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Quantitative Reverse Transcriptase (quantitative + reverse_transcriptase)
Terms modified by Quantitative Reverse Transcriptase Selected AbstractsNovel markers of inflammation identified in tumor necrosis factor receptor,associated periodic syndrome (TRAPS) by transcriptomic analysis of effects of TRAPS-associated tumor necrosis factor receptor type I mutations in an endothelial cell lineARTHRITIS & RHEUMATISM, Issue 1 2009Susana L. Rebelo Objective To analyze the effects of tumor necrosis factor receptor,associated periodic syndrome (TRAPS),associated mutant tumor necrosis factor receptor type I (TNFRI) expression in a cell type directly relevant to the inflammation in TRAPS, and to identify novel markers associated with mutant TNFRI expression. Methods Transcriptome analysis on 30,000 human genes was performed on SK-Hep-1 human endothelial cells transfected with either wild-type (WT) or TRAPS-associated mutant TNFRI. Quantitative reverse transcriptase,polymerase chain reaction and protein expression levels measured by enzyme-linked immunosorbent assay verified transcriptional changes for selected genes both in supernatants from cells expressing mutant TNFRI and in patient plasma. Results Cells expressing mutant TNFRI showed up-regulation of multiple proinflammatory genes relative to WT transfectants, including genes for pentraxin 3, granulocyte,macrophage colony-stimulating factor, granulocyte colony-stimulating factor, CCL2, and CCL5, which were also expressed as proteins. In addition, the expression of most of these markers was increased in the plasma and peripheral blood mononuclear cells from TRAPS patients relative to those from healthy controls. The cysteine mutations (C33Y and C52F), which are associated with a more severe clinical phenotype, induced more genes than the low-penetrance mutation R92Q, which is associated with a milder phenotype. The expression of most genes was induced by a death domain (DD),dependent mechanism, since they were not induced by expression of TNFRI mutants with an inactivated DD. Conclusion TRAPS-associated TNFRI mutants induce the expression of multiple genes encoding inflammatory molecules, cellular receptors, transcription factors, and regulators of apoptosis in endothelial cells that require the cytoplasmic signaling properties of the receptor. Different mutants have specific expression profiles, indicating mutation-specific effects. The expression of some of these markers was also elevated in samples from TRAPS patients. [source] Transcriptional response to aging and caloric restriction in heart and adipose tissueAGING CELL, Issue 5 2007Nancy J. Linford Summary Sustained caloric restriction (CR) extends lifespan in animal models but the mechanism and primary tissue target(s) have not been identified. Gene expression changes with aging and CR were examined in both heart and white adipose tissue (WAT) of Fischer 344 (F344) male rats using Affymetrix® RAE 230 arrays and validated by quantitative reverse transcriptase,polymerase chain reaction (qRT-PCR) on 18 genes. As expected, age had a substantial effect on transcription on both tissues, although only 21% of cardiac age-associated genes were also altered in WAT. Gene set enrichment analysis revealed coordinated small magnitude changes in ribosomal, proteasomal, and mitochondrial genes with similarities in aging between heart and WAT. CR had very different effects on these two tissues at the transcriptional level. In heart, very few age-associated expression changes were affected by CR, while in WAT, CR suppressed a substantial subset of the age-associated changes. Genes unaltered by aging but altered by CR were identified in WAT but not heart. Most interestingly, we identified a gene expression signature associated with mammalian target of rapamycin (mTOR) activity that was down-regulated with age but preserved by CR in both WAT and heart. In addition, lipid metabolism genes, particularly those associated with peroxisome proliferator-activated receptor , (PPAR,)-mediated adipogenesis were reduced with age but preserved with CR in WAT. These results highlight tissue-specific differences in the gene expression response to CR and support a role for CR-mediated preservation of mTOR activity and adipogenesis in aging WAT. [source] Innate and adaptive immune activation in the brain of MPS IIIB mouse modelJOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2009Julianne DiRosario Abstract Mucopolysaccharidosis (MPS) IIIB is a lysosomal storage disease with severe neurological manifestations due to ,- N -acetylglucosaminidase (NaGlu) deficiency. The mechanism of neuropathology in MPS IIIB is unclear. This study investigates the role of immune responses in neurological disease of MPS IIIB in mice. By means of gene expression microarrays and real-time quantitative reverse transcriptase,polymerase chain reaction, we demonstrated significant up-regulation of numerous immune-related genes in MPS IIIB mouse brain involving a broad range of immune cells and molecules, including T cells, B cells, microglia/macrophages, complement, major histocompatibility complex class I, immunoglobulin, Toll-like receptors, and molecules essential for antigen presentation. The significantly enlarged spleen and lymph nodes in MPS IIIB mice were due to an increase in splenocytes/lymphocytes, and functional assays indicated that the T cells were activated. An autoimmune component to the disease was further suggested by the presence of putative autoantigen or autoantigens in brain extracts that reacted specifically with serum IgG from MPS IIIB mice. We also demonstrated for the first time that immunosuppression with prednisolone alone can significantly slow the central nervous system disease progression. Our data indicate that immune responses contribute greatly to the neuropathology of MPS IIIB and should be considered as an adjunct treatment in future therapeutic developments for optimal therapeutic effect. © 2008 Wiley-Liss, Inc. [source] Prostaglandin D2 pathway and peroxisome proliferator-activated receptor ,-1 expression are induced by mechanical loading in an osteoblastic cell lineJOURNAL OF PERIODONTAL RESEARCH, Issue 2 2006Chitpol Siddhivarn Objective:, The hypothesis underlying the current study was that the arachidonic acid cascade, specifically activation of the prostaglandin (PG) D2 pathway in osteoblasts, is an anabolic signal induced by mechanical loading. Background:, Previous studies have shown that mechanical loading of osteoblasts triggers cyclooxygenase (COX)-2, PGE2 and prostacyclin (PGI2) synthesis. Since modest mechanical loading of osteoblasts promotes bone formation, we sought to determine whether mechanical stress activates the osteoblastic PGD2 pathway resulting in the synthesis of osteogenic cyclopentenones, including ,12PGJ2. Methods:, Osteoblast monolayers were stretched using a Bioflex apparatus at a frequency of 1 Hz with 1% elongation. Cells and cell media were collected at various time points: 5, 10, 15, 30 min; and 1, 4, 16, 24 h. RNA was extracted for quantitative reverse transcriptase,polymerase chain reaction (RT,PCR). In certain experiments, cells were pre-labeled with 14C arachidonic acid prior to stretching. Radiolabeled metabolites in cell media were identified by reverse-phase high performance liquid chromatography (RP-HPLC). Osteoblasts were evaluated for an induction in bone nodule formation by stretching. Results:, Mechanical strain significantly increased mRNA expression of COX-1, COX-2, PGD2 synthase and peroxisome proliferator-activated receptor (PPAR) ,-1, but not of PPAR,-2 as compared to control unstretched cells (p < 0.05). Mechanical loading stimulated the release of PGE2, PGD2 and the PGD2 metabolite ,12PGJ2. Mechanical strain resulted in the induction of bone nodules. Conclusions:, This report indicates that mechanical loading of osteoblasts results in activation of PGD2 and the concomitant expression of transcription factor PPAR,-1 mRNA. The coordinated synthesis of ,12PGJ2, a natural ligand for PPAR,-1, with the increased expression of PPAR,-1, suggests that biomechanical transduction pathways that initially involve the activation of cyclooxygenases may also involve the activation of the ,12PGJ2,PPAR pathway. [source] PRIMARY CARBON AND NITROGEN METABOLIC GENE EXPRESSION IN THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE): DIEL PERIODICITY AND EFFECTS OF INORGANIC CARBON AND NITROGEN,JOURNAL OF PHYCOLOGY, Issue 5 2009Espen Granum Diel periodicity and effects of inorganic carbon (Ci) and NO3, on the expression of 11 key genes for primary carbon and nitrogen metabolism, including potential C4 photosynthesis, in the marine diatom Thalassiosira pseudonana Hasle et Heimdal were investigated. Target gene transcripts were measured by quantitative reverse transcriptase,PCR, and some of the gene-encoded proteins were analyzed by Western blotting. The diatom was grown with a 12 h photoperiod at two different Ci concentrations maintained by air-equilibration with either 380 ,L · L,1 (near-ambient) or 100 ,L · L,1 (low) CO2. Transcripts of the principal Ci and NO3, assimilatory genes RUBISCO LSU (rbcL) and nitrate reductase displayed very strong diel oscillations with peaks at the end of the scotophase. Considerable diel periodicities were also exhibited by the ,-carboxylase genes phosphoenolpyruvate carboxylase (PEPC1 and PEPC2) and phosphoenolpyruvate carboxykinase (PEPCK), and the Benson,Calvin cycle gene sedoheptulose,bisphosphatase (SBPase), with peaks during mid- to late scotophase. In accordance with the transcripts, there were substantial diel periodicities in PEPC1, PEPC2, PEPCK, and especially rbcL proteins, although they peaked during early to mid-photophase. Inorganic carbon had some transient effects on the ,-carboxylase transcripts, and glycine decarboxylase P subunit was highly up-regulated by low Ci concentration, indicating increased capacity for photorespiration. Nitrogen-starved cells had reduced amounts of carbon metabolic gene transcripts, but the PEPC1, PEPC2, PEPCK, and rbcL transcripts increased rapidly when NO3, was replenished. The results suggest that the ,-carboxylases in T. pseudonana play key anaplerotic roles but show no clear support for C4 photosynthesis. [source] Effect of Chronic Ethanol Ingestion and Gender on Heart Left Ventricular p53 Gene ExpressionALCOHOLISM, Issue 8 2005Heidi Jänkälä Background: Although the beneficial effects of mild to moderate ethanol consumption have been implied with respect to heart, alcohol abuse has proven to be a major cause of nonischemic cardiomyopathy in Western society. However, the biochemical and molecular mechanisms, which mediate the pathologic cardiac effects of ethanol, remain largely unknown. The aim of the present study was to explore the effects of chronic ethanol exposure on cardiac apoptosis and expression of some of the genes associated with cardiac remodeling in vivo. Methods: Alcohol-avoiding Alko Non Alcohol rats of both sexes were used. The ethanol-exposed rats (females, n= 6; males, n= 8) were given 12% (v/v) ethanol as the only available fluid from age of three to 24 months of age. The control rats (females, n= 7; males, n= 5) had only water available. At the end of the experiment, free walls of left ventricles of hearts were immediately frozen. Cytosolic DNA fragmentation, reflecting apoptosis, was measured using a commercial quantitative sandwich enzyme-linked immunosorbent assay kit, and mRNA levels were analyzed using a quantitative reverse transcriptase,polymerase chain reaction method. Results: Ethanol treatment for two years increased cardiac left ventricular p53 mRNA levels significantly (p= 0.014) compared with control rats. The gene expression was also dependent on the gender (p= 0.001), so that male rats had higher left ventricular p53 mRNA levels than female rats. However, no significant differences in levels of DNA fragmentation were detected. Conclusions: Chronic ethanol exposure in vivo induces rat cardiac left ventricular p53 gene expression. Expression of p53 is also gender-dependent, males having higher p53 mRNA levels than females. This preliminary finding suggests a role for the p53 gene in ethanol-induced cardiac remodeling. The results might also have some relevance for the known gender-dependent differences in propensity to cardiovascular disease. [source] Ethanol and red wine polyphenols induce the short-term downregulation of PAI-1 gene expression in vivo in rat aortic endotheliumJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 9 2007Hernan E Grenett Abstract Moderate alcohol or red wine consumption reduces the risk of cardiovascular mortality. This cardiovascular protection is likely due to the additive, combined and/or synergistic effects of alcohol itself or other components of wine, in particular polyphenols. Experiments were carried out to determine whether ethanol/polyphenols also decrease plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in vivo, using the rat as an animal model. Male Sprague,Dawley rats were gavaged with ethanol, the individual polyphenols catechin and quercetin or saline vehicle. The in vivo effect of ethanol or individual polyphenols on PAI-1 mRNA was then assessed by in situ hybridisation and quantitative reverse transcriptase (RT) polymerase chain reaction (RT-PCR). PAI-1 mRNA expression was significantly reduced in the endothelial and smooth muscle cells of the thoracic aorta of all experimental rats. RT-PCR analysis of PAI-1 mRNA levels in vascular tissue showed a ,55% reduction in PAI-1 mRNA consistent with the decrease in aortic endothelium PAI-1 mRNA observed with in situ hybridisation. This decrease may enhance endothelial cell (EC)-mediated fibrinolytic activity in vivo. The cardioprotection afforded by moderate red wine consumption can therefore be attributed in part to the combined effects of ethanol and individual polyphenols on EC fibrinolysis. Copyright © 2007 Society of Chemical Industry [source] Phylogenetic analysis, genomic organization, and expression analysis of multi-copper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolorNEW PHYTOLOGIST, Issue 3 2009P. E. Courty Summary ,,In forest soils, ectomycorrhizal and saprotrophic Agaricales differ in their strategies for carbon acquisition, but share common gene families encoding multi-copper oxidases (MCOs). These enzymes are involved in the oxidation of a variety of soil organic compounds. ,,The MCO gene family of the ectomycorrhizal fungus Laccaria bicolor is composed of 11 genes divided into two distinct subfamilies corresponding to laccases (lcc) sensu stricto (lcc1 to lcc9), sharing a high sequence homology with the coprophilic Coprinopsis cinerea laccase genes, and to ferroxidases (lcc10 and lcc11) that are not present in C. cinerea. The fet3 -like ferroxidase genes lcc10 and lcc11 in L. bicolor are each arranged in a mirrored tandem orientation with an ftr gene coding for an iron permease. Unlike C. cinerea, L. bicolor has no sid1/sidA gene for siderophore biosynthesis. ,,Transcript profiling using whole-genome expression arrays and quantitative reverse transcriptase,polymerase chain reaction (qRT-PCR) revealed that some transcripts were very abundant in ectomycorrhizas (lcc3 and lcc8), in fruiting bodies (lcc7) or in the free-living mycelium grown on agar medium (lcc9 and lcc10), suggesting a specific function of these MCOs. ,,The amino acid composition of the MCO substrate binding sites suggests that L. bicolor MCOs interact with substrates different from those of saprotrophic fungi. [source] Function of plastidial pyruvate kinases in seeds of Arabidopsis thaliana,THE PLANT JOURNAL, Issue 3 2007Sébastien Baud Summary Pyruvate kinase (PK) catalyses the irreversible synthesis of pyruvate and ATP, which are both used in multiple biochemical pathways. These compounds are essential for sustained fatty acid production in the plastids of maturing Arabidopsis embryos. Using a real-time quantitative reverse transcriptase (RT)-PCR approach, the three genes encoding putative plastidial PKs (PKps) in Arabidopsis, namely PKp1 (At3g22960), PKp2 (At5g52920) and PKp3 (At1g32440), were shown to be ubiquitously expressed. However, only PKp1 and PKp2 exhibited significant expression in maturing seeds. The activity of PKp1 and PKp2 promoters was consistent with this pattern, and the study of the PKp1:GFP and PKp2:GFP fusion proteins confirmed the plastidial localization of these enzymes. To further investigate the function of these two PKp isoforms in seeds comprehensive functional analyses were carried out, including the cytological, biochemical and molecular characterization of two pkp1 and two pkp2 alleles, together with a pkp1pkp2 double mutant. The results obtained outlined the importance of these PKps for fatty acid synthesis and embryo development. Mutant seeds were depleted of oil, their fatty acid content was drastically modified, embryo elongation was retarded and, finally, seed germination was also affected. Together, these results provide interesting insights concerning the carbon fluxes leading to oil synthesis in maturing Arabidopsis seeds. The regulation of this metabolic network by the WRINKLED1 transcription factor is discussed, and emphasizes the role of plastidial metabolism and the importance of its tight regulation. [source] Deficiency of CXCR2, but not other chemokine receptors, attenuates autoantibody-mediated arthritis in a murine modelARTHRITIS & RHEUMATISM, Issue 7 2010Jonathan P. Jacobs Objective Chemokines coordinate leukocyte trafficking in homeostasis and during immune responses. Prior studies of their role in arthritis have used animal models with both an initial adaptive immune response and an inflammatory effector phase. We undertook analysis of chemokines and their receptors in the effector phase of arthritis using the K/BxN mouse serum,transfer model. Methods A time-course microarray analysis of serum-transferred arthritis was performed, examining ankle tissue, synovial fluid, and peripheral blood leukocytes. Up-regulation of chemokines was confirmed by quantitative reverse transcriptase,polymerase chain reaction. The functional relevance of chemokine induction was assessed by transferring serum into mice deficient in CCR1,7, CCR9, CXCR2, CXCR3, CXCR5, CX3CR1, CCL2, or CCL3. Further mechanistic analysis of CXCR2 involved treatment of arthritic mice with a CXCR2 antagonist, bone marrow (BM) cell transfers with CXCR2+/, and CXCR2,/, donors and recipients, flow cytometry of synovial cells, and competition experiments measuring enrichment of CXCR2-expressing neutrophils in arthritic joints of mice with mixed CXCR2+/+ and CXCR2,/, BM cells. Results Gene expression profiling revealed up-regulation of the CXCR2 ligands CXCL1, CXCL2, and CXCL5 in the joint in parallel with disease activity. CXCR2,/, mice had attenuated disease relative to CXCR2+/, littermates, as did mice receiving the CXCR2 inhibitor, while deficiency of other chemokine receptors did not affect arthritis severity. CXCR2 was required only on hematopoietic cells and was widely expressed on synovial neutrophils. CXCR2-expressing neutrophils were preferentially recruited to arthritic joints in the presence of CXCR2-deficient neutrophils. Conclusion CXCR2 (but not other chemokine receptors) is critical for the development of autoantibody-mediated arthritis, exhibiting a cell-autonomous role in neutrophil recruitment to inflamed joints. [source] Control of Dkk-1 ameliorates chondrocyte apoptosis, cartilage destruction, and subchondral bone deterioration in osteoarthritic kneesARTHRITIS & RHEUMATISM, Issue 5 2010Lin-Hsiu Weng Objective Perturbation of Wnt signaling components reportedly regulates chondrocyte fate and joint disorders. The Wnt inhibitor Dkk-1 mediates remodeling of various tissue types. We undertook this study to examine whether control of Dkk-1 expression prevents joint deterioration in osteoarthritic (OA) knees. Methods Anterior cruciate ligament transection,and collagenase-induced OA in rat knees was treated with end-capped phosphorothioate Dkk-1 antisense oligonucleotide (Dkk-1,AS). Articular cartilage destruction, cartilage degradation markers, bone mineral density (BMD), and subchondral trabecular bone volume of injured knee joints were measured using Mankin scoring, enzyme-linked immunosorbent assay, dual x-ray absorptiometry, and histomorphometry. Dkk-1,responsive molecule expression and apoptotic cells in knee tissue were detected by quantitative reverse transcriptase,polymerase chain reaction, immunoblotting, and TUNEL staining. Results Up-regulated Dkk-1 expression was associated with increased Mankin score and with increased serum levels of cartilage oligomeric matrix protein and C-telopeptide of type II collagen (CTX-II) during OA development. Dkk-1,AS treatment alleviated OA-associated increases in Dkk-1 expression, Mankin score, cartilage fibrillation, and serum cartilage degradation markers. Dkk-1,AS also alleviated epiphyseal BMD loss and subchondral bone exposure associated with altered serum levels of osteocalcin and CTX-I. The treatment abrogated chondrocyte/osteoblast apoptosis and subchondral trabecular bone remodeling in OA. Dkk-1 knockdown increased levels of nuclear ,-catenin and phosphorylated Ser473 -Akt but attenuated expression of inflammatory factors (Toll-like receptor 4 [TLR-4], TLR-9, interleukin-1,, and tumor necrosis factor ,), the apoptosis regulator Bax, matrix metalloproteinase 3, and RANKL in OA knee joints. Conclusion Interference with the cartilage- and bone-deleterious actions of Dkk-1 provides therapeutic potential for alleviating cartilage destruction and subchondral bone damage in OA knee joints. [source] Adeno-associated virus type 5,mediated intraarticular administration of tumor necrosis factor small interfering RNA improves collagen-induced arthritisARTHRITIS & RHEUMATISM, Issue 3 2010Maroun Khoury Objective RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis. Methods We developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor , (TNF,) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNF, gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 × 109 particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase,polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay. Results After a single injection of rAAV5-shTNF into inflamed joints, local TNF, gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-, production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNF, at the protein level, either locally or systemically. Conclusion Our data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis. [source] Scavenger receptor class A type I/II determines matrix metalloproteinase,mediated cartilage destruction and chondrocyte death in antigen-induced arthritisARTHRITIS & RHEUMATISM, Issue 10 2009P. L. E. M. van Lent Objective Scavenger receptor class A type I (SR-AI) and SR-AII are expressed by macrophages in particular and bind and internalize a broad range of molecules (including endotoxins, apoptotic bodies, and oxidized low-density lipoprotein). This study was undertaken to investigate the role of SR-AI/II in mediating severe cartilage destruction in antigen-induced arthritis (AIA). Methods AIA was induced in the knee joints of SR-AI/II,/, mice and wild-type (WT) controls. Joint inflammation and cartilage destruction (chondrocyte death) were measured by examining the histology of total knee joints. Matrix metalloproteinase (MMP),mediated neoepitopes were measured by immunolocalization using anti-VDIPEN antibodies and chondrocyte activation with anti-S100A8 antibodies. Messenger RNA (mRNA) levels were determined in inflamed synovium using microarray analysis and quantitative reverse transcriptase,polymerase chain reaction. In synovial washouts, cytokines (interleukin-1, [IL-1,], IL-10, and tumor necrosis factor ,) and S100A8/S100A9 were measured using Luminex and enzyme-linked immunosorbent assay. Results Levels of SR-AI/II mRNA were strongly elevated in inflamed synovium in AIA. On days 2, 8, and 14 after AIA induction, joint inflammation (exudates/infiltrate) was similar between the 2 groups. In WT mice, severe cartilage destruction was found in multiple cartilage surfaces of the inflamed knee joint on day 14 after AIA induction. MMP-mediated matrix destruction ranged between 40% and 60%, and chondrocyte death was prominent in 40,75% of the cartilage surfaces. In striking contrast, in SR-AI/II,/, mice, despite comparable joint inflammation, pronounced cartilage destruction was almost completely absent. Levels of IL-1, and S100A8/S100A9 were significantly lower on days 7 and 14 after AIA induction, but levels of mRNA for various MMPs (MMP-2, MMP-3, MMP-9, and MMP-13) were comparable. Conclusion Our findings indicate that SR-AI and SR-AII are crucial receptors involved in mediating severe cartilage destruction in AIA. [source] Fibroblast growth factor 2 is an intrinsic chondroprotective agent that suppresses ADAMTS-5 and delays cartilage degradation in murine osteoarthritisARTHRITIS & RHEUMATISM, Issue 7 2009Shi-Lu Chia Objective We have previously identified in articular cartilage an abundant pool of the heparin-binding growth factor, fibroblast growth factor 2 (FGF-2), which is bound to the pericellular matrix heparan sulfate proteoglycan, perlecan. This pool of FGF-2 activates chondrocytes upon tissue loading and is released following mechanical injury. In vitro, FGF-2 suppresses interleukin-1,driven aggrecanase activity in human cartilage explants, suggesting a chondroprotective role in vivo. We undertook this study to investigate the in vivo role of FGF-2 in murine cartilage. Methods Basal characteristics of the articular cartilage of Fgf2,/, and Fgf2+/+ mice were determined by histomorphometry, nanoindentation, and quantitative reverse transcriptase,polymerase chain reaction. The articular cartilage was graded histologically in aged mice as well as in mice in which osteoarthritis (OA) had been induced by surgical destabilization of the medial meniscus. RNA was extracted from the joints of Fgf2,/, and Fgf2+/+ mice following surgery and quantitatively assessed for key regulatory molecules. The effect of subcutaneous administration of recombinant FGF-2 on OA progression was assessed in Fgf2,/, mice. Results Fgf2,/, mice were morphologically indistinguishable from wild-type (WT) animals up to age 12 weeks; the cartilage thickness and proteoglycan staining were equivalent, as was the mechanical integrity of the matrix. However, Fgf2,/, mice exhibited accelerated spontaneous and surgically induced OA. Surgically induced OA in Fgf2,/, mice was suppressed to levels in WT mice by subcutaneous administration of recombinant FGF-2. Increased disease in Fgf2,/, mice was associated with increased expression of messenger RNA of Adamts5, the key murine aggrecanase. Conclusion These data identify FGF-2 as a novel endogenous chondroprotective agent in articular cartilage. [source] Modulation of lubricin biosynthesis and tissue surface properties following cartilage mechanical injuryARTHRITIS & RHEUMATISM, Issue 1 2009Aled R. C. Jones Objective To evaluate the effects of injurious compression on the biosynthesis of lubricin at different depths within articular cartilage and to examine alterations in structure and function of the articular surface following mechanical injury. Methods Bovine cartilage explants were subdivided into level 1, with intact articular surface, and level 2, containing middle and deep zone cartilage. Following mechanical injury, lubricin messenger RNA (mRNA) levels were monitored by quantitative reverse transcriptase,polymerase chain reaction, and soluble or cartilage-associated lubricin protein was analyzed by Western blotting and immunohistochemistry. Cartilage morphology was assessed by histologic staining, and tissue functionality was assessed by friction testing. Results Two days after injury, lubricin mRNA expression was up-regulated ,3-fold for level 1 explants and was down-regulated for level 2 explants. Lubricin expression in level 1 cartilage returned to control levels after 6 days in culture. Similarly, lubricin protein synthesis and secretion increased in response to injury for level 1 explants and decreased for level 2 cartilage. Histologic staining revealed changes in the articular surface of level 1 explants following injury, with respect to glycosaminoglycan and collagen content. Injured level 1 explants displayed an increased coefficient of friction relative to controls. Conclusion Our findings indicate that increased lubricin biosynthesis appears to be an early transient response of surface-layer cartilage to injurious compression. However, distinct morphologic changes occur with injury that appear to compromise the frictional properties of the tissue. [source] | |||||||||||||