Hypertrophy of the ligamentum flavum (HLF) is one of the common causes of lumbar spinal stenosis (LSS). The key molecules and mechanisms responsible for HLF remain unclear. Here, we used an integrated transcriptome and proteomics analysis of human ligamentum flavum (LF), and subsequent immunohistochemistry and real-time PCR assays, to show upregulation of CRLF1 to be the dominant response to HLF. TGF-β1 significantly increased mRNA expression of CRLF1 through SMAD3 pathway. CRLF1 enhanced LF fibrosis via ERK signaling pathway at the post-transcriptional level and was required for the pro-fibrotic effect of TGF-β1. Knockdown of CRLF1 was shown here to reduce fibrosis caused by inflammatory cytokines and mechanical stress. Furthermore, we found that bipedal standing posture can cause HLF and upregulation of CRLF1 expression in mice LF. Overexpression of CRLF1 was indicated to cause HLF in vivo, whereas CRLF1 knockdown impeded the formation of HLF in bipedal standing mice. These results revealed a crucial role of CRLF1 in LF hypertrophy. We propose that inhibition of CRLF1 is a potential therapeutic strategy to treat HLF.

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Objective: To explore the main causes of hypertrophied ligamentum flavum (HLF) and the possibility of using bipedal standing mouse model to simulate the pathological changes in human HLF.Methods: Thirty-two 8-week-old C57BL/6 male mice were randomly assigned to the experimental group (n = 16) and control group (n = 16). In the experimental group, mice were induced to adopt a bipedal standing posture by their hydrophobia. The experimental mice were maintained bipedal standing for 8 h a day with an interval of 2 h to consume food and water. The control mice were placed in a similar environment without bipedal standing. Eight 18-month-old C57BL/6 male mice were compared to evaluate the LF degeneration due to aging factor. Three-dimensional (3D) reconstruction and finite element models were carried out to analyze the stress and strain distribution of the mouse LF in sprawling and bipedal standing postures. Hematoxylin and Eosin (HE), Verhoeff-Van Gieson (VVG), and immunohistochemistry (IHC) staining were used to evaluate the LF degeneration of mice and humans. RT-qPCR and immunofluorescence analysis were used to evaluate the expressions of fibrosis-related factors and inflammatory cytokines of COL1A1, COL3A1, α-SMA, MMP2, IL-1β, and COX-2. Results:The von Mises stress (8.85 Â 10 À2 MPa) and maximum principal strain (6.64 Â 10 À1 ) in LF were increased 4944 and 7703 times, respectively, in bipedal standing mice. HE staining showed that the mouse LF area was greater in the bipedal standing 10-week-old group ([10.01 AE 2.93] Â 10 4 μm 2 ) than that in the control group ([3.76 AE 1.87] Â 10 4 μm 2 ) and 18-month-old aged group ([6.09 AE 2.70] Â 10 4 μm 2 ). VVG staining showed that the HLF of mice (3.23 AE 0.58) and humans (2.23 AE 0.31) had a similar loss of elastic fibers and an increase in collagen fibers. The cell density was higher during the process of HLF in mice (39.63 AE 4.81) and humans (23.25 AE 2.05). IHC staining showed that the number of α-SMA positive cells were significantly increased in HLF of mice (1.63 AE 0.74) and humans (3.50 AE 1.85). The expressions of inflammatory cytokines and fibrosis-related factors of COL1A1, COL3A1, α-SMA, MMP2, IL-1β, and COX-2 were consistently higher in bipedal standing group than the control group. Conclusion:Our study suggests that 3D finite element models can help analyze the abnormal stress and strain distributions of LF in modeling mice. Mechanical stress is the main cause of hypertrophied ligamentum flavum compared to aging. The bipedal standing mice model can reflect the pathological characteristics of human HLF. The bipedal standing mice model can provide a standardized condition to elucidate the molecular mechanisms of mechanical stress-induced HLF in vivo.

Lumbar spinal stenosis (LSS) is a common condition in elderly patients, which is associated with back pain, lower extremity pain, and neurogenic claudication owing to degenerative changes. 1 Ligamentum flavum hypertrophy (LFH) has been considered as a major cause of LSS development. 2 LFH results from ligamentum flavum tissue fibrosis brought about by inflammation and stress. [3][4][5][6][7] Pathological LFH exhibits the loss of elastic fibers and an increase in collagen fibers, suggesting the fibrotic changes. 3 Several inflammatory cytokines, including transforming growth factor-β1 (TGF-β1),

Ligamentum flavum hypertrophy (LFH) is a major cause of lumbar spinal canal stenosis (LSCS). The pathomechanisms for LFH have not been fully elucidated. Isobaric tags for relative and absolute quantitation (iTRAQ) technology, proteomics assessments of human ligamentum flavum (LF), and successive assays were performed to explore the effect of clusterin (CLU) upregulation on LFH pathogenesis. LFH samples exhibited higher cell positive rates of the CLU, TGF-β1, α-SMA, ALK5 and p-SMAD3 proteins than non-LFH samples. Mechanical stress and TGF-β1 initiated CLU expression in LF cells. Notably, CLU inhibited the expression of mechanical stress-stimulated and TGF-β1-stimulated COL1A2 and α-SMA. Mechanistic studies showed that CLU inhibited mechanical stress-stimulated and TGF-β1-induced SMAD3 activities through suppression of the phosphorylation of SMAD3 and by inhibiting its nuclear translocation by competitively binding to ALK5. PRKD3 stabilized CLU protein by inhibiting lysosomal distribution and degradation of CLU. CLU attenuated mechanical stress-induced LFH in vivo. In summary, the findings showed that CLU attenuates mechanical stress-induced LFH by modulating the TGF-β1 pathways in vitro and in vivo. These findings imply that CLU is induced by mechanical stress and TGF-β1 and inhibits LF fibrotic responses via negative feedback regulation of the TGF-β1 pathway. These findings indicate that CLU is a potential treatment target for LFH.

ObjectiveMechanical stress is an important risk factor for intervertebral disc degeneration (IVDD). Angiopoietin‐2 (ANG‐2) is regulated by mechanical stress and is widely involved in the regulation of extracellular matrix metabolism. In addition, the signaling cascade between HIF‐1α and NF‐κB is critical in matrix degradation. This study aims to investigate the role and molecular mechanism of ANG‐2 in regulating the degeneration of annulus fibrosus (AF) through the HIF‐1α/NF‐κB signaling pathway.MethodsThe bipedal standing mice IVDD model was constructed, and histological experiments were used to evaluate the degree of IVDD and the expression of ANG‐2 in the AF. Mouse primary AF cells were extracted in vitro and subjected to mechanical stretching experiments. Western blot assay was used to detect the effect of mechanical stress on ANG‐2, and the role of the ANG‐2‐mediated HIF‐1α/NF‐κB pathway in matrix degradation. In addition, the effect of inhibiting ANG‐2 expression by siRNA or monoclonal antibody on delaying IVDD was investigated at in vitro and in vivo levels. One‐way ANOVA with the least significant difference method was used for pairwise comparison of the groups with homogeneous variance, and Dunnett's method was used to compare the groups with heterogeneous variance.ResultsIn IVDD, the expressions of catabolic biomarkers (mmp‐13, ADAMTS‐4) and ANG‐2 were significantly increased in AF. In addition, p65 expression was increased while HIF‐1α expression was significantly decreased. The results of western blot assay showed mechanical stress significantly up‐regulated the expression of ANG‐2 in AF cells, and promoted matrix degradation by regulating the activity of HIF‐1α/NF‐κB pathway. Exogenous addition of Bay117082 and CoCl2 inhibited matrix degradation caused by mechanical stress. Moreover, injection of neutralizing antibody or treatment with siRNA to inhibit the expression of ANG‐2 improved the matrix metabolism of AF and inhibited IVDD progression by regulating the HIF‐1α/NF‐κB signaling pathway.ConclusionIn IVDD, mechanical stress could regulate the HIF‐1α/NF‐κB signaling pathway and matrix degradation by mediating ANG‐2 expression in AF degeneration.