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Biomedical subjects

C Farquharson

Publications and source records attributed to C Farquharson.

At least 19 recordsLinked to original sources
PubMed2006

The growth plate sparing effects of the selective glucocorticoid receptor modulator, AL-438.

Long-term use of glucocorticoids (GC) can cause growth retardation in children due to their actions on growth plate chondrocytes. AL-438, a non-steroidal anti-inflammatory agent that acts through the glucocorticoid receptor (GR) retains full anti-inflammatory efficacy but has reduced negative effects on osteoblasts compared to those elicited by prednisolone (Pred) or dexamethasone (Dex). We have used the murine chondrogenic ATDC5 cell line to compare the effects of AL-438 with those of Dex and Pred on chondrocyte dynamics. Dex and Pred caused a reduction in cell proliferation and proteoglycan synthesis, whereas exposure to AL-438 had no effect. LPS-induced IL-6 production in ATDC5 cells was reduced by Dex or AL-438, showing that AL-438 has similar anti-inflammatory efficacy to Dex in these cells. Fetal mouse metatarsals grown in the presence of Dex were shorter than control bones whereas AL-438 treated metatarsals paralleled control bone growth. These results indicate that the adverse effects Dex or Pred have on chondrocyte proliferation and bone growth were attenuated following AL-438 exposure, suggesting that AL-438 has a reduced side effect profile on chondrocytes compared to other GCs. This could prove important in the search for new anti-inflammatory treatments for children.

Animals↗
PubMed2006

The restricted potential for recovery of growth plate chondrogenesis and longitudinal bone growth following exposure to pro-inflammatory cytokines.

Childhood chronic inflammatory disease can be associated with transient and permanent growth retardation. This study examined the potential for spontaneous growth recovery following pro-inflammatory cytokine exposure. Murine ATDC5 chondrogenic cells and postnatal metatarsals were exposed to interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha (TNFalpha), and their growth and proliferative capacity were determined following recovery. TNFalpha and IL-1beta reduced chondrocyte proliferation and aggrecan and collagen types II and X expression at minimum concentrations of 10 ng/ml and 0.1 ng/ml respectively. TNFalpha but not IL-1beta exposure led to increased caspase-3 activity and altered cellular morphology, consistent with reduced viability. Cytokine exposure particularly inhibited proteoglycan synthesis. This effect was dose and duration dependent. Compared with the control, IL-1beta and TNFalpha led to a 71% and 45% reduction in metatarsal growth after 8 days of exposure respectively (P < 0.05). An additive effect of IL-1beta combined with TNFalpha was observed (110% decrease; P < 0.05). Metatarsals exposed to IL-1beta or TNFalpha individually for a 2-day period, and allowed to recover spontaneously in the absence of cytokines for a further 6 days, showed normal growth trajectories. In combination, growth was 59% lower (P < 0.01) compared with control metatarsals at the end of the recovery period. Exposure to the combination for 4 days followed by a 4-day recovery period resulted in 87% decrement compared with controls (P < 0.05). IL-6 did not alter any parameter studied. IL-1beta and TNFalpha exert diverse inhibitory effects on ATDC5 chondrocyte dynamics and metatarsal growth. The extent of recovery following cytokine exposure depends on the duration of exposure, and may be incomplete following longer periods of exposure.

Animals↗
PubMed2006

Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.

Animals↗
PubMed2006

Cytokine actions in growth disorders associated with pediatric chronic inflammatory diseases (review).

Growth disorders are commonly observed in children suffering from chronic inflammatory diseases such as Juvenile Idiopathic Arthritis (JIA) and Inflammatory Bowel Disease (IBD). These disorders range from general growth retardation to local acceleration of growth in the affected limb and are associated with the increased production of pro-inflammatory cytokines. In this article, we review how cytokines influence child growth by exerting a local effect at the level of the growth plate, and through systemic effects throughout the whole body.

Arthritis, Juvenile↗
PubMed2005

The pathophysiology of the growth plate in juvenile idiopathic arthritis.

Children with chronic inflammatory diseases, such as juvenile idiopathic arthritis (JIA), suffer from a variety of growth disorders. These range from general growth retardation to local acceleration of growth in the affected limb. These disorders are associated with the increased production of proinflammatory cytokines, which may influence growth through a local effect in the growth plates of long bones and/or systemic effects throughout the whole body. In this article we review these aspects and also discuss the evidence for interaction between the inflammatory cytokine and growth-signalling pathways.

Arthritis, Juvenile↗
PubMed2004

Insulin-like growth factor-I augments chondrocyte hypertrophy and reverses glucocorticoid-mediated growth retardation in fetal mice metatarsal cultures.

The study aims were to improve our understanding of the mechanisms of glucocorticoid-induced growth retardation at the growth plate and determine whether IGF-I could ameliorate the effects. Fetal mouse metatarsals were cultured for up to 10 d with dexamethasone (Dex; 10(-6) m) and/or IGF-I and GH (both at 100 ng/ml). Both continuous and alternate-day Dex treatment inhibited bone growth to a similar degree, whereas IGF-I alone or together with Dex caused an increase in bone growth. GH had no effects. These observations may be explained at the cellular level; cell proliferation within the growing bone was decreased by Dex and increased by IGF-I and these effects were more marked in the cells of the perichondrium than those in the growth plate. However, the most prominent observation was noted in the hypertrophic zone where all treatments containing IGF-I significantly increased (3-fold) the length of this zone, whereas Dex alone had no significant effect. In conclusion, Dex impaired longitudinal growth by inhibiting chondrocyte proliferation, whereas IGF-I stimulated chondrocyte hypertrophy and reversed the growth-inhibitory Dex effects. However, the IGF-I-mediated improvement in growth was at the expense of altering the balance between proliferating and hypertrophic chondrocytes within the metatarsal.

Alkaline Phosphatase↗
PubMed2003

Bone strength during growth: influence of growth rate on cortical porosity and mineralization.

Although it is well accepted that bone architecture adapts to withstand the loads placed on it, the manner in which this occurs in the immature growing skeleton is not fully understood. To investigate the possible mechanisms, we have compared morphometric differences between tibiae from chickens with fast and those with slow growth potential and also distinguished between the effects of genetic potential and growth rate on their impact on bone quality. Two different fast-growing (ad lib modern) strains, one additionally feed-restricted and one slow-growing (control) strain of chicken, were compared at 15 and 42 days of age. The ad lib modern strains had similar final body weights and were approximately twice the weight of the control and restricted-fed birds. Tibiae from the control and restricted birds had a higher ash content and lower porosity than the ad lib modern strain at 42 days. The porosity was a result of rapid primary osteon formation at the periosteal surface and incomplete infilling of the resultant canal by osteoblasts. When adjusted to average body weight of contemporaries, bones from the control strain and the restricted-fed modern birds were stiffer and at least as strong as those from the fast growing ad lib-fed birds. In conclusion, rapid bone deposition at the periosteal surface was associated with decreased mineralization, increased cortical porosity, and altered biomechanical properties. Our results also indicate that growth rate, and not genetic potential, of the fast growing birds was responsible for the rapid periosteal bone deposition.

Acid Phosphatase↗
PubMed2003

Expression of type X collagen, Indian hedgehog and parathyroid hormone related-protein in normal and tibial dyschondroplastic chick growth plates.

Tibial dyschondroplasia (TD) is a form of aberrant endochondral ossification in chickens, in that a plug of avascular cartilage (TD lesion) is formed within the growth plate. Histologically, the lesion is filled with apparently transitional chondrocytes that have been unable to differentiate to hypertrophic chondrocytes. We have examined the spatial expression of mRNAs for type X collagen, Indian hedgehog (Ihh) and Parathyroid Hormone-related protein (PTHrP) in the TD growth plate by in situ hybridization in order to ascertain at which stage chondrocyte differentiation is arrested in TD. In the normal growth plate, type X collagen mRNA was expressed by both prehypertrophic and hypertrophic chondrocytes. Indian Hedgehog mRNA was detected in a band of prehypertrophic chondrocytes and PTHrP expression was localized to a narrow band of prehypertrophic chondrocytes and in osteoblasts within the diaphysis. In TD sections, collagen X expression was seen within differentiating cells, within a small number of lesion cells, and within hypertrophic chondrocytes on the diaphyseal side of the lesion. Ihh expression was also seen within the differentiating cells and throughout the lesion. These data indicate that chondrocyte differentiation is arrested at the transitional stage just prior to hypertrophy. Contrary to the previously reported PTHrP expression patterns in TD chicks by immunohistochemistry, PTHrP mRNA was not detected in the TD lesion. This observation probably reflects the cessation of PTHrP gene expression by chondrocytes in the more severe TD lesions. The results from the present study also imply that the arrest of cell differentiation in TD is independent of PTHrP and that endochondral ossification in the post-hatch avian growth plate may involve additional regulatory pathways.

Animals↗
PubMed2002

Chromosomal localization of the chicken and mammalian orthologues of the orphan phosphatase PHOSPHO1 gene.

PHOSPHO1 is a recently identified phosphatase expressed at high levels in the chicken growth plate and which may be involved in generating inorganic phosphate for skeletal matrix mineralization. Using a degenerate RT-PCR approach a fragment of human PHOSPHO1 was cloned. This enabled the identification of the human orthologue on HSA17q21, and the mouse orthologue on a region of MMU11 that exhibits conservation of synteny with HSA17q21. Chicken PHOSPHO1 was mapped by SSCP analysis to position 44 cM on GGA27, adjacent to the HOXB@ (44 cM) and COL1A1 (36 cM) loci. Comparison of genes on GGA27 with their orthologues on the preliminary draft of the human genome identifies regions of conserved synteny equivalent to 25 Mb on HSA17q21.2-23.3 and approximately 20 Mb on GGA27 in which the gene order appears to be conserved. Mapping of the PHOSPHO1 genes to regions of HSA17q21.3, MMU11 and GGA27 that exhibit conservation of synteny provides strong evidence that they are orthologous.

Amino Acid Sequence↗
PubMed2002

Glucocorticoid effects on chondrogenesis, differentiation and apoptosis in the murine ATDC5 chondrocyte cell line.

Glucocorticoids (GC) are used extensively in children and may cause growth retardation, which is in part due to the direct effects of GC on the growth plate. We characterised the ATDC5 chondrocyte cell line, which mimics the in vivo process of longitudinal bone growth, to examine the effects of dexamethasone (Dex) and prednisolone (Pred) during two key time points in the chondrocyte life cycle - chondrogenesis and terminal differentiation. Additionally, we studied the potential for recovery following Dex exposure. During chondrogenesis, Dex and Pred exposure at 10(-8) M, 10(-7) M and 10(-6) M resulted in a significant mean reduction in cell number (28% vs 20%), cell proliferation (27% vs 24%) and proteoglycan synthesis (47% vs 43%) and increased alkaline phosphatase (ALP) activity (106% vs 62%), whereas the incidence of apoptosis was unaltered. Minimal effects were noted during terminal differentiation with both GC although all concentrations of Dex lowered apoptotic cell number. To assess catch-up growth the cells were incubated for a total of 14 days which included 1, 3, 7, 10 or 14 days exposure to 10(-6) M Dex, prior to the recovery period. Recovery of proteoglycan synthesis was irreversibly impaired following just one day exposure to Dex. Although cell number showed a similar pattern, significant impairment was only achieved following 14 days exposure. Irreversible changes in ALP activity were only noticed following 10 days exposure to Dex. In conclusion, GC have maximal effects during chondrogenesis; Dex is more potent than Pred and cells exposed to Dex recover but this may be restricted due to differential effects of GC on specific chondrocyte phenotypes.

Animals↗
PubMed2001

Regulation of chondrocyte terminal differentiation in the postembryonic growth plate: the role of the PTHrP-Indian hedgehog axis.

Chondrocyte differentiation during embryonic bone growth is controlled by interactions between PTHrP and Indian hedgehog. We have now determined that the major components of this signaling pathway are present in the postembryonic growth plate. PTHrP was immunolocalized throughout the growth plate, and semiquantitative RT-PCR analysis of maturationally distinct chondrocyte fractions indicated that PTHrP, Indian hedgehog, and the PTH/PTHrP receptor were expressed at similar levels throughout the growth plate. However, patched, the hedgehog receptor, was more highly expressed in proliferating chondrocytes. Although all fractionated cells responded to PTHrP in culture by increasing thymidine incorporation and cAMP production and decreasing alkaline phosphatase activity, the magnitude of response was greatest in the proliferative chondrocytes. Bone morphogenetic proteins are considered likely intermediates in PTHrP signaling. Expression of bone morphogenetic protein-2 and 4--7 was detected within the growth plate, and PTHrP inhibited the expression of bone morphogenetic protein-4 and 6. Although organ culture studies indicated a possible paracrine role for epiphyseal chondrocyte-derived PTHrP in regulating growth plate chondrocyte differentiation, the presence within the postembryonic growth plate of functional components of the PTHrP-Indian hedgehog pathway suggests that local mechanisms intrinsic to the growth plate exist to control the rate of endochondral ossification.

Animals↗
PubMed2000

Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia.

Tibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought.

Animals↗
PubMed2000

Identification of a family of noncanonical ubiquitin-conjugating enzymes structurally related to yeast UBC6.

Ubiquitin-conjugating enzymes (UBCs) selectively target proteins for proteasomal degradation by the covalent attachment of ubiquitin moieties. Yeast UBC6 is unusual in having an active site distinct from all other UBCs and in possessing a transmembrane domain that anchors it to the cytoplasmic surface of the endoplasmic reticulum. During a differential display analysis on chick growth plate chondrocytes we isolated a cDNA encoding a noncanonical ubiquitin-conjugating enzyme (NCUBE1) structurally similar to yeast UBC6. Chick NCUBE1 transcripts were detected in all tissues examined and decreased threefold during chondrocyte terminal differentiation. Database searches identified other related proteins; the human and mouse orthologues of NCUBE1, a second human homologue of yeast UBC6 (NCUBE2), and related proteins from S. pombe, C. elegans, and P. mariana. Together with yeast UBC6 these proteins constitute a distinct family of UBCs sharing a conserved noncanonical active site sequence and a C-terminal transmembrane domain. By analogy with yeast UBC6 they are likely to be localised to the endoplasmic reticulum where they may be involved in targeting retrotranslocated, ER-associated proteins for proteasomal degradation.

Amino Acid Sequence↗
PubMed2000

Dietary effects on bone quality and turnover, and Ca and P metabolism in chickens.

The responses of skeletal health and blood calcium (Ca) and phosphorus (P) status to different dietary calcium and available P (avP) contents were examined in 2-week-old chickens. High avP content resulted in more inorganic P (P(i)) and less ionised Ca (Ca(2+)) in the blood. Birds on low Ca and high avP diets showed a higher incidence of tibial dyschondroplasia and hypocalcaemic rickets and were therefore assumed to be hypocalcaemic. However the molar Ca:P ratio in bone was closer to the expected 1.67:1 in diets high in avP. No significant dietary mineral content effect was found on bone mineral content, or breaking strength, within the range of diets used. Results suggest that variations in molar Ca:P ratios in bone have little effect on mechanical competence, and that diets for this strain should be higher in Ca, and could be lower in avP, than current recommendations.

Amino Acids↗
PubMed2000

Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation.

Ex-FABP, extracellular fatty acid binding protein, is a 21 kDa lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryo development and in granulocytes. Ex-FABP synthesis was increased in chondrocyte and myoblast cultures by inflammatory agents (LPS; IL6) and repressed by antiinflammatory agents. Expression of Ex-FABP and specific gelatinases is paralleled in hypertrophic cartilage; LPS specifically induced high molecular weight gelatinase ( > 200 kDa). LPS-treated hypertrophic chondrocytes showed increased chemotactic activity for endothelial cells paralleled by increased expression of transferrin. A high amount of Ex-FABP was expressed in adult pathological cartilage both in dyschondroplastic and osteoarthritic chickens. Controls were negative. Ex-FABP could represent a stress protein physiologically expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions. We also suggest that during endochondral bone formation other responses characteristic of a local inflammatory status, such as gelatinase production and angiogenic factor secretion, are "physiologically" activated.

Acute-Phase Reaction↗
PubMed2000

Skeletal development in the meat-type chicken.

1. This study ascertained how bone of modern meat-type chickens develops under typical commercial conditions and compares development with that in genetic precursor stock. 2. A modern fast-growing selected strain and a slower-growing control strain were used. Birds were weighed weekly. A random sample was taken from each population at a range of ages up to 39 d. 3. A tibiotarsus from each bird was X-rayed and its dimensions and estimated resistance to bending were determined. Cortical bone samples were ashed to measure total mineral, calcium and phosphorus content. Cortical samples were also taken for porosity assessment. 4. As expected, the selected strain grew faster and heavier than the control strain. Despite this, both strains demonstrated similar periods of rapid bone formation (days 4 to 18) and mineralisation (days 4 to 11), and achieved similar estimates of resistance to bending. 5. However, cortical bone of the selected strain was less well mineralised and more porous than that of the control strain and showed a significant increase in the molar Ca:P ratios above the expected range of values during the first 2 to 3 weeks of life. 6. Despite production of bones with the correct dimensions for load support, the relatively poor density and mineral content of bone in the selected strain is likely to reduce effective breaking strength of the tibiotarsus. Possible reasons may be either inadequate dietary supply of Ca and P or impaired utilisation of the minerals due to a rapid growth rate or genetic factors.

Animals↗
PubMed2000

Chondrocytes and longitudinal bone growth: the development of tibial dyschondroplasia.

Growth plate cartilage is central to the process of bone elongation. Chondrocytes originating within the resting zone of the growth plate proceed through a series of intermediate phenotypes: proliferating, prehypertrophic and hypertrophic, before reaching a terminally differentiated state. Disruption of this chondrocyte maturational sequence causes many skeletal abnormalities in poultry such as tibial dyschondroplasia (TD), which is a common cause of deformity and lameness in the broiler chicken. Cell and matrix components of the growth plate have been studied in order to determine the cause(s) of the premature arrest of chondrocyte differentiation and retention of prehypertrophic chondrocytes observed in TD. Chondrocyte proliferation proceeds normally in TD, but markers of the differentiated phenotype, local growth factors, and the vitamin D receptor are abnormally expressed within the prehypertrophic chondrocytes above, and within, the lesion. Tibial dyschondroplasia is also associated with a reduced incidence of apoptosis, suggesting that the lesion contains an accumulation of immature cells that have outlived their normal life span. Immunolocalization studies of matrix components suggest an abnormal distribution within the TD growth plate that is consistent with a failure of the chondrocytes to fully hypertrophy. In addition, the collagen matrix of the TD lesion is highly crosslinked, which may make the formed lesion more impervious to vascular invasion and osteoclastic resorption. Recent studies have applied the techniques of differential display and semiquantitative reverse transcriptase-polymerase chain reaction to RNA obtained from discrete populations of growth plate chondrocytes of different maturational phenotypes. This strategy has allowed us to compare phenotypically identical cell fractions from normal and TD growth plates in an attempt to identify possible candidate genes for TD.

Animals↗