Although mechanical stress as a result of spinal instability is known to cause hypertrophy of the ligameiitum flavum resulting in degenerative spinal canal stenosis, the mechanism of the ligament hypertrophy is not well understood. In the present study, we investigated the effect of mechanical stretching force on collagen synthesis and transforming growth factor-/3 1 (TGF-PI) production using ligament cells isolated from human ligamentum flavum in vitro. Ligamenturn flavum cells (LFCs) were isolated from human ligamenturn flavum obtained from patients who underwent lumbar spine surgery. The LFCs were subjected to a mechanical stretching force using a cominercially available stretching device that physically deformed the cells. Collagen synthesis and TGF-PI production levels in the LFCs were then examined. Notable increases were observed in the gene expressions of collagen types I, 111, and V in LFCs subjected to mechanical stretching force. The increase in collagen gene expression of LFCs was inhibited in the presence of anti-TGF-pl antibodies. Production of TGF-PI by the LFCs also increased significantly by the mechanical stretching force. Exogenous application of TGF-PI was confirmed to increase collagen synthesis of the LFCs. This data indicated that mechanical stretching force can promote TGF-Dl production by LFCs, resulting in hypertrophy of the ligament.

Loss of Sirt1 in chondrocytes led to the accelerated development of OA in mice. Our observations suggest that SIRT1 has a preventive role against the development of OA.

Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

In this study, we examined the effects of overexpression of SIRT1 on IL-1b-induced gene expression changes in human chondrocytes to explore a protective role of SIRT1 in human chondrocytes. SIRT1 was overexpressed in human chondrocytes by expression plasmid under stimulation with IL-1b. SIRT1 was also inhibited by siRNA under stimulation with IL-1b. Gene expression changes were examined by real-time PCR. The interaction of SIRT1 and p65 (NF-kB) were examined by Western blotting. SIRT1, MMP-13, and ADAMTS-5 expressions in human cartilage were examined by immunohistochemistry. IL-1b stimulation significantly up-regulated MMP-1, 2, 9, and 13 and ADAMTS-5. Overexpression of SIRT1 significantly inhibited the up-regulation of those genes caused by IL-1b while the inhibition of SIRT1 further increased them. In addition, the overexpression of SIRT1 markedly reduced the IL-1b-induced acetylation of p65. SIRT1 expression was clearly detected in the non-OA cartilage while MMP-13 and ADAMTS-5 were undetectable. In contrast, in the OA cartilage, SIRT1 expression was decreased while MMP-13 and ADAMTS-5 were increased. Our observations suggested that SIRT1 can play a protective role by suppressing IL-1b-induced expressions of cartilage-degrading enzymes partially through the modulation of the NF-kB pathway. SIRT1 overexpression might be a new therapeutic approach for OA. ß