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Biomedical subjects

E J Mackie

Publications and source records attributed to E J Mackie.

At least 19 recordsLinked to original sources
PubMed2006

Physiological death of hypertrophic chondrocytes.

OBJECTIVE: Post-proliferative chondrocytes in growth cartilage are present in two forms, light and dark cells. These cells undergo hypertrophy and die by a mechanism that is morphologically distinct from apoptosis, but has not been characterized. The aims of the current study were to document the ultrastructural appearance of dying hypertrophic chondrocytes, and to establish a culture system in which the mechanism of their death can be examined. DESIGN: Growth cartilage from fetal and growing postnatal horses was examined by electron microscopy. Chondrocytes were isolated from epiphyseal cartilage from fetal horses and grown in pellet culture, then examined by light and electron microscopy, and quantitative polymerase chain reaction. RESULTS: In tissue specimens, it was observed that dying dark chondrocytes underwent progressive extrusion of cytoplasm into the extracellular space, whereas light chondrocytes appeared to disintegrate within the cellular membrane. Pellets cultured in 0.1% fetal calf serum (FCS) contained dying light and dark chondrocytes similar to those seen in vivo. Transforming growth factor-beta1 or 10% FCS increased the proportion of dark cells and induced cell death. Triiodothyronine increased the differentiation of dark and light cells and induced their death. Dark cells were associated with higher levels of matrix metalloproteinase-13 expression than light cells, and light cells were associated with higher levels of type II collagen expression. CONCLUSIONS: Light and dark hypertrophic chondrocytes each undergo a distinctive series of non-apoptotic morphological changes as they die. Pellet culture can be used as a model of the two forms of physiological death of hypertrophic chondrocytes.

Animals↗
PubMed2005

Thrombin is a pro-fibrotic factor for rat renal fibroblasts in vitro.

BACKGROUND: Generation of thrombin occurs in response to parenchymal injury. Thrombin not only converts plasma fibrinogen into an insoluble fibrin clot, but also potentially augments inflammation through receptor-mediated activity. This study examines whether thrombin may potentially exacerbate fibrosis by upregulating the function of interstitial fibroblasts in vitro. METHODS: Fibroblasts were isolated by explant outgrowth culture of rat kidneys. Subcultured cells were grown in DMEM+10% FCS supplemented with 0.1-0.5 U/ml thrombin. Functional parameters examined included kinetics (thymidine incorporation and change in cell number), differentiation (Western blotting for alpha-smooth muscle actin; alphaSMA), expression of procollagen alpha1(I) (Northern blotting) and contraction of collagen I lattices. RT-PCR was used to characterise expression of protease-activated receptors (PAR) previously implicated in thrombin's cellular effects. RESULTS: Cell population growth was increased 66 +/- 41 and 47 +/- 41% by 0.1 and 0.5 U/ml thrombin respectively (both p < 0.05 vs. basal). Likewise, 0.5 U/ml thrombin increased corrected procollagen alpha1(I) expression 2.4-fold (p < 0.05 vs. basal) and exacerbated the ability of fibroblasts to contract collagen matrix (p < 0.05 vs. basal). These effects were not associated with any change in expression of the myofibroblast marker alphaSMA. Effects on cell number were inhibited by treatment with (D)-Phe-Pro-Arg-chloromethylketone HCl (PPACK) suggesting that functional effects were mediated by serine protease activity. PAR-1 was the only fully functional known thrombin receptor expressed by these cells. CONCLUSION: Thrombin is a potential unrecognised fibroblast agonist in renal disease. Further studies of thrombin and its receptors may yield valuable insights into the pathogenesis of interstitial fibrosis.

Actins↗
PubMed2005

Studies on the receptors mediating responses of osteoblasts to thrombin.

The serine protease thrombin stimulates proliferation in osteoblasts, but decreases alkaline phosphatase (ALP) activity, a marker of osteoblast differentiation. Three thrombin receptors have been identified, protease activated receptor (PAR)-1, PAR-3 and PAR-4; we have previously demonstrated that mouse osteoblasts express PAR-1 and PAR-4. The effect of thrombin on osteoblast proliferation and differentiation was studied to determine which of the thrombin receptors is responsible for the primary effects of thrombin. Primary mouse calvarial osteoblasts from PAR-1-null and wild-type mice, and synthetic peptides that specifically activate PAR-1 (TFFLR-NH2) and PAR-4 (AYPGKF-NH2) were used. Both the PAR-1-activating peptide and thrombin stimulated incorporation of 5-bromo-2'-deoxyuridine (two to four-fold, P < 0.001) and reduced alkaline phosphatase activity (approximately three-fold, P < 0.05) in cells from wild-type mice. The PAR-4-activating peptide, however, had no effect on either alkaline phosphatase activity or proliferation in these cells. Neither thrombin nor PAR-4-activating peptide was able to affect osteoblast proliferation or alkaline phosphatase activity in cells isolated from PAR-1-null mice. The results demonstrate that thrombin stimulates proliferation and inhibits differentiation of osteoblasts through activation of PAR-1. No other thrombin receptor appears to be involved in these effects.

Animals↗
PubMed2004

Acute sixth-nerve palsy after vincristine therapy.

The chemotherapeutic effectiveness of vincristine was first reported in 1962, and with its increased use the incidence of reported neurologic side effects has also increased. We reported a case of acute sixth-nerve palsy occurring soon after administration of vincristine for acute lymphoblastic leukemia (ALL), a previously unreported side effect.

Abducens Nerve↗
PubMed2003

Establishment of a model of cortical bone repair in mice.

A model of cortical bone repair has been established for use in mice. The cortical defect consisted of a hole drilled through the entire diameter of the tibial diaphysis. The hematoma that initially filled the drill site was invaded by cells of mesenchymal appearance within 5 days of injury. Trabeculae of mineralized woven bone were present throughout the drill site by day 9. A reaction in the periosteum adjacent to the drill site, consisting of both new bone and cartilage formation, preceded deposition of bone tissue in the drill site. New woven bone was modeled to restore the marrow cavity to normal by 4 weeks after injury, and almost normal cortical structure was achieved by 6 weeks after injury. Immunohistochemical studies indicated that type III collagen was expressed within the drill site by day 5, reached a peak at day 7, and was diminished by day 9. In contrast, type I collagen was first detectable in the drill site at day 7, and staining was more intense by day 9. Osteopontin expression in the drill site coincided with the process of mineralization of new bone in this location. The model of bone repair described here provides a method for inducing reproducible bone lesions in a readily identifiable location in mice. It will be useful in the investigation of bone cell function in mouse strains that have been subjected to genetic manipulation.

Animals↗
PubMed2003

Osteoblasts: novel roles in orchestration of skeletal architecture.

Osteoblasts are located on bone surfaces and are the cells responsible for bone formation through secretion of the organic components of bone matrix. Osteoblasts are derived from mesenchymal osteoprogenitor cells found in bone marrow and periosteum. Following a period of secretory activity, osteoblasts undergo either apoptosis or terminal differentiation to form osteocytes surrounded by bone matrix. Osteoblasts secrete a characteristic mixture of extracellular matrix proteins including type I collagen as the major component as well as proteoglycans, glycoproteins and gamma-carboxylated proteins. Cells of the osteoblast lineage also provide factors essential for differentiation of osteoclasts (bone-resorbing cells). By regulating osteoclast differentiation and activity in response to systemic influences, osteoblasts not only play a central role in regulation of skeletal architecture, but also in calcium homeostasis. Inadequate osteoblastic bone formation in relation to osteoclastic resorption results in osteoporosis, a disease characterised by enhanced skeletal fragility. Cellfacts: Osteoblasts are the cells responsible for bone formation. Osteoblasts indirectly control levels of bone resorption. Osteoblasts play a key role in the pathophysiology of osteoporosis and the resulting fractures, which constitute a major public health burden in developed countries.

Animals↗
PubMed2002

Dissection of protease-activated receptor-1-dependent and -independent responses to thrombin in skeletal myoblasts.

Thrombin exerts a number of effects on skeletal myoblasts in vitro. It stimulates proliferation and intracellular calcium mobilization and inhibits differentiation and apoptosis induced by serum deprivation in these cells. Many cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Expression of PAR-1 is present in mononuclear myoblasts in vitro, but repressed when fusion occurs to form myotubes. In the current study, we used PAR-1-null mice to determine which of thrombin's effects on myoblasts are mediated by PAR-1. Thrombin inhibited fusion almost as effectively in cultures prepared from the muscle of PAR-1-null myoblasts as in cultures prepared from wild-type mice. Apoptosis was inhibited as effectively in PAR-1-null myoblasts as in wild-type myoblasts. These effects in PAR-1-null myoblasts were mediated by a secreted inhibitor of apoptosis and fusion, as demonstrated previously for normal rat myoblasts. Thrombin failed to induce an intracellular calcium response in PAR-1-null myoblast cultures, although these cells were able to mobilize intracellular calcium in response to activation of other receptors. PAR-1-null myoblasts also failed to proliferate in response to thrombin. These results demonstrate that thrombin's effects on myoblast apoptosis and fusion are not mediated by PAR-1 and that PAR-1 is the only thrombin receptor capable of inducing proliferation and calcium mobilization in neonatal mouse myoblasts.

Animals↗
PubMed2002

Protease-activated receptors: a means of converting extracellular proteolysis into intracellular signals.

Protease-activated receptors (PARs) mediate cellular responses to a variety of extracellular proteases. The four known PARs constitute a subgroup of the family of seven-transmembrane domain G protein-coupled receptors and activate intracellular signalling pathways typical for this family of receptors. Activation of PARs involves proteolytic cleavage of the extracellular domain, resulting in formation of a new N terminus, which acts as a tethered ligand. PAR-1, -3, and -4 are relatively selective for activation by thrombin whereas PAR-2 is activated by a variety of proteases, including trypsin and tryptase. Recent studies in mice genetically incapable of expressing specific PARs have defined roles for PAR-1 in vascular development, and for PAR-3 and -4 in platelet activation, which plays a fundamental role in blood coagulation. PAR-1 has also been implicated in a variety of other biological processes including inflammation, and brain and muscle development. Responses mediated by PAR-2 include contraction of intestinal smooth muscle, epithelium-dependent smooth muscle relaxation in the airways and vasculature, and potentiation of inflammatory responses. The area of PAR research is rapidly expanding our understanding of how cells communicate and control biological functions, in turn increasing our knowledge of disease processes and providing potential targets for therapeutic intervention.

Animals↗
PubMed2001

Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion.

Periodontitis is a chronic inflammatory disease affecting oral tissues. Oral epithelial cells represent the primary barrier against bacteria causing the disease. We examined the responses of such cells to an arginine-specific cysteine proteinase (RgpB) produced by a causative agent of periodontal disease, Porphyromonas gingivalis. This protease caused an intracellular calcium transient in an oral epithelial cell line (KB), which was dependent on its enzymatic activity. Since protease-activated receptors (PARs) might mediate such signaling, reverse transcription-PCR was used to characterize the range of these receptors expressed in the KB cells. The cells were found to express PAR-1, PAR-2, and PAR-3, but not PAR-4. In immunohistochemical studies, human gingival epithelial cells were found to express PAR-1, PAR-2, and PAR-3 on their surface, but not PAR-4, indicating that the cell line was an effective model for the in vivo situation. PAR-1 and PAR-2 expression was confirmed in intracellular calcium mobilization assays by treatment of the cells with the relevant receptor agonist peptides. Desensitization experiments strongly indicated that signaling of the effects of RgpB was occurring through PAR-1 and PAR-2. Studies with cells individually transfected with each of these two receptors confirmed that they were both activated by RgpB. Finally, it was shown that, in the oral epithelial cell line, PAR activation by the bacterial protease-stimulated secretion of interleukin-6. This induction of a powerful proinflammatory cytokine suggests a mechanism whereby cysteine proteases from P. gingivalis might mediate inflammatory events associated with periodontal disease on first contact with a primary barrier of cells.

Adhesins, Bacterial↗
PubMed2000

Evidence for the activation of PAR-2 by the sperm protease, acrosin: expression of the receptor on oocytes.

Proteinase-activated receptor-2 (PAR-2) is a member of a family of G-protein-coupled, seven-transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin-like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR-2. This substrate was cleaved with kinetics similar to those of the known PAR-2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR-2, most probably due to activation of the receptor. Immunohistochemical studies using PAR-2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR-2 on oocytes.

Acrosin↗
PubMed2000

Tenascin-C induced stimulation of chondrogenesis is dependent on the presence of the C-terminal fibrinogen-like globular domain.

The relationship between structure of tenascin-C (Tn-C), a multi-domain extracellular matrix protein, and its stimulation of chondrogenesis was examined using recombinant Tn-C isoforms (full length or with specific domains deleted) as substrata for undifferentiated chicken mesenchymal cells. Of the Tn-C variants tested, only Tn-C lacking the fibrinogen-like domain or Tn-C comprised solely of fibrinogen-like domains failed to stimulate chondrogenesis. The ability of variants to stimulate chondrogenesis was not dependent on their ability to support adhesion or stimulate proliferation. These results demonstrate that the fibrinogen-like domain of Tn-C is necessary but not sufficient for induction of chondrogenesis.

Animals↗
PubMed2000

Expression of protease-activated receptor-2 during embryonic development.

Protease-activated receptor-2 (PAR-2) is the second member of a novel family of G-protein-coupled receptors, activated through proteolytic cleavage within the extracellular domain to reveal a newly formed amino terminus that acts as a tethered ligand causing receptor activation. PAR-2 is expressed in a number of adult tissues, but its distribution during development has not been characterized. Knowledge of the tissue distribution of PAR-2 during development will provide clues as to its function(s) in vivo. In the current immunohistochemical study, a polyclonal antibody raised against a peptide corresponding to the post-cleavage amino terminal sequence of PAR-2 was used to localize PAR-2 expression in developing mouse tissues. In the developing central nervous system and cardiac muscle, PAR-2 expression was detectable at embryonic day 12 and persisted throughout embryogenesis. At embryonic day 14, PAR-2 expression was strong in peripheral nerves, but either weak or absent in skin, bone, skeletal muscle, and blood vessels. In embryonic day 17 and postnatal day 1 hindlimbs, however, PAR-2 staining was observed throughout the layers of the epidermis, in osteoblasts, muscle fibers, and in vascular smooth muscle and endothelium. The pattern of PAR-2 expression observed during embryonic development and the association of expression with differentiation in certain tissues suggest compelling physiological roles for this novel receptor.

Amino Acid Sequence↗
PubMed2000

Expression of protease-activated receptor-2 by osteoblasts.

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.

Adhesins, Bacterial↗
PubMed2000

Protease-activated receptor-2 mediates proliferative responses in skeletal myoblasts.

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by proteases within the N terminus, exposing a new tethered ligand that binds and activates the receptor. Activators of PAR-2 include trypsin and mast cell tryptase. Skeletal myoblasts are known to express PAR-1, a thrombin receptor. The current study was undertaken to determine whether myoblasts express PAR-2. Primary neonatal rat and mouse skeletal myoblast cultures were shown to express PAR-2 in polymerase chain reaction and immunocytochemical studies. Expression of PAR-2 was also demonstrated by immunohistochemistry in developing mouse skeletal muscle in vivo. Trypsin or a synthetic peptide corresponding to the rat PAR-2 tethered ligand caused a dose-dependent elevation in intracellular calcium in cultured rat myoblasts, with an EC(50) of 13 nM or 56 microM, respectively. Studies aimed at identifying the function of PAR-2 in myoblasts demonstrated no effect of the receptor-activating peptide on survival or fusion in serum-deprived myoblasts. The PAR-2-activating peptide did, however, stimulate proliferation of serum-deprived myoblasts. These results demonstrate that skeletal muscle cells express PAR-2, activation of which leads to stimulation of myoblast proliferation.

Amino Acid Sequence↗
PubMed1999

Thrombin, a survival factor for cultured myoblasts.

Three members of the family of protease-activated receptors (PARs), PARs-1, -3 and -4, have been identified as thrombin receptors. PAR-1 is expressed by primary myoblast cultures, and expression is repressed once myoblasts fuse to form myotubes. The current study was undertaken to investigate the hypothesis that thrombin inhibits myoblast fusion. Primary rodent myoblast cultures were deprived of serum to promote myoblast fusion and then cultured in the presence or absence of thrombin. Thrombin inhibited myoblast fusion, but another notable effect was observed; 50% of control cells were apoptotic within 24 h of serum deprivation, whereas less than 15% of thrombin-treated cells showed signs of apoptosis. Proteolysis was required for the effect of thrombin, but no other serine protease tested mimicked the action of thrombin. Neither a PAR-1- nor a PAR-4-activating peptide inhibited apoptosis or fusion, and myoblast cultures were negative for PAR-3 expression. Myoblasts exposed to thrombin for 1 h and then changed to medium without thrombin accumulated apoptosis inhibitory activity in their medium over the subsequent 20 h. Thus the protective action of thrombin appears to be effected through cleavage of an unidentified thrombin receptor, leading to secretion of a downstream apoptosis inhibitory factor. These results demonstrate that thrombin functions as a survival factor for myoblasts and is likely to play an important role in muscle development and repair.

Animals↗
PubMed1999

Cholera toxin and forskolin stimulate formation of osteoclast-like cells in mouse marrow cultures and cultured mouse calvarial bones.

Osteoclasts are hematopoietic in origin and formed by proliferation, differentiation and fusion of osteoclast progenitor cells. However, the signal transducing mechanisms involved in generation of osteoclasts are not clear. We have used two well-known adenylate cyclase stimulators to examine the effect of cyclic AMP (cAMP) on the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells in cultured mouse calvarial bones and in mouse bone marrow cultures. The effects of forskolin and cholera toxin were compared with those of parathyroid hormone (PTH) and 1,25(OH)2vitaminD3 (1,25(OH)2D3). PTH, as well as forskolin and cholera toxin, increased the number of osteoclast profiles/mm bone in 24-h and 120-h cultures of mouse calvarial bones. In mouse bone marrow cultures, 1,25(OH)2D3 or PTH stimulated formation of TRAP-positive multinucleated cells. Moreover, forskolin or cholera toxin produced dose-dependent stimulation of these cells at a range of concentrations correlating with their effect on cAMP production. The osteoclastic phenotype of the TRAP-positive cells was demonstrated by autoradiography of 125I-labelled calcitonin binding and by the bone-resorbing activity of the cells. The sustained presence (0-9 d) of forskolin or PTH was required to obtain maximal formation of osteoclasts. However, the presence of 1,25(OH)2D3 was required only for the last 3 d of culture for maximal osteoclast formation. We conclude that PTH may stimulate osteoclast generation using the adenylate cyclase cAMP system as a signal transduction mechanism.

Acid Phosphatase↗