoptosis. This study provides the first evidence that AhR expression is essential for the physiological regulation of cellular miRNA levels- including miR-196a. Future experiments designed to elucidate the functional relationship between the AhR and miR-196a may delineate additional novel ligand-independent roles for the AhR. - Highlights: • The AhR controls proliferation and apoptosis in lung cells. • The AhR regulates the

In Vitro Vitamin K 3 Effect on Conjunctival Fibroblast Migration and Proliferation

OpenAIRE

Pinilla, I.; Izaguirre, L. B.; Gonzalvo, F. J.; Piazuelo, E.; Garcia-Gonzalez, M. A.; Sanchez-Cano, A. I.; Sopeña, F.

2014-01-01

Purpose. To evaluate the dose effect of vitamin K3 on wound healing mechanisms. Methods. Conjunctival fibroblasts were incubated for 24 hours. An artificial wound was made and the cells were incubated with fresh medium plus doses of vitamin K3 to be tested. Wound repair was monitored at 0, 18, 24, and 48 hours. Proliferation was measured in actively dividing cells by [3H]thymidine uptake. Six different groups were tested: group 1/no drugs added, group 2/ethanol 0.1%, group 3/vitamin K3 1 mg/L...

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

International Nuclear Information System (INIS)

Matsumura, Kaori; Taketomi, Takaharu; Yoshizaki, Keigo; Arai, Shinsaku; Sanui, Terukazu; Yoshiga, Daigo; Yoshimura, Akihiko; Nakamura, Seiji

2011-01-01

Research highlights: → Sprouty2-deficient mice exhibit cleft palate as a result of failure of palatal shelf elevation. → We examined palate cell proliferation in Sprouty2-deficient mice. → Palate mesenchymal cell proliferation was increased in Sprouty2 KO mice. → Sprouty2 plays roles in murine palatogenesis by regulating cell proliferation. -- Abstract: Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.

Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

Energy Technology Data Exchange (ETDEWEB)

Matsumura, Kaori [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Taketomi, Takaharu, E-mail: taketomi@dent.kyushu-u.ac.jp [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yoshizaki, Keigo [Section of Orthodontics, Division of Oral Health, Growth and Development, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Arai, Shinsaku [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Sanui, Terukazu [Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yoshiga, Daigo [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan); Yoshimura, Akihiko [Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582 (Japan); Japan Science and Technology Agency (JST), CREST, Chiyoda-ku, Tokyo 102-0075 (Japan); Nakamura, Seiji [Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582 (Japan)

2011-01-28

Research highlights: {yields} Sprouty2-deficient mice exhibit cleft palate as a result of failure of palatal shelf elevation. {yields} We examined palate cell proliferation in Sprouty2-deficient mice. {yields} Palate mesenchymal cell proliferation was increased in Sprouty2 KO mice. {yields} Sprouty2 plays roles in murine palatogenesis by regulating cell proliferation. -- Abstract: Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.

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

Science.gov (United States)

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.

Random mtDNA mutations modulate proliferation capacity in mouse embryonic fibroblasts

International Nuclear Information System (INIS)

Kukat, Alexandra; Edgar, Daniel; Bratic, Ivana; Maiti, Priyanka; Trifunovic, Aleksandra

2011-01-01

Highlights: → Increased mtDNA mutations in MEFs lead to high level of spontaneous immortalization. → This process is independent of endogenous ROS production. → Aerobic glycolysis significantly contributes to spontaneous immortalization of MEFs. -- Abstract: An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O 2 ) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

8,12;8,20-diepoxy-8,14-secopregnane glycosides from roots of Asclepias tuberosa and their effect on proliferation of human skin fibroblasts.

Science.gov (United States)

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.

Human Umbilical Cord Blood Serum Has Higher Potential in Inducing Proliferation of Fibroblast than Fetal Bovine Serum

Directory of Open Access Journals (Sweden)

Ferry Sandra

2017-09-01

Full Text Available Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS.  Human umbilical cord blood (hUCB has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS could be potential and were investigated in current study. Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay. Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000. Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000. Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast. Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation

A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

DEFF Research Database (Denmark)

Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

1992-01-01

major brands migrating at apparent Mr of 38,000, 45,000, and 80,000, in addition to many minor bands between Mr 45,000 and 97,000, including Mr 52,000. The Mr 45,000 and 38,000 were associated with the cell membrane and Mr 52,000 as well as Mr 38,000 were associated with the lysosomes. The 1B10......Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...

Connective tissue growth factor stimulates the proliferation, migration and differentiation of lung fibroblasts during paraquat-induced pulmonary fibrosis.

Science.gov (United States)

Yang, Zhizhou; Sun, Zhaorui; Liu, Hongmei; Ren, Yi; Shao, Danbing; Zhang, Wei; Lin, Jinfeng; Wolfram, Joy; Wang, Feng; Nie, Shinan

2015-07-01

It is well established that paraquat (PQ) poisoning can cause severe lung injury during the early stages of exposure, finally leading to irreversible pulmonary fibrosis. Connective tissue growth factor (CTGF) is an essential growth factor that is involved in tissue repair and pulmonary fibrogenesis. In the present study, the role of CTGF was examined in a rat model of pulmonary fibrosis induced by PQ poisoning. Histological examination revealed interstitial edema and extensive cellular thickening of interalveolar septa at the early stages of poisoning. At 2 weeks after PQ administration, lung tissue sections exhibited a marked thickening of the alveolar walls with an accumulation of interstitial cells with a fibroblastic appearance. Masson's trichrome staining revealed a patchy distribution of collagen deposition, indicating pulmonary fibrogenesis. Western blot analysis and immunohistochemical staining of tissue samples demonstrated that CTGF expression was significantly upregulated in the PQ-treated group. Similarly, PQ treatment of MRC-5 human lung fibroblast cells caused an increase in CTGF in a dose-dependent manner. Furthermore, the addition of CTGF to MRC-5 cells triggered cellular proliferation and migration. In addition, CTGF induced the differentiation of fibroblasts to myofibroblasts, as was evident from increased expression of α-smooth muscle actin (α-SMA) and collagen. These findings demonstrate that PQ causes increased CTGF expression, which triggers proliferation, migration and differentiation of lung fibroblasts. Therefore, CTGF may be important in PQ-induced pulmonary fibrogenesis, rendering this growth factor a potential pharmacological target for reducing lung injury.

Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression

DEFF Research Database (Denmark)

Gagliardi, Mark; Maynard, Scott; Miyake, Tetsuaki

2003-01-01

in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF......Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C....../EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had...

Fibroblast response to initial attachment and proliferation on titanium and zirconium surfaces.

Directory of Open Access Journals (Sweden)

Araceli Meza-Rodríguez

2016-08-01

Full Text Available Introduction: In recent decades, dental implants have become one of the best options for comprehensive dental restoration; their placement is a multidisciplinary task that requires a solid understanding of biological, periodontal, surgical and prosthetic principles. Objective: The aim of this study was to quantify in vitro the adhesion and proliferation of human gingival fibroblasts (HGF response on titanium (Ti and zirconia (Zr surfaces. Methodology: Samples of Ti and Zr were observed under atomic force microscopy (AFM. HGFs were inoculated in each sample to determine adhesion and cell proliferation. The reagent MTT was mixed with medium DMEM and inoculated in each plate; formazan was dissolved with dimethyl sulfoxide and analyzed at 540nm in a microplate spectrophotometer. The test was performed with three independent experiments. Data were analyzed with Kolmogorov-Smirnov tests (Lilliefors, Kruskal-Wallis tests and Mann-Whitney test comparisons. Results: Topography of the Zr plates showed greater roughness (Ra=0.39μm than Ti (Ra=0.049μm. Quantification of HGF adhesion was significantly higher (p<0.05 in Ti, while proliferation showed no statistically significant differences between the groups. Conclusion: It is noteworthy that, even though Ti initially showed increased cell adhesion on the surface, after 24h Zr samples showed similar proliferation; this demonstrates that both surfaces have a comparable biological response.

Cerium oxide nanoparticles stimulate proliferation of primary mouse embryonic fibroblasts in vitro

Energy Technology Data Exchange (ETDEWEB)

Popov, Anton L., E-mail: antonpopovleonid@gmail.com [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation); Popova, Nelly R. [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation); Selezneva, Irina I. [Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region (Russian Federation); Pushchino State Institute of Natural sciences, Pushchino, Moscow region (Russian Federation); Akkizov, Azamat Y. [Kabardino-Balkarian State University, Nalchik (Russian Federation); Ivanov, Vladimir K. [Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, Moscow (Russian Federation); National Research Tomsk State University, Tomsk (Russian Federation)

2016-11-01

The increasing application of cell therapy technologies in the treatment of various diseases requires the development of new effective methods for culturing primary cells. The major limitation for the efficient use of autologous cell material is the low rate of cell proliferation. Successful cell therapy requires sufficient amounts of cell material over a short period of time with the preservation of their differentiation and proliferative potential. In this regard, the development of novel, highly efficient stimulators of proliferative activity in stem cells is a truly urgent task. In this paper we have demonstrated that citrate-stabilized cerium oxide nanoparticles (nanoceria) enhance the proliferative activity of primary mouse embryonic fibroblasts in vitro. Cerium oxide nanoparticles stimulate cell proliferation in a wide range of concentrations (10{sup −3} M–10{sup −9} M) through reduction of intracellular levels of reactive oxygen species (ROS) during the lag phase of cell growth and by modulating the expression level of the major antioxidant enzymes. We found the optimal concentration of nanoceria, which provides the greatest acceleration of cell proliferation in vitro, while maintaining the levels of intracellular ROS and mRNA of antioxidant enzymes in the physiological range. Our results confirm that nanocrystalline ceria can be considered as a basis for effective and inexpensive supplements in cell culturing. - Highlights: • Citrate-stabilized cerium oxide nanoparticles are shown to stimulate proliferation of primary embryonic cells in vitro. • Some of mechanisms involved in stimulating of the proliferation by CeO{sub 2} have been uncovered. • The most effective (optimal) concentration of CeO{sub 2} nanoparticles for stimulation of proliferation was determined.

Platelet-rich plasma (PRP) and adipose-derived mesenchymal stem cells: stimulatory effects on proliferation and migration of fibroblasts and keratinocytes in vitro.

Science.gov (United States)

Stessuk, Talita; Puzzi, Maria Beatriz; Chaim, Elinton Adami; Alves, Paulo César Martins; de Paula, Erich Vinicius; Forte, Andresa; Izumizawa, Juliana Massae; Oliveira, Carolina Caliári; Frei, Fernando; Ribeiro-Paes, João Tadeu

2016-09-01

The clinical use of tissue engineering associated with cell therapy is considered a new alternative therapy for the repair of chronic lesions with potential application in different medical areas, mostly in orthopedic and dermatological diseases. Platelet-rich plasma (PRP) is a rich source of growth factors and cytokines important for wound healing. Adipose-derived mesenchymal stem cells (ADSCs) have shown potential to accelerate the resolution of ulcers, to stimulate cell proliferation, and to benefit the quality of skin repair. This study aims to determine the effect of PRP and conditioned medium (CM) from ADSC on fibroblast and keratinocyte proliferation in vitro. Migration and proliferation assays were performed to evaluate the growth of fibroblasts and keratinocytes in the presence of PRP, CM, and CM + PRP. Significant proliferative stimulation was observed after 48 h of culture (p PRP, 100 % CM, and 25 % PRP + 25 % CM, if compared with control. Keratinocyte proliferation was stimulated after 48 h in cultures with 25, 50, and 100 % CM, and growth was compared with controls. The migration assay detected a significant migratory stimulus in fibroblasts cultured with 10 % PRP + 10 % CM after 48 h. These in vitro results suggest that PRP and ADSC have therapeutic potential for healing and re-epithelialization of chronic wounds in vivo.

Repression of TSC1/TSC2 mediated by MeCP2 regulates human embryo lung fibroblast cell differentiation and proliferation.

Science.gov (United States)

Wang, Yuanyuan; Chen, Chen; Deng, Ziyu; Bian, Erbao; Huang, Cheng; Lei, Ting; Lv, Xiongwen; Liu, Liping; Li, Jun

2017-03-01

Pulmonary fibrosis (PF) is a severe inflammatory disease with limited effective treatments. It is known that the transdifferentiation of human embryo lung fibroblast (HELF) cells from pulmonary fibroblasts into myofibroblasts, contributes to the progression of pulmonary fibrogenesis. The tuberous sclerosis proteins TSC1 and TSC2 are two key signaling factors which can suppress cell growth and proliferation. However, the roles of TSC1 and TSC2 in lung fibroblast are unclear. Here, we developed a PF model with bleomycin (BLM) in mice and conducted several simulation experiments in HELF cells. Our study shows that the expression of TSC1 and TSC2 in fibrotic mice lung was reduced and stimulation of HELF cells with TGF-β1 resulted in a down-regulation of TSC1 and TSC2. In addition, overexpression of TSC1 or TSC2 decreased cell proliferation and differentiation. Furthermore, we found that reduced expression of TSC1 and TSC2 caused by TGF-β1 is associated with the promoter methylation status of TSC1 and TSC2. MeCP2, controls an epigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis. We found that expression of TSC1 and TSC2 can be repressed by MeCP2, which regulates HELF cell differentiation and proliferation as myofibroblasts and lead to PF ultimately. Copyright © 2016. Published by Elsevier B.V.

Comparison of the effect of activated or non-activated PRP in various concentrations on osteoblast and fibroblast cell line proliferation.

Science.gov (United States)

Vahabi, Surena; Yadegari, Zahra; Mohammad-Rahimi, Hossein

2017-09-01

Platelet-rich plasma (PRP) contains growth factors which positively affect cell proliferation, cell differentiation, chemotaxis and intracellular matrix synthesis. All these processes are involved in wound healing and tissue regeneration; thus, PRP as a source of growth factors can be used in periodontal regenerative therapies. The purpose of the present study was to assess the effect of various concentrations of activated and non-activated PRP on proliferation of osteoblasts and fibroblasts in vitro. PRP was obtained from three healthy volunteers. 75, 50, 25, and 10% concentrations of f PRP were prepared by dilution in Dulbecco's modified Eagle's medium. In activated PRP groups, PRP concentrations were activated by adding calcium gluconate. Human gingival fibroblast (HGF) cell line and MG-63 (osteosarcoma) human osteoblast-like cell line were used in the study. The MTT proliferation assay was used to assess the effect of different types of PRP concentrates on proliferation of HGF and MG-63 cells, in 24, 48 and 72 h. After 24, 48, and 72 h, the proliferation rate of both cell lines was higher in the positive control group, except in 72 h in HGF cell lines, that 10% non-activated PRP group and 10 and 25% activated PRP groups has higher proliferation rate than the positive control group, which it was not significant. Proliferation rate in cells with 10% activated PRP was highest among samples containing PRP. The current study failed to show the significant effect of activated or non-activated PRP on proliferation of HGFs or MG-63 osteoblast-like cells. However, our results showed that activated PRP had a greater effect than non-activated PRP.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

Science.gov (United States)

Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

1990-09-01

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

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

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Jaguin, Marie [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Fardel, Olivier [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France); Pôle Biologie, Centre Hospitalier Universitaire (CHU) Rennes, 2 rue Henri Le Guilloux, 35033 Rennes Cedex (France); Lecureur, Valérie, E-mail: valerie.lecureur@univ-rennes1.fr [UMR INSERM U1085, Institut de Recherche sur la Santé, l' Environnement et le Travail (IRSET), Université de Rennes 1, 2 Avenue du Pr Léon Bernard, 35043 Rennes Cedex (France)

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 significantly 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.

Hypotonic stress promotes ATP release, reactive oxygen species production and cell proliferation via TRPV4 activation in rheumatoid arthritis rat synovial fibroblasts

International Nuclear Information System (INIS)

Hu, Fen; Hui, Zhenhai; Wei, Wei; Yang, Jianyu; Chen, Ziyuan; Guo, Bu; Xing, Fulin; Zhang, Xinzheng; Pan, Leiting; Xu, Jingjun

2017-01-01

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune-disease with complex and unclear etiology. Hypotonicity of synovial fluid is a typical characteristic of RA, which may play pivotal roles in RA pathogenesis. In this work, we studied the responses of RA synovial fibroblasts to hypotonic stress in vitro and further explored the underlying mechanisms. Data showed that hyposmotic solutions significantly triggered increases in cytosolic calcium concentration ([Ca 2+ ] c ) of synoviocytes. Subsequently, it caused rapid release of ATP, as well as remarkable production of intracellular reactive oxygen species (ROS). Meanwhile, hypotonic stimulus promoted the proliferation of synovial fibroblasts. These effects were almost abolished by calcium-free buffer and significantly inhibited by gadolinium (III) chloride (a mechanosensitive Ca 2+ channel blocker) and ruthenium red (a transient receptor potential vanilloid 4 (TRPV4) blocker). 4α-phorbol 12,13-didecanoate, a specific agonist of TRPV4, also mimicked hypotonic shock-induced responses shown above. In contrast, voltage-gated channel inhibitors verapamil and nifedipine had little influences on these responses. Furthermore, RT-PCR and western blotting evidently detected TRPV4 expression at mRNA and protein level in isolated synoviocytes. Taken together, our results indicated that hypotonic stimulus resulted in ATP release, ROS production, and cell proliferation depending on Ca 2+ entry through activation of TRPV4 channel in synoviocytes. - Highlights: • Hypotonic stress evokes Ca 2+ entry in rheumatoid arthritis synovial fibroblasts. • Hypotonic stress induces rapid ATP release and ROS production in synoviocytes. • Hypotonic stimulation promotes the proliferation of synovial fibroblasts. • TRPV4 controls hypotonic-induced responses in synoviocytes.

Fibroblast spheroids as a model to study sustained fibroblast quiescence and their crosstalk with tumor cells

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Salmenperä, Pertteli, E-mail: pertteli.salmenpera@helsinki.fi [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Karhemo, Piia-Riitta [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Räsänen, Kati [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland); Laakkonen, Pirjo [Research Programs Unit, Translational Cancer Biology, and Institute of Biomedicine, University of Helsinki, P.O. Box 63, FIN-00014 (Finland); Vaheri, Antti [Department of Virology, Medicum, Faculty of Medicine, University of Helsinki, P.O. Box 21, FIN-00014 (Finland)

2016-07-01

Stromal fibroblasts have an important role in regulating tumor progression. Normal and quiescent fibroblasts have been shown to restrict and control cancer cell growth, while cancer-associated, i. e. activated fibroblasts have been shown to enhance proliferation and metastasis of cancer cells. In this study we describe generation of quiescent fibroblasts in multicellular spheroids and their effects on squamous cell carcinoma (SCC) growth in soft-agarose and xenograft models. Quiescent phenotype of fibroblasts was determined by global down-regulation of expression of genes related to cell cycle and increased expression of p27. Interestingly, microarray analysis showed that fibroblast quiescence was associated with similar secretory phenotype as seen in senescence and they expressed senescence-associated-β-galactosidase. Quiescent fibroblasts spheroids also restricted the growth of RT3 SCC cells both in soft-agarose and xenograft models unlike proliferating fibroblasts. Restricted tumor growth was associated with marginally increased tumor cell senescence and cellular differentiation, showed with senescence-associated-β-galactosidase and cytokeratin 7 staining. Our results show that the fibroblasts spheroids can be used as a model to study cellular quiescence and their effects on cancer cell progression. - Highlights: • Fibroblasts acquire a sustained quiescence when grown as multicellular spheroids. • This quiescence is associated with drastic change in gene expression. • Fibroblasts spheroids secrete various inflammation-linked cytokines and chemokines. • Fibroblasts spheroids reduced growth of RT3 SCC cells in xenograft model.

Improved fibronectin-immobilized fibrinogen microthreads for the attachment and proliferation of fibroblasts

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Rajangam T

2013-03-01

Full Text Available Thanavel Rajangam, Seong Soo A AnDepartment of Bionanotechnology, Gachon University, Seongnam, South KoreaAbstract: The aim of this study was to fabricate fibrinogen (Fbg microfibers with different structural characteristics for the development of 3-D tissue-engineering scaffolds. Fabricated Fbg microfibers were investigated for their biomolecule encapsulation, cell adhesion, and proliferations. Microfibers with three different concentrations of Fbg (5, 10, and 15 wt% were prepared by a gel solvent-extraction method using a silicone rubber tube. Fbg microfibers were covalently modified with fibronectin (FN by using water-soluble 1-ethyl-3-(3-dimethylaminopropyl carbodiimide as the cross-linking agent. Fbg microfibers were characterized by their FN cross-linking properties, structural morphology, and in vitro degradation. Furthermore, FN/Fbg microfibers were evaluated for cell attachment and proliferation. The biocompatibility and cell proliferation of the microfibers were assessed by measuring adenosine triphosphate activity in C2C12 fibroblast cells. Cell attachment and proliferation on microfibers were further examined using fluorescence and scanning electron microscopic images. FN loading on the microfibers was confirmed by fluorescence and infrared spectroscopy. Surface morphology was characterized by scanning electron microscopy, and showed highly aligned nanostructures for fibers made with 15 wt% Fbg, a more porous structure for fibers made with 10 wt% Fbg, and a less porous structure for those made with 5 wt% Fbg. Controlled biodegradation of the fiber was observed for 8 weeks by using an in vitro proteolytic degradation assay. Fbg microfibers with highly aligned nanostructures (15 wt% showed enhanced biomolecule encapsulation, as well as higher cell adhesion and proliferation than another two types of FN/Fbg fibers (5 and 10 wt% and unmodified Fbg fibers. The promising results obtained from the present study reveal that optimal structure

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

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Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

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. In 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

Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

International Nuclear Information System (INIS)

Han, Yanfu; Chai, Jiake; Sun, Tianjun; Li, Dongjie; Tao, Ran

2011-01-01

Highlights: → Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. → Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. → We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. → Collagen type I and collagen type III mRNA level was higher in differentiated cells. → 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. In 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-engineered dermis.

Mechanistic study of DNA damage and radioprotection of small molecule treatment in the irradiated proliferating and quiescent human lung fibroblast cells

International Nuclear Information System (INIS)

Dai, Jiawen; Baskar, Rajamanickam

2014-01-01

Ionizing radiation is an invaluable diagnostic and treatment tool used in various clinical applications and also in cancer control. However, assessing normal tissue injury is of a great interest. Since radiation sensitivity varies with different phases of cell cycle, understanding how these cells differ in their sensitivity will help to prevent or reduce the radiation injury. We have used both proliferating and quiescent human normal lung fibroblast cells and investigated key proteins involved in the cell cycle, DNA damage and death, further the radioprotective role of small molecule after low doses (d ≤1Gy) of radiation exposure. Among the cell cycle/death proteins investigated, p53 and phosphorylation of p53 (Ser-15) were induced in both the proliferating and quiescent phases of cells when studied at different time intervals. In the proliferating cells after irradiation along with p53, cyclin dependent kinase (CDK) inhibitors p21, p27 were induced. However, similarly in the quiescent cells along with the p53, p21 and p27 were also induced. The DNA damage assessed by phosphorylation of histone H2AX expression showed an increase even in the non-dividing quiescent cells after 1 Gy of radiation exposure. Whereas cell cycle proteins Cyclin A and E and cell death proteins Bax and cytochrome-c did not show any increase in the quiescent cells. In conclusion, human normal lung fibroblast cells that are not actively dividing are also showed similar radiation response as of proliferating cells. Furthermore, proliferating and quiescent cells treated with small molecules attenuate p53 and its downstream target protein p21 indicating radioprotection of the cells. The specific activation of p53, phosphorylation of p53 (Ser-15), p21 and phosphorylation of histone H2AX following radiation doses of d ≤ 1 Gy in the quiescent cells demonstrated in this study may give us a better understanding about the radiation response of non-dividing fibroblast cells, which is present in many

Effects of Monoclonal Antibody Cetuximab on Proliferation of Non-small Cell Lung Cancer Cell lines

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Zhen CHEN

2010-08-01

Full Text Available Background and objective The epidermal growth factor receptor (EGFR monoclonal antibody cetuximab has been used widely in non-small cell lung cancer patients. The aim of this study is to explore the effect of lung cancer cells (A549, H460, H1299, SPC-A-1 which were treated by cetuximab in vitro. Methods We studied the effects of increasing concentrations of cetuximab (1 nmol/L-625 nmol/L in four human lung cancer cell lines (A549, SPC-A-1, H460, H1229. CCK8 measured the inhibition of cell proliferation in each group. A549, SPC-A-1 were marked by PI and the statuses of apoptosis were observed. Western blot were used to detect the proliferation-related signaling protein and apoptosis-related protein in A549. Results The treatment with cetuximab resulted in the effect on cell proliferation and apoptosis in a time- and dosedependent manner. The expression of activated key enzymes (p-AKT, p-EGFR, p-MAPK in EGFR signaling transduction pathway were down-regulated more obviously. Conclusion Cetuximab is an effective targeted drug in the treatment of lung cancer cell lines, tissues, most likely to contribute to the inhibition of key enzymes in EGFR signaling transduction pathway.

Mesenchymal stem cells induce dermal fibroblast responses to injury

International Nuclear Information System (INIS)

Smith, Andria N.; Willis, Elise; Chan, Vincent T.; Muffley, Lara A.; Isik, F. Frank; Gibran, Nicole S.; Hocking, Anne M.

2010-01-01

Although bone marrow-derived mesenchymal stem cells have been shown to promote repair when applied to cutaneous wounds, the mechanism for this response remains to be determined. The aim of this study was to determine the effects of paracrine signaling from mesenchymal stem cells on dermal fibroblast responses to injury including proliferation, migration and expression of genes important in wound repair. Dermal fibroblasts were co-cultured with bone marrow-derived mesenchymal stem cells grown in inserts, which allowed for paracrine interactions without direct cell contact. In this co-culture model, bone marrow-derived mesenchymal stem cells regulate dermal fibroblast proliferation, migration and gene expression. When co-cultured with mesenchymal stem cells, dermal fibroblasts show increased proliferation and accelerated migration in a scratch assay. A chemotaxis assay also demonstrated that dermal fibroblasts migrate towards bone marrow-derived mesenchymal stem cells. A PCR array was used to analyze the effect of mesenchymal stem cells on dermal fibroblast gene expression. In response to mesenchymal stem cells, dermal fibroblasts up-regulate integrin alpha 7 expression and down-regulate expression of ICAM1, VCAM1 and MMP11. These observations suggest that mesenchymal stem cells may provide an important early signal for dermal fibroblast responses to cutaneous injury.

Enhancing proliferation and optimizing the culture condition for human bone marrow stromal cells using hypoxia and fibroblast growth factor-2

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Jung-Seok Lee

2018-04-01

Full Text Available This study aimed to determine the cellular characteristics and behaviors of human bone marrow stromal cells (hBMSCs expanded in media in a hypoxic or normoxic condition and with or without fibroblast growth factor-2 (FGF-2 treatment. hBMSCs isolated from the vertebral body and expanded in these four groups were evaluated for cellular proliferation/migration, colony-forming units, cell-surface characterization, in vitro differentiation, in vivo transplantation, and gene expression. Culturing hBMSCs using a particular environmental factor (hypoxia and with the addition of FGF-2 increased the cellular proliferation rate while enhancing the regenerative potential, modulated the multipotency-related processes (enhanced chondrogenesis-related processes/osteogenesis, but reduced adipogenesis, and increased cellular migration and collagen formation. The gene expression levels in the experimental samples showed activation of the hypoxia-inducible factor-1 pathway and glycolysis in the hypoxic condition, with this not being affected by the addition of FGF-2. The concurrent application of hypoxia and FGF-2 could provide a favorable condition for culturing hBMSCs to be used in clinical applications associated with bone tissue engineering, due to the enhancement of cellular proliferation and regenerative potential. Keywords: Bone marrow stromal cells, Hypoxia, Fibroblast growth factor, Tissue regeneration, Microenvironment interactions

LXA4 actions direct fibroblast function and wound closure

International Nuclear Information System (INIS)

Herrera, Bruno S.; Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice; Leung, Kai P.; Van Dyke, Thomas E.

2015-01-01

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A 4 (LXA 4 ), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA 4 on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA 4 receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA 4 receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA 4 slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA 4 tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA 4 in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF-β1 up-regulates LXA 4 receptor (ALX

Enhancement of hybridoma formation, clonability and cell proliferation in a nanoparticle-doped aqueous environment

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Karnieli Ohad

2008-01-01

Full Text Available Abstract Background The isolation and production of human monoclonal antibodies is becoming an increasingly important pursuit as biopharmaceutical companies migrate their drug pipelines away from small organic molecules. As such, optimization of monoclonal antibody technologies is important, as this is becoming the new rate-limiting step for discovery and development of new pharmaceuticals. The major limitations of this system are the efficiency of isolating hybridoma clones, the process of stabilizing these clones and optimization of hybridoma cell secretion, especially for large-scale production. Many previous studies have demonstrated how perturbations in the aqueous environment can impact upon cell biology. In particular, radio frequency (RF irradiation of solutions can have dramatic effects on behavior of solutions, cells and in particular membrane proteins, although this effect decays following removal of the RF. Recently, it was shown that nanoparticle doping of RF irradiated water (NPD water produced a stabilized aqueous medium that maintained the characteristic properties of RF irradiated water for extended periods of time. Therefore, the ordering effect in water of the RF irradiation can now be studied in systems that required prolonged periods for analysis, such as eukaryotic cell culture. Since the formation of hybridoma cells involves the formation of a new membrane, a process that is affected by the surrounding aqueous environment, we tested these nanoparticle doped aqueous media formulations on hybridoma cell production. Results In this study, we tested the entire process of isolation and production of human monoclonal antibodies in NPD water as a means for further enhancing human monoclonal antibody isolation and production. Our results indicate an overall enhancement of hybridoma yield, viability, clonability and secretion. Furthermore, we have demonstrated that immortal cells proliferate faster whereas primary human fibroblasts

Antigen-specific cytotoxic T cell and antigen-specific proliferating T cell clones can be induced to cytolytic activity by monoclonal antibodies against T3

NARCIS (Netherlands)

Spits, H.; Yssel, H.; Leeuwenberg, J.; de Vries, J. E.

1985-01-01

T3 is a human differentiation antigen expressed exclusively on mature T cells. In this study it is shown that anti-T3 monoclonal antibodies, in addition to their capacity to induce T cells to proliferate, are able to induce antigen-specific cytotoxic T lymphocyte clones to mediate antigen

Effects of Mechanical Stretch on Cell Proliferation and Matrix Formation of Mesenchymal Stem Cell and Anterior Cruciate Ligament Fibroblast

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Liguo Sun

2016-01-01

Full Text Available Mesenchymal stem cells (MSCs and fibroblasts are two major seed cells for ligament tissue engineering. To understand the effects of mechanical stimulation on these cells and to develop effective approaches for cell therapy, it is necessary to investigate the biological effects of various mechanical loading conditions on cells. In this study, fibroblasts and MSCs were tested and compared under a novel Uniflex/Bioflex culture system that might mimic mechanical strain in ligament tissue. The cells were uniaxially or radially stretched with different strains (5%, 10%, and 15% at 0.1, 0.5, and 1.0 Hz. The cell proliferation and collagen production were compared to find the optimal parameters. The results indicated that uniaxial stretch (15% at 0.5 Hz; 10% at 1.0 Hz showed positive effects on fibroblast. The uniaxial strains (5%, 10%, and 15% at 0.5 Hz and 10% strain at 1.0 Hz were favorable for MSCs. Radial strain did not have significant effect on fibroblast. On the contrary, the radial strains (5%, 10%, and 15% at 0.1 Hz had positive effects on MSCs. This study suggested that fibroblasts and MSCs had their own appropriate mechanical stimulatory parameters. These specific parameters potentially provide fundamental knowledge for future cell-based ligament regeneration.

Effects of extremely low-frequency magnetotherapy on proliferation of human dermal fibroblasts.

Science.gov (United States)

Pasi, Francesca; Sanna, Samuele; Paolini, Alessandro; Alquati, Marco; Lascialfari, Alessandro; Corti, Maurizio Enrico; Liberto, Riccardo Di; Cialdai, Francesca; Monici, Monica; Nano, Rosanna

2016-01-01

Extremely low-frequency electromagnetic fields (ELF-EMFs) applied in magnetotherapy have frequency lower than 100 Hz and magnetic field intensity ranging from 0.1 to 20 mT. For many years, the use of magnetotherapy in clinics has been increasing because of its beneficial effects in many processes, e.g., skin diseases, inflammation and bone disorders. However, the understanding of the microscopic mechanisms governing such processes is still lacking and the results of the studies on the effects of ELF-EMFs are controversial because effects derive from different conditions and from intrinsic responsiveness of different cell types.In the present study, we studied the biological effects of 1.5 h exposure of human dermal fibroblasts to EMFs with frequencies of 5 and 50 Hz and intensity between 0.25 and 1.6 mT. Our data showed that the magnetic treatment did not produce changes in cell viability, but gave evidence of a sizeable decrease in proliferation at 24 h after treatment. In addition, immunofluorescence experiments displayed an increase in tubulin expression that could foreshadow changes in cell motility or morphology. The decrease in proliferation with unchanged viability and increase in tubulin expression could be consistent with the triggering of a transdifferentiation process after the exposure to ELF-EMFs.

Cathelicidin suppresses colon cancer development by inhibition of cancer associated fibroblasts

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Cheng M

2014-12-01

Full Text Available Michelle Cheng,1,* Samantha Ho,1,* Jun Hwan Yoo,1,2,* Deanna Hoang-Yen Tran,1,* Kyriaki Bakirtzi,1 Bowei Su,1 Diana Hoang-Ngoc Tran,1 Yuzu Kubota,1 Ryan Ichikawa,1 Hon Wai Koon1 1Center for Inflammatory Bowel Diseases, Division of Digestive Diseases, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, USA; 2Digestive Disease Center, CHA University Bundang Medical Center, Seongnam, Republic of Korea *These authors share co-first authorship Background: Cathelicidin (LL-37 in humans and mCRAMP in mice represents a family of endogenous antimicrobial and anti-inflammatory peptides. Cancer-associated fibroblasts can promote the proliferation of colon cancer cells and growth of colon cancer tumors. Methods: We examined the role of cathelicidin in the development of colon cancer, using subcutaneous human HT-29 colon-cancer-cell-derived tumor model in nude mice and azoxymethane- and dextran sulfate-mediated colon cancer model in C57BL/6 mice. We also determined the indirect antitumoral mechanism of cathelicidin via the inhibition of epithelial–mesenchymal transition (EMT of colon cancer cells and fibroblast-supported colon cancer cell proliferation. Results: Intravenous administration of cathelicidin expressing adeno-associated virus significantly reduced the size of tumors, tumor-derived collagen expression, and tumor-derived fibroblast expression in HT-29-derived subcutaneous tumors in nude mice. Enema administration of the mouse cathelicidin peptide significantly reduced the size and number of colonic tumors in azoxymethane- and dextran sulfate-treated mice without inducing apoptosis in tumors and the adjacent normal colonic tissues. Cathelicidin inhibited the collagen expression and vimentin-positive fibroblast expression in colonic tumors. Cathelicidin did not directly affect HT-29 cell viability, but did significantly reduce tumor growth factor-ß1-induced EMT of colon cancer cells. Media conditioned by the

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

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Ribeiro-Resende Victor

2012-07-01

Full Text Available Abstract Background Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC, satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2 is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS. Results Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL region of the lumbar spinal cord (LSC in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. Conclusion We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.

LXA{sub 4} actions direct fibroblast function and wound closure

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Herrera, Bruno S. [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Kantarci, Alpdogan; Zarrough, Ahmed; Hasturk, Hatice [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States); Leung, Kai P., E-mail: kai.p.leung.civ@mail.mil [Microbiology Branch, US Army Dental and Trauma Research Detachment, Institute of Surgical Research, JBSA Fort Sam Houston, TX (United States); Van Dyke, Thomas E., E-mail: tvandyke@forsyth.org [Department of Applied Oral Sciences, Center for Periodontology, The Forsyth Institute, Cambridge, MA (United States)

2015-09-04

Timely resolution of inflammation is crucial for normal wound healing. Resolution of inflammation is an active biological process regulated by specialized lipid mediators including the lipoxins and resolvins. Failure of resolution activity has a major negative impact on wound healing in chronic inflammatory diseases that is manifest as excess fibrosis and scarring. Lipoxins, including Lipoxin A{sub 4} (LXA{sub 4}), have known anti-fibrotic and anti-scarring properties. The goal of this study was to elucidate the impact of LXA{sub 4} on fibroblast function. Mouse fibroblasts (3T3 Mus musculus Swiss) were cultured for 72 h in the presence of TGF-β1, to induce fibroblast activation. The impact of exogenous TGF-β1 (1 ng/mL) on LXA{sub 4} receptor expression (ALX/FPR2) was determined by flow cytometry. Fibroblast proliferation was measured by bromodeoxyuridine (BrdU) labeling and migration in a “scratch” assay wound model. Expression of α-smooth muscle actin (α-SMA), and collagen types I and III were measured by Western blot. We observed that TGF-β1 up-regulates LXA{sub 4} receptor expression, enhances fibroblast proliferation, migration and scratch wound closure. α-SMA levels and Collagen type I and III deposition were also enhanced. LXA{sub 4} slowed fibroblast migration and scratch wound closure at early time points (24 h), but wound closure was equal to TGF-β1 alone at 48 and 72 h. LXA{sub 4} tended to slow fibroblast proliferation at both concentrations, but had no impact on α-SMA or collagen production by TGF-β1 stimulated fibroblasts. The generalizability of the actions of resolution molecules was examined in experiments repeated with resolvin D2 (RvD2) as the agonist. The activity of RvD2 mimicked the actions of LXA{sub 4} in all assays, through an as yet unidentified receptor. The results suggest that mediators of resolution of inflammation enhance wound healing and limit fibrosis in part by modulating fibroblast function. - Highlights: • TGF

Basic fibroblast growth factor enhances cell proliferation in the dentate gyrus of neonatal rats following hypoxic-ischemic brain damage.

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Zhu, Huan; Qiao, Lixing; Sun, Yao; Yin, Liping; Huang, Li; Jiang, Li; Li, Jiaqing

2018-04-23

Perinatal hypoxic-ischemic insult is considered a major contributor to child mortality and morbidity and leads to neurological deficits in newborn infants. There has been a lack of promising neurotherapeutic interventions for hypoxic-ischemic brain damage (HIBD) for clinical application in infants. The present study aimed to investigate the correlation between neurogenesis and basic fibroblast growth factor (bFGF) in the hippocampal dentate gyrus (DG) region in neonatal rats following HIBD. Cell proliferation was examined by detecting BrdU signals, and the role of bFGF in cell proliferation in the DG region following neonatal HIBD was investigated. Cell proliferation was induced by HIBD in the hippocampal DG of neonatal rats. Furthermore, bFGF gene expression was upregulated in the hippocampus in neonatal rats, particularly between 7 and 14 days after HIBD. Moreover, intraperitoneal injection of exogenous bFGF enhanced cell proliferation in the hippocampal DG following neonatal HIBD. Taken together, these data indicate that cell proliferation in the DG could be induced by neonatal HIBD, and bFGF promotes proliferation following neonatal HIBD. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma.

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Le, Tran; New, Jacob; Jones, Joel W; Usman, Shireen; Yalamanchali, Sreeya; Tawfik, Ossama; Hoover, Larry; Bruegger, Dan E; Thomas, Sufi Mary

2017-10-01

Juvenile nasopharyngeal angiofibroma (JNA) is a benign tumor that presents in adolescent males. Although surgical excision is the mainstay of treatment, recurrences complicate treatment. There is a need to develop less invasive approaches for management. JNA tumors are composed of fibroblasts and vascular endothelial cells. We identified fibroblast growth factor receptor (FGFR) and vascular endothelial growth factor (VEGF) expression in JNA-derived fibroblasts. FGFR influences fibroblast proliferation and VEGF is necessary for angiogenesis. We hypothesized that targeting FGFR would mitigate JNA fibroblast proliferation, invasion, and migration, and that targeting the VEGF receptor would attenuate endothelial tubule formation. After informed consent, fibroblasts from JNA explants of 3 patients were isolated. Fibroblasts were treated with FGFR inhibitor AZD4547, 0 to 25 μg/mL for 72 hours and proliferation was quantified using CyQuant assay. Migration and invasion of JNA were assessed using 24-hour transwell assays with subsequent fixation and quantification. Mitigation of FGFR and downstream signaling was evaluated by immunoblotting. Tubule formation was assessed in human umbilical vein endothelial cells (HUVECs) treated with vehicle control (dimethylsulfoxide [DMSO]) or semaxanib (SU5416) as well as in serum-free media (SFM) or JNA conditioned media (CM). Tubule length was compared between treatment groups. Compared to control, AZD4547 inhibited JNA fibroblast proliferation, migration, and invasion through inhibition of FGFR and downstream signaling, specifically phosphorylation of - p44/42 mitogen activated protein kinase (p44/42 MAPK). JNA fibroblast CM significantly increased HUVEC tubule formation (p = 0.0039). AZD4547 effectively mitigates FGFR signaling and decreases JNA fibroblast proliferation, migration, and invasion. SU5416 attenuated JNA fibroblast-induced tubule formation. AZD4547 may have therapeutic potential in the treatment of JNA. © 2017 ARS

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

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Fan, Rong-hui, E-mail: fan_ronghuixa@163.com [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Zhu, Xiu-mei; Sun, Yao-wen [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Peng, Hui-zi [Department of Cosmetology Plastic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Wu, Hang-li; Gao, Wen-jie [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China)

2016-07-08

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Administration of PDE4 Inhibitors Suppressed the Pannus-Like Inflammation by Inhibition of Cytokine Production by Macrophages and Synovial Fibroblast Proliferation

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Katsuya Kobayashi

2007-01-01

Full Text Available A marked proliferation of synovial fibroblasts in joints leads to pannus formation in rheumatoid arthritis (RA. Various kinds of cytokines are produced in the pannus. The purpose of this study is to elucidate the effects of phosphodiesterase 4 (PDE4 inhibitors in a new animal model for the evaluation of pannus formation and cytokine production in the pannus. Mice sensitized with methylated bovine serum albumin (mBSA were challenged by subcutaneous implantation of a membrane filter soaked in mBSA solution in the back of the mice. Drugs were orally administered for 10 days. The granuloma formed around the filter was collected on day 11. It was chopped into pieces and cultured in vitro for 24 hr. The cytokines were measured in the supernatants. The type of cytokines produced in the granuloma was quite similar to those produced in pannus in RA. Both PDE4 inhibitors, KF66490 and SB207499, suppressed the production of IL-1β, TNF-α, and IL-12, and the increase in myeloperoxidase activity, a marker enzyme for neutrophils and hydroxyproline content. Compared to leflunomide, PDE4 inhibitors more strongly suppressed IL-12 production and the increase in myeloperoxidase activity. PDE4 inhibitors also inhibited lipopolysaccharide-induced TNF-α and IL-12 production from thioglycolate-induced murine peritoneal macrophages and the proliferation of rat synovial fibroblasts. These results indicate this model makes it easy to evaluate the effect of drugs on various cytokine productions in a granuloma without any purification step and may be a relevant model for evaluating novel antirheumatic drugs on pannus formation in RA. PDE4 inhibitors could have therapeutic effects on pannus formation in RA by inhibition of cytokine production by macrophages and synovial fibroblast proliferation.

Administration of PDE4 inhibitors suppressed the pannus-like inflammation by inhibition of cytokine production by macrophages and synovial fibroblast proliferation.

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Kobayashi, Katsuya; Suda, Toshio; Manabe, Haruhiko; Miki, Ichiro

2007-01-01

A marked proliferation of synovial fibroblasts in joints leads to pannus formation in rheumatoid arthritis (RA). Various kinds of cytokines are produced in the pannus. The purpose of this study is to elucidate the effects of phosphodiesterase 4 (PDE4) inhibitors in a new animal model for the evaluation of pannus formation and cytokine production in the pannus. Mice sensitized with methylated bovine serum albumin (mBSA) were challenged by subcutaneous implantation of a membrane filter soaked in mBSA solution in the back of the mice. Drugs were orally administered for 10 days. The granuloma formed around the filter was collected on day 11. It was chopped into pieces and cultured in vitro for 24 hr. The cytokines were measured in the supernatants. The type of cytokines produced in the granuloma was quite similar to those produced in pannus in RA. Both PDE4 inhibitors, KF66490 and SB207499, suppressed the production of IL-1beta, TNF-alpha, and IL-12, and the increase in myeloperoxidase activity, a marker enzyme for neutrophils and hydroxyproline content. Compared to leflunomide, PDE4 inhibitors more strongly suppressed IL-12 production and the increase in myeloperoxidase activity. PDE4 inhibitors also inhibited lipopolysaccharide-induced TNF-alpha and IL-12 production from thioglycolate-induced murine peritoneal macrophages and the proliferation of rat synovial fibroblasts. These results indicate this model makes it easy to evaluate the effect of drugs on various cytokine productions in a granuloma without any purification step and may be a relevant model for evaluating novel antirheumatic drugs on pannus formation in RA. PDE4 inhibitors could have therapeutic effects on pannus formation in RA by inhibition of cytokine production by macrophages and synovial fibroblast proliferation.

Evaluation of the effect of laser radiation on fibroblast proliferation in repair of skin wounds of rats with iron deficiency anemia

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DeCastro, Isabele C. V.; Oliveira-Sampaio, Susana C. P.; Monteiro, Juliana S. de C.; Ferreira, Maria de Fátima L.; Cangussu, Maria T.; N. dos Santos, Jean; Pinheiro, Antonio Luiz B.

2011-03-01

The aim of this study was to assess the effect of low- level laser therapy (LLLT) on fibroblast proliferation on wound repair of rats with Iron deficiency anemia since there is no reports on literature about this subject. Iron deficiency anemia was induced on 36 newborn rats then an excisional wound was created on the dorsum of the animals which were divided into four groups: (I) - non-anemic, (II) - Anemic, (III) - non-anemic + LLLT, (IV) Anemic+ LLLT. The animals in each group were sacrificed at 7, 14 and 21 days. Laser irradiation was performed on each group (λ660nm,40Mw,CW) by contact mode with a dose of 2,5J/ cm2 in four points on the area of the wound and total of 10J/cm2 per session. Data were evaluated by analysis of variance (ANOVA) followed by Paired t-test. The results showed LLLT was able to stimulate fibroblastic proliferation in rats with iron deficiency anemia at the 21st day while at control group (III) no statistically significant differences was found.

Impact of matrix stiffness on fibroblast function

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El-Mohri, Hichem; Wu, Yang; Mohanty, Swetaparna; Ghosh, Gargi, E-mail: gargi@umich.edu

2017-05-01

Chronic non-healing wounds, caused by impaired production of growth factors and reduced vascularization, represent a significant burden to patients, health care professionals, and health care system. While several wound dressing biomaterials have been developed, the impact of the mechanical properties of the dressings on the residing cells and consequently on the healing of the wounds is largely overlooked. The primary focus of this study is to explore whether manipulation of the substrate mechanics can regulate the function of fibroblasts, particularly in the context of their angiogenic activity. A photocrosslinkable hydrogel platform with orthogonal control over gel modulus and cell adhesive sites was developed to explore the quantitative relationship between ECM compliance and fibroblast function. Increase in matrix stiffness resulted in enhanced fibroblast proliferation and stress fiber formation. However, the angiogenic activity of fibroblasts was found to be optimum when the cells were seeded on compliant matrices. Thus, the observations suggest that the stiffness of the wound dressing material may play an important role in the progression of wound healing. - Highlights: • Proliferation and stress fiber formation of fibroblasts increase with increasing matrix mechanics. • Cell area correlates with the growth of fibroblasts. • Angiogenic activity of fibroblasts optimum when cells seeded on compliant gels.

Investigating the role of c-Jun N-terminal kinases in the proliferation of Werner syndrome fibroblasts using diaminopyridine inhibitors

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Davis Terence

2011-12-01

Full Text Available Abstract Fibroblasts derived from the progeroid Werner syndrome show reduced replicative lifespan and a "stressed" morphology, both alleviated using the MAP kinase inhibitor SB203580. However, interpretation of these data is problematical because although SB203580 has the stress-activated kinases p38 and JNK1/2 as its preferred targets, it does show relatively low overall kinase selectivity. Several lines of data support a role for both p38 and JNK1/2 activation in the control of cellular proliferation and also the pathology of diseases of ageing, including type II diabetes, diseases to which Werner Syndrome individuals are prone, thus making the use of JNK inhibitors attractive as possible therapeutics. We have thus tested the effects of the widely used JNK inhibitor SP600125 on the proliferation and morphology of WS cells. In addition we synthesised and tested two recently described aminopyridine based inhibitors. SP600125 treatment resulted in the cessation of proliferation of WS cells and resulted in a senescent-like cellular phenotype that does not appear to be related to the inhibition of JNK1/2. In contrast, use of the more selective aminopyridine CMPD 6o at concentrations that fully inhibit JNK1/2 had a positive effect on cellular proliferation of immortalised WS cells, but no effect on the replicative lifespan of primary WS fibroblasts. In addition, CMPD 6o corrected the stressed WS cellular morphology. The aminopyridine CMPD 6r, however, had little effect on WS cells. CMDP 6o was also found to be a weak inhibitor of MK2, which may partially explain its effects on WS cells, since MK2 is known to be involved in regulating cellular morphology via HSP27 phosphorylation, and is thought to play a role in cell cycle arrest. These data suggest that total JNK1/2 activity does not play a substantial role in the proliferation control in WS cells.

mTOR complex 2 phosphorylates IMP1 cotranslationally to promote IGF2 production and the proliferation of mouse embryonic fibroblasts

DEFF Research Database (Denmark)

Dai, Ning; Christiansen, Jan; Nielsen, Finn

2013-01-01

uncover a new mechanism by which mTOR regulates organismal growth by promoting IGF2 production in the mouse embryo through mTORC2-catalyzed cotranslational IMP1/IMP3 phosphorylation. Inasmuch as TORC2 is activated by association with ribosomes, the present results indicate that mTORC2-catalyzed...... production, and diminished proliferation. The proliferation of the IMP1-null fibroblasts can be restored to wild-type levels by IGF2 in vitro or by re-expression of IMP1, which corrects the defects in IGF2 RNA splicing and translation. The ability of IMP1 to correct these defects is dependent on IMP1...

Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

Science.gov (United States)

Vahabi, Surena; Vaziri, Shahram; Torshabi, Maryam

2015-01-01

Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF) and platelet-rich fibrin (PRF) on proliferation and viability of human gingival fibroblasts (HGFs). Materials and Methods: Anitua’s PRGF and Choukran’s PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-valuesPRGF treatment induced statistically significant (PPRGF than in PRF group (PPRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF. PMID:26877740

MicroRNA-124 controls the proliferative, migratory, and inflammatory phenotype of pulmonary vascular fibroblasts.

Science.gov (United States)

Wang, Daren; Zhang, Hui; Li, Min; Frid, Maria G; Flockton, Amanda R; McKeon, B Alexandre; Yeager, Michael E; Fini, Mehdi A; Morrell, Nicholas W; Pullamsetti, Soni S; Velegala, Sivareddy; Seeger, Werner; McKinsey, Timothy A; Sucharov, Carmen C; Stenmark, Kurt R

2014-01-03

Pulmonary hypertensive remodeling is characterized by excessive proliferation, migration, and proinflammatory activation of adventitial fibroblasts. In culture, fibroblasts maintain a similar activated phenotype. The mechanisms responsible for generation/maintenance of this phenotype remain unknown. We hypothesized that aberrant expression of microRNA-124 (miR-124) regulates this activated fibroblast phenotype and sought to determine the signaling pathways through which miR-124 exerts effects. We detected significant decreases in miR-124 expression in fibroblasts isolated from calves and humans with severe pulmonary hypertension. Overexpression of miR-124 by mimic transfection significantly attenuated proliferation, migration, and monocyte chemotactic protein-1 expression of hypertensive fibroblasts, whereas anti-miR-124 treatment of control fibroblasts resulted in their increased proliferation, migration, and monocyte chemotactic protein-1 expression. Furthermore, the alternative splicing factor, polypyrimidine tract-binding protein 1, was shown to be a direct target of miR-124 and to be upregulated both in vivo and in vitro in bovine and human pulmonary hypertensive fibroblasts. The effects of miR-124 on fibroblast proliferation were mediated via direct binding to the 3' untranslated region of polypyrimidine tract-binding protein 1 and subsequent regulation of Notch1/phosphatase and tensin homolog/FOXO3/p21Cip1 and p27Kip1 signaling. We showed that miR-124 directly regulates monocyte chemotactic protein-1 expression in pulmonary hypertension/idiopathic pulmonary arterial hypertension fibroblasts. Furthermore, we demonstrated that miR-124 expression is suppressed by histone deacetylases and that treatment of hypertensive fibroblasts with histone deacetylase inhibitors increased miR-124 expression and decreased proliferation and monocyte chemotactic protein-1 production. Stable decreases in miR-124 expression contribute to an epigenetically reprogrammed, highly

[Isolation, purification and primary culture of adult mouse cardiac fibroblasts].

Science.gov (United States)

Li, Rujun; Gong, Kaizheng; Zhang, Zhengang

2017-01-01

Objective To establish a method for primary culture of adult mouse cardiac fibroblasts. Methods Myocardial tissues from adult mice were digested with 1 g/L trypsin and 0.8 g/L collagenase IV by oscillating water bath for a short time repeatedly. Cardiac fibroblasts and myocardial cells were isolated with differential adhesion method. Immunofluorescence staining was used to assess the purity of cardiac fibroblasts. The cell morphology was observed under an inverted phase contrast microscope. The proliferation of cardiac fibroblasts was analyzed by growth curve and CCK-8 assay. The Smad2/3 phosphorylation induced by TGF-β1 was detected by Western blotting. Results After 90 minutes of differential adhesion, adherent fibroblasts formed spherical cell mass and after 3 days, cells were spindle-shaped and proliferated rapidly. Cells were confluent after 5 days and the growth curve presented nearly "S" shape. The positive expression rate of vimentin was 95%. CCK-8 assay showed that the optimal cell proliferating activity was found from day 3 to day 5. The level of phosphorylated Smad2/3 obviously increased at the second passage induced by TGF-β1. Conclusion This method is economical and stable to isolate cardiac fibroblasts with high activity and high purity from adult mice.

Fibroblast-matrix interplay: Nintedanib and pirfenidone modulate the effect of IPF fibroblast-conditioned matrix on normal fibroblast phenotype.

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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.

Inhibition of lectin-like oxidized low-density lipoprotein receptor-1 reduces cardiac fibroblast proliferation by suppressing GATA Binding Protein 4

Energy Technology Data Exchange (ETDEWEB)

Liu, Bin; Liu, Ning-Ning; Liu, Wei-Hua; Zhang, Shuang-Wei; Zhang, Jing-Zhi; Li, Ai-Qun [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China); Liu, Shi-Ming, E-mail: gzliushiming@126.com [Department of Cardiology, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou (China); Guangzhou Institute of Cardiovascular Disease, Guangzhou (China)

2016-07-08

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and GATA Binding Protein 4 (GATA4) are important for the growth of cardiac fibroblasts (CFs). When deregulated, LOX-1 and GATA4 can cause cardiac remodeling. In the present study, we found novel evidence that GATA4 was required for the LOX-1 regulation of CF proliferation. The inhibition of LOX-1 by RNA interference LOX-1 lentivirus resulted in the loss of PI3K/Akt activation and GATA4 protein expression. The overexpression of LOX-1 by lentivirus rescued CF proliferation, PI3K/Akt activation, and GATA4 protein expression. Moreover, GATA4 overexpression enhanced CF proliferation with LOX-1 inhibition. We also found that the inhibition of PI3K/Akt activation by LY294002, a PI3K inhibitor, reduced cell proliferation and protein level of GATA4. In summary, GATA4 may play an important role in the LOX-1 and PI3K/Akt regulation of CF proliferation. -- Highlights: •GATA4 is regulated by LOX-1 signaling in CFs. •GATA4 is involved in LOX-1 regulating CF proliferation. •GATA4 is regulated by PI3K/Akt signaling in CFs.

Anti-proliferative effect of olmesartan on Tenon's capsule fibroblasts

Directory of Open Access Journals (Sweden)

Xuan Wang

2016-05-01

Full Text Available AIM: To evaluate the inhibitive effect of olmesartan to fibroblast proliferation and the anti-scarring effect in Tenon’s capsule, both in vitro and in vivo. METHODS: Human primary Tenon’s capsule fibroblasts were cultured in vitro, treated with up titrating concentrations of olmesartan. The rate of inhibition was tested with methyl thiazol tetrazolium (MTT method. Real-time PCR was performed to analyze changes in mRNA expressions of the fibrosis-related factors: matrix metalloproteinase-2 (MMP-2, tissue inhibitor of metalloproteinase (TIMP-1,2 and proliferating cell nuclear antigen (PCNA. Thirty rabbits were divided into 5 groups (3, 7, 14, 21, and 28d. A rabbit conjunctiva flap model was created in each eye. Olmesartan solution was injected subconjunctivally and then evaluated its anti-proliferation and anti-fibrosis effects through the histological morphology and immunohistochemistry of MMP-2 and PCNA in each group. Only the 7d group was treated with Masson’s trichrome to compare the neovascularization in the subconjunctiva area. RESULTS: In vitro, cultured Tenon's capsule human fibroblasts showed a dose dependent inhibition by olmesartan in MTT. Olmesartan reduced mRNA expressions of MMP-2 and PCNA but increased mRNA expressions of TIMP-1 and TIMP-2. In vivo, the rabbit eyes treated with olmesartan at 3rd, 7th, 14th and 21st days demonstrated a significant reduced expressions of MMP-2 and PCNA compared with control eye, no significant difference observed in 28th day group. The cellular proliferation and neovascularization was suppressed by olmesartan in Masson’s trichrome observation. CONCLUSION: By inhibiting fibroblasts in vitro and in vivo, olmesartan prevents the proliferation and activity of fibroblasts in scar tissue formation, which might benefit glaucoma filtering surgery.

Plasma rich in growth factors (PRGF-Endoret) stimulates proliferation and migration of primary keratocytes and conjunctival fibroblasts and inhibits and reverts TGF-beta1-Induced myodifferentiation.

Science.gov (United States)

Anitua, Eduardo; Sanchez, Mikel; Merayo-Lloves, Jesus; De la Fuente, Maria; Muruzabal, Francisco; Orive, Gorka

2011-08-01

Plasma rich in growth factors (PRGF-Endoret) technology is an autologous platelet-enriched plasma obtained from patient's own blood, which after activation with calcium chloride allows the release of a pool of biologically active proteins that influence and promote a range of biological processes including cell recruitment, and growth and differentiation. Because ocular surface wound healing is mediated by different growth factors, we decided to explore the potential of PRGF-Endoret technology in stimulating the biological processes related with fibroblast-induced tissue repair. Furthermore, the anti-fibrotic properties of this technology were also studied. Blood from healthy donors was collected, centrifuged and, whole plasma column (WP) and the plasma fraction with the highest platelet concentration (F3) were drawn off, avoiding the buffy coat. Primary human cells including keratocytes and conjunctival fibroblasts were used to perform the "in vitro" investigations. The potential of PRGF-Endoret in promoting wound healing was evaluated by means of a proliferation and migration assays. Fibroblast cells were induced to myofibroblast differentiation after the treatment with 2.5 ng/mL of TGF-β1. The capability of WP and F3 to prevent and inhibit TGF-β1-induced differentiation was evaluated. Results show that this autologous approach significantly enhances proliferation and migration of both keratocytes and conjunctival fibroblasts. In addition, plasma rich in growth factors prevents and inhibits TGF-β1-induced myofibroblast differentiation. No differences were found between WP and F3 plasma fractions. These results suggest that PRGF-Endoret could reduce scarring while stimulating wound healing in ocular surface. F3 or whole plasma column show similar biological effects in keratocytes and conjunctival fibroblast cells.

HGF is released from buccal fibroblasts after smokeless tobacco stimulation

DEFF Research Database (Denmark)

Dabelsteen, S; Christensen, S; Gron, B

2005-01-01

on exposure time and on concentration of the tobacco extract. High concentration increased production of HGF 4-fold. KGF production was doubled when high concentration of tobacco was used, low concentration did not stimulate cells. GM-CSF production was low in both stimulated and non-stimulated cells......To investigate the effect of smokeless tobacco (ST) on (1) HGF, KGF and GM-CSF expression by buccal fibroblasts and (2) on keratinocyte and fibroblast proliferation. Buccal fibroblasts were stimulated with different concentrations of ST extracts in a double dilution from 0.50% w/v to 0.03% w....... Keratinocytes and fibroblasts showed no increase in proliferation after stimulation with increased concentrations of ST. The results suggest that HGF and KGF may play an important role as a paracrine growth factor in epithelial hyperplasia in ST lesions....

Engineered electrospun poly(caprolactone)/polycaprolactone-g-hydroxyapatite nano-fibrous scaffold promotes human fibroblasts adhesion and proliferation

Energy Technology Data Exchange (ETDEWEB)

Keivani, F. [Biology Department, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shokrollahi, P., E-mail: p.shokrolahi@ippi.ac.ir [Department of Biomaterials, Faculty of Science, Iran Polymer and Petrochemical Institute, Tehran (Iran, Islamic Republic of); Zandi, M. [Department of Biomaterials, Faculty of Science, Iran Polymer and Petrochemical Institute, Tehran (Iran, Islamic Republic of); Irani, S. [Biology Department, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of); Shokrolahi, F. [Department of Biomaterials, Faculty of Science, Iran Polymer and Petrochemical Institute, Tehran (Iran, Islamic Republic of); Khorasani, S.C. [Biology Department, Science and Research Branch, Islamic Azad University, Tehran (Iran, Islamic Republic of)

2016-11-01

Polycaprolactone (PCL)/hydroxyapatite nano-composites are among the best candidates for tissue engineering. However, interactions between nHAp and PCL are difficult to control leading to inhomogeneous dispersion of the bio-ceramic particles. Grafting of polymer chains at high density/chain length while promotes the phase compatibility may result in reduced HAp exposed surface area and therefore, bioactivity is compromised. This issue is addressed here by grafting PCL chains onto HAp nano-particles through ring opening polymerization of ε-caprolactone (PCL-g-HAp). FTIR and TGA analysis showed that PCL (6.9 wt%), was successfully grafted on the HAp. PCL/PCL-g-HAp nano-fibrous scaffold showed up to 10 and 33% enhancement in tensile strength and modulus, respectively, compared to those of PCL/HAp. The effects of HAp on the in vitro HAp formation were investigated for both the PCL/HAp and PCL/PCL-g-HAp scaffolds. Precipitation of HAp on the nano-composite scaffolds observed after 15 days incubation in simulated body fluid (SBF), as confirmed by scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX). Human fibroblasts were seeded on PCL, PCL/HAp and PCL/PCL-g-HAp scaffolds. According to MTT assay, the highest cell proliferation was recorded for PCL/PCL-g-HAp nano-composite, at all time intervals (1–21 days, P < 0.001). Fluorescent microscopy (of DAPI stained samples) and electron microscopy images showed that all nano-fibrous scaffolds (PCL, PCL/HAp, and PCL/PCL-g-HAp), were non-toxic against cells, while more cell adhesion, and the most uniform cell distribution observed on the PCL/PCL-g-HAp. Overall, grafting of relatively short chains of PCL on the surface of HAp nano-particles stimulates fibroblasts adhesion and proliferation on the PCL/PCL-g-HAp nano-composite. - Highlights: • PCL chains were grafted on HAp nano-particles at relatively low density, through ROP of ε-caprolactone (PCL-g-HAp) • PCL-g-HAp featured a relatively high

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca).

Science.gov (United States)

Wang, Jun-Jie; Liu, Yu-Liang; Sun, Yuan-Chao; Ge, Wei; Wang, Yong-Yong; Dyce, Paul W; Hou, Rong; Shen, Wei

2015-01-01

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca)

Science.gov (United States)

Wang, Jun-Jie; Liu, Yu-Liang; Sun, Yuan-Chao; Ge, Wei; Wang, Yong-Yong; Dyce, Paul W.; Hou, Rong; Shen, Wei

2015-01-01

It has been widely known that the giant panda (Ailuropoda melanoleuca) is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs) is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF), a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM) has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU) labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK) signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro. PMID:26375397

Basic Fibroblast Growth Factor Stimulates the Proliferation of Bone Marrow Mesenchymal Stem Cells in Giant Panda (Ailuropoda melanoleuca.

Directory of Open Access Journals (Sweden)

Jun-Jie Wang

Full Text Available It has been widely known that the giant panda (Ailuropoda melanoleuca is one of the most endangered species in the world. An optimized platform for maintaining the proliferation of giant panda mesenchymal stem cells (MSCs is very necessary for current giant panda protection strategies. Basic fibroblast growth factor (bFGF, a member of the FGF family, is widely considered as a growth factor and differentiation inducer within the stem cell research field. However, the role of bFGF on promoting the proliferation of MSCs derived from giant panda bone marrow (BM has not been reported. In this study, we aimed to investigate the role of bFGF on the proliferation of BM-MSCs derived from giant panda. MSCs were cultured for cell proliferation analysis at 24, 48 and 72 hrs following the addition of bFGF. With increasing concentrations of bFGF, cell numbers gradually increased. This was further demonstrated by performing 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide (MTT cell proliferation assay, 5-Bromo-2-deoxyUridine (BrdU labeling and cell cycle testing. Furthermore, the percentage of MSCs that were OCT4 positive increased slightly following treatment with 5 ng/ml bFGF. Moreover, we demonstrated that the extracellular signal-regulated kinase (ERK signaling pathway may play an important role in the proliferation of panda MSCs stimulated by bFGF. In conclusion, this study suggests that giant panda BM-MSCs have a high proliferative capacity with the addition of 5 ng/ml bFGF in vitro.

Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts

OpenAIRE

Chen, Joseph C.; Johnson, Brittni A.; Erikson, David W.; Piltonen, Terhi T.; Barragan, Fatima; Chu, Simon; Kohgadai, Nargis; Irwin, Juan C.; Greene, Warner C.; Giudice, Linda C.; Roan, Nadia R.

2014-01-01

STUDY QUESTION How does seminal plasma (SP) affect the transcriptome of human primary endometrial epithelial cells (eEC) and stromal fibroblasts (eSF)? SUMMARY ANSWER Exposure of eEC and eSF to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death. WHAT IS KNOWN ALREADY Studies in both humans and animals suggest that SP can access and induce physiological changes in the upper female reproductiv...

Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

Directory of Open Access Journals (Sweden)

Francesca Saporito

2018-02-01

Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells

Directory of Open Access Journals (Sweden)

Xinyue Liang

2016-01-01

Full Text Available Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR. In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK/extracellular signal-regulated kinase (ERK and phosphatidylinositol 3′ -kinase(PI3K-Akt (PI3K/AKT phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy. In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

Effects of Plasma Rich in Growth Factors and Platelet-Rich Fibrin on Proliferation and Viability of Human Gingival Fibroblasts

Directory of Open Access Journals (Sweden)

Surena Vahabi

2016-01-01

Full Text Available Objectives: Platelet preparations are commonly used to enhance bone and soft tissue regeneration. Considering the existing controversies on the efficacy of platelet products for tissue regeneration, more in vitro studies are required. The aim of the present study was to compare the in vitro effects of plasma rich in growth factors (PRGF and platelet-rich fibrin (PRF on proliferation and viability of human gingival fibroblasts (HGFs.Materials and Methods: Anitua's PRGF and Choukran's PRF were prepared according to the standard protocols. After culture periods of 24, 48 and 72 hours, proliferation of HGFs was evaluated by the methyl thiazol tetrazolium assay. Statistical analysis was performed using one-way ANOVA followed by Tukey-Kramer’s multiple comparisons and P-values<0.05 were considered statistically significant.Results: PRGF treatment induced statistically significant (P<0.001 proliferation of HGF cells compared to the negative control (100% viability at 24, 48 and 72 hours in values of 123%±2.25%, 102%±2.8% and 101%±3.92%, respectively. The PRF membrane treatment of HGF cells had a statistically significant effect on cell proliferation (21%±1.73%, P<0.001 at 24 hours compared to the negative control. However, at 48 and 72 hours after treatment, PRF had a negative effect on HGF cell proliferation and caused 38% and 60% decrease in viability and proliferation compared to the negative control, respectively. The HGF cell proliferation was significantly higher in PRGF than in PRF group (P< 0.001.Conclusion: This study demonstrated that PRGF had a strong stimulatory effect on HGF cell viability and proliferation compared to PRF.

Adiponectin Is Involved in Connective Tissue Growth Factor-Induced Proliferation, Migration and Overproduction of the Extracellular Matrix in Keloid Fibroblasts.

Science.gov (United States)

Luo, Limin; Li, Jun; Liu, Han; Jian, Xiaoqing; Zou, Qianlei; Zhao, Qing; Le, Qu; Chen, Hongdou; Gao, Xinghua; He, Chundi

2017-05-12

Adiponectin, an adipocyte-derived hormone, exerts pleiotropic biological effects on metabolism, inflammation, vascular homeostasis, apoptosis and immunity. Recently, adiponectin has been suggested to attenuate the progression of human dermal fibrosis. Connective tissue growth factor (CTGF) is induced in keloids and is thought to be participated in the formation of keloid fibrosis. However, the roles played by adiponectin in keloids remain unclear. In this study, we explored the effects of adiponectin on CTGF-induced cell proliferation, migration and the deposition of extracellular matrix (ECM) and their associated intracellular signalling pathways in keloid fibroblasts (KFs). We also explored possible mechanisms of keloid pathogenesis. Primary fibroblast cultures were established from foreskin biopsies and skin biopsies from patients with keloids. The expression of adiponectin and adiponectin receptors (adipoRs) was evaluated by reverse transcription-PCR (RT-PCR), quantitative real-time RT-PCR, immunofluorescence staining, and immunohistochemical analysis. Next, KFs and normal dermal fibroblasts (NFs) were treated with CTGF in the presence or absence of adiponectin. A cell counting kit-8 (CCK-8) and the Transwell assay were used to examine cell proliferation and migration. The level of the collagen I, fibronectin (FN) and α-smooth muscle actin (α-SMA) mRNAs and proteins were determined by quantitative real-time RT-PCR and western blotting. The effects of RNA interference (RNAi) targeting the adipoR genes were detected. Phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase-protein kinase (PI3K-Akt) were examined by western blotting to further investigate the signalling pathways. Furthermore, inhibitors of signal transduction pathways were investigated. The expression levels of adiponectin and adipoRs were significantly decreased in keloids compared with those

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Benamer, Najate [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Fares, Nassim [Laboratoire de Physiologie, Faculte de Medecine, Universite Saint Joseph, Beyrouth (Lebanon); Bois, Patrick [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France); Faivre, Jean-Francois, E-mail: Jean-Francois.Faivre@univ-poitiers.fr [UMR CNRS/Universite de Poitiers No. 6187, Pole Biologie Sante Bat B36, BP 633, 1 rue Georges Bonnet, 86022 Poitiers (France)

2011-04-29

Highlights: {yields} In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. {yields} S1P increases cell proliferation through SUR2/Kir6.1 activation. {yields} S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. {yields} S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

International Nuclear Information System (INIS)

Benamer, Najate; Fares, Nassim; Bois, Patrick; Faivre, Jean-Francois

2011-01-01

Highlights: → In cardiac fibroblasts, SUR2/Kir6.1 channel is activated by S1P via the S1P3R. → S1P increases cell proliferation through SUR2/Kir6.1 activation. → S1P decreases collagen and IL-6 secretion through SUR2/Kir6.1 activation. → S1P stimulates fibroblast migration independently from SUR2/Kir6.1 channel. -- Abstract: The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.

The use of chitosan-dextran gel shows anti-inflammatory, antibiofilm, and antiproliferative properties in fibroblast cell culture.

Science.gov (United States)

Paramasivan, Sathish; Jones, Damien; Baker, Leonie; Hanton, Lyall; Robinson, Simon; Wormald, Peter J; Tan, Lorwai

2014-01-01

Chitosan-dextran gel has been used as an antihemostatic agent and antiadhesive agent after endoscopic sinus surgery. Because Staphylococcus aureus biofilms have been implicated in recalcitrant chronic rhinosinusitis, this study aimed to further investigate the (i) anti-inflammatory, (ii) bacterial biofilm inhibition, (iii) antiproliferative effects, and (iv) wound-healing properties of chitosan and chitosan-dextran gel. Fibroblasts were isolated from human nasal tissue and were used to determine the effects of chitosan and chitosan-dextran gel on (i) cell proliferation, (ii) wound healing, (iii) inflammation in fibroblast cultures challenged with superantigens S. aureus enterotoxin B (SEB) and toxic shock syndrome toxin (TSST), and (iv) on S. aureus biofilms. Chitosan was highly effective at reducing IL-8 expression after TSST and SEB challenge. Chitosan was also effective at reducing IL-8 expression of nonchallenged fibroblasts showing its anti-inflammatory effects on fibroblasts in a diseased state. Chitosan-dextran gel showed strong antibiofilm properties at 50% (v/v) concentration in vitro. Dextran, on its own, showed antibiofilm properties at 1.25% (w/v) concentration. Chitosan, on its own, reduced proliferation of fibroblasts to 82% of control proliferation and chitosan-dextran gel reduced proliferation of the fibroblasts to 0.04% of control proliferation. Relative to the no treatment controls, chitosan-dextran gel significantly delayed the wound-healing rate over the first 48 hours of the experiment. Chitosan-dextran gel reduced fibroblast proliferation and wound-healing time, showing a possible mechanism of reducing adhesions in the postsurgical period. Chitosan reduced IL-8 levels, showing its anti-inflammatory properties. Chitosan-dextran gel and dextran treatment showed antibiofilm properties in our model.

Chromosome aberration induction in human diploid fibroblast and epithelial cells

International Nuclear Information System (INIS)

Scott, D.

1986-01-01

The relative sensitivity of cultured human fibroblasts and epithelial cells to radiation-induced chromosomal aberrations was investigated. Lung fibroblast and kidney epithelial cells from the same fetus were compared, as were skin fibroblasts and epithelial keratinocytes from the same foreskin sample. After exposure of proliferating fetal cells to 1.5 Gy X-rays there was a very similar aberration yield in the fibroblasts and epithelial cells. Observations of either little or no difference in chromosomal sensitivity between human fibroblasts and epithelial cells give added confidence that quantitative cytogenetic data obtained from cultured fibroblasts are relevant to the question of sensitivity of epithelial cells which are the predominant cell type in human cancers. (author)

[Regulation of airway stem cell proliferation in idiopathic pulmonary fibrosis].

Science.gov (United States)

Yang, S X; Wu, Q; Sun, X; Li, X; Li, K; Xu, L; Li, Y; Zhang, Q Y; Zhang, Y C; Chen, H Y

2016-09-01

To investigate the effect of fibroblasts on regulating airway stem cell proliferation in idiopathic pulmonary fibrosis. Lung cell suspension was prepared from β-actin-GFP mice. Airway stem cells were obtained by fluorescence activated cell sorting and co-cultured with lung fibroblasts. The fibroblasts were treated with TGF-β inhibitor SB43142. The expression of growth factors FGF1/2 and the effect of FGF1/2 on stem cell proliferation were observed. The cloning efficiency of airway stem cells, when co-cultured with normal lung fibroblast cells for 8 days, was (3.5±1.1)%, while the cloning efficiency was reduced to (0.04±0.04)% when co-cultured with lung fibroblasts from idiopathic pulmonary fibrosis patients. The difference between the 2 groups was statistically significant(P=0.002 5). TGF-β receptor inhibitor SB431542 increased lung fibroblast growth factors FGF1/2 expression.FGF1 mRNA expression was increased to the experimental group 0.005 5 from 0.000 2 in the control group.FGF2 mRNA expression of the amount raised to the experimental group 0.000 15 from 0.000 8 in the control group.FGF1/2 promoted the growth of airway stem cells. After FGF1/2 was co-cultured with normal lung fibroblast cells for 8 days, the cloning efficiency of airway stem cells was (0.3±0.1)%. During the development of idiopathic pulmonary fibrosis, fibroblast secreted FGF1/2 regulate airway stem cell proliferation.

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

International Nuclear Information System (INIS)

Hirata, Eri; Takita, Hiroko; Watari, Fumio; Yokoyama, Atsuro; Ménard-Moyon, Cécilia; Venturelli, Enrica; Bianco, Alberto

2013-01-01

Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF–CNT) showed the same effect as FGF alone. In addition, FGF–CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF–CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF–CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications. (paper)

Carbon nanotubes functionalized with fibroblast growth factor accelerate proliferation of bone marrow-derived stromal cells and bone formation

Science.gov (United States)

Hirata, Eri; Ménard-Moyon, Cécilia; Venturelli, Enrica; Takita, Hiroko; Watari, Fumio; Bianco, Alberto; Yokoyama, Atsuro

2013-11-01

Multi-walled carbon nanotubes (MWCNTs) were functionalized with fibroblast growth factor (FGF) and the advantages of their use as scaffolds for bone augmentation were evaluated in vitro and in vivo. The activity of FGF was assessed by measuring the effect on the proliferation of rat bone marrow stromal cells (RBMSCs). The presence of FGF enhanced the proliferation of RBMSCs and the FGF covalently conjugated to the nanotubes (FGF-CNT) showed the same effect as FGF alone. In addition, FGF-CNT coated sponges were implanted between the parietal bone and the periosteum of rats and the formation of new bone was investigated. At day 14 after implantation, a larger amount of newly formed bone was clearly observed in most pores of FGF-CNT coated sponges. These findings indicated that MWCNTs accelerated new bone formation in response to FGF, as well as the integration of particles into new bone during its formation. Scaffolds coated with FGF-CNT could be considered as promising novel substituting materials for bone regeneration in future tissue engineering applications.

The influence of glancing angle deposited nano-rough platinum surfaces on the adsorption of fibrinogen and the proliferation of primary human fibroblasts

International Nuclear Information System (INIS)

Dolatshahi-Pirouz, A; Foss, M; Chevallier, J; Besenbacher, F; Pennisi, C P; Yoshida, K; Skeldal, S; Andreasen, P; Zachar, V

2009-01-01

We have used the glancing angle deposition (GLAD) method as a simple and fast method to generate nano-rough surfaces for protein adsorption experiments and cell assays. The surface roughness and the detailed geometrical surface morphology of the thin films were characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). As the GLAD deposition angle approaches grazing incidence, sharp and whisker-like columnar protrusions are formed. Smaller and less sharp surface features appear for the thin films synthesized at higher deposition angles. By changing the GLAD deposition angle together with the total amount of mass deposited per area on the respective surfaces, the size of the surface features can be varied on the nanoscale. Using the GLAD topographies as model surfaces, we have investigated the influence of the nano-roughness on fibrinogen adsorption and on the proliferation of primary human fibroblasts. It is found that fibrinogen, an important blood protein, preferentially adheres on the whisker-like nano-rough substrates in comparison to a flat surface. Furthermore, the proliferation of the human fibroblasts is significantly reduced on the nano-rough substrates. These results demonstrate that the GLAD technique can be used to fabricate nano-rough surface morphologies that significantly influence both protein and cellular adhesion to surfaces and are therefore well suited for biological assays.

Heat Shock Protein 90 Inhibitor (17-AAG) Induces Apoptosis and Decreases Cell Migration/Motility of Keloid Fibroblasts.

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Yun, In Sik; Lee, Mi Hee; Rah, Dong Kyun; Lew, Dae Hyun; Park, Jong-Chul; Lee, Won Jai

2015-07-01

The regulation of apoptosis, proliferation, and migration of fibroblasts is altered in keloids. The 90-kDa heat shock protein (heat shock protein 90) is known to play a key role in such regulation. Therefore, the authors investigated whether the inhibition of heat shock protein 90 in keloid fibroblasts could induce apoptosis and attenuate keloid fibroblast proliferation and migration. The authors evaluated heat shock protein 90 expression in keloid tissues with immunohistochemistry. The authors used cell viability [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and annexin V/propidium iodide staining for apoptosis, a wound healing model and cell tracking system to assess cell migration, and Akt Western blotting analysis in keloid fibroblasts after inhibition of heat shock protein 90 with 17-allylaminodemethoxygeldanamycin (17-AAG). The expression of heat shock protein 90 in keloid tissues was significantly increased compared with normal tissues. The 17-AAG-treated keloid fibroblasts showed significantly decreased proliferation, promotion of apoptosis, and decreased expression of Akt. Furthermore, a dose-dependent decrease in cell migration was noted after 17-AAG treatment of keloid fibroblasts. The 17-AAG-treated keloid fibroblasts had less directionality to the wound center and migrated a shorter distance. The authors confirmed that the inhibition of heat shock protein 90 in keloid fibroblasts could promote apoptosis and attenuate proliferation and migration of keloid fibroblasts. Therefore, the authors think that the inhibition of heat shock protein 90 is a key factor in the regulation of biological processes in keloids. With further preclinical study, the authors will be able to apply these results clinically for keloid treatment.

Sialylation regulates myofibroblast differentiation of human skin fibroblasts.

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Sasaki, Norihiko; Itakura, Yoko; Toyoda, Masashi

2017-04-18

Fibroblasts are key players in maintaining skin homeostasis and in orchestrating physiological tissue repair and skin regeneration. Dysfunctions in fibroblasts that occur with aging and the senescent process lead to the delayed healing observed in elderly people. The molecular mechanisms leading to fibroblast dysfunction during aging and the senescent process have not yet been clarified. Previously, changes in patterns of glycosylation were observed in fibroblasts in aging and the senescent process, but the effect of these changes on the function of fibroblasts has not been well documented. Here, we investigated whether changes in glycosylation during the process to senescence may have functional effects on fibroblasts. The changes in cell surface glycans on skin fibroblasts during the process to senescence were examined in early-passage (EP) and late-passage (LP) skin fibroblasts by fluorescence-activated cell sorting analysis using lectins. The contributors to the changes in cell surface glycans were examined by real-time polymerase chain reaction or Western blot analysis. The effects of changes in glycosylation on proliferation, migration, induction of cellular senescence, and myofibroblast differentiation induced by transforming growth factor (TGF)-β1 stimulation were examined in EP fibroblasts. The changes in glycosylation were performed by GalNAc-α-O-benzyl or sialidase treatment. A decrease in sialylation of glycoproteins and an increase in sialidase NEU1 were observed in LP fibroblasts. The reduction of sialylation did not have any effect on proliferation, migration, or induction of cellular senescence. On the other hand, myofibroblast differentiation was inhibited by the reduction of sialylation, indicating that sialylation is important for myofibroblast differentiation. The localization of CD44 in lipid rafts, which is required for myofibroblast differentiation, was inhibited by the reduction of sialylation. Furthermore, reduced myofibroblast

Senescent phenotypes of skin fibroblasts from patients with Tangier disease

International Nuclear Information System (INIS)

Matsuura, Fumihiko; Hirano, Ken-ichi; Ikegami, Chiaki; Sandoval, Jose C.; Oku, Hiroyuki; Yuasa-Kawase, Miyako; Tsubakio-Yamamoto, Kazumi; Koseki, Masahiro; Masuda, Daisaku; Tsujii, Ken-ichi; Ishigami, Masato; Nishida, Makoto; Shimomura, Iichiro; Hori, Masatsugu; Yamashita, Shizuya

2007-01-01

Tangier disease (TD) is characterized by a deficiency of high density lipoprotein (HDL) in plasma and patients with TD have an increased risk for coronary artery disease (CAD). Recently, we reported that fibroblasts from TD exhibited large and flattened morphology, which is often observed in senescent cells. On the other hand, data have accumulated to show the relationship between cellular senescence and development of atherosclerotic CAD. The aim of the present study was to investigate whether TD fibroblasts exhibited cellular senescence. The proliferation of TD fibroblasts was gradually decreased at population doubling level (PDL) ∼10 compared with control cells. TD cells practically ceased proliferation at PDL ∼30. DNA synthesis was markedly decreased in TD fibroblasts. TD cells exhibited a higher positive rate for senescence-associated β-galactosidase (SA-β-gal), which is one of the biomarkers of cellular senescence in vitro. These data showed that TD cells reached cellular senescence at an earlier PDL compared with controls. Although, there was no difference in the telomere length of fibroblasts between TD and controls at the earlier passage (PDL 6), the telomere length of TD cells was shorter than that of controls at the late passage (PDL 25). Taken together, the current study demonstrates that the late-passaged TD fibroblasts showed senescent phenotype in vitro, which might be related to the increased cardiovascular manifestations in TD patients

The importance of the nuclear glutathione in the Cell Proliferation

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Markovic, Jelena

2009-01-01

The present thesis offers an insight in the importance of nuclear GSH in cell proliferation. The research was performed in three different cellular models of diverse proliferating activity: immortalized mouse embryonic fibroblasts 3T3, mammary adenocarcinoma cell line MCF7 and primary embryonic neuralonal culture. The results presented here provide evidence that suggest that the relationship between GSH level and telomerase activity, previously described by our group for 3T3 fibroblasts is a ...

Allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture

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Restu Syamsul Hadi

2014-08-01

Full Text Available Background Transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. Their potential for regenerative therapy of chronic wounds should be evaluated. The aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (PBMC. Methods In this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. We used one intervention group of allogeneic fibroblasts co-cultured with PBMC and 2 control groups of separate fibroblast and PBMC cultures.Fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (OCT4 expression. Viability was evaluated using water soluble tetrazolium-1 (WST-1 proliferation assay. Absorbances were measured at 450 nm. Data analysis was performed by student’s paired t-test. Results Dermal fibroblasts were shown to secrete collagen, express OCT4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with PBMC. Co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05. Calculated viable cell numbers increased 1.8 and 5.1-fold, respectively, at days 2 and 4 in vitro. Both groups showed comparable doubling times at days 2 and 4 in vitro. PBMC did not interfere with allogeneic fibroblast viability and proliferative capacity Conclusions Allogeneic fibroblasts remain viable and proliferate in the presence of host PBMC. Future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. Dermal fibroblasts are a potential source for cell therapy in chronic wound management.

Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation

International Nuclear Information System (INIS)

Vezeridis, Peter S.; Semeins, Cornelis M.; Chen Qian; Klein-Nulend, Jenneke

2006-01-01

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and L-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation

Helium generated cold plasma finely regulates activation of human fibroblast-like primary cells.

Directory of Open Access Journals (Sweden)

Paola Brun

Full Text Available Non-thermal atmospheric pressure plasmas are being developed for a wide range of health care applications, including wound healing. However in order to exploit the potential of plasma for clinical applications, the understanding of the mechanisms involved in plasma-induced activation of fibroblasts, the cells active in the healing process, is mandatory. In this study, the role of helium generated plasma in the tissue repairing process was investigated in cultured human fibroblast-like primary cells, and specifically in hepatic stellate cells and intestinal subepithelial myofibroblasts. Five minutes after treatment, plasma induced formation of reactive oxygen species (ROS in cultured cells, as assessed by flow cytometric analysis of fluorescence-activated 2',7'-dichlorofluorescein diacetate probe. Plasma-induced intracellular ROS were characterized by lower concentrations and shorter half-lives with respect to hydrogen peroxide-induced ROS. Moreover ROS generated by plasma treatment increased the expression of peroxisome proliferator activated receptor (PPAR-γ, nuclear receptor that modulates the inflammatory responses. Plasma exposure promoted wound healing in an in vitro model and induced fibroblast migration and proliferation, as demonstrated, respectively, by trans-well assay and partitioning between daughter cells of carboxyfluorescein diacetate succinimidyl ester fluorescent dye. Plasma-induced fibroblast migration and proliferation were found to be ROS-dependent as cellular incubation with antioxidant agents (e.g. N-acetyl L-cysteine cancelled the biological effects. This study provides evidence that helium generated plasma promotes proliferation and migration in liver and intestinal fibroblast-like primary cells mainly by increasing intracellular ROS levels. Since plasma-evoked ROS are time-restricted and elicit the PPAR-γ anti-inflammatory molecular pathway, this strategy ensures precise regulation of human fibroblast activation and

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

Energy Technology Data Exchange (ETDEWEB)

Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Zhang, Yong, E-mail: zhangyong1956@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China); Gao, Ming-Qing, E-mail: gaomingqing@nwsuaf.edu.cn [College of Veterinary Medicine, Northwest A& F University, Yangling 712100, Shaanxi (China); Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A& F University, Yangling 712100, Shaanxi (China)

2016-11-15

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis

International Nuclear Information System (INIS)

Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

2016-01-01

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. - Highlights: • Inflammatory BMEs affect the properties of BMFs during mastitis. • BMEs inhibited the proliferation and promoted the migration of BMFs. • BMEs enhanced secretion of inflammatory mediators and deposition of ECM in BMFs. • Changes of the properties of BMFs were mediated by specific signal molecules.

Effect of polysaccharides of ganoderma lucidum (PGL) on cell cycle and proliferation of NIH3T3 fibroblast exposed to γ-rays

International Nuclear Information System (INIS)

Ji Xiuqing; Wu Shiling; Zhou Yinghui; Xu Lan

2001-01-01

Objective: To study the effects of γ-irradiation on cell cycle and proliferation of fibroblasts and the radioprotective effect of PGL in term of cellular level. Methods: The cells in the control group and PGL (50 mg/L. 100 mg/L. 150 mg/L) groups were irradiated with 15 Gy γ-rays. The cell cycle and proliferation of NIH3T3 cells were determined by flow cytometry after irradiation within 12 h. Results: The percentage of G 0 -G 1 phase was 52.76% in the positive control group. Compared with the negative control group (90.9%), the proliferation of NIH3T3 was promoted significantly (P 0 -G 1 phases in the GPL (50 mg/L, 100 mg/L, 150 mg/L) groups were 66.17%, 71.73%, 76.10% respectively, the proliferation of NIH3T3 cells was inhibited by adding GPL. compared with the positive control group, in the 50 mg/L group P < 0.05, in the 100 mg/L and 150 mg/L groups P < 0.01. It was found that the apoptosis of NIH3T3 cells had little change after comparing non-irradiated group with the irradiated groups. Conclusion: Radiation with γ-rays at a dose of 15 Gy, can significantly stimulate proliferation of NIH3T3 cells, but no induced apoptosis can not be achieve within 12 h after irradiation. PGL can inhibit cell proliferation, so PGL has radioprotective activity on cell levels

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

Science.gov (United States)

Demirci, Selami; Doğan, Ayşegül; Apdik, Hüseyin; Tuysuz, Emre Can; Gulluoglu, Sukru; Bayrak, Omer Faruk; Şahin, Fikrettin

2018-01-01

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Inflammatory responses of stromal fibroblasts to inflammatory epithelial cells are involved in the pathogenesis of bovine mastitis.

Science.gov (United States)

Zhang, Wenyao; Li, Xuezhong; Xu, Tong; Ma, Mengru; Zhang, Yong; Gao, Ming-Qing

2016-11-15

Hypernomic secretion of epithelial cytokines has several effects on stromal cells. The contributions of inflammatory epithelial cells to stromal fibroblasts in bovine mammary glands with mastitis remain poorly understood. Here, we established an inflammatory epithelial cell model of bovine mastitis with gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. We characterized immune responses of mammary stromal fibroblasts induced by inflammatory epithelial cells. Our results showed that inflammatory epithelial cells affected stromal fibroblast characteristics by increasing inflammatory mediator expression, elevating extracellular matrix protein deposition, decreasing proliferation capacity, and enhancing migration ability. The changes in stromal fibroblast proliferation and migration abilities were mediated by signal molecules, such as WNT signal pathway components. LPS- and LTA-induced inflammatory epithelial cells triggered different immune responses in stromal fibroblasts. Thus, in mastitis, bovine mammary gland stromal fibroblasts were affected by inflammatory epithelial cells and displayed inflammation-specific changes, suggesting that fibroblasts play crucial roles in bovine mastitis. Copyright © 2016 Elsevier Inc. All rights reserved.

TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

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Chen, Xiao-Qing; Zhang, Dao-Liang [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Jiang, Wei-Feng; Zhou, Li [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Zhao, Liang, E-mail: zhaol_zg@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China); Wang, Quan-Xing, E-mail: wqxejd@126.com [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Liu, Xu, E-mail: liuxu_xk@163.com [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University, Shanghai (China)

2015-08-14

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage.

TRIF promotes angiotensin II-induced cross-talk between fibroblasts and macrophages in atrial fibrosis

International Nuclear Information System (INIS)

Chen, Xiao-Qing; Zhang, Dao-Liang; Zhang, Ming-Jian; Guo, Meng; Zhan, Yang-Yang; Liu, Fang; Jiang, Wei-Feng; Zhou, Li; Zhao, Liang; Wang, Quan-Xing; Liu, Xu

2015-01-01

Aims: Atrial fibroblasts and macrophages have long been thought to participate in atrial fibrillation (AF). However, which specific mediator may regulate the interaction between them remains unclear. Methods and results: We provided the evidence for the involvement of Toll/IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF), an important inflammation-related molecule, in the pathophysiology of AF. Patients with AF showed higher levels of angiotensin II (AngII) and TRIF expression and larger number of macrophages infiltration in left atria appendage than individuals with sinus rhythm (SR). In the cell study, AngII induced chemokines expressions in mouse atrial fibroblasts and AngII-stimulated atrial fibroblasts induced the chemotaxis of macrophages, which were reduced by losartan and TRIF siRNA. Meanwhile, AngII-stimulated atrial fibroblasts proliferation was enhanced by macrophages. Conclusions: Our data demonstrated that TRIF may be a crucial factor promoting the interaction between atrial fibroblasts and macrophages, leading to atrial fibrosis. - Highlights: • Compared with SR, AF showed higher TRIF expression in left atrial appendage. • TRIF siRNA reversed macrophage chemotaxis induced by AngII-treated fibroblast. • TRIF siRNA reversed chemokines expressions induced by AngII in fibroblast. • AngII-stimulated atrial fibroblast proliferation was enhanced by macrophage

Attachment, Proliferation, and Morphological Properties of Human Dermal Fibroblasts on Ovine Tendon Collagen Scaffolds: A Comparative Study.

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Busra, Fauzi Mh; Lokanathan, Yogeswaran; Nadzir, Masrina Mohd; Saim, Aminuddin; Idrus, Ruszymah Bt Hj; Chowdhury, Shiplu Roy

2017-03-01

Collagen type I is widely used as a biomaterial for tissue-engineered substitutes. This study aimed to fabricate different three-dimensional (3D) scaffolds using ovine tendon collagen type I (OTC-I), and compare the attachment, proliferation and morphological features of human dermal fibroblasts (HDF) on the scaffolds. This study was conducted between the years 2014 to 2016 at the Tissue Engineering Centre, UKM Medical Centre. OTC-I was extracted from ovine tendon, and fabricated into 3D scaffolds in the form of sponge, hydrogel and film. A polystyrene surface coated with OTC-I was used as the 2D culture condition. Genipin was used to crosslink the OTC-I. A non-coated polystyrene surface was used as a control. The mechanical strength of OTC-I scaffolds was evaluated. Attachment, proliferation and morphological features of HDF were assessed and compared between conditions. The mechanical strength of OTC-I sponge was significantly higher than that of the other scaffolds. OTC-I scaffolds and the coated surface significantly enhanced HDF attachment and proliferation compared to the control, but no differences were observed between the scaffolds and coated surface. In contrast, the morphological features of HDF including spreading, filopodia, lamellipodia and actin cytoskeletal formation differed between conditions. OTC-I can be moulded into various scaffolds that are biocompatible and thus could be suitable as scaffolds for developing tissue substitutes for clinical applications and in vitro tissue models. However, further study is required to determine the effect of morphological properties on the functional and molecular properties of HDF.

Influence on proliferation and adhesion of human gingival fibroblasts from different titanium surface decontamination treatments: An in vitro study.

Science.gov (United States)

Cao, Jie; Wang, Tong; Pu, Yinfei; Tang, Zhihui; Meng, Huanxin

2018-03-01

To investigate the effects of different decontamination treatments on microstructure of titanium (Ti) surface as well as proliferation and adhesion of human gingival fibroblasts (HGFs). Ti discs with machined (M) and sand blasted, acid etched (SAE) surfaces were treated with five different decontamination treatments: (1) stainless steel curette (SSC), ultrasonic system with (2) straight carbon fiber tip (UCF) or (3) metal tip (UM), (4) rotating Ti brush (RTB), and (5) Er:YAG laser (30 mJ/pulse at 30 Hz). Surface roughness was analyzed under optical interferometry. HGFs were cultured on each disc. Proliferation and adhesive strength were analyzed. qRT-PCR and ELISA were performed to detect the RNA and protein expression of FAK, ITGB1, COL1A1, and FN1 respectively from different Ti surfaces. Surface roughness increased on M surface. Proliferation, adhesive strength and gene expression were higher on M surface than SAE surface. Decontamination treatments affected surface parameters significantly (P < 0.001), making M surface less smooth while SAE surface became less rough. SSC, UCF, UM and RTB decreased proliferation on M surfaces significantly (P < 0.05). UCF, RTB and laser increased proliferation on SAE surface significantly (P < 0.05). UM decreased adhesive strength on M surface significantly and laser increased adhesive strength on SAE surface significantly (P < 0.05). Gene expression increased with time and was altered by decontamination treatments significantly (P < 0.001). Decontamination treatments influence surface roughness and cell behavior of HGFs. Laser might be an optimal decontamination treatment which has the least negative effect on M surface and the most positive effect on SAE surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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.

Cellular response of pulp fibroblast to single or multiple photobiomodulation applications

Science.gov (United States)

Fernandes, Amanda; Lourenço Neto, Natalino; Teixeira Marques, Nadia Carolina; Lourenço Ribeiro Vitor, Luciana; Tavares Oliveira Prado, Mariel; Cardoso Oliveira, Rodrigo; Moreira Machado, Maria Aparecida Andrade; Marchini Oliveira, Thais

2018-06-01

This study aimed to evaluate in vitro the effects of single or multiple photobiomodulation (PBM) applications on the viability and proliferation of pulp fibroblasts. Pulp fibroblasts from human deciduous teeth were obtained from a biorepository, plated into 96-well plates, and irradiated according to the experimental groups. At 24 h, 48 h, and 72 h after irradiation, cell viability and proliferation were assessed through MTT and Crystal Violet assays, respectively. The intragroup comparison revealed statistically significant differences for 2.5 J cm‑2 (3×) with increasing viability at 72 h over 48 h (p  =  0.027). The intergroup analysis showed a greater viability of the multiple PBM applications 2.5 J cm‑2 (3×) over the single application 7.5 J cm‑2 (1×) at 72 h. The application of 5 J cm‑2 (1×) exhibited greater proliferation than the application of 7.5 J cm‑2 (1×), 2.5 J cm‑2 (2×) and 2.5 J cm‑2 (3×). Single or multiple PBM applications demonstration different stimulatory effects on pulp fibroblast. The results show that the group submitted to multiple irradiation presented significantly higher cell viability than the groups with single irradiation at 72 h. However, the photobiomodulation therapy with single irradiations was more effective on cell proliferation at 24 h.

IL1β-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

International Nuclear Information System (INIS)

Zhu, Yingting; Zhu, Min; Lance, Peter

2012-01-01

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1β in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1β, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1β. Further analysis indicated that the major COX-2 product, prostaglandin E 2 , directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1β. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1β in the fibroblasts.

FRS2α is Essential for the Fibroblast Growth Factor to Regulate the mTOR Pathway and Autophagy in Mouse Embryonic Fibroblasts

OpenAIRE

Xiang Lin, Yongyou Zhang, Leyuan Liu, Wallace L. McKeehan, Yuemao Shen, Siyang Song, Fen Wang

2011-01-01

Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/A...

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

International Nuclear Information System (INIS)

Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B.; Altran, Silvana C.; Isaac, Cesar

2011-01-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

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Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

2011-07-01

Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

ZnO nanorod–templated well-aligned ZrO2 nanotube arrays for fibroblast adhesion and proliferation

International Nuclear Information System (INIS)

Lu, Zhisong; Hu, Weihua; Ming Li, Chang; Zhu, Zhihong; Liu, Jinping

2014-01-01

Cellular responses to porous tubular structures have recently been investigated in highly ordered ZrO 2 nanotube arrays fabricated with anodization. However, the potential applications of the nanotube arrays are hindered by instrument requirements and substrate limitations, as well as by the complicated processes needed for synthesis. In this work, ZrO 2 nanotube arrays were synthesized by in situ hydrolysis of zirconium propoxide with a zinc oxide nanorod array–based template. Fibroblast cells were able to grow on the nanotube array surface with produced elongated filopodia. Studies of the capability of cell growth and the expression of adhesion- and proliferation-related genes reveal that ZrO 2 nanotube arrays may provide a better environment for cell adhesion and growth than a flat titanium surface. These findings not only provide fundamental insight into cell response to nanostructures but also provide an opportunity to use a unique approach to fabricate ZrO 2 nanotube array structures for potential implant applications. (papers)

Pentadecapeptide BPC 157 Enhances the Growth Hormone Receptor Expression in Tendon Fibroblasts

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Chung-Hsun Chang

2014-11-01

Full Text Available BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

Mutagenesis and lethality following S phase irradiation of xeroderma pigmentosum and normal human diploid fibroblasts with ultraviolet light

International Nuclear Information System (INIS)

Grosovsky, A.J.; Little, J.B.

1983-01-01

The mutagenic and lethal effects of u.v. light exposure in the DNA synthetic phase of the cell cycle were determined in xeroderma pigmentosum complementation group A (XP-A), hereditary adenomatosis of the colon and rectum (ACR), and a normal, foreskin derived cell strain (AG1522). For AG1522, an increased sensitivity to the cytotoxic effects of u.v. light was observed as compared to previous findings for confluent, non-proliferating cultures. XP-A fibroblasts were markedly hypersensitive and ACR fibroblasts exhibited an intermediate response. The mutagenic response of ACR fibroblasts, however, was similar to normal fibroblasts. A threshold of 1.5-2 J/m 2 was observed for u.v. induced mutagenesis in normal and ACR fibroblasts. XP fibroblasts, on the other hand, were strikingly hypermutable and demonstrated little or no threshold. When S phase mutagenesis was considered as a function of survival level rather than u.v. light dose, XP fibroblasts remained significantly hypermutable as compared with normal fibroblasts at all survival levels. Previous mutagenesis results with confluent, non-proliferating cultures of XP and normal fibroblasts were reanalyzed as a function of cytotoxicity; XP hypermutability at all survival levels was also observed. (author)

Evaluation of monoclonal antibodies for the development of breast cancer immunotoxins

International Nuclear Information System (INIS)

Bjorn, M.J.; Ring, D.; Frankel, A.

1985-01-01

Eighty-five antibodies recognizing breast cancer-selective antigens were conjugated to ricin toxin A-chain using a disulfide linkage. The cytotoxicities of the resulting immunotoxins were determined on breast cancer cells and normal human fibroblasts. Twenty-four antibodies formed immunotoxins that were toxic to at least one breast cancer cell line at concentrations of 10 nM or less but were nontoxic to human fibroblast lines used as negative controls. Some of the breast tumor-selective immunotoxins were as toxic as a conjugate between monoclonal anti-transferrin receptor and ricin toxin A-chain (50% inhibition of cellular protein synthesis at approximately 0.1 nM). Another set of four immunotoxins were indiscriminately toxic to human breast tumor cell lines, two human fibroblast cell lines, and a human lymphoblastoid line. Several of the antibodies the toxin conjugates of which specifically killed breast cancer cell lines may be useful in cancer therapy, since they show a wide range of binding to individual breast tumors and cell lines and a limited range of binding to normal tissue types

Scleral fibroblast response to experimental glaucoma in mice

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Tezel, Gülgün; Cone-Kimball, Elizabeth; Steinhart, Matthew R.; Jefferys, Joan; Pease, Mary E.; Quigley, Harry A.

2016-01-01

Purpose To study the detailed cellular and molecular changes in the mouse sclera subjected to experimental glaucoma. Methods Three strains of mice underwent experimental bead-injection glaucoma and were euthanized at 3 days and 1, 3, and 6 weeks. Scleral protein expression was analyzed with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using 16O/18O labeling for quantification in 1- and 6-week tissues. Sclera protein samples were also analyzed with immunoblotting with specific antibodies to selected proteins. The proportion of proliferating scleral fibroblasts was quantified with Ki67 and 4’,6-diamidino-2-phenylindole (DAPI) labeling, and selected proteins were studied with immunohistochemistry. Results Proteomic analysis showed increases in molecules involved in integrin-linked kinase signaling and actin cytoskeleton signaling pathways at 1 and 6 weeks after experimental glaucoma. The peripapillary scleral region had more fibroblasts than equatorial sclera (p=0.001, n=217, multivariable regression models). There was a sixfold increase in proliferating fibroblasts in the experimental glaucoma sclera at 1 week and a threefold rise at 3 and 6 weeks (p=0.0005, univariate regression). Immunoblots confirmed increases for myosin, spectrin, and actinin at 1 week after glaucoma. Thrombospondin-1 (TSP-1), HINT1, vimentin, actinin, and α-smooth muscle actin were increased according to immunohistochemistry. Conclusions Scleral fibroblasts in experimental mouse glaucoma show increases in actin cytoskeleton and integrin-related signaling, increases in cell division, and features compatible with myofibroblast transition. PMID:26900327

Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

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Wei, Hongbo; Zhao, Lingzhou; Chen, Bangdao; Bai, Shizhu; Zhao, Yimin

2014-01-01

Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant. PMID:25054322

Improved Fibroblast Functionalities by Microporous Pattern Fabricated by Microelectromechanical Systems

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Hongbo Wei

2014-07-01

Full Text Available Fibroblasts, which play an important role in biological seal formation and maintenance, determine the long-term success of percutaneous implants. In this study, well-defined microporous structures with micropore diameters of 10–60 µm were fabricated by microelectromechanical systems and their influence on the fibroblast functionalities was observed. The results show that the microporous structures with micropore diameters of 10–60 µm did not influence the initial adherent fibroblast number; however, those with diameters of 40 and 50 µm improved the spread, actin stress fiber organization, proliferation and fibronectin secretion of the fibroblasts. The microporous structures with micropore diameters of 40–50 µm may be promising for application in the percutaneous part of an implant.

Heart Development, Diseases, and Regeneration　- New Approaches From Innervation, Fibroblasts, and Reprogramming.

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Ieda, Masaki

2016-09-23

It is well known that cardiac function is tightly controlled by neural activity; however, the molecular mechanism of cardiac innervation during development and the relationship with heart disease remain undetermined. My work has revealed the molecular networks that govern cardiac innervation and its critical roles in heart diseases such as silent myocardial ischemia and arrhythmias. Cardiomyocytes proliferate during embryonic development, but lose their proliferative capacity after birth. Cardiac fibroblasts are a major source of cells during fibrosis and induce cardiac hypertrophy after myocardial injury in the adult heart. Despite the importance of fibroblasts in the adult heart, the role of fibroblasts in embryonic heart development was previously not determined. I demonstrated that cardiac fibroblasts play important roles in myocardial growth and cardiomyocyte proliferation during embryonic development, and I identified key paracrine factors and signaling pathways. In contrast to embryonic cardiomyocytes, adult cardiomyocytes have little regenerative capacity, leading to heart failure and high mortality rates after myocardial infarction. Leveraging the knowledge of developmental biology, I identified cardiac reprogramming factors that can directly convert resident cardiac fibroblasts into cardiomyocytes for heart regeneration. These findings greatly improved our understanding of heart development and diseases, and provide a new strategy for heart regenerative therapy. (Circ J 2016; 80: 2081-2088).

Effect of 660 nm Light-Emitting Diode on the Wound Healing in Fibroblast-Like Cell Lines

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Myung-Sun Kim

2015-01-01

Full Text Available Light in the red to near-infrared (NIR range (630–1000 nm, which is generated using low energy laser or light-emitting diode (LED arrays, was reported to have a range of beneficial biological effects in many injury models. NIR via a LED is a well-accepted therapeutic tool for the treatment of infected, ischemic, and hypoxic wounds as well as other soft tissue injuries in humans and animals. This study examined the effects of exposure to 660 nm red LED light at intensities of 2.5, 5.5, and 8.5 mW/cm2 for 5, 10, and 20 min on wound healing and proliferation in fibroblast-like cells, such as L929 mouse fibroblasts and human gingival fibroblasts (HGF-1. A photo illumination-cell culture system was designed to evaluate the cell proliferation and wound healing of fibroblast-like cells exposed to 600 nm LED light. The cell proliferation was evaluated by MTT assay, and a scratched wound assay was performed to assess the rate of migrating cells and the healing effect. Exposure to the 660 nm red LED resulted in an increase in cell proliferation and migration compared to the control, indicating its potential use as a phototherapeutic agent.

Botulinum Toxin Type A Inhibits α-Smooth Muscle Actin and Myosin II Expression in Fibroblasts Derived From Scar Contracture.

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Chen, Minliang; Yan, Tongtong; Ma, Kui; Lai, Linying; Liu, Chang; Liang, Liming; Fu, Xiaobing

2016-09-01

Scar contracture (SC) is one of the most common complications resulting from major burn injuries. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Recent reports suggest that botulinum toxin type A (BTXA) is effective at reducing SC clinically, but the molecular mechanism for this action is unknown. α-Smooth muscle actin (α-SMA) and myosin II are the main components of stress fibers, which are the contractile structures of fibroblasts. The effects of BTXA on α-SMA and myosin II in SC are still unknown. This study aimed to explore the effect of BTXA on α-SMA and myosin II expression in fibroblasts derived from SC and to elucidate its actual mechanism further. Fibroblasts were isolated from tissue specimens of SC. Fibroblasts were cultured in Dulbecco modified Eagle medium with different concentrations of BTXA and their proliferation was analyzed through the tetrazolium-based colorimetric method at 1, 4, and 7 days. Proteins of α-SMA and myosin II were checked using Western blot in fibroblasts treated with different concentrations of BTXA at 1, 4, and 7 days. Fibroblasts without BTXA treatment had a higher proliferation than that in other groups, which indicated that the proliferation of fibroblasts was significantly inhibited by BTXA (P < 0.05). Proteins of α-SMA and myosin II between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (P < 0.05). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from SC and reduced the expression of α-SMA and myosin II, which provided theoretical support for the application of BTXA to control SC.

Factor XIIIa is expressed by fibroblasts in fibrovascular tumors.

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Nemeth, A J; Penneys, N S

1989-10-01

Factor XIIIa (FXIIIa), a blood and intracellularly produced coagulation factor, has been found in a variety of cell types including fibroblast-like mesenchymal cells, and has been shown to stimulate the proliferation of fibroblasts and some neoplastic cells in vitro. We have already shown that the dendritic fibroblasts composing the fibrous papule contain this factor. We hypothesized that histopathologically similar fibrovascular tumors may also express FXIIIa and, in this report, show that the large stellate fibroblasts found in acquired digital fibrokeratomas, angiofibromas (adenoma sebaceum of Pringle), and oral fibroma (oral fibrous hyperplasia) also express FXIIIa. We postulate that FXIIIa, possibly acting as a growth factor, may be a common denominator in the pathogenesis of these tumors. Another possibility is that these tumors may be the consequence of a local overproduction of FXIIIa in response to an, as yet, unidentified stimulus.

Responses of fibroblasts and glial cells to nanostructured platinum surfaces

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Pennisi, C P; Sevcencu, C; Yoshida, K [Center for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg (Denmark); Dolatshahi-Pirouz, A; Foss, M; Larsen, A Nylandsted; Besenbacher, F [Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus (Denmark); Hansen, J Lundsgaard [Department of Physics and Astronomy, Aarhus University, Aarhus (Denmark); Zachar, V, E-mail: cpennisi@hst.aau.d [Laboratory for Stem Cell Research, Aalborg University (Denmark)

2009-09-23

The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

Differential gene expression in human fibroblasts after alpha-particle emitter (211)At compared with (60)Co irradiation

DEFF Research Database (Denmark)

Danielsson, Anna; Claesson, Kristina; Parris, Toshima Z

2013-01-01

trastuzumab monoclonal antibody (0.25, 0.5, and 1 Gy) and (60)Co (1, 2, and 3 Gy). Results: We report gene expression profiles that distinguish the effect different radiation qualities and absorbed doses have on cellular functions in human fibroblasts. In addition, we identified commonly expressed transcripts...

Potensi Terapeutik Fibroblast Growth Factor 21 terhadap Resistensi Insulin

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Kurniasari Kurniasari

2015-12-01

Full Text Available Fibroblast growth factor 21 (FGF21 merupakan salah satu dari anggota FGF yang berperansebagai faktor endokrin. Hepar dan jaringan adiposa merupakan tempat kerja utama FGF21.Ekspresi FGF21 di hepar diatur oleh peroxisome proliferator activated receptor alpha (PPARαsedangkan di jaringan adiposa diatur oleh peroxisome proliferator activated receptor gamma(PPARγ. Kedua faktor transkripsi tersebut terlibat dalam metabolisme karbohidrat dan lipid. Padaresistensi insulin terdapat hiperglikemia, hiperinsulinemia, dan dislipidemia. Pemberian FGF21pada berbagai studi in vivo dan in vitro telah menunjukan potensi FGF21 dalam mengatasi kelainanakibat resistensi insulin sekaligus meningkatkan sensitivitas jaringan terhadap insulin. Kata kunci: FGF21, PPARγ, PPARα, resistensi insulin Fibroblast Growth Factor 21 (FGF21 Potension in InsulinResistance Treatment Abstract Fibroblast growth factor 21 (FGF21 is a member of FGF family that plays a role as endocrinefactor. Liver and adipose tissue are major target of FGF21. The expression of FGF21 in liveris regulated by peroxisome proliferator activated receptor alpha (PPARα, while peroxisomeproliferator activated receptor gamma (PPARγ regulate FGF21 expression in adipose tissue.Both transcription factors are involved in carbohydrate and lipid metabolism. Hyperglycemia,hyperinsulinemia, and dyslipidemia are observed in insulin resistance. Treatment with FGF21 inin vitro and in vivo study showed that FGF21 have the potential to overcome insulin resistance aswell as increasing tissue’s sensitivity towards insulin. Keywords: FGF21, PPARγ, PPARα, insulin resistance Normal 0 false false false IN X-NONE X-NONE

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

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Hoban PR

2006-06-01

Full Text Available Abstract Background Idiopathic Pulmonary Fibrosis (IPF is a debilitating disease characterized by exaggerated extracellular matrix deposition and aggressive lung structural remodeling. Disease pathogenesis is driven by fibroblastic foci formation, consequent on growth factor overexpression and myofibroblast proliferation. We have previously shown that both CTGF overexpression and myofibroblast formation in IPF cell lines are dependent on RhoA signaling. As RhoA-mediated regulation is also involved in cell cycle progression, we hypothesise that this pathway is key to lung fibroblast turnover through modulation of cyclin D1 kinetic expression. Methods Cyclin D1 expression was compared in primary IPF patient-derived fibroblasts and equivalent normal control cells. Quantitative real time PCR was employed to examine relative expression levels of cyclin D1 mRNA; protein expression was confirmed by western blotting. Effects of Rho signaling were investigated using transient transfection of constitutively active and dominant negative RhoA constructs as well as pharmacological inhibitors. Cellular proliferation of lung fibroblasts was determined by BrdU incorporation ELISA. To further explore RhoA regulation of cyclin D1 in lung fibroblasts and associated cell cycle progression, an established Rho inhibitor, Simvastatin, was incorporated in our studies. Results Cyclin D1 expression was upregulated in IPF compared to normal lung fibroblasts under exponential growth conditions (p Conclusion These findings report for the first time that cyclin D1 expression is deregulated in IPF through a RhoA dependent mechanism that influences lung fibroblast proliferation. This potentially unravels new molecular targets for future anti-IPF strategies; accordingly, Simvastatin inhibition of Rho-mediated cyclin D1 expression in IPF fibroblasts merits further exploitation.

Decreased Fibroblast and Increased Osteoblast Functions on Ionic Plasma Deposited Nanostructured Ti Coatings

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Storey Dan

2007-01-01

Full Text Available AbstractBioactive coatings are in high demand to control cellular functions for numerous medical devices. The objective of this in vitro study was to characterize for the first time fibroblast (fibrous scar tissue forming cells adhesion and proliferation on an important polymeric biomaterial (silicone coated with titanium using a novel ionic plasma deposition (IPD process. Fibroblasts are one of the first anchorage-dependent cells to arrive at an implant surface during the wound healing process. Persistent excessive functions of fibroblasts have been linked to detrimental fibrous tissue formation which may cause implant failure. The IPD process creates a surface-engineered nanostructure (with features usually below 100 nm by first using a vacuum to remove all contaminants, then guiding charged metallic ions or plasma to the surface of a medical device at ambient temperature. Results demonstrated that compared to currently used titanium and uncoated silicone, silicone coated with titanium using IPD significantly decreased fibroblast adhesion and proliferation. Results also showed competitively increased osteoblast (bone-forming cells over fibroblast adhesion on silicone coated with titanium; in contrast, osteoblast adhesion was not competitively increased over fibroblast adhesion on uncoated silicone or titanium controls. In this manner, this study strongly suggests that IPD should be further studied for biomaterial applications in which fibrous tissue encapsulation is undesirable (such as for orthopedic implants, cardiovascular components, etc..

Anti-fibrotic effects of theophylline on lung fibroblasts

International Nuclear Information System (INIS)

Yano, Yukihiro; Yoshida, Mitsuhiro; Hoshino, Shigenori; Inoue, Koji; Kida, Hiroshi; Yanagita, Masahiko; Takimoto, Takayuki; Hirata, Haruhiko; Kijima, Takashi; Kumagai, Toru; Osaki, Tadashi; Tachibana, Isao; Kawase, Ichiro

2006-01-01

Theophylline has been used in the management of bronchial asthma and chronic obstructive pulmonary disease for over 50 years. It has not only a bronchodilating effect, but also an anti-inflammatory one conducive to the inhibition of airway remodeling, including subepithelial fibrosis. To date however, whether theophylline has a direct inhibitory effect on airway fibrosis has not been established. To clarify this question, we examined whether theophylline affected the function of lung fibroblasts. Theophylline suppressed TGF-β-induced type I collagen (COL1) mRNA expression in lung fibroblasts and also inhibited fibroblast proliferation stimulated by FBS and TGF-β-induced α-SMA protein. A cAMP analog also inhibited TGF-β-induced COL1 mRNA expression in lung fibroblasts. A PKA inhibitor reduced the inhibitory effect of theophylline on TGF-β-induced COL1 mRNA expression. These results indicate that theophylline exerts anti-fibrotic effects, at least partly, through the cAMP-PKA pathway

Fibroblasts in fibrosis: novel roles and mediators

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Ryan Thomas Kendall

2014-05-01

Full Text Available Fibroblasts are the most common cell type of the connective tissues found throughout the body and the principal source of the extensive extracellular matrix (ECM characteristic of these tissues. They are also the central mediators of the pathological fibrotic accumulation of ECM and the cellular proliferation and differentiation that occurs in response to prolonged tissue injury and chronic inflammation. The transformation of the fibroblast cell lineage involves classical developmental signaling programs and includes a surprisingly diverse range of precursor cell types—most notably, myofibroblasts that are the apex of the fibrotic phenotype. Myofibroblasts display exaggerated ECM production; constitutively secrete and are hypersensitive to chemical signals such as cytokines, chemokines, and growth factors; and are endowed with a contractile apparatus allowing them to manipulate the ECM fibers physically to close open wounds. In addition to ECM production, fibroblasts have multiple concomitant biological roles, such as in wound healing, inflammation, and angiogenesis, which are each interwoven with the process of fibrosis. We now recognize many common fibroblast-related features across various physiological and pathological protracted processes. Indeed, a new appreciation has emerged for the role of noncancerous fibroblast interactions with tumors in cancer progression. Although the predominant current clinical treatments of fibrosis involve nonspecific immunosuppressive and anti-proliferative drugs, a variety of potential therapies under investigation specifically target fibroblast biology.

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

Science.gov (United States)

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.

Quantification of epithelial cells in coculture with fibroblasts by fluorescence image analysis.

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Krtolica, Ana; Ortiz de Solorzano, Carlos; Lockett, Stephen; Campisi, Judith

2002-10-01

To demonstrate that senescent fibroblasts stimulate the proliferation and neoplastic transformation of premalignant epithelial cells (Krtolica et al.: Proc Natl Acad Sci USA 98:12072-12077, 2001), we developed methods to quantify the proliferation of epithelial cells cocultured with fibroblasts. We stained epithelial-fibroblast cocultures with the fluorescent DNA-intercalating dye 4,6-diamidino-2-phenylindole (DAPI), or expressed green fluorescent protein (GFP) in the epithelial cells, and then cultured them with fibroblasts. The cocultures were photographed under an inverted microscope with appropriate filters, and the fluorescent images were captured with a digital camera. We modified an image analysis program to selectively recognize the smaller, more intensely fluorescent epithelial cell nuclei in DAPI-stained cultures and used the program to quantify areas with DAPI fluorescence generated by epithelial nuclei or GFP fluorescence generated by epithelial cells in each field. Analysis of the image areas with DAPI and GFP fluorescences produced nearly identical quantification of epithelial cells in coculture with fibroblasts. We confirmed these results by manual counting. In addition, GFP labeling permitted kinetic studies of the same coculture over multiple time points. The image analysis-based quantification method we describe here is an easy and reliable way to monitor cells in coculture and should be useful for a variety of cell biological studies. Copyright 2002 Wiley-Liss, Inc.

Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

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S Montagnani

2009-12-01

Full Text Available Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

Effects of ascorbate and B-aminopropionitrile on tendon fibroblast migration in vitro

International Nuclear Information System (INIS)

Nelson, J.M.; Cohen, I.K.; Diegelmann, R.F.

1986-01-01

Ascorbate (Asc) stimulates collagen synthesis and hydroxylation whereas beta-aminopropionitrile (BAPN) inhibits collagen cross-link formation. In this study, an in vitro model employing isolated chicken tendon biopsies (2 mm in dia.) in a fibrin clot has been used to examine the effect of Asc and BAPN on tendon fibroblast migration and proliferation. After 5 days in culture with either Asc (0.1 mM), or BAPN (0.5, 1.0, 2.0 mM), cell migration was measured using an automatic planimeter and cell proliferation was quantitated by 125 IUDR incorporation into DNA. In cultures treated with Asc alone, cell migration was enhanced by approximately 33% compared to controls without ascorbate (5.9 mm 2 vs. 4.4 mm 2 ; p < 0.05) with no significant effect on cell proliferation. In contrast, cultures incubated with increasing concentrations of BAPN (0.5 - 2.0 mM) displayed a dose-dependent decrease (up to 9.6-fold at 2 mM) in fibroblast migration into the clot. The inhibitory effect of BAPN on cell migration was not due to a corresponding inhibition of fibroblast proliferation. These observations suggest that Asc enhanced collagen formation and thus allowed greater cell migration into the fibrin matrix. In contrast, in the absence of a mature, cross-linked collagen matrix following exposure to BAPN, cell migration was sub-optimal. These in vitro studies support the hypothesis that modulation of the collagen matrix may be a useful means of regulating tissue repair in vivo

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

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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.

Effect of triptolide on proliferation and apoptosis of angiotensin II ...

African Journals Online (AJOL)

Background: The effect of triptolide (TPL) on cardiac fibroblasts (CFbs) and cardiac fibrosis remain unknown till now. This study was conducted to explore the effects of TPL on proliferation and apoptosis of angiotensin II (Ang II)-induced CFbs. Materials and Methods: Ang II was used to promote proliferation of CFbs.

In vitro blood and fibroblast responses to BisGMA-TEGDMA/bioactive glass composite implants.

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Abdulmajeed, Aous A; Kokkari, Anne K; Käpylä, Jarmo; Massera, Jonathan; Hupa, Leena; Vallittu, Pekka K; Närhi, Timo O

2014-01-01

This in vitro study was designed to evaluate both blood and human gingival fibroblast responses to bisphenol A-glycidyl methacrylate-triethyleneglycol dimethacrylate (BisGMA-TEGDMA)/bioactive glass (BAG) composite, aimed to be used as composite implant abutment surface modifier. Three different types of substrates were investigated: (a) plain polymer (BisGMA 50 wt%-TEGDMA 50 wt%), (b) BAG-composite (50 wt% polymer + 50 wt% fraction of BAG-particles, <50 μm), and (c) plain BAG plates (100 wt% BAG). The blood response, including the blood-clotting ability and platelet adhesion morphology were evaluated. Human gingival fibroblasts were plated and cultured on the experimental substrates for up to 10 days, then the cell proliferation rate was assessed using AlamarBlue assay™. The BAG-composite and plain BAG substrates had a shorter clotting time than plain polymer substrates. Platelet activation and aggregation were most extensive, qualitatively, on BAG-composite. Analysis of the normalized cell proliferation rate on the different surfaces showed some variations throughout the experiment, however, by day 10 the BAG-composite substrate showed the highest (P < 0.001) cell proliferation rate. In conclusion, the presence of exposed BAG-particles enhances fibroblast and blood responses on composite surfaces in vitro.

A theoretical investigation of the effect of proliferation and adhesion on monoclonal conversion in the colonic crypt

KAUST Repository

Mirams, Gary R.

2012-11-01

The surface epithelium lining the intestinal tract renews itself rapidly by a coordinated programme of cell proliferation, migration and differentiation events that is initiated in the crypts of Lieberkühn. It is generally believed that colorectal cancer arises due to mutations that disrupt the normal cellular dynamics of the crypts. Using a spatially structured cell-based model of a colonic crypt, we investigate the likelihood that the progeny of a mutated cell will dominate, or be sloughed out of, a crypt. Our approach is to perform multiple simulations, varying the spatial location of the initial mutation, and the proliferative and adhesive properties of the mutant cells, to obtain statistical distributions for the probability of their domination. Our simulations lead us to make a number of predictions. The process of monoclonal conversion always occurs, and does not require that the cell which initially gave rise to the population remains in the crypt. Mutations occurring more than one to two cells from the base of the crypt are unlikely to become the dominant clone. The probability of a mutant clone persisting in the crypt is sensitive to dysregulation of adhesion. By comparing simulation results with those from a simple one-dimensional stochastic model of population dynamics at the base of the crypt, we infer that this sensitivity is due to direct competition between wild-type and mutant cells at the base of the crypt. We also predict that increases in the extent of the spatial domain in which the mutant cells proliferate can give rise to counter-intuitive, non-linear changes to the probability of their fixation, due to effects that cannot be captured in simpler models. © 2012 Elsevier Ltd.

Neuropeptide substance P stimulates the formation of osteoclasts via synovial fibroblastic cells

International Nuclear Information System (INIS)

Matayoshi, Takaaki; Goto, Tetsuya; Fukuhara, Eiji; Takano, Hiroshi; Kobayashi, Shigeru; Takahashi, Tetsu

2005-01-01

The present study was designed to evaluate the effects of neuropeptide substance P (Sp) on the formation of osteoclasts via synovial fibroblastic cells. Synovial fibroblastic cells derived from rat knee joint expressed the Sp receptor, neurokinin-1 receptor (NK 1 -R). The addition of Sp stimulated the proliferation of synovial fibroblastic cells and this effect was inhibited by Sp or NK 1 -R antagonists. Increased expression of the receptor activator of nuclear factor κB ligand (Rankle) in synovial fibroblastic cells after the addition of Sp was demonstrated by reverse transcriptase-polymerase chain reaction and immunofluorescence staining. Osteoprotegerin expression in synovial fibroblastic cells was decreased after incubation with SP. In co-cultures of synovial fibroblastic cells and rat peripheral blood monocytes, SP stimulated osteoclastogenesis. These results suggest that SP in the joint cavity may cause both hypertrophy of the synovium and induction of increased osteoclast formation through the increased expression of RANKL in the synovium

Kaempferol inhibits fibroblast collagen synthesis, proliferation and activation in hypertrophic scar via targeting TGF-β receptor type I.

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Li, Hongwei; Yang, Liu; Zhang, Yuebing; Gao, Zhigang

2016-10-01

Hypertrophic scar (HPS) formation is a debilitating condition that results in pain, esthetic symptom and loss of tissue function. So far, no satisfactory therapeutic approach has been available for HPS treatment. In this study, we discovered that a natural small molecule, kaempferol, could significantly inhibit HPS formation in a mechanical load-induced mouse model. Our results also demonstrated that kaempferol remarkably attenuated collagen synthesis, proliferation and activation of fibroblasts in vitro and in vivo. Western blot analysis further revealed that kaempferol significantly down-regulated Smad2 and Smad3 phosphorylation in a dose-dependent manner. At last, we found that such bioactivity of kaempferol which resulted from the inhibition of TGF-β1/Smads signaling was induced by the selective binding of kaempferol to TGF-β receptor type I (TGFβRI). These findings suggest that kaempferol could be developed into a promising agent for the treatment of HPS or other fibroproliferative disorders. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Total glucosides of paeony inhibit the proliferation of fibroblast-like synoviocytes through the regulation of G proteins in rats with collagen-induced arthritis.

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Jia, Xiao-Yi; Chang, Yan; Sun, Xiao-Jing; Wu, Hua-Xun; Wang, Chun; Xu, Hong-Mei; Zhang, Lei; Zhang, Ling-Ling; Zheng, Yong-Qiu; Song, Li-Hua; Wei, Wei

2014-01-01

The aim of this study was to investigate the expression of G proteins in fibroblast-like synoviocytes (FLSs) from rats with collagen-induced arthritis (CIA) and to determine the effect of total glucosides of paeony (TGP). CIA rats were induced with chicken type II collagen (CCII) in Freund's complete adjuvant. The rats with experimental arthritis were randomly separated into five groups and then treated with TGP (25, 50, and 100mg/kg) from days 14 to 35 after immunization. The secondary inflammatory reactions were evaluated through the polyarthritis index and histopathological changes. The level of cyclic adenosine monophosphate (cAMP) was measured by radioimmunoassay. The FLS proliferation response was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The toxin-catalyzed ADP-ribosylation of G proteins was performed through autoradiography. The results show that TGP (25, 50, and 100mg/kg) significantly decreased the arthritis scores of CIA rats and improved the histopathological changes. TGP inhibited the proliferation of FLSs and increased the level of cAMP. Moreover, the FLS proliferation and the level of Gαi expression were significantly increased, but the level of Gαs expression was decreased after stimulation with IL-1β (10ng/ml) in vitro. TGP (12.5 and 62.5μg/ml) significantly inhibited the FLS proliferation and regulated the balance between Gαi and Gαs. These results demonstrate that TGP may exert its anti-inflammatory effects through the suppression of FLS proliferation, which may be associated with its ability to regulate the balance of G proteins. Thus, TGP may have potential as a therapeutic agent for the treatment of rheumatoid arthritis. © 2013.

Involvement of H- and N-Ras isoforms in transforming growth factor-β1-induced proliferation and in collagen and fibronectin synthesis

International Nuclear Information System (INIS)

Martinez-Salgado, Carlos; Fuentes-Calvo, Isabel; Garcia-Cenador, Begona; Santos, Eugenio; Lopez-Novoa, Jose M.

2006-01-01

Transforming growth factor β1 (TGF-β1) has a relevant role in the origin and maintenance of glomerulosclerosis and tubule-interstitial fibrosis. TGF-β and Ras signaling pathways are closely related: TGF-β1 overcomes Ras mitogenic effects and Ras counteracts TGF-β signaling. Tubule-interstitial fibrosis is associated to increases in Ras, Erk, and Akt activation in a renal fibrosis model. We study the role of N- and H-Ras isoforms, and the involvement of the Ras effectors Erk and Akt, in TGF-β1-mediated extracellular matrix (ECM) synthesis and proliferation, using embrionary fibroblasts from double knockout (KO) mice for H- and N-Ras (H-ras -/- /N-ras -/- ) isoforms and from heterozygote mice (H-ras +/- /N-ras +/- ). ECM synthesis is increased in basal conditions in H-ras -/- /N-ras -/- fibroblasts, this increase being higher after stimulation with TGF-β1. TGF-β1-induced fibroblast proliferation is smaller in H-ras -/- /N-ras -/- than in H-ras +/- /N-ras +/- fibroblasts. Erk activation is decreased in H-ras -/- /N-ras -/- fibroblasts; inhibition of Erk activation reduces fibroblast proliferation. Akt activation is higher in double KO fibroblasts than in heterozygotes; inhibition of Akt activation also inhibits ECM synthesis. We suggest that H- and N-Ras isoforms downregulate ECM synthesis, and mediate proliferation, in part through MEK/Erk activation. PI3K-Akt pathway activation may be involved in the increase in ECM synthesis observed in the absence of H- and N-Ras

Agminated Fibroblastic Conective Tissue Nevus: A New Clinical Presentation.

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Downey, Camila; Requena, Luis; Bagué, Silvia; Sánchez Martínez, Miquel Ángel; Lloreta, Josep; Baselga, Eulalia

2016-07-01

Connective tissue nevi are benign hamartomatous lesions in which one or several of the components of the dermis (collagen, elastin, glicosaminoglycans) show predominance or depletion. Recently, de Feraudy et al broadened the spectrum of connective tissue nevus, describing fibroblastic connective tissue nevus (FCTN), which is characterized by proliferation of CD34(+) cells of fibroblastic and myofibroblastic lineage. Only solitary papules and nodules have been described. We present the first case of FCTN with multiple agminated lesions on the leg of an infant and the difficulties encountered in the differential diagnosis with dermatofibrosarcoma protuberans. © 2016 Wiley Periodicals, Inc.

Toll-like receptor 9 mediated responses in cardiac fibroblasts.

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Ingrid Kristine Ohm

Full Text Available Altered cardiac Toll-like receptor 9 (TLR9 signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β. Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088 or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

Differences between Solution and Membrane Forms of Chitosan on the In Vitro Activity of Fibroblasts

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Bahar Uslu

2015-03-01

Full Text Available Background: Chitosan, a linear polysaccharide, has been recently used in biomedical applications. In vitro studies have demonstrated its effect on cellular growth and its stimulatory action on cellular layer formation. Aims: The present study aims to compare the proliferative effects of chitosan in two forms, membranous and solution forms, on Swiss 3T3 mouse embryonic fibroblasts. Study Design: In vitro study. Methods: Three experimental groups were formed: cells were cultured in a normal medium without chitosan (Control Group; cells were cultured either in a medium containing 2.0% chitosan in membranous form (Membrane Group or chitosan solution at a concentration of 2.0% (Solution Group.Two different methods were used in the experiments: cells cultured on the medium containing chitosan in solution or membranous forms (method 1; and chitosan solution or membranous forms were added into the medium containing previously cultured cells (method 2. Results: Scanning electron microscopic investigations of the experimental groups revealed cells with well-defined cellular projections, intact cellular membranes and tight intercellular junctions. They were especially prominent in the membrane group of method 1 and in the membrane and solution groups of method 2. Mouse monoclonal anti-collagen 1 primary antibody was used to indicate collagen synthesis. Prominent collagen synthesis was detected in the membrane groups on the 10th day of culture for both methods. Bromodeoxyuridine (BrdU and MTT assays were performed in order to assess cellular proliferation and viability, respectively. BrdU labelling tests indicated a higher proliferation index in the membrane group of method 1 on the 5th and 10th days. For the second method, the membranous form on the 10th day and solution form on the 5th day were the most effective groups in terms of cellular proliferation. MTT results reflected a high cellular viability in method 1 on the 5th day of treatment with the

Modeling triple-negative breast cancer heterogeneity: effects of stromal macrophages, fibroblasts and tumor vasculature.

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Norton, Kerri-Ann; Jin, Kideok; Popel, Aleksander S

2018-05-08

A hallmark of breast tumors is its spatial heterogeneity that includes its distribution of cancer stem cells and progenitor cells, but also heterogeneity in the tumor microenvironment. In this study we focus on the contributions of stromal cells, specifically macrophages, fibroblasts, and endothelial cells on tumor progression. We develop a computational model of triple-negative breast cancer based on our previous work and expand it to include macrophage infiltration, fibroblasts, and angiogenesis. In vitro studies have shown that the secretomes of tumor-educated macrophages and fibroblasts increase both the migration and proliferation rates of triple-negative breast cancer cells. In vivo studies also demonstrated that blocking signaling of selected secreted factors inhibits tumor growth and metastasis in mouse xenograft models. We investigate the influences of increased migration and proliferation rates on tumor growth, the effect of the presence on fibroblasts or macrophages on growth and morphology, and the contributions of macrophage infiltration on tumor growth. We find that while the presence of macrophages increases overall tumor growth, the increase in macrophage infiltration does not substantially increase tumor growth and can even stifle tumor growth at excessive rates. Copyright © 2018. Published by Elsevier Ltd.

The role of γ-ray-induced fibroblast apoptosis in inhibiting biliary duct hypertrophic scar formation in dogs

International Nuclear Information System (INIS)

He Guijin; Zhang Hong; Gao Xinyi; Xu Shuhe; Gao Hong; Jiang Weiguo; Jiangtao; Dai Xianwei; Ma Kai

2005-01-01

Objective: To investigate the role of γ-ray-induced fibroblast apoptosis in the inhibition of biliary duct hypertrophic scar formation in dogs. Methods: γ-radiation-induced apoptotic fibroblast cells were analysed by using transmission electron microscopy and DNA from frozen biliary duct tissue was extracted with phenol chloroform. DNA ladder profile after extraction of RNA was observed, and apoptosis cells in paraffinem-bedded biliary duct tissue sections were examined used immuno-histochemical method. Dog biliary duct cross-sections were stained with hematoxylin-erosin, Masson's trichrome, and Verhoeff-van Giesen stains. Muscle formation area, lumen circumference, and stenosis degree were determined by a computer-assisted image analysis system. Results: 103 Pd radioactive stent significantly inhibited fibroblast proliferation. The features of fibroblast apoptosis (e.g, apoptic bodies, DNA ladder band) could be seen in the 103 Pd radioactive stent group. The fibroblast apoptotic rate was significantly increased in the 103 Pd radioactive stent group than in the control group (P 103 Pd radioactive stent significantly reduced biliary muscular formation. Conclusion: 103 Pd radioactive stent could have the effect of inhibiting the proliferation of scar-forming fibroblast, and thus could be used for treatment and (or) prevention of hypertrophic scar formation in biliary duct. (authors)

Doubling potential of fibroblasts from different species after ionising radiation

International Nuclear Information System (INIS)

Macieira-Coelho, A.; Diatloff, C.; Malaise, E.

1976-01-01

It is stated that whereas chicken fibroblasts invariably die after a certain number of doublings in vitro, and this fact is never altered by chemical or physical agents, mouse fibroblasts invariably acquire spontaneously an infinite growth potential. In the human species fibroblasts never acquire spontaneously the capacity to divide for ever, although they can become permanent cell lines after treatment with certain viruses. This behaviour of fibroblasts in vitro has been attributed to different nutritional requirements. Experiments are described with human and mouse fibroblasts in which it was found that the response to ionising radiation matches the relative tendencies of the fibroblasts to yield permanent cell lines. Irradiation was commenced during the phase of active proliferation. Human fibroblast cultures irradiated with 100 R stopped dividing earlier than the controls, whereas cultures irradiated with 200, 300 and 500 R had the same lifespan as the control cultures. Cultures irradiated with 400 R showed the longest survival. With mouse fibroblasts the growth curves of the irradiated cells were of the same type as in the controls, but recovery occurred earlier. The results indicated that ionising radiation accelerates a natural phenomenon; in cells with a limited growth potential (chicken) it shortens the lifespan, whereas in cells that can acquire an unlimited growth potential (mouse) it accelerates acquisition of this potential; human fibroblasts showed an intermediate response, since ionising radiation neither established the cultures as with mouse cells nor reduced the number of cells produced as with chicken fibroblasts. Possible explanations for the different behaviour of the species are offered. (U.K.)

The intriguing role of fibroblasts and c-Jun in the chemopreventive and therapeutic effect of finasteride on xenograft models of prostate cancer

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Yi-Nong Niu

2016-01-01

Full Text Available In a large clinical trial, finasteride reduced the rate of low-grade prostate cancer (PCa while increasing the incidence of high-grade cancer. Whether finasteride promotes the development of high-grade tumors remains controversial. We demonstrated the role of fibroblasts and c-Jun in chemopreventive and therapeutic effect of finasteride on xenograft models of PCa. LNCaP (PC3 cells or recombinants of cancer cells and fibroblasts were implanted in male athymic nude mice treated with finasteride. Tumor growth, cell proliferation, apoptosis, p-Akt, and p-ERK1/2 were evaluated. In LNCaP (PC3 mono-grafted models, finasteride did not change the tumor growth. In recombinant-grafted models, fibroblasts and c-Jun promoted tumor growth; finasteride induced proliferation of LNCaP cells and repressed PC3 cell apoptosis. When c-Jun was knocked out, fibroblasts and/or finasteride did not promote the tumor growth. Finasteride inhibited p-Akt and p-ERK1/2 in mono-culture cancer cells while stimulating the same signaling molecules in the presence of fibroblasts. Reduced p-Akt and p-ERK1/2 were noted in the presence of c-Jun−/− fibroblasts. Fibroblasts and c-Jun promote PCa growth; finasteride further stimulates tumor growth with promoted proliferation, repressed apoptosis, and up-regulated pro-proliferative molecular pathway in the presence of fibroblasts and c-Jun. Stromal-epithelial interactions play critical roles in finasteride′s therapeutic effects on PCa. Our findings have preliminary implications in using finasteride as a chemopreventive or therapeutic agent for PCa patients.

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

Science.gov (United States)

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.

FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

Science.gov (United States)

Gebarowski, Tomasz; Gebczak, Katarzyna; Wiatrak, Benita; Kulma, Anna; Pelc, Katarzyna; Czuj, Tadeusz; Szopa, Jan; Gasiorowski, Kazimierz

2017-03-01

Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

Monoclonal antibody

International Nuclear Information System (INIS)

Oyamada, Hiyoshimaru

1987-01-01

Some aspects of monoclonal antibodies are described, centering on studies made by the author and those presented at the Second International Conference on Monoclonal Antibody Immunoconjugates for Cancer held in March this year (1987). The history of immuno-nuclear medicine and procedures for producing monoclonal antibodies are briefly outlined. Monoclonal antibodies are immunoglobulins. Here, the structure of IgG, which is used most frequently, is described. An IgG is composed of two antigen binding fragments (Fab) and one crystallizable fragment (Fc). The end portion of a Fab reacts with an antigen. One of the major applications of immuno-nuclear medicine is the diagnosis of cancer. As label nucleides, 131 I and 111 I were selected in most cases in the past while 123 I and 99m Tc are currently used more often. Advantages and disadvantages of this diagnosis method is discussed citing studies presented at the First (1986) and Second (1987) International Conference on Monoclonal Antibody Immunoconjugates for Cancer. The present status of the application of monoclonal antibodies to treatment of cancer is also described. (Nogami, K.)

PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

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Mao-lin HUANG

2014-09-01

Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

DNA repair synthesis in human fibroblasts requires DNA polymerase delta

International Nuclear Information System (INIS)

Nishida, C.; Reinhard, P.; Linn, S.

1988-01-01

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

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Jon M Carthy

Full Text Available Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts.The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin β1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-β signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2.Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

Nemosis, a novel way of fibroblast activation, in inflammation and cancer

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Vaheri, Antti, E-mail: antti.vaheri@helsinki.fi [Haartman Institute, POB 21, FI-00014 University of Helsinki (Finland); Enzerink, Anna; Raesaenen, Kati; Salmenperae, Pertteli [Haartman Institute, POB 21, FI-00014 University of Helsinki (Finland)

2009-06-10

Malignant cells when grown in suspension, as a rule, proliferate and can form spheroids that have been used as a model of tumor nodules, micrometastases and avascular tumors. In contrast, normal adherent cells cannot be stimulated to grow as multicellular aggregates. Now, recent results show that normal fibroblasts if forced to cluster (spheroid formation) do not grow but undergo a new pathway of cell activation (nemosis) leading to a massive proinflammatory, proteolytic and growth factor response. The clustering and activation are initiated by fibronectin-integrin interaction. The activated fibroblasts are able to modulate the behavior of cancer cells and, furthermore malignant cells boost this activation even further. In this model, the activation of fibroblasts terminates in programmed necrosis-like cell death. Activation of the tumor stroma, especially of fibroblasts, is of critical importance for tumor progression, although mechanisms leading to their activation are still largely uncharacterized. In summary, our results suggest that this kind of fibroblast activation (nemosis) may be involved in pathological conditions such as inflammation and cancer.

Nemosis, a novel way of fibroblast activation, in inflammation and cancer

International Nuclear Information System (INIS)

Vaheri, Antti; Enzerink, Anna; Raesaenen, Kati; Salmenperae, Pertteli

2009-01-01

Malignant cells when grown in suspension, as a rule, proliferate and can form spheroids that have been used as a model of tumor nodules, micrometastases and avascular tumors. In contrast, normal adherent cells cannot be stimulated to grow as multicellular aggregates. Now, recent results show that normal fibroblasts if forced to cluster (spheroid formation) do not grow but undergo a new pathway of cell activation (nemosis) leading to a massive proinflammatory, proteolytic and growth factor response. The clustering and activation are initiated by fibronectin-integrin interaction. The activated fibroblasts are able to modulate the behavior of cancer cells and, furthermore malignant cells boost this activation even further. In this model, the activation of fibroblasts terminates in programmed necrosis-like cell death. Activation of the tumor stroma, especially of fibroblasts, is of critical importance for tumor progression, although mechanisms leading to their activation are still largely uncharacterized. In summary, our results suggest that this kind of fibroblast activation (nemosis) may be involved in pathological conditions such as inflammation and cancer.

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

Science.gov (United States)

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.

Stromal fibroblasts derived from mammary gland of bovine with mastitis display inflammation-specific changes.

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Chen, Qing; He, Guiliang; Zhang, Wenyao; Xu, Tong; Qi, Hongliang; Li, Jing; Zhang, Yong; Gao, Ming-Qing

2016-06-07

Fibroblasts are predominant components of mammary stromal cells and play crucial roles in the development and involution of bovine mammary gland; however, whether these cells contribute to mastitis has not been demonstrated. Thus, we have undertaken biological and molecular characterization of inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis and normal fibroblasts (NFs) from slaughtered dairy cows because of fractured legs during lactation. The functional contributions of INFs to normal epithelial cells were also investigated by using an in vitro co-culture model. We present evidence that the INFs were activated fibroblasts and showed inflammation-related features. Moreover, INFs significantly inhibited the proliferation and β-casein secretion of epithelial cells, as well as upregulated the expression of tumor necrosis factor-α and interleukin-8 in epithelial cells. These findings indicate that functional alterations can occur in stromal fibroblasts within the bovine mammary gland during mastitis, demonstrating the importance of stromal fibroblasts in bovine mastitis and its treatment.

Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

DEFF Research Database (Denmark)

Persson, H.; Købler, Carsten; Mølhave, Kristian

2013-01-01

beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA......Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain...... largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion...

Antifibrotic effects of crocetin in scleroderma fibroblasts and in bleomycin-induced sclerotic mice

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Yinghua Song

2013-10-01

Full Text Available OBJECTIVE: To investigate the antifibrotic effects of crocetin in scleroderma fibroblasts and in sclerotic mice. METHODS: Skin fibroblasts that were isolated from three systemic scleroderma (SSc patients and three healthy subjects were treated with crocetin (0.1, 1 or 10 μM. Cell proliferation was measured with an MTT assay. Alpha-smooth muscle actin was detected via an immunohistochemical method. Alpha 1 (I procollagen (COL1A1, alpha 1 (III procollagen (COL3A1, matrix metalloproteinase (MMP-1 and tissue inhibitor of matrix metalloproteinase (TIMP-1 mRNA levels were measured using real-time PCR. SSc mice were established by the subcutaneous injection of bleomycin. Crocetin (50 mg/kg/d was injected intraperitoneally for 14 days. Dermal thickness and lung fibrosis were assessed with Masson's trichrome staining. Plasma ET-1 was detected with an enzyme-linked immunosorbent assay (ELISA. Skin and lung ET-1 and COL1A1 mRNA levels were measured via real-time PCR. RESULTS: Crocetin inhibited the proliferation of SSc and normal fibroblasts, an effect that increased with crocetin concentration and incubation time. Crocetin decreased the expression of α-SMA and the levels of mRNA for COL1A1, COL3A1 and matrix metalloproteinase-1, while crocetin increased TIMP-1 mRNA levels in both SSc and normal fibroblasts. Skin and lung fibrosis was induced, and the levels of ET-1 in the plasma, skin and lungs were elevated in bleomycin-injected mice. Crocetin alleviated the thickening of the dermis and lung fibrosis; decreased COL1A1 mRNA levels in the skin and lung; and simultaneously decreased ET-1 concentrations in the plasma and ET-1 mRNA levels in the skin and lungs of the bleomycin-induced sclerotic mice, especially during the early phase (weeks 1-3. CONCLUSION: Crocetin inhibits cell proliferation, differentiation and collagen production in SSc fibroblasts. Crocetin alleviates skin and lung fibrosis in a bleomycin-induced SSc mouse model, in part due to a

Thermoreversible gelation polymer as an embolic material for aneurysm treatment: a delivery device for dermal fibroblasts and basic fibroblast growing factor into experimental aneurysms in rats.

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Dobashi, Hisashi; Akasaki, Yasuharu; Yuki, Ichiro; Arai, Takao; Ohashi, Hiroki; Murayama, Yuichi; Takao, Hiroyuki; Abe, Toshiaki

2013-11-01

This study evaluates whether thermoreversible gelation polymer (TGP) can be used as a delivery device to deploy dermal fibroblasts and cytokines into experimental aneurysms in rats. The right common iliac artery of rats was surgically ligated and an experimental aneurysm was created by applying exogenous elastase. Seven days later, two aneurysms were harvested and used as controls (Group A), two were embolized with pure TGP (Group B), two were embolized with TGP and basic fibroblast growth factor (bFGF) (Group C) and two were embolized with TGP loaded with rat dermal fibroblasts (Group D). The aneurysms were also embolized with TGP mixed with dermal fibroblasts and bFGF at different concentrations (10 ng/ml: Group E (n=2), 100 ng/ml: Group F (n=2), 1000 ng/ml: Group G (n=2)). Each aneurysm sample was harvested after 7 days and histologic analyses were performed. The most advanced thrombus organization in the aneurysm, such as prominent fibroblast proliferation and collagen deposition, was observed in Groups E, F and G, although there was no noticeable difference between the groups. Moderate thrombus organization was seen in Group D and minimal thrombus organization was seen in Groups B and C. TGP mixed with both dermal fibroblasts and bFGF induced the most advanced thrombus organization in the experimental aneurysms followed by TGP mixed only with dermal fibroblasts. TGP may be useful as a delivery device to deploy fibroblasts and cytokines into aneurysms.

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

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

2017-08-01

membrane. MPC proliferation, differentiation and fusion were assessed from cells stained for BrdU, desmin and myogenin. On biopsy cross-sections, fibroblast number was seen to increase, along with myogenic cell number, by d7 and increase further by d30, where fibroblasts were observed to be preferentially located immediately surrounding regenerating muscle fibres. In vitro, the presence of fibroblasts in direct contact with MPCs was found to moderately stimulate MPC proliferation and strongly stimulate both MPC differentiation and MPC fusion. It thus appears, in humans, that fibroblasts exert a strong positive regulatory influence on MPC activity, in line with observations during in vivo skeletal muscle regeneration. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Proliferative and inductive effects of Cyclosporine a on gingival fibroblast of child and adult

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Bahareh Nazemi Salman

2013-01-01

Conclusions: The mechanism of a CsA-induced fibroblast overgrowth may converge on the steps involving fibroblast proliferation and cytokine network including IL-6, IL-8, IL-1β, TGF-β1, and PGE 2 , in both adults and pediatrics. As the prevalence and intensity of drug-induced gingival overgrowth is more serious in the pediatrics. As group than in adults, we suggest that more studies be conducted on the pediatric group.

Advanced Research of Fibroblast Growth Factor Receptor 
in Non-small Cell Lung Cancer

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Dan PU

2013-11-01

Full Text Available Lung cancer is severely threatening human health. In recent years, the treatment for lung adenocarcinoma has made a great progress, targeted therapy has been widely applied in clinic, and benefits amount of patients. However, in squamous cell lung cancer, the incidence of epidermal growth factor receptor (EGFR gene mutant and ALK fusion gene are low,and targeted therapy like Tarceva and crizotinib, can hardly work. Since the fibroblast growth factors (fibroblast growth factor, FGF pathway is considered to be related to tumor cell proliferation, metastasis and angiogenesis, more and more researches proved the amplification of fibroblast growth factor receptor (FGFR in squamous cell lung cancer. Experiments in vivo and in vitro found that blocking FGF pathway could reduce the proliferation of tumor cells and inhibit metastasis. The FGF pathway might be a new target for treatment of squamous cell lung cancer. This article reviews the effect of FGFR in tumorigenesis,as well as the prospect as a therapeutic target in non-small cell lung cancer.

Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors

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Yi Zhao

2017-04-01

Full Text Available IntroductionMany antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells.MethodsCultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS in the presence of dead and dying cells, their supernatants (SNs, or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo.ResultsThe stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment.ConclusionInosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

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

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

2004-07-14

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

Effects of chitosan-coated fibers as a scaffold for three-dimensional cultures of rabbit fibroblasts for ligament tissue engineering.

Science.gov (United States)

Sarukawa, Junichiro; Takahashi, Masaaki; Abe, Masashi; Suzuki, Daisuke; Tokura, Seiichi; Furuike, Tetsuya; Tamura, Hiroshi

2011-01-01

Material selection in tissue-engineering scaffolds is one of the primary factors defining cellular response and matrix formation. In this study, we fabricated chitosan-coated poly(lactic acid) (PLA) fiber scaffolds to test our hypothesis that PLA fibers coated with chitosan highly promoted cell supporting properties compared to those without chitosan. Both PLA fibers (PLA group) and chitosan-coated PLA fibers (PLA-chitosan group) were fabricated for this study. Anterior cruciate ligament (ACL) fibroblasts were isolated from Japanese white rabbits and cultured on scaffolds consisting of each type of fiber. The effects of cell adhesivity, proliferation, and synthesis of the extracellular matrix (ECM) for each fiber were analyzed by cell counting, hydroxyproline assay, scanning electron microscopy and quantitative RT-PCR. Cell adhesivity, proliferation, hydroxyproline content and the expression of type-I collagen mRNA were significantly higher in the PLA-chitosan group than in the PLA group. Scanning electron microscopic observation showed that fibroblasts proliferated with a high level of ECM synthesis around the cells. Chitosan coating improved ACL fibroblast adhesion and proliferation, and had a positive effect on matrix production. Thus, the advantages of chitosan-coated PLA fibers show them to be a suitable biomaterial for ACL tissue-engineering scaffolds.

Fibroblast growth factor 10-fibroblast growth factor receptor 2b mediated signaling is not required for adult glandular stomach homeostasis.

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Allison L Speer

Full Text Available The signaling pathways that are essential for gastric organogenesis have been studied in some detail; however, those that regulate the maintenance of the gastric epithelium during adult homeostasis remain unclear. In this study, we investigated the role of Fibroblast growth factor 10 (FGF10 and its main receptor, Fibroblast growth factor receptor 2b (FGFR2b, in adult glandular stomach homeostasis. We first showed that mouse adult glandular stomach expressed Fgf10, its receptors, Fgfr1b and Fgfr2b, and most of the other FGFR2b ligands (Fgf1, Fgf7, Fgf22 except for Fgf3 and Fgf20. Fgf10 expression was mesenchymal whereas FGFR1 and FGFR2 expression were mostly epithelial. Studying double transgenic mice that allow inducible overexpression of Fgf10 in adult mice, we showed that Fgf10 overexpression in normal adult glandular stomach increased epithelial proliferation, drove mucous neck cell differentiation, and reduced parietal and chief cell differentiation. Although a similar phenotype can be associated with the development of metaplasia, we found that Fgf10 overexpression for a short duration does not cause metaplasia. Finally, investigating double transgenic mice that allow the expression of a soluble form of Fgfr2b, FGF10's main receptor, which acts as a dominant negative, we found no significant changes in gastric epithelial proliferation or differentiation in the mutants. Our work provides evidence, for the first time, that the FGF10-FGFR2b signaling pathway is not required for epithelial proliferation and differentiation during adult glandular stomach homeostasis.

Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis.

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François Huaux

Full Text Available Morbi-mortality in cystic fibrosis (CF is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

Effects of platelet rich plasma (PRP) on human gingival fibroblast, osteoblast and periodontal ligament cell behaviour.

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Kobayashi, Eizaburo; Fujioka-Kobayashi, Masako; Sculean, Anton; Chappuis, Vivianne; Buser, Daniel; Schaller, Benoit; Dőri, Ferenc; Miron, Richard J

2017-06-02

The use of platelet rich plasma (PRP, GLO) has been used as an adjunct to various regenerative dental procedures. The aim of the present study was to characterize the influence of PRP on human gingival fibroblasts, periodontal ligament (PDL) cells and osteoblast cell behavior in vitro. Human gingival fibroblasts, PDL cells and osteoblasts were cultured with conditioned media from PRP and investigated for cell migration, proliferation and collagen1 (COL1) immunostaining. Furthermore, gingival fibroblasts were tested for genes encoding TGF-β, PDGF and COL1a whereas PDL cells and osteoblasts were additionally tested for alkaline phosphatase (ALP) activity, alizarin red staining and mRNA levels of osteoblast differentiation markers including Runx2, COL1a2, ALP and osteocalcin (OCN). It was first found that PRP significantly increased cell migration of all cells up to 4 fold. Furthermore, PRP increased cell proliferation at 3 and 5 days of gingival fibroblasts, and at 3 days for PDL cells, whereas no effect was observed on osteoblasts. Gingival fibroblasts cultured with PRP increased TGF-β, PDGF-B and COL1 mRNA levels at 7 days and further increased over 3-fold COL1 staining at 14 days. PDL cells cultured with PRP increased Runx2 mRNA levels but significantly down-regulated OCN mRNA levels at 3 days. No differences in COL1 staining or ALP staining were observed in PDL cells. Furthermore, PRP decreased mineralization of PDL cells at 14 days post seeding as assessed by alizarin red staining. In osteoblasts, PRP increased COL1 staining at 14 days, increased COL1 and ALP at 3 days, as well as increased ALP staining at 14 days. No significant differences were observed for alizarin red staining of osteoblasts following culture with PRP. The results demonstrate that PRP promoted gingival fibroblast migration, proliferation and mRNA expression of pro-wound healing molecules. While PRP induced PDL cells and osteoblast migration and proliferation, it tended to have

Imaging of multiple myeloma and related monoclonal plasma cell diseases. An update

International Nuclear Information System (INIS)

Weber, Marc-Andre; Delorme, Stefan; Hillengass, Jens

2014-01-01

Multiple myeloma is a hematologic disorder characterized by the infiltration and proliferation of monoclonal plasma cells mainly in the bone marrow. The main symptoms are hypercalcemia, renal impairment, cytopenia/anemia and bone disease - summarized as CRAB-criteria. Symptomatic multiple myeloma is consistently preceded by asymptomatic premalignant stages called monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. Staging of multiple myeloma is based on the measurement of the monoclonal protein in serum and urine as well as the assessment of impairment of hematopoiesis, renal function and mineralized bone. In the last decade the development of novel therapeutic agents has led to an increase in response rates and survival time of patients with multiple myeloma, which further stresses the value of response assessment by imaging. Cross sectional imaging like MRI and CT is currently replacing conventional radiological surveys in the initial work-up and follow-up of patients with monoclonal plasma cell diseases. The added value of MRI is to improve initial staging by unraveling a diffuse infiltration of bone marrow by plasma cells, a focal pattern or a combination of both. Furthermore, a complete remission of myeloma confirmed by MRI and CT goes along with a better prognosis compared to a complete response based only on serological parameters.

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

International Nuclear Information System (INIS)

Bruzzone, Santina; Battaglia, Florinda; Mannino, Elena; Parodi, Alessia; Fruscione, Floriana; Basile, Giovanna; Salis, Annalisa; Sturla, Laura; Negrini, Simone; Kalli, Francesca; Stringara, Silvia; Filaci, Gilberto

2012-01-01

Highlights: ► ABA is an endogenous hormone in humans, regulating different cell responses. ► ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. ► UV-B irradiation increases ABA content in SSc cultures. ► 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 the 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-β (TGF-β). Conversely, migration toward ABA, but not toward TGF-β, 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.

The effect of well-characterized, very low-dose x-ray radiation on fibroblasts.

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Truong, Katelyn; Bradley, Suzanne; Baginski, Bryana; Wilson, Joseph R; Medlin, Donald; Zheng, Leon; Wilson, R Kevin; Rusin, Matthew; Takacs, Endre; Dean, Delphine

2018-01-01

The purpose of this study is to determine the effects of low-dose radiation on fibroblast cells irradiated by spectrally and dosimetrically well-characterized soft x-rays. To achieve this, a new cell culture x-ray irradiation system was designed. This system generates characteristic fluorescent x-rays to irradiate the cell culture with x-rays of well-defined energies and doses. 3T3 fibroblast cells were cultured in cups with Mylar® surfaces and were irradiated for one hour with characteristic iron (Fe) K x-ray radiation at a dose rate of approximately 550 μGy/hr. Cell proliferation, total protein analysis, flow cytometry, and cell staining were performed on fibroblast cells to determine the various effects caused by the radiation. Irradiated cells demonstrated increased proliferation and protein production compared to control samples. Flow cytometry revealed that a higher percentage of irradiated cells were in the G0/G1 phase of the cell cycle compared to control counterparts, which is consistent with other low-dose studies. Cell staining results suggest that irradiated cells maintained normal cell functions after radiation exposure, as there were no qualitative differences between the images of the control and irradiated samples. The result of this study suggest that low-dose soft x-ray radiation might cause an initial pause, followed by a significant increase, in proliferation. An initial "pause" in cell proliferation could be a protective mechanism of the cells to minimize DNA damage caused by radiation exposure. The new cell irradiation system developed here allows for unprecedented control over the properties of the x-rays given to the cell cultures. This will allow for further studies on various cell types with known spectral distribution and carefully measured doses of radiation, which may help to elucidate the mechanisms behind varied cell responses to low-dose x-rays reported in the literature.

Wound healing morbidity in STS patients treated with preoperative radiotherapy in relation to in vitro skin fibroblast radiosensitivity, proliferative capacity and TGF-β activity

International Nuclear Information System (INIS)

Akudugu, John M.; Bell, Robert S.; Catton, Charles; Davis, Aileen M.; Griffin, Anthony M.; O'Sullivan, Brian; Waldron, John N.; Ferguson, Peter C.; Wunder, Jay S.; Hill, Richard P.

2006-01-01

Background and purpose: In a recent study, we demonstrated that the ability of dermal fibroblasts, obtained from soft tissue sarcoma (STS) patients, to undergo initial division in vitro following radiation exposure correlated with the development of wound healing morbidity in the patients following their treatment with preoperative radiotherapy. Transforming growth factor beta (TGF-β) is thought to play an important role in fibroblast proliferation and radiosensitivity both of which may impact on wound healing. Thus, in this study we examined the interrelationship between TGF-β activity, radiosensitivity and proliferation of cultured fibroblasts and the wound healing response of STS patients after preoperative radiotherapy to provide a validation cohort for our previous study and to investigate mechanisms. Patients and methods: Skin fibroblasts were established from skin biopsies of 46 STS patients. The treatment group consisted of 28 patients who received preoperative radiotherapy. Eighteen patients constituted a control group who were either irradiated postoperatively or did not receive radiation treatment. Fibroblast cultures were subjected to the colony forming and cytokinesis-blocked binucleation assays (low dose rate: ∼0.02 Gy/min) and TGF-β assays (high dose-rate: ∼1.06 Gy/min) following γ-irradiation. Fibroblast radiosensitivity and initial proliferative ability were represented by the surviving fraction at 2.4 Gy (SF 2.4 ) and binucleation index (BNI), respectively. Active and total TGF-β levels in fibroblast cultures were determined using a biological assay. Wound healing complication (WHC), defined as the requirement for further surgery or prolonged deep wound packing, was the clinical endpoint examined. Results: Of the 28 patients treated with preoperative radiotherapy, 8 (29%) had wound healing difficulties. Fibroblasts from patients who developed WHC showed a trend to retain a significantly higher initial proliferative ability after

Endostatin induces proliferation of oral carcinoma cells but its effect on invasion is modified by the tumor microenvironment

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Alahuhta, Ilkka [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Aikio, Mari [Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu (Finland); Oulu Center for Cell-Matrix Research, University of Oulu (Finland); Väyrynen, Otto; Nurmenniemi, Sini [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Suojanen, Juho [Department of Oral and Maxillo-facial Diseases, University of Helsinki, Helsinki University Central Hospital (Finland); Teppo, Susanna [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Pihlajaniemi, Taina; Heljasvaara, Ritva [Biocenter Oulu and Faculty of Biochemistry and Molecular Medicine, University of Oulu (Finland); Oulu Center for Cell-Matrix Research, University of Oulu (Finland); Salo, Tuula [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland); Department of Oral and Maxillo-facial Diseases, University of Helsinki, Helsinki University Central Hospital (Finland); Department of Oral Diagnosis, School of Dentistry, State University of Campinas, Piracicaba, Sao Paolo (Brazil); Nyberg, Pia, E-mail: pia.nyberg@oulu.fi [Research Group of Cancer and Translational Medicine, Faculty of Medicine, University of Oulu (Finland); Medical Research Center, Oulu University Hospital, Oulu (Finland)

2015-08-01

The turnover of extracellular matrix liberates various cryptic molecules with novel biological activities. Endostatin is an endogenous angiogenesis inhibitor that is derived from the non-collagenous domain of collagen XVIII. Although there are a large number of studies on its anti-tumor effects, the molecular mechanisms are not yet completely understood, and the reasons why endostatin has not been successful in clinical trials are unclear. Research has mostly focused on its anti-angiogenic effect in tumors. Here, we aimed to elucidate how endostatin affects the behavior of aggressive tongue HSC-3 carcinoma cells that were transfected to overproduce endostatin. Endostatin inhibited the invasion of HSC-3 cells in a 3D collagen–fibroblast model. However, it had no effect on invasion in a human myoma organotypic model, which lacks vital fibroblasts. Recombinant endostatin was able to reduce the Transwell migration of normal fibroblasts, but had no effect on carcinoma associated fibroblasts. Surprisingly, endostatin increased the proliferation and decreased the apoptosis of cancer cells in organotypic models. Also subcutaneous tumors overproducing endostatin grew bigger, but showed less local invasion in nude mice xenografts. We conclude that endostatin affects directly to HSC-3 cells increasing their proliferation, but its net effect on cancer invasion seem to depend on the cellular composition and interactions of tumor microenvironment. - Highlights: • Endostatin affects not only angiogenesis, but also carcinoma cells and fibroblasts. • Endostatin increased carcinoma cell proliferation, but decreased 3D invasion. • The invasion inhibitory effect was sensitive to the microenvironment composition. • Fibroblasts may be a factor regulating the fluctuating roles of endostatin.

Basic calcium phosphate crystal-induced Egr-1 expression stimulates mitogenesis in human fibroblasts

International Nuclear Information System (INIS)

Zeng, Xiao R.; Sun Yubo; Wenger, Leonor; Cheung, Herman S.

2005-01-01

Previously, we have reported that basic calcium phosphate (BCP) crystals stimulate mitogenesis and synthesis of matrix metalloproteinases in cultured human foreskin and synovial fibroblasts. However, the detailed mechanisms involved are still unclear. In the present study, using RT-PCR and Egr-1 promoter analysis we showed that BCP crystals could stimulate early growth response gene Egr-1 transcription through a PKCα-dependent p44/p42 MAPK pathway. Using a retrovirus gene expression system (Clontech) to overexpress Egr-1 in human fibroblast BJ-1 cells resulted in promotion of mitogenesis measured either by MTT cell proliferation analysis or by direct cell counting. The results demonstrate that Egr-1 may play a key role in mediating BCP crystal-induced synovial fibroblast mitogenesis

The Biological Behaviors of Rat Dermal Fibroblasts Can Be Inhibited by High Levels of MMP9

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Sheng-Neng Xue

2012-01-01

Full Text Available Aims. To explore the effects of the high expression of MMP9 on biological behaviors of fibroblasts. Methods. High glucose and hyperhomocysteine were used to induce MMP9 expression in skin fibroblasts. Cell proliferation was detected by flow cytometry and cell viability by CCK-8. ELISA assay was used to detect collagen (hydroxyproline secretion. Scratch test was employed to evaluate horizontal migration of cells and transwell method to evaluate vertical migration of cells. Results. The mRNA and protein expressions of MMP9 and its protease activity were significantly higher in cells treated with high glucose and hyperhomocysteine than those in control group. At the same time, the S-phase cell ratio, proliferation index, cell viability, collagen (hydroxyproline secretion, horizontal migration rate, and the number of vertical migration cells decreased in high-glucose and hyperhomocysteine-treated group. Tissue inhibitor of metalloproteinase 1 (TIMP1, which inhibits the activity of MMP9, recovered the above biological behaviors. Conclusions. High expression of MMP9 in skin fibroblasts could be induced by cultureing in high glucose and hyperhomocysteine medium, which inhibited cell biological behaviors. Inhibitions could be reversed by TIMP1. The findings suggested that MMP9 deters the healing of diabetic foot ulcers by inhibiting the biological behaviors of fibroblasts.

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

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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.

Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

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Shih, Chi-Jen, E-mail: cjshih@kmu.edu.tw [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan (China); Lu, Pei-Shan [Department of Fragrance and Cosmetic Science, College of Pharmacy, Kaohsiung Medical University, 100 Shi-Chuan 1st Road, Kaohsiung 80708, Taiwan (China); Hsieh, Chih-Hsin [Department of Orthopaedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Chen, Wen-Cheng [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Chen, Jian-Chih [Department of Orthopaedics, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China); Department of Orthopaedics, College of Medicine, Kaohsiung Medical University, Kaohsiung 80708, Taiwan (China)

2014-09-30

Highlights: • Fluorescent microscopy images show that BG-M has excellent cellular affinity. • Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. • Cells cultured on BG-M had approximately 1.4 times higher proliferation activity. - Abstract: The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity.

Effects of bioglass powders with and without mesoporous structures on fibroblast and osteoblast responses

International Nuclear Information System (INIS)

Shih, Chi-Jen; Lu, Pei-Shan; Hsieh, Chih-Hsin; Chen, Wen-Cheng; Chen, Jian-Chih

2014-01-01

Highlights: • Fluorescent microscopy images show that BG-M has excellent cellular affinity. • Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. • Cells cultured on BG-M had approximately 1.4 times higher proliferation activity. - Abstract: The main objective of this study was to compare the responses of fibroblasts and osteoblasts to bioglass (BG) and bioglass-containing mesoporous structure (BG-M) powders. The BG-M powders exhibited specific surface areas approximately three times larger than those of the BG powders. The formation of a hysteresis loop also signified the presence of mesoporous structures in the BG-M samples; however, a hysteresis loop was not observed for the BG samples, resulting in 1/5 the pore volume of the BG-M samples. The viabilities of the fibroblasts and osteoblasts cultured in media containing the BG-M powders for 1, 2, and 3 days were greater than 90%. Importantly, the results of fluorescent microscopy images show that BG-M has excellent cellular affinity. Both the BG and BG-M substrates had positive effects on the proliferation of the osteoblastic cells. However, cells cultured on BG-M had approximately 1.4 times higher proliferation activity

Comparison between human cord blood serum and platelet-rich plasma supplementation for Human Wharton's Jelly Stem Cells and dermal fibroblasts culture

Directory of Open Access Journals (Sweden)

Hashemi SS

2016-08-01

Full Text Available We carried out a side-by-side comparison of the effects of Human cord blood serum (HcbS versus embryonic PRP on Human Wharton's Jelly Stem Cells(hWMSCand dermal fibroblasts proliferation. Human umbilical cord blood was collected to prepare activated serum (HCS and platelet-rich plasma (CPRP.Wharton's Jelly Stem Cells and dermal fibroblasts were cultured in complete medium with10% CPRP, 10%HCSor 10% fetal bovine serumand control (serum-free media.The efficiency of the protocols was evaluated in terms of the number of adherent cells and their expansion and Cell proliferation. We showed that proliferation of fibroblasts and mesenchymal stem cells in the presence of cord blood serum and platelet-rich plasma significantly more than the control group (p≤0/05. As an alternative to FBS, cord blood serum has been proved as an effective component in cell tissue culture applications and embraced a vast future in clinical applications of regenerative medicine. However, there is still a need to explore the potential of HCS and its safe applications in humanized cell therapy or tissue engineering.

Estimation of low-dose radiation-responsive proteins in the absence of genomic instability in normal human fibroblast cells.

Science.gov (United States)

Yim, Ji-Hye; Yun, Jung Mi; Kim, Ji Young; Nam, Seon Young; Kim, Cha Soon

2017-11-01

Low-dose radiation has various biological effects such as adaptive responses, low-dose hypersensitivity, as well as beneficial effects. However, little is known about the particular proteins involved in these effects. Here, we sought to identify low-dose radiation-responsive phosphoproteins in normal fibroblast cells. We assessed genomic instability and proliferation of fibroblast cells after γ-irradiation by γ-H2AX foci and micronucleus formation analyses and BrdU incorporation assay, respectively. We screened fibroblast cells 8 h after low-dose (0.05 Gy) γ-irradiation using Phospho Explorer Antibody Microarray and validated two differentially expressed phosphoproteins using Western blotting. Cell proliferation proceeded normally in the absence of genomic instability after low-dose γ-irradiation. Phospho antibody microarray analysis and Western blotting revealed increased expression of two phosphoproteins, phospho-NFκB (Ser536) and phospho-P70S6K (Ser418), 8 h after low-dose radiation. Our findings suggest that low-dose radiation of normal fibroblast cells activates the expression of phospho-NFκB (Ser536) and phospho-P70S6K (Ser418) in the absence of genomic instability. Therefore, these proteins may be involved in DNA damage repair processes.

Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis

OpenAIRE

Bianchessi, Marta; Chen, Yuwen; Durgam, Sushmitha; Pondenis, Holly; Stewart, Matthew

2015-01-01

Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assa...

Biphasic effect of arsenite on cell proliferation and apoptosis is associated with the activation of JNK and ERK1/2 in human embryo lung fibroblast cells

International Nuclear Information System (INIS)

He Xiaoqing; Chen Rui; Yang Ping; Li Aiping; Zhou Jianwei; Liu Qizhan

2007-01-01

Biphasic dose-response relationship induced by environmental agents is often characterized with the effect of low-dose stimulation and high-dose inhibition. Some studies showed that arsenite may induce cell proliferation and apoptosis via biphasic dose-response relationship in human cells; however, mechanisms underlying this phenomenon are not well understood. In the present study, we aimed at investigating the relationship between biphasic effect of arsenite on cell proliferation and apoptosis and activation of JNK and ERK1/2 in human embryo lung fibroblast (HELF) cells. Our results demonstrated that cell proliferation may be stimulated at lower concentrations (0.1 and 0.5 μM) arsenite but inhibited at higher concentrations (5 and 10 μM). When cell apoptosis was used as the endpoint, the concentration-response curves were changed to U-shapes. During stimulation phospho-JNK levels were significantly increased at 3, 6, and 12 h after 0.1 or 0.5 μM arsenite exposure. Phospho-ERK1/2 levels were increased with different concentrations (0.1-10 μM) of arsenite at 6, 12, and 24 h. Blocking of JNK pathway with 20 μM SP600125 or ERK1/2 by 100 μM PD98059 significantly inhibited biphasic effect of arsenite in cells. Data in the present study suggest that activation of JNK and ERK1/2 may be involved in biphasic effect of arsenite when measuring cell proliferation and apoptosis in HELF cells. JNK activation seems to play a more critical role than ERK1/2 activation in the biphasic process

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

International Nuclear Information System (INIS)

Liu, Xueting; Fang, Shencun; Liu, Haijun; Wang, Xingang; Dai, Xiaoniu; Yin, Qing; Yun, Tianwei; Wang, Wei; Zhang, Yingming; Liao, Hong; Zhang, Wei; Yao, Honghong; Chao, Jie

2015-01-01

Background: Silicosis is a systemic disease caused by inhaling silicon dioxide (SiO 2 ). Phagocytosis of SiO 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 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 2 treatment resulted 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 2 -induced increase in cell activation and migration in both 2D and 3D culture systems; and 3) RNA interference targeting CCR2 prevented the SiO 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 2 . CCR2 was also up-regulated in response to SiO 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 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 2 induced MCP-1 release from cultured human pulmonary fibroblast (HPF-a). • SiO 2 directly activated HPF-a via the MCP-1/CCR2 pathway. • SiO 2 increased HPF-a migration in both 2D and 3D model via the MCP-1/CCR2 pathway. • RNA-i of MCP-1/CCR2

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

Energy Technology Data Exchange (ETDEWEB)

Liu, Xueting [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Fang, Shencun [Nine Department of Respiratory Medicine, Nanjing Chest Hospital, Nanjing, Jiangsu 210029 (China); Liu, Haijun [Neurobiology Laboratory, New Drug Screening Centre, China Pharmaceutical University, Nanjing, Jiangsu 210009 (China); Wang, Xingang; Dai, Xiaoniu; Yin, Qing; Yun, Tianwei [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Wang, Wei; Zhang, Yingming [Nine Department of Respiratory Medicine, Nanjing Chest Hospital, Nanjing, Jiangsu 210029 (China); Liao, Hong [Neurobiology Laboratory, New Drug Screening Centre, China Pharmaceutical University, Nanjing, Jiangsu 210009 (China); Zhang, Wei [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Yao, Honghong [Department of Pharmacology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China); Chao, Jie, E-mail: chaojie@seu.edu.cn [Department of Physiology, Medical School of Southeast University, Nanjing, Jiangsu 210009 (China)

2015-10-15

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 resulted 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 and 3D

Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

Energy Technology Data Exchange (ETDEWEB)

Bruzzone, Santina, E-mail: santina.bruzzone@unige.it [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Battaglia, Florinda [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Mannino, Elena [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Parodi, Alessia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Fruscione, Floriana [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Basile, Giovanna [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Salis, Annalisa; Sturla, Laura [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Negrini, Simone; Kalli, Francesca; Stringara, Silvia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Filaci, Gilberto [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Department of Internal Medicine, Viale Benedetto XV 6, 16132 Genova (Italy); and others

2012-05-25

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 the 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.

Tubule-Derived Wnts Are Required for Fibroblast Activation and Kidney Fibrosis.

Science.gov (United States)

Zhou, Dong; Fu, Haiyan; Zhang, Lu; Zhang, Ke; Min, Yali; Xiao, Liangxiang; Lin, Lin; Bastacky, Sheldon I; Liu, Youhua

2017-08-01

Cell-cell communication via Wnt ligands is necessary in regulating embryonic development and has been implicated in CKD. Because Wnt ligands are ubiquitously expressed, the exact cellular source of the Wnts involved in CKD remains undefined. To address this issue, we generated two conditional knockout mouse lines in which Wntless (Wls), a dedicated cargo receptor that is obligatory for Wnt secretion, was selectively ablated in tubular epithelial cells or interstitial fibroblasts. Blockade of Wnt secretion by genetic deletion of Wls in renal tubules markedly inhibited myofibroblast activation and reduced renal fibrosis after unilateral ureteral obstruction. This effect associated with decreased activation of β -catenin and downstream gene expression and preserved tubular epithelial integrity. In contrast, fibroblast-specific deletion of Wls exhibited little effect on the severity of renal fibrosis after obstructive or ischemia-reperfusion injury. In vitro , incubation of normal rat kidney fibroblasts with tubule-derived Wnts promoted fibroblast proliferation and activation. Furthermore, compared with kidney specimens from patients without CKD, biopsy specimens from patients with CKD also displayed increased expression of multiple Wnt proteins, predominantly in renal tubular epithelium. These results illustrate that tubule-derived Wnts have an essential role in promoting fibroblast activation and kidney fibrosis via epithelial-mesenchymal communication. Copyright © 2017 by the American Society of Nephrology.

Fibrosis of Two: Epithelial Cell-Fibroblast Interactions in Pulmonary Fibrosis

Science.gov (United States)

Sakai, Norihiko; Tager, Andrew M.

2013-01-01

Idiopathic pulmonary fibrosis (IPF) is characterized by the progressive and ultimately fatal accumulation of fibroblasts and extracellular matrix in the lung that distorts its architecture and compromises its function. IPF is now thought to result from wound-healing processes that, although initiated to protect the host from injurious environmental stimuli, lead to pathological fibrosis due to these processes becoming aberrant or over-exuberant. Although the environmental stimuli that trigger IPF remain to be identified, recent evidence suggests that they initially injure the alveolar epithelium. Repetitive cycles of epithelial injury and resultant alveolar epithelial cell death provoke the migration, proliferation, activation and myofibroblast differentiation of fibroblasts, causing the accumulation of these cells and the extracellular matrix that they synthesize. In turn, these activated fibroblasts induce further alveolar epithelial cell injury and death, thereby creating a vicious cycle of pro-fibrotic epithelial cell-fibroblast interactions. Though other cell types certainly make important contributions, we focus here on the “pas de deux” (steps of two), or perhaps more appropriate to IPF pathogenesis, the “folie à deux” (madness of two) of epithelial cells and fibroblasts that drives the progression of pulmonary fibrosis. We describe the signaling molecules that mediate the interactions of these cell types in their “fibrosis of two”, including transforming growth factor-β, connective tissue growth factor, sonic hedgehog, prostaglandin E2, angiotensin II and reactive oxygen species. PMID:23499992

Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts

Directory of Open Access Journals (Sweden)

Zhang Dongwei

2011-11-01

Full Text Available Abstract Background Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, a polyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferation and reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However, there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin on the expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases. Methods We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice and human fetal lung fibroblasts (HFL-1 on the expression of cathepsins K and L which have been implicated in matrix degradation, TGF-β1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K and L, collagen, and TGF-β1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition on cell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-β1. Results Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompanied by increased expression levels of cathepsins L (25%-39% and K (41%-76% and a 30% decrease in TGF-β1 expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumin treatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here, cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and the expression of TGF-β1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation and migration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects were partly reversed by a potent cathepsin inhibitor. Conclusion This study demonstrates that

One in vitro model for visceral adipose-derived fibroblasts in chronic inflammation

International Nuclear Information System (INIS)

Yue Guiping; Du Lirui; Xia Tao; He Xianhui; Qiu Huan; Xu Lihui; Chen Xiaodong; Feng Shengqiu; Yang Zaiqing

2005-01-01

One pathogenesis of the obesity-associated complications is that consistent with increased body fat mass, the elevation of adipose tissue-derived cytokines inflicts a low-grade chronic inflammation, which ultimately leads to metabolic disorders. Adipocytes and macrophages in visceral adipose (VA) have been confirmed to contribute to the chronic inflammation; however, the role of the resident fibroblasts is still unknown. We established one VA fibroblast cell line, termed VAFC. Morphological analysis indicated that there were large numbers of pits at the cell plasma membrane. In vitro VAFC cells promoted bone marrow cells to differentiate into macrophages and protected them from apoptosis in the serum-free conditions. Additionally, they also interfered in lymphocytes proliferation. On the basis of these results, this cell line might be an in vitro model for understanding the role of adipose-derived fibroblasts in obesity-associated chronic inflammation

Carcinoma-Associated Fibroblasts Are a Promising Therapeutic Target

International Nuclear Information System (INIS)

Togo, Shinsaku; Polanska, Urszula M.; Horimoto, Yoshiya; Orimo, Akira

2013-01-01

Human carcinomas frequently exhibit significant stromal reactions such as the so-called “desmoplastic stroma” or “reactive stroma”, which is characterised by the existence of large numbers of stromal cells and extracellular matrix proteins. Carcinoma-associated fibroblasts (CAFs), which are rich in activated fibroblast populations exemplified by myofibroblasts, are among the predominant cell types present within the tumour-associated stroma. Increased numbers of stromal myofibroblasts are often associated with high-grade malignancies with poor prognoses in humans. CAF myofibroblasts possess abilities to promote primary tumour development, growth and progression by stimulating the processes of neoangiogenesis as well as tumour cell proliferation, survival, migration and invasion. Moreover, it has been demonstrated that CAFs serve as a niche supporting the metastatic colonisation of disseminated carcinoma cells in distant organs. Their contribution to primary and secondary malignancies makes these fibroblasts a potential therapeutic target and they also appear to be relevant to the development of drug resistance and tumour recurrence. This review summarises our current knowledge of tumour-promoting CAFs and discusses the therapeutic feasibility of targeting these cells as well as disrupting heterotypic interactions with other cell types in tumours that may improve the efficacy of current anti-tumour therapies

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

Energy Technology Data Exchange (ETDEWEB)

Berndt, Alexander, E-mail: alexander.berndt@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Büttner, Robert, E-mail: Robert-Buettner@gmx.net [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany); Gühne, Stefanie, E-mail: stefanie_guehne@gmx.net [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Gleinig, Anna, E-mail: annagleinig@yahoo.com [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Richter, Petra, E-mail: P.Richter@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Chen, Yuan, E-mail: Yuan.Chen@med.uni-jena.de [Center for Molecular Biomedicine, Institute of Pathology, Jena University Hospital, 07740 Jena (Germany); Franz, Marcus, E-mail: Marcus.Franz@med.uni-jena.de [Clinic of Internal Medicine I, Jena University Hospital, 07740 Jena (Germany); Liebmann, Claus, E-mail: Claus.Liebmann@uni-jena.de [Institute of Biochemistry and Biophysics, Friedrich Schiller University Jena, 07740 Jena (Germany)

2014-04-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM{sub TGF}, FCM{sub PDGF}) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM{sub B}). FCM{sub TGF} stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM{sub TGF}≫FCM{sub PDGF} induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM{sub TGF}>FCM{sub PDGF}) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin

Effects of activated fibroblasts on phenotype modulation, EGFR signalling and cell cycle regulation in OSCC cells

International Nuclear Information System (INIS)

Berndt, Alexander; Büttner, Robert; Gühne, Stefanie; Gleinig, Anna; Richter, Petra; Chen, Yuan; Franz, Marcus; Liebmann, Claus

2014-01-01

Crosstalk between carcinoma associated fibroblasts (CAFs) and oral squamous cell carcinoma (OSCC) cells is suggested to mediate phenotype transition of cancer cells as a prerequisite for tumour progression, to predict patients’ outcome, and to influence the efficacy of EGFR inhibitor therapies. Here we investigate the influence of activated fibroblasts as a model for CAFs on phenotype and EGFR signalling in OSCC cells in vitro. For this, immortalised hTERT-BJ1 fibroblasts were activated with TGFβ1 and PDGFAB to generate a myofibroblast or proliferative phenotype, respectively. Conditioned media (FCM TGF , FCM PDGF ) were used to stimulate PE/CA-PJ15 OSCC cells. Results were compared to the effect of conditioned media of non-stimulated fibroblasts (FCM B ). FCM TGF stimulation leads to an up-regulation of vimentin in the OSCC cells and an enhancement of invasive behaviour, indicating EMT-like effects. Similarly, FCM TGF ≫FCM PDGF induced up-regulation of EGFR, but not of ErbB2/ErbB3. In addition, we detected an increase in basal activities of ERK, PI3K/Akt and Stat3 (FCM TGF >FCM PDGF ) accompanied by protein interaction of vimentin with pERK. These effects are correlated with an increased proliferation. In summary, our results suggest that the activated myofibroblast phenotype provides soluble factors which are able to induce EMT-like phenomena and to increase EGFR signalling as well as cell proliferation in OSCC cells. Our results indicate a possible influence of activated myofibroblasts on EGFR-inhibitor therapy. Therefore, CAFs may serve as promising novel targets for combined therapy strategies. - Highlights: • A cell culture model for cancer associated fibroblasts is described. • The mutual interaction with OSCC cells leads to up-regulation of EGFR in tumour cells. • mCAF induces EGFR downstream signalling with increased proliferation in OSCC. • Erk activation is associated with protein interaction with vimentin as sign of EMT. • Results qualify

Biological Differences between Hanwoo longissimus dorsi and semimembranosus Muscles in Collagen Synthesis of Fibroblasts.

Science.gov (United States)

Subramaniyan, Sivakumar Allur; Hwang, Inho

2017-01-01

Variations in physical toughness between muscles and animals are a function of growth rate and extend of collagen type I and III. The current study was designed to investigate the ability of growth rate, collagen concentration, collagen synthesizing and degrading genes on two different fibroblast cells derived from Hanwoo m. longissimus dorsi (LD) and semimembranosus (SM) muscles. Fibroblast cell survival time was determined for understanding about the characteristics of proliferation rate between the two fibroblasts. We examined the collagen concentration and protein expression of collagen type I and III between the two fibroblasts. The mRNA expression of collagen synthesis and collagen degrading genes to elucidate the molecular mechanisms on toughness and tenderness through collagen production between the two fibroblast cells. From our results the growth rate, collagen content and protein expression of collagen type I and III were significantly higher in SM than LD muscle fibroblast. The mRNA expressions of collagen synthesized genes were increased whereas the collagen degrading genes were decreased in SM than LD muscle. Results from confocal microscopical investigation showed increased fluorescence of collagen type I and III appearing stronger in SM than LD muscle fibroblast. These results implied that the locomotion muscle had higher fibroblast growth rate, leads to produce more collagen, and cause tougher than positional muscle. This in vitro study mirrored that background toughness of various muscles in live animal is likely associated with fibroblast growth pattern, collagen synthesis and its gene expression.

RNA-seq analysis of synovial fibroblasts brings new insights into rheumatoid arthritis

Directory of Open Access Journals (Sweden)

Heruth Daniel P

2012-12-01

Full Text Available Abstract Background Rheumatoid arthritis (RA is a chronic autoimmune-disease of unknown origin that primarily affects the joints and ultimately leads to their destruction. Growing evidence suggests that synvovial fibroblasts play important roles in the initiation and the perpetuation of RA but underlying molecular mechanisms are not understood fully. In the present study, Illumina RNA sequencing was used to profile two human normal control and two rheumatoid arthritis synvovial fibroblasts (RASFs transcriptomes to gain insights into the roles of synvovial fibroblasts in RA. Results We found that besides known inflammatory and immune responses, other novel dysregulated networks and pathways such as Cell Morphology, Cell-To-Cell Signaling and Interaction, Cellular Movement, Cellular Growth and Proliferation, and Cellular Development, may all contribute to the pathogenesis of RA. Our study identified several new genes and isoforms not previously associated with rheumatoid arthritis. 122 genes were up-regulated and 155 genes were down-regulated by at least two-fold in RASFs compared to controls. Of note, 343 known isoforms and 561 novel isoforms were up-regulated and 262 known isoforms and 520 novel isoforms were down-regulated by at least two-fold. The magnitude of difference and the number of differentially expressed known and novel gene isoforms were not detected previously by DNA microarray. Conclusions Since the activation and proliferation of RASFs has been implicated in the pathogenesis of rheumatoid arthritis, further in-depth follow-up analysis of the transcriptional regulation reported in this study may shed light on molecular pathogenic mechanisms underlying synovial fibroblasts in arthritis and provide new leads of potential therapeutic targets.

Amelogenin is phagocytized and induces changes in integrin configuration, gene expression and proliferation of cultured normal human dermal fibroblasts

DEFF Research Database (Denmark)

Almqvist, Sofia; Werthén, Maria; Johansson, Anna

2010-01-01

Fibroblasts are central in wound healing by expressing important mediators and producing and remodelling extracellular matrix (ECM) components. This study aimed at elucidating possible mechanisms of action of the ECM protein amelogenin on normal human dermal fibroblasts (NHDF). Amelogenin at 100...

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

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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.

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

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Jaroslaw Suchanski

Full Text Available In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first

Podoplanin increases the migration of human fibroblasts and affects the endothelial cell network formation: A possible role for cancer-associated fibroblasts in breast cancer progression.

Science.gov (United States)

Suchanski, Jaroslaw; Tejchman, Anna; Zacharski, Maciej; Piotrowska, Aleksandra; Grzegrzolka, Jedrzej; Chodaczek, Grzegorz; Nowinska, Katarzyna; Rys, Janusz; Dziegiel, Piotr; Kieda, Claudine; Ugorski, Maciej

2017-01-01

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such

Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition

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L. V. Altukhova

2015-09-01

the burn area, and mutual stimulation of auto-fibroblasts and auto-keratinocytes to proliferate and to synthesize biologically active substances, i.e. cytokines and growth factors.

Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro.

Science.gov (United States)

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

2017-06-01

Papillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation. The aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts. To study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix. Conditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents. The findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.

Assembly of fibronectin into the extracellular matrix of early and late passage human skin fibroblasts

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Mann, D.M.

1987-01-01

The specific binding of soluble 125 I-human plasma fibronectin ( 125 I-HFN-P) to confluent cultures of early and late passage human skin fibroblasts was investigated. Previous studies HFN-P bound to fibroblast cell layers indicated that HNF-P was present in the cultures in two separate pools, distinguishable on the basis of their solubility in 1% deoxycholate. Examination of the kinetics of 125 I-HFN-P binding to Pool I of early and late passage cultures revealed that both cultures required 2-4 h to approach steady-state conditions. Other kinetic studies showed that the rates of low of 125 I-HFN-P from either Pool I or Pool II were similar for both cultures. Further, Scatchard analysis revealed a single class of Pool I binding sites with apparent dissociation constants (K/sub d/) of 5.3 x 10 -8 M (early passage) and 4.2 x 10 -8 M (late passage). These results indicate that early and late passage cultures of human fibroblasts exhibit differences in the number of cell surface biding sites for soluble fibronectin, and in the extent to which they incorporate soluble fibronectin into the extracellular matrix. Parameters which affect the fibronectin matrix assembly system of human skin fibroblasts were also examined. In addition, several monoclonal anti-fibronectin antibodies were characterized and developed as experimental probes for fibronectin structure and function

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

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Dawson, R A; Goberdhan, N J; Freedlander, E; MacNeil, S

1996-03-01

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

Inhibition of fibroblast growth by Notch1 signaling is mediated by induction of Wnt11-dependent WISP-1.

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Zhao-Jun Liu

Full Text Available Fibroblasts are an integral component of stroma and important source of growth factors and extracellular matrix (ECM. They play a prominent role in maintaining tissue homeostasis and in wound healing and tumor growth. Notch signaling regulates biological function in a variety of cells. To elucidate the physiological function of Notch signaling in fibroblasts, we ablated Notch1 in mouse (Notch1(Flox/Flox embryonic fibroblasts (MEFs. Notch1-deficient (Notch1(-/- MEFs displayed faster growth and motility rate compared to Notch1(Flox/Flox MEFs. Such phenotypic changes, however, were reversible by reconstitution of Notch1 activation via overexpression of the intracellular domain of Notch1 (NICD1 in Notch1-deficient MEFs. In contrast, constitutive activation of Notch1 signaling by introducing NICD1 into primary human dermal fibroblasts (FF2441, which caused pan-Notch activation, inhibited cell growth and motility, whereas cellular inhibition was relievable when the Notch activation was countered with dominant-negative mutant of Master-mind like 1 (DN-MAML-1. Functionally, "Notch-activated" stromal fibroblasts could inhibit tumor cell growth/invasion. Moreover, Notch activation induced expression of Wnt-induced secreted proteins-1 (WISP-1/CCN4 in FF2441 cells while deletion of Notch1 in MEFs resulted in an opposite effect. Notably, WISP-1 suppressed fibroblast proliferation, and was responsible for mediating Notch1's inhibitory effect since siRNA-mediated blockade of WISP-1 expression could relieve cell growth inhibition. Notch1-induced WISP-1 expression appeared to be Wnt11-dependent, but Wnt1-independent. Blockade of Wnt11 expression resulted in decreased WISP-1 expression and liberated Notch-induced cell growth inhibition. These findings indicated that inhibition of fibroblast proliferation by Notch pathway activation is mediated, at least in part, through regulating Wnt1-independent, but Wnt11-dependent WISP-1 expression.

Nuclear medicine: Monoclonal antibodies

International Nuclear Information System (INIS)

Endo, K.; Sakahara, H.; Koizumi, M.; Kawamura, Y.; Torizuka, K.; Yokoyama, A.

1986-01-01

Antitumor monoclonal antibody was successfully labeled with Tc-99m by using dithiosemicarbazone (DTS) as a bifunctional chelating agent. In the first step, DTS was coupled to antibody without loss of immunoreactivity; the compound then efficiently formed a neutral 1:1 chelate with pentavalent or tetravalent Tc-99m. Imaging with Tc-99m-labeled monoclonal antibody to human osteosarcoma (OST-7) clearly displayed a small tumor in nude mice at 6 and 24 hours after intravenous administration. The tumor-to-blood ratio of the Tc-99m-labeled monoclonal antibody was higher than that of a radioiodinated antibody and similar to that of an In-111-labeled antibody. Thus, conjugation of DTS to monoclonal antibody followed by radiometalation is a simple and efficient method of preparing Tc-99m-labeled monoclonal antibody

Peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist inhibits collagen synthesis in human hypertrophic scar fibroblasts by targeting Smad3 via miR-145

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Zhu, Hua-Yu; Li, Chao; Zheng, Zhao; Zhou, Qin; Guan, Hao; Su, Lin-Lin; Han, Jun-Tao; Zhu, Xiong-Xiang; Wang, Shu-yue; Li, Jun, E-mail: lijunfmmu@163.com; Hu, Da-Hai, E-mail: hudahaifmmu@aliyun.com

2015-03-27

The transcription factor peroxisome proliferator-activated receptor-γ (PPAR-γ) functions to regulate cell differentiation and lipid metabolism. Recently, its agonist has been documented to regulate extracellular matrix production in human dermal fibroblasts. This study explored the underlying molecular mechanisms and gene interactions in hypertrophic scar fibroblasts (HSFBs) in vitro. HSFBs were cultured and treated with or without PPAR-γ agonist or antagonist for gene expression. Bioinformatical analysis predicted that miR-145 could target Smad3 expression. Luciferase assay was used to confirm such an interaction. The data showed that PPAR-γ agonist troglitazone suppressed expression of Smad3 and Col1 in HSFBs. PPAR-γ agonist induced miR-145 at the gene transcriptional level, which in turn inhibited Smad3 expression and Col1 level in HSFBs. Furthermore, ELISA data showed that Col1 level in HSFBs was controlled by a feedback regulation mechanism involved in PPAR-γ agonist and antagonist-regulated expression of miR-145 and Smad3 in HSFBs. These findings indicate that PPAR-γ-miR-145-Smad3 axis plays a role in regulation of collagen synthesis in HSFBs. - Highlights: • PPAR-γ agonist inhibits collagen synthesis in HSFBs. • Smad3 and type I collagen expression are decreased by PPAR-γ agonist. • miR-145 expression is increased by PPAR-γ agonist in HSFBs. • Increased miR-145 inhibits collagen synthesis by targeting Smad3. • miR-145 regulates collagen synthesis.

Ergosterol peroxide from Cordyceps cicadae ameliorates TGF-β1-induced activation of kidney fibroblasts.

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Zhu, Rong; Zheng, Rong; Deng, Yueyi; Chen, Yiping; Zhang, Shuwei

2014-02-15

Chronic kidney disease is a growing public health problem with an urgent need for new pharmacological agents. Ergosterol peroxide (EP) is the major sterol produced by Cordyceps cicadae Shing (C. cicadae), a widely used traditional Chinese medicine. C. cicadae has been used to treat many kinds of diseases and has a potential benefit on renoprotection. This study aimed to investigate the anti-fibrotic effects of EP as well as the underlying mechanisms. A normal rat kidney fibroblast cell line (NRK-49F) was stimulated to undergo fibroblast activation by transforming growth factor-β1 (TGF-β1) and EP treatment was applied to explore its potential anti-fibrotic effects. Cell proliferation was investigated using MTT analysis. Fibrosis-associated protein expression was analyzed using immunohistochemistry and/or Western blotting. EP treatment attenuated TGF-β1-induced renal fibroblast proliferation, expression of cytoskeleton protein and CTGF, as well as ECM production. Additionally, EP blocked TGF-β1-stimulated phosphorylation of ERK1/2, p38 and JNK pathway. Moreover, the TGF-β1-induced expression of fibronectin was attenuated by either inhibition of MAPKs or by EP treatment. In conclusion, our findings demonstrate that EP is able to suppress TGF-β1-induced fibroblasts activation in NRK-49F. This new information provides a line of theoretical evidence supporting the use of C. cicadae in the intervention of kidney disease and suggests that EP has the potential to be developed as a therapeutic agent to prevent renal fibrosis. Copyright © 2013 Elsevier GmbH. All rights reserved.

Anti-fibrotic effect of pirfenidone in muscle derived-fibroblasts from Duchenne muscular dystrophy patients.

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Zanotti, Simona; Bragato, Cinzia; Zucchella, Andrea; Maggi, Lorenzo; Mantegazza, Renato; Morandi, Lucia; Mora, Marina

2016-01-15

Tissue fibrosis, characterized by excessive deposition of extracellular matrix proteins, is the end point of diseases affecting the kidney, bladder, liver, lung, gut, skin, heart and muscle. In Duchenne muscular dystrophy (DMD), connective fibrotic tissue progressively substitutes muscle fibers. So far no specific pharmacological treatment is available for muscle fibrosis. Among promising anti-fibrotic molecules, pirfenidone has shown anti-fibrotic and anti-inflammatory activity in animal and cell models, and has already been employed in clinical trials. Therefore we tested pirfenidone anti-fibrotic properties in an in vitro model of muscle fibrosis. We evaluated effect of pirfenidone on fibroblasts isolated from DMD muscle biopsies. These cells have been previously characterized as having a pro-fibrotic phenotype. We tested cell proliferation and migration, secretion of soluble collagens, intracellular levels of collagen type I and fibronectin, and diameter of 3D fibrotic nodules. We found that pirfenidone significantly reduced proliferation and cell migration of control and DMD muscle-derived fibroblasts, decreased extracellular secretion of soluble collagens by control and DMD fibroblasts, as well as levels of collagen type I and fibronectin, and, in DMD fibroblasts only, reduced synthesis and deposition of intracellular collagen. Furthermore, pirfenidone was able to reduce the diameter of fibrotic-nodules in our 3D model of in vitro fibrosis. These pre-clinical results indicate that pirfenidone has potential anti-fibrotic effects also in skeletal muscle fibrosis, urging further studies in in vivo animal models of muscular dystrophy in order to translate the drug into the treatment of muscle fibrosis in DMD patients. Copyright © 2015 Elsevier Inc. All rights reserved.

In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

Science.gov (United States)

Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

2008-02-01

Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast or fibroblast. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

Evodiamine attenuates TGF-β1-induced fibroblast activation and endothelial to mesenchymal transition.

Science.gov (United States)

Wu, Qing-Qing; Xiao, Yang; Jiang, Xiao-Han; Yuan, Yuan; Yang, Zheng; Chang, Wei; Bian, Zhou-Yan; Tang, Qi-Zhu

2017-06-01

The aim of this study is to investigate the effect of evodiamine on fibroblast activation in cardiac fibroblasts and endothelial to mesenchymal transition (EndMT) in human umbilical vein endothelial cells (HUVECs). Neonatal rat cardiac fibroblasts were stimulated with transforming growth factor beta 1 (TGF-β1) to induce fibroblast activation. After co-cultured with evodiamine (5, 10 μM), the proliferation and pro-fibrotic proteins expression of cardiac fibroblasts were evaluated. HUVECs were also stimulated with TGF-β1 to induce EndMT and treated with evodiamine (5, 10 μM) at the same time. The EndMT response in the HUVECs was evaluated as well as the capacity of the transitioned endothelial cells migrating to surrounding tissue. As a result, Evodiamine-blunted TGF-β1 induced activation of cardiac fibroblast into myofibroblast as assessed by the decreased expressions of α-SMA. Furthermore, evodiamine reduced the increased protein expression of fibrosis markers in neonatal and adult rat cardiac fibroblasts induced by TGF-β1. HUVECs stimulated with TGF-β1 exhibited lower expression levels of CD31, CD34, and higher levels of α-SMA, vimentin than the control cells. This phenotype was eliminated in the HUVECs treated with both 5 and 10 μM evodiamine. Evodiamine significantly reduced the increase in migration ability that occurred in response to TGF-β1 in HUVECs. In addition, the activation of Smad2, Smad3, ERK1/2, and Akt, and the nuclear translocation of Smad4 in both cardiac fibroblasts and HUVEC were blocked by evodiamine treatment. Thus, evodiamine could prevent cardiac fibroblasts from activation into myofibroblast and protect HUVEC against EndMT. These effects may be mediated by inhibition of the TGFβ pathway in both cardiac fibroblasts and HUVECs.

Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

International Nuclear Information System (INIS)

Adhikari, Ananta Raj; Geranpayeh, Tanya; Chu, Wei Kan; Otteson, Deborah C.

2016-01-01

In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1 × 10 12 to 1 × 10 14 ions/cm 2 ), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. - Highlights: • Argon irradiation modifies surface chemistry and increases hydrophilicity of poly(lactic-glycolic) acid (PLGA) films. • Both native and irradiated PLGA films were not cytotoxic for mouse fibroblasts. • Fibroblast proliferation increased on PLGA substrates modified with higher doses of Argon irradiation. • Surface modification with Argon irradiation increases biocompatibility of PLGA films.

Lysophosphatidic acid signaling through its receptor initiates profibrotic epithelial cell fibroblast communication mediated by epithelial cell derived connective tissue growth factor.

Science.gov (United States)

Sakai, Norihiko; Chun, Jerold; Duffield, Jeremy S; Lagares, David; Wada, Takashi; Luster, Andrew D; Tager, Andrew M

2017-03-01

The expansion of the fibroblast pool is a critical step in organ fibrosis, but the mechanisms driving expansion remain to be fully clarified. We previously showed that lysophosphatidic acid (LPA) signaling through its receptor LPA 1 expressed on fibroblasts directly induces the recruitment of these cells. Here we tested whether LPA-LPA 1 signaling drives fibroblast proliferation and activation during the development of renal fibrosis. LPA 1 -deficient (LPA 1 -/- ) or -sufficient (LPA 1 +/+ ) mice were crossed to mice with green fluorescent protein expression (GFP) driven by the type I procollagen promoter (Col-GFP) to identify fibroblasts. Unilateral ureteral obstruction-induced increases in renal collagen were significantly, though not completely, attenuated in LPA 1 -/- Col-GFP mice, as were the accumulations of both fibroblasts and myofibroblasts. Connective tissue growth factor was detected mainly in tubular epithelial cells, and its levels were suppressed in LPA 1 -/- Col-GFP mice. LPA-LPA 1 signaling directly induced connective tissue growth factor expression in primary proximal tubular epithelial cells, through a myocardin-related transcription factor-serum response factor pathway. Proximal tubular epithelial cell-derived connective tissue growth factor mediated renal fibroblast proliferation and myofibroblast differentiation. Administration of an inhibitor of myocardin-related transcription factor/serum response factor suppressed obstruction-induced renal fibrosis. Thus, targeting LPA-LPA 1 signaling and/or myocardin-related transcription factor/serum response factor-induced transcription could be promising therapeutic strategies for renal fibrosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Effect of phenytoin and age on gingival fibroblast enzymes.

Directory of Open Access Journals (Sweden)

Surena Vahabi

2014-06-01

Full Text Available The alteration of cytokine balance is stated to exert greater influence on gingival overgrowth compared to the direct effect of the drug on the regulation of extracellular matrix metabolism. The current study evaluated the effect of phenytoin on the regulation of collagen, lysyl oxidase and elastin in gingival fibroblasts.Normal human gingival fibroblasts (HGFs were obtained from 4 healthy children and 4 adults. Samples were cultured with phenytoin. MTT test was used to evaluate the proliferation and ELISA was performed to determine the level of IL1β and PGE2 production by HGFs. Total RNA of gingival fibroblasts was extracted and RT-PCR was performed on samples. Mann-Whitney U test was used to analyze the data with an alpha error level less than 0.05.There was a significant difference in the expression of elastin between the controls and treated samples in both adult and pediatric groups and also in the lysyl oxidase expression of adult controls and treated adults. No significant difference was found between collagen expression in adults.The significant difference in elastin and lysyl oxidase expression between adult and pediatric samples indicates the significant effect of age on their production.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

International Nuclear Information System (INIS)

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

2012-01-01

Highlights: ► We identify a function of the YIGSR peptide to enhance collagen synthesis in Hs27. ► YIGSR peptide enhanced collagen type 1 synthesis both of gene and protein levels. ► There were no changes in cell proliferation and MMP-1 level in YIGSR treatment. ► The YIGSR effect on collagen synthesis mediated activation of FAK, pyk2 and ERK. ► The YIGSR-induced FAK and ERK activation was modulated by FAK and MEK inhibitors. -- Abstract: The dermal ECM is synthesized from fibroblasts and is primarily compromised of fibrillar collagen and elastic fibers, which support the mechanical strength and resiliency of skin, respectively. Laminin, a major glycoprotein located in the basement membrane, promotes cell adhesion, cell growth, differentiation, and migration. The laminin tyrosine-isoleucine-glycine-serine-arginine (YIGSR) peptide, corresponding to the 929–933 sequence of the β1 chain, is known to be a functional motif with effects on the inhibition of tumor metastasis, the regulation of sensory axonal response and the inhibition of angiogenesis through high affinity to the 67 kDa laminin receptor. In this study, we identified a novel function of the YIGSR peptide to enhance collagen synthesis in human dermal fibroblasts. To elucidate this novel function regarding collagen synthesis, we treated human dermal fibroblasts with YIGSR peptide in both a time- and dose-dependent manner. According to subsequent experiments, we found that the YIGSR peptide strongly enhanced collagen type 1 synthesis without changing cell proliferation or cellular MMP-1 level. This YIGSR peptide-mediated collagen type 1 synthesis was modulated by FAK inhibitor and MEK inhibitor. This study clearly reveals that YIGSR peptide plays a novel function on the collagen type 1 synthesis of dermal fibroblasts and also suggests that YIGSR is a strong candidate peptide for the treatment of skin aging and wrinkles.

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour

Energy Technology Data Exchange (ETDEWEB)

Irving, Michael; Murphy, Mark F; Lilley, Francis; French, Paul W; Burton, David R [General Engineering Research Institute, Liverpool John Moores University, Liverpool, L3 3AF (United Kingdom); Dixon, Simon [Biomer Technology LTD, 10 Seymour Court, Tudor Road, Manor Park, Runcorn, Cheshire, WA7 1SY (United Kingdom); Sharp, Martin C [General Engineering Research Institute, Liverpool John Moores University, Liverpool, L3 3AF (United Kingdom)

2017-02-01

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72 h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods. - Highlights: • Abrasive polishing can generate nano-scratches on stainless steel to cast polymer films for cell

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour

International Nuclear Information System (INIS)

Irving, Michael; Murphy, Mark F; Lilley, Francis; French, Paul W; Burton, David R; Dixon, Simon; Sharp, Martin C

2017-01-01

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72 h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods. - Highlights: • Abrasive polishing can generate nano-scratches on stainless steel to cast polymer films for cell

Irradiated murine fibroblasts as feeder layer used in human cell culture

International Nuclear Information System (INIS)

Almeida, Tiago L.; Klingbeil, Fatima G.; Yoshito, Daniele; Caproni, Priscila; Mathor, Monica B.; Herson, Marisa R.

2007-01-01

In 1975, Rheinwald and Green published an in vitro model for keratinocyte cell cultures in which the use of murine fibroblasts, as a feeder layer was introduced. These cells are modified fibroblasts, which presence render keratinocyte cells to remain proliferative for longer periods of time. This optimization of culture outputs has allowed for several clinical applications of confluent keratinocyte cultures as skin substitutes or wound dressings in situations such as post burn extensive skin loss, loss of oral mucosa, and other skin disorders. Nevertheless, proliferation of fibroblast in co-culture with keratinocytes must be controlled by anti-proliferative measures such as irradiation; at the same time, keratinocytes require specific nutrients in the culture medium, which may interfere with the fibroblast feeder layer viability. Therefore, the thorough understanding of the impact of different issues such as culture media composition, irradiation dose and pre-plating storage conditions of irradiated fibroblast to be used as feeder layer in these co-culture systems is important. In this work, changes as far as viability and proliferative rates of irradiated fibroblasts in culture were evaluated in relation to the type of culture medium used, dose of gamma radiation exposure, storage and timing of cell plating post irradiation. Results indicate that the type of culture medium used and time-lag between irradiation, refrigeration and plating of irradiated cells do not have significant impact in culture outcomes. However, the dose of gamma radiation administered to the cells may influence the final quality of these cells if to be used as a feeder layer. (author)

Protective role of microRNA-29a in denatured dermis and skin fibroblast cells after thermal injury

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Jie Zhou

2016-03-01

Full Text Available Our previous study has suggested that downregulated microRNA (miR-29a in denatured dermis might be involved in burn wound healing. However, the exact role of miR-29a in healing of burn injury still remains unclear. Here, we found that expression of miR-29a was notably upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury, and thereafter gradually downregulated compared with control group. By contrast, the expression of collagen, type I, alpha 2 (COL1A2 and vascular endothelial growth factor (VEGF-A were first reduced and subsequently upregulated in denatured dermis tissues and skin fibroblast cells after thermal injury. We further identified COL1A2 as a novel target of miR-29a, which is involved in type I collagen synthesis, and showed that miR-29a negatively regulated the expression level of COL1A2 in skin fibroblast cells. In addition, VEGF-A, another target gene of miR-29a, was also negatively mediated by miR-29a in skin fibroblast cells. Inhibition of miR-29a expression significantly promoted the proliferation and migration of skin fibroblast cells after thermal injury, and knockdown of COL1A2 and VEGF-A reversed the effects of miR-29a on the proliferation and migration of skin fibroblast cells. Furthermore, we found that Notch2/Jagged2 signaling was involved in miR-29a response to burn wound healing. Our findings suggest that downregulated miR-29a in denatured dermis may help burn wound healing in the later phase, probably via upregulation of COL1A2 and VEGF-A expression, which can further enhance type I collagen synthesis and angiogenesis.

Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor

International Nuclear Information System (INIS)

Eardley, D.D.; Makrides, V.

1986-01-01

Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks 35 S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications.

Science.gov (United States)

Sahoo, Sambit; Ang, Lay-Teng; Cho-Hong Goh, James; Toh, Siew-Lok

2010-02-01

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications. 2009 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

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

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Sivapragasam Gothai

2016-03-01

Full Text Available 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 treatment of cuts, wounds and burns. Moringa oleifera is an herb used as 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 M. oleifera leaves extract are completely unknown. Methods: In the current study, ethyl acetate fraction of Moringa oleifera 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 M. oleifera leaves showed remarkable proliferative and migratory effect on normal human dermal fibroblasts. Conclusion: The present study suggested that ethyl acetate fraction of M. oleifera 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. [J Complement Med Res 2016; 5(1.000: 1-6

Effects of nanostructurized silicon on proliferation of stem and cancer cell.

Science.gov (United States)

Osminkina, L A; Luckyanova, E N; Gongalsky, M B; Kudryavtsev, A A; Gaydarova, A Kh; Poltavtseva, R A; Kashkarov, P K; Timoshenko, V Yu; Sukhikh, G T

2011-05-01

In vitro experiments showed that stem and cancer cells retained their viability on the surface of porous silicon with 10-100 nm nanostructures, but their proliferation was inhibited. Silicon nanoparticles of 100 nm in size obtained by mechanical grinding of porous silicon films or crystal silicon plates in a concentration below 1 mg/ml in solution did not modify viability and proliferation of mouse fibroblast and human laryngeal cancer cells. Additional ultrasonic exposure of cancer cells in the presence of 1 mg/ml silicon nanoparticles added to nutrient medium led to complete destruction of cells or to the appearance of membrane defects blocking their proliferation and initiating their apoptotic death.

Shape-dependent regulation of proliferation in normal and malignant human cells and its alteration by interferon

International Nuclear Information System (INIS)

Kulesh, D.A.; Greene, J.J.

1986-01-01

The relationship between cell morphology, proliferation, and contact inhibition was studied in normal and malignant human cells which varied in their sensitivity to contact inhibition. Their ability to proliferate was examined under conditions where the cells were constrained into different shapes by plating onto plastic surfaces coated with poly(2-hydroxyethyl methacrylate). Poly(2-hydroxyethyl methacrylate) can precisely vary the shape of cells without toxicity. Cell proliferation was quantitated by cell counts and labeling indices were determined by autoradiography. The normal JHU-1 foreskin fibroblasts and IMR-90 lung fibroblasts exhibited contact-inhibited growth with a saturation density of 2.9 X 10(5) and 2.0 X 10(5) cells/cm2, respectively. These cells also exhibited stringent dependency on cell shape with a mitotic index of less than 3% at poly(2-hydroxyethyl methacrylate) concentrations at which the cells were rounded versus a labeling index of 75-90% when the cells were flat. The malignant bladder carcinoma line RT-4 exhibited partial contact-inhibited growth. Its dependency on cell shape was less stringent than that of normal cells with a mitotic index of 37-40% when rounded and 79% when flat. The malignant fibrosarcoma line, HT1080, was not contact inhibited and was entirely shape independent with a mitotic index of 70-90% regardless of cell shape. Treatment of HT1080 cells with low concentration of human fibroblast interferon (less than 40 units/ml) restored shape-dependent proliferation while having little effect on normal cells. Subantiproliferative doses of interferon were also shown to restore contact-inhibited proliferation control to malignant cells previously lacking it

In-vitro examination of the biocompatibility of fibroblast cell lines on alloplastic meshes and sterilized polyester mosquito mesh.

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Wiessner, R; Kleber, T; Ekwelle, N; Ludwig, K; Richter, D-U

2017-06-01

The use of alloplastic implants for tissue strengthening when treating hernias is an established therapy worldwide. Despite the high incidence of hernias in Africa and Asia, the implantation of costly mesh netting is not financially feasible. Because of that various investigative groups have examined the use of sterilized mosquito netting. The animal experiments as well as the clinical trials have both shown equivalent short- and long-term results. The goal of this paper is the comparison of biocompatibility of human fibroblasts on the established commercially available nets and on sterilized polyester mosquito mesh over a period of 12 weeks. Three commercially available plastic mesh types and a gas-sterilized mosquito polyethylenterephtalate (polyester) mesh were examined. Human fibroblasts from subcutaneous healthy tissue were used. Various tests for evaluating the growth behavior and the cell morphology of human fibroblasts were conducted. The semi-quantitative (light microscopy) and qualitative (scanning electron microscopy) analyses were performed after 1 week and then again after 12 weeks. The cell proliferation and cytotoxicity of the implants were investigated with the help of the 5'-bromo-2'-deoxyuridine (BrdU)-cell proliferation test and the LDH-cytotoxicity test. The number of live cells per ml was determined with the Bürker counting chamber. In addition, analyses were made of the cell metabolism (oxidative stress) by measuring the pH value, hydrogen peroxide, and glycolysis. After 12 weeks, a proliferation of fibroblasts on all mesh is documented. No mesh showed a complete apoptosis of the cells. This qualitative observation could be confirmed quantitatively in a biochemical assay by marking the proliferating cells with BrdU. The biochemical analysis brought the proof that the materials used, including the polyester of the mosquito mesh, are not cytotoxic for the fibroblasts. The vitality of the cells was between 94 and 98%. The glucose metabolism

Multivalent conjugates of basic fibroblast growth factor enhance in vitro proliferation and migration of endothelial cells.

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Zbinden, Aline; Browne, Shane; Altiok, Eda I; Svedlund, Felicia L; Jackson, Wesley M; Healy, Kevin E

2018-05-01

Growth factors hold great promise for regenerative therapies. However, their clinical use has been halted by poor efficacy and rapid clearance from tissue, necessitating the delivery of extremely high doses to achieve clinical effectiveness which has raised safety concerns. Thus, strategies to either enhance growth factor activity at low doses or to increase their residence time within target tissues are necessary for clinical success. In this study, we generated multivalent conjugates (MVCs) of basic fibroblast growth factor (bFGF), a key growth factor involved in angiogenesis and wound healing, to hyaluronic acid (HyA) polymer chains. Multivalent bFGF conjugates (mvbFGF) were fabricated with minimal non-specific interaction observed between bFGF and the HyA chain. The hydrodynamic radii of mvbFGF ranged from ∼50 to ∼75 nm for conjugation ratios of bFGF to HyA chains at low (10 : 1) and high (30 : 1) feed ratios, respectively. The mvbFGF demonstrated enhanced bioactivity compared to unconjugated bFGF in assays of cell proliferation and migration, processes critical to angiogenesis and tissue regeneration. The 30 : 1 mvbFGF outperformed the 10 : 1 conjugate, which could be due to either FGF receptor clustering or interference with receptor mediated internalization and signal deactivation. This study simultaneously investigated the role of both protein to polymer ratio and multivalent conjugate size on their bioactivity, and determined that increasing the protein-to-polymer ratio and conjugate size resulted in greater cell bioactivity.

[The process of heme synthesis in bone marrow mesenchymal stem cells cultured under fibroblast growth factor bFGF and hypoxic conditions].

Science.gov (United States)

Poleshko, A G; Lobanok, E S; Mezhevikina, L M; Fesenko, E E; Volotkovskiĭ, I D

2014-01-01

It was demonstrated that fibroblast growth factor bFGF influences the process of heme synthesis, the proliferation activity and viability of bone marrow mesenchymal stem cells in culture under hypoxic conditions. The addition of fibroblast growth factor bFGF (7 ng/ml) to the medium under above conditions led to the accumulation of aminolevulinic acid--an early porphyrin and heme precursor, an increase in CD 71 expression--a transferrin receptor, and also a decrease in porphyrin pigments and heme contents--a late precursor and end products of heme synthesis, respectively. It was found that cultivation of the cells under hypoxic conditions and bFGF is an optimum to maintain high viability and proliferation capacity of the mesenchymal stem cells.

Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

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Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

2015-01-01

Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

Poly(ADP-ribose) polymerase inhibition reduces tumor necrosis factor-induced inflammatory response in rheumatoid synovial fibroblasts

NARCIS (Netherlands)

García, S.; Bodaño, A.; Pablos, J. L.; Gómez-Reino, J. J.; Conde, C.

2008-01-01

To investigate the effect of poly(ADP-ribose) polymerase (PARP) inhibition on the production of inflammatory mediators and proliferation in tumour necrosis factor (TNF)-stimulated fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). Cultured FLS from patients with RA were

Asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts

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Linda Yuliati

2015-12-01

Asiaticoside induces HDF proliferation and type I and III collagen synthesis in a time- and dose-dependent pattern. Asiaticoside has a similar effect as retinoic acid on type I and type III collagen synthesis.

Determination of the wound healing effect of Calendula extracts using the scratch assay with 3T3 fibroblasts.

Science.gov (United States)

Fronza, M; Heinzmann, B; Hamburger, M; Laufer, S; Merfort, I

2009-12-10

PHARMACOLOGICAL RELEVANCE: Presentation of the scratch assay as a convenient and inexpensive in vitro tool to gain first insights in the wound healing potential of plant extracts and natural compounds. The present study deals with the optimization of the scratch assay which can be used as an in vitro model for quantification of fibroblast migration to and proliferation into the wounded area. It is suitable for the first evaluation of the wound re-epithelialization potential of crude herbal extracts, isolated compounds and pharmaceutical preparations. As a proof of concept three preparations from traditional medicinal plants were investigated. Swiss 3T3 albino mouse fibroblasts were used in monolayers and platelet derived growth factor as positive control. Hexane and ethanolic extracts from Calendula officinalis and Matricaria recutita, Hypericum oil as well as the triterpenoids faradiol myristate and palmitate were studied. To differentiate between proliferation and migration antimitotic mitomycin C was added. Both extracts of Calendula officinalis stimulated proliferation and migration of fibroblasts at low concentrations, e.g. 10 microg/ml enhanced cell numbers by 64.35% and 70.53%, respectively. Inhibition of proliferation showed that this effect is mainly due to stimulation of migration. Faradiol myristate and palmitate gave comparable stimulation rates at an almost 50 microg/ml concentration, indicating that they contribute partially, but not most significantly to the wound healing effects of Calendula preparations. Extracts from Matricaria recutita were only moderately active. Hypericum oil was cytotoxic at concentrations higher than 0.5 microg/ml. The scratch assay in the present form can be used as a promising scientific approach and platform to differentiate between plant extracts known for their wound healing and their anti-inflammatory properties.

Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome

International Nuclear Information System (INIS)

Tatano, Yutaka; Fujinawa, Reiko; Kozutsumi, Yasunori; Takahashi, Tsutomu; Tsuji, Daisuke; Takeuchi, Naohiro; Tsuta, Kohji; Takada, Goro; Sakuraba, Hitoshi; Itoh, Kohji

2008-01-01

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1β (IL-1β), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1β expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression

System-wide analysis reveals a complex network of tumor-fibroblast interactions involved in tumorigenicity.

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Megha Rajaram

Full Text Available Many fibroblast-secreted proteins promote tumorigenicity, and several factors secreted by cancer cells have in turn been proposed to induce these proteins. It is not clear whether there are single dominant pathways underlying these interactions or whether they involve multiple pathways acting in parallel. Here, we identified 42 fibroblast-secreted factors induced by breast cancer cells using comparative genomic analysis. To determine what fraction was active in promoting tumorigenicity, we chose five representative fibroblast-secreted factors for in vivo analysis. We found that the majority (three out of five played equally major roles in promoting tumorigenicity, and intriguingly, each one had distinct effects on the tumor microenvironment. Specifically, fibroblast-secreted amphiregulin promoted breast cancer cell survival, whereas the chemokine CCL7 stimulated tumor cell proliferation while CCL2 promoted innate immune cell infiltration and angiogenesis. The other two factors tested had minor (CCL8 or minimally (STC1 significant effects on the ability of fibroblasts to promote tumor growth. The importance of parallel interactions between fibroblasts and cancer cells was tested by simultaneously targeting fibroblast-secreted amphiregulin and the CCL7 receptor on cancer cells, and this was significantly more efficacious than blocking either pathway alone. We further explored the concept of parallel interactions by testing the extent to which induction of critical fibroblast-secreted proteins could be achieved by single, previously identified, factors produced by breast cancer cells. We found that although single factors could induce a subset of genes, even combinations of factors failed to induce the full repertoire of functionally important fibroblast-secreted proteins. Together, these results delineate a complex network of tumor-fibroblast interactions that act in parallel to promote tumorigenicity and suggest that effective anti

Different effects of 25-kDa amelogenin on the proliferation, attachment and migration of various periodontal cells

International Nuclear Information System (INIS)

Li, Xiting; Shu, Rong; Liu, Dali; Jiang, Shaoyun

2010-01-01

Previous studies have assumed that amelogenin is responsible for the therapeutic effect of the enamel matrix derivative (EMD) in periodontal tissue healing and regeneration. However, it is difficult to confirm this hypothesis because both the EMD and the amelogenins are complex mixtures of multiple proteins. Further adding to the difficulties is the fact that periodontal tissue regeneration involves various types of cells and a sequence of associated cellular events including the attachment, migration and proliferation of various cells. In this study, we investigated the potential effect of a 25-kDa recombinant porcine amelogenin (rPAm) on primarily cultured periodontal ligament fibroblasts (PDLF), gingival fibroblasts (GF) and gingival epithelial cells (GEC). The cells were treated with 25-kDa recombinant porcine amelogenin at a concentration of 10 μg/mL. We found that rPAm significantly promoted the proliferation and migration of PDLF, but not their adhesion. Similarly, the proliferation and adhesion of GF were significantly enhanced by treatment with rPAm, while migration was greatly inhibited. Interestingly, this recombinant protein inhibited the growth rate, cell adhesion and migration of GEC. These data suggest that rPAm may play an essential role in periodontal regeneration through the activation of periodontal fibroblasts and inhibition of the cellular behaviors of gingival epithelial cells.

Monoclonal antibodies and cancer

International Nuclear Information System (INIS)

Haisma, H.J.

1987-01-01

The usefulness of radiolabeled monoclonal antibodies for imaging and treatment of human (ovarian) cancer was investigated. A review of tumor imaging with monoclonal antibodies is presented. Special attention is given to factors that influence the localization of the antibodies in tumors, isotope choice and methods of radiolabeling of the monoclonal antibodies. Two monoclonal antibodies, OC125 and OV-TL3, with high specificity for human epithelial ovarian cancer are characterized. A simple radio-iodination technique was developed for clinical application of the monoclonal antibodies. The behavior of monoclonal antibodies in human tumor xenograft systems and in man are described. Imaging of tumors is complicated because of high background levels of radioactivity in other sites than the tumor, especially in the bloodpool. A technique was developed to improve imaging of human tumor xenographs in nude mice, using subtraction of a specific and a non-specific antibody, radiolabeled with 111 In, 67 Ga and 131 I. To investigate the capability of the two monoclonal antibodies, to specifically localize in human ovarian carcinomas, distribution studies in mice bearing human ovarian carcinoma xenografts were performed. One of the antibodies, OC125, was used for distribution studies in ovarian cancer patients. OC125 was used because of availability and approval to use this antibody in patients. The same antibody was used to investigate the usefulness of radioimmunoimaging in ovarian cancer patients. The interaction of injected radiolabeled antibody OC125 with circulating antigen and an assay to measure the antibody response in ovarian cancer patients after injection of the antibody is described. 265 refs.; 30 figs.; 19 tabs

Comparisons of phenotype and immunomodulatory capacity among rhesus bone-marrow-derived mesenchymal stem/stromal cells, multipotent adult progenitor cells, and dermal fibroblasts

Science.gov (United States)

Wang, Qi; Clarkson, Christina; Graham, Melanie; Donahue, Robert; Hering, Bernhard J.; Verfaillie, Catherine M.; Bansal-Pakala, Pratima; O'Brien, Timothy D.

2015-01-01

Background Potent immunomodulatory effects have been reported for mesenchymal stem/stromal cells (MSCs), multipotent adult progenitor cells (MAPCs), and fibroblasts. However, side-by-side comparisons of these cells specifically regarding immunophenotype, gene expression, and suppression of proliferation of CD4+ and CD8+ lymphocyte populations have not been reported. Methods We developed MAPC and MSC lines from rhesus macaque bone marrow and fibroblast cell lines from rhesus dermis and assessed phenotypes based upon differentiation potential, flow cytometric analysis of immunophenotype, and quantitative RT-PCR analysis of gene expression. Using allogeneic lymphocyte proliferation assays, we compared the in vitro immunomodulatory potency of each cell type. Results and Conclusions Extensive phenotypic similarities exist among each cell type, although immunosuppressive potencies are distinct. MAPCs are most potent, and fibroblasts are the least potent cell type. All three cell types demonstrated immunomodulatory capacity such that each may have potential therapeutic applications such as in organ transplantation, where reduced local immune response is desirable. PMID:24825538

ETOH inhibits embryonic neural stem/precursor cell proliferation via PLD signaling

International Nuclear Information System (INIS)

Fujita, Yuko; Hiroyama, Masami; Sanbe, Atsushi; Yamauchi, Junji; Murase, Shoko; Tanoue, Akito

2008-01-01

While a mother's excessive alcohol consumption during pregnancy is known to have adverse effects on fetal neural development, little is known about the underlying mechanism of these effects. In order to investigate these mechanisms, we investigated the toxic effect of ethanol (ETOH) on neural stem/precursor cell (NSC) proliferation. In cultures of NSCs, phospholipase D (PLD) is activated following stimulation with epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). Exposure of NSCs to ETOH suppresses cell proliferation, while it has no effect on cell death. Phosphatidic acid (PA), which is a signaling messenger produced by PLD, reverses ETOH inhibition of NSC proliferation. Blocking the PLD signal by 1-butanol suppresses the proliferation. ETOH-induced suppression of NSC proliferation and the protective effect of PA for ETOH-induced suppression are mediated through extracellular signal-regulated kinase signaling. These results indicate that exposure to ETOH impairs NSC proliferation by altering the PLD signaling pathway

Effect of monoclonal antibodies (MoAb) to class I and class II HLA antigens on lectin- and MoAb OKT3-induced lymphocyte proliferation.

Science.gov (United States)

Akiyama, Y; Zicht, R; Ferrone, S; Bonnard, G D; Herberman, R B

1985-04-01

We have examined the effect of several monoclonal antibodies (MoAb) to monomorphic determinants of class II HLA antigens, and MoAb to monomorphic determinants of class I HLA antigens and to beta-2-microglobulin (beta 2-mu) on lectin- and MoAb OKT3-induced proliferation of human peripheral blood mononuclear cells (PBMNC) and cultured T cells (CTC). Some, but not all, anti-class II HLA MoAb inhibited the proliferative response of PBMNC to MoAb OKT3 and pokeweed mitogen (PWM). The degree of inhibitory effect varied considerably. This effect was not limited to anti-class II HLA MoAb since anti-class I HLA MoAb and anti-beta 2-mu MoAb also inhibited MoAb OKT3- or PWM-induced proliferative responses. In contrast, the response of PBMNC to phytohemagglutinin (PHA) and concanavalin A (Con A) was not blocked by any anti-class II HLA MoAb. However, some anti-class II HLA MoAb also inhibited the proliferative response of CTC plus allogeneic peripheral blood adherent accessory cells (AC) to PHA or Con A as well as to MoAb OKT3 or PWM. This may be attributable to the substantially greater class II HLA antigen expression by CTC than by fresh lymphocytes. Pretreatment of either CTC or AC with anti-class II HLA MoAb inhibited OKT3-induced proliferation. In contrast, pretreatment of CTC, but not AC, with anti-class I HLA MoAb inhibited the proliferative response of CTC to OKT3. Pretreatment of CTC with anti-class I HLA MoAb inhibited PHA-, Con A and PWM-induced proliferation, to a greater degree than the anti-class II HLA MoAb. It appears as if lymphocyte activation by different mitogens exhibits variable requirements for the presence of cells expressing major histocompatibility determinants. Binding of Ab to membrane markers may interfere with lymphocyte-AC cooperation, perhaps by inhibiting binding of mitogens to their receptors or by interfering with lymphocyte and AC function. We also have examined the role of class II HLA antigens on CTC by depleting class II HLA-positive cells

MiR-34a/miR-93 target c-Ski to modulate the proliferaton of rat cardiac fibroblasts and extracellular matrix deposition in vivo and in vitro.

Science.gov (United States)

Zhang, Chengliang; Zhang, Yanfeng; Zhu, Hong; Hu, Jiajia; Xie, Zhongshang

2018-06-01

Cardiac fibrosis is associated with diverse heart diseases. In response to different pathological irritants, cardiac fibroblasts may be induced to proliferate and differentiate into cardiac myofibroblasts, thus contributing to cardiac fibrosis. TGF-β signaling is implicated in the development of heart failure through the induction of cardiac fibrosis. C-Ski, an inhibitory regulator of TGF-β signaling, has been reported to suppress TGF-β1-induced human cardiac fibroblasts' proliferation and ECM protein increase; however, the underlying molecular mechanism needs further investigation. In the present study, we demonstrated that c-Ski could ameliorate isoproterenol (ISO)-induced rat myocardial fibrosis model and TGF-β1-induced primary rat cardiac fibroblasts' proliferation, as well as extracellular matrix (ECM) deposition. The protein level of c-Ski was dramatically decreased in cardiac fibrosis and TGF-β1-stimulated primary rat cardiac fibroblasts. In recent decades, a family of small non-coding RNA, namely miRNAs, has been reported to regulate gene expression by interacting with diverse mRNAs and inducing either translational suppression or mRNA degradation. Herein, we selected miR-34a and miR-93 as candidate miRNAs that might target to regulate c-Ski expression. After confirming that miR-34a/miR-93 targeted c-Ski to inhibit its expression, we also revealed that miR-34a/miR-93 affected TGF-β1-induced fibroblasts' proliferation and ECM deposition through c-Ski. Taken together, we demonstrated a miR-34a/miR-93-c-Ski axis which modulates TGF-β1- and ISO-induced cardiac fibrosis in vitro and in vivo; targeting the inhibitory factors of c-Ski to rescue its expression may be a promising strategy for the treatment of cardiac fibrosis. Copyright © 2018 Elsevier Inc. All rights reserved.

Improved cellular response of ion modified poly(lactic acid-co-glycolic acid) substrates for mouse fibroblast cells

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Adhikari, Ananta Raj, E-mail: aa8381@gmail.com [Department of Sciences, Wentworth Institute of Technology, Boston MA 02115 (United States); Geranpayeh, Tanya [Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States); Chu, Wei Kan [Texas Center for Superconductivity, University of Houston, Houston, TX 77204 (United States); Department of Physics, University of Houston, Houston, TX 77204 (United States); Otteson, Deborah C. [Department of Biology and Biochemistry, University of Houston, Houston, TX 77204 (United States); Department of Basic and Vision Sciences, College of Optometry, University of Houston, Houston, TX 77204 (United States)

2016-03-01

In this report, the effects of argon (Ar) ion irradiation on poly(lactic acid-co-glycolic acid) (PLGA) substrates on biocompatibility were studied. PLGA scaffold substrates were prepared by spin coating glass surfaces with PLGA dissolved in anhydrous chloroform. Previously, we showed that surface modifications of PLGA films using ion irradiation modulate the inherent hydrophobicity of PLGA surface. Here we show that with increasing ion dose (1 × 10{sup 12} to 1 × 10{sup 14} ions/cm{sup 2}), hydrophobicity and surface roughness decreased. Biocompatibility for NIH3T3 mouse fibroblast cells was increased by argon irradiation of PLGA substrates. On unirradiated PLGA films, fibroblasts had a longer doubling time and cell densities were 52% lower than controls after 48 h in vitro. Argon irradiated PLGA substrates supported growth rates similar to control. Despite differences in cell cycle kinetics, there was no detectible cytotoxicity observed on any substrate. This demonstrates that argon ion irradiation can be used to tune the surface microstructure and generate substrates that are more compatible for the cell growth and proliferation. - Highlights: • Argon irradiation modifies surface chemistry and increases hydrophilicity of poly(lactic-glycolic) acid (PLGA) films. • Both native and irradiated PLGA films were not cytotoxic for mouse fibroblasts. • Fibroblast proliferation increased on PLGA substrates modified with higher doses of Argon irradiation. • Surface modification with Argon irradiation increases biocompatibility of PLGA films.

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

Science.gov (United States)

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.

Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia.

Science.gov (United States)

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

2003-01-01

Although it is known that systemic diseases such as diabetes result in impaired wound healing, the mechanism for this impairment is not understood. Because fibroblasts are essential for wound repair, we compared the in vitro behavior of fibroblasts cultured from diabetic, leptin receptor-deficient (db/db) mice with wild-type fibroblasts from mice of the same genetic background in processes important during tissue repair. Adult diabetic mouse fibroblast migration exhibited a 75% reduction in migration compared to normal fibroblasts (P under basal or hypoxic conditions, confirming that the results from db/db fibroblasts in mature mice resulted from the diabetic state and were not because of alterations in the leptin-leptin receptor axis. Markers of cellular viability including proliferation and senescence were not significantly different between diabetic and wild-type fibroblasts. We conclude that, in vitro, diabetic fibroblasts show 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.

Detection of human cancer in an animal model using radio-labelled tumour-associated monoclonal antibodies

International Nuclear Information System (INIS)

Epenetos, A.A.; Arklie, J.; Knowles, R.W.; Bodmer, W.F.

1982-01-01

Monoclonal antibodies to epithelial-cell antigenic determinants, labelled with 123 I and 125 I, were administered parenterally to immunodeficient mice bearing human tumours derived from a human cancer cell line. Anterior, posterior and lateral radioscans of the body were taken with a gamma scintillation camera at various times from immediately to 65 days after injection. Visual displays of the images were processed by standard computer techniques. The model used a human colon-cancer cell line, HT29, and the monoclonal antibody, AUAl, which is specific to an epithelial proliferating antigen. Tumour detection was achieved in all the mice. The smallest tumour detectable appeared to be about 1 mm in diameter. The degree of antibody uptake in a tumour depended on its size and the blood supply of its surrounding tissues. (author)

Quiescence does not affect p53 and stress response by irradiation in human lung fibroblasts

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Dai, Jiawen [Molecular Radiobiology Laboratory, Division of Cellular and Molecular Research (Singapore); Itahana, Koji, E-mail: koji.itahana@duke-nus.edu.sg [Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School (Singapore); Baskar, Rajamanickam, E-mail: r.baskar@nccs.com.sg [Molecular Radiobiology Laboratory, Division of Cellular and Molecular Research (Singapore); Department of Radiation Oncology, National Cancer Centre (Singapore)

2015-02-27

Cells in many organs exist in both proliferating and quiescent states. Proliferating cells are more radio-sensitive, DNA damage pathways including p53 pathway are activated to undergo either G{sub 1}/S or G{sub 2}/M arrest to avoid entering S and M phase with DNA damage. On the other hand, quiescent cells are already arrested in G{sub 0}, therefore there may be fundamental difference of irradiation response between proliferating and quiescent cells, and this difference may affect their radiosensitivity. To understand these differences, proliferating and quiescent human normal lung fibroblasts were exposed to 0.10–1 Gy of γ-radiation. The response of key proteins involved in the cell cycle, cell death, and metabolism as well as histone H2AX phosphorylation were examined. Interestingly, p53 and p53 phosphorylation (Ser-15), as well as the cyclin-dependent kinase inhibitors p21 and p27, were induced similarly in both proliferating and quiescent cells after irradiation. Furthermore, the p53 protein half-life, and expression of cyclin A, cyclin E, proliferating cell nuclear antigen (PCNA), Bax, or cytochrome c expression as well as histone H2AX phosphorylation were comparable after irradiation in both phases of cells. The effect of radioprotection by a glycogen synthase kinase 3β inhibitor on p53 pathway was also similar between proliferating and quiescent cells. Our results showed that quiescence does not affect irradiation response of key proteins involved in stress and DNA damage at least in normal fibroblasts, providing a better understanding of the radiation response in quiescent cells, which is crucial for tissue repair and regeneration. - Highlights: • p53 response by irradiation was similar between proliferating and quiescent cells. • Quiescent cells showed similar profiles of cell cycle proteins after irradiation. • Radioprotection of GSK-3β inhibitor caused similar effects between these cells. • Quiescence did not affect p53 response despite its

Quiescence does not affect p53 and stress response by irradiation in human lung fibroblasts

International Nuclear Information System (INIS)

Dai, Jiawen; Itahana, Koji; Baskar, Rajamanickam

2015-01-01

Cells in many organs exist in both proliferating and quiescent states. Proliferating cells are more radio-sensitive, DNA damage pathways including p53 pathway are activated to undergo either G 1 /S or G 2 /M arrest to avoid entering S and M phase with DNA damage. On the other hand, quiescent cells are already arrested in G 0 , therefore there may be fundamental difference of irradiation response between proliferating and quiescent cells, and this difference may affect their radiosensitivity. To understand these differences, proliferating and quiescent human normal lung fibroblasts were exposed to 0.10–1 Gy of γ-radiation. The response of key proteins involved in the cell cycle, cell death, and metabolism as well as histone H2AX phosphorylation were examined. Interestingly, p53 and p53 phosphorylation (Ser-15), as well as the cyclin-dependent kinase inhibitors p21 and p27, were induced similarly in both proliferating and quiescent cells after irradiation. Furthermore, the p53 protein half-life, and expression of cyclin A, cyclin E, proliferating cell nuclear antigen (PCNA), Bax, or cytochrome c expression as well as histone H2AX phosphorylation were comparable after irradiation in both phases of cells. The effect of radioprotection by a glycogen synthase kinase 3β inhibitor on p53 pathway was also similar between proliferating and quiescent cells. Our results showed that quiescence does not affect irradiation response of key proteins involved in stress and DNA damage at least in normal fibroblasts, providing a better understanding of the radiation response in quiescent cells, which is crucial for tissue repair and regeneration. - Highlights: • p53 response by irradiation was similar between proliferating and quiescent cells. • Quiescent cells showed similar profiles of cell cycle proteins after irradiation. • Radioprotection of GSK-3β inhibitor caused similar effects between these cells. • Quiescence did not affect p53 response despite its known role in

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

International Nuclear Information System (INIS)

Fuchigami, Takao; Kibe, Toshiro; Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio; Nishizawa, Yoshiaki; Hijioka, Hiroshi; Fujii, Tomomi; Ueda, Masahiro; Nakamura, Norifumi; Kiyono, Tohru; Kishida, Michiko

2014-01-01

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

Regulation of IL-6 and IL-8 production by reciprocal cell-to-cell interactions between tumor cells and stromal fibroblasts through IL-1α in ameloblastoma

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Fuchigami, Takao [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kibe, Toshiro [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Koyama, Hirofumi; Kishida, Shosei; Iijima, Mikio [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Nishizawa, Yoshiaki [Kagoshima University Faculty of Medicine, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Hijioka, Hiroshi; Fujii, Tomomi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Ueda, Masahiro [Natural Science Centre for Research and Education, Kagoshima University, 1-21-24 Koorimoto, Kagoshima 890-8580 (Japan); Nakamura, Norifumi [Department of Oral and Maxillofacial Surgery, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kiyono, Tohru [Department of Virology, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuouku, Tokyo 104-0045 (Japan); Kishida, Michiko, E-mail: kmichiko@m2.kufm.kagoshima-u.ac.jp [Department of Biochemistry and Genetics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)

2014-09-05

Highlights: • We studied the interaction between tumor cells and fibroblasts in ameloblastoma. • AM-3 ameloblastoma cells secreted significantly high IL-1α levels. • IL-1α derived from AM-3 cells promoted IL-6 and IL-8 secretion of fibroblasts. • IL-6 and IL-8 activated the cellular motility and proliferation of AM-3 cells. - Abstract: Ameloblastoma is an odontogenic benign tumor that occurs in the jawbone, which invades bone and reoccurs locally. This tumor is treated by wide surgical excision and causes various problems, including changes in facial countenance and mastication disorders. Ameloblastomas have abundant tumor stroma, including fibroblasts and immune cells. Although cell-to-cell interactions are considered to be involved in the pathogenesis of many diseases, intercellular communications in ameloblastoma have not been fully investigated. In this study, we examined interactions between tumor cells and stromal fibroblasts via soluble factors in ameloblastoma. We used a human ameloblastoma cell line (AM-3 ameloblastoma cells), human fibroblasts (HFF-2 fibroblasts), and primary-cultured fibroblasts from human ameloblastoma tissues, and analyzed the effect of ameloblastoma-associated cell-to-cell communications on gene expression, cytokine secretion, cellular motility and proliferation. AM-3 ameloblastoma cells secreted higher levels of interleukin (IL)-1α than HFF-2 fibroblasts. Treatment with conditioned medium from AM-3 ameloblastoma cells upregulated gene expression and secretion of IL-6 and IL-8 of HFF-2 fibroblasts and primary-cultured fibroblast cells from ameloblastoma tissues. The AM3-stimulated production of IL-6 and IL-8 in fibroblasts was neutralized by pretreatment of AM-3 cells with anti-IL-1α antibody and IL-1 receptor antagonist. Reciprocally, cellular motility of AM-3 ameloblastoma cells was stimulated by HFF-2 fibroblasts in IL-6 and IL-8 dependent manner. In conclusion, ameloblastoma cells and stromal fibroblasts behave

The in vitro viability and growth of fibroblasts cultured in the presence of different bone grafting materials (NanoBone and Straumann Bone Ceramic).

Science.gov (United States)

Kauschke, E; Rumpel, E; Fanghänel, J; Bayerlein, T; Gedrange, T; Proff, P

2006-02-01

Different clinical applications, including dentistry, are making increasing demands on bone grafting material. In the present study we have analysed the viability, proliferation and growth characteristics of fibroblasts cultured in vitro together with two different bone grafting materials, NanoBone and Straumann Bone Ceramic, over a period of 24 and 28 days respectively. Viability was measured at least every 72 hours by using the alamarBlue assay, a test that measures quantitatively cell proliferation and viability but does not require cell fixation or extraction. After one week of culture fibroblast viability was as high as in controls for both grafting materials and remained high (> 90%) for the duration of the experiment. Cell growth was evaluated microscopically. Scanning electron microscopy revealed a dense fibroblast growth at the surface of both bone grafting materials after three weeks of in vitro culture. Generally, our in vitro analyses contribute to further insights into cell - scaffold interactions.

Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

Science.gov (United States)

Gache, Yannick; Brellier, Florence; Rouanet, Sophie; Al-Qaraghuli, Sahar; Goncalves-Maia, Maria; Burty-Valin, Elodie; Barnay, Stéphanie; Scarzello, Sabine; Ruat, Martial; Sevenet, Nicolas; Avril, Marie-Françoise; Magnaldo, Thierry

2015-01-01

Basal cell carcinoma (BCC) is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH). PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS), a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH) expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis. Our findings

Fibroblastic and myofibroblastic tumors of the head and neck: Comprehensive imaging-based review with pathologic correlation

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Hourani, Roula, E-mail: rh64@aub.edu.lb [Department of Diagnostic Radiology, American University of Beirut Medical Center, Beirut (Lebanon); Taslakian, Bedros, E-mail: bt05@aub.edu.lb [Department of Diagnostic Radiology, American University of Beirut Medical Center, Beirut (Lebanon); Shabb, Nina S., E-mail: ns04@aub.edu.lb [Department of Pathology and Laboratory Medicine, American University of Beirut Medical Center, Beirut (Lebanon); Nassar, Lara, E-mail: ln07@aub.edu.lb [Department of Diagnostic Radiology, American University of Beirut Medical Center, Beirut (Lebanon); Hourani, Mukbil H., E-mail: mh17@aub.edu.lb [Department of Diagnostic Radiology, American University of Beirut Medical Center, Beirut (Lebanon); Moukarbel, Roger, E-mail: rm17@aub.edu.lb [Department of Otolaryngology – Head and Neck Surgery, American University of Beirut Medical Center, Beirut (Lebanon); Sabri, Alain, E-mail: as71@aub.edu.lb [Department of Otolaryngology – Head and Neck Surgery, American University of Beirut Medical Center, Beirut (Lebanon); Rizk, Toni, E-mail: tonirisk@hotmail.com [Department of Neurosurgery, Hôtel-Dieu de France, Saint-Joseph University, Beirut (Lebanon)

2015-02-15

Highlights: • Almost all fibroblastic tumors are evaluated with non-invasive imaging. • Radiologists should be familiar with the imaging appearance of fibroblastic tumors. • Most appropriate initial examination when fibromatosis coli suspected is ultrasound. • Most common location of ossifying fibromas is the tooth-bearing regions. - Abstract: Fibroblastic and myofibroblastic tumors of the head and neck are a heterogeneous group of disorders characterized by the proliferation of fibroblasts, myofibroblasts, or both. These tumors may be further subclassified on the basis of their behavior as benign, intermediate with malignant potential, or malignant. There are different types of fibroblastic and myofibroblastic tumors that can involve the head and neck including desmoid-type fibromatosis, solitary fibrous tumor, myofibroma/myofibromatosis, nodular fasciitis, nasopharyngeal angiofibroma, fibrosarcoma, dermatofibrosarcoma protuberans, fibromatosis coli, inflammatory myofibroblastic tumor, ossifying fibroma, fibrous histiocytoma, nodular fasciitis, fibromyxoma, hyaline fibromatosis and fibrous hamartoma. Although the imaging characteristics of fibroblastic and myofibroblastic tumors of the head and neck are nonspecific, imaging plays a pivotal role in the noninvasive diagnosis and characterization of these tumors, providing information about the constitution of tumors, their extension and invasion of adjacent structures. Correlation with the clinical history may help limit the differential diagnosis and radiologists should be familiar with the imaging appearance of these tumors to reach an accurate diagnosis.

Fibroblastic and myofibroblastic tumors of the head and neck: Comprehensive imaging-based review with pathologic correlation

International Nuclear Information System (INIS)

Hourani, Roula; Taslakian, Bedros; Shabb, Nina S.; Nassar, Lara; Hourani, Mukbil H.; Moukarbel, Roger; Sabri, Alain; Rizk, Toni

2015-01-01

Highlights: • Almost all fibroblastic tumors are evaluated with non-invasive imaging. • Radiologists should be familiar with the imaging appearance of fibroblastic tumors. • Most appropriate initial examination when fibromatosis coli suspected is ultrasound. • Most common location of ossifying fibromas is the tooth-bearing regions. - Abstract: Fibroblastic and myofibroblastic tumors of the head and neck are a heterogeneous group of disorders characterized by the proliferation of fibroblasts, myofibroblasts, or both. These tumors may be further subclassified on the basis of their behavior as benign, intermediate with malignant potential, or malignant. There are different types of fibroblastic and myofibroblastic tumors that can involve the head and neck including desmoid-type fibromatosis, solitary fibrous tumor, myofibroma/myofibromatosis, nodular fasciitis, nasopharyngeal angiofibroma, fibrosarcoma, dermatofibrosarcoma protuberans, fibromatosis coli, inflammatory myofibroblastic tumor, ossifying fibroma, fibrous histiocytoma, nodular fasciitis, fibromyxoma, hyaline fibromatosis and fibrous hamartoma. Although the imaging characteristics of fibroblastic and myofibroblastic tumors of the head and neck are nonspecific, imaging plays a pivotal role in the noninvasive diagnosis and characterization of these tumors, providing information about the constitution of tumors, their extension and invasion of adjacent structures. Correlation with the clinical history may help limit the differential diagnosis and radiologists should be familiar with the imaging appearance of these tumors to reach an accurate diagnosis

Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

International Nuclear Information System (INIS)

Yoshito, Daniele

2011-01-01

For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

An in vitro evaluation of cytotoxicity of curcumin against human dental pulp fibroblasts

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Praveenkumar S Mandrol

2016-01-01

Full Text Available Objective: The objective of this study was to evaluate the cytotoxicity of curcumin to primary dental pulp fibroblasts in vitro. Materials and Methods: Dental pulp fibroblasts from primary maxillary central incisors were cultured and used for cytotoxicity tests after the fourth passage. Ninety-five percent curcumin was diluted with dimethylsulfoxide to prepare 100%, 50%, and 25% concentrations. Each concentration of curcumin was added in triplicate into 96-well microtiter plate containing the fibroblast culture at 104/well. Cells without treatment served as a control group. The number of viable cells after 48 hrs incubation at 37°C in a humidified atmosphere of 5 % CO2 and 95 % air was determined by the 3-(4, 5-dimethyl-thiazol-2-yl-2, 5-diphenyl-tetrazolium bromide (MTT assay. The relative viability of pulp cells was expressed as color intensity of the number in the experimental wells relative to that of the control group. Absorbances were read at 492 nm on a microplate reader with a background subtraction at 620 nm. Results: Cell viability of primary dental pulp fibroblasts to 25%, 50%, and 100% curcumin concentration was 174%, 310%, and 317%, respectively. Conclusions: Curcumin promotes cell viability and induces proliferation of primary dental pulp fibroblasts and has the potential to be developed into an economical and reliable medicament for vital pulp therapy.

EDA-containing fibronectin increases proliferation of embryonic stem cells.

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Noelia Losino

Full Text Available Embryonic stem cells (ESC need a set of specific factors to be propagated. They can also grow in conditioned medium (CM derived from a bovine granulosa cell line BGC (BGC-CM, a medium that not only preserves their main features but also increases ESC´s proliferation rate. The mitogenic properties of this medium were previously reported, ascribing this effect to an alternative spliced generated fibronectin isoform that contains the extra domain A (FN EDA(+. Here, we investigated if the FN EDA(+ isoform increased proliferation of mouse and human ES cells. We analyzed cell proliferation using conditioned media produced by different mouse embryonic fibroblast (MEF lines genetically engineered to express FN constitutively including or excluding the EDA domain (FN EDA(-, and in media supplemented with recombinant peptides containing or not the EDA. We found that the presence of EDA in the medium increased mouse and human ESC's proliferation rate. Here we showed for the first time that this FN isoform enhances ESC's proliferation. These findings suggest a possible conserved behavior for regulation of ES cells proliferation by this FN isoform and could contribute to improve their culturing conditions both for research and cell therapy.

Sol-gel derived bioactive coating on zirconia: Effect on flexural strength and cell proliferation.

Science.gov (United States)

Shahramian, Khalil; Leminen, Heidi; Meretoja, Ville; Linderbäck, Paula; Kangasniemi, Ilkka; Lassila, Lippo; Abdulmajeed, Aous; Närhi, Timo

2017-11-01

The purpose of this study was to evaluate the effect of sol-gel derived bioactive coatings on the biaxial flexural strength and fibroblast proliferation of zirconia, aimed to be used as an implant abutment material. Yttrium stabilized zirconia disc-shaped specimens were cut, ground, sintered, and finally cleansed ultrasonically in each of acetone and ethanol for 5 minutes. Three experimental groups (n = 15) were fabricated, zirconia with sol-gel derived titania (TiO 2 ) coating, zirconia with sol-gel derived zirconia (ZrO 2 ) coating, and non-coated zirconia as a control. The surfaces of the specimens were analyzed through images taken using a scanning electron microscope (SEM), and a non-contact tapping mode atomic force microscope (AFM) was used to record the surface topography and roughness of the coated specimens. Biaxial flexural strength values were determined using the piston-on-three ball technique. Human gingival fibroblast proliferation on the surface of the specimens was evaluated using AlamarBlue assay™. Data were analyzed using a one-way analysis of variance (ANOVA) followed by Tukey's post-hoc test. Additionally, the biaxial flexural strength data was also statistically analyzed with the Weibull distribution. The biaxial flexural strength of zirconia specimens was unaffected (p > 0.05). Weibull modulus of TiO 2 coated and ZrO 2 coated groups (5.7 and 5.4, respectively) were lower than the control (8.0). Specimens coated with ZrO 2 showed significantly lower fibroblast proliferation compared to other groups (p sol-gel derived coatings have no influence on the flexural strength of zirconia. ZrO 2 coated specimens showed significantly lower cell proliferation after 12 days than TiO 2 coated or non-coated control. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2401-2407, 2017. © 2016 Wiley Periodicals, Inc.

Uses of monoclonal antibody 8H9

Science.gov (United States)

Cheung, Nai-Kong V.

2013-04-09

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

Kallikrein-related peptidase 4 induces cancer-associated fibroblast features in prostate-derived stromal cells.

Science.gov (United States)

Kryza, Thomas; Silva, Lakmali M; Bock, Nathalie; Fuhrman-Luck, Ruth A; Stephens, Carson R; Gao, Jin; Samaratunga, Hema; Lawrence, Mitchell G; Hooper, John D; Dong, Ying; Risbridger, Gail P; Clements, Judith A

2017-10-01

The reciprocal communication between cancer cells and their microenvironment is critical in cancer progression. Although involvement of cancer-associated fibroblasts (CAF) in cancer progression is long established, the molecular mechanisms leading to differentiation of CAFs from normal fibroblasts are poorly understood. Here, we report that kallikrein-related peptidase-4 (KLK4) promotes CAF differentiation. KLK4 is highly expressed in prostate epithelial cells of premalignant (prostatic intraepithelial neoplasia) and malignant lesions compared to normal prostate epithelia, especially at the peristromal interface. KLK4 induced CAF-like features in the prostate-derived WPMY1 normal stromal cell line, including increased expression of alpha-smooth muscle actin, ESR1 and SFRP1. KLK4 activated protease-activated receptor-1 in WPMY1 cells increasing expression of several factors (FGF1, TAGLN, LOX, IL8, VEGFA) involved in prostate cancer progression. In addition, KLK4 induced WPMY1 cell proliferation and secretome changes, which in turn stimulated HUVEC cell proliferation that could be blocked by a VEGFA antibody. Importantly, the genes dysregulated by KLK4 treatment of WPMY1 cells were also differentially expressed between patient-derived CAFs compared to matched nonmalignant fibroblasts and were further increased by KLK4 treatment. Taken together, we propose that epithelial-derived KLK4 promotes tumour progression by actively promoting CAF differentiation in the prostate stromal microenvironment. © 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Enriched Astaxanthin Extract from Haematococcus pluvialis Augments Growth Factor Secretions to Increase Cell Proliferation and Induces MMP1 Degradation to Enhance Collagen Production in Human Dermal Fibroblasts

Directory of Open Access Journals (Sweden)

Hsin-Yu Chou

2016-06-01

Full Text Available Among many antioxidants that are used for the repairing of oxidative stress induced skin damages, we identified the enriched astaxanthin extract (EAE from Haematococcus pluvialis as a viable ingredient. EAE was extracted from the red microalgae through supercritical fluid carbon dioxide extraction. To compare the effectiveness, EAE wastreated on human dermal fibroblasts with other components, phorbol 12-myristate 13-acetate (PMA, and doxycycline. With sirius red staining and quantitative real-time polymerase chain reaction (qRT-PCR, we found that PMA decreased the collagen concentration and production while overall the addition of doxycycline and EAE increased the collagen concentration in a trial experiments. EAE increased collagen contents through inhibited MMP1 and MMP3 mRNA expression and induced TIMP1, the antagonists of MMPs protein, gene expression. As for when tested for various proteins through western blotting, it was seen that the addition of EAE increased the expression of certain proteins that promote cell proliferation. Testing those previous solutions using growth factor assay, it was noticeable that EAE had a positive impact on cell proliferation and vascular endothelial growth factor (VEGF than doxycycline, indicating that it was a better alternative treatment for collagen production. To sum up, the data confirmed the possible applications as medical cosmetology agentsand food supplements.

Scleroderma keratinocytes promote fibroblast activation independent of transforming growth factor beta.

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McCoy, Sara S; Reed, Tamra J; Berthier, Celine C; Tsou, Pei-Suen; Liu, Jianhua; Gudjonsson, Johann E; Khanna, Dinesh; Kahlenberg, J Michelle

2017-11-01

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oup.com

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

Science.gov (United States)

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.

Laminin peptide YIGSR induces collagen synthesis in Hs27 human dermal fibroblasts

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Yoon, Jong Hyuk; Kim, Jaeyoon; Lee, Hyeongjoo [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, So Young [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Jang, Hwan-Hee [Functional Food and Nutrition Division, Department of Agrofood Resources, Rural Development Administration, Suwon 441-853 (Korea, Republic of); Ryu, Sung Ho [Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784 (Korea, Republic of); Kim, Beom Joon [Department of Dermatology, Chung-Ang University College of Medicine, Seoul 156-756 (Korea, Republic of); Department of Convergence Medicine and Pharmaceutical Biosciences, Graduate School, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Taehoon G., E-mail: taehoon@novacelltech.com [NovaCell Technology Inc., Pohang, Kyungbuk 790-784 (Korea, Republic of)

2012-11-23

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

Differential Gene Expression of Fibroblasts: Keloid versus Normal

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Michael F. Angel

2002-11-01

Full Text Available Abstract: This study investigated gene regulation and unique gene products in both keloid (KDF and normal (NDF dermal fibroblasts in established cell lines. For gene regulation, NDF versus KDF were compared using Clontech's Atlas™ Human cDNA Expression Array while unique gene products were studied using RNA Fingerprinting Kit. RNA from each sample was converted to cDNA using oligo-dT primers. Down-regulated genes using Atlas Array in KDF were 1 60 S ribosomal protein, 2 Thioredoxin dependent peroxidase, 3 Nuclease sensitive element DNA binding protein, 4 c-myc purine-binding transcription factor, 5 c-AMP dependent protein kinase, and, 6 Heat Shock Protein 90 kDa. Genes that are up regulated in KDF were 1 Tubulin and 2 Heat Shock Protein 27 kDa. With the differential display, we found 17 bands unique to both KDF and NDF. The specific gene and the manner in which they were differentially regulated have direct implications to understanding keloid fibroblast proliferation.

Redox-active cerium oxide nanoparticles protect human dermal fibroblasts from PQ-induced damage

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Claudia von Montfort

2015-04-01

Full Text Available 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.

Prognosis in monoclonal proteinaemia

NARCIS (Netherlands)

Schaar, Cornelis Gerardus

2006-01-01

Monoclonal proteinaemia (M-proteinemia) is associated with multiple myeloma (MM) or other hematological malignancies. In the absence of these diseases the term MGUS (Monoclonal Gammopathy of Undetermined Significance) is used. During 1991-1993 1464 patients with newly diagnosed M-proteinemia in the

Uses of monoclonal antibody 8H9

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Cheung, Nai-Kong V.

2018-04-10

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

International Nuclear Information System (INIS)

Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y.

1991-01-01

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of [3H]thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3) and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of [3H]thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity

Regulation of Hepatic Stellate Cells and Fibrogenesis by Fibroblast Growth Factors

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Justin D. Schumacher

2016-01-01

Full Text Available Fibroblast growth factors (FGFs are a family of growth factors critically involved in developmental, physiological, and pathological processes, including embryogenesis, angiogenesis, wound healing, and endocrine functions. In the liver, several FGFs are produced basally by hepatocytes and hepatic stellate cells (HSCs. Upon insult to the liver, expression of FGFs in HSCs is greatly upregulated, stimulating hepatocyte regeneration and growth. Various FGF isoforms have also been shown to directly induce HSC proliferation and activation thereby enabling autocrine and paracrine regulation of HSC function. Regulation of HSCs by the endocrine FGFs, namely, FGF15/19 and FGF21, has also recently been identified. With the ability to modulate HSC proliferation and transdifferentiation, targeting FGF signaling pathways constitutes a promising new therapeutic strategy to treat hepatic fibrosis.

Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

Science.gov (United States)

Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

2016-01-13

The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds.

Action of low-power laser irradiation on the proliferation of human gingival fibroblasts in vitro

Science.gov (United States)

Almeida-Lopes, Luciana; Jaeger, Marcia M. M.; Brugnera, Aldo, Jr.; Rigau, Josepa

1998-04-01

The low level power laser has been used in dental treatments aiming to improve tissue healing. An in vitro study was performed to analyze the laser influence on gingival fibroblast. A human gingival fibroblast culture (LMF) was produced in DME medium with 10% bovine fetal serum (BFS) cells (LMF) were allocated in Petri plates and cultured in different SFB concentrations (0%, 5% e 10%). After 48 hours the plates were divided in 9 groups: 3 control: 3 irradiated by 635 nm laser; and 3 irradiated by 780 nm laser. The cultured cells received 4 applications, in 12 hours intervals, with energy dosage of 2 joules for each plate, by means of a punctual technique. The growth curves showed that the growth levels were lower in low BFS concentrations. The irradiation with laser accelerated the growth rate in all groups. Additionally, the number of cells developed in low BFS concentration (5%) and irradiated was similar to the number of control cells developed in ideal conditions (10% BFS). There was no statistically significant differences between the effects of the two types of laser studied.

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

Science.gov (United States)

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.

In-vitro inhibition of IFNγ+ iTreg mediated by monoclonal antibodies against cell surface determinants essential for iTreg function

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Daniel Volker

2012-08-01

Full Text Available Abstract Background IFNγ-producing CD4+CD25+Foxp3+ PBL represent a subtype of iTreg that are associated with good long-term graft outcome in renal transplant recipients and suppress alloresponses in-vitro. To study the mechanism of immunosuppression, we attempted to block cell surface receptors and thereby inhibited the function of this iTreg subset in-vitro using monoclonal antibodies and recombinant proteins. Methods PBL of healthy control individuals were stimulated polyclonally in-vitro in the presence of monoclonal antibodies or recombinant proteins against/of CD178, CD152, CD279, CD28, CD95, and HLA-DR. Induction of IFNγ+ iTreg and proliferation of effector cells was determined using four-color fluorescence flow cytometry. Blockade of iTreg function was analyzed using polyclonally stimulated co-cultures with separated CD4+CD25+CD127-IFNγ+ PBL. Results High monoclonal antibody concentrations inhibited the induction of CD4+CD25+Foxp3+IFNγ+ PBL (anti-CD152, anti-CD279, anti-CD95: p +CD25+CD127-IFNγ+ PBL (anti-CD178, anti-CD152, anti-CD279, anti-CD95: p +CD25+Foxp3+IFNγ+ PBL (rCD152 and rCD95: p +CD25+CD127-IFNγ+ PBL showed lower cell proliferation than co-cultures with CD4+CD25+CD127-IFNγ- PBL (p +CD25+CD127-IFNγ- PBL-containing co-cultures in the presence of monoclonal antibody (anti-CD28, anti-CD152, anti-CD279: p +CD25+CD127-IFNγ+ PBL (with the exception anti-CD28 monoclonal antibody: p +CD25+CD127-IFNγ- PBL but do not efficiently block suppressive iTreg function in co-cultures with CD4+CD25+CD127-IFNγ+ PBL. Conclusions CD178, CD152, CD279, CD28, CD95, and HLA-DR determinants are important for induction and suppressive function of IFNγ+ iTreg.

Effect of low-power red light laser irradiation on the viability of human skin fibroblast

Energy Technology Data Exchange (ETDEWEB)

Bednarska, K.; Rozga, B.; Leyko, W.; Bryszewska, M. [Institute of Biophysics, University of Lodz (Poland); Kolodziejczyk, K.; Szosland, D. [Diabetological Clinic, Medical Academy of Lodz (Poland)

1998-10-01

Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm{sup 2}). The energy densities were 2 J/cm{sup 2} and 12 J/cm{sup 2}, respectively, and the irradiation was carried out at a temperature of 22 C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 C, in the presence of 5% CO{sub 2}, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation. (orig.) With 4 figs., 2 tabs., 13 refs.

Colorectal cancer cell-derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway.

Science.gov (United States)

Dai, Guangyao; Yao, Xiaoguang; Zhang, Yubin; Gu, Jianbin; Geng, Yunfeng; Xue, Fei; Zhang, Jingcheng

2018-04-01

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs. Copyright © 2018 Société Française du Cancer. Published by Elsevier Masson SAS. All rights reserved.

Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma.

Science.gov (United States)

Wu, Xin; Serna, Vanida A; Thomas, Justin; Qiang, Wenan; Blumenfeld, Michael L; Kurita, Takeshi

2017-12-15

Recent genomic studies have identified subtypes of uterine leiomyoma (LM) with distinctive genetic alterations. Here, we report the elucidation of the biological characteristics of the two most prevalent uterine leiomyoma subtypes, MED12-mutant (MED12-LM) and HMGA2-overexpressing (HMGA2-LM) uterine leiomyomas. Because each tumor carries only one genetic alteration, both subtypes are considered to be monoclonal. Approximately 90% of cells in HMGA2-uterine leiomyoma were smooth muscle cells (SMC) with HMGA2 overexpression. In contrast, MED12-LM consisted of similar numbers of SMC and non-SMC, which were mostly tumor-associated fibroblasts (TAF). Paradoxically, TAF carried no mutations in MED12, suggesting an interaction between SMC and TAF to coordinate their growth. The higher amount of extracellular matrix in MED12-LM than HMGA2-LM was partially due to the high concentration of collagen-producing TAF. SMC growth in a xenograft assay was driven by progesterone in both uterine leiomyoma subtypes. In contrast, TAF in MED12-LM proliferated in response to estradiol, whereas progesterone had no effect. The high concentration of estrogen-responsive TAF in MED12-LM explains the inconsistent discoveries between in vivo and in vitro studies on the mitogenic effect of estrogen and raises questions regarding the accuracy of previous studies utilizing MED12-LM cell culture. In addition, the differential effects of estradiol and progesterone on these uterine leiomyoma subtypes emphasize the importance of subtypes and genotypes in designing nonsurgical therapeutic strategies for uterine leiomyoma. Cancer Res; 77(24); 6891-901. ©2017 AACR . ©2017 American Association for Cancer Research.

Quantitative morphological analysis of proliferating and nonproliferating subpopulations of IMR-90 fibroblasts during aging in vitro

International Nuclear Information System (INIS)

Pool, T.B.; Heitman, T.O.; Buck, M.A.

1982-01-01

Early-, mid- and late-passage cultures (population doubling levels 12, 35, and 51, respectively) of IMR-90 fibroblasts were exposed to 3 H-thymidine for 48 h prior to fixation in situ for morphometric analysis in order to determine quantitatively what ultrastructural changes accompany the loss of proliferative capacity during aging in vitro. Analysis of autoradiographs, both at the light and electron microscopic levels, with an image analyzer followed by ANOVA statistical scrutiny demonstrated that a significant increase in relative cell area, an indicator of cell size, was characteristic of cells unable to incorporate 3 H-TdR at both mid- and late-passage, but not at early-passage levels. Nuclear size also increased significantly with progressive passage level but was not related to proliferative capacity. No significant difference in the area fraction of nucleoli per unit area of nucleus or of mitochondria, Golgi, or lysosomes was seen in either subpopulation at any passage level. Dilated cisternae of rough endoplasmic reticulum in early-passage cells were seen if cells were harvested with trypsin and fixed either before or after centrifugation, but were not seen in labeled or unlabeled cells from any passage level when cultures were fixed in situ. We conclude that a significant increase in cell size is the only significant morphological change associated with the loss of proliferative capacity of IRM-90 fibroblasts. Furthermore, our data indicate that there is no accumulation of secondary lysosomes in human diploid fibroblasts during aging in vitro; we therefore cannot support any hypothesis of aging or proliferative decline that is based mechanistically upon this phenomenon

Remodeling by fibroblasts alters the rate-dependent mechanical properties of collagen.

Science.gov (United States)

Babaei, Behzad; Davarian, Ali; Lee, Sheng-Lin; Pryse, Kenneth M; McConnaughey, William B; Elson, Elliot L; Genin, Guy M

2016-06-01

The ways that fibroblasts remodel their environment are central to wound healing, development of musculoskeletal tissues, and progression of pathologies such as fibrosis. However, the changes that fibroblasts make to the material around them and the mechanical consequences of these changes have proven difficult to quantify, especially in realistic, viscoelastic three-dimensional culture environments, leaving a critical need for quantitative data. Here, we observed the mechanisms and quantified the mechanical effects of fibroblast remodeling in engineered tissue constructs (ETCs) comprised of reconstituted rat tail (type I) collagen and human fibroblast cells. To study the effects of remodeling on tissue mechanics, stress-relaxation tests were performed on ETCs cultured for 24, 48, and 72h. ETCs were treated with deoxycholate and tested again to assess the ECM response. Viscoelastic relaxation spectra were obtained using the generalized Maxwell model. Cells exhibited viscoelastic damping at two finite time constants over which the ECM showed little damping, approximately 0.2s and 10-30s. Different finite time constants in the range of 1-7000s were attributed to ECM relaxation. Cells remodeled the ECM to produce a relaxation time constant on the order of 7000s, and to merge relaxation finite time constants in the 0.5-2s range into a single time content in the 1s range. Results shed light on hierarchical deformation mechanisms in tissues, and on pathologies related to collagen relaxation such as diastolic dysfunction. As fibroblasts proliferate within and remodel a tissue, they change the tissue mechanically. Quantifying these changes is critical for understanding wound healing and the development of pathologies such as cardiac fibrosis. Here, we characterize for the first time the spectrum of viscoelastic (rate-dependent) changes arising from the remodeling of reconstituted collagen by fibroblasts. The method also provides estimates of the viscoelastic spectra of

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

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Zhu, Zhong Xin [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Sun, Cong Cong [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Zheng, Jia Yong [Wenzhou People' s Hospital, Wenzhou, Zhejiang (China); Zhou, Xuan [Ningbo First Hospital, Ningbo, Zhejiang (China); Cong, Wei Tao [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Li, Xiao Kun, E-mail: proflxk@163.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China); Jin, Li Tai, E-mail: jin_litai@126.com [School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang (China)

2017-06-15

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. Western 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.

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

International Nuclear Information System (INIS)

Zhu, Zhong Xin; Sun, Cong Cong; Ting Zhu, Yu; Wang, Ying; Wang, Tao; Chi, Li Sha; Cai, Wan Hui; Zheng, Jia Yong; Zhou, Xuan; Cong, Wei Tao; Li, Xiao Kun; Jin, Li Tai

2017-01-01

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. Western 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.

Cleaved CD147 shed from the surface of malignant melanoma cells activates MMP2 produced by fibroblasts.

Science.gov (United States)

Hatanaka, Miho; Higashi, Yuko; Fukushige, Tomoko; Baba, Naoko; Kawai, Kazuhiro; Hashiguchi, Teruto; Su, Juan; Zeng, Weiqi; Chen, Xiang; Kanekura, Takuro

2014-12-01

Cluster of differentiation 147 (CD147)/basigin on the malignant tumor cell surface is critical for tumor proliferation, invasiveness, metastasis, and angiogenesis. CD147 expressed on malignant melanoma cells can induce tumor cell invasion by stimulating the production of matrix metalloproteinases (MMPs) by surrounding fibroblasts. Membrane vesicles, microvesicles and exosomes have attracted attention, as vehicles of functional molecules and their association with CD147 has been reported. Cleaved CD147 fragments released from tumor cells were reported to interact with fibroblasts. We investigated the intercellular mechanisms by which CD147 stimulates fibroblasts to induce MMP2 activity. CD147 was knocked-down using short hairpin RNA (shRNA). The stimulatory effect of CD147 in cell culture supernatants, microvesicles, and exosomes on the enzymatic activity of MMP2 was examined by gelatin zymography. Supernatants from A375 control cells induced increased enzymatic activity of fibroblasts; such activity was significantly lower in CD147 knock-down cells. Cleaved CD147 plays a pivotal role in stimulating fibroblasts to induce MMP2 activity. Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

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

Science.gov (United States)

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

Therapeutic Recombinant Monoclonal Antibodies

Science.gov (United States)

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

Epitope Mapping of Monoclonal Antibody PMab-38 Against Dog Podoplanin.

Science.gov (United States)

Chang, Yao-Wen; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is extensively expressed by normal lymphatic endothelial cells, renal podocytes, and pulmonary type I alveolar cells. Nevertheless, increased expression of PDPN in malignant tumors not only associates with poor prognosis but also facilitates hematogenous metastasis through interaction with C-type lectin-like receptor-2 presented on platelets, followed by PDPN-mediated platelet activation. We previously reported a novel PMab-38 antibody, an anti-dog PDPN (dPDPN) monoclonal antibody, which specifically recognizes PDPN in squamous cell carcinomas melanomas and cancer-associated fibroblasts in canine cancer tissues. However, the specific binding with the epitope of PMab-38 remains undefined. In this study, flow cytometry was utilized to investigate the epitope of PMab-38, which was determined using a series of deletion or point mutants of dPDPN. The results revealed that the critical epitope of PMab-38 is Tyr67 and Glu68 of dPDPN.

Establishment of the first humpback whale fibroblast cell lines and their application in chemical risk assessment

International Nuclear Information System (INIS)

Burkard, Michael; Whitworth, Deanne; Schirmer, Kristin; Nash, Susan Bengtson

2015-01-01

Highlights: • We established and characterised the first humpback whale fibroblast cell lines. • Cell lines have a stable karyotype with 2n = 44. • Exposure to p,p′-DDE resulted in a concentration-dependent loss of cell viability. • p,p′-DDE sensitivity differed considerably from human fibroblasts. • Exposure to a whale blubber extract showed higher sensitivity than to p,p′-DDE alone. - Abstract: This paper reports the first successful derivation and characterization of humpback whale fibroblast cell lines. Primary fibroblasts were isolated from the dermal connective tissue of skin biopsies, cultured at 37 °C and 5% CO_2 in the standard mammalian medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS). Of nine initial biopsies, two cell lines were established from two different animals and designated HuWa1 and HuWa2. The cells have a stable karyotype with 2n = 44, which has commonly been observed in other baleen whale species. Cells were verified as being fibroblasts based on their spindle-shaped morphology, adherence to plastic and positive immunoreaction to vimentin. Population doubling time was determined to be ∼41 h and cells were successfully cryopreserved and thawed. To date, HuWa1 cells have been propagated 30 times. Cells proliferate at the tested temperatures, 30, 33.5 and 37 °C, but show the highest rate of proliferation at 37 °C. Short-term exposure to para,para′-dichlorodiphenyldichloroethylene (p,p′-DDE), a priority compound accumulating in southern hemisphere humpback whales, resulted in a concentration-dependent loss of cell viability. The effective concentration which caused a 50% reduction in HuWa1 cell viability (EC_5_0 value) was approximately six times greater than the EC_5_0 value for the same chemical measured with human dermal fibroblasts. HuWa1 exposed to a natural, p,p′-DDE-containing, chemical mixture extracted from whale blubber showed distinctively higher sensitivity than to p,p′-DDE alone. Thus, we

Establishment of the first humpback whale fibroblast cell lines and their application in chemical risk assessment

Energy Technology Data Exchange (ETDEWEB)

Burkard, Michael, E-mail: Michael.burkard@eawag.ch [Griffith University, Environmental Futures Research Institute, Southern Ocean Persistent Organic Pollutants Program, Brisbane, QLD (Australia); Eawag, Swiss Federal Institute of Technology, Dübendorf (Switzerland); Whitworth, Deanne [The University of Queensland, School of Veterinary Science, Gatton, QLD (Australia); Schirmer, Kristin [Eawag, Swiss Federal Institute of Technology, Dübendorf (Switzerland); ETH Zürich, Institute of Biogechemistry and Pollutant Dynamics, Zürich (Switzerland); EPF Lausanne, School of Architecture, Civil and Environmental Engineering, Lausanne (Switzerland); Nash, Susan Bengtson [Griffith University, Environmental Futures Research Institute, Southern Ocean Persistent Organic Pollutants Program, Brisbane, QLD (Australia)

2015-10-15

Highlights: • We established and characterised the first humpback whale fibroblast cell lines. • Cell lines have a stable karyotype with 2n = 44. • Exposure to p,p′-DDE resulted in a concentration-dependent loss of cell viability. • p,p′-DDE sensitivity differed considerably from human fibroblasts. • Exposure to a whale blubber extract showed higher sensitivity than to p,p′-DDE alone. - Abstract: This paper reports the first successful derivation and characterization of humpback whale fibroblast cell lines. Primary fibroblasts were isolated from the dermal connective tissue of skin biopsies, cultured at 37 °C and 5% CO{sub 2} in the standard mammalian medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS). Of nine initial biopsies, two cell lines were established from two different animals and designated HuWa1 and HuWa2. The cells have a stable karyotype with 2n = 44, which has commonly been observed in other baleen whale species. Cells were verified as being fibroblasts based on their spindle-shaped morphology, adherence to plastic and positive immunoreaction to vimentin. Population doubling time was determined to be ∼41 h and cells were successfully cryopreserved and thawed. To date, HuWa1 cells have been propagated 30 times. Cells proliferate at the tested temperatures, 30, 33.5 and 37 °C, but show the highest rate of proliferation at 37 °C. Short-term exposure to para,para′-dichlorodiphenyldichloroethylene (p,p′-DDE), a priority compound accumulating in southern hemisphere humpback whales, resulted in a concentration-dependent loss of cell viability. The effective concentration which caused a 50% reduction in HuWa1 cell viability (EC{sub 50} value) was approximately six times greater than the EC{sub 50} value for the same chemical measured with human dermal fibroblasts. HuWa1 exposed to a natural, p,p′-DDE-containing, chemical mixture extracted from whale blubber showed distinctively higher sensitivity than to p,p′-DDE alone

Bioactive reagents used in mesotherapy for skin rejuvenation in vivo induce diverse physiological processes in human skin fibroblasts in vitro- a pilot study.

Science.gov (United States)

Jäger, Claudia; Brenner, Christiane; Habicht, Jüri; Wallich, Reinhard

2012-01-01

The promise of mesotherapy is maintenance and/or recovery of a youthful skin with a firm, bright and moisturized texture. Currently applied medications employ microinjections of hyaluronic acid, vitamins, minerals and amino acids into the superficial layer of the skin. However, the molecular and cellular processes underlying mesotherapy are still elusive. Here we analysed the effect of five distinct medication formulas on pivotal parameters involved in skin ageing, that is collagen expression, cell proliferation and morphological changes using normal human skin fibroblast cultures in vitro. Whereas in the presence of hyaluronic acid, NCTF135(®) and NCTF135HA(®) , cell proliferation was comparable to control cultures; however, with higher expression of collagen type-1, matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1, addition of Soluvit(®) N and Meso-BK led to apoptosis and/or necrosis of human fibroblasts. The data indicate that bioactive reagents currently applied for skin rejuvenation elicit strikingly divergent physiological processes in human skin fibroblast in vitro. © 2011 John Wiley & Sons A/S.

Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers.

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Aschermann, Ilknur; Noor, Seema; Venturelli, Sascha; Sinnberg, Tobias; Mnich, Christian D; Busch, Christian

2017-01-01

Chronic leg ulcers (CLUs) are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT) has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred "from bench to bedside", and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial) were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the treatment of chronic, therapy-refractory ulcers. © 2017 The Author

Extracorporal Shock Waves Activate Migration, Proliferation and Inflammatory Pathways in Fibroblasts and Keratinocytes, and Improve Wound Healing in an Open-Label, Single-Arm Study in Patients with Therapy-Refractory Chronic Leg Ulcers

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Ilknur Aschermann

2017-02-01

Full Text Available Background/Aims: Chronic leg ulcers (CLUs are globally a major cause of morbidity and mortality with increasing prevalence. Their treatment is highly challenging, and many conservative, surgical or advanced therapies have been suggested, but with little overall efficacy. Since the 1980s extracorporal shock wave therapy (ESWT has gained interest as treatment for specific indications. Here, we report that patients with CLU showed wound healing after ESWT and investigated the underlying molecular mechanisms. Methods: We performed cell proliferation and migration assays, FACS- and Western blot analyses, RT-PCR, and Affymetrix gene expression analyses on human keratinocytes and fibroblasts, and a tube formation assay on human microvascular endothelial cells to assess the impact of shock waves in vitro. In vivo, chronic therapy-refractory leg ulcers were treated with ESWT, and wound healing was assessed. Results: Upon ESWT, we observed morphological changes and increased cell migration of keratinocytes. Cell-cycle regulatory genes were upregulated, and proliferation induced in fibroblasts. This was accompanied by secretion of pro-inflammatory cytokines from keratinocytes, which are known to drive wound healing, and a pro-angiogenic activity of endothelial cells. These observations were transferred “from bench to bedside”, and 60 consecutive patients with 75 CLUs with different pathophysiologies (e.g. venous, mixed arterial-venous, arterial were treated with ESWT. In this setting, 41% of ESWT-treated CLUs showed complete healing, 16% significant improvement, 35% improvement, and 8% of the ulcers did not respond to ESWT. The induction of healing was independent of patient age, duration or size of the ulcer, and the underlying pathophysiology. Conclusions: The efficacy of ESWT needs to be confirmed in controlled trials to implement ESWT as an adjunct to standard therapy or as a stand-alone treatment. Our results suggest that EWST may advance the

Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF).

Science.gov (United States)

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 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 division stimulation. It also suggests that the effects of bFGF on different cell types with

Fc receptors for mouse IgG1 on human monocytes: polymorphism and role in antibody-induced T cell proliferation.

Science.gov (United States)

Tax, W J; Hermes, F F; Willems, R W; Capel, P J; Koene, R A

1984-09-01

In previous studies, it was shown that there is polymorphism in the mitogenic effect of mouse IgG1 monoclonal antibodies against the T3 antigen of human T cells. This polymorphism implies that IgG1 anti-T3 antibodies are not mitogenic for T cells from 30% of healthy individuals. The present results demonstrate that this polymorphism is caused by polymorphism of an Fc receptor for mouse IgG1, present on human monocytes. The Fc receptor for murine IgG1 could be detected by a newly developed rosetting assay on monocytes from all individuals responsive to the mitogenic effect of IgG1 anti-T3 antibodies. This Fc receptor was not detectable on monocytes from those individuals exhibiting no mitogenic responses to IgG1 anti-T3 monoclonal antibodies. Cross-linking of T3 antigens appears to be essential for antibody-induced mitosis of T cells, because mononuclear cells that did not proliferate in response to WT 31 (an IgG1 antibody against T3 antigen) showed a proliferative response to Sepharose beads coated with WT 31. The Fc receptor--if functionally present--may be involved in the cross-linking of T3 antigens through anti-T3 antibodies. Further evidence for the involvement of this Fc receptor in antibody-induced T cell proliferation was provided by inhibition studies. Immune complexes containing IgG1 antibodies were able to inhibit the proliferative response to IgG1 anti-T3 antibodies. This inhibition by immune complexes appears to be mediated through the monocyte Fc receptor for mouse IgG1. These findings are important for the interpretation of previously described inhibitory effects of anti-T cell monoclonal antibodies on T cell proliferation, and show that such inhibitory effects may be monocyte-mediated (via immune complexes) rather than caused by a direct involvement of the respective T cell antigens in T cell mitosis. The Fc receptor for mouse IgG1 plays a role in antibody-induced T cell proliferation. Its polymorphism may have important implications for the

Comparison of fibroblast cell regeneration in three different concentrations of Wharton’s Jelly mesenchymal stem cells conditioned medium (WJMSCs-CM)

Science.gov (United States)

Untoro, E. G.; Asrianti, D.; Usman, M.; Meidyawati, R.; Margono, A.

2017-08-01

Wharton’s Jelly-derived mesenchymal stem cells (WJMSCs) have gained interest as an alternative source of stem cells for regenerative medicine. Although many studies have characterized Wharton’s Jelly biologically, the effects of different concentrations in a cultured medium have not yet been compared. Damaged fibroblasts, the primary components of irreversible dental pulpitis, irreversibly impair the ability to regenerate and lead to the disruption of extracellular matrix. This study was performed to evaluate the potency of three WJMSCs-CM concentrations in improving serum-starved fibroblasts. Fibroblasts were cultivated in five passages, and divided into four groups. The first group (the control group) consisted of fibroblast cells that had been treated using starvation methods. The other groups (the treatment groups) were treated with various concentration of WJMSCs-CM (50%, 25% and 12.5%). Proliferative ability was evaluated using a cell count method and analyzed with a one-way ANOVA. Cultivation of serum-starved fibroblasts produced significantly higher cell counts in 12.5% WJMSCs-CM compared to the 50% group. It can be concluded that 12.5% WJMSCs-CM is the most efficient concentration for fibroblast proliferation.

Fibroblast and T cells conditioned media induce maturation dendritic cell and promote T helper immune response

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Masoumeh Asadi

2012-06-01

Full Text Available Dendritic cells (DCs induce pathogen-specific T cell responses. We comprehensively studied the effects of addition of maturation stimulus, fibroblasts (fibroblast conditioned medium, PHA activated T cells (T cell conditioned medium, and mixture of fibroblast & PHA activated T cells (FCM-TCCM conditioned media on maturation of DCs. Monocytes were cultured with GM-CSF and IL-4 for five days. Maturation factors included MCM and TNF-α as control group. FCM and TCCM, or FCM-TCCM supernatant were considered as the treatment group. Tumor antigens were added at day five. Matured DCs were harvested at day seven. Phenotypic and functional analyses were carried out using anti (CD14, CD80, CD86, CD83 and HLA-DR monoclonal antibodies. Phagocytic activity, mixed lymphocyte reaction (MLR and cytokine production were also evaluated. At the end of culturing period, significantly fully matured DCs with large amount cytoplasm and copious dendritic projections were found in the presence of MCM, TNF-α with or without FCM, TCCM, FCM as well as TCCM. Flow cytometric analysis revealed that expression of CD14 decreased in particular in treated DCs, at the 5th day and expression of CD80, CD86 and HLA-DR was higher when FCM, TCCM, FCM plus TCCM were added to maturation factor. This study demonstrated that DCs matured with these methods had optimum function in comparison with either factor alone.

Low rate doses effects of gamma radiation on glycoproteins of transmembrane junctions in fibroblasts

International Nuclear Information System (INIS)

Bringas, J.E.; Caceres, J.L.

1996-01-01

Glycoproteins of trans-membrane junctions are molecules that help to bind cells with the extracellular matrix. Integrins are the most important trans-membrane molecules among others. The damage of gamma radiation on those proteins could be an important early event that causes membrane abnormalities which may lead to cell malfunction and cancer induced by radiation due to cell dissociation. Randomized blocks with 3 repetitions of mouse embryo fibroblast cultures, were irradiated with Cobalt-60 gamma rays, during 20 days. Biological damage to glycoproteins and integrins was evaluated by cellular growth and fibroblast proliferative capacity. Integrins damage was studied by isolation by column immunoaffinity chromatography migrated on SDS-Page under reducing and non reducing conditions, and inhibition of integrins extracellular matrix adhesion by monoclonal antibodies effect. The dose/rate (0.05 Gy/day-0.2 Gy/day) of gamma given to cells did not show damage evidence on glycoproteins and integrins. If damage happened, it was repaired by cells very soon, was delayed by continuous cellular division or by glycoproteins characteristic of being multiple extracellular ligatures. Bio effects became more evident with an irradiation time greater than 20 days or a high dose/rate. (authors). 6 refs

Direct lineage reprogramming of mouse fibroblasts to functional midbrain dopaminergic neuronal progenitors

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Han-Seop Kim

2014-01-01

Full Text Available The direct lineage reprogramming of somatic cells to other lineages by defined factors has led to innovative cell-fate-change approaches for providing patient-specific cells. Recent reports have demonstrated that four pluripotency factors (Oct4, Sox2, Klf4, and c-Myc are sufficient to directly reprogram fibroblasts to other specific cells, including induced neural stem cells (iNSCs. Here, we show that mouse fibroblasts can be directly reprogrammed into midbrain dopaminergic neuronal progenitors (DPs by temporal expression of the pluripotency factors and environment containing sonic hedgehog and fibroblast growth factor 8. Within thirteen days, self-renewing and functional induced DPs (iDPs were generated. Interestingly, the inhibition of both Jak and Gsk3β notably enhanced the iDP reprogramming efficiency. We confirmed the functionality of the iDPs by showing that the dopaminergic neurons generated from iDPs express midbrain markers, release dopamine, and show typical electrophysiological profiles. Our results demonstrate that the pluripotency factors-mediated direct reprogramming is an invaluable strategy for supplying functional and proliferating iDPs and may be useful for other neural progenitors required for disease modeling and cell therapies for neurodegenerative disorders.

Reversal of drug-induced gingival overgrowth by UV-mediated apoptosis of gingival fibroblasts - an in vitro study.

Science.gov (United States)

Ritchhart, Casey; Joy, Anita

2018-05-01

Gingival overgrowth (GO) is an undesirable result of certain drugs like Cyclosporine A (CsA). Histopathology of GO shows hyperplasia of gingival epithelium, expansion of connective tissue with increased collagen, or a combination. Factors such as age, gender, oral hygiene, duration, and dosage also influence onset and severity of GO. One of the mechanisms behind uncontrolled cell proliferation in drug-induced GO is inhibition of apoptotic pathways, with a consequent effect on normal cell turnover. Our objective was to determine if UV photo-treatment would activate apoptosis in the gingival fibroblast component. Human gingival fibroblast cells (HGF-1) were exposed to 200ng/ml or 400ng/ml CsA and maintained for 3, 6, and 9 days, followed by UV radiation for 2, 5, or 10min (N=6). Naïve (no CsA or UV), negative (UV, no CsA), and positive controls (CsA, no UV) were designated. Prior to UV treatment, growth media was replaced with 1M PBS to prevent absorption of UV radiation by serum proteins, and cells were incubated in growth media for 24h post-UV before processing for TUNEL assay, cell proliferation assays, or immunofluorescence. Data showed a temporal increase in proliferation of HGF-1 cells under the influence of CsA. The 200ng/ml dose was more effective in causing over-proliferation. UV treatment for 10min resulted in significant reduction in cell numbers, as evidenced by counts and proliferation assays. Our study is a first step to further evaluate UV-mediated apoptosis as a mechanism to control certain forms of GO. Copyright © 2018 Elsevier GmbH. All rights reserved.

Astragaloside IV inhibits pathological functions of gastric cancer-associated fibroblasts.

Science.gov (United States)

Wang, Zhen-Fei; Ma, Da-Guang; Zhu, Zhe; Mu, Yong-Ping; Yang, Yong-Yan; Feng, Li; Yang, Hao; Liang, Jun-Qing; Liu, Yong-Yan; Liu, Li; Lu, Hai-Wen

2017-12-28

To investigate the inhibitory effect of astragaloside IV on the pathological functions of cancer-associated fibroblasts, and to explore the underlying mechanism. Paired gastric normal fibroblast (GNF) and gastric cancer-associated fibroblast (GCAF) cultures were established from resected tissues. GCAFs were treated with vehicle control or different concentrations of astragaloside IV. Conditioned media were prepared from GNFs, GCAFs, control-treated GCAFs, and astragaloside IV-treated GCAFs, and used to culture BGC-823 human gastric cancer cells. Proliferation, migration and invasion capacities of BGC-823 cells were determined by MTT, wound healing, and Transwell invasion assays, respectively. The action mechanism of astragaloside IV was investigated by detecting the expression of microRNAs and the expression and secretion of the oncogenic factor, macrophage colony-stimulating factor (M-CSF), and the tumor suppressive factor, tissue inhibitor of metalloproteinase 2 (TIMP2), in different groups of GCAFs. The expression of the oncogenic pluripotency factors SOX2 and NANOG in BGC-823 cells cultured with different conditioned media was also examined. GCAFs displayed higher capacities to induce BGC-823 cell proliferation, migration, and invasion than GNFs ( P GNFs, GCAFs expressed a lower level of microRNA-214 ( P < 0.01) and a higher level of microRNA-301a ( P < 0.01). Astragaloside IV treatment significantly up-regulated microRNA-214 expression ( P < 0.01) and down-regulated microRNA-301a expression ( P < 0.01) in GCAFs. Reestablishing the microRNA expression balance subsequently suppressed M-CSF production ( P < 0.01) and secretion ( P < 0.05), and elevated TIMP2 production ( P < 0.01) and secretion ( P < 0.05). Consequently, the ability of GCAFs to increase SOX2 and NANOG expression in BGC-823 cells was abolished by astragaloside IV. Astragaloside IV can inhibit the pathological functions of GCAFs by correcting their dysregulation of microRNA expression, and it is

Pulsed-low intensity ultrasound enhances extracellular matrix production by fibroblasts encapsulated in alginate

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Siti PM Bohari

2012-12-01

Full Text Available In this study, the effect of pulsed-low intensity ultrasound on cell proliferation, collagen production and glycosaminoglycan deposition by 3T3 fibroblasts encapsulated in alginate was evaluated. Hoechst 33258 assay for cell number, hydroxyproline assay for collagen content and dimethylamine blue assay for glycosaminoglycan content were performed on samples from cell cultures treated with pulsed-low intensity ultrasound and a control group. Pulsed-low intensity ultrasound shows no effect on cell proliferation, while collagen and glycosaminoglycan contents were consistently higher in the samples treated with pulsed-low intensity ultrasound, showing a statistically significant difference (p < 0.05 on day 10. Alcian blue staining showed that glycosaminoglycans were deposited around the cells in both groups. These results suggest that pulsed-low intensity ultrasound shows no effect on cell proliferation but has potential for inducing collagen and glycosaminoglycan production in cells cultured in alginate gels.

Effect of botulinum toxin type A on transforming growth factor beta1 in fibroblasts derived from hypertrophic scar: a preliminary report.

Science.gov (United States)

Xiao, Zhibo; Zhang, Fengmin; Lin, Weibin; Zhang, Miaobo; Liu, Ying

2010-08-01

Hypertrophic scar is a common dermal disease. Numerous treatments are currently available but they do not always yield excellent therapeutic results. Hence, alternatives are needed. Recent basic and clinical research has shown that botulinum toxin type A (BTXA) has antihypertrophic scar properties but the molecular mechanism for this action is unknown. The aim of this study was to explore the effect of BTXA on transforming growth factor beta1 (TGF-beta1) in fibroblasts derived from hypertrophic scar and further elucidate its actual mechanism. Fibroblasts were isolated from tissue specimens of hypertrophic scar. Fibroblasts were treated with BTXA and the difference in proliferation between treated and nontreated cells was analyzed through the MTT method from the first to the fifth day after treatment. Proteins of TGF-beta1 were checked using ELISA in fibroblasts with BTXA and without BTXA from the first to the fifth day. The growth of the fibroblast treated with BTXA was obviously slower than that of the fibroblast without BTXA treatment (p < 0.01), which showed that BTXA effectively inhibited the growth of fibroblasts. Proteins of TGF-beta1 between fibroblasts with BTXA and fibroblasts without BTXA are statistically significant (p < 0.01). These results suggest that BTXA effectively inhibited the growth of fibroblasts derived from hypertrophic scar and in turn caused a decrease in TGF-beta1 protein, indicating that BTXA-based therapies for hypertrophic scar are promising and worth investigating further.

Cigarette Smoke-Exposed Candida albicans Increased Chitin Production and Modulated Human Fibroblast Cell Responses

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Humidah Alanazi

2014-01-01

Full Text Available The predisposition of cigarette smokers for development of respiratory and oral bacterial infections is well documented. Cigarette smoke can also contribute to yeast infection. The aim of this study was to investigate the effect of cigarette smoke condensate (CSC on C. albicans transition, chitin content, and response to environmental stress and to examine the interaction between CSC-pretreated C. albicans and normal human gingival fibroblasts. Following exposure to CSC, C. albicans transition from blastospore to hyphal form increased. CSC-pretreated yeast cells became significantly (P<0.01 sensitive to oxidation but significantly (P<0.01 resistant to both osmotic and heat stress. CSC-pretreated C. albicans expressed high levels of chitin, with 2- to 8-fold recorded under hyphal conditions. CSC-pretreated C. albicans adhered better to the gingival fibroblasts, proliferated almost three times more and adapted into hyphae, while the gingival fibroblasts recorded a significantly (P<0.01 slow growth rate but a significantly higher level of IL-1β when in contact with CSC-pretreated C. albicans. CSC was thus able to modulate both C. albicans transition through the cell wall chitin content and the interaction between C. albicans and normal human gingival fibroblasts. These findings may be relevant to fungal infections in the oral cavity in smokers.

Cyclic GMP-AMP Synthase Is Required for Cell Proliferation and Inflammatory Responses in Rheumatoid Arthritis Synoviocytes

OpenAIRE

Wang, Yan; Su, Guo-Hua; Zhang, Fang; Chu, Jing-Xue; Wang, Yun-Shan

2015-01-01

Rheumatoid arthritis (RA) is characterized by inflammatory cell infiltration, fibroblast-like synoviocytes (FLS) invasive proliferation, and joint destruction. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces immune activation. In this study, we examined whether cGAS plays a role in RA FLS. In this study, cGAS was overexpressed in RA-FLS compared with OA FLS. TNFα stimulation induced cGAS expression in RA FLS. Overexpression of cGAS promoted the proliferation and knockdow...

The endogenous fluorescence of fibroblast in collagen gels as indicator of stiffness of the extracellular matrix

Science.gov (United States)

Padilla-Martinez, J. P.; Ortega-Martinez, A.; Franco, W.

2016-03-01

The stiffness or rigidity of the extracellular matrix (ECM) regulates cell response. Established mechanical tests to measure stiffness, such as indentation and tensile tests, are invasive and destructive to the sample. Endogenous or native molecules to cells and ECM components, like tryptophan and cross-links of collagen, display fluorescence upon irradiation with ultraviolet light. Most likely, the concentration of these endogenous fluorophores changes as the stiffness of the ECM changes. In this work we investigate the endogenous fluorescence of collagen gels containing fibroblasts as a non-invasive non-destructive method to measure stiffness of the ECM. Human fibroblast cells were cultured in three-dimensional gels of type I collagen (50,000 cells/ml). This construct is a simple model of tissue contraction. During contraction, changes in the excitation-emission matrix (a fluorescence map in the 240-520/290-530 nm range) of constructs were measured with a spectrofluoremeter, and changes in stiffness were measured with a standard indentation test over 16 days. Results show that a progressive increase in fluorescence of the 290/340 nm excitation-emission pair correlates with a progressive increase in stiffness (r=0.9, α=0.5). The fluorescence of this excitation-emission pair is ascribed to tryptophan and variations in the fluorescence of this pair correlate with cellular proliferation. In this tissue model, the endogenous functional fluorescence of proliferating fibroblast cells is a biomechanical marker of stiffness of the ECM.

N-cadherin is overexpressed in Crohn's stricture fibroblasts and promotes intestinal fibroblast migration.

LENUS (Irish Health Repository)

Burke, John P

2012-02-01

BACKGROUND: Intestinal fibroblasts mediate stricture formation in Crohn\\'s disease (CD). Transforming growth factor-beta (TGF-beta) is important in fibroblast activation, while cell attachment and migration is regulated by the adhesion molecule N-cadherin. The aim of this study was to investigate the expression and function of N-cadherin in intestinal fibroblasts in patients with fibrostenosing CD. METHODS: Intestinal fibroblasts were cultured from seromuscular biopsies from patients undergoing resection for terminal ileal fibrostenosing CD (n = 14) or controls patients (n = 8). N-cadherin expression was assessed using Western blot and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Fibroblasts were stimulated with TGF-beta and selective pathway inhibitors Y27632, PD98050, and LY294002 were used to examine the Rho\\/ROCK, ERK-1\\/2, and Akt signaling pathways, respectively. Cell migration was assessed using a scratch wound assay. N-cadherin was selectively overexpressed using a plasmid. RESULTS: Fibroblasts from fibrostenosing CD express increased constitutive N-cadherin mRNA and protein and exhibit enhanced basal cell migration relative to those from directly adjacent normal bowel. Control fibroblasts treated with TGF-beta induced N-cadherin in a dose-dependent manner which was inhibited by Rho\\/ROCK and Akt pathway modulation. Control fibroblasts exhibited enhanced cell migration in response to treatment with TGF-beta or transfection with an N-cadherin plasmid. CONCLUSIONS: Fibroblasts from strictures in CD express increased constitutive N-cadherin and exhibit enhanced basal cell migration. TGF-beta is a potent inducer of N-cadherin in intestinal fibroblasts resulting in enhanced cell migration. The TGF-beta-mediated induction of N-cadherin may potentiate Crohn\\'s stricture formation.

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

Science.gov (United States)

Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

Modeling cell adhesion and proliferation: a cellular-automata based approach.

Science.gov (United States)

Vivas, J; Garzón-Alvarado, D; Cerrolaza, M

Cell adhesion is a process that involves the interaction between the cell membrane and another surface, either a cell or a substrate. Unlike experimental tests, computer models can simulate processes and study the result of experiments in a shorter time and lower costs. One of the tools used to simulate biological processes is the cellular automata, which is a dynamic system that is discrete both in space and time. This work describes a computer model based on cellular automata for the adhesion process and cell proliferation to predict the behavior of a cell population in suspension and adhered to a substrate. The values of the simulated system were obtained through experimental tests on fibroblast monolayer cultures. The results allow us to estimate the cells settling time in culture as well as the adhesion and proliferation time. The change in the cells morphology as the adhesion over the contact surface progress was also observed. The formation of the initial link between cell and the substrate of the adhesion was observed after 100 min where the cell on the substrate retains its spherical morphology during the simulation. The cellular automata model developed is, however, a simplified representation of the steps in the adhesion process and the subsequent proliferation. A combined framework of experimental and computational simulation based on cellular automata was proposed to represent the fibroblast adhesion on substrates and changes in a macro-scale observed in the cell during the adhesion process. The approach showed to be simple and efficient.

The role of hormones and growth factors in the cellular proliferation control in mammals

International Nuclear Information System (INIS)

Armelin, H.A.

1978-01-01

A review is done about fibroblast proliferation, its control by classic hormones and hormonal growth factors, showing their main implications and the stage of this research at present. The control exerted on fibronlast proliferation by hormonal growth factors and classic hormones is demonstrated. The existence of basic mechanisms valid for all types of cells is suggested. Experiences are carried out with the aim of finding growth mutants useful in the elucidation of the biochemical mechanisms involved in growth regulation. Radiactive precursors and autoradiographic techniques are used in the research. (M.A.) [pt

Monoclonal antibodies to Pneumocystis carinii

DEFF Research Database (Denmark)

Kovacs, J A; Halpern, J L; Lundgren, B

1989-01-01

To increase understanding of the antigenic structure of Pneumocystis carinii, we developed monoclonal antibodies to rat and human P. carinii. The specificity of the antibodies was demonstrated by immunofluorescence and immunoblot studies. Only one of five monoclonal antibodies to rat P. carinii r...

Quantitative Evaluation of Myostatin Gene in Stably Transfected Caprine Fibroblast Cells by Anti-Myostatin shRNA.

Science.gov (United States)

Jain, Sudhir Kumar; Jain, Hemlata; Kumar, Dharmendra; Bedekar, Megha Kadam; Pandey, Akhilesh Kumar; Sarkhel, Bikash Chandra

2015-09-01

Skeletal muscle is the major component of lean tissue that is used for consumption, and myostatin is a negative regulator of skeletal muscle growth. Downregulation of this gene therefore offers a strategy for developing superior animals with enhanced muscle growth. Knockdown of myostatin was achieved by RNA interference technology. The anti-myostatin shRNA were designed and stably transfected in caprine fibroblast cells. The reduced expression of target gene was achieved and measured in clonal fibroblast cells by real-time PCR. Two single-cell clones induced significant decrease of myostatin gene expression by 73.96 and 72.66 %, respectively (P < 0.05). To ensure the appropriate growth of transfected cell, seven media were tested. The best suited media was used for transfected fibroblast cell proliferation. The findings suggest that shRNA provides a novel potential tool for gene knockdown and these stably transfected cells can be used as the donor cells for animal cloning.

A proteomic analysis of the functional effects of fatty acids in NIH 3T3 fibroblasts

LENUS (Irish Health Repository)

Magdalon, Juliana

2011-11-24

Abstract Previous studies have demonstrated that long chain fatty acids influence fibroblast function at sub-lethal concentrations. This study is the first to assess the effects of oleic, linoleic or palmitic acids on protein expression of fibroblasts, as determined by standard proteomic techniques. The fatty acids were not cytotoxic at the concentration used in this work as assessed by membrane integrity, DNA fragmentation and the MTT assay but significantly increased cell proliferation. Subsequently, a proteomic analysis was performed using two dimensional difference gel electrophoresis (2D-DIGE) and MS based identification. Cells treated with 50 μM oleic, linoleic or palmitic acid for 24 h were associated with 24, 22, 16 spots differentially expressed, respectively. Among the identified proteins, α-enolase and far upstream element binding protein 1 (FBP-1) are of importance due to their function in fibroblast-associated diseases. However, modulation of α-enolase and FBP-1 expression by fatty acids was not validated by the Western blot technique.

Atelocollagen sponge and recombinant basic fibroblast growth factor combination therapy for resistant wounds with deep cavities.

Science.gov (United States)

Nakanishi, Asako; Hakamada, Arata; Isoda, Ken-ichi; Mizutani, Hitoshi

2005-05-01

Recent advances in bioengineering have introduced materials that enhance wound healing. Even with such new tools, some deep ulcers surrounded by avascular tissues, including bone, tendon, and fascia, are resistant to various therapies and easily form deep cavities with loss of subcutaneous tissue. Atelocollagen sponges have been used as an artificial dermis to cover full-thickness skin defects. Topical recombinant human basic fibroblast growth factor has been introduced as a growth factor to induce fibroblast proliferation in skin ulcers. We applied these materials in combination in two patients with deep resistant wounds: one with a cavity reaching the mediastinum through a divided sternum and one with deep necrotic wounds caused by electric burns. These wounds did not respond to the topical basic fibroblast growth factor alone. In contrast, the combination therapy closed the wounds rapidly without further surgical treatment. This combination therapy is a potent treatment for resistant wounds with deep cavities.

Prevalence and type of monoclonal gammopathy of undetermined significance in an apparently healthy Nigerian population: a cross sectional study.

Science.gov (United States)

Onwah, A Lawretta; Adeyemo, Titilope A; Adediran, Adewumi; Ajibola, Sarah O; Akanmu, Alani S

2012-06-28

The prevalence of monoclonal gammopathy of undetermined significance (MGUS), a premalignant plasma-cell disorder has not been determined in our geographic area Nigeria. A cross sectional survey was carried on apparently healthy Nigerians selected by multistage sampling technique from the cosmopolitan city of Lagos, Nigeria. Subjects enrolled into the study had 2-step screening for the presence, type and concentration of monoclonal band. Agarose-gel electrophoresis was performed on all serum samples, and any serum sample with a discrete band of monoclonal protein or thought to have a localized band was subjected to Immunofixation. Subjects were also evaluated for Bence jones proteinuria, haematological and biochemical parameters. Four hundred and ten subjects with a mean age of 45.68 ± 10.3 years, a median of 45.00 years and a range of 20 to 80 years were enrolled into the study. MGUS was identified in only one (0.24 percent) of the 410 study subject. This subject was demonstrated to have a double monoclonal gammopathy; IgGλ at 16.9 g/L and IgAκ at 8.5 g/L. None of them including the sole subject with MGUS had a monoclonal urinary light chain. Among residents of Lagos, Nigeria, MGUS was found in only 0.24% percent of apparently normal persons with a median age of 45 years. This suggests that MGUS which represents the earliest stage of monoclonal plasma/lymphoid cell proliferation is not a common finding in the relatively young population of Nigeria. Future epidemiologic studies dealing with plasma cell disorders in older people are required to carefully examine the relationship between environmental factors and prevalence of MGUS and its ultimate progression to MM.

[Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts].

Science.gov (United States)

Paco, Karen; Ponce-Soto, Luis Alberto; Lopez-Ilasaca, Marco; Aguilar, José L

2016-01-01

To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François, E-mail: fberthia@rci.rutgers.edu

2015-02-27

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.

Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts

International Nuclear Information System (INIS)

Faulknor, Renea A.; Olekson, Melissa A.; Nativ, Nir I.; Ghodbane, Mehdi; Gray, Andrea J.; Berthiaume, François

2015-01-01

During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived soluble factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β 1 (TGF-β 1 )-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β 1 at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β 1 is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β 1

Effects of cholera toxin and isobutylmethylxanthine on growth of human fibroblasts

International Nuclear Information System (INIS)

Espinoza, B.; Wharton, W.

1986-01-01

Cholera toxin produced a dose-dependent decrease in the restimulation of G 0 /G 1 traverse in density-arrested human fibroblasts but did not inhibit the stimulation of cells arrested in G 0 after serum starvation at low density. In addition, cholera toxin did not inhibit the proliferation of sparse logarithmically growing human fibroblasts, even when low concentrations of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) were also present. However, the final density to which sparse cells grew was limited by cholera toxin, when added either alone or together with low concentrations of IBMX. In contrast, high concentrations of the phosphodiesterase inhibitor alone produced a profound inhibition in the growth of sparse human fibrobasts. IBMX produced an inhibition both in the G 1 and in the G 2 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

Stromal COX-2 signaling activated by deoxycholic acid mediates proliferation and invasiveness of colorectal epithelial cancer cells

International Nuclear Information System (INIS)

Zhu, Yingting; Zhu, Min; Lance, Peter

2012-01-01

Highlights: ► Human colonic cancer associated fibroblasts are major sources of COX-2 and PGE 2 . ► The fibroblasts interact with human colonic epithelial cancer cells. ► Activation of COX-2 signaling in the fibroblasts affects behavior of the epithelia. ► Protein Kinase C controls the activation of COX-2 signaling. -- Abstract: COX-2 is a major regulator implicated in colonic cancer. However, how COX-2 signaling affects colonic carcinogenesis at cellular level is not clear. In this article, we investigated whether activation of COX-2 signaling by deoxycholic acid (DCA) in primary human normal and cancer associated fibroblasts play a significant role in regulation of proliferation and invasiveness of colonic epithelial cancer cells. Our results demonstrated while COX-2 signaling can be activated by DCA in both normal and cancer associated fibroblasts, the level of activation of COX-2 signaling is significantly greater in cancer associated fibroblasts than that in normal fibroblasts. In addition, we discovered that the proliferative and invasive potential of colonic epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts pre-treated with DCA than with normal fibroblasts pre-treated with DCA. Moreover, COX-2 siRNA attenuated the proliferative and invasive effect of both normal and cancer associate fibroblasts pre-treated with DCA on the colonic cancer cells. Further studies indicated that the activation of COX-2 signaling by DCA is through protein kinase C signaling. We speculate that activation of COX-2 signaling especially in cancer associated fibroblasts promotes progression of colonic cancer.

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

OpenAIRE

1982-01-01

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular ...

The effect of pH on cell viability, cell migration, cell proliferation, wound closure, and wound reepithelialization

DEFF Research Database (Denmark)

Kruse, Carla R; Singh, Mansher; Targosinski, Stefan

2017-01-01

primary keratinocyte and fibroblast function in vitro and on wound healing in vivo. In vitro, primary human keratinocytes and fibroblasts were cultured in different levels of pH (5.5-12.5) and the effect on cell viability, proliferation, and migration was studied. A rat full-thickness wound model was used...... to investigate the effect of pH (5.5-9.5) on wound healing in vivo. The effect of pH on inflammation was monitored by measuring IL-1 α concentrations from wounds and cell cultures exposed to different pH environments. Our results showed that both skin cell types tolerated wide range of pH very well. They further...... demonstrated that both acidic and alkaline environments decelerated cell migration in comparison to neutral environments and interestingly alkaline conditions significantly enhanced cell proliferation. Results from the in vivo experiments indicated that a prolonged, strongly acidic wound environment prevents...

Cell Surface Glycoprotein of Reactive Stromal Fibroblasts as a Potential Antibody Target in Human Epithelial Cancers

Science.gov (United States)

Garin-Chesa, Pilar; Old, Lloyd J.; Rettig, Wolfgang J.

1990-09-01

The F19 antigen is a cell surface glycoprotein (M_r, 95,000) of human sarcomas and proliferating, cultured fibroblasts that is absent from resting fibroblasts in normal adult tissues. Normal and malignant epithelial cells are also F19^-. The present immunohistochemical study describes induction of F19 in the reactive mesenchyme of epithelial tumors. F19^+ fibroblasts were found in primary and metastatic carcinomas, including colorectal (18 of 18 cases studied), breast (14/14), ovarian (21/21), bladder (9/10), and lung carcinomas (13/13). In contrast, the stroma of benign colorectal adenomas, fibrocystic disease and fibroadenomas of breast, benign prostate hyperplasia, in situ bladder carcinomas, and benign ovarian tumors showed no or only moderate numbers of F19^+ fibroblasts. Analysis of dermal incision wounds revealed that F19 is strongly induced during scar formation. Comparison of F19 with the extracellular matrix protein tenascin, a putative marker of tumor mesenchyme, showed a cellular staining pattern for F19 vs. the extracellular matrix pattern for tenascin and widespread expression of tenascin in F19^- normal tissues and benign tumors. Our results suggest that the F19^+ phenotype correlates with specialized fibroblast functions in wound healing and malignant tumor growth. Because of its abundance in tumor mesenchyme, F19 may serve as a target for antibodies labeled with radioisotopes or toxic agents, or inflammatogenic antibodies, in carcinoma patients.

Astragaloside IV suppresses transforming growth factor-β1 induced fibrosis of cultured mouse renal fibroblasts via inhibition of the MAPK and NF-κB signaling pathways

Energy Technology Data Exchange (ETDEWEB)

Che, Xiajing; Wang, Qin; Xie, Yuanyuan; Xu, Weijia; Shao, Xinghua; Mou, Shan, E-mail: shan_mou@126.com; Ni, Zhaohui, E-mail: doctor_nzh@126.com

2015-09-04

Renal fibrosis, a progressive process characterized by the accumulation of extracellular matrix (ECM) leading to organ dysfunction, is a characteristic of chronic kidney diseases. Among fibrogenic factors known to regulate the renal fibrotic process, transforming growth factor-β (TGF-β) plays a central role. In the present study, we examined the effect of Astragaloside IV (AS-IV), a component of the traditional Chinese medicinal plant Astragalus membranaceus, on the processes associated with renal fibrosis in cultured mouse renal fibroblasts treated with TGF-β1. RT-PCR, western blotting, immunofluorescence staining and collagen assays showed that AS-IV suppressed TGF-β1 induced fibroblast proliferation, transdifferentiation, and ECM production in a dose-dependent manner. Examination of the underlying mechanisms showed that the effect of AS-IV on the inhibition of fibroblast differentiation and ECM formation were mediated by its modulation of the activity of the MAPK and NF-κB signaling pathways. Taken together, our results indicate that AS-IV alleviates renal interstitial fibrosis via a mechanism involving the MAPK and NF-κB signaling pathways and demonstrate the therapeutic potential of AS-IV for the treatment of chronic kidney diseases. - Highlights: • AS-IV suppressed TGF-β1 induced renal fibroblast proliferation. • AS-IV suppressed TGF-β1 induced renal fibroblast transdifferentiation. • AS-IV suppressed TGF-β1 induced ECM production. • AS-IV alleviates renal fibrosis via the MAPK and NF-κB signaling pathways.

A method for the isolation and characterization of functional murine monoclonal antibodies by single B cell cloning.

Science.gov (United States)

Carbonetti, Sara; Oliver, Brian G; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Sack, Brandon; Bergl, Emilee; Kappe, Stefan H I; Sather, D Noah

2017-09-01

Monoclonal antibody technologies have enabled dramatic advances in immunology, the study of infectious disease, and modern medicine over the past 40years. However, many monoclonal antibody discovery procedures are labor- and time-intensive, low efficiency, and expensive. Here we describe an optimized mAb discovery platform for the rapid and efficient isolation, cloning and characterization of monoclonal antibodies in murine systems. In this platform, antigen-binding splenic B cells from immunized mice are isolated by FACS and cocultured with CD40L positive cells to induce proliferation and mAb production. After 12days of coculture, cell culture supernatants are screened for antigen, and IgG positivity and RNA is isolated for reverse-transcription. Positive-well cDNA is then amplified by PCR and the resulting amplicons can be cloned into ligation-independent expression vectors, which are then used directly to transfect HEK293 cells for recombinant antibody production. After 4days of growth, conditioned medium can be screened using biolayer interferometry for antigen binding and affinity measurements. Using this method, we were able to isolate six unique, functional monoclonal antibodies against an antigen of the human malaria parasite Plasmodium falciparum. Importantly, this method incorporates several important advances that circumvent the need for single-cell PCR, restriction cloning, and large scale protein production, and can be applied to a wide array of protein antigens. Copyright © 2017 Elsevier B.V. All rights reserved.

Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

International Nuclear Information System (INIS)

Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

1982-01-01

The metabolism of [1,2,6,7-3H]testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC

Monoclonal antibodies in oncology

International Nuclear Information System (INIS)

Chan, S.Y.T.; Sikora, K.

1986-01-01

Monoclonal antibodies (MCAs) can be used to differentiate between normal and neoplastic cells and thus exploited for diagnostic and, ultimately, therapeutic gain. The evidence for the existence of human tumour antigens is reviewed. Several areas of diagnosis are already benefiting from the application of the monoclonal technology. Immunohistology can help the pathologist with difficult diagnostic problems. New classifications of lymphoma and leukaemia can be based on specific surface molecules. Similarly, the detection of shed tumour antigens is already established as part of the routine assessment of many patients with common solid tumours. Isotopically labeled monoclonal antibodies have been used to localise primary and metastatic tumours. The use of antibodies in this way is not only a promising diagnostic tool but also the first step in studying the possibility of arming antibodies to provide therapeutic agents. Such trials are currently in progress. (Auth.)

Stromal COX-2 signaling activated by deoxycholic acid mediates proliferation and invasiveness of colorectal epithelial cancer cells

Energy Technology Data Exchange (ETDEWEB)

Zhu, Yingting, E-mail: yitizhu@yahoo.com [Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724 (United States); Tissue Tech Inc., Miami, FL 33173 (United States); Zhu, Min; Lance, Peter [Arizona Cancer Center, The University of Arizona, Tucson, AZ 85724 (United States)

2012-08-31

Highlights: Black-Right-Pointing-Pointer Human colonic cancer associated fibroblasts are major sources of COX-2 and PGE{sub 2}. Black-Right-Pointing-Pointer The fibroblasts interact with human colonic epithelial cancer cells. Black-Right-Pointing-Pointer Activation of COX-2 signaling in the fibroblasts affects behavior of the epithelia. Black-Right-Pointing-Pointer Protein Kinase C controls the activation of COX-2 signaling. -- Abstract: COX-2 is a major regulator implicated in colonic cancer. However, how COX-2 signaling affects colonic carcinogenesis at cellular level is not clear. In this article, we investigated whether activation of COX-2 signaling by deoxycholic acid (DCA) in primary human normal and cancer associated fibroblasts play a significant role in regulation of proliferation and invasiveness of colonic epithelial cancer cells. Our results demonstrated while COX-2 signaling can be activated by DCA in both normal and cancer associated fibroblasts, the level of activation of COX-2 signaling is significantly greater in cancer associated fibroblasts than that in normal fibroblasts. In addition, we discovered that the proliferative and invasive potential of colonic epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts pre-treated with DCA than with normal fibroblasts pre-treated with DCA. Moreover, COX-2 siRNA attenuated the proliferative and invasive effect of both normal and cancer associate fibroblasts pre-treated with DCA on the colonic cancer cells. Further studies indicated that the activation of COX-2 signaling by DCA is through protein kinase C signaling. We speculate that activation of COX-2 signaling especially in cancer associated fibroblasts promotes progression of colonic cancer.

Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

Directory of Open Access Journals (Sweden)

Paul P Bonvallet

Full Text Available Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone electrospun scaffold (70:30 col/PCL containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM, and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344 rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14% over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold. Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration

Improved wound healing in pressure-induced decubitus ulcer with controlled release of basic fibroblast growth factor

International Nuclear Information System (INIS)

Jiang Wei; Wang Hailun; Jin Faguang; Yu Chunyan; Chu Dongling; Wang Lin; Lu Xian

2008-01-01

The purpose was to evaluate the efficacy of the wound dressing containing basic fibroblast growth factor (bFGF)-loaded microspheres on promoting healing in pressure-induced decubitus ulcer. In this study, the pressure-induced ulcer in swine was used as a model to demonstrate the hypothesis that controlled release of bFGF has the potential to provide optimal healing milieu for chronic wounds in the repair process. Average size of the microspheres was 14.36 ± 3.56 μm and the network gelatin sponges were characterized with an average pore size of 80-160 μm. Both the in vitro release efficiency and the protein bioactivity revealed that bFGF was released from the microspheres in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of fibroblasts. Pressure-induced ulcer was created at 500 g/cm 2 pressure loaded on swine dorsal skin 12 h daily for 2 consecutive days. After removal of the pressure load, the gelatin sponge containing bFGF gelatin microspheres or bFGF in solution was implanted into the wound. Swine were sacrificed at 7, 14, and 21 days after implantation, and a full-thickness biopsy was taken and stained for histological analysis. It was observed that controlled release of bFGF provided an accelerated recovery in the wound areas. Histological investigations showed that the dressings were biocompatible and had capability of proliferating fibroblasts and inducing neovascularisation. The present study implied the clinical potential of gelatin sponge with bFGF microspheres to promote the healing in pressure-induced decubitus ulcer

Improved wound healing in pressure-induced decubitus ulcer with controlled release of basic fibroblast growth factor

Energy Technology Data Exchange (ETDEWEB)

Jiang Wei [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Wang Hailun [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Jin Faguang [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China)], E-mail: nidewenzhang@163.com; Yu Chunyan [Department of Dermatology, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Chu Dongling [Department of Respiratory Diseases, Tangdu Hospital, Fourth Military Medical University, Xi' an 710038 (China); Wang Lin [Department of Internal Medicine, 316 Hospital of PLA, Beijing 100093 (China); Lu Xian [93942 Unit Hospital of PLA, Xianyang 710012 (China)

2008-07-14

The purpose was to evaluate the efficacy of the wound dressing containing basic fibroblast growth factor (bFGF)-loaded microspheres on promoting healing in pressure-induced decubitus ulcer. In this study, the pressure-induced ulcer in swine was used as a model to demonstrate the hypothesis that controlled release of bFGF has the potential to provide optimal healing milieu for chronic wounds in the repair process. Average size of the microspheres was 14.36 {+-} 3.56 {mu}m and the network gelatin sponges were characterized with an average pore size of 80-160 {mu}m. Both the in vitro release efficiency and the protein bioactivity revealed that bFGF was released from the microspheres in a controlled manner and it was biologically active as assessed by its ability to induce the proliferation of fibroblasts. Pressure-induced ulcer was created at 500 g/cm{sup 2} pressure loaded on swine dorsal skin 12 h daily for 2 consecutive days. After removal of the pressure load, the gelatin sponge containing bFGF gelatin microspheres or bFGF in solution was implanted into the wound. Swine were sacrificed at 7, 14, and 21 days after implantation, and a full-thickness biopsy was taken and stained for histological analysis. It was observed that controlled release of bFGF provided an accelerated recovery in the wound areas. Histological investigations showed that the dressings were biocompatible and had capability of proliferating fibroblasts and inducing neovascularisation. The present study implied the clinical potential of gelatin sponge with bFGF microspheres to promote the healing in pressure-induced decubitus ulcer.

Prevalence and type of monoclonal gammopathy of undetermined significance in an apparently healthy Nigerian population: a cross sectional study

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Onwah A

2012-06-01

Full Text Available Abstract Background The prevalence of monoclonal gammopathy of undetermined significance (MGUS, a premalignant plasma-cell disorder has not been determined in our geographic area Nigeria. Methods A cross sectional survey was carried on apparently healthy Nigerians selected by multistage sampling technique from the cosmopolitan city of Lagos, Nigeria. Subjects enrolled into the study had 2-step screening for the presence, type and concentration of monoclonal band. Agarose-gel electrophoresis was performed on all serum samples, and any serum sample with a discrete band of monoclonal protein or thought to have a localized band was subjected to Immunofixation. Subjects were also evaluated for Bence jones proteinuria, haematological and biochemical parameters. Results Four hundred and ten subjects with a mean age of 45.68 ± 10.3 years, a median of 45.00 years and a range of 20 to 80 years were enrolled into the study. MGUS was identified in only one (0.24 percent of the 410 study subject. This subject was demonstrated to have a double monoclonal gammopathy; IgGλ at 16.9 g/L and IgAκ at 8.5 g/L. None of them including the sole subject with MGUS had a monoclonal urinary light chain. Conclusion Among residents of Lagos, Nigeria, MGUS was found in only 0.24% percent of apparently normal persons with a median age of 45 years. This suggests that MGUS which represents the earliest stage of monoclonal plasma/lymphoid cell proliferation is not a common finding in the relatively young population of Nigeria. Future epidemiologic studies dealing with plasma cell disorders in older people are required to carefully examine the relationship between environmental factors and prevalence of MGUS and its ultimate progression to MM.

Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells.

Science.gov (United States)

Khor, Hwei Ling; Kuan, Yujun; Kukula, Hildegard; Tamada, Kaoru; Knoll, Wolfgang; Moeller, Martin; Hutmacher, Dietmar W

2007-05-01

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.

MFG-E8 Reprogramming of Macrophages Promotes Wound Healing by Increased bFGF Production and Fibroblast Functions.

Science.gov (United States)

Laplante, Patrick; Brillant-Marquis, Frédéric; Brissette, Marie-Joëlle; Joannette-Pilon, Benjamin; Cayrol, Romain; Kokta, Victor; Cailhier, Jean-François

2017-09-01

Macrophages are essential for tissue repair. They have a crucial role in cutaneous wound healing, participating actively in the inflammation phase of the process. Unregulated macrophage activation may, however, represent a source of excessive inflammation, leading to abnormal wound healing and hypertrophic scars. Our research group has shown that apoptotic endothelial and epithelial cells secrete MFG-E8, which has the ability to reprogram macrophages from an M1 (proinflammatory) to an M2 (anti-inflammatory, pro-repair) phenotype. Hence, we tested whether modulation of macrophage reprogramming would promote tissue repair. Using a mouse model of wound healing, we showed that the presence and/or addition of MFG-E8 favors wound closure associated with an increase in CD206-positive cells and basic fibroblast growth factor production in healing tissues. More importantly, adoptive transfer of ex vivo MFG-E8-treated macrophages promoted wound closure. We also observed that MFG-E8-treated macrophages produced basic fibroblast growth factor that is responsible for fibroblast migration and proliferation. Taken together, our results strongly suggest that MFG-E8 plays a key role in macrophage reprogramming in tissue healing through induction of an anti-inflammatory M2 phenotype and basic fibroblast growth factor production, leading to fibroblast migration and wound closure. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Keratinocytes from APP/APLP2-deficient mice are impaired in proliferation, adhesion and migration in vitro

International Nuclear Information System (INIS)

Siemes, Christina; Quast, Thomas; Kummer, Christiane; Wehner, Sven; Kirfel, Gregor; Mueller, Ulrike; Herzog, Volker

2006-01-01

Growing evidence shows that the soluble N-terminal form (sAPPα) of the amyloid precursor protein (APP) represents an epidermal growth factor fostering keratinocyte proliferation, migration and adhesion. APP is a member of a protein family including the two mammalian amyloid precursor-like proteins APLP1 and APLP2. In the mammalian epidermis, only APP and APLP2 are expressed. APP and APLP2-deficient mice die shortly after birth but do not display a specific epidermal phenotype. In this report, we investigated the epidermis of APP and/or APLP2 knockout mice. Basal keratinocytes showed reduced proliferation in vivo by about 40%. Likewise, isolated keratinocytes exhibited reduced proliferation rates in vitro, which could be completely rescued by either exogenously added recombinant sAPPα, or by co-culture with dermal fibroblasts derived from APP knockout mice. Moreover, APP-knockout keratinocytes revealed reduced migration velocity resulting from severely compromised cell substrate adhesion. Keratinocytes from double knockout mice died within the first week of culture, indicating essential functions of APP-family members for survival in vitro. Our data indicate that sAPPα has to be considered as an essential epidermal growth factor which, however, in vivo can be functionally compensated to a certain extent by other growth factors, e.g., factors released from dermal fibroblasts

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Science.gov (United States)

Anwar, Adil; Li, Min; Frid, Maria G.; Kumar, Binod; Gerasimovskaya, Evgenia V.; Riddle, Suzette R.; McKeon, B. Alexandre; Thukaram, Roopa; Meyrick, Barbara O.; Fini, Mehdi A.

2012-01-01

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a “constitutively activated” phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the “activated” highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, αVβ3 and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH. PMID:22582113

Antibodies and Selection of Monoclonal Antibodies.

Science.gov (United States)

Hanack, Katja; Messerschmidt, Katrin; Listek, Martin

Monoclonal antibodies are universal binding molecules with a high specificity for their target and are indispensable tools in research, diagnostics and therapy. The biotechnological generation of monoclonal antibodies was enabled by the hybridoma technology published in 1975 by Köhler and Milstein. Today monoclonal antibodies are used in a variety of applications as flow cytometry, magnetic cell sorting, immunoassays or therapeutic approaches. First step of the generation process is the immunization of the organism with appropriate antigen. After a positive immune response the spleen cells are isolated and fused with myeloma cells in order to generate stable, long-living antibody-producing cell lines - hybridoma cells. In the subsequent identification step the culture supernatants of all hybridoma cells are screened weekly for the production of the antibody of interest. Hybridoma cells producing the antibody of interest are cloned by limited dilution till a monoclonal hybridoma is found. This is a very time-consuming and laborious process and therefore different selection strategies were developed since 1975 in order to facilitate the generation of monoclonal antibodies. Apart from common automation of pipetting processes and ELISA testing there are some promising approaches to select the right monoclonal antibody very early in the process to reduce time and effort of the generation. In this chapter different selection strategies for antibody-producing hybridoma cells are presented and analysed regarding to their benefits compared to conventional limited dilution technology.

Effect of periodontal dressings on human gingiva fibroblasts in vitro

International Nuclear Information System (INIS)

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

1989-01-01

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

A simple and effective protocol for fast isolation of human Tenon's fibroblasts from a single trabeculectomy biopsy - a comparison of cell behaviour in different culture media.

Science.gov (United States)

Przekora, Agata; Zarnowski, Tomasz; Ginalska, Grazyna

2017-01-01

Human Tenon's fibroblasts (HTFs) play a crucial role in wound healing. They cause postoperative scarring of the filtering bleb and are thus responsible for trabeculectomy failure. This study aimed to find an effective and fast protocol for HTF isolation from trabeculectomy biopsies. The protocol was compared with the commonly recommended HTF isolation procedure, which uses Dulbecco's modified Eagle's medium (DMEM). We used Eagle's minimum essential medium (EMEM) enriched with fibroblast growth factor (FGF), which selectively promoted the proliferation of HTF cells. A secondary goal was to compare HTF morphology, metabolism and growth during parallel cultivation of the isolated cells in FGF-enriched EMEM and DMEM. Standard procedures for HTF isolation from tissue biopsies require a 20- to 30-day culture of the explants to obtain the first monolayer. Our protocol yielded the first monolayer after approx. 15 days. More importantly, the majority of the cells were fibroblasts with only individual epithelium-derived cells present. Using FGF-enriched EMEM allowed 1.3 × 10 6 vimentin-positive fibroblasts to be obtained from a single biopsy within approx. 25 days. Using DMEM resulted in isolation failure and required exchange to FGF-enriched medium to recover the fibroblast culture. HTFs maintained in FGF-enriched EMEM also showed faster proliferation and a different type I collagen production ability compared to HTFs cultured in DMEM. Thus, FGF-enriched EMEM is recommended for fast propagation of HTFs unless the aim of the study is to assess the effect of a tested agent on proliferation ability or type I collagen production. Our fast protocol for HTF isolation allows easy setup of cell banks by researchers under laboratory conditions and could be very useful during testing of novel ophthalmologic anti-fibrotic agents in vitro. Molecular analysis of HTFs isolated from patients with known treatment histories may provide valuable information on the effects of some

Enriched glucose and dextrin mannitol-based media modulates fibroblast behavior on bacterial cellulose membranes

Energy Technology Data Exchange (ETDEWEB)

Stumpf, Taisa R.; Pértile, Renata A.N. [Integrated Technologies Laboratory, Department of Chemical and Food Engineering (Brazil); Rambo, Carlos R., E-mail: rambo@intelab.ufsc.br [Department of Electrical Engineering, Federal University of Santa Catarina, Florianópolis 88040-900 (Brazil); Porto, Luismar M. [Integrated Technologies Laboratory, Department of Chemical and Food Engineering (Brazil)

2013-12-01

Bacterial cellulose (BC) produced by Gluconacetobacter hansenii is a suitable biopolymer for biomedical applications. In order to modulate the properties of BC and expand its use as substrate for tissue engineering mainly in the form of biomembranes, glucose or dextrin were added into a BC fermentation mannitol-based medium (BCGl and BCDe, respectively) under static culture conditions. SEM images showed effects on fiber density and porosity on both sides of the BC membranes. Both enriched media decreased the BET surface area, water holding capacity, and rehydration rate. Fourier transform infrared (attenuated total reflectance mode) spectroscopy (FTIR-ATR) analysis revealed no change in the chemical structure of BC. L929 fibroblast cells were seeded on all BC-based membranes and evaluated in aspects of cell adhesion, proliferation and morphology. BCG1 membranes showed the highest biological performance and hold promise for the use in tissue engineering applications. - Highlights: • Glucose and dextrin were used to modify culture media for BC production. • Microarchitecture of BC was different depending on the enriching agent. • Fibroblasts adhered on the surface of BC modified microarchitectures. • Fibroblasts adhered on glucose modified BC exhibited healthy cell morphology.

Enriched glucose and dextrin mannitol-based media modulates fibroblast behavior on bacterial cellulose membranes

International Nuclear Information System (INIS)

Stumpf, Taisa R.; Pértile, Renata A.N.; Rambo, Carlos R.; Porto, Luismar M.

2013-01-01

Bacterial cellulose (BC) produced by Gluconacetobacter hansenii is a suitable biopolymer for biomedical applications. In order to modulate the properties of BC and expand its use as substrate for tissue engineering mainly in the form of biomembranes, glucose or dextrin were added into a BC fermentation mannitol-based medium (BCGl and BCDe, respectively) under static culture conditions. SEM images showed effects on fiber density and porosity on both sides of the BC membranes. Both enriched media decreased the BET surface area, water holding capacity, and rehydration rate. Fourier transform infrared (attenuated total reflectance mode) spectroscopy (FTIR-ATR) analysis revealed no change in the chemical structure of BC. L929 fibroblast cells were seeded on all BC-based membranes and evaluated in aspects of cell adhesion, proliferation and morphology. BCG1 membranes showed the highest biological performance and hold promise for the use in tissue engineering applications. - Highlights: • Glucose and dextrin were used to modify culture media for BC production. • Microarchitecture of BC was different depending on the enriching agent. • Fibroblasts adhered on the surface of BC modified microarchitectures. • Fibroblasts adhered on glucose modified BC exhibited healthy cell morphology

Monoclonal gammopathy: a diagnosis for to keep in mind; Gammapatia monoclonal: un diagnostico a tener en cuenta

Energy Technology Data Exchange (ETDEWEB)

Howland Alvarez, Ivon; Figueredo Peguero, Yrving; Luna Conde, Clara, E-mail: ihowlanda@infomed.sld.cu [Centro de Investigaciones Medico Quirurgicas, La Habana (Cuba); others, and

2011-07-01

How to identify monoclonal gammopathies at risk for progression has been studied for the last year. 40 patients were studied in which a monoclonal band had been detected, in some of the cases de novo. The electrophoresis was performed in the Hydrasys system. Of the total of electrophoresis carried out, the 14% was monoclonal gammopathy. In 36% a diagnostic assumption was not stated. Most frequent diagnosis in the group of patients with a diagnosis was multiple myeloma. Average age of patients was 61.5 years and there were differences among percentages for sex.

Imaging of multiple myeloma and related monoclonal plasma cell diseases. An update; Bildgebung des multiplen Myeloms und verwandter monoklonaler Plasmazellerkrankungen. Ein Update

Energy Technology Data Exchange (ETDEWEB)

Weber, Marc-Andre [Universitaetsklinikum, Heidelberg (Germany). Abt. Diagnostische und Interventionelle Radiologie; Delorme, Stefan [Deutsches Krebsforschungszentrum, Heidelberg (Germany). Abt. Radiologie; Hillengass, Jens [Universitaetsklinikum, Heidelberg (Germany). Sektion Multiples Myelom

2014-09-15

Multiple myeloma is a hematologic disorder characterized by the infiltration and proliferation of monoclonal plasma cells mainly in the bone marrow. The main symptoms are hypercalcemia, renal impairment, cytopenia/anemia and bone disease - summarized as CRAB-criteria. Symptomatic multiple myeloma is consistently preceded by asymptomatic premalignant stages called monoclonal gammopathy of undetermined significance and smoldering multiple myeloma. Staging of multiple myeloma is based on the measurement of the monoclonal protein in serum and urine as well as the assessment of impairment of hematopoiesis, renal function and mineralized bone. In the last decade the development of novel therapeutic agents has led to an increase in response rates and survival time of patients with multiple myeloma, which further stresses the value of response assessment by imaging. Cross sectional imaging like MRI and CT is currently replacing conventional radiological surveys in the initial work-up and follow-up of patients with monoclonal plasma cell diseases. The added value of MRI is to improve initial staging by unraveling a diffuse infiltration of bone marrow by plasma cells, a focal pattern or a combination of both. Furthermore, a complete remission of myeloma confirmed by MRI and CT goes along with a better prognosis compared to a complete response based only on serological parameters.

MCM - 2 and Ki - 67 as proliferation markers in renal cell carcinoma: A quantitative and semi - quantitative analysis.

Science.gov (United States)

Mehdi, Muhammad Zain; Nagi, Abdul Hanan; Naseem, Nadia

2016-01-01

Fuhrman nuclear grade is the most important histological parameter to predict prognosis in a patient of renal cell carcinoma (RCC). However, it suffers from inter-observer and intra-observer variation giving rise to need of a parameter that not only correlates with nuclear grade but is also objective and reproducible. Proliferation is the measure of aggressiveness of a tumour and it is strongly correlated with Fuhrman nuclear grade, clinical survival and recurrence in RCC. Ki-67 is conventionally used to assess proliferation. Mini-chromosome maintenance 2 (MCM-2) is a lesser known marker of proliferation and identifies a greater proliferation faction. This study was designed to assess the prognostic significance of MCM-2 by comparing it with Fuhrman nuclear grade and Ki-67. n=50 cases of various ages, stages, histological subtypes and grades of RCC were selected for this study. Immunohistochemical staining using Ki-67(MIB-1, Mouse monoclonal antibody, Dako) and MCM-2 (Mouse monoclonal antibody, Thermo) was performed on the paraffin embedded blocks in the department of Morbid anatomy and Histopathology, University of Health Sciences, Lahore. Labeling indices (LI) were determined by two pathologists independently using quantitative and semi-quantitative analysis. Statistical analysis was carried out using SPSS 20.0. Kruskall-Wallis test was used to determine a correlation of proliferation markers with grade, and Pearson's correlate was used to determine correlation between the two proliferation markers. Labeling index of MCM-2 (median=24.29%) was found to be much higher than Ki-67(median=13.05%). Both markers were significantly related with grade (p=0.00; Kruskall-Wallis test). LI of MCM-2 was found to correlate significantly with LI of Ki-67(r=0.0934;p=0.01 with Pearson's correlate). Results of semi-quantitative analysis correlated well with quantitative analysis. Both Ki-67 and MCM-2 are markers of proliferation which are closely linked to grade. Therefore, they

Adiponectin promotes hyaluronan synthesis along with increases in hyaluronan synthase 2 transcripts through an AMP-activated protein kinase/peroxisome proliferator-activated receptor-α-dependent pathway in human dermal fibroblasts

International Nuclear Information System (INIS)

Yamane, Takumi; Kobayashi-Hattori, Kazuo; Oishi, Yuichi

2011-01-01

Highlights: ► Adiponectin promotes hyaluronan synthesis along with an increase in HAS2 transcripts. ► Adiponectin also increases the phosphorylation of AMPK. ► A pharmacological activator of AMPK increases mRNA levels of PPARα and HAS2. ► Adiponectin-induced HAS2 mRNA expression is blocked by a PPARα antagonist. ► Adiponectin promotes hyaluronan synthesis via an AMPK/PPARα-dependent pathway. -- Abstract: Although adipocytokines affect the functions of skin, little information is available on the effect of adiponectin on the skin. In this study, we investigated the effect of adiponectin on hyaluronan synthesis and its regulatory mechanisms in human dermal fibroblasts. Adiponectin promoted hyaluronan synthesis along with an increase in the mRNA levels of hyaluronan synthase 2 (HAS2), which plays a primary role in hyaluronan synthesis. Adiponectin also increased the phosphorylation of AMP-activated protein kinase (AMPK). A pharmacological activator of AMPK, 5-aminoimidazole-4-carboxamide-1β-ribofuranoside (AICAR), increased mRNA levels of peroxisome proliferator-activated receptor-α (PPARα), which enhances the expression of HAS2 mRNA. In addition, AICAR increased the mRNA levels of HAS2. Adiponectin-induced HAS2 mRNA expression was blocked by GW6471, a PPARα antagonist, in a concentration-dependent manner. These results show that adiponectin promotes hyaluronan synthesis along with increases in HAS2 transcripts through an AMPK/PPARα-dependent pathway in human dermal fibroblasts. Thus, our study suggests that adiponectin may be beneficial for retaining moisture in the skin, anti-inflammatory activity, and the treatment of a variety of cutaneous diseases.

Angiotensin II increases CTGF expression via MAPKs/TGF-{beta}1/TRAF6 pathway in atrial fibroblasts

Energy Technology Data Exchange (ETDEWEB)

Gu, Jun [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China); Liu, Xu, E-mail: xkliuxu@yahoo.cn [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China); Wang, Quan-xing, E-mail: shmywqx@126.com [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Tan, Hong-wei [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China); Guo, Meng [National Key Laboratory of Medical Immunology, Second Military Medical University, Shanghai (China); Jiang, Wei-feng; Zhou, Li [Department of Cardiology, Shanghai Chest Hospital, Shanghai Jiaotong University School of medicine, Shanghai (China)

2012-10-01

The activation of transforming growth factor-{beta}1(TGF-{beta}1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGF{beta}1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGF{beta}-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-{beta}1/non-Smad signaling pathways. In the present study, we explored the role of TGF-{beta}1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 {mu}M) provoked the activation of P38 mitogen activated protein kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 {mu}M) also promoted TGF{beta}1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGF{beta}1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGF{beta}1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis. -- Highlights: Black-Right-Pointing-Pointer MAPKs/TGF{beta}1/TRAF6 participates in AngII-induced CTGF expression in atrial fibroblasts. Black-Right-Pointing-Pointer TGF{beta}1/TRAF6 participates in AngII-induced atrial fibroblasts proliferation. Black-Right-Pointing-Pointer TRAF6 may represent a new target for reversing Ang II-induced atrial fibrosis.

Tumor imaging with monoclonal antibodies

International Nuclear Information System (INIS)

Haisma, H.; Hilgers, J.

1987-01-01

Many monoclonal antibodies directed against tumor-associated antigens have been identified, but so far none of these are tumor specific. Polyclonal and monoclonal antibodies have been used for imaging of a wide variety of tumors with success. Radiolabeling of antibody is usually done with iodine isotopes of which 123 I is the best candidate for radioimmunodetection purposes. The labeling of antibodies through chelates makes it possible to use metal radioisotopes like 111 In, which is the best radioisotope for imaging with monoclonal antibodies due to its favorable half-life of 2.5 days. Usually imaging cannot be performed within 24 h after injection, but clearance of antibody can be increased by using F(ab) 2 of Fab. Another approach is to clear non-bound antibody by a second antibody, directed against the first. The detection limit of immunoimaging is about 2 cm, but will be improved by tomography or SPECT. There is still a high false positive and false negative rate, which makes it impossible to use radioimmunodetection as the only technique for diagnosis of tumors. In combination with other detection techniques, tumor imaging with monoclonal antibodies can improve diagnosis. 44 refs.; 3 tabs

Biodegradable poly (lactic acid-co-glycolic acid scaffolds as carriers for genetically-modified fibroblasts.

Directory of Open Access Journals (Sweden)

Tatjana Perisic

Full Text Available Recent advances in gene delivery into cells allow improved therapeutic effects in gene therapy trials. To increase the bioavailability of applied cells, it is of great interest that transfected cells remain at the application site and systemic spread is minimized. In this study, we tested clinically used biodegradable poly(lactic acid-co-glycolic acid (PLGA scaffolds (Vicryl & Ethisorb as transient carriers for genetically modified cells. To this aim, we used human fibroblasts and examined attachment and proliferation of untransfected cells on the scaffolds in vitro, as well as the mechanical properties of the scaffolds at four time points (1, 3, 6 and 9 days of cultivation. Furthermore, the adherence of cells transfected with green fluorescent protein (GFP and vascular endothelial growth factor (VEGF165 and also VEGF165 protein secretion were investigated. Our results show that human fibroblasts adhere on both types of PLGA scaffolds. However, proliferation and transgene expression capacity were higher on Ethisorb scaffolds most probably due to a different architecture of the scaffold. Additionally, cultivation of the cells on the scaffolds did not alter their biomechanical properties. The results of this investigation could be potentially exploited in therapeutic regiments with areal delivery of transiently transfected cells and may open the way for a variety of applications of cell-based gene therapy, tissue engineering and regenerative medicine.

Effect of microemulsions on cell viability of human dermal fibroblasts

Science.gov (United States)

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.

Production of Monoclonal Antibodies specific for Progesterone

OpenAIRE

YÜCEL, Fatıma

2014-01-01

Progesterone levels in milk and serum are indicators of pregnancy in cattle. The progesterone level reaches a peak on the 21 st and 22 nd days of pregnancy. Monoclonal antibodies specific to progesterone could be used for the immunodetection of milk and serum progesterone levels. We report here the development of hybrid cells prdoducing monoclonal antibodies specific for progesterone using hybridoma technology. Hybridoma cells secreting monoclonal antibodies against progesterone (MAM 2H1...

Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

DEFF Research Database (Denmark)

Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders

2002-01-01

The production of hepatocyte growth factor (HGF) and keratinocyte growth factor (KGF) in subepithelial fibroblasts from buccal mucosa, periodontal ligament, and skin was determined after co-culture with keratinocytes. The purpose was to detect differences between the fibroblast subpopulations...... days by ELISA. When cultured on polystyrene, the constitutive level of KGF and HGF in periodontal fibroblasts was higher than the level in buccal and skin fibroblasts. In the presence of keratinocytes, all three types of fibroblasts in general increased their HGF and KGF production 2-3 times. When...... cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...

Leptin Regulates Proliferation and Apoptosis in Human Prostate

Directory of Open Access Journals (Sweden)

Eduardo Leze

2012-01-01

Full Text Available This paper aimed to evaluate the leptin role on the cellular proliferation and the expression of fibroblast growth factor 2, aromatase enzyme, and apoptotic genes in the human prostate tissue. Methods. Fifteen samples of hyperplasic prostate tissue were divided in four symmetric parts maintained in RPMI medium supplemented with 10% fetal bovine serum, 1 ng/mL of gentamicin, and added with 50 ng/mL leptin (L or not (C. After 3 hours of incubation, gene expression was evaluated by real time RT-PCR. Cellular proliferation was evaluated by immunohistochemistry for PCNA. Results. The leptin treatment led to an increase cellular proliferation (C=21.8±0.5; L=64.8±0.9; P<0.0001 and in the expression of Bax (C=0.4±0.1; L=0.9±0.2; P<0.05 while Bcl-2 (C=19.9±5.6; L=5.6±1.8; P<0.05, Bcl-x (C=0.2±0.06; L=0.07±0.02; P<0.05, and aromatase expressions (C=1.9±0.6; L=0.4±0.1; P<0.04 were significantly reduced. Conclusion. Leptin has an important role in maintaining the physiological growth of the prostate since it stimulates both cellular proliferation and apoptosis, with the decrement in the aromatase gene expression.

Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

International Nuclear Information System (INIS)

Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

2010-01-01

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

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

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Jaroslav Zelenka

2015-01-01

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

Knockdown of versican 1 blocks cigarette-induced loss of insoluble elastin in human lung fibroblasts.

Science.gov (United States)

Xu, Lu-lu; Lu, Yun-tao; Zhang, Jing; Wu, Lian; Merrilees, Mervyn J; Qu, Jie-ming

2015-08-15

COPD lung is characterized by loss of alveolar elastic fibers and an increase in the chondroitin sulfate (CS) matrix proteoglycan versican V1 (V1). V1 is a known inhibitor of elastic fiber deposition and this study investigates the effects of knockdown of V1, and add-back of CS, on CCL-210 lung fibroblasts treated with cigarette smoke extract (CSE) as a model for COPD. CSE inhibited fibroblast proliferation, viability, tropoelastin synthesis, and elastin deposition, and increased V1 synthesis and secretion. V1 siRNA decreased V1 and constituent CS, did not affect tropoelastin production, but blocked the CSE-induced loss in insoluble elastin. Exogenous CS reduced insoluble elastin, even in the presence of V1 siRNA. These findings confirm that V1 and CS impair the assembly of tropoelastin monomers into insoluble fibers, and further demonstrate that specific knockdown of V1 alleviates the impaired assembly of elastin seen in cultures of pulmonary fibroblasts exposed to CSE, indicating a regulatory role for this protein in the pathophysiology of COPD. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell proliferation and migration are modulated by Cdk-1-phosphorylated endothelial-monocyte activating polypeptide II.

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Margaret A Schwarz

Full Text Available Endothelial-Monocyte Activating Polypeptide (EMAP II is a secreted protein with well-established anti-angiogenic activities. Intracellular EMAP II expression is increased during fetal development at epithelial/mesenchymal boundaries and in pathophysiologic fibroproliferative cells of bronchopulmonary dysplasia, emphysema, and scar fibroblast tissue following myocardial ischemia. Precise function and regulation of intracellular EMAP II, however, has not been explored to date.Here we show that high intracellular EMAP II suppresses cellular proliferation by slowing progression through the G2M cell cycle transition in epithelium and fibroblast. Furthermore, EMAP II binds to and is phosphorylated by Cdk1, and exhibits nuclear/cytoplasmic partitioning, with only nuclear EMAP II being phosphorylated. We observed that extracellular secreted EMAP II induces endothelial cell apoptosis, where as excess intracellular EMAP II facilitates epithelial and fibroblast cells migration.Our findings suggest that EMAP II has specific intracellular effects, and that this intracellular function appears to antagonize its extracellular anti-angiogenic effects during fetal development and pulmonary disease progression.

The hallmarks of fibroblast ageing.

Science.gov (United States)

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.

Expression of platelet-derived growth factor and its receptors in proliferative disorders of fibroblastic origin.

OpenAIRE

Smits, A.; Funa, K.; Vassbotn, F. S.; Beausang-Linder, M.; af Ekenstam, F.; Heldin, C. H.; Westermark, B.; Nistér, M.

1992-01-01

Platelet-derived growth factor (PDGF) is known to stimulate the proliferation of connective tissue-derived cells in vitro. Less is known about its functions in vivo, and the role of PDGF in the development of human tumors has not been clarified. The authors have investigated the occurrence of PDGF and PDGF receptors in a series of proliferative disorders of fibroblastic origin using immunohistochemical and in situ hybridization techniques. High expression of PDGF beta-receptor mRNA and protei...

The influence of SrO and CaO in silicate and phosphate bioactive glasses on human gingival fibroblasts.

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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.

The effect of tranilast on fibroblast activation protein α (FAP-α expression in normal and keloid fibroblasts in vitro

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Paweł P. Antończak

2017-07-01

Full Text Available Introduction . Tranilast (N-(3’,4’-demethoxycinnamoyl-anthranilic acid is an anti-allergic drug. Its mechanism of action is based on the inhibition of antigen-induced release of chemical mediators from mast cells and basophils. It also reveals antifibroproliferative activities. These properties of tranilast are used in the treatment of hypertrophic scars and keloids. Keloids are characterized by incorrect extracellular matrix components turnover. Fibroblasts derived from keloids reveal overproduction of collagen type I and decreased degradation of extracellular matrix in comparison with normal fibroblasts. Fibroblast activation protein α (FAP-α may play an important role in remodeling of extracellular matrix and the invasive properties of keloids. Objective . In the present study, the effect of tranilast on expression of FAP-α gene and its protein was evaluated in normal human dermal fibroblasts and fibroblasts derived from keloids cultured in vitro . Materials and methods. In the first stage of the study, the influence of tranilast on cell viability was estimated. The second stage of the study included the quantitative evaluation of FAP-α mRNA expression in normal and keloid fibroblasts treated with tranilast. The third stage of the study comprised fibroblast activation protein α expression analysis in the examined cells treated with tranilast. Results and conclusions . The expression of FAP-α gene and fibroblast activation protein α is higher in keloid fibroblasts. Tranilast at concentrations of 3 μM and 30 μM up-regulated mRNA FAP-α expression in normal fibroblasts but did not influence keloid fibroblasts. The drug, at concentrations of 30 μM and 300 μM up-regulated fibroblast activation protein α expression in normal fibroblasts and did not influence keloid fibroblasts. Tranilast antiproliferative effect is not associated with FAP-α expression in keloid fibroblasts.

Fibroblast growth factor receptors in breast cancer.

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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.

Radiolabeled monoclonal antibodies: a review

International Nuclear Information System (INIS)

Toledo e Souza, I.T. de; Okada, H.

1990-05-01

Since the description by Kohler and Milstein 1975 of their technique for producing monoclonal antibodies of predefined specificity, it has become a mainstay in most laboratories that utilize immunochemical techniques to study problems in basic, applied or clinical research. Paradoxically, the very success of monoclonal antibodies has generated a literature which is now so vast and scattered that it has become difficult to obtain a perspective. This brief review represents the distillation of many publications relating to the production and use of monoclonaal antibodies as radiopharmaceuticals. Significant advances were made possible in the last few years by combined developments in the fields of tumor-associated antigens and of monoclonal antibodies. In fact monoclonal antibodies against some well defined tumor-associated antigens, has led to significantly greater practical possibilities for producing highly specific radiolabeled antibodies as radiopharmaceuticals for diagnosis and therapy of human tumors. One of the main requirements of this methodology is the availability of stable radiopharmaceutical reagents which after labeling in vivo injection retain the capacity of specific interaction with the defined antigen and their molecular integrity. Since injection into human is the objetive of this kind of study all the specifications of radiopharmaceutical have to be fulfilled e.g. sterility, apirogenicity and absence of toxicity. (author) [pt

Telomere length analysis in monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia Binet A

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F.M. Furtado

Full Text Available Monoclonal B-cell lymphocytosis (MBL is an asymptomatic clinical entity characterized by the proliferation of monoclonal B cells not meeting the diagnosis criteria for chronic lymphocytic leukemia (CLL. MBL may precede the development of CLL, but the molecular mechanisms responsible for disease progression and evolution are not completely known. Telomeres are usually short in CLL and their attrition may contribute to disease evolution. Here, we determined the telomere lengths of CD5+CD19+ cells in MBL, CLL, and healthy volunteers. Twenty-one CLL patients, 11 subjects with high-count MBL, and 6 with low-count MBL were enrolled. Two hundred and sixty-one healthy volunteers aged 0 to 88 years were studied as controls. After diagnosis confirmation, a flow cytometry CD19+CD5+-based cell sorting was performed for the study groups. Telomere length was determined by qPCR. Telomere length was similar in the 3 study groups but shorter in these groups compared to normal age-matched subjects that had been enrolled in a previous study from our group. These findings suggest that telomere shortening is an early event in CLL leukemogenesis.

Control of cell proliferation by a porous chitosan scaffold with multiple releasing capabilities

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Cai, Shu-Jyun; Li, Ching-Wen; Weihs, Daphne; Wang, Gou-Jen

2017-01-01

Abstract The aim of this study was to develop a porous chitosan scaffold with long-acting drug release as an artificial dressing to promote skin wound healing. The dressing was fabricated by pre-freezing at different temperatures (−20 and −80 °C) for different periods of time, followed by freeze-drying to form porous chitosan scaffolds with different pore sizes. The chitosan scaffolds were then used to investigate the effect of the controlled release of fibroblast growth factor-basic (bFGF) and transforming growth factor-β1 (TGFβ1) on mouse fibroblast cells (L929) and bovine carotid endothelial cells (BEC). The biocompatibility of the prepared chitosan scaffold was confirmed with WST-1 proliferation and viability assay, which demonstrated that the material is suitable for cell growth. The results of this study show that the pore sizes of the porous scaffolds prepared by freeze-drying can change depending on the pre-freezing temperature and time via the formation of ice crystals. In this study, the scaffolds with the largest pore size were found to be 153 ± 32 μm and scaffolds with the smallest pores to be 34 ± 9 μm. Through cell culture analysis, it was found that the concentration that increased proliferation of L929 cells for bFGF was 0.005 to 0.1 ng/mL, and the concentration for TGFβ1 was 0.005 to 1 ng/mL. The cell culture of the chitosan scaffold and growth factors shows that 3.75 ng of bFGF in scaffolds with pore sizes of 153 ± 32 μm can promote L929 cell proliferation, while 400 pg of TGFβ1 in scaffolds with pore size of 34 ± 9 μm can enhance the proliferation of L929 cells, but also inhibit BEC proliferation. It is proposed that the prepared chitosan scaffolds can form a multi-drug (bFGF and TGFβ1) release dressing that has the ability to control wound healing via regulating the proliferation of different cell types. PMID:29230255

Feasibility study of a biocompatible pneumatic dispensing system using mouse 3T3-J2 fibroblasts

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Lee, Sangmin; Kim, Hojin; Kim, Joonwon

2017-12-01

This paper presents results for dispensing living cells using a pneumatic dispensing system to verify the feasibility of using this system to fabricate biomaterials. Living cells (i.e., mouse 3T3-J2 fibroblast) were dispensed with different dispensing pressures in order to evaluate the effect of dispensing process on cell viability and proliferation. Based on the results of a live-dead assay, more than 80% of cell viability has been confirmed which was reasonably similar to that in the control group. Furthermore, measurement of cell metabolic activity after dispensing confirmed that the dispensed cell proliferated at a rate comparable to that of the control group. These results demonstrate that the pneumatic dispensing system is a promising tool for fabrication of biomaterials.

Test of Antifibrotic Drugs in a Cellular Model of Fibrosis Based on Muscle-Derived Fibroblasts from Duchenne Muscular Dystrophy Patients.

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Zanotti, Simona; Mora, Marina

2018-01-01

An in vitro model of muscle fibrosis, based on the use of primary human fibroblasts isolated from muscle biopsies of patients affected by Duchenne muscular dystrophies (DMD) and cultivated in monolayer and 3D conditions, is used to test the potential antifibrotic activity of pirfenidone (PFD). This in vitro model may be usefully also to evaluate the toxicity and efficacy of other candidate molecules for the treatment of fibrosis. The drug toxicity is evaluated using a colorimetric assay based on the conversion of tetrazolium salt (MTT) to insoluble formazan, while the effect of the drug on cell proliferation is measured with the bromodeoxyuridine incorporation assay. The efficacy of the drug is evaluated in fibroblast monolayers by quantitating synthesis and deposition of intracellular collagen with a spectrophotometric picrosirius red-based assay, and by quantitating cell migration using a "scratch" assay. The efficacy of PFD as antifibrotic drug is also evaluated in a 3D fibroblast model by measuring diameters and number of nodules.

Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

KAUST Repository

Kashuba, Elena

2015-05-12

We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells. Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.

[Effects of rhynchophylla alkaloids on vascular adventitial fibroblast apoptosis and proliferation in the thoracic aorta of spontaneously hypertensive rats].

Science.gov (United States)

Dai, Guo-Hua; Sun, Jing-Chang; Qi, Dong-Mei

2012-09-01

To study the effects of rhynchophylline, isorhynchophylline, and rhynchophylla alkaloids on the vascular adventitial fibroblasts (VAF) apoptosis and proliferation in thoracic aorta of spontaneously hypertensive rats (SHR), and on the Bcl-2, Bax, c-Fos, c-Myc, laminin (LN), and fibronectin (FN). Forty 8-week old male SHR were randomly divided into five groups, i. e., the model group, the captopril group (17.5 mg/kg), the isorhynchophylline group (5.0 mg/kg), the rhynchophylline group (5.0 mg/kg), and the rhynchophylla alkaloids group (50.0 mg/kg), 8 in each group. In addition, eight 8-week old male Wistar rats were selected as the normal group. Equal volume of normal saline was given to rats in the normal group and the model group by gastrogavage. Rats in the rest groups were perfused with isovolumic medication solution (10 mL/kg), six days per week for eight successive weeks. The dosage of drugs was adjusted according to the change of body weight. The VAF apoptosis rate of the thoracic aorta was measured by Annexin V-FITC combined with PI dyeing and flow cytometry. The protein expressions of thoracic aortic Bcl-2, Bax, c-Myc, c-Fos, FN, and LN were detected by immunohistochemical assay. The adventitial transforming growth factor beta1 (TGF-beta1) mRNA expression in the thoracic aorta was detected by in situ hybridization method. Compared with the model group, the tail arterial systolic pressure decreased, the VAF apoptosis and the protein expression of Bax increased, Bcl-2, c-Fos, FN, LN, and TGF-beta1 mRNA all decreased in the thoracic aorta of SHR in each treatment group after 4-and 8-week of intervention. Rhynchophylline, isorhynchophylline, and rhynchophylla alkaloids could inhibit the protein expression of c-Myc with statistical difference (Prhynchophylla alkaloids group (P>0.05). There was statistical difference in increased VAF apoptosis and decreased protein expressions of Bcl-2, c-Myc, and LN (Prhynchophylla alkaloids group (P>0.05). Rhynchophylline

Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

International Nuclear Information System (INIS)

Rutz, H. Peter; Little, John B.

1995-01-01

Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO 2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G 2 /M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles in proliferating and nonproliferating mammalian cells

International Nuclear Information System (INIS)

Korbelik, M.; Osmak, M.; Suhar, A.; Turk, V.; Skrk, J.

1990-01-01

Dynamics of postirradiation intracellular cysteine and aspartic proteinases profiles were examined in proliferating and nonproliferating Chinese hamster fibroblasts (V 79). The results show that there are significant alterations in cysteine and aspartic intracellular proteinases activity already in the early postirradiation period, which are different in proliferating and nonproliferating cells. Irradiation of the cells examined to low doses and up to 15 Gy induced an increase in cysteine proteinases activity in the early postexposure period, while at higher irradiation doses applied, the activity of these proteinases was decreased. These observations suggest that intracellular proteinases are actively participating in process involving recovery from radiation injury or cell killing. (orig.) [de

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts

International Nuclear Information System (INIS)

Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

2006-01-01

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation

A cytoskeleton-associated protein, TMAP/CKAP2, is involved in the proliferation of human foreskin fibroblasts.

Science.gov (United States)

Jeon, Sang-Min; Choi, Bongkun; Hong, Kyung Uk; Kim, Eunhee; Seong, Yeon-Sun; Bae, Chang-Dae; Park, Joobae

2006-09-15

Previously, we reported the cloning of a cytoskeleton-associated protein, TMAP/CKAP2, which was up-regulated in primary human gastric cancers. Although TMAP/CKAP2 has been found to be expressed in most cancer cell lines examined, the function of CKAP2 is not known. In this study, we found that TMAP/CKAP2 was not expressed in G0/G1 arrested HFFs, but that it was expressed in actively dividing cells. After initiating the cell cycle, TMAP/CKAP2 levels remained low throughout most of the G1 phase, but gradually increased between late G1 and G2/M. Knockdown of TMAP/CKAP2 reduced pRB phosphorylation and increased p27 expression, and consequently reduced HFF proliferation, whereas constitutive TMAP/CKAP2 expression increased pRB phosphorylation and enhanced proliferation. Our results show that this novel cytoskeleton-associated protein is expressed cell cycle dependently and that it is involved in cell proliferation.

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

Science.gov (United States)

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

2016-03-01

Advanced age is characterized by impairments in wound healing, and evidence is accumulating that this may be due in part to a concomitant increase in oxidative stress. Extended exposure to reactive oxygen species (ROS) is thought to lead to cellular dysfunction and organismal death via the destructive oxidation of intra-cellular proteins, lipids and nucleic acids. Extracellular superoxide dismutase (ecSOD/SOD3) is a prime antioxidant enzyme in the extracellular space that eliminates ROS. Here, we demonstrate that reduced SOD3 levels contribute to healing impairments in aged mice. These impairments include delayed wound closure, reduced neovascularization, impaired fibroblast proliferation and increased neutrophil recruitment. We further establish that SOD3 KO and aged fibroblasts both display reduced production of TGF-β1, leading to decreased differentiation of fibroblasts into myofibroblasts. Taken together, these results suggest that wound healing impairments in ageing are associated with increased levels of ROS, decreased SOD3 expression and impaired extracellular oxidative stress regulation. Our results identify SOD3 as a possible target to correct age-related cellular dysfunction in wound healing. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Quantum Dots-Based Immunofluorescent Imaging of Stromal Fibroblasts Caveolin-1 and Light Chain 3B Expression and Identification of Their Clinical Significance in Human Gastric Cancer

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Honglei Chen

2012-10-01

Full Text Available Caveolin-1 (Cav-1 expression deficiency and autophagy in tumor stromal fibroblasts (hereafter fibroblasts are involved in tumor proliferation and progression, particularly in breast and prostate cancer. The aim of this study was to detect the expression of fibroblastic Cav-1 and LC3B, markers of autophagy, in gastric cancer (GC and to analyze their clinical significances. Furthermore, because Epstein-Barr virus (EBV-associated GC (EBVaGC is a unique subtype of GC; we compared the differential expression of fibroblastic Cav-1 and LC3B in EBVaGC and non-EBVaGC. Quantum dots (QDs-based immunofluorescence histochemistry was used to examine the expression of fibroblastic Cav-1 and LC3B in 118 cases of GC with adequate stroma. QDs-based double immunofluorescence labeling was performed to detect the coexpression of Cav-1 and LC3B proteins. EBV-encoded small RNA was detected by QDs-based fluorescence in situ hybridization to identify EBVaGC. Multivariate analysis indicated that low fibroblastic Cav-1 level was an independent prognosticator (p = 0.029 that predicted poorer survival of GC patients. Positive fibroblastic LC3B was correlated with lower invasion (p = 0.032 and was positively associated with Cav-1 expression (r = 0.432, p < 0.001. EBV infection did not affect fibroblastic Cav-1 and LC3B expression. In conclusion, positive fibroblastic LC3B correlates with lower invasion, and low expression of fibroblastic Cav-1 is a novel predictor of poor GC prognosis.

The effect of mitomycin-c in keloid fibroblast cultures

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Ishandono Dachlan

2016-12-01

Full Text Available ABSTRACT Keloid occurs due to hyperactivity of keloid fibroblast (KF in proliferation, migration, collagen deposition, together with low rates of collagen degradation. These are under the responsibility of TGF-b. Mitomycin C (MC is used for treating keloid by a topical application during surgery at the level of 0.02% to 0.08%. Unfortunately, the lowest effective level of MC for keloid has not been determined yet. We aimed to determine the lowest effective level of MC in the suppression of KF activities. Various levels of MC diluted in growth medium were administered on KF that were isolated from six patients. After 24 hours and 72 hours of incubation, cellular proliferation, collagen deposition, cellular migration and level of TGF-b, were analyzed. Application of 120 uM MC on KF culture for 24 hours could significantly reduce TGF-b production from 1265.74 ± 274.81 pg/mL to 265.17 ± 12.20 pg/mL; proliferation index from 100% to 84.01 ± 12.91%; inhibit cellular migration to 64.38 ± 3.66%; but reduce collagen depositions from 100% to only 91.13 ± 10.19%. The lowest MC level is on 30 uM or equal with 0.001%. In conclusion, the lowest level of MC can suppress the activities of KF is 0.001%. Moreover, due to low activity in inhibiting collagen deposition, MC would be better as an adjuvant drug for keloid surgery.

Generation of a Monoclonal Antibody against Mycoplasma spp. following Accidental Contamination during Production of a Monoclonal Antibody against Lawsonia intracellularis

OpenAIRE

Hwang, Jeong-Min; Lee, Ji-Hye; Yeh, Jung-Yong

2012-01-01

This report describes Mycoplasma contamination of Lawsonia intracellularis cultures that led to the unintended acquisition of a monoclonal antibody against Mycoplasma spp. during the attempted generation of a monoclonal antibody against L. intracellularis.

Macrophage migration inhibitory factor stimulated by Helicobacter pylori increases proliferation of gastric epithelial cells

Science.gov (United States)

Xia, Harry Hua-Xiang; Lam, Shiu Kum; Chan, Annie O.O.; Lin, Marie Chia Mi; Kung, Hsiang Fu; Ogura, Keiji; Berg, Douglas E.; Wong, Benjamin C. Y.

2005-01-01

AIM: Helicobacter pylori (H pylori) is associated with increased gastric inflammatory and epithelial expression of macrophage migration inhibitory factor (MIF) and gastric epithelial cell proliferation. This study aimed at determining whether H pylori directly stimulates release of MIF in monocytes, whether the cag pathogenicity island (PAI) is involved for this function, and whether MIF stimulated by H pylori increases gastric epithelial cell proliferation in vitro. METHODS: A cytotoxic wild-type H pylori strain (TN2)and its three isogenic mutants (TN2△cag, TN2△cagA and TN2△cagE) were co-cultured with cells of a human monocyte cell line, THP-1, for 24 h at different organism/cell ratios. MIF in the supernatants was measured by an ELISA. Cells of a human gastric cancer cell line, MKN45, were then co-cultured with the supernatants, with and without monoclonal anti-MIF antibody for 24 h. The cells were further incubated for 12 h after addition of 3H-thymidine, and the levels of incorporation of 3H-thymidine were measured with a liquid scintillation counter. RESULTS: The wild-type strain and the isogenic mutants, TN2△cagA and TN2△cagE, increased MIF release at organism/cell ratios of 200/1 and 400/1, but not at the ratios of 50/1 and 100/1. However, the mutant TN2△cag did not increase the release of MIF at any of the four ratios. 3H-thymidine readings for MKN-45 cells were significantly increased with supernatants derived from the wild-type strain and the mutants TN2△cagA and TN2△cagE, but not from the mutant TN2△cag. Moreover, in the presence of monoclonal anti-MIF antibody, the stimulatory effects of the wild-type strain on cell proliferation disappeared. CONCLUSION: H pylori stimulates MIF release in monocytes, likely through its cag PAI, but not related to cagA or cagE. H pylori-stimulated monocyte culture supernatant increases gastric cell proliferation, which is blocked by anti-MIF antibody, suggesting that MIF plays an important role in H

SDF-1 in Mammary Fibroblasts of Bovine with Mastitis Induces EMT and Inflammatory Response of Epithelial Cells.

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He, Guiliang; Ma, Mengru; Yang, Wei; Wang, Hao; Zhang, Yong; Gao, Ming-Qing

2017-01-01

Fibroblasts constitute the majority of the stromal cells within bovine mammary gland, yet the functional contributions of these cells to mastitis and fibrosis and the mechanism are poorly understood. In this study, we demonstrate that inflammation-associated fibroblasts (INFs) extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix (ECM) proteins, chemokines and cytokines compared to normal fibroblasts (NFs) from dairy cows during lactation. The INFs induced epithelial-mesenchymal transition (EMT) and inflammatory responses of mammary epithelial cells in a vitro co-culture model. These functional contributions of INFs to normal epithelial cells were mediated through their ability to secrete stromal cell-derived factor 1 (SDF-1). SDF-1 was highly secreted/expressed by INFs, lipopolysaccharide (LPS) -treated NFs, lipoteichoic acid (LTA) -treated NFs, as well as mastitic tissue compared to their counterparts. Exogenous SDF-1 promoted EMT on epithelial cells through activating NF-κB pathway, induced inflammation response and inhibited proliferation of epithelial cells. In addition, SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland, which was attenuated by a specific inhibitor of the receptor of SDF-1. Our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1, which could result in the inflammation spread and fibrosis within mammary gland.

A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

International Nuclear Information System (INIS)

Pan Hui; Kantharia, Sarah; Jiang Hongliang; Chen Weiliam

2011-01-01

Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

A fibroblast/macrophage co-culture model to evaluate the biocompatibility of an electrospun Dextran/PLGA scaffold and its potential to induce inflammatory responses

Energy Technology Data Exchange (ETDEWEB)

Pan Hui; Kantharia, Sarah [Department of Biomedical Engineering, State University of New York-Stony Brook, Stony Brook, NY 11794-2580 (United States); Jiang Hongliang [Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027 (China); Chen Weiliam, E-mail: weiliam.chen@nyumc.org [Division of Wound Healing and Regenerative Medicine, Department of Surgery, New York University School of Medicine, New York, NY 10016 (United States)

2011-12-15

Fibroblasts and macrophages are the two major types of cells responding to implanted biomaterials. They play crucial roles in inflammatory responses, host-material interactions and tissue remodeling. However, the synergistic interactions of these two cell types with biomaterials are not fully understood. In this investigation, an in vitro fibroblast/macrophage co-culture system was utilized to examine the biocompatibility and the potential to induce inflammatory responses of an electrospun Dextran/PLGA scaffold. The scaffold did not affect the morphologies, attachments, proliferations and viabilities of both the fibroblasts and macrophages, cultured separately or together. Moreover, it only activated a small subset of the macrophages implicating a low potential to induce either severe acute or chronic inflammatory response. Additionally, fibroblasts played a role in prolonging macrophage activation in the presence of the scaffolds. Using antibody arrays, IL-10, SDF-1, MIP-1 gamma and RANTES were found to be up-regulated when the cells were incubated with the scaffolds. The results of subdermal implantation of the Dextran/PLGA scaffolds confirmed its biocompatibility and low inflammatory potential.

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

International Nuclear Information System (INIS)

Ma, Jane; Bishoff, Bridget; Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane; Castranova, Vincent

2017-01-01

The emission of cerium oxide nanoparticles (CeO 2 ) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO 2 induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO 2 -induced fibrosis. Male Sprague-Dawley rats were exposed to CeO 2 (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO 2 (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO 2 -exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO 2 exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO 2 -exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO 2 exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO 2 nanoparticle exposure. - Highlights: • CeO 2 exposure induced lung fibrosis. • CeO 2 were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO 2 caused ATII cell hypertrophy and hyperplasia and altered fibroblast function

Role of epithelial-mesenchymal transition (EMT) and fibroblast function in cerium oxide nanoparticles-induced lung fibrosis

Energy Technology Data Exchange (ETDEWEB)

Ma, Jane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Bishoff, Bridget [Mylan Pharmaceuticals, Morganntown, WV (United States); Mercer, R.R.; Barger, Mark; Schwegler-Berry, Diane [Health Effects Laboratory Division, NIOSH, Morgantown, WV (United States); Castranova, Vincent, E-mail: vcastran@hsc.wvu.edu [School of Pharmacy, West Virginia University, Morgantown, WV (United States)

2017-05-15

The emission of cerium oxide nanoparticles (CeO{sub 2}) from diesel engines, using cerium compounds as a catalyst to lower the diesel exhaust particles, is a health concern. We have previously shown that CeO{sub 2} induced pulmonary inflammation and lung fibrosis. The objective of the present study was to investigate the modification of fibroblast function and the role of epithelial-mesenchymal transition (EMT) in CeO{sub 2}-induced fibrosis. Male Sprague-Dawley rats were exposed to CeO{sub 2} (0.15 to 7 mg/kg) by a single intratracheal instillation and sacrificed at various times post-exposure. The results show that at 28 days after CeO{sub 2} (3.5 mg/kg) exposure, lung fibrosis was evidenced by increased soluble collagen in bronchoalveolar lavage fluid, elevated hydroxyproline content in lung tissues, and enhanced sirius red staining for collagen in the lung tissue. Lung fibroblasts and alveolar type II (ATII) cells isolated from CeO{sub 2}-exposed rats at 28 days post-exposure demonstrated decreasing proliferation rate when compare to the controls. CeO{sub 2} exposure was cytotoxic and altered cell function as demonstrated by fibroblast apoptosis and aggregation, and ATII cell hypertrophy and hyperplasia with increased surfactant. The presence of stress fibers, expressed as α-smooth muscle actin (SMA), in CeO{sub 2}-exposed fibroblasts and ATII cells was significantly increased compared to the control. Immunohistofluorescence analysis demonstrated co-localization of TGF-β or α-SMA with prosurfactant protein C (SPC)-stained ATII cells. These results demonstrate that CeO{sub 2} exposure affects fibroblast function and induces EMT in ATII cells that play a role in lung fibrosis. These findings suggest potential adverse health effects in response to CeO{sub 2} nanoparticle exposure. - Highlights: • CeO{sub 2} exposure induced lung fibrosis. • CeO{sub 2} were detected in lung tissue, alveolar type II (ATII) cells and fibroblasts. • CeO{sub 2} caused ATII

Regulation of cell proliferation and apoptosis in neuroblastoma cells by ccp1, a FGF2 downstream gene

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Inman Gareth J

2010-11-01

Full Text Available Abstract Background Coiled-coil domain containing 115 (Ccdc115 or coiled coil protein-1 (ccp1 was previously identified as a downstream gene of Fibroblast Growth Factor 2 (FGF2 highly expressed in embryonic and adult brain. However, its function has not been characterised to date. Here we hypothesized that ccp1 may be a downstream effecter of FGF2, promoting cell proliferation and protecting from apoptosis. Methods Forced ccp1 expression in mouse embryonic fibroblast (MEF and neuroblastoma SK-N-SH cell line, as well as down-regulation of ccp1 expression by siRNA in NIH3T3, was used to characterize the role of ccp1. Results Ccp1 over-expression increased cell proliferation, whereas down-regulation of ccp1 expression reduced it. Ccp1 was able to increase cell proliferation in the absence of serum. Furthermore, ccp1 reduced apoptosis upon withdrawal of serum in SK-N-SH. The mitogen-activated protein kinase (MAPK or ERK Kinase (MEK inhibitor, U0126, only partially inhibited the ccp1-dependent BrdU incorporation, indicating that other signaling pathway may be involved in ccp1-induced cell proliferation. Induction of Sprouty (SPRY upon FGF2 treatment was accelerated in ccp1 over-expressing cells. Conclusions All together, the results showed that ccp1 regulates cell number by promoting proliferation and suppressing cell death. FGF2 was shown to enhance the effects of ccp1, however, it is likely that other mitogenic factors present in the serum can also enhance the effects. Whether these effects are mediated by FGF2 influencing the ccp1 function or by increasing the ccp1 expression level is still unclear. At least some of the proliferative regulation by ccp1 is mediated by MAPK, however other signaling pathways are likely to be involved.

Gamopatias monoclonais: critérios diagnósticos e diagnósticos diferenciais Monoclonal gammopathies: diagnosis criteria and differential diagnosis

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Rosa Malena D. Faria

2007-03-01

Full Text Available As gamopatias monoclonais constituem um grupo de desordens caracterizado pela proliferação monoclonal de plasmócitos, que produzem e secretam imunoglobulina ou fragmento de imunoglobulina monoclonal (proteína M . Este artigo propõe uma revisão dos critérios diagnósticos das principais gamopatias monoclonais e diagnósticos diferenciais, uma vez que é comum a sobreposição de muitas características clínicas entre suas variantes. A gamopatia monoclonal de significado indeterminado (MGUS é definida pela presença de proteína M sérica 30% ou plasmocitoma documentado por biópsia. Se a lesão óssea decorre de plasmocitoma solitário ou somente osteoporose, sem fratura, a plasmocitose medular também precisa ser > 30%, para preencher critérios de MM. As gamopatias monoclonais podem estar associadas a diversas doenças, incluindo desordens linfoproliferativas, reumatológicas, neurológicas, dermatológicas e infecciosas. A definição das características clínicas e laboratoriais de cada entidade, maligna ou benigna, facilita o diagnóstico das gamopatias monoclonais e, como conseqüência, seu manejo clínico pelos médicos assistentes.Monoclonal gammopathies are a group of disorders characterized by proliferation of monoclonal plasma cells, which produce and secrete monoclonal immunoglobulin or fragments of monoclonal immunoglobulin (M protein. This paper proposes to review diagnostic criteria of the most important monoclonal gammopathies and their differential diagnosis, because superposition of many clinical characteristics is common between variants. The monoclonal gammopathy of undetermined significance (MGUS is defined by the presence of serum M protein < 3g/dL and/or urinary M protein < 1g/24h, bone marrow plasma cell < 10%, and absence of organ and tissue damage. Asymptomatic multiple myeloma (MM is characterized by the presence of M protein, bone marrow or tissue biopsy plasma cell infiltration, and non-compliance of the

Growth inhibitory effects of endotoxins from Bacteroides gingivalis and intermedius on human gingival fibroblasts in vitro

International Nuclear Information System (INIS)

Layman, D.L.; Diedrich, D.L.

1987-01-01

Purified endotoxin or lipopolysaccharide from Bacteroides gingivalis and Bacteroides intermedius caused a similar dose-dependent inhibition of growth of cultured human gingival fibroblasts as determined by 3 H-thymidine incorporation and direct cell count. Approximately 200 micrograms/ml endotoxin caused a 50% reduction in 3 H-thymidine uptake of logarithmically growing cells. Inhibition of growth was similar in cultures of fibroblasts derived from either healthy or diseased human gingiva. When examining the change in cell number with time of exposure in culture, the rate of proliferation was significantly suppressed during the logarithmic phase of growth. However, the cells recovered so that the rate of proliferation, although reduced, was sufficient to produce a cell density similar to the control cells with prolonged culture. The endotoxins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The profiles of the Bacteroides endotoxins were different. B. gingivalis endotoxin showed a wide range of distinct bands indicating a heterogeneous distribution of molecular species. Endotoxin from B. intermedius exhibited a few discrete low molecular weight bands, but the majority of the lipopolysaccharides electrophoresed as a diffuse band of high molecular weight material. The apparent heterogeneity of the two Bacteroides endotoxins and the similarity in growth inhibitory capacity suggest that growth inhibitory effects of these substances cannot be attributed to any polysaccharide species of endotoxin

KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms.

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Garry L Coles

2015-10-01

Full Text Available The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that Kinesin family member 7 (Kif7 promotes Hedgehog (Hh signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. Kif7 encodes a microtubule interacting protein that controls Hh signaling through regulation of microtubule dynamics within the primary cilium. However, whether Kif7 has a function in nonciliated cells remains largely unknown. The role Kif7 plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that Kif7 is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation in vivo and in vitro. Immunostaining and transmission electron microscopy experiments show that Kif7dda/dda mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and Kif7dda/dda mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that Kif7 may function as a general regulator of cellular proliferation. We ascertained that in G1, Kif7 and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that Kif7 may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein.

Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts

International Nuclear Information System (INIS)

Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Qin, Haiyan; Hu, Qingang; Zhang, Junfeng; Dong, Lei

2015-01-01

Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson’s staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson’s, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

Sphere-shaped nano-hydroxyapatite/chitosan/gelatin 3D porous scaffolds increase proliferation and osteogenic differentiation of human induced pluripotent stem cells from gingival fibroblasts.

Science.gov (United States)

Ji, Jun; Tong, Xin; Huang, Xiaofeng; Wang, Tiancong; Lin, Zitong; Cao, Yazhou; Zhang, Junfeng; Dong, Lei; Qin, Haiyan; Hu, Qingang

2015-07-08

Hydroxyapatite (HA) is an important component of human bone and bone tissue engineering scaffolds. A plethora of bone tissue engineering scaffolds have been synthesized so far, including nano-HA/chitosan/gelatin (nHA/CG) scaffolds; and for seeding cells, stem cells, especially induced pluripotent stem cells (iPSCs), have been a promising cell source for bone tissue engineering recently. However, the influence of different HA nano-particle morphologies on the osteogenic differentiation of human iPSCs (hiPSCs) from human gingival fibroblasts (hGFs) is unknown. The purpose of this study was to investigate the osteogenic differentiation of hiPSCs from hGFs seeded on nHA/CG scaffolds with 2 shapes (rod and sphere) of nHA particles. Firstly, hGFs isolated from discarded normal gingival tissues were reprogrammed into hiPSCs. Secondly, hiPSCs were seeded on rod-like nHA/CG (rod-nHA/CG) and sphere-shaped nHA/CG (sphere-nHA/CG) scaffolds respectively and then cell/scaffold complexes were cultured in vitro. Scanning electron microscope, hematoxyline and eosin (HE) staining, Masson's staining, and quantitative real-time polymerase chain reaction techniques were used to examine hiPSC morphology, proliferation, and differentiation on rod-nHA/CG and sphere-nHA/CG scaffolds. Finally, hiPSCs composited with 2 kinds of nHA/CG were transplanted in vivo in a subcutaneous implantation model for 12 weeks; pure scaffolds were also transplanted as a blank control. HE, Masson's, and immunohistochemistry staining were applied to detect new bone regeneration ability. The results showed that sphere-nHA/CG significantly increased hiPSCs from hGF proliferation and osteogenic differentiation in vitro. hiPSCs and sphere-nHA/CG composities generated large bone, whereas hiPSCs and rod-nHA/CG composities produced tiny bone in vivo. Moreover, pure scaffolds without cells almost produced no bone. In conclusion, our work provided a potential innovative bone tissue engineering approach using

Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

Science.gov (United States)

Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

2017-06-01

Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

Mesenchymal stem cell-conditioned medium accelerates skin wound healing: An in vitro study of fibroblast and keratinocyte scratch assays

International Nuclear Information System (INIS)

Walter, M.N.M.; Wright, K.T.; Fuller, H.R.; MacNeil, S.; Johnson, W.E.B.

2010-01-01

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-β1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

A radioligand immunoassay for 1,25-dihydroxyvitamin D3 receptors using monoclonal antibody: detection of a phenotypic receptor variant in vitamin D-dependency rickets (type II) which does not bind hormone

International Nuclear Information System (INIS)

Pike, J.W.; Dokoh, Shigeharu; Liberman, U.A.; Eil, C.; Haussler, M.R.; Marx, S.J.

1984-01-01

Vitamin D-dependency rickets, type II (VDDRII), is a well recognized heritable disorder characterized by peripheral target organ resistance to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the hormonally active form of the vitamin. Recently, cultured skin fibroblasts obtained from a number of patients with VDDRII have been utilized to characterize the underlying molecular defects associated with this malady. Recently monoclonal antibodies to the vitamin D receptor have been generated, and a radioligand immunoassay (RLIA) for the detection of this molecule has been developed which is independent of its hormone-binding capacity. This report describes the application of the immunoassay in the detection of receptor-like molecules in fibroblasts derived from patients with VDDRII. The results indicate that the molecule is generally present in all patients, and provides a mechanism for individual responsiveness to pharmacologic treatment with vitamin D 3 metabolites. 8 refs.; 3 figs.; 1 table

Tamarind Seed Xyloglucans Promote Proliferation and Migration of Human Skin Cells through Internalization via Stimulation of Proproliferative Signal Transduction Pathways

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W. Nie

2013-01-01

Full Text Available Xyloglucans (XGs of Tamarindus indica L. Fabaceae are used as drug vehicles or as ingredients of cosmetics. Two xyloglucans were extracted from T. indica seed with cold water (TSw and copper complex precipitation (TSc. Both were analyzed in regard to composition and influence on cell viability, proliferation, cell cycle progression, migration, MAPK phosphorylation, and gene expression of human skin keratinocytes (NHEK and HaCaT and fibroblasts (NHDF in vitro. TSw and TSc differed in molecular weight, rhamnose content, and ratios of xylose, arabinose, galactose, and glucose. Both XGs improved keratinocytes and fibroblast proliferation, promoted the cell cycle, and stimulated migration and intracellular enzyme activity of NHDF after endosomal uptake. Only TSw significantly enhanced HaCaT migration and extracellular enzyme activity of NHDF and HaCaT. TSw and TSc predominantly enhanced the phosphorylation of molecules that referred to Erk signaling in NHEK. In NHDF parts of the integrin signaling and SAPK/JNK pathway were affected. Independent of cell type TSw marginally regulated the expression of genes, which referred to membrane proteins, cytoskeleton, cytokine signaling, and ECM as well as to processes of metabolism and transcription. Results show that T. indica xyloglucans promote skin regeneration by a direct influence on cell proliferation and migration.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN-depleted head and neck cancer tumor cells.

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Liu, Zhiyong; Hartman, Yolanda E; Warram, Jason M; Knowles, Joseph A; Sweeny, Larissa; Zhou, Tong; Rosenthal, Eben L

2011-08-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma-mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer, there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here, we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were cocultured with fibroblasts or inoculated with fibroblasts into severe combined immunodeficient mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Coculture experiments showed fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN-silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN-silenced cells compared with control vector-transfected cells, whereas inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast coculture, suggesting the importance of FGFR2 signaling in fibroblast-mediated tumor growth. Analysis of xenografted tumors revealed that EMMPRIN-silenced tumors had a larger stromal compartment compared with control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast-independent tumor growth.

Fibroblast growth factor receptor mediates fibroblast-dependent growth in EMMPRIN depleted head and neck cancer tumor cells

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Liu, Zhiyong; Hartman, Yolanda E.; Warram, Jason M.; Knowles, Joseph A.; Sweeny, Larrisa; Zhou, Tong; Rosenthal, Eben L.

2011-01-01

Head and neck squamous cell carcinoma tumors (HNSCC) contain a dense fibrous stroma which is known to promote tumor growth, although the mechanism of stroma mediated growth remains unclear. As dysplastic mucosal epithelium progresses to cancer there is incremental overexpression of extracellular matrix metalloprotease inducer (EMMPRIN) which is associated with tumor growth and metastasis. Here we present evidence that gain of EMMPRIN expression allows tumor growth to be less dependent on fibroblasts by modulating fibroblast growth factor receptor-2 (FGFR2) signaling. We show that silencing EMMPRIN in FaDu and SCC-5 HNSCC cell lines inhibits cell growth, but when EMMPRIN-silenced tumor cells were co-cultured with fibroblasts or inoculated with fibroblasts into SCID mice, the growth inhibition by silencing EMMPRIN was blunted by the presence of fibroblasts. Co-culture experiments demonstrated fibroblast-dependent tumor cell growth occurred via a paracrine signaling. Analysis of tumor gene expression revealed expression of FGFR2 was inversely related to EMMPRIN expression. To determine the role of FGFR2 signaling in EMMPRIN silenced tumor cells, ligands and inhibitors of FGFR2 were assessed. Both FGF1 and FGF2 enhanced tumor growth in EMMPRIN silenced cells compared to control vector transfected cells, while inhibition of FGFR2 with blocking antibody or with a synthetic inhibitor (PD173074) inhibited tumor cell growth in fibroblast co-culture, suggesting the importance of FGFR2 signaling in fibroblast mediated tumor growth. Analysis of xenografted tumors revealed EMMPRIN silenced tumors had a larger stromal compartment compared to control. Taken together, these results suggest that EMMPRIN acquired during tumor progression promotes fibroblast independent tumor growth. PMID:21665938

Upregulation of NOXA by 10-Hydroxycamptothecin plays a key role in inducing fibroblasts apoptosis and reducing epidural fibrosis

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Jihang Dai

2017-01-01

Full Text Available The fibrosis that develops following laminectomy or discectomy often causes serious complications, and the proliferation of fibroblasts is thought to be the major cause of epidural fibrosis. 10-Hydroxycamptothecin (HCPT has been proven to be efficient in preventing epidural fibrosis, but the exact mechanism is still unclear. NOXA is a significant regulator of cell apoptosis, which has been reported to be beneficial in the treatment of fibrosis. We performed a series of experiments, both in vitro and in vivo, to explore the intrinsic mechanism of HCPT that underlies the induction of apoptosis in fibroblasts, and also to investigate whether HCPT has positive effects on epidural fibrosis following laminectomy in rats. Fibroblasts were cultured in vitro and stimulated by varying concentrations of HCPT (0, 1, 2, 4 µg/ml for various durations (0, 24, 48, 72 h; the effect of HCPT in inducing the apoptosis of fibroblasts was investigated via Western blots and TUNEL assay. Our results showed that HCPT could induce apoptosis in fibroblasts and up-regulate the expression of NOXA. Following the knockdown of NOXA in fibroblasts, the results of Western blot analysis showed that the level of apoptotic markers, such as cleaved-PARP and Bax, was decreased. The results from the TUNEL assay also showed a decreased rate of apoptosis in NOXA-knocked down fibroblasts. For the in vivo studies, we performed a laminectomy at the L1-L2 levels in rats and applied HCPT of different concentrations (0.2, 0.1, 0.05 mg/ml and saline locally; the macroscopic histological assessment, hydroxyproline content analysis and histological staining were performed to evaluate the effect of HCPT on reducing epidural fibrosis. The TUNEL assay in epidural tissues showed that HCPT could obviously induce apoptosis in fibroblasts in a dose-dependent manner. Also, immunohistochemical staining showed that the expression of NOXA increased as the concentrations of HCPT increased. Our findings are

Monoclonal antibody PAL-E specific for endothelium

NARCIS (Netherlands)

Schlingemann, R. O.; Dingjan, G. M.; Emeis, J. J.; Blok, J.; Warnaar, S. O.; Ruiter, D. J.

1985-01-01

A monoclonal antibody, PAL-E, is described that is specific for endothelial cells. The monoclonal antibody, an IgG2a, markedly stains endothelium of capillaries, medium-sized and small veins, and venules in frozen sections of human and some animal tissues tested. It reacts not at all or only weakly

Monoclonal for cancer detection and therapy

International Nuclear Information System (INIS)

Baldwin, R.W.; Byers, V.S.

1985-01-01

This book contains 18 chapters. Some of the chapter titles are: Monoclonal Antibodies to Breast Cancer and Their Application; Clinical Applications of Radioimmunolocalisation; Localisation of Cancer of the Ovary and Metastases Using 123 I-labelled Monoclonal Antibody HMFG-2 Compared to Surgical Findings; Interest of Globotriaosylceramide Membrane Antigen as Target for Immunotoxins; and Analysis, Results and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy

Long Noncoding RNA PANDA Positively Regulates Proliferation of Osteosarcoma Cells.

Science.gov (United States)

Kotake, Yojiro; Goto, Taiki; Naemura, Madoka; Inoue, Yasutoshi; Okamoto, Haruna; Tahara, Keiichiro

2017-01-01

A long noncoding RNA, p21-associated ncRNA DNA damage-activated (PANDA), associates with nuclear transcription factor Y subunit alpha (NF-YA) and inhibits its binding to promoters of apoptosis-related genes, thereby repressing apoptosis in normal human fibroblasts. Here, we show that PANDA is involved in regulating proliferation in the U2OS human osteosarcoma cell line. U2OS cells were transfected with siRNAs against PANDA 72 h later and they were subjected to reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR and cell-cycle analysis. PANDA was highly expressed in U2OS cells, and its expression was induced by DNA damage. Silencing PANDA caused arrest at the G 1 phase of the cell cycle, leading to inhibition of cell proliferation. Quantitative RT-PCR showed that silencing PANDA increased mRNA levels of the cyclin-dependent kinase inhibitor p18, which caused G 1 phase arrest. These results suggest that PANDA promotes G 1 -S transition by repressing p18 transcription, and thus promotes U2OS cell proliferation. Copyright© 2017 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Connective tissue growth factor/CCN2-null mouse embryonic fibroblasts retain intact transforming growth factor-β responsiveness

International Nuclear Information System (INIS)

Mori, Yasuji; Hinchcliff, Monique; Wu, Minghua; Warner-Blankenship, Matthew; Lyons, Karen M.; Varga, John

2008-01-01