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Inflammation plays an important role in the development of several cancers. Inflammatory cytokines, including tumor necrosis factor-α (TNF-α), are associated with the induction of inflammation. Chronic inflammation contributes to the progression of cancer through several mechanisms, including increased cytokine production and activation of transcription factors, such as nuclear factor-κB (NF-κB). Zerumbone (ZER), a component of subtropical ginger (Zingiber zerumbet Smith), seems to have anti-inflammatory, anti-cancer, and antioxidant activities. In this study, we aimed to explore the protective function and mechanisms of ZER against TNF-α-induced cancer-promoting cytokines. We found that the viability of stimulated human fibroblast cell lines was reduced after treatment with ZER (IC 50 =18 µmol/L), compared to un-stimulated fibroblasts (IC 50 =40 µmol/L). Besides, ZER inhibited mRNA expression and protein secretion of transforming growth factor-β (TGF-β), interleukin-33 (IL-33), monocyte chemoattractant protein-1 (MCP-1), and stromal cell-derived factor 1 (SDF-1), which were produced by TNF-α-induced fibroblasts, as measured by quantitative real time-PCR (qRT-PCR) and ELISA assays. The mRNA expression levels of TGF-β, IL-33, SDF-1, and MCP-1 showed 8, 5, 2.5, and 4-fold reductions, respectively. Moreover, secretion of TGF-β, IL-33, SDF-1, and MCP-1 was reduced to 3.65±0.34 ng/mL, 6.3±0.26, 1703.6±295.2, and 5.02±0.18 pg/mL, respectively, compared to the untreated group. In addition, the conditioned media (CM) of TNF-α-stimulated fibroblasts increased the NF-κB expression in colorectal cancer cell lines (HCT-116 and Sw48), while in the vicinity of ZER, the expression of NF-κB was reversed. Considering the significant effects of ZER, this component can be used as an appropriate alternative herbal treatment for cancer-related chronic inflammation.

We studied the effect of platelet lysate and platelet-poor plasma from patients with trophic ulcers with and without type 2 diabetes mellitus on proliferation, migration, and apoptosis of human dermal fibroblast, mesenchymal stem cells, and endothelial cells. It is shown that plasma obtained from patients with type 2 diabetes mellitus produced inhibitory effects.

Chemically induced cell fate conversion, including reprogramming to pluripotent stem cells and direct reprogramming to somatic cells, has been proved to be an alternative strategy with many advantages, in comparison with conventional transcription factors- or microRNAs-enabled cell reprogramming. Many functional and desirable cells have been generated via the chemically induced reprogramming. Neural stem cells (NSCs) hold great potential in basic research and clinical application. Here, we describe a detailed protocol for converting mouse fibroblasts into NSCs by a cocktail of chemical compounds.

Adipose-derived stem cells (ADSCs) and their derivatives have aroused intense interest in fields of dermatological and aesthetic medicine. As a major component detected in ADSCs secretome, platelet-derived growth factor AA (PDGF-AA) has been reported mediating extracellular matrix deposition and remodeling, thus might contribute to its anti-aging effect. On the basis of establishing an experimental model that simulate actual skin aging by exposing HDFs to both intrinsic and extrinsic aging factors, we pretreated human dermal fibroblasts (HDFs) with ADSC-conditioned medium (ADSC-CM) before being irradiated, aiming at exploring preventive effects of ADSCs secretome against aging damages. 48 h after irradiation, we detected cellular proliferation; β-galactosidase stain; mRNA expressions of MMP-1, MMP-9, and TIMP-1; and protein expressions of collagen I, collagen III, and elastin. Moreover, we detected related protein expression of PI3K/Akt signal pathway, which can be activated by PDGF-AA and was newly found to promote extracellular matrix protein synthesis. Concentration of PDGF-AA in the prepared ADSC-CM decreased over time and maintained excellent bioactivity at low temperature until the 11th week. ADSC-CM pretreatment can slightly or significantly improve cellular proliferative activity and reduce cellular senescence in irradiated HDFs. Besides, ADSC-CM pretreatment increased collagen I, collagen III, elastin, and TIMP-1 expressions but decreased MMP-1 and MMP-9 expressions both in irradiated and nonirradiated HDFs. ADSC-CM pretreatment significantly increased pAkt protein expression, and ECM protein expression greatly decreased in case of LY294002 application. The results were similar in three generations of HDFs, yet varied with different degrees. Generally, ADSC-CM we prepared demonstrates a certain degree of positive role in preventing HDFs from intrinsic and extrinsic aging damages and that PDGF-AA may contribute to making it become effective with some other components in ADSC-CM.

Aim: We investigated the healing effects of conditioned medium (CM) derived from a physiological 3D culture system engineered to use an extracellular matrix/stromal vascular fraction (SVF) gel enriched for adipose on diabetic wounds in rats. This CM (Gel-CM) was compared with that from a 2D culture system that used SVF cells (SVF-CM). Materials & methods: Keratinocytes, fibroblasts and wounds were treated with Gel-CM and SVF-CM, and cytokine levels in the CM types were quantified. Results: Proliferation and migration of keratinocytes and fibroblasts were significantly higher after treatment with Gel-CM than with SVF-CM. Collagen secretion by fibroblasts and wound closure were highly stimulated by Gel-CM. Proteomic analyses revealed a higher concentration of growth factors in Gel-CM than in SVF-CM. Conclusion: Gel-CM is a promising therapeutic option for treating diabetic wounds.

Skin substitutes are one of the main treatments for skin loss, and a skin substitute that is readily available would be the best treatment option. However, most cell-based skin substitutes require long production times, and therefore, patients endure long waiting times. The proteins secreted from the cells and tissues play vital roles in promoting wound healing. Thus, we aimed to develop an acellular three-dimensional (3D) skin patch with dermal fibroblast conditioned medium (DFCM) and collagen hydrogel for immediate treatment of skin loss. Fibroblasts from human skin samples were cultured using serum-free keratinocyte-specific media (KM1 or KM2) and serum-free fibroblast-specific medium (FM) to obtain DFCM-KM1, DFCM-KM2, and DFCM-FM, respectively. The acellular 3D skin patch was soft, semi-solid, and translucent. Collagen mixed with DFCM-KM1 and DFCM-KM2 showed higher protein release compared to collagen plus DFCM-FM. In vitro and in vivo testing revealed that DFCM and collagen hydrogel did not induce an immune response. The implantation of the 3D skin patch with or without DFCM on the dorsum of BALB/c mice demonstrated a significantly faster healing rate compared to the no-treatment group 7 days after implantation, and all groups had complete re-epithelialization at day 17. Histological analysis confirmed the structure and integrity of the regenerated skin, with positive expression of cytokeratin 14 and type I collagen in the epidermal and dermal layer, respectively. These findings highlight the possibility of using fibroblast secretory factors together with collagen hydrogel in an acellular 3D skin patch that can be used allogeneically for immediate treatment of full-thickness skin loss.

Recently, multipotent mesenchymal stromal cell (MSC) treatment has attracted special attention as a new alternative strategy for stimulating regeneration. Irradiation myocardial fibrosis (IMF) is a major complication associated with total body irradiation for hematopoietic stem cell transplantation, nuclear accidents, and thoracic radiotherapy for lung cancer, esophageal cancer, proximal gastric cancer, breast cancer, thymoma, and lymphoma. The aim of the present study was to assess the therapeutic paracrine effects of human umbilical cord-derived mesenchymal stromal cells (UC-MSCs) in the cell model of IMF. For this purpose, primary human cardiac fibroblasts (HCF) cells were irradiated and cultured with the conditioned medium of UC-MSCs (MSCCM). MSCCM promoted cell viability, reduced collagen deposition as measured by Sircol assay and qPCR (Col1A1 and Col1A2), prevented oxidative stress and increased antioxidant status (as measured by malondialdehyde content and the activities and mRNA levels of antioxidant enzymes), and reduced pro-fibrotic TGF-β1, IL-6 and IL-8 levels (as examined by ELISA kit and qPCR). Pretreatment with inhibitor of NF-κB led to a decrease in the levels of TGF-β1 in cell lysate of HCF cells by ELISA kit. Furthermore, we also found that MSCCM prevented NF-κB signaling pathway activation for its proinflammatory actions induced by irradiation. Taken together, our data suggest that MSCCM could reduce irradiation-induced TGF-β1 production through inhibition of the NF-κB signaling pathway. These data provide new insights into the functional actions of MSCCM on irradiation myocardial fibrosis.

Background: Human adipose tissue-derived mesenchymal stem cells (ASCs) have potential utility as modulators of the regeneration of tissue that is inflamed or scarred secondary to injuries such as burns or trauma. However, the effect of ASCs on one particular type of scarring, keloidal disease, remains unknown. The absence of an optimal model for investigation has hindered the development of an effective therapy using ASCs for keloids., Objective: To investigate the influence of ASCs on angiogenesis, extracellular matrix deposition, and inflammatory cell influx in keloids., Methods: We analyzed the proliferation, migration, and apoptosis of human keloid-derived fibroblasts treated with a starvation-induced, conditioned medium from ASCs (ASCs-CM). This was achieved by Brdu proliferation assay, a validated co-culture migration assay, and flow cytometry, respectively. To assess the change in phenotype to a pro-fibrotic state, fibroblasts were analyzed by real-time PCR and contraction assay. A keloid implantation animal model was used to assess the paracrine effect of ASCs histochemically and immunohistochemically on scar morphology, collagen deposition, inflammatory cell composition, and blood vessel density. In tandem, an antibody-based array was used to identify protein concentration in the presence of ASCs-CM at time point 0, 24, and 48h., Results: ASCs-CM inhibited the proliferation and collagen synthesis of human keloid-derived fibroblasts. ASCs-CM was associated with reduced inflammation and fibrosis in the keloid implantation model. Thirty-four cytokines were differentially regulated by ASCs-CM at 24h. These included molecules associated with apoptosis, matrix metalloproteases, and their inhibitors. The same molecules were present at relatively higher concentrations at the 48h timepoint., Conclusion: These results suggest that ASCs are associated with the inhibition of fibrosis in keloids by a paracrine effect. This phenomenon may have utility as a therapeutic approach in the clinical environment., (Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.)

Background: A keloid is a fibroproliferative disorder occurring in wounds characterized by an exaggerated response to injury. To date, no effective cure has been identified. As multipotent stem cells, human adipose-derived stem cells (ADSCs) may show the possibility for curing diseases such as fibrosis. This study sought to explore the potential role of human ADSCs in curing keloids., Methods: After culture in conditioned medium, gene and protein expression of keloid fibroblasts was examined using real-time polymerase chain reaction (RT-PCR) and Western blotting, while analysis of the cell cycle was used to measure the proliferative properties of the cells. Furthermore, ex vivo explant cultures were used to test the effects of ADSC-conditioned medium (ADSC-CM) on CD31 + and CD34 + expression in keloid tissue., Results: Our experimental results show that ADSC-CM was able to attenuate extracellular matrix-related gene expression as well as decrease protein expression. Cell proliferation was significantly suppressed in our study. CD31 + and CD34 + vessels in ex vivo explants were reduced by 55% and 57% in treatment groups compared with control groups., Conclusions: Human ADSC-CM significantly inhibited keloid fibroblast-related bioactivities.

Background: Mesenchymal stem cells are a valuable cell source in regenerative medicine, and conditioned medium obtained from mesenchymal stem cells reportedly inhibits inflammation. Keloids are characterized by abnormal fibrosis, caused by fibroblasts in response to inflammation. In this study, the authors evaluated whether conditioned medium obtained from amnion-derived mesenchymal stem cells suppressed activation of keloid fibroblasts., Methods: Keloid (n = 7), mature (n = 5), and normal (n = 5) fibroblasts were harvested from patients. Fibroblasts were stimulated with transforming growth factor (TGF)-β, and the effects of conditioned medium obtained from amnion-derived mesenchymal stem cells on cell proliferation, activation, and expression of extracellular matrix-related genes were analyzed. The effect of concentrating the conditioned medium by ultrafiltration on fibroblast activation was also analyzed., Results: Conditioned medium obtained from amnion-derived mesenchymal stem cells significantly up-regulated proliferation of mature fibroblasts but tended to suppress that of keloid fibroblasts. Conditioned medium obtained from amnion-derived mesenchymal stem cells significantly suppressed the TGF-β-induced up-regulation of α-smooth muscle actin in keloid and normal fibroblasts and collagen I in keloid fibroblasts, but not in mature fibroblasts. The conditioned medium obtained from amnion-derived mesenchymal stem cells concentrated by ultrafiltration and the filtrate significantly suppressed TGF-β-induced α-smooth muscle actin expression., Conclusion: Conditioned medium obtained from amnion-derived mesenchymal stem cells prevents proliferation and activation of keloid fibroblasts and is a promising keloid treatment for administration as a topical agent., Clinical Question/level of Evidence: Therapeutic, V.

Background: Carcinoma associated fibroblasts (CAFs or myofibroblasts) are activated fibroblasts which participate in breast tumor growth, angiogenesis, invasion, metastasis and therapy resistance. As such, recent efforts have been directed toward understanding the factors responsible for activation of the phenotype. In this study, we have investigated how changes in the mechanical stiffness of a 3D hydrogel alter the behavior and myofibroblast-like properties of human mammary fibroblasts (HMFs)., Results: Here, we utilized microbial transglutaminase (mTG) to mechanically tune the stiffness of gelatin hydrogels and used rheology to show that increasing concentrations mTG resulted in hydrogels with greater elastic moduli (G'). Upon encapsulation of HMFs in 200 (compliant), 300 (moderate) and 1100 Pa (stiff) mTG hydrogels, it was found that the HMFs remained viable and proliferated over the 7 day culture period. Specifically, rates of proliferation were greatest for HMFs in moderate hydrogels. Regarding morphology, HMFs in compliant and moderate hydrogels exhibited a spindle-like morphology while HMFs in stiff hydrogels exhibited a rounded morphology with several large cellular protrusions. Quantification of cell morphology revealed that HMFs cultured in all mTG hydrogels overall assumed a more elongated phenotype over time in culture; however, few significant differences in morphology were observed between HMFs in each of the hydrogel conditions. To determine whether matrix stiffness upregulated expression of ECM and myofibroblast markers, western blot was performed on HMFs in compliant, moderate and stiff hydrogels. It was found that ECM and myofibroblast proteins varied in expression during both the culture period and according to matrix stiffness with no clear correlation between matrix stiffness and a myofibroblast phenotype. Finally, TGF-β levels were quantified in the conditioned media from HMFs in compliant, moderate and stiff hydrogels. TGF-β was significantly greater for HMFs encapsulated in stiff hydrogels., Conclusions: Overall, these results show that while HMFs are viable and proliferate in mTG hydrogels, increasing matrix stiffness of mTG gelatin hydrogels doesn't support a robust myofibroblast phenotype from HMFs. These results have important implications for further understanding how modulating 3D matrix stiffness affects fibroblast morphology and activation into a myofibroblast phenotype.

Background: The main goal of bone tissue engineering has been the generation of healthy bone in order to replace affected tissue. Therefore, optimized biomaterials are needed which allow the survival and growth of mesenchymal stem cells. Until now the key challenge in the clinical application of cell-based tissue engineering bone implants was poor diffusion of oxygen into the tissue, making functional blood vessel networks a necessity. With their ability to evolve into different cell types, to expand extensively in vitro, and to release paracrine soluble factors, bone marrow stromal cells (BMSC) are highly attractive for tissue engineering. During the last years hypoxia became a proven method to control proliferation, differentiation, and pluripotency of BMSC. Here we applied different methods to characterize metabolically conditioned media (MCM) in comparison to hypoxia conditioned media (HCM) and evaluated their ability to attract BMSC in 2-D migration assays., Methods: BMSC and fibroblasts of human origin were isolated and cultivated to obtain HCM and MCM. Both media were characterized by angiogenesis arrays, cytokine arrays, and ELISA for selected factors. 2-D migration tests were performed with Corning Transwell®-96 permeable support chambers with porous polyester membranes with a pore size of 8.0 μm., Results: Characterization of HCM and MCM revealed that the concentration of angiogenic factors was higher in MCM than in HCM. However, the chemoattractive capacity of MCM for BMSC was equivalent to that of HCM. HCM and MCM produced by human skin fibroblasts attracted human BMSC as efficiently as HCM and MCM produced by human BMSC., Conclusions: HCM and MCM have a high chemoattractive capacity for BMSC. Both conditioned media harbor high concentrations of angiogenic factors which are important for angiogenesis and cell migration. Both chemoattracting conditioned media can also be derived from skin fibroblasts which can easily be obtained from patients in individualized therapy approaches.

Spectroscopic markers characteristic of reference glycosaminoglycan molecules were identified previously based on their vibrational signatures. Infrared spectral signatures of glycosaminoglycans in fixed cells were also recently demonstrated but probing live cells still remains challenging. Raman microspectroscopy is potentially interesting to perform studies under physiological conditions. The aim of the present work was to identify the Raman spectral signatures of GAGs in fixed and live cells and in their conditioned media. Biochemical and Raman analyses were performed on five cell types: chondrocytes, dermal fibroblasts, melanoma (SK-MEL-28), wild type CHO, and glycosaminoglycan-defective mutant CHO-745 cells. The biochemical assay of sulfated GAGs in conditioned media was only possible for chondrocytes, dermal fibroblasts, and wild type CHO due to the detection limit of the test. In contrast, Raman microspectroscopy allowed probing total glycosaminoglycan content in conditioned media, fixed and live cells and the data were analysed by principal component analysis. Our results showed that the Raman technique is sensitive enough to identify spectral markers of glycosaminoglycans that were useful to characterise the conditioned media of the five cell types. The results were confirmed at the single cell level on both live and fixed cells with a good differentiation between the cell types. Furthermore, the principal component loadings revealed prominent glycosaminoglycan-related spectral information. Raman microspectroscopy allows monitoring of the glycosaminoglycan profiles of single live cells and could therefore be developed for cell screening purposes and holds promise for identifying glycosaminoglycan signatures as a marker of cancer progression in tissues.

Interaction between keratinocytes and fibroblasts plays a critical role in maintaining skin integrity under both normal and pathological conditions. We have previously demonstrated that keratinocyte-releasable factors influence the expression of key extracellular matrix components, such as collagen and matrix metalloproteinases in dermal fibroblasts. In this study, we utilized DNA microarray analysis to examine the effects of keratinocyte-releasable factors on the expression of several cytokines in human dermal fibroblasts. The results revealed significantly higher granulocyte colony-stimulating factor (G-CSF) expression in fibroblasts co-cultured with keratinocytes relative to mono-cultured cells, which was verified by RT-PCR and western blot. G-CSF is an important hematopoietic factor also thought to play a beneficial role in wound healing through stimulating keratinocyte proliferation. To partially characterize the keratinocyte-releasable factors responsible for stimulating G-CSF production, keratinocyte-conditioned medium (KCM) was subjected to thermal treatment and ammonium sulfate precipitation before treating fibroblasts. The results showed that keratinocyte-releasable G-CSF-stimulating factors remain stable at 56°C and upon 50% ammonium sulfate precipitation. Knowing that keratinocytes release IL-1, which stimulates G-CSF expression in various immune cells, several experiments were conducted to ask whether this might also be the case for fibroblasts. The results showed that the addition of recombinant IL-1 markedly increased G-CSF expression in fibroblasts; however, IL-1 receptor antagonist only partially abrogated KCM-stimulated G-CSF expression, indicating the role of additional keratinocyte-releasable factors. These findings underline the importance of cross-talk between keratinocytes and fibroblasts, suggesting that communication between these cells in vivo modulates the production of cytokines required for cutaneous wound healing and maintenance. J. Cell. Biochem. 118: 308-317, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

There is increasing evidence that cancers are heterogeneous and contain a hierarchical organization consisting of cancer stem cells and their differentiated cell progeny. These cancer stem cells are at the core of the tumor as they represent the clonogenic cells within a tumor. Moreover, these cells are considered to contain selective therapy resistance, which suggests a pivotal role in therapy resistance and tumor relapse. Here we show that differentiated cells can re-acquire stemness through factors secreted from fibroblasts. This induced CSC state also coincides with re-acquisition of resistance to chemotherapy. Resistance induced in newly formed CSCs is mediated by the anti-apoptotic molecule BCLXL and inhibition of BCLXL with the BH3 mimetic ABT-737 sensitizes these cancer cells toward chemotherapy. These data point to an important interplay between tumor cells and their microenvironment in the regulation of stemness and therapy resistance.

Objective: To investigate the effects of culture supernatant of human amnion mesenchymal stem cells (hAMSCs-CS) on biological characteristics of human fibroblasts., Methods: (1) hAMSCs were isolated from deprecated human fresh amnion tissue of placenta and then sub-cultured. The morphology of hAMSCs on culture day 3 and hAMSCs of the third passage were observed with inverted phase contrast microscope. (2) Two batches of hAMSCs of the third passage were obtained, then the expression of vimentin of cells was observed with immunofluorescence method, and the expression of cell surface marker CD90, CD73, CD105, and CD45 was detected by flow cytometer. (3) hAMSCs-CS of the third passage at culture hour 72 were collected, and the content of insulin-like growth factor Ⅰ (IGF-Ⅰ), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were detected by enzyme-linked immunosorbent assay. (4) Human fibroblasts were isolated from deprecated human fresh prepuce tissue of circumcision and then sub-cultured. Human fibroblasts of the third passage were used in the following experiments. Cells were divided into blank control group and 10%, 30%, 50%, and 70% hAMSCs-CS groups according to the random number table (the same grouping method below), with 48 wells in each group. Cells in blank control group were cultured with DMEM/F12 medium containing 2% fetal bovine serum (FBS), while cells in the latter 4 groups were cultured with DMEM/F12 medium containing corresponding volume fraction of hAMSCs-CS and 2% FBS. The proliferation activity of cells was detected by cell counting kit 8 and microplate reader at culture hour 12, 24, 48, and 72, respectively, and corresponding volume fraction of hAMSCs-CS which causing the best proliferation activity of human fibroblasts was used in the following experiments. (5) Human fibroblasts were divided into blank control group and 50% hAMSCs-CS group and treated as in (4), with 4 wells in each group, at post scratch hour (PSH) 0 (immediately after scratch), 12, 24, 48, and 72, the migration distance of cells was observed and measured with inverted phase contrast microscope. (6) Human fibroblasts were grouped and treated as in (5), with 3 battles in each group, and apoptosis rate of cells was detected by flow cytometer. Data were processed with analysis of variance of factorial design, analysis of variance for repeated measurement, one-way analysis of variance, LSD test, and t test., Results: (1) On culture day 3, most hAMSCs were in large form, and spindle-shaped with much prominences like fibroblasts or in flat polygonal shape. hAMSCs of the third passage were spindle-shaped. The expression of vimentin of hAMSCs of the third passage was strongly positive, and the expressions of surface markers CD90, CD73, and CD105 of the cells were positive, while the expression of CD45 of the cells was negative. (2) The content of IGF-Ⅰ, VEGF, EGF, and bFGF in hAMSCs-CS were respectively (11.7±1.0), (316±68), (6.1±0.4), and (1.49±0.05) pg/mL. (3) At culture hour 12-72, the proliferation activity of human fibroblasts in each hAMSCs-CS group was significantly higher than that in blank control group (with P values below 0.01), and the proliferation activity of human fibroblasts in 50% hAMSCs-CS group was the highest. (4) The width of scratch in two groups was nearly the same at PSH 0. The migration distance of cells in 50% hAMSCs-CS group was significantly longer than that in blank control group at PSH 12-72 (with P values below 0.01). (5) The apoptosis rate of human fibroblasts in blank control group was (16.2±2.4)%, which was significantly higher than that in 50% hAMSCs-CS group [(7.4±3.6)%, t=6.710, P<0.01]., Conclusions: hAMSCs-CS can promote proliferation and migration of human fibroblasts and inhibit the apoptosis of human fibroblasts.

AGEs accumulation in the skin affects extracellular matrix (ECM) turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE) has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE's proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.

Silk protein sericins, in the recent years, find application in cosmetics and pharmaceuticals and as biomaterials. We investigate the potential of sericin, extracted from both mulberry Bombyx mori and different non-mulberry sources, namely, tropical tasar, Antheraea mylitta; muga, Antheraea assama; and eri, Samia ricini, as growth supplement in serum-free culture medium. Sericin supplemented media containing different concentrations of sericins from the different species are examined for attachment, growth, proliferation, and morphology of fibrosarcoma cells. The optimum sericin supplementation seems to vary with the source of sericins. The results indicate that all the sericins promote the growth of L929 cells in serum-free culture media; however, S. ricini sericin seems to promote better growth of cells amongst other non-mulberry sericins.

Background: Carcinoma associated fibroblasts (CAFs), an important component of tumor microenvironment, are capable of enhancing tumor cells invasion and migration through initiation of epithelial-mesenchymal transition (EMT). MRC-5 fibroblasts are one of the CAFs expressing alpha-smooth muscle actin. It is ascertained that medium conditioned by MRC-5 fibroblasts stimulate motility and invasion of breast cancer cells. However, its role in hepatocellular carcinoma (HCC) is less clear. The aim of our study was to investigate the effect of MRC-5-CM on HCC and explore the underlying mechanisms., Methods and Results: Using a combination of techniques, the role of MRC-5-CM in HCC was evaluated. We determined that MRC-5-CM induced the non-classical EMT in Bel-7402 and MHCC-LM3 cell lines. Initiation of the non-classical EMT was mainly via quintessential redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, rather than up-regulation of typical EMT-related transcription factors (i.e., Snail, Twist1, ZEB-1 and ZEB2). We also found that MRC-5-CM potentiated both the migration and invasion of Bel-7402 and MHCC-LM3 cells in mesenchymal movement mode through activation of the α6, β3, β4, β7 integrin/FAK pathway and upregulation of MMP2. The flow cytometric analysis showed that MRC-5-CM induced G1 phase arrest in Bel-7402 cells with a concomitant reduction of S phase cells. In contrast, MRC-5-CM induced S phase arrest in MHCC-LM3 cells with a concomitant reduction of cells in the G2/M phase. MRC-5-CM also inhibited apoptosis in Bel-7402 cells while inducing apoptosis in MHCC-LM3 cells., Conclusion: Collectively, MRC-5-CM promoted HCC cell motility and invasiveness through initiation of the non-classical EMT, including redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, activation of the integrin/FAK pathway, and upregulation of MMP2. Hence, MRC-5-CM exerted distinct roles in Bel-7402 and MHCC-LM3 cell viability by regulating cyclins, cyclin dependent kinases (CDKs), CDK inhibitors (CKIs), Bcl-2 family proteins and other unknown mechanosensors.

Intervertebral disc degeneration (IVD) is one of the important causes of low back pain and is associated with inflammation induced by interaction between macrophages and the human annulus fibrosus (AF) cells. Low-level light therapy (LLLT) has been widely known to regulate inflammatory reaction. However, the effect of LLLT on macrophage-mediated inflammation in the AF cells has not been studied till date. The aim of this study is to mimic the inflammatory microenvironment and to investigate the anti-inflammatory effect of LLLT at a range of wavelengths (405, 532 and 650 nm) on the AF treated with macrophage-like THP-1 cells conditioned medium (MCM) containing proinflammatory cytokines and chemokines (interleukin-1beta, tumor necrosis factor-alpha, interleukin-6 and 8). We observed that AF cells exposed to MCM secrete significantly higher concentrations of IL-6, IL-8, IL-1β and TNF-α. LLLT markedly inhibited secretion of IL-6 at 405 nm in a time-dependent manner. Level of IL-8 was significantly decreased at all wavelengths in a time-dependent manner. We showed that MCM can induce the inflammatory microenvironment in AF cells and LLLT selectively suppressed IL-6 and 8 levels. The results indicate that LLLT is a potential method of IVD treatment and provide insights into further investigation of its anti-inflammation effect on IVD., (© 2015 The American Society of Photobiology.)

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

The development and spread of mammary carcinomas require synergetic interplay between tumor cells and their microenvironment through paracrine secretions, which are still not well defined. We have shown here that interleukin-6 (IL-6), either recombinant or secreted from highly invasive breast cancer cells, down-regulates the tumor suppressor proteins p16(INK4A), p21(WAF1), and p53 and activates breast stromal fibroblasts in a paracrine manner. The formation of myofibroblasts requires p16(INK4A) down-regulation and the activation of the JAK2/STAT3 pathway. Indeed, the transcription factor STAT3 positively controls the expression of the three major myofibroblast markers, SDF-1, α-smooth muscle actin (α-SMA), and TGF-β1, and mediates IL-6-related down-regulation of p16(INK4A), p21(WAF1), and p53 as well as the activation of stromal fibroblasts. Importantly, these effects were mediated through STAT3-dependent up-regulation of the mRNA-binding protein AUF1, whose promoter contains three canonical STAT3 binding sites. AUF1 binds the SDF-1, α-SMA, TGF-β1, and IL-6 mRNAs and reduces their turnover. Consequently, specific AUF1 down-regulation inhibits IL-6-dependent activation of breast stromal fibroblasts, whereas AUF1 ectopic expression of p37(AUF1) activated these cells and enhanced their paracrine induction of epithelial-to-mesenchymal transition in breast cancer cells, which shows a non-cell-autonomous oncogenic function of AUF1. Together, these results demonstrate a major role of IL-6 in activating breast stromal fibroblasts through STAT3-dependent AUF1 induction., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Previous studies have demonstrated that senescent cancer-associated fibroblasts (CAFs) derived from genetically unstable oral squamous cell carcinomas (GU-OSCC), unlike non-senescent CAFs from genetically stable carcinomas (GS-OSCC), promoted keratinocyte invasion in vitro in a paracrine manner. The mechanism by which this occurs is unclear., Methods: Previous work to characterise the senescent-associated secretory phenotype (SASP) has used antibody arrays, technology that is limited by the availability of suitable antibodies. To extend this work in an unbiased manner, we used 2D gel electrophoresis and mass spectroscopy for protein identification. Matrix metalloproteinases (MMPs) were investigated by gelatin zymography and western blotting. Neutralising antibodies were used to block key molecules in the functional assays of keratinocyte adhesion and invasion., Results: Among a variety of proteins that were differentially expressed between CAFs from GU-OSCC and GS-OSCC, MMP-2 was a major constituent of senescent CAF-CM derived from GU-OSCC. The presence of active MMP-2 was confirmed by gelatine zymography. MMP-2 derived from senescent CAF-CM induced keratinocyte dis-cohesion and epithelial invasion into collagen gels in a TGF-β-dependent manner., Conclusions: Senescent CAFs from GU-OSCC promote a more aggressive oral cancer phenotype by production of active MMP-2, disruption of epithelial adhesion and induction of keratinocyte invasion.

Chronic exposure to solar radiation is the primary cause of photoaging and benign and malignant skin tumors. A conditioned serum-free medium (SFM) was prepared from umbilical cord mesenchymal stem cells (UC-MSCs) and its anti-photoaging effect, following chronic UV irradiation in vitro and in vivo, was evaluated. UC-MSC SFM had a stimulatory effect on human dermal fibroblast proliferation and reduced UVA-induced cell death. In addition, UC-MSC SFM blocked UVA inhibition of superoxide dismutase activity. Topical application of UC-MSC SFM to mouse skin prior to UV irradiation blocked the inhibition of superoxide dismutase and glutathione peroxidase activities, and prevented the upregulation of malonaldehyde. UC-MSC SFM thus protects against photoaging induced by UVA and UVB radiation and is a promising candidate for skin anti-photoaging treatments.

Dermal wounds can heal detrimentally by formation of excess fibrosis or hypertrophic scarring. These phenomena are normally absent in the oral mucosa. Macrophages play an important role in wound repair, have a marked heterogeneity and are thought to contribute to fibrosis. To investigate to what extend macrophages are involved in the occurrence of fibrosis, the effect of differently activated macrophages on dermal and gingival fibroblasts was studied in vitro. Macrophages were differentiated into a classical (M1) or alternative (M2) phenotype, which was assessed by receptor expression (CD40/mannose receptor) and cytokine secretion (interleukin-4 and -12). Fibroblasts were exposed to these macrophages and/or conditioned medium (cm), and differentiation into α-SMA-expressing myofibroblasts was quantified. M2, but not M1 macrophages induced α-SMA expression in both dermal and gingival fibroblasts. Blocking of transforming growth factor-β1 did not decrease the α-SMA expression mediated by M2 macrophages. It appeared that this induction was mediated by platelet derived growth factor-CC (PDGF-CC), produced by M2 macrophages. The expression and role of this growth factor was confirmed by ELISA, RT-PCR, and blocking experiments. Our results indicate that M2 macrophages are able to induce myofibroblast differentiation via production of PDGF-CC. Based on our findings we conclude that PDGF-CC may play a hitherto unknown role in the differentiation of myofibroblasts., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Cellular senescence suppresses cancer by eliminating potentially oncogenic cells, participates in tissue repair, contributes to cancer therapy, and promotes organismal aging. Numerous activities of senescent cells depend on the aptitude of these cells to secrete myriads of bioactive molecules, a behavior termed the senescence-associated secretory phenotype (SASP). The SASP supports cell-autonomous functions like the senescence-associated growth arrest, and mediates paracrine interactions between senescent cells and their surrounding microenvironment. The biological functions and the regulation of the SASP are beginning to emerge, and current SASP assessment techniques include the analysis of SASP factors at the mRNA level, the direct measurement of factors inside or outside the cell (i.e., secreted), and the detection of SASP-provoked cellular responses. Here, we focus on a simple approach to collect SASP-conditioned media in order to directly measure secreted SASP factors using sandwich enzyme-linked immunosorbent assay. As an example, we discuss the assessment of the major SASP factor interleukin-6 in senescent human fibroblasts. Supplemental notes are provided to easily adapt this procedure to other SASP factors, change cell types, or scale the techniques for different volumes or high-throughput measurements. These techniques should facilitate the discovery of novel functions and regulators of the SASP.

Neurogenesis via the activation of endogenous neural progenitor cells is a potential treatment strategy for brain injury, including intracerebral hemorrhage (ICH). We assessed the efficacy of combined cell and brain-derived neurotrophic factor (BDNF) treatment in a mouse model of ICH induced by intracerebral collagenase injection. Complementary DNAs of mouse BDNF were transfected into cell lines of 3T3 fibroblasts. The expression and bioactivity of BDNF were analyzed by immunocytochemistry, Western blot, ELISA, and functional assays. Hematoma area and brain tissue loss were assessed by magnetic resonance imaging. The BDNF-transfected or nontransfected 3T3 fibroblasts were implanted as a growth factor source in mice with ICH. Neurogenesis and functional recovery were evaluated 15 days after ICH. The BDNF-treated mice had the most doublecortin-positive cells near lesions and the least brain tissue loss in all groups. Both cell treatment groups had abundant newly proliferative glial fibrillary acidic protein-positive cells and better functional improvement than controls. These results indicate that fibroblast transplantation, together with recombinant BDNF treatment, after ICH is beneficial in mice. The early functional recovery may result from the growth factors that are provided or evoked by the implanted grafts. These results suggest a potential approach for combining gene and cell therapy for ICH treatment.

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)(1) can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial. The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium(2). Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs(3). Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)(4). Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions(5-7). Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established(8). To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells(9), adult skin cells(10), foreskin fibroblasts(11-12), amniotic mesenchymal cells(13)). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A(14). Obviously, there is an evident difference in growth factor production by mouse and human feeder cells. Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgfβ1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs(15-16). For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs. Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

We have investigated the use of BMSC (bone marrow stromal cell) as a feeder cell for improving culture efficiency of ESC (embryonic stem cell). B6CBAF1 blastocysts or ESC stored after their establishment were seeded on to a feeder layer of either SCA-1+/CD45-/CD11b- BMSC or MEF (mouse embryonic fibroblast). Feeder cell activity in promoting ESC establishment from the blastocysts and in supporting ESC maintenance did not differ significantly between BMSC and MEF feeders. However, the highest efficiency of colony formation after culturing of inner cell mass cells of blastocysts was observed with the BMSC line that secreted the largest amount of LIF (leukaemia inhibitory factor). Exogenous LIF was essential for the ESC establishment on BMSC feeder, but not for ESC maintenance. Neither change in stem cell-specific gene expression nor increase in stem cell aneuploidy was detected after the use of BMSC feeder. We conclude that BMSC can be utilized as the feeder of ESC, which improves culture efficiency.

Cigarette smoking causes apoptotic death, senescence, and impairment of repair functions in lung fibroblasts, which maintain the integrity of alveolar structure by producing extracellular matrix (ECM) proteins. Therefore, recovery of lung fibroblasts from cigarette smoke-induced damage may be crucial in regeneration of emphysematous lung resulting from degradation of ECM proteins and subsequent loss of alveolar cells. Recently, we reported that bone marrow-derived mesenchymal stem cell-conditioned media (MSC-CM) led to angiogenesis and regeneration of lung damaged by cigarette smoke. In this study, to further investigate reparative mechanisms for MSC-CM-mediated lung repair, we attempted to determine whether MSC-CM can recover lung fibroblasts from cigarette smoke-induced damage. In lung fibroblasts exposed to cigarette smoke extract (CSE), MSC-CM, not only inhibited apoptotic death, but also induced cell proliferation and reversed CSE-induced changes in the levels of caspase-3, p53, p21, p27, Akt, and p-Akt. MSC-CM also restored expression of ECM proteins and collagen gel contraction while suppressing CSE-induced expression of cyclooxygenase-2 and microsomal PGE(2) synthase-2. The CSE-opposing effects of MSC-CM on cell fate, expression of ECM proteins, and collagen gel contraction were partially inhibited by LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor. In rats, MSC-CM administration also resulted in elevation of p-Akt and restored proliferation of lung fibroblasts, which was suppressed by exposure to cigarette smoke. Taken together, these data suggest that MSC-CM may recover lung fibroblasts from cigarette smoke-induced damage, possibly through inhibition of apoptosis, induction of proliferation, and restoration of lung fibroblast repair function, which are mediated in part by the PI3K/Akt pathway.

Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-β (TGF-β) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease α-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-β-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: The aim of this work was to determine the relevance of Choukroun's platelet-rich fibrin (PRF) in dental implantology by determining the in vitro effects of soluble factors released by PRF clot. We used 3 different cell lines implicated in dental implantology: osteoblast, keratinocyte, and fibroblast., Methods: Cellular viability, cell proliferation, and gene expression were analyzed using PRF conditioned medium. Three different cells lines were used: SaOS2 (osteoblast), MRC5 (fibroblast), and KB (epithelial cell)., Results: The sulforhodamine B assay showed a significant increase in cell number for the undiluted and 1:3 diluted conditioned medium after 24 and 48 hours. There was no effect for the 1:9 dilution. Cell cycle analysis by flow cytometry confirmed the viability test results. After 48 hours, PRF conditioned medium induced gene expression in osteoblasts. Expression of osteopontin and osteocalcin, late osteogenic markers, was observed using reverse transcriptase-polymerase chain reaction (RT-PCR)., Conclusions: This study establishes a model to evaluate, in vitro, the effects of soluble growth factors released by PRF clot. Our work confirmed PRF is useful in stimulating tissue healing and bone regeneration. This work should recommend Choukroun's PRF in numerous implantology clinical applications.

Objective: This study evaluated the cytotoxicity of GuttaFlow and EndoSequence BC sealers and compared them with AH Plus and Tubli-Seal sealers., Study Design: Samples (0.5 mg) of freshly mixed or set BC, GuttaFlow, AH Plus, and Tubli-Seal sealers were eluted with 300, 600, and 1,000 μL cell culture medium for 24 and 72 hours. L929 cells were seeded into 96-well plates at 3 × 10(4) cells/well and cultured with 100 μL eluate from each eluate group. Cells cultured only with culture medium served as control. After 24 hours' incubation, the cytotoxicity was evaluated by MTT assay. Cell viability was calculated as the percentage of the control group, and the results were analyzed with a one-way analysis of variance., Results: For the freshly mixed sealer, cell viability in the AH Plus group was less than in all of the other 3 sealer groups. The Tubli-Seal sealer group had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. For the set sealer, the Tubli-Seal and AH Plus groups had less cell viability than the EndoSequence BC and GuttaFlow sealer groups. There was no cell viability difference between the EndoSequence BC and GuttaFlow sealer groups in the either freshly mixed or set sealer group., Conclusions: The GuttaFlow and EndoSequence BC sealers have lower cytotoxicity than the AH Plus and Tubli-Seal sealers., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO., ((c) 2010 Wiley-Liss, Inc.)

Aims: To investigate the effects of conditioned media from mesenchymal stem cells (MSC) on the wound healing activities of corneal stromal fibroblasts., Methods: Cell cycle analysis and early stage activation of apoptosis, chemotactic chambers and fibroblast-populated type I collagen gels were used to assess corneal stromal fibroblast proliferation, migration and contraction, respectively. Fibroblasts were obtained from human donor corneas and MSC from fresh rat bone marrow. MSC conditioned media and fibroblast culture medium (FCM), with and without calf serum supplementation, were compared., Results: MSC conditioned media and serum-free FCM had an inhibitory effect on the progression of corneal fibroblasts through the cell cycle. There was a significant increase in the number of cells in the G0-G1 phase for MSC conditioned media and serum-free FCM (p=0.001, p=0.97 respectively). Fibroblast migration and relaxed and stressed gel contraction were significantly inhibited by MSC conditioned media and serum-free FCM compared with FCM with serum (all p=0.001). Glucose and lactate analysis confirmed that these factors were not contributing to this effect., Conclusion: MSC conditioned media was found to inhibit the wound healing activities of corneal stromal fibroblasts in vitro. Putative factors secreted by MSC could be developed for therapeutic use in corneal repair.

Objective: Hydrolytic activity is increased in conditioned media from human gingival and pulp fibroblasts in response to exposure to triethyleneglycol dimethacrylate (TEGDMA). The purpose of this study was to determine if this conditioned media with hydrolytic activity could cause the biodegradation of a dental resin material., Methods: Resin material specimens were stored for 30 days at 37 degrees C in distilled water, unconditioned media, artificial serum, conditioned media from human gingival and pulp fibroblasts and conditioned media from human pulp and gingival fibroblasts that were exposed to TEGDMA. The media was exchanged every other day. The specimens were subjected to Fourier transform infrared spectroscopy (FTIR) before and after storage. The area under the carbonyl peak was calculated for all the specimens to determine the extent of the degradation., Results: Differences before and after storage in the area under the carbonyl peak were statistically significant for the specimens stored in the conditioned media from the fibroblasts that were exposed to TEGDMA. All of the other specimens did not produce differences in the area under the carbonyl peak that were statistically significant when comparing the before and after storage results., Significance: The biodegradation demonstrated here could contribute to the marginal leaking seen clinically with the use of resin materials in dental restorations.

There is increased interest in the effects of secretory products from aged cells on promoting both benign and malignant cell growth. We identified a human fibroblast line, AG04382, from an aged donor that naturally demonstrated senescence-associated features and whose conditioned media significantly induced proliferation of benign prostatic hyperplasia (BPH1) cells. Candidate cytokines mediating this effect were identified with protein arrays and validated by ELISA. We found that the AG04382 fibroblast line secreted high levels of CXCL5, CCL5, and CCL2, but relative to the other lines, its conditioned media was unique in its increased expression of CCL5. Blocking studies using specific antibodies against CXCL5, CCL5, and CCL2 in the conditioned media of AG04382 showed that only CCL5 contributed significantly to BPH1 proliferation. Stimulation of BPH1 cells with rhuCCL5 resulted in increased proliferation and migration, as well as significant changes in the expression of genes that influence angiogenesis. These data suggest that CCL5 is a candidate chemokine secreted by aged cells that promotes prostate growth and regulates angiogenesis.

Background: The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined., Methods and Results: We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-alpha (TNF-alpha) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-alpha and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-beta and NO., Conclusion: The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.

Purpose: To explore whether interactions between normal epithelial and mesenchymal cells can modulate the extent of radiation-induced DNA damage in one or both types of cells., Methods and Materials: Human primary thyrocytes (PT), diploid fibroblasts BJ, MRC-5, and WI-38, normal human mammary epithelial cells (HMEC), and endothelial human umbilical cord vein endothelial cells (HUV-EC-C), cultured either individually or in co-cultures or after conditioned medium transfer, were irradiated with 0.25 to 5 Gy of gamma-rays and assayed for the extent of DNA damage., Results: The number of gamma-H2AX foci in co-cultures of PT and BJ fibroblasts was approximately 25% lower than in individual cultures at 1 Gy in both types of cells. Reciprocal conditioned medium transfer to individual cultures before irradiation resulted in approximately a 35% reduction of the number gamma-H2AX foci at 1 Gy in both types of cells, demonstrating the role of paracrine soluble factors. The DNA-protected state of cells was achieved within 15 min after conditioned medium transfer; it was reproducible and reciprocal in several lines of epithelial cells and fibroblasts, fibroblasts, and endothelial cells but not in epithelial and endothelial cells. Unlike normal cells, human epithelial cancer cells failed to establish DNA-protected states in fibroblasts and vice versa., Conclusions: The results imply the existence of a network of reciprocal interactions between normal epithelial and some types of mesenchymal cells mediated by soluble factors that act in a paracrine manner to protect DNA from genotoxic stress.

Cyclooxygenase-2 (COX-2) overexpression is associated with cancer. One potential mechanism is DNA damage caused by COX-2 derived oxidants. Since DNA in proliferating cells is highly vulnerable to oxidative damage and mutation, we propose that COX-2 transactivation by exogenous stimuli is suppressed in proliferating cells compared to quiescent cells. In this review, we provide evidence for reduced COX-2 transcriptional expression in response to phorbol esters (PMA), lipopolysaccharide (LPS), interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). Our results show that COX-2 transcription in proliferating fibroblasts is suppressed by a small molecular weight compound produced by proliferating cells. By contrast, COX-2 expression in response to exogenous stimuli is robust in quiescent cells. The quiescent cells in human body may play a primary role in mounting response to exogenous stimuli. Salicylate inhibits COX-2 transcriptional activation in quiescent cells but not in serum-driven proliferating cells by blocking C/EBPbeta DNA binding. These studies suggest that COX-2 expressions in quiescent and proliferating cells are regulated by different mechanisms. Further investigations into their transcriptional control mechanisms will have great impact on the fundamental understanding of the division of cell functions between quiescent and proliferating cells and the design of novel therapeutic strategies.

Thymic stromal lymphopoietin (TSLP) is an interleukin (IL)-7-like cytokine produced by epithelial cells and triggers dendritic cell-mediated Th2 type allergic inflammatory responses. This study investigated whether Toll-like receptor (TLR) ligands, lipopolysaccharide (LPS) and poly-IC affect TSLP production in synovial fibroblasts. Enzyme-linked immunosorbent assay showed that LPS and poly-IC upregulated TSLP production in synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, we found that nuclear factor (NF)-kappaB inhibitor IMD-0354, dexamethasone, and interferon (IFN)-gamma inhibited the LPS- and poly-IC-induced TSLP production in RA and OA synovial fibroblasts. Thus, LPS and poly-IC can upregulate TSLP via a NF-kappaB pathway in synovial fibroblasts, which is downregulated by dexamethasone and interferon (IFN)-gamma. The current findings suggest that TSLP may be involved in the pathophysiology of inflammatory arthritis as well as allergic disease.

Saiko is predominantly contained in Saireito, a Chinese herbal medicine. The present study was conducted to determine whether or not Saiko is involved in the inhibition by Saireito of nifedipine-induced proliferation and collagen synthesis in gingival fibroblasts. Nifedipine (10 microM) significantly enhanced the proliferation starting on day 5 of the culture period. When added together with nifedipine, Saiko at concentrations of 0.05%-0.2% (w/v) dose-dependently inhibited the nifedipine-induced proliferation, and at the highest concentration tested (0.2%), Saiko inhibited the nifedipine-induced proliferation by about 40%. Moreover, Saiko (0.2%) also inhibited the normal proliferation at days 11 and 14. Sole application of nifedipine (10 microM) augmented the release of bFGF, and Saiko concentration-dependently reduced the level of bFGF in the nifedipine-containing culture medium. Nifedipine (10 microM) increased the production of type I collagen to almost twice that of the control (normal medium), and Saiko at concentrations above 0.1% significantly reduced the nifedipineinduced production of collagen. In conclusion, the present findings demonstrate that Saiko inhibited the nifedipine-induced proliferation of gingival fibroblasts by reducing the release of bFGF and that Saiko is involved in the Saireito-induced inhibition of nifedipine-stimulated proliferation and collagen synthesis in gingival fibroblasts.

Our previous study demonstrated that a laminin-like molecule produced by periodontal ligament fibroblasts (PLFs) induces gingival epithelial cell chemotaxis. The aim of this study was to identify the laminin isoforms that are expressed by PLFs. Proteins in PLF-conditioned medium from serum-free cultures were separated by gel filtration followed by gelatin-affinity chromatography to remove fibronectin. Protein expression of laminin isoforms was determined using Western blotting, and mRNA expression was examined by RT-PCR. Partially purified laminin evoked gingival epithelial cell chemotaxis, and this activity was blocked by anti-integrin alpha3, alpha6, and beta1 antibodies. Although RT-PCR analysis showed PLFs expressed laminin alpha1 to alpha5, beta1 to beta3, gamma1, and gamma2 chain mRNAs, the predominant laminin chains detected by Western blotting were alpha4, alpha2, beta1, beta2, and gamma1. These results suggest that PLFs secrete mainly laminin-8/9 (alpha4beta1gamma1/alpha4beta2gamma1) and laminin-2/4 (alpha2beta1gamma1/alpha2beta2gamma1). PLF-derived laminins may be involved in the pathogenesis and progression of periodontitis by inducing apical migration of epithelial cells in certain circumstances.

Fibroblasts are the most ubiquitous cell types within our body. They produce various factors to maintain the texture and structure of a particular organ or tissue. To identify protein factors secreted by fibroblasts and alteration of these protein factors upon oxidative stress, HCA3 human skin diploid fibroblasts were exposed to a sublethal dose of H2O2, which induces a prematurely senescent phenotype. Conditioned media from prematurely senescent cells versus control cells were analyzed for proteins using an LC-MS/MS-based proteomic technique. Collagen alpha1(VI), collagen alpha2(I), fibronectin, lumican, and matrix metalloproteinase 2 were among the proteins consistently detected from control and H2O2-treated cells. Insulin-like growth factor-binding protein-6 (IGFBP-6) consistently showed up in the conditioned medium of H2O2-treated cells but not from untreated cells. Increased IGFBP-6 production due to H2O2 treatment was confirmed by RT-PCR and Western blot analyses. While H2O2 induced a dose-dependent elevation of IGFBP-6 mRNA, Western blot analyses detected elevated levels of IGFBP-6 protein in the conditioned medium of H2O2-treated cells. In comparison, fibronectin or matrix metalloproteinase 2 did not show changes at the mRNA level in cell lysates or at the protein level in the conditioned medium by H2O2 treatment. Using several types of toxins at sublethal doses, including cis-platin, hydroxyurea, colchicine, L-mimosine, rhodamine, dithiothreitol, or N-ethylmaleimide, we found that these agents induced increases of IGFBP-6 at mRNA and protein levels. An increased level of IGFBP-6 protein was detected in the plasma of aging mice and of young mice treated with doxorubicin. These data suggest that IGFBP-6 may serve as a sensitive biomarker of cell degeneration or injury in vitro and in vivo.

Objective: The highly osteolytic interface tissue between the bone and loosening total hip prosthesis is characterized by low pH, formation of foreign body giant cells, osteoclasts, and production of receptor activator of nuclear factor-kappaB (RANKL) and cathepsin K. We hypothesized that fibroblasts in the interface tissue may form a source for RANKL production., Methods: Primary interface tissue fibroblasts, fibrous joint capsule fibroblasts, and trabecular bone osteoblasts were stimulated with tumor necrosis factor-alpha (TNF-alpha), interleukin 1beta (IL-1beta), IL-6, IL-11, or 1alpha,25-(OH)2 vitamin D3. Cellular RANKL and released cathepsin K were detected by Western blotting. RANKL in cell lysates and osteoprotegerin (OPG) in cell culture medium were measured by ELISA. RANKL, OPG, and cathepsin K mRNA were measured with quantitative reverse transcriptase polymerase chain reaction., Results: Interface tissue fibroblasts were found to produce RANKL. 1alpha,25-(OH)2 vitamin D3 stimulation increased RANKL mRNA expression. TNF-alpha was found to be the most potent OPG inducer in interface tissue fibroblasts. Cathepsin K mRNA production in fibroblasts was upregulated roughly 3-fold (p < 0.01) after 1alpha,25-(OH)2D3 stimulation, and both pro- and active cathepsin K protein was released to fibroblast culture media., Conclusion: Interface tissue fibroblasts are able to produce RANKL, OPG, and cathepsin K and may contribute indirectly and directly to pathologic periprosthetic collagenolysis and bone destruction.

Objective: To perform the proteome analysis of conditioned medium prepared from mouse embryonic fibroblast feeder layers by 2-dimensional (2D) electrophoresis and mass spectrometry and to find out the possible differentiation-inhibitory factor in conditioned medium., Methods: Feeder layers were prepared by 60Co gamma-irradiation on mouse embryonic fibroblast. Insulin-transferrin-sodium selenite supplemented medium was used to culture the feeder layers for 24 hours. The condioned medium prepared from mouse embryonic fibroblast feeder layers were made into powder by lyophilization, the redissolved solution was applied to Sephadex G-50 gel filtration chromatography, and then cold acetone was used to precipitate the proteins in the eluted solution. The protein samples were applied to 2D electrophoresis. The 2D images were analyzed by 2D image analysis software. Selected protein spots were digested by trypsin, analyzed by mass spectrometry, and then searched against the NCBInr batabase using Mascot MS/MS Ions Search., Results: The protein samples extracted from mouse embryonic fibroblast feeder layers conditioned medium could be used for 2D electrophoresis. On 2D images, there were (221+/-67) spots. Most of the proteins were located in the region of MW 20 approximately 70 kD, pI 4 approximately 8. Using mass spectrometry, we preliminarily identified 13 spots: 3 keratins, 3 transferrins, 1 trypsin precursor, 2 unknown proteins (3 spots), 1 connexin 46, 1 beta-galactoside binding protein, and 1 secreted protein, acidic and rich in cysteine., Conclusion: Conditioned medium prepared from mouse embryonic fibroblast feeder layers contain beta-galactoside binding protein and secreted protein, acidic and rich in cysteine.

Calcific aortic valve stenosis, the main heart valve disease in the elderly, is based on progressive calcification and fibrous thickening of the valve. Several reports addressed the pathogenesis of tissue calcification in this disorder, but few data exist on the molecular mechanisms of the fibrosis and remodeling of the extracellular matrix. The cytokine "receptor activator of nuclear factor kappa B ligand" (RANKL), is expressed in stenotic aortic valves and involved in valvular calcification during calcific aortic valve stenosis. The present study aimed to assess the influence of RANKL on the molecular mechanisms of connective tissue remodeling. In an established cell culture model of primary human aortic valve myofibroblasts, stimulation with RANKL increased cell proliferation as compared to medium alone. Matrix metalloproteinase (MMP)-1 was detectable time-dependently in conditioned media from RANKL-stimulated cells, but absent in media from control cells. MMP-1 activity was increased by RANKL, as measured by collagenase activity assay. Zymography showed an increase in active MMP-2 in RANKL-stimulated cells. These results support the concept that MMPs are involved in the connective tissue remodeling during calcific aortic valve stenosis. RANKL might regulate this process by promoting cell proliferation and MMP expression and activation.

Pulsed electromagnetic fields (PEMF) have been shown to be clinically beneficial, but their mechanism of action remains unclear. The present study examined the impact of PEMF on angiogenesis, a process critical for successful healing of various tissues. PEMF increased the degree of endothelial cell tubulization (sevenfold) and proliferation (threefold) in vitro. Media from PEMF cultures had a similar stimulatory effect, but heat denaturation ablated this activity. In addition, conditioned media was able to induce proliferative and chemotactic changes in both human umbilical vein endothelial cells and fibroblasts, but had no effect on osteoblasts. Angiogenic protein screening demonstrated a fivefold increase in fibroblast growth factor beta-2 (FGF-2), as well as smaller increases in other angiogenic growth factors (angiopoietin-2, thrombopoietin, and epidermal growth factor). Northern blot analysis demonstrated an increase in FGF-2 transcription, and FGF-2 neutralizing antibody inhibited the effects of PEMF. In vivo, PEMF exposure increased angiogenesis more than twofold. We conclude that PEMF augments angiogenesis primarily by stimulating endothelial release of FGF-2, inducing paracrine and autocrine changes in the surrounding tissue. These findings suggest a potential role for PEMF in therapeutic angiogenesis.

Neovascularization is involved in beneficial and detrimental processes of tendon pathology. We investigated the influence of repetitive motion on the expression of the most important angiogenic factor, the vascular endothelial growth factor (VEGF) in the 3T3 NIH fibroblast cell line and in cultures of rat Achilles tendon fibroblasts. Monolayers of subconfluently grown cells were stretched in rectangular silicone dishes with cyclic uniaxial movement. Strain was applied over 24 h varying the frequency (0.5-1 Hz). Fibroblasts (3T3 fibroblasts and rat Achilles tendon cultures) cultivated without the application of cyclic strain released measurable VEGF amounts into their culture supernatants. Cyclic stretching of the cells with a frequency of 1 Hz resulted in an increased expression of VEGF. A low frequency (0.5 Hz) reduced VEGF expression to control levels. RT PCR revealed VEGF 121 and VEGF 165 as the only splice forms that were induced by cyclic stretching. Western blot experiments could further show that cyclic stretching induced activation of the transcription factor HIF-1alpha. These results demonstrate that mechanical factors are involved in the regulation of VEGF expression in tendon tissue.

Indoleamine 2,3-dioxygenase (IDO), a tryptophan-catabolizing enzyme, is an intracellular enzyme possessing various immunosuppressive properties. Here, we report the possible use of this enzyme to suppress proliferation of immune cells cocultured with IDO-expressing fibroblasts of an allogenic skin substitute. Fetal skin fibroblasts embedded within bovine collagen were treated with cytokine interferon-gamma (IFN-gamma) to induce expression of IDO mRNA and protein. Expression of IDO mRNA was evaluated by Northern analysis. IDO enzyme activity was evaluated by measurement of kynurenine and tryptophan levels in the IFN-gamma untreated and treated fibroblasts. The results of Northern analysis showed a dose-dependent increase in expression of IDO mRNA in response to various concentrations of IFN-gamma used. The levels of kynurenine and tryptophan measured, as the bioactivity of IDO, were significantly different in the IFN-gamma treated fibroblasts, compared to those of controls (P < 0.001). In a lasting effect experiment, the expression of IDO mRNA was gradually reduced to an undetectable level within 32 h of IFN-gamma removal. The results of Western blot analysis, however, revealed a significantly longer (192 h) lasting effect of IFN-gamma on IDO protein level, relative to that of mRNA expression. To demonstrate immunosuppressive effects of IDO on proliferation of immune cells, IDO-expressing fibroblasts were cocultured with peripheral blood mononuclear cells (PBMC) for a period of 5 days. The results of (3)H-thymidine incorporation showed a significant reduction in proliferation of PBMC when cocultured with IDO-expressing fibroblasts, compared to those cocultured with non-IDO-expressing fibroblasts (P < 0.001). Furthermore, addition of IDO-inhibitor (1-methyl-d-tryptophan) reversed the suppressive effects of IDO on PBMC proliferation in a dose-dependant fashion. To test the viability of immune cells cocultured with IDO-expressing fibroblasts, FACS analysis of the PI stained PBMC was conducted and no significant difference was found between these cells and the controls. In another set of experiments, we showed that migration rate and subsequent proliferation of IDO-expressing fibroblasts are also the same as those of control cells. In conclusion, IDO-expressing allogenic fibroblasts embedded within collagen gel suppress the proliferation of allogenic immune cells, while they still remain viable in this IDO-induced tryptophan-deficient culture environment., (Copyright 2003 Wiley-Liss, Inc.)