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Matrix Production (matrix + production)
Selected AbstractsDifferentiation of human mesenchymal stem cells and articular chondrocytes: Analysis of chondrogenic potential and expression pattern of differentiation-related transcription factorsJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 2 2007Camilla Karlsson Abstract Mesenchymal stem cells (MSCs) are a candidate for replacing chondrocytes in cell-based repair of cartilage lesions. However, it has not been clarified if these cells can acquire the hyaline phenotype, and whether chondrocytes and MSCs show the same expression patterns of critical control genes in development. In order to study this, articular chondrocytes and iliac crest derived MSCs were allowed to differentiate in pellet mass cultures. Gene expression of markers for the cartilage phenotype, helix-loop-helix (HLH) transcription factors, and chondrogenic transcription factors were analyzed by real-time PCR. Matrix production was assayed using biochemical analysis for hydroxyproline, glycosaminoglycans, and immunohistochemistry for collagen types I and II. Significantly decreased expression of collagen type I was accompanied by increased expression of collagen types IIA and IIB during differentiation of chondrocytes, indicating differentiation towards a hyaline phenotype. Chondrogenesis in MSCs on the other hand resulted in up-regulation of collagen types I, IIA, IIB, and X, demonstrating differentiation towards cartilage of a mixed phenotype. Expression of HES1 increased significantly during chondrogenesis in chondrocytes while expression in MSCs was maintained at a low level. The HLH gene HES5 on the other hand was only detected in chondrocytes. Expression of ID1 decreased significantly in chondrocytes while the opposite was seen in MSCs. These findings suggest that chondrocytes and MSCs differentiated and formed different subtypes of cartilage, the hyaline and a mixed cartilage phenotype, respectively. Differentially regulated HLH genes indicated the possibility for HLH proteins in regulating chondrogenic differentiation. This information is important to understand the potential use of MSCs in cartilage repair. © 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:152,163, 2007 [source] Buried alive: How osteoblasts become osteocytesDEVELOPMENTAL DYNAMICS, Issue 1 2006Tamara A. Franz-Odendaal Abstract During osteogenesis, osteoblasts lay down osteoid and transform into osteocytes embedded in mineralized bone matrix. Despite the fact that osteocytes are the most abundant cellular component of bone, little is known about the process of osteoblast-to-osteocyte transformation. What is known is that osteoblasts undergo a number of changes during this transformation, yet retain their connections to preosteoblasts and osteocytes. This review explores the osteoblast-to-osteocyte transformation during intramembranous ossification from both morphological and molecular perspectives. We investigate how these data support five schemes that describe how an osteoblast could become entrapped in the bone matrix (in mammals) and suggest one of the five scenarios that best fits as a model. Those osteoblasts on the bone surface that are destined for burial and destined to become osteocytes slow down matrix production compared to neighbouring osteoblasts, which continue to produce bone matrix. That is, cells that continue to produce matrix actively bury cells producing less or no new bone matrix (passive burial). We summarize which morphological and molecular changes could be used as characters (or markers) to follow the transformation process. Developmental Dynamics 235:176,190, 2006. © 2005 Wiley-Liss, Inc. [source] Endothelin receptor selectivity in chronic kidney disease: rationale and review of recent evidenceEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 2009W. Neuhofer Abstract Endothelin (ET) is a potent vasoconstrictory peptide with proinflammatory and profibrotic properties that exerts its biological effects through two pharmacologically distinct receptor subtypes, namely ETA and ETB. In addition to its substantial contribution to normal renal function, a large body of evidence suggests that derangement of the renal ET system is involved in the initiation and progression of chronic kidney disease (CKD) in diabetes, hypertension and glomerulonephritis. Thus, the use of ET receptor antagonists (ERAs) may offer potential novel treatment strategies in CKD. Recent literature on the role of the renal ET system in the healthy kidney was reviewed. In addition, an unbiased PubMed search was performed for studies published during the last 5 years that addressed the effects of ERAs in CKD. A particular objective was to extract information regarding whether selective or nonselective ERAs may have therapeutic potential in humans. ET-1 acts primarily as an autocrine or paracrine factor in the kidney. In normal physiology, ET-1 promotes diuresis and natriuresis by local production and action through ETB receptors in the renal medulla. In pathology, ET-1 mediates vasoconstriction, mesangial-cell proliferation, extracellular matrix production and inflammation, effects that are primarily conveyed by ETA receptors. Results obtained in animal models and in humans with the use of ERAs in CKD are encouraging; nevertheless, it is still under debate which receptor subtype should be targeted. According to most studies, selective inhibition of ETA receptors appears superior compared with nonselective ERAs because this approach does not interfere with the natriuretic, antihypertensive and ET clearance effects of ETB receptors. Although preliminary data in humans are promising, the potential role of ERAs in patients with CKD and the question of which receptor subtype should be targeted can only be clarified in randomized clinical trials. [source] Earliest Mineral and Matrix Changes in Force-Induced Musculoskeletal Disease as Revealed by Raman Microspectroscopic Imaging,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 1 2004Catherine P Tarnowski Abstract Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common human birth defect in the skull. Raman microspectroscopy was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Raman imaging revealed decreased relative mineral content in skulls undergoing craniosynostosis compared with unloaded specimens. Introduction: Raman microspectroscopy, a nondestructive vibrational spectroscopic technique, was used to examine the composition, relative amounts, and locations of the mineral and matrix produced in mouse skulls undergoing force-induced craniosynostosis. Craniosynostosis, premature fusion of the skull bones at the sutures, is the second most common birth defect in the face and skull. The calvaria, or flat bones that comprise the top of the skull, are most often affected, and craniosynostosis is a feature of over 100 human syndromes and conditions. Materials and Methods: Raman images of the suture, the tips immediately adjacent to the suture (osteogenic fronts), and mature parietal bones of loaded and unloaded calvaria were acquired. Images were acquired at 2.6 × 2.6 ,m spatial resolution and ranged in a field of view from 180 × 210 ,m to 180 × 325 ,m. Results and Conclusions: This study found that osteogenic fronts subjected to uniaxial compression had decreased relative mineral content compared with unloaded osteogenic fronts, presumably because of new and incomplete mineral deposition. Increased matrix production in osteogenic fronts undergoing craniosynostosis was observed. Understanding how force affects the composition, relative amounts, and location of the mineral and matrix provides insight into musculoskeletal disease in general and craniosynostosis in particular. This is the first report in which Raman microspectroscopy was used to study musculoskeletal disease. These data show how Raman microspectroscopy can be used to study subtle changes that occur in disease. [source] Src is a major signaling component for CTGF induction by TGF-,1 in osteoblasts,JOURNAL OF CELLULAR PHYSIOLOGY, Issue 3 2010X. Zhang Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor ,1 (TGF-,1) where it acts as a downstream mediator of TGF-,1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF-,1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-,1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-,1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF-,1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-,1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-,1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. J. Cell. Physiol. 224: 691,701, 2010. © 2010 Wiley-Liss, Inc. [source] Constitutive expression of thrombospondin 1 in MC3T3-E1 osteoblastic cells inhibits mineralizationJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2006Akemichi Ueno Thrombospondin 1 (TSP1) is a multifunctional extracellular glycoprotein present mainly in the fetal and adult skeleton. Although an inhibitory effect of TSP1 against pathological mineralization in cultured vascular pericytes has been shown, its involvement in physiological mineralization by osteoblasts is still unknown. To determine the role of TSP1 in biomineralization, mouse osteoblastic MC3T3-E1 cells were cultured in the presence of antisense phosphorothioate oligodeoxynucleotides complementary to the TSP1 sequence. The 18- and 24-mer antisense oligonucleotides caused concentration-dependent increases in the number of mineralized nodules, acid-soluble calcium deposition in the cell/matrix layer, and alkaline phosphatase activity within 9 days, without affecting cell proliferation. The corresponding sense or scrambled oligonucleotides did not affect these parameters. In the antisense oligonucleotide-treated MC3T3-E1 cells, thickened extracellular matrix, well-developed cell processes, increased intracellular organelles, and collagen fibril bundles were observed. On the other hand, the addition of TSP1 to the culture decreased the production of a mineralized matrix by MC3T3-E1 cells. Furthermore, MC3T3-E1 clones overexpressing mouse TSP1 were established and assayed for TSP1 protein and their capacity to mineralize. TSP1 dose-dependently inhibited mineralization by these cells both in vitro and in vivo. These results indicate that TSP1 functions as an inhibitory regulator of bone mineralization and matrix production by osteoblasts to sustain bone homeostasis. J. Cell. Physiol. 209: 322,332, 2006. © 2006 Wiley-Liss, Inc. [source] Effect of cell seeding concentration on the quality of tissue engineered constructs loaded with adult human articular chondrocytesJOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, Issue 1 2008Sebastian Concaro Abstract Many aspects of the process of in vitro differentiation of chondrocytes in three-dimensional (3D) scaffolds need to be further investigated. Chitosan scaffolds were produced by freeze-drying 3% w/v 90% DDA chitosan gels. The effect of the cell seeding concentration was evaluated by culturing human adult chondrocytes in chitosan scaffolds After the first passage, cells were seeded into chitosan scaffolds with a diameter of 8 mm. The final cell seeding concentration per cm3 of chitosan scaffold was: Group A, 3 × 106; Group B, 6 × 106; Group C, 12 × 106; and Group D, 25 × 106 cells. After 14 and 28 days in 3D culture, the constructs were assesed for collagen, glucosaminoglycans and DNA content. The mechanical properties of the constructs were determined using a dynamic oscillatory shear test. The histological aspect of the constructs was evaluated using the Bern score. The collagen and GAG concentration increased, varying the cell seeding concentration. There was a significant increase in proteoglycan and hydroxyproline production between groups C and D. The sulphated GAG content increased significantly in the group D as compared to the other groups. The mechanical properties of the different constructs increased over time, from 9.6 G,/kPa at 14 days of 3D culture to 14.6 G,/kPa at 28 days under the same culture conditions. In this study we were able to determine that concentrations of 12,25 million cells/cm2 are needed to increase the matrix production and mechanical properties of human adult chondrocytes under static conditions. Copyright © 2008 John Wiley & Sons, Ltd. [source] Transforming growth factor-, and Smad signalling in kidney diseasesNEPHROLOGY, Issue 1 2005Review Article SUMMARY: Extensive studies have demonstrated that transforming growth factor-beta (TGF-,) plays an important role in the progression of renal diseases. TGF-, exerts its biological functions mainly through its downstream signalling molecules, Smad2 and Smad3. It is now clear that Smad3 is critical for TGF-,'s pro-fibrotic effect, whereas the functions of Smad2 in fibrosis in response to TGF-, still need to be determined. Our recent studies have demonstrated that Smad signalling is also a critical pathway for renal fibrosis induced by other pro-fibrotic factors, such as angiotensin II and advanced glycation end products (AGE). These pro-fibrotic factors can activate Smads directly and independently of TGF-,. They can also cause renal fibrosis via the ERK/p38 MAP kinase,Smad signalling cross-talk pathway. In contrast, blockade of Smad2/3 activation by overexpression of an inhibitory Smad7 prevents collagen matrix production induced by TGF-,, angiotensin II, high glucose and AGE and attenuates renal fibrosis in various animal models including rat obstructive kidney, remnant kidney and diabetic kidney diseases. Results from these studies indicate that Smad signalling is a key and final common pathway of renal fibrosis. In addition, TGF-, has anti-inflammatory and immune-regulatory properties. Our most recent studies demonstrated that TGF-, transgenic mice are protected against renal inflammation in mouse obstructive and diabetic models. Upregulation of renal Smad7, thereby blocking NF.,B activation via induction of I,B,, is a central mechanism by which TGF-, inhibits renal inflammation. In conclusion, TGF-, signals through Smad2/3 to mediate renal fibrosis, whereas induction of Smad7 inhibits renal fibrosis and inflammation. Thus, targeting Smad signalling by overexpression of Smad7 may have great therapeutic potential for kidney diseases. [source] Spatiotemporal expression of NGFR during pre-natal human tooth developmentORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 2 2002KB Becktor Structured Abstract Authors, Becktor KB, Hansen BF, Nolting D, Kjær I. Objectives, The relation between nerve growth factor receptor (NGFR) in the human pre-natal tooth buds and the dental follicle was investigated. In particular, we sought to determine if there is a specific pattern of p75NGFR expression in developing human tooth buds and their surrounding tissue. Setting and Sample Population, The Department of Orthodontics at Copenhagen University, Denmark. Histological sections from 11 fetuses, aged 11,21 gestational weeks. Method, The sections were studied by conventional immunohistochemistry. Results, Specific spatiotemporal patterns of p75NGFR reactions were observed in the tooth buds and dental follicle: Before matrix production by the ameloblasts, the entire inner enamel epithelium and the entire dental follicle display p75NGFR immunoreactivity; after matrix production is initiated, the immunoreactivity of the matrix producing cells is lost, as is that of the dental follicle adjacent to these matrix-producing cells. Conclusion, A unique spatiotemporal distribution of NGFR in the pre-eruptive human tooth bud was demonstrated. [source] Rho kinase,dependent activation of SOX9 in chondrocytesARTHRITIS & RHEUMATISM, Issue 1 2010Dominik R. Haudenschild Objective The transcription factor SOX9 directly regulates the expression of the major proteoglycans and collagens comprising the cartilage extracellular matrix. The DNA binding activity and cellular localization of SOX9 is controlled through posttranslational modifications, including phosphorylation. The activity of Rho kinase (ROCK) has profound effects on the actin cytoskeleton, and these effects are instrumental in determining the phenotype and differentiation of chondrocytes. However, the mechanisms linking ROCK to altered chondrocyte gene expression remain unknown. The purpose of the present study was to test for a direct interaction between ROCK and SOX9. Methods Human SW1353 chondrosarcoma cells were transfected with constructs coding for RhoA, ROCK, Lim kinase, and SOX9. The interaction between ROCK and SOX9 was tested on purified proteins, and was verified within a cellular context using induced overexpression and activation of the Rho pathway. The effects of SOX9 transcriptional activation were quantified with a luciferase reporter plasmid containing SOX9 binding sites from the COL2A1 enhancer element. Results SOX9 was found to contain a consensus phosphorylation site for ROCK. In vitro, ROCK directly phosphorylated SOX9 at Ser181, and the overexpression of ROCK or the activation of the RhoA pathway in SW1353 chondrosarcoma cells increased SOX9Ser181 phosphorylation. ROCK caused a dose-dependent increase in the transcription of a SOX9-luciferase reporter construct, and increased phosphorylation and nuclear accumulation of SOX9 protein in response to transforming growth factor , treatment and mechanical compression. Conclusion These results demonstrate a new interaction that directly links ROCK to increased cartilage matrix production via activation of SOX9 in response to mechanical and growth factor stimulation. [source] A role for ,/, T cells in a mouse model of fracture healingARTHRITIS & RHEUMATISM, Issue 6 2009Nona T. Colburn Objective Fractures can initiate an immune response that disturbs osteoblastic and osteoclastic cellular homeostasis through cytokine production and release. The aim of our study was to investigate ,/, T cells, innate lymphocytes known to be involved in tissue repair, as potential cellular components of the osteoimmune system's response to an in vivo model of bone injury. The absence of such cells or their effector cytokines influences the fate of other responder cells in proliferation, differentiation, matrix production, and ultimate callus formation. Methods Tibia fractures were created in 60 ,/, T cell,deficient mice (also called , T cell receptor [TCR],knockout mice) and 60 control C57BL/6 mice. Analysis included radiographs, basic histology, mechanical testing, flow cytometry, and immunohistochemical localization of ,/, TCR,positive subsets from control animals and of CD44 expression from both groups, as well as enzyme-linked immunosorbent assay for the effector cytokines interleukin-2 (IL-2), interferon-, (IFN,), and IL-6. Results Animals deficient in ,/, T cells demonstrated more mature histologic elements and quantitative increases in the expression of major bone (bone sialoprotein) and cartilage (type II collagen) matrix proteins and in the expression of bone morphogenetic protein 2 at a critical reparative phase. Moreover, only ,/, T cell,deficient animals had a decrease in the osteoprogenitor antiproliferative cytokines IL-6 and IFN, at the reparative phase. The result was improved stability at the repair site and an overall superior biomechanical strength in ,/, T cell,deficient mice compared with controls. Conclusion The evidence for a role of ,/, T cells in the context of skeletal injury demonstrates the importance of the immune system's effect on bone biology, which is relevant to the field of osteoimmunology, and offers a potential molecular platform from which to develop essential therapeutic strategies. [source] Blocking vascular endothelial growth factor with soluble Flt-1 improves the chondrogenic potential of mouse skeletal muscle,derived stem cellsARTHRITIS & RHEUMATISM, Issue 1 2009Seiji Kubo Objective To investigate the effect of vascular endothelial growth factor (VEGF) stimulation and the effect of blocking VEGF with its antagonist, soluble Flt-1 (sFlt-1), on chondrogenesis, using muscle-derived stem cells (MDSCs) isolated from mouse skeletal muscle. Methods The direct effect of VEGF on the in vitro chondrogenic ability of mouse MDSCs was tested using a pellet culture system, followed by real-time quantitative polymerase chain reaction (PCR) and histologic analyses. Next, the effect of VEGF on chondrogenesis within the synovial joint was tested, using genetically engineered MDSCs implanted into rat osteochondral defects. In this model, MDSCs transduced with a retroviral vector to express bone morphogenetic protein 4 (BMP-4) were coimplanted with MDSCs transduced to express either VEGF or sFlt-1 (a VEGF antagonist) to provide a gain- and loss-of-function experimental design. Histologic scoring was used to compare cartilage formation among the treatment groups. Results Hyaline-like cartilage matrix production was observed in both VEGF-treated and VEGF-blocked (sFlt-1,treated) pellet cultures, but quantitative PCR revealed that sFlt-1 treatment improved the expression of chondrogenic genes in MDSCs that were stimulated to undergo chondrogenic differentiation with BMP-4 and transforming growth factor ,3 (TGF,3). In vivo testing of articular cartilage repair showed that VEGF-transduced MDSCs caused an arthritic change in the knee joint, and sFlt-1 improved the MDSC-mediated repair of articular cartilage, compared with BMP-4 alone. Conclusion Soluble Flt-1 gene therapy improved the BMP-4, and TGF,3-induced chondrogenic gene expression of MDSCs in vitro and improved the persistence of articular cartilage repair by preventing vascularization and bone invasion into the repaired articular cartilage. [source] Adenosine A2A receptors in diffuse dermal fibrosis: Pathogenic role in human dermal fibroblasts and in a murine model of sclerodermaARTHRITIS & RHEUMATISM, Issue 8 2006E. S. L. Chan Objective Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis. Methods Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor,deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. Results Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor,mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor,deficient and A2A receptor antagonist,treated mice were protected from developing bleomycin-induced dermal fibrosis. Conclusion These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma. [source] The Response of Articular Chondrocytes to Type II Collagen,Au NanocompositesARTIFICIAL ORGANS, Issue 12 2007Shan-hui Hsu Abstract:, The nanocomposites (denoted "CII,Au") of porcine type II collagen (CII) with 0.05, 0.1, 0.5, 1, or 2.5% (wt/wt) Au nanoparticles (,5 nm) were fabricated for potential use in cartilage tissue engineering. Au formed clusters on the surface of all nanocomposites and appeared to distribute along the collagen fibrils inside the matrix. The addition of Au at low concentrations (,0.5%) increased the modulus and viscosity, as well as the free radical-scavenging ability. These effects decreased at higher concentrations of Au. The chondrocytes on CII,Au became spindle-like with lamellipodia formation. Cell proliferation on CII,Au 0.1% was promoted. Nitric oxide (NO) in the culture medium was reduced by CII,Au 0.05% and CII,Au 0.1%. Type I collagen, aggrecan, and Sox 9 gene expressions increased with an increased Au content, but slightly decreased at 2.5% Au. There was no significant difference in the CII gene expression. The cellular uptake of Au was observed but less than that which occurred when 10 ppm of Au was added in culture medium. Chondrocytes cultured with ,10 ppm of Au nanoparticles showed neither cytotoxicity nor change in gene expression. Au at an appropriate amount could be well dispersed in CII, and enhanced the material modulus, antioxidant effect, as well as the chondrocyte growth and matrix production. [source] Expression and regulation of connective tissue growth factor by transforming growth factor , and tumour necrosis factor , in fibroblasts isolated from strictures in patients with Crohn's diseaseBRITISH JOURNAL OF SURGERY (NOW INCLUDES EUROPEAN JOURNAL OF SURGERY), Issue 10 2006D. Beddy Background: Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and extracellular matrix production. Fibroblasts may initiate stricture formation in Crohn's disease through overexpression of CTGF. Stricturing that occurs in patients with Crohn's disease after treatment with anti-tumour necrosis factor (TNF) , may be due to dysregulation of CTGF homeostasis. The aim of this study was to examine CTGF expression and regulation in fibroblasts isolated from patients with Crohn's disease. Methods: Fibroblasts were isolated by a primary explant technique from serosal biopsies of strictured segments of bowel in eight patients undergoing resection for Crohn's disease and from normal colon in seven patients having resection for benign or malignant colorectal disease. Cells were stimulated with transforming growth factor (TGF) , and TNF-,. CTGF protein and mRNA expression were measured by western blotting and real-time polymerase chain reaction respectively. Results: Mean(s.d.) CTGF protein expression in strictured Crohn's fibroblasts was higher than that in normal fibroblasts (56·5(9·7) versus 17·0(10·0) respectively; P = 0·011). In normal and strictured Crohn's fibroblasts, culture with TGF-, increased CTGF protein and mRNA expression. Co-culture of normal fibroblasts with TNF-, suppressed TGF-,-stimulated CTGF expression. Conclusion: Increased expression of CTGF in strictured Crohn's fibroblasts underlies its role in fibrosis. TNF-, suppresses fibrosis by downregulating fibroblast CTGF expression, an effect that may be lost following anti-TNF-, treatment, thereby promoting stricture formation. Copyright © 2006 British Journal of Surgery Society Ltd. Published by John Wiley & Sons, Ltd. [source] CP27 affects viability, proliferation, attachment and gene expression in embryonic fibroblastsCELL PROLIFERATION, Issue 4 2002X. Luan CP27 is a gene that has been cloned from an E11 early embryonic library and has been suggested to mediate early organogenesis (Diekwisch et al., 1999, Gene 235, 19). We have hypothesized that CP27 exhibits its effects on organogenesis by affecting individual cell function. Based on the CP27 expression pattern we have selected the CP27 expressing embryonic fibroblast cell line BALB/c 3T3 to determine the effects of CP27 on cell function. CP27 loss of function strategies were performed by adding 5, 12.5 or 25 µg/ml anti-CP27 antibody to cultured BALB/c 3T3 cells and comparing the results to controls in which identical concentrations of rabbit serum were added to the culture medium. Other controls included an antibody against another extracellular matrix protein amelogenin (negative control) and anti-CP27 antibodies directed against other areas of the CP27 molecule (positive control). Following cell culture, cell viability, apoptosis, cell proliferation, cell shape, cellular attachment and fibronectin matrix production were assayed using MTT colourimetric assay, BrdU staining, morphometry, immunostaining and western blot analysis. Block of CP27 function using an antibody strategy resulted in the following significant changes: (i) reduced viability, (ii) increased number of apoptotic cells, (iii) reduced proliferation, (iv) alterations in cell shape, (v) loss of attachment, and (vi) reduction in fibronectin matrix production. There was also a redistribution in fibronectin matrix organization demonstrated by immunohistochemistry. We conclude that CP27 plays an important role in the maintance of normal cell function and that CP27 block leads to significant changes in cellular behaviour. [source] | |||||||||||||