Accelerated cellular senescence in degenerate intervertebral discs: a possible role in the pathogenesis of intervertebral disc degeneration

Current evidence implicates intervertebral disc degeneration as a major cause of low back pain, although its pathogenesis is poorly understood. Numerous characteristic features of disc degeneration mimic those seen during ageing but appear to occur at an accelerated rate. We hypothesised that this is due to accelerated cellular senescence, which causes fundamental changes in the ability of disc cells to maintain the intervertebral disc (IVD) matrix, thus leading to IVD degeneration. Cells isolated from non-degenerate and degenerate human tissue were assessed for mean telomere length, senescence-associated β-galactosidase (SA-β-gal), and replicative potential. Expression of P16 ^INK4A (increased in cellular senescence) was also investigated in IVD tissue by means of immunohistochemistry. RNA from tissue and cultured cells was used for real-time polymerase chain reaction analysis for matrix metalloproteinase-13, ADAMTS 5 (a disintegrin and metalloprotease with thrombospondin motifs 5), and P16 ^INK4A . Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. In non-degenerate discs, there was an age-related increase in cellular expression of P16 ^INK4A . Cells from degenerate discs (even from young patients) exhibited increased expression of P16 ^INK4A , increased SA-β-gal staining, and a decrease in replicative potential. Importantly, there was a positive correlation between P16 ^INK4A and matrix-degrading enzyme gene expression. Our findings indicate that disc cell senescence occurs in vivo and is accelerated in IVD degeneration. Furthermore, the senescent phenotype is associated with increased catabolism, implicating cellular senescence in the pathogenesis of IVD degeneration.     

Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1β and TNFα expression profile

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263–275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263–275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy.     

Catabolic cytokine expression in degenerate and herniated human intervertebral discs: IL-1beta and TNFalpha expression profile

Low back pain is a common and debilitating disorder. Current evidence implicates intervertebral disc (IVD) degeneration and herniation as major causes, although the pathogenesis is poorly understood. While several cytokines have been implicated in the process of IVD degeneration and herniation, investigations have predominately focused on Interleukin 1 (IL-1) and tumor necrosis factor alpha (TNFalpha). However, to date no studies have investigated the expression of these cytokines simultaneously in IVD degeneration or herniation, or determined which may be the predominant cytokine associated with these disease states. Using quantitative real time PCR and immunohistochemistry we investigated gene and protein expression for IL-1beta, TNFalpha and their receptors in non-degenerate, degenerate and herniated human IVDs. IL-1beta gene expression was observed in a greater proportion of IVDs than TNFalpha (79% versus 59%). Degenerate and herniated IVDs displayed higher levels of both cytokines than non-degenerate IVDs, although in degenerate IVDs higher levels of IL-1beta gene expression (1,300 copies/100 ng cDNA) were observed compared to those of TNFalpha (250 copies of TNFalpha/100 ng cDNA). Degenerate IVDs showed ten-fold higher IL-1 receptor gene expression compared to non-degenerate IVDs. In addition, 80% of degenerate IVD cells displayed IL-1 receptor immunopositivity compared to only 30% of cells in non-degenerate IVDs. However, no increase in TNF receptor I gene or protein expression was observed in degenerate or herniated IVDs compared to non-degenerate IVDs. We have demonstrated that although both cytokines are produced by human IVD cells, IL-1beta is expressed at higher levels and in more IVDs, particularly in more degenerate IVDs (grades 4 to 12). Importantly, this study has highlighted an increase in gene and protein production for the IL-1 receptor type I but not the TNF receptor type I in degenerate IVDs. The data thus suggest that although both cytokines may be involved in the pathogenesis of IVD degeneration, IL-1 may have a more significant role than TNFalpha, and thus may be a better target for therapeutic intervention.     

TM-25659-Induced Activation of FGF21 Level Decreases Insulin Resistance and Inflammation in Skeletal Muscle via GCN2 Pathways

The TAZ activator 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2′-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) inhibits adipocyte differentiation by interacting with peroxisome proliferator-activated receptor gamma. TM-25659 was previously shown to decrease weight gain in a high fat (HF) diet-induced obesity (DIO) mouse model. However, the fundamental mechanisms underlying the effects of TM-25659 remain unknown. Therefore, we investigated the effects of TM-25659 on skeletal muscle functions in C2 myotubes and C57BL/6J mice. We studied the molecular mechanisms underlying the contribution of TM-25659 to palmitate (PA)-induced insulin resistance in C2 myotubes. TM-25659 improved PA-induced insulin resistance and inflammation in C2 myotubes. In addition, TM-25659 increased FGF21 mRNA expression, protein levels, and FGF21 secretion in C2 myotubes via activation of GCN2 pathways (GCN2-phosphoeIF2α-ATF4 and FGF21). This beneficial effect of TM-25659 was diminished by FGF21 siRNA. C57BL/6J mice were fed a HF diet for 30 weeks. The HF-diet group was randomly divided into two groups for the next 14 days: the HF-diet and HF-diet + TM-25659 groups. The HF diet + TM-25659-treated mice showed improvements in their fasting blood glucose levels, insulin sensitivity, insulin-stimulated Akt phosphorylation, and inflammation, but neither body weight nor food intake was affected. The HF diet + TM-25659-treated mice also exhibited increased expression of both FGF21 mRNA and protein. These data indicate that TM-25659 may be beneficial for treating insulin resistance by inducing FGF21 in models of PA-induced insulin resistance and HF diet-induced insulin resistance.     

Synthesis of cinnamoyl ketoamides as hybrid structures of antioxidants and calpain inhibitors

The excessive calpain activation causes serious cellular damage or even cell death in neurological disorders such as stroke and Alzheimer’s disease. Oxidative stress has also been implicated in the initiation or progression of neurodegenerative diseases. In the present studies, a series of cinnamoyl ketoamides 4a-4j were synthesized as hybrid structures of antioxidants and calpain inhibitors. Cinnamoyl ketoamides, possessing an alkyl chain at the α-position, showed potent μ-calpain inhibitory activities indicating that the cinnamoyl skeleton can be regarded as an acyclic variant of calpain inhibitory chromone carboxamide 2. Among synthesized, compound 4e was the most potent inhibitor of μ-calpain (IC₅₀ = 0.13 μM) and also exhibited strong antioxidant activities in DPPH and superoxide anion radical scavenging and lipid peroxidation inhibition assay systems.     

Large-scale plantlets conversion from cotyledonary somatic embryos of Kalopanax septemlobus tree using bioreactor cultures

A suitable bioreactor system for large scale embryo-to-plantlets conversion of Kalopanax septemlobus was established. In temporary immersion with net (TIN) bioreactor, 85% of embryos successfully produced plantlets whereas in continuous immersion with net (CIN) bioreactor, only conversion rate of 29.3% was obtained. Embryos cultured in TIN bioreactor produced more vigorous plantlets in terms of fresh weight, height, root length, roots and leaves quantity. In CIN bioreactor, Kalopanax plantlets showed high malondialdehyde (MDA) content and increased activities of reactive oxygen species (ROS)-processing enzymes, such as ascorbate peroxidase (APX) and glutathione reductase (GR) indicating the occurrence of oxidative stress. However, superoxide dismutase (SOD) and catalase (CAT) showed similar activities in plantlets grown in different bioreactors. Kalopanax plantlets grown in both TIN and CIN bioreactors were harvested and transferred to greenhouse for their acclimatization. Plantlets grown in CIN bioreactor exhibited low survival rate (75.8%) compared to those grown in TIN bioreactor (100%). MDA content decreased with progression of acclimatization indicating a decrease in oxidative stress. However, MDA level in CIN derived plantlets was higher than TIN derived plantlets. In TIN derived plantlets, an increase in SOD and GR activities were observed after 1 week and thereafter decreased. CAT activity decreased while APX activity started to increase after 1 week of acclimatization. The results indicated that Kalopanax plantlets were able to overcome oxidative stress mainly through SOD activity. However, levels of antioxidant enzyme activities were higher in CIN derived plantlets than TIN derived plantlets. Kalopanax plantlets obtained from TIN bioreactor performed better during the acclimatization phase and showed higher survival rate than material obtained on CIN bioreactor or conventional culture systems.     

Production of cephalosporin C using crude glycerol in fed-batch culture of <Emphasis Type="Italic">Acremonium chrysogenum</Emphasis> M35

In this study, cephalosporin C production by Acremonium chrysogenum M35 cultured with crude glycerol instead of rice oil and methionine was investigated. The addition of crude glycerol increased cephalosporin C production by 6-fold in shake-flask culture, and also the amount of cysteine. In fed-batch culture without methionine, crude glycerol resulted only in overall improvement in cephalosporin C production (about 700%). In addition, A. chrysogenum M35 became highly differentiated in fed-batch culture with crude glycerol, compared with the differentiation in batch culture. The results presented here suggest that crude glycerol can replace methionine and plant oil as cysteine and carbon sources during cephalosporin C production by A. chrysogenum M35.