CLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 9 2005
Jennifer Martin
SUMMARY 1.,Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase inhibitors (statins) reduce mortality after myocardial infarction (MI). Although this may be predominantly due to their known anti-ischaemic actions, these drugs are known to have other beneficial effects. 2.,Because pathological deposition of extracellular matrix (ECM) material is a key component of remodelling after MI, we sought to determine whether atorvastatin could inhibit ECM production in vitro. 3.,The addition of atorvastatin to rat cardiac fibroblasts stimulated with either transforming growth factor (TGF)-,1 (TGF-,1) or angiotensin (Ang) II reduced collagen synthesis in a dose-dependent manner (3.7-fold reduction (95% confidence interval (CI) 1.8,15; P < 0.01) and 5.3-fold reduction (95% CI 1.8,7.7; P < 0.01), respectively, compared with stimulant alone). Similar observations were made in human cardiac fibroblast cell culture. Atorvastatin also dose-dependently reduced TGF-,1 and AngII-induced increases in ,(I)-procollagen mRNA (P < 0.01 for both), as well as gene expression of the profibrotic peptide connective tissue growth factor. 4.,Atorvastatin appears to directly inhibit collagen production by cardiac fibroblasts. This antifibrotic action may contribute to the antiremodelling effect of statins. [source]

Physicochemical and biological evaluation of plasma-induced graft polymerization of acrylamide onto polydimethylsiloxane

JOURNAL OF APPLIED POLYMER SCIENCE, Issue 4 2008
A. Parvin
Abstract Polydimethylsiloxane (PDMS) rubbers exhibit good mechanical properties for biomedical and industrial applications, but their inherently high hydrophobicity limits biomedical applications of this material despite its favorable mechanical properties. In this work, surface modification of PDMS by radio-frequency glow discharge and subsequently graft polymerization of acrylamide was studied. PAAm-grafted, oxygen plasma-treated, and control (untreated) PDMS rubbers were characterized using attenuated total reflectance Fourier transform infrared, scanning electron microscopy, dynamic mechanical thermal analyses, zeta potential, and contact angle techniques. Fibroblast (L929) cell attachment and growth onto these surfaces were examined by optical microscopy. The data from in vitro assays showed that cell attachment onto control surface was very negligible while significant cell attachment and growth was observed onto oxygen plasma-treated and PAAm-grafted PDMS surfaces. The method developed in this work offers a convenient way of surface modifications of biomaterials to improve attachment of cells onto substrates. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2008 [source]

Increased Glycosaminoglycans Production in Sclerosing Basal Cell Carcinoma-Derived Fibroblasts and Stimulation of Normal Skin Fibroblast Glycosaminoglycans Production by a Cytokine-Derived from Sclerosing Basal Cell Carcinoma

DERMATOLOGIC SURGERY, Issue 11 2000
Ronald L. Moy MD
Sclerosing basal cell carcinoma (S-BCC) is characterized by an abundant stroma. There is evidence that some tumor cells secrete cytokines that are mitogenic for stromal fibroblasts (FBs). From this study we report increased glycosaminoglycan (GAG) production by cultures of S-BCC FBs in comparison to cultures of nodular BCC (N-BCC) FBs and normal skin FBs. GAG production was measured by cetylpyridinium chloride precipitation of incorporated [3H]-glucosamine. The sclerosing BCC FBs demonstrated a significant increase in production of GAG over control FBs (P < .001) and over N-BCC FBs (P < .001). Values reported as a mean percentage ± SEM for GAG production by S-BCC over control normal skin FBs are 359 ± 28 and over N-BCC FBs are 266 ± 27. In additional experiments, cell extract dilutions from S-BCC tumor, normal dermis, and normal epidermis were incubated with cultures of normal skin FBs. S-BCC-conditioned media was also incubated with normal FBs and GAG production was measured. For both S-BCC extracts and conditioned media, a dose response curve was established showing increased GAG production by normal FBs in relation to increasing the concentration of S-BCC extract or conditioned media. When S-BCC extract was added to normal FBs there was increased GAG production in comparison to normal FBs incubated with dermal or epidermal extracts (P < .001) for both. Two growth factors, transforming growth factor-, (TGF-,) and platelet-derived growth factor (PDGF), already known to be mitogenic for FBs, were incubated with N-BCC and normal FBs in an effort to elucidate the potential cytokine(s) released by S-BCC, causing increased GAG production by surrounding FBs. Neither of these cytokines proved to be effective in promoting a significant increase in GAG production. Our findings support the hypothesis that BCCs release factors that alter stromal FB production of GAG. [source]

Augmentation of all- trans -retinoic acid concentration in plasma by preventing inflammation responses induced by atRA-loaded microspheres with concurrent treatment of dexamethasone

DRUG DEVELOPMENT RESEARCH, Issue 4 2004
Kyeongsoon Park
Abstract All- trans retinoic acid (atRA)-loaded microspheres severely induce inflammatory responses after microsphere implantation. Fibroblasts and a thick band of fibrous capsule resulting from the inflammatory responses could hamper drug permeation to the bloodstream because fibroblasts actively metabolize atRA into polar metabolites and the thick fibrous capsule acts as a diffusion barrier. In the present study, we investigated whether the fibroblast proliferation and collagen deposition induced by atRA released from microspheres might affect the atRA concentration in plasma and atRA metabolism with or without treatment of dexamethasone as an anti-inflammatory drug. After subcutaneous injection of atRA-loaded microspheres in rats, it was observed that atRA-loaded microspheres induced severe inflammatory responses and stimulated fibroblast proliferation and collagen deposition in fibrous capsules. On the other hand, the orally treated dexamethasone effectively prevented inflammatory responses in a dose-dependent manner and suppressed about 49% of the number of fibroblasts and collagen deposition in fibrous capsules at 14 days. In addition, after the treatment of dexamethasone, the atRA concentration in plasma was increased, and its metabolism was decreased approximately by 40% at 7 days, compared to the group treated alone with atRA-loaded microspheres. In conclusion, the concurrent treatment of dexmethasone with atRA-loaded microspheres could prevent inflammatory responses and metabolism of atRA, thereby maintaining the atRA concentration in plasma for longer periods in the therapeutic range. Drug Dev. Res. 61:197,206, 2004. © 2004 Wiley-Liss, Inc. [source]

Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardium

EUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 6 2009
H. J. Ankersmit
Abstract Background, Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c-kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium. Methods, Immune function of viable peripheral blood mononuclear cells (PBMC) was evaluated after co-culture with irradiated apoptotic PBMC (IA-PBMC) in vitro. Viable PBMC, IA-PBMC and culture supernatants (SN) thereof were obtained after 24 h. Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were utilized to quantify interleukin-8 (IL-8), vascular endothelial growth factor, matrix metalloproteinase-9 (MMP9) in PBMC, SN and SN exposed fibroblasts. Cell suspensions of viable- and IA-PBMC were infused in an experimental rat AMI model. Immunohistological analysis was performed to detect inflammatory and pro-angiogenic cells within 72 h post-infarction. Functional data and determination of infarction size were quantified by echocardiography and Elastica van Gieson staining. Results, The IA-PBMC attenuated immune reactivity and resulted in secretion of pro-angiogenic IL-8 and MMP9 in vitro. Fibroblasts exposed to viable and IA-PBMC derived SN caused RNA increment of IL-8 and MMP9. AMI rats that were infused with IA-PBMC cell suspension evidenced enhanced homing of endothelial progenitor cells within 72 h as compared to control (medium alone, viable-PBMC). Echocardiography showed a significant reduction in infarction size and improvement in post AMI remodelling as evidenced by an attenuated loss of ejection fraction. Conclusion, These data indicate that infusion of IA-PBMC cell suspension in experimental AMI circumvented inflammation, caused preferential homing of regenerative EPC and replaced infarcted myocardium. [source]

Phase Reversion-Induced Nanograined/Ultrafine-Grained Structures in Austenitic Stainless Steel and their Significance in Modulating Cellular Response: Biochemical and Morphological Study with Fibroblasts,

ADVANCED ENGINEERING MATERIALS, Issue 12 2009
R. Devesh Kumar Misra
Materials science, engineering, and biological sciences have been combined to improve the tissue compatibility of medical devices. In this regard, nano/ultrafine structuring of austenitic stainless steel obtained using an innovative approach of "phase-reversion" has been evaluated for modulation of cellular activity. The biochemical and morphology study with fibroblasts point toward the improvement of tissue compatibility on comparison with coarse-grained structures, strengthening the foundation of nanostructured materials for bio-medical applications. [source]

Cytotoxicity and Cell Cycle Effects of Bare and Poly(vinyl alcohol)-Coated Iron Oxide Nanoparticles in Mouse Fibroblasts

ADVANCED ENGINEERING MATERIALS, Issue 12 2009
Morteza Mahmoudi
Super-paramagnetic iron oxide nanoparticles (SPIONs) are recognized as powerful biocompatible materials for use in various biomedical applications, such as drug delivery, magnetic-resonance imaging, cell/protein separation, hyperthermia and transfection. This study investigates the impact of high concentrations of SPIONs on cytotoxicity and cell-cycle effects. The interactions of surface-saturated (via interactions with cell medium) bare SPIONs and those coated with poly(vinyl alcohol) (PVA) with adhesive mouse fibroblast cells (L929) are investigated using an MTT assay. The two SPION formulations are synthesized using a co-precipitation method. The bare and coated magnetic nanoparticles with passivated surfaces both result in changes in cell morphology, possibly due to clustering through their magnetostatic effect. At concentrations ranging up to 80,×,10,3,M, cells exposed to the PVA-coated nanoparticles demonstrate high cell viability without necrosis and apoptosis. In contrast, significant apoptosis is observed in cells exposed to bare SPIONs at a concentration of 80,×,10,3,M. Nanoparticle exposure (20,80,×,10,3,M) leads to variations in both apoptosis and cell cycle, possibly due to irreversible DNA damage and repair of oxidative DNA lesions, respectively. Additionally, the formation of vacuoles within the cells and granular cells indicates autophagy cell death rather than either apoptosis or necrosis. [source]

Human skin fibroblasts: From mesodermal to hepatocyte-like differentiation,

HEPATOLOGY, Issue 5 2007
Philippe A. Lysy
The phenotypic homology of fibroblasts and mesenchymal stem cells (MSCs) has been recently described. Our study investigated the in vitro potential of human skin fibroblasts to differentiate into mesodermal (osteocyte and adipocyte) and endodermal (hepatocyte) cell lineages by comparison with human bone marrow (hBM) MSCs. The endodermal potential of fibroblasts was then explored in vivo in a mouse model of liver injury. Fibroblasts were able to acquire osteocyte and adipocyte phenotypes as assessed by cytochemistry and gene expression analyses. After exposure to a specific differentiation cocktail, these cells presented hepatocyte-like morphology and acquired liver-specific markers on protein and gene expression levels. Furthermore, these fibroblast-derived hepatocyte-like cells (FDHLCs) displayed the ability to store glycogen and synthesize small amounts of urea. By gene expression analysis, we observed that fibroblasts remained in a mesenchymal-epithelial transition state after hepatocyte differentiation. Moreover, FDHLCs lost their hepatocyte-like phenotype after dedifferentiation. In vivo, human fibroblasts infused directly into the liver of hepatectomized severe combined immunodeficient (SCID) mice engrafted in situ and expressed hepatocyte markers (albumin, alpha-fetoprotein, and cytokeratin 18) together with the mesodermal marker fibronectin. Despite lower liver-specific marker expression, the in vitro and in vivo differentiation profile of fibroblasts was comparable to that of mesenchymal-derived hepatocyte-like cells (MDHLCs). In conclusion, our work demonstrates that human skin fibroblasts are able to display mesodermal and endodermal differentiation capacities and provides arguments that these cells share MSCs features both on the phenotypic and functional levels. (HEPATOLOGY 2007;46:1574,1585.) [source]

Development of a swine model of secondary liver tumor from a genetically induced swine fibroblast cell line

HPB, Issue 3 2008
R. Abbas
Abstract Aim. Metastatic disease is the most common liver tumor. Although alternative therapies have been developed for non-surgical candidates, those therapies lacked ideal testing prior to clinical application because of a paucity of large animal models. The purpose of the present study was to develop a model for secondary liver tumor in a large animal. Material and methods. Fibroblasts were isolated from swine ear lobules and then transfected with amphotrophic retroviruses encoding human or murine genetic material (hTERT, p53DD, cyclinD-1, CDK4R24C, Myc T58A, RasG12V). Transformed cell lines were finally inoculated subcutaneously (s.c.) into: 1) immunodeficient mice (nude), 2) immunocompetent mice (wild type), 3) immunosuppressed swine (under tacrolimus or corticosteroids), 4) immunocompetent swine, and 5) into the liver and portal circulation of swine under steroid-based immunosuppression. Results. In the murine model, tumor growth was evident in 100% of the nude mice (n=5), with a peak size of 20 mm (15.22±4.5 mm; mean±SD) at the time of sacrifice (3 weeks). Tumor growth was evident in 71% of the wild mice (n=21), with a peak size of 7.8 mm (4.19±1.1 mm) by the third week of growth. In the swine model, tumor growth was evident in 75% (3/4 ears; n=2) of swine under tacrolimus-based immunosuppression versus 50% of swine under steroids-based immunosuppression (n=2). Tumor growth was slow in two animals, while in one animal the tumor was larger with a peak growth of 42 mm at 3 weeks. The tumor pattern in the ear lobules was characterized by slow growth, with a peak size of 6,8 mm in the immunocompetent swine at 3 weeks. All tumors were shown to be malignant by histology. In contrast, inoculums of the transformed fibroblast cell line in swine livers showed no evidence of tumor growth at 3 weeks. Conclusions. Development of a transformed swine fibroblast cell line was successful, resulting in an in vivo malignant tumor. Cell line inoculums had tumorigenic properties in nude mice, wild-type mice, and immunosuppressed swine, as judged by uncontrolled cell growth, invasion of surrounding tissue, neoangiogenesis, and invasion of normal vasculature, resulting in the formation of tumor nodules. Such properties were not observed in swine upon inoculation into the liver/portal circulation. [source]

Cell Proliferation of Human Fibroblasts on Alumina and Hydroxyapatite-Based Ceramics with Different Surface Treatments,

INTERNATIONAL JOURNAL OF APPLIED CERAMIC TECHNOLOGY, Issue 2 2010
Juliana Marchi
Biocompatibility is an important characteristic of dental implant material, and in vitro tests are required to elucidate the interaction between these materials and human tissues. Cell proliferation assays were done with fibroblasts plated on the surface of alumina and hydroxyapatite sintered samples, each with a different surface treatment (sintered, rectified, or polished). After 1, 2, and three days, the samples were prepared for scanning electron microscopy observations. The data were compared by analysis of variance followed by Tukey's test. It was concluded that neither the hydroxyapatite or alumina substrate is cytotoxic, and hydroxyapatite is more biocompatible than alumina. [source]

Substrate adhesion affects contraction and mechanical properties of fibroblast populated collagen lattices

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2008
Meng-Yi Chen
Abstract Fibroblasts can condense a hydrated collagen lattice to a tissue-like structure. The purpose of this study was to evaluate the effect of substrate adhesion on the contraction and mechanical properties of fibroblast populated collagen lattices. Bacteriological grade polystyrene (BGPS) plates and tissue culture polystyrene (TCPS) plates were used as substrates for incubation of fibroblast populated collagen lattices. Hydrophobicity of the polystyrene surfaces was measured by the static sessile contact angle method. Collagen lattice contraction was recorded for 2 weeks, after which the lattices were mechanically tested. The BGPS culture plate had a significantly larger contact angle and was more hydrophobic than the TCPS culture plate. Both hydrophobicity and peripheral detachment of the collagen gel significantly decreased the time lag before initiation of gel contraction and increased the strength of the fibroblast populated collagen lattices. Substrate adhesion affects the contractility and strength of cell seeded collagen gels. This information may be useful in developing tissue engineered tendons and ligaments. © 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 2008 [source]

Periodontal growth factors and tissue carriers: Biocompatibility and mitogenic efficacy in vitro

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH, Issue 1 2006
Claudio Cesari
Clinical research has long been testing techniques of integrating biomaterials with many external factors, such as simple proteins or more complicated devices, in order to achieve the restitutio ad integrum of periodontium. This study assessed the in vitro effectiveness of platelet derivate growth factor-BB (PDGF) and insulin growth factor I (IGF); the biocompatibility of materials like Paroguide, Oclastim membranes, Gingistat sponges, Surgiplaster, and Capset; and their efficacy as carriers for the platelet derivate growth factor-BB (PDGF) and insulin growth factor I (IGF). Fibroblasts from the human periodontal ligament were incubated with growth factors free or vehiculated. Mitogenic effect was evaluated by measuring the growth rate and biocompatibility by observing cell morphology at SEM. PDGF was the most effective in stimulating cell proliferation both in solution (p < 0.001) and vehiculated (p < 0.01). Surgiplaster and Capset were more biocompatible; however, final analysis to assess their efficacy as carriers failed to disclose significant differences between experimental findings and control. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2006 [source]

Compound Heterozygous Mutations in the Vitamin D Receptor in a Patient With Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets With Alopecia,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 4 2009
Yulin Zhou
Abstract Hereditary vitamin D-resistant rickets (HVDRR) is a rare recessive genetic disorder caused by mutations in the vitamin D receptor (VDR). In this study, we examined the VDR in a young girl with clinical features of HVDRR including rickets, hypophosphatemia, and elevated serum 1,25(OH)2D. The girl also had total alopecia. Two mutations were found in the VDR gene: a nonsense mutation (R30X) in the DNA-binding domain and a unique 3-bp in-frame deletion in exon 6 that deleted the codon for lysine at amino acid 246 (,K246). The child and her mother were both heterozygous for the 3-bp deletion, whereas the child and her father were both heterozygous for the R30X mutation. Fibroblasts from the patient were unresponsive to 1,25(OH)2D3 as shown by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 responsiveness. [3H]1,25(OH)2D3 binding and immunoblot analysis showed that the patient's cells expressed the VDR,K246 mutant protein; however, the amount of VDR,K246 mutant protein was significantly reduced compared with wildtype controls. In transactivation assays, the recreated VDR,K246 mutant was unresponsive to 1,25(OH)2D3. The ,K246 mutation abolished heterodimerization of the mutant VDR with RXR, and binding to the coactivators DRIP205 and SRC-1. However, the ,K246 mutation did not affect the interaction of the mutant VDR with the corepressor Hairless (HR). In summary, we describe a patient with compound heterozygous mutations in the VDR that results in HVDRR with alopecia. The R30X mutation truncates the VDR, whereas the ,K246 mutation prevents heterodimerization with RXR and disrupts coactivator interactions. [source]

Oxidative stress in NPC1 deficient cells: protective effect of allopregnanolone

JOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 9b 2009
Stefania Zampieri
Abstract Niemann-Pick C disease (NPC) is an autosomal recessive neurodegenerative disorder caused by the abnormal function of NPC1 or NPC2 proteins, leading to an accumulation of unesterified cholesterol and glycosphingolipids (GSLs) in the lysosomes. The mechanisms underlying the pathophysiology in NPC disease are not clear. Oxidative damage is implicated in the pathophysiology of different neurological disorders and the effect of GSL accumulation on the intracellular redox state has been documented. Therefore, we determined whether the intracellular redox state might contribute to the NPC disease pathophysiology. Because the treatment of NPC mice with allopregnanolone (ALLO) increases their lifespan and delays the onset of neurological impairment, we analysed the effect of ALLO on the oxidative damage in human NPC fibroblasts. Concentrations of reactive oxygen species (ROS) and lipid peroxidation were higher in fibroblasts from NPC patients than in fibroblasts from normal subjects. Fibroblasts from NPC patients were more susceptible to cell death through apoptosis after an acute oxidative insult. This process is mediated by activation of the NF-,B signalling pathway. Knockdown of NPC1 mRNA both in normal fibroblasts and in human SH-SY5Y neuroblastoma cells caused increased ROS concentrations. ALLO treatment of fibroblasts from NPC patients or NPC1 knockdown cells reduced the levels of ROS and lipid peroxidation and prevented peroxide-induced apoptosis and NF-kB activation. Thus, these findings suggest that oxidative stress might contribute to the NPC disease and ALLO might be beneficial in the treatment of the disease, at least in part, due to its ability to restore the intracellular redox state. [source]

The origin of fibroblasts and mechanism of cardiac fibrosis

JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010
Guido Krenning
Fibroblasts are at the heart of cardiac function and are the principal determinants of cardiac fibrosis. Nevertheless, cardiac fibroblasts remain poorly characterized in molecular terms. Evidence is evolving that the cardiac fibroblast is a highly heterogenic cell population, and that such heterogeneity is caused by the distinct origins of fibroblasts in the heart. Cardiac fibroblasts can derive either from resident fibroblasts, from endothelial cells via an endothelial,mesenchynmal transition or from bone marrow-derived circulating progenitor cells, monocytes and fibrocytes. Here, we review the function and origin of fibroblasts in cardiac fibrosis.NB. The information given is correct. J. Cell. Physiol. 225: 631,637, 2010. © 2010 Wiley-Liss, Inc. [source]

Triclosan reduces microsomal prostaglandin E synthase-1 expression in human gingival fibroblasts

JOURNAL OF CLINICAL PERIODONTOLOGY, Issue 1 2005
M. Mustafa
Abstract Objective: The effect of triclosan (2,4,4,-trichloro-2,-hydroxydiphenyl ether) on the expression of cyclooxygenase-2 (COX-2) and microsomal prostaglandin E synthase-1 (mPGES-1) and on the translocation of the nuclear factor- ,B (NF- ,B) in relation to prostaglandin E2 (PGE2) production was investigated in human gingival fibroblasts challenged with tumor necrosis factor , (TNF,). Methods: Fibroblasts were established from gingival biopsies obtained from six children. COX-2 mRNA and protein expression was quantified using mRNA quantitation and enzyme immunometric assay kits. mPGES-1 mRNA was analysed by RT-PCR, mPGES-1 protein and NF-,B translocation by immunoblotting. PGE2 was determined by radioimmunoassay. Results: The cytokine TNF, enhanced the expression of mRNA as well as the protein levels of both COX-2 and mPGES-1 and subsequently the production of PGE2 in gingival fibroblasts. Treatment of gingival fibroblasts with triclosan (1 ,g/ml) significantly reduced the stimulatory effect of TNF, (10 ng/ml) on the expression of mPGES-1 at both the mRNA and the protein level by an average of 21% and 43%, respectively, and subsequently the production of PGE2 (p<0.01). Triclosan did not, however, affect the translocation of NF- ,B or the expression of COX-2 in TNF,- stimulated cells. Conclusion: The results show that triclosan reduces the augmented biosynthesis of PGE2 by inhibiting the mRNA and the protein expression of mPGES-1 in gingival fibroblasts. This finding may partly explain the anti-inflammatory effect of the agent previously reported in clinical studies. [source]

Epithelioid cell histiocytoma , histogenetic and kinetics analysis of dermal microvascular unit dendritic cell subpopulations

JOURNAL OF CUTANEOUS PATHOLOGY, Issue 7 2003
Jeffrey S. Silverman
Background:, Epithelioid cell histiocytoma (ECH), also known as epithelioid fibrous histiocytoma, is a peculiar dermal tumor, which can mimic melanocytic, vascular, epithelial, or other histiocytic lesions. Thought to arise from dermal dendrocytes, most ECH contain approximately 50% FXIIIa+ histiocytic dendrocytes, but not all lesional cells express FXIIIa. A putative fibroblastic component has not been characterized. Methods:, We analyzed the differentiation and cell kinetics of dermal microvascular unit cells in 12 previously reported ECH using antibodies to FXIIIa, CD68 (KP1), CD34, CD117, CD31, smooth muscle actin, collagen type 1 aminopropeptide, and MIB-1, using single and double immunostains. Results:, In ECH, many variably sized CD34/CD31+ tumor vessels with actin+ myopericytes were surrounded by epithelioid-to-dendritic cells of three types. About 5,80% were dendritic histiocytes that expressed FXIIIa but not CD31 or KP1. Fibroblasts, in some cases showing mild nuclear pleomorphism, were usually collagen type 1+, but CD34 and actin, in 11/12 cases. One ,early' ECH had 40% CD34+ epithelioid cells, admixed with 50% FXIIIa+ histiocytes. Most ECH had about 2,20% KP1+, CD117+ mast cells. Mast cell numbers increased with FXIIIa+ histiocyte numbers and the intensity of FXIIIa expression. MIB-1/FXIIIa double-labeling showed only rare cycling histiocytes, with numerous cycling fibroblasts and endothelial cells. Conclusions:, Our findings support the impression that ECH is a vascular fibrous histiocytoma. The constituent cells appear to arise from the activation of resident microvascular CD34+ dermal fibroblasts and the accumulation of FXIIIa+ dendritic stromal assembly histiocytes. The CD34+ cells appear to differentiate toward collagenous fibrocytes in association with histiocytes and mast cells in forming collagenous stroma and vessels. ECH is a tumor composed of all requisite cell types consistent with the origin from the dermal microvascular unit. [source]

Stress-induced responses of human skin fibroblasts in vitro reflect human longevity

AGING CELL, Issue 5 2009
Pim Dekker
Summary Unlike various model organisms, cellular responses to stress have not been related to human longevity. We investigated cellular responses to stress in skin fibroblasts that were isolated from young and very old subjects, and from offspring of nonagenarian siblings and their partners, representatives of the general population. Fibroblasts were exposed to rotenone and hyperglycemia and assessed for senescence-associated ,-galactosidase (SA-,-gal) activity by flow cytometry. Apoptosis/cell death was measured with the Annexin-V/PI assay and cell-cycle analysis (Sub-G1 content) and growth potential was determined by the colony formation assay. Compared with fibroblasts from young subjects, baseline SA-,-gal activity was higher in fibroblasts from old subjects (P = 0.004) as were stress-induced increases (rotenone: P < 0.001, hyperglycemia: P = 0.027). For measures of apoptosis/cell death, fibroblasts from old subjects showed higher baseline levels (Annexin V+/PI+ cells: P = 0.040, Sub-G1: P = 0.014) and lower stress-induced increases (Sub-G1: P = 0.018) than fibroblasts from young subjects. Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from young subjects (P = 0.017 and 0.006, respectively). Baseline levels of SA-,-gal activity and apoptosis/cell death were not different between fibroblasts from offspring and partner. Stress-induced increases were lower for SA-,-gal activity (rotenone: P = 0.064, hyperglycemia: P < 0.001) and higher for apoptosis/cell death (Annexin V+/PI, cells: P = 0.041, Annexin V+/PI+ cells: P = 0.008). Numbers and total size of colonies under nonstressed conditions were higher for fibroblasts from offspring (P = 0.001 and 0.024, respectively) whereas rotenone-induced decreases were lower (P = 0.008 and 0.004, respectively). These data provide strong support for the hypothesis that in vitro cellular responses to stress reflect the propensity for human longevity. [source]

Delayed kinetics of DNA double-strand break processing in normal and pathological aging

AGING CELL, Issue 1 2008
Olga A. Sedelnikova
Summary Accumulation of DNA damage may play an essential role in both cellular senescence and organismal aging. The ability of cells to sense and repair DNA damage declines with age. However, the underlying molecular mechanism for this age-dependent decline is still elusive. To understand quantitative and qualitative changes in the DNA damage response during human aging, DNA damage-induced foci of phosphorylated histone H2AX (,-H2AX), which occurs specifically at sites of DNA double-strand breaks (DSBs) and eroded telomeres, were examined in human young and senescing fibroblasts, and in lymphocytes of peripheral blood. Here, we show that the incidence of endogenous ,-H2AX foci increases with age. Fibroblasts taken from patients with Werner syndrome, a disorder associated with premature aging, genomic instability and increased incidence of cancer, exhibited considerably higher incidence of ,-H2AX foci than those taken from normal donors of comparable age. Further increases in ,-H2AX focal incidence occurred in culture as both normal and Werner syndrome fibroblasts progressed toward senescence. The rates of recruitment of DSB repair proteins to ,-H2AX foci correlated inversely with age for both normal and Werner syndrome donors, perhaps due in part to the slower growth of ,-H2AX foci in older donors. Because genomic stability may depend on the efficient processing of DSBs, and hence the rapid formation of ,-H2AX foci and the rapid accumulation of DSB repair proteins on these foci at sites of nascent DSBs, our findings suggest that decreasing efficiency in these processes may contribute to genome instability associated with normal and pathological aging. [source]

Senescence-associated ,-galactosidase is lysosomal ,-galactosidase

AGING CELL, Issue 2 2006
Bo Yun Lee
Summary Replicative senescence limits the proliferation of somatic cells passaged in culture and may reflect cellular aging in vivo. The most widely used biomarker for senescent and aging cells is senescence-associated ,-galactosidase (SA-,-gal), which is defined as ,-galactosidase activity detectable at pH 6.0 in senescent cells, but the origin of SA-,-gal and its cellular roles in senescence are not known. We demonstrate here that SA-,-gal activity is expressed from GLB1, the gene encoding lysosomal ,-D-galactosidase, the activity of which is typically measured at acidic pH 4.5. Fibroblasts from patients with autosomal recessive GM1 -gangliosidosis, which have defective lysosomal ,-galactosidase, did not express SA-,-gal at late passages even though they underwent replicative senescence. In addition, late passage normal fibroblasts expressing small-hairpin interfering RNA that depleted GLB1 mRNA underwent senescence but failed to express SA-,-gal. GLB1 mRNA depletion also prevented expression of SA-,-gal activity in HeLa cervical carcinoma cells induced to enter a senescent state by repression of their endogenous human papillomavirus E7 oncogene. SA-,-gal induction during senescence was due at least in part to increased expression of the lysosomal ,-galactosidase protein. These results also indicate that SA-,-gal is not required for senescence. [source]

Application of in situ detection techniques to determine the systemic condition of lymphocystis disease virus infection in cultured gilt-head seabream, Sparus aurata L.

JOURNAL OF FISH DISEASES, Issue 2 2009
I Cano
Abstract Immunohistochemistry (IHC) and in situ hybridization (ISH) techniques have been used for the detection of lymphocystis disease virus (LCDV) in formalin-fixed, paraffin-embedded tissues from gilt-head seabream, Sparus aurata L. Diseased and recovered fish from the same population were analysed. IHC was performed with a polyclonal antibody against a 60-kDa viral protein. A specific digoxigenin-labelled probe, obtained by PCR amplification of a 270-bp fragment of the gene coding the LCDV major capsid protein, was used for ISH. LCDV was detected in skin dermis and gill lamellae, as well as in several internal organs such as the intestine, liver, spleen and kidney using both techniques. Fibroblasts, hepatocytes and macrophages seem to be target cells for virus replication. The presence of lymphocystis cells in the dermis of the skin and caudal fin, and necrotic changes in the epithelium of proximal renal tubules were the only histological alterations observed in fish showing signs of the disease. [source]

Interface membrane fibroblasts around aseptically loosened endoprostheses express MMP-13

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2008
Susanne Wagner
Abstract The objective of this article was to assess whether matrix metalloproteinase-13 (MMP-13) is produced by cells of the peri-implant interface tissues and to further characterize these cells. Tissue specimens were collected from the bone,prosthesis interface at the time of revision surgery of clinically loosened hip and knee arthroplasties (n,=,27). Synovial tissues from osteoarthritic patients and young patients with mild joint deformity were used as controls (n,=,6). Tissue samples were fixed in 4% PFA, decalcified with EDTA, and embedded in paraffin. Sections (4 µm) were stained with hematoxylin/eosin and for the osteoclastic marker enzyme tartrate resistant acid phosphatase. Monocytes/macrophages were characterized with a monoclonal antibody against CD68 and mRNAs encoding MMP-13 and ,1 collagen I (COL1A1) were detected by in situ hybridization. Cells expressing transcripts encoding MMP-13 were found in 70% of the interface tissues. These cells colocalized with a cell population expressing COL1A1 mRNA, and were fibroblastic in appearance. MMP-13 expressing cells were found in the close vicinity of osteoclasts and multinuclear giant cells. No signals for transcripts encoding MMP-13 were detected in multinuclear giant cells or in osteoclasts. Control tissues were negative for transcripts encoding MMP-13 mRNA. Fibroblasts of the interface from aseptically loosened endoprostheses selectively express MMP-13. By the expression and the release of MMP-13, these fibroblastic cells may contribute to the local degradation of the extracellular matrix and to bone resorption. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:143,152, 2008 [source]

Arthroplasty membrane-derived fibroblasts directly induce osteoclast formation and osteolysis in aseptic loosening

JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 3 2005
A. Sabokbar
Abstract Purpose: Both macrophages and fibroblasts are the main cell types found in periprosthetic tissues surrounding failed joint arthroplasties. These fibroblasts are known to express RANKL and to produce TNF,, factors which promote osteoclast formation and bone resorption. In this study we have analysed the role that arthroplasty membrane-derived fibroblasts (AFb) play in inducing the generation of bone resorbing osteoclasts. Methods: Fibroblasts were isolated from periprosthetic tissues and co-cultured with human monocytes in an osteoclast differentiation assay in the presence or absence of M-CSF and inhibitors of RANKL (OPG) and/or TNF,. RANKL expression by AFbs was determined by RT-PCR and the extent of osteoclast differentiation by the expression of TRAP, VNR and evidence of lacunar resorption. Results: In the presence of M-CSF, large numbers of TRAP+ and VNR+ multinucleated cells capable of lacunar resorption, were noted in co-cultures of monocytes and RANKL-expressing AFbs. Cell-cell contact was required for osteoclast formation. The addition of OPG and anti-TNF, alone significantly reduced but did not abolish the extent of osteoclast formation, whereas the addition of both together abolished osteoclast formation and lacunar resorption. Conclusion: Our results indicate that fibroblasts in periprosthetic tissues are capable of inducing the differentiation of normal human peripheral blood mononuclear cells to mature osteoclasts by a mechanism that involves both RANKL and TNF,. Suppression of both RANKL and inflammatory cytokines is likely to be required to control periprosthetic osteolysis. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source]

Gingival fibroblasts grown from cyclosporin-treated patients show a reduced production of matrix metalloproteinase-1 (MMP-1) compared with normal gingival fibroblasts, and cyclosporin down-regulates the production of MMP-1 stimulated by pro-inflammatory cytokines

JOURNAL OF PERIODONTAL RESEARCH, Issue 6 2007
T. Z. Sukkar
Background and Objective:, Cyclosporin-induced gingival overgrowth arises from an alteration in collagen homeostasis and is enhanced by inflammatory changes in the gingival tissues. The aim of this study was to investigate the interaction among interleukin-1, oncostatin M, cyclosporin and nifedipine in promoting the up-regulation of matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinase by gingival fibroblasts. Material and Methods:, Fibroblast cultures (n = 5) were obtained from healthy controls and from patients with cyclosporin-induced gingival overgrowth, and cells were harvested between the fourth and ninth passages. Cells were stimulated with interleukin-1 and oncostatin M, alone or in combination, and with different concentrations of cyclosporin (0,2000 ng/mL) and nifedipine (0,200 ng/mL). MMP-1 and tissue inhibitor of metalloproteinase-1 production was determined using an enzyme-linked immunosorbent assay technique. A CyQuant cell proliferation assay was used to determine the DNA concentration in the sample. Results:, Fibroblasts obtained from patients with cyclosporin-induced gingival overgrowth produced significantly lower levels of MMP-1 than control fibroblasts (p < 0.001); tissue inhibitor of metalloproteinase-1 levels were significantly lower (p < 0.05), and the ratio of MMP-1 to tissue inhibitor of metalloproteinase-1 was reduced, in the conditioned medium of patients with cyclosporin-induced gingival overgrowth compared with controls. Interleukin-1 and oncostatin M produced a significant increase in the up-regulation of MMP-1, which was reversed when cyclosporin and nifedipine were added to the cell cultures (p < 0.05). Conclusion:, Pro-inflammatory cytokines significantly up-regulate MMP-1 in cultured gingival fibroblasts. Up-regulation is attenuated by both cyclosporin and nifedipine. The interaction may account for the synergism between inflammation and cyclosporin-induced gingival overgrowth. [source]

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

JOURNAL OF PERIODONTAL RESEARCH, Issue 4 2000
H. K. Kendall
Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOSII) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-,) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-, and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-, alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis. [source]

Electrospun Composite Mats of Poly[(D,L -lactide) -co- glycolide] and Collagen with High Porosity as Potential Scaffolds for Skin Tissue Engineering

MACROMOLECULAR MATERIALS & ENGINEERING, Issue 9 2009
Ye Yang
Abstract Electrospun composite mats of poly[(D,L -lactide) -co- glycolide] and collagen with high porosities of 85,90% and extended pore sizes of 90,130,µm were prepared to mimic the ECM morphologically and chemically. The existence of collagen molecules on the fiber surface was confirmed, enabling the cells to find enhanced binding sites for their integrin receptors. The mechanical data for the blended fibrous mats indicated that they were sufficiently durable for dermal tissue engineering. Fibroblasts derived from GFP transgenic C57BL/6 mice were used to directly observe cell proliferation, and the inoculation of collagen enhanced cell attachment, proliferation and extracellular matrix secretion, which were found to be dependent on the amount of collagen in the composite scaffold. [source]

Ultrastructural changes in feline dental pulp with periodontal disease

MICROSCOPY RESEARCH AND TECHNIQUE, Issue 5 2003
Jamileh Ghoddusi
Abstract A light and transmission electron microscopic study was conducted on dental pulp on cats suffering periodontal disease. After extraction, pulp tissues were fixed and embedded in Epon-Araldite. Thick layers of predentin (50 ,m) and odontoblasts (30 ,m) were observed. In thin sections, odontoblasts showed many mitochondria and secretary vesicles. Some capillaries with several fenestrations were located within the odontoblastic layer. All the sections of pulp examined displayed a generalized infiltration of chronic inflammatory cells. Fibroblasts displayed lytic changes in some areas. These findings imply that the pulp is significantly affected by periodontal disease and furcation-involved teeth should be a carefully considered factor when dental treatment is planned. Microsc. Res. Tech. 61:423,427, 2003. © 2003 Wiley-Liss, Inc. [source]

Involvement of Reactive Oxygen Species in TGF-,1-induced Tropoelastin Expression by Human Dermal Fibroblasts

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 6 2009
Won Seon Choi
Chronic exposure to solar UV radiation causes marked changes in the dermal extracellular matrix that underlie the loss of resiliency and increased laxity observed in photoaged skin. In particular, the dermal elastin content increases substantially and the normal, well-organized elastic fibers are replaced by amorphous elastotic material. Transforming growth factor-,1 (TGF-,1) stimulates synthesis of elastin by dermal fibroblasts and may mediate the increase in elastin in chronically photodamaged skin. We investigated pathways involved in the TGF,,1-induced increase in tropoelastin (TE), the soluble elastin monomer and assessed the role of reactive oxygen species (ROS) in the regulation of TE mRNA. Antioxidants and an inhibitor of NADPH oxidase blocked TGF,,1-induced TE mRNA increase even when added 1.5 h after TGF-,1, although ROS were detected for only 30 min. The TE mRNA increase required activation of Smad4, shown using Smad4 siRNA, and also involved the ERK1/2, p38 and JNK MAP kinases but not PI3K. ROS did not enhance signaling through Smad2 but did enhance activation of p38 and ERK1/2 at 10 min after TGF-,1. These results indicate that Smad and MAPK pathways mediate TGF,,1-induced TE expression and that ROS are required for both early signal transduction and later steps that increase elastin. [source]

Mitochondrial Responses of Normal and Injured Human Skin Fibroblasts Following Low Level Laser Irradiation,An In Vitro Study

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 4 2009
Innocent L. Zungu
Laser irradiation has proved to be very efficient in speeding and improving the quality of healing in pathological conditions of diverse etiologies. However, the mechanisms by which the beneficial effects are attained are not clear. Mitochondria are the primary phototargets during irradiation. The study aimed to establish if laser irradiation had an effect on hypoxic and acidotic cells. The study also aimed to use existing information regarding the possible mechanism of action (established in wounded cells) and apply these principles to acidic and hypoxic irradiated cells to determine whether laser has a stimulatory or inhibitory effect. Cell cultures were modified to simulate conditions of hypoxia (hypoxic gas mixture 95% N2 and 5% O2) and acidosis (pH 6.7) whereas the central scratch model was used to simulate a wound. Cells were irradiated with a helium,neon (632.8 nm, 3 mW cm,2) laser using 5 or 16 J cm,2 on days 1 and 4. Mitochondrial responses were measured 1 or 24 h after laser irradiation by assessing changes in mitochondrial membrane potential (MMP), cyclic AMP, intracellular Ca2+ and adenosine triphosphate (ATP) cell viability. Hypoxia and acidosis significantly reduced MMP when compared with normal nonirradiated control cells. Wounded, hypoxic and acidotic cells irradiated with 5 J cm,2 showed an increase in mitochondrial responses when compared with nonirradiated cells while 16 J cm,2 showed a significant decrease. The study confirmed that laser irradiation with 5 J cm,2 stimulated an increase in intracellular Ca2+ which resulted in an increase in MMP, ATP and cAMP, which ultimately results in photobiomodulation to restore homeostasis of injured cells. [source]

Zeolite Encapsulation Decreases TiO2 -photosensitized ROS Generation in Cultured Human Skin Fibroblasts,

PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2006
Biao Shen
ABSTRACT Sunscreens protect skin against sunburn. However, studies have demonstrated that UV-irradiated sunscreen components such as titanium dioxide (TiO2) promote the photogeneration of reactive oxygen species (ROS). Because encapsulation of TiO2 within zeolites alters its photocatalytic activity, supra-molecular composites based on NaY zeolite hosts containing TiO2 guests were prepared, and the effects on ROS formation in cells under UVA-irradiation evaluated. DCFH-DA (2,,7,-dichlorofluorescein diacetate) was used as a profluorescent probe to monitor intracellular ROS. The detection of in-tracellular 2,,7,-dichlorofluorescein (DCF) fluorescence by confocal microscopy revealed that DCFH-DA was taken up, hydrolyzed and oxidized by yeast cells and cultured human skin fibroblasts within 20 and 6 min, respectively. Higher DCF fluorescence was observed in fibroblasts following UVA irra-diation in the absence but not in the presence of the radical nitroxide, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpipery-dine-1-oxyl), which exhibits superoxide dismutase-mimetic and catalase-mimetic activity. UVA-induced fluorescence increased by -50% in the presence of 32-nm anatase TiO2 particles and decreased by essentially an equal amount in the presence of TiO2 encapsulated within NaY zeolites (TiO2@NaY). Addition of the uncomplexed NaY host also decreased (by ,30%) the amount of UVA-induced fluorescence but, un-expectedly, the combination of the free guest and host (TiO2@NaY) caused a doubling of the fluorescence. Protection of cells against TiO2 -induced intracellular ROS by encapsulation suggests that supramolecular species may be beneficial in photoprotection of the skin. In contrast, the potentiation of TiO2 -induced ROS by uncomplexed NaY points to a critical role for formulation when free TiO2 is used as a sun screen ingredient. [source]