Mesenchymal stem cells (MSCs) are multipotent cells capable of developing along the chondrogenic, osteogenic and adipogenic lineages. As such, they have received interest as a potential cell source for tissue engineering strategies. Cartilage is an avascular tissue and thus resides in a microenvironment with reduced oxygen tension. The aim of this study was to examine the effect of a low oxygen environment on MSC differentiation along the chondrogenic route. In MSCs exposed to chondrogenic growth factors, transforming growth factor-beta and dexamethasone, in a hypoxic environment (2% oxygen), the induction of collagen II expression and proteoglygan deposition was significantly greater than that observed when cells were exposed to the chondrogenic growth factors under normoxic (20% oxygen) conditions. The transcription factor, hypoxia-inducible factor-1alpha (HIF-1alpha), is a crucial mediator of the cellular response to hypoxia. Following exposure of MSCs to hypoxia (2% oxygen), HIF-1alpha translocated from the cytosol to the nucleus and bound to its target DNA consensus sequence. Similarly, hypoxia evoked an increase in phosphorylation of both AKT and p38 mitogen activated protein kinase, upstream of HIF-1alpha activation. Furthermore, the PI3 kinase/AKT inhibitor, LY294002, and p38 inhibitor, SB 203580, prevented the hypoxia-mediated stabilisation of HIF-1alpha. To assess the role of HIF-1alpha in the hypoxia-induced increase in chondrogenesis, we employed an siRNA knockdown approach. In cells exposed to HIF-1alpha siRNA, the hypoxia-induced enhancement of chondrogenesis, as evidenced by upregulation of collagen II, sox-9 and proteoglycan deposition, was absent. This provides evidence for HIF-1alpha being a key mediator of the beneficial effect of a low oxygen environment on chondrogenesis.

Lipopolysaccharide, a component of the cell wall of Gram-negative bacteria, may be responsible for at least some of the pathophysiological sequelae of bacterial infections, probably by inducing an increase in interleukin-1␤ (IL-1␤) concentration. We report that intraperitoneal injection of lipopolysaccharide increased hippocampal caspase-1 activity and IL-1␤ concentration; these changes were associated with increased activity of the stress-activated kinase c-Jun NH 2 -terminal kinase, decreased glutamate release, and impaired long term potentiation. The degenerative changes in hippocampus and entorhinal cortical neurones were consistent with apoptosis because translocation of cytochrome c and poly(ADP-ribose) polymerase cleavage were increased. Inhibition of caspase-1 blocked these changes, suggesting that IL-1␤ mediated the lipopolysaccharide-induced changes.There is increasing awareness of the existence of bidirectional communication between the immune and nervous systems. The proinflammatory cytokine, interleukin-1␤ (IL-1␤), 1 is one molecule that may play a pivotal role in integrating neuronal immune responses with those of the endocrine system because it exerts significant effects in all systems, for example in response to stressors such as infection. Gram-negative bacterial infections are associated with multiple pathophysiological changes; it is widely accepted that these changes are stimulated by lipopolysaccharide (LPS), a component of the outer membrane of most Gram-negative bacteria. These changes, which include fever, changes in sleep pattern, and anorexia (1), are mimicked by, and therefore thought to be mediated through production of, IL-1␤. Thus LPS, injected centrally or peripherally, increases IL-1␤ concentrations (2, 3) and IL-1␤ mRNA expression (4) in rat brain.Although it appears that in certain circumstances IL-1␤ may be neuroprotective, the consensus is that prolonged exposure, or exposure of tissue to high concentrations of IL-1␤, results in degenerative changes (5). Therefore it is significant that increased IL-1 concentrations in different brain areas have been correlated with neurodegenerative disorders such as Down syndrome, Alzheimer's disease (6), and Parkinson's disease (7), whereas in experimental models, IL-1␤ is considered to be responsible for the cell damage associated with ischemia (8) and excitotoxicity (9) and is increased after experimental traumatic lesions (10). A striking example of a neuronal deficit induced by IL-1␤ is the impairment in long term potentiation (LTP) in the hippocampus in vitro (11-13) and in vivo (14 -16).IL-1␤ is produced by glia (17, 18) and neurones (19,20) in response to tissue stress. It is cleaved from the inactive percursor, pro-IL-1␤, by the action of caspase-1, a member of a large family of cysteine proteases that have been implicated in apoptotic cell death (21-25). It might be predicted therefore that any trigger such as LPS, which induces an increase in IL-1␤, will do so by increasing activity of caspase-1.Our objective was to investigate th...

Adult mesenchymal stem cells have the proclivity to differentiate along multiple lineages giving rise to new bone, cartilage, muscle, or fat. Collagen, a normal constituent of bone, provides strength and structural stability and is therefore a potential candidate for use as a substrate on which to engineer bone and cartilage from their respective mesenchymal-derived precursors. In this study, a collagen- glycosaminoglycan scaffold was used to provide a suitable three-dimensional (3-D) environment on which to culture adult rat mesenchymal stem cells and induce differentiation along the osteogenic and chondrogenic lineages. The results demonstrate that adult rat mesenchymal stem cells can undergo osteogenesis when grown on the collagen-glycosaminoglycan scaffold and stimulated with osteogenic factors (dexamethasone, ascorbic acid, beta-glycerophosphate), as evaluated by the temporal induction of the bone-specific proteins, collagen I and osteocalcin, and subsequent matrix mineralization. The osteogenic factors were coupled to activation of the extracellular-regulated protein kinase (ERK), and this kinase was found to play a role in the osteogenic process. As well as supporting osteogenesis, when the cell-seeded scaffold was exposed to chondrogenic factors (dexamethasone and TGF-1beta), collagen II immunoreactivity was increased, providing evidence that the scaffold can also provide a suitable 3-D environment that supports chondrogenesis.

The objective of this study was to investigate the hypothesis that the application of dynamic compression following transforming growth factor-beta3 (TGF-beta3) induced differentiation will further enhance chondrogenesis of mesenchymal stem cells (MSCs). Porcine MSCs were encapsulated in agarose hydrogels and cultured in a chemically defined medium with TGF-beta3 (10 ng/mL). Dynamic compression (1 Hz, 10% strain, 1 h/day) was initiated at either day 0 or day 21 and continued until day 42 of culture; with TGF-beta3 withdrawn from some groups at day 21. Biochemical and mechanical properties of the MSC-seeded constructs were evaluated up to day 42. The application of dynamic compression from day 0 inhibited chondrogenesis of MSCs. This inhibition of chondrogenesis in response to dynamic compression was not observed if MSC-seeded constructs first underwent 21 days of chondrogenic differentiation in the presence of TGF-beta3. Spatial differences in sGAG accumulation in response to both TGF-beta3 stimulation and dynamic compression were observed within the constructs. sGAG release from the engineered construct into the surrounding culture media was also dependent on TGF-beta3 stimulation, but was not effected by dynamic compression. Continued supplementation with TGF-beta3 appeared to be a more potent chondrogenic stimulus than the application of 1 h of daily dynamic compression following cytokine initiated differentiation. In the context of cartilage tissue engineering, the results of this study suggest that MSC seeded constructs should be first allowed to undergo chondrogenesis in vitro prior to implantation in a load bearing environment.

The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.

Among the several changes that occur in the aged brain is an increase in the concentration of the proinflammatory cytokine interleukin-1␤ that is coupled with a deterioration in cell function. This study investigated the possibility that treatment with the polyunsaturated fatty acid eicosapentaenoic acid might prevent interleukin-1␤-induced deterioration in neuronal function. Assessment of four markers of apoptotic cell death, cytochrome c translocation, caspase-3 activation, poly-(ADP-ribose) polymerase cleavage, and terminal dUTP nick-end staining, revealed an age-related increase in each of these measures, and the evidence presented indicates that treatment of aged rats with eicosapentaenoate reversed these changes as well as the accompanying increases in interleukin-1␤ concentration and p38 activation. The data are consistent with the idea that activation of p38 plays a significant role in inducing the changes described since interleukin-1␤-induced activation of cytochrome c translocation and caspase-3 activation in cortical tissue in vitro were reversed by the p38 inhibitor SB203580. The age-related increases in interleukin-1␤ concentration and p38 activation in cortex were mirrored by similar changes in hippocampus. These changes were coupled with an age-related deficit in long term potentiation in perforant path-granule cell synapses, while eicosapentaenoate treatment was associated with reversal of age-related changes in interleukin-1␤ and p38 and with restoration of long term potentiation. Increased expression of the proinflammatory cytokine interleukin-1␤ (IL-1␤)1 has been linked with neurodegenerative disorders like Down's syndrome, Alzheimer's disease, and Parkinson's disease (1, 2). Consistent with the view that IL-1␤ plays a role in deterioration of cell function are the findings that IL-1␤ expression is increased, in parallel with cell damage, in experimental models of ischemia (3), excitotoxicity (4), and traumatic lesions (5). Indeed, IL-1␤ has been shown to trigger cell death in primary cultures of human fetal neurons (6) and inhibition of caspase-1, which leads to formation of active IL-1␤, and blocks lipopolysaccharide-induced changes in cell morphology, which are consistent with cell death (7).IL-1␤ has been shown to stimulate the mitogen-activated protein kinases p38 and c-Jun NH 2 -terminal kinase (8, 9), and activation of both c-Jun NH 2 -terminal kinase (10, 11) and p38 (10, 12-16) has been closely linked with apoptotic cell death. Significantly, an increase in p38 activity has been coupled with apoptotic changes in Alzheimer's disease (17, 18). Concomitant increases in IL-1␤ concentration and p38 activity have been reported in the aged rat brain (19 -21); in hippocampus these changes are correlated with compromised synaptic function and with an age-related impairment in long term potentiation (LTP) (19 -22), while consistent with the high expression of IL-1␤ and IL-1RI in hippocampus is the finding that the cytokine depresses LTP in dentate gyrus (8,19,20,23,24). Significantly, we have ...

Amyloid-␤ (A␤) is a major constituent of the neuritic plaque found in the brain of Alzheimer's disease patients, and a great deal of evidence suggests that the neuronal loss that is associated with the disease is a consequence of the actions of A␤. In the past few years, it has become apparent that activation of c-Jun N-terminal kinase (JNK) mediates some of the effects of A␤ on cultured cells; in particular, the evidence suggests that A␤-triggered JNK activation leads to cell death. In this study, we investigated the effect of intracerebroventricular injection of A␤ (1-40) on signaling events in the hippocampus and on long term potentiation in Schaffer collateral CA1 pyramidal cell synapses in vivo. We report that A␤ One of the pathological hallmarks of Alzheimer's disease (AD) 1 is an accumulation of plaques consisting predominately of amyloid-␤ (A␤) peptide, which is processed from amyloid precursor protein by the action of ␤-and ␥-secretase (1). Neuronal cell loss is one feature of AD, and evidence from analysis of changes in cultured cells suggests that A␤ acts as the executioner. Thus, neuronal cultures exposed to A␤ demonstrate signs of apoptosis (2-4), and previous evidence from this laboratory has revealed that cultured cortical neurons exposed to A␤ exhibited increased expression of the tumor suppressor p53; increased activation of caspase-3, a marker of apoptotic cell death; and increased TUNEL reactivity (5). The evidence is consistent with the idea that activation of the stress-activated protein kinase, c-Jun N-terminal kinase (JNK) played a significant role, because depletion of JNK1 following exposure to antisense oligonucleotide prevented the effects of A␤ (5). Similarly, Morishima et al. (6) reported that A␤ increased phosphorylation of JNK and c-Jun in cultured cortical neurons and that these changes were associated with expression of the death inducer Fas ligand (FasL). Others have reported findings that support a role for JNK activation in mediating at least certain effects of A␤. For instance, A␤-induced parallel increases in JNK activation and TUNEL reactivity in PC12 cells (7), whereas activation of JNK was shown to be localized to amyloid deposits in 7-and 12-month-old mice that overexpress amyloid precursor protein (8).It has emerged in several experimental models that increased JNK phosphorylation is associated with deficits in synaptic function; for instance, increased activation of JNK has been reported in the hippocampi of aged rats (9, 10), rats exposed to whole body irradiation (11), and rats injected with the proinflammatory cytokine, interleukin (IL)-1␤ (12) or lipopolysaccharide (13), and in all cases glutamate release was decreased. In each of these experimental conditions, long term potentiation (LTP), a model of synaptic plasticity, was markedly impaired, and this impairment was coupled with an increased hippocampal concentration of IL-1␤.A number of groups have reported that A␤ administration exerts an inhibitory effect on LTP. For instance, A␤ peptides (14 -16) and natu...

A role for mechanical stimulation in the control of cell fate has been proposed and mechanical conditioning of mesenchymal stem cells (MSCs) is of interest in directing MSC behavior for tissue engineering applications. This study investigates strain-induced differentiation and proliferation of MSCs, and investigates the cellular mechanisms of mechanotransduction. MSCs were seeded onto a collagen-coated silicone substrate and exposed to cyclic tensile mechanical strain of 2.5% at 0.17 Hz for 1-14 days. To examine mechanotransduction, cells were strained in the presence of the stretch-activated cation channel (SACC) blocker, gadolinium chloride (GdCl(3)); the extracellular regulated kinase (ERK) inhibitor, U0126; the p38 inhibitor, SB203580; and the phosphatidylinosito1 3-kinase (PI3-kinase) inhibitor, LY294002. Following exposure to strain, the osteogenic markers Cbfalpha1, collagen type I, osteocalcin, and BMP2 were temporally expressed. Exposure to strain in the presence of GdCl(3) (10 microM) reduced the induction of collagen I expression, thus identifying a role for SACC, at least in part, as mechanosensors in strain-induced MSC differentiation. The strain-induced synthesis of BMP2 was found to be reduced by inhibitors of the kinases, ERK, p38, and PI3 kinase. Additionally, mechanical strain reduced the rate of MSC proliferation. The identification of the mechanical control of MSC proliferation and the molecular link between mechanical stimulation and osteogenic differentiation has consequences for regenerative medicine through the development of a functional tissue engineering approach.