Transcriptomic profiling of chondrocytes and tenocytes in de- and re-differentiation relative to native tissue
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ABSTRACT:
ORGANISM(S): Rattus norvegicus
SUBMITTER: Elizabeth Laird
PROVIDER: E-MTAB-4800 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Autologous chondrocyte transplantation (ACT) is a routine technique to regenerate focal cartilage lesions. However, patients with osteoarthritis (OA) are lacking an appropriate long-lasting treatment alternative, partly since it is not known if chondrocytes from OA patients have the same chondrogenic differentiation potential as chondrocytes from donors not affected by OA. Articular chondrocytes from patients with OA undergoing total knee replacement (Mankin Score >3, Ahlbäck Score >2) and from patients undergoing ACT, here referred to as normal donors (ND), were isolated applying protocols used for ACT. Their chondrogenic differentiation potential was evaluated both in high-density pellet and scaffold (Hyaff-11) cultures by histological proteoglycan assessment (Bern Score) and immunohistochemistry for collagen types I and II. Chondrocytes cultured in monolayer and scaffolds were subjected to gene expression profiling using genome-wide oligonucleotide microarrays. Expression data were verified by using quantitative RT-PCR. Chondrocytes from ND and OA donors demonstrated accumulation of comparable amounts of cartilage matrix components, including sulphated proteoglycans and collagen types I and II. The mRNA expression of cartilage markers (COL2A1, COMP, aggrecan, CRTL1, SOX9) and genes involved in matrix synthesis (biglycan, COL9A2, COL11A1, TIMP4, CILP2) was highly induced in 3D cultures of chondrocytes from both donor groups. Genes associated with hypertrophic or OA cartilage (COL10A1, RUNX2, periostin, ALP, PTHR1, MMP13, COL1A1, COL3A1) were not significantly regulated between the two groups of donors. The expression of 661 genes, including COMP, FN1, and SOX9, were differentially regulated between OA and ND chondrocytes cultured in monolayer. During scaffold culture, the differences diminished between the OA and ND chondrocytes, and only 184 genes were differentially regulated. Only few genes were differentially expressed between OA and ND chondrocytes in Hyaff-11 culture. The risk of differentiation into hypertrophic cartilage does not seem to be increased for OA chondrocytes. Our findings suggest that the chondrogenic capacity is not significantly affected by OA and OA chondrocytes fulfill the requirements for matrix-associated ACT. Experiment Overall Design: Gene expression profiles of monolayer cultures (ML; passage 2) and Hyaff-11 scaffold cultures (3D; 14 days in vitro) of chondrocytes from 3 normal donors (ND; underwent ACT treatment) and 3 donors suffering from Osteoarthritis (OA; underwent knee replacement surgery) were determined. Comparative analyses between 3D and ML cultures (3D vs. ML) were performed to assess differentiation capacity of ND and OA chondrocytes. Furthermore, OA-related differences were determined comparing OA and ND monolayers as well as scaffold cultures (each OA vs. ND).
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Project description:Glucosamine proved to be a potent, broad-spectrum inhibitor of IL-1beta. Of the 2,813 genes whose transcription was altered by IL-1beta stimulation (p<0.0001), glucosamine significantly blocked the response in 2,055 (~73%). Glucosamine fully protected the chondrocytes from IL-1-induced expression of inflammatory cytokines, chemokines and growth factors as well as proteins involved in PGE2 and NO synthesis. It also blocked the IL-1-induced expression of matrix specific proteases such as MMPs -3,-9,-10,-12 and ADAMTS-1. Experiment Overall Design: Articular cartilage was isolated from the femoral heads of male Wistar rats under aseptic conditions. Chondrocytes were obtained by sequential digestion of the cartilage with pronase and type II collagenase. After filtration to remove tissue debris, the cells were cultured in 75-cm2 flasks in complete Dulbeccoâs Modified Eagle Medium (DMEM; supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Experiments were subsequently performed with second-passage cultures whereby the cells from the large cultures were trypsinized, pooled and seeded into twenty 25 cm2 flasks. These were then divided into 4 treatment groups to evaluate the effects of glucosamine and IL-1 on global transcription patterns.To the culture medium in half of the flasks, glucosamine, HCl was added to a final concentration of 20 mM. Six hours later, IL-1beta was added at 10 ng/ml to 5 of the flasks receiving glucosamine and to 5 of the untreated flasks. Fourteen hours post IL-1beta stimulation, total RNA was isolated individually from the respective cultures and microarray experiments processed.
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Project description:Altered Ca2+ handling has both immediate physiological effects and long-term genomic effects on vascular smooth muscle function. Previously we have shown that elevation of cytoplasmic Ca2+ through voltage-dependent Ca2+ channels (VDCCs) or store-operated Ca2+ channels (SOCCs) results in phosphorylation of the Ca2+/cAMP response element binding protein (CREB) in cerebral arteries. Here we demonstrate that stimulation of these different Ca2+ influx pathways results in transcriptional activation of a distinct, yet overlapping set of genes, and that the induction of selected CRE-regulated genes is prevented by the addition of corresponding Ca2+ channel blockers. Using oligonucleotide array analysis, changes in mRNA levels were quantified following membrane depolarization with K+ or depletion of intracellular Ca2+ stores with thapsigargin in human cerebral vascular smooth muscle cells. Array results for differentially regulated genes containing a CRE were confirmed by quantitative RT-PCR, and corresponding changes in protein expression were shown by Western blot analysis and immunofluorescence. Membrane depolarization induced a transient increase in c-fos mRNA and a sustained increase in early growth response-1 (Egr-1) mRNA and protein that were inhibited by application of the VDCC blocker, nimodipine, and the SOCC inhibitor, 2-aminoethoxydiphenylborate (2-APB). Thapsigargin induced a sustained increase in c-fos mRNA and MAP kinase phosphatase-1 (MKP-1) mRNA and protein, and these effects were decreased by 2-APB but not by nimodipine. Our findings thus indicate that Ca2+ entry through VDCCs and SOCCs can differentially regulate CRE-containing genes in vascular smooth muscle and imply that signals involved in growth modulation are both temporally and spatially regulated by Ca2+. Experiment Overall Design: each of three experiments cell cultures were split three ways; one of the resulting samples was left untreated (C), another was treated with thapsigargin (TG), and the third was treated with elevated K+ (K). The resulting nine samples were used for triplicate estimates of the response of each gene to TG and K treatments. Experiment Overall Design: We tested the prospective hypothesis that genes having a CRE are differentially expressed after TG or K treatment using a permutation test: each of the 22,283 probe sets on the Affymetrix GeneChip was categorized two ways based on 1) whether or not it contains a CRE or not . Independence of CRE and threshold differential expression was rejected by Fisherâs exact test for both TG treatment ( ) and K treatment ( ). Target genes (c-fos, egr-1, and mkp-1 ) were identified based on ranking.
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