CTSD upregulation as a key driver of spinal ligament abnormalities in spinal stenosis.

Spinal stenosis (SS) is frequently caused by spinal ligament abnormalities, such as ossification and hypertrophy, which narrow the spinal canal and compress the spinal cord or nerve roots, leading to myelopathy or sciatic symptoms; however, the underlying pathological mechanism is poorly understood, hampering the development of effective nonsurgical treatments. Our study aims to investigate the role of co-expression hub genes in patients with spinal ligament ossification and hypertrophy. To achieve this, we conducted an integrated analysis by combining RNA-seq data of ossification of the posterior longitudinal ligament (OPLL) and microarray profiles of hypertrophy of the ligamentum flavum (HLF), consistently pinpointing CTSD as an upregulated hub gene in both OPLL and HLF. Subsequent RT-qPCR and IHC assessments confirmed the heightened expression of CTSD in human OPLL, ossification of the ligamentum flavum (OLF), and HLF samples. We observed an increase in CTSD expression in human PLL and LF primary cells during osteogenic differentiation, as indicated by western blotting (WB). To assess CTSD's impact on osteogenic differentiation, we manipulated its expression levels in human PLL and LF primary cells using siRNAs and lentivirus, as demonstrated by WB, ALP staining, and ARS. Our findings showed that suppressing CTSD hindered the osteogenic differentiation potential of PLL and LF cells, while overexpressing CTSD activated osteogenic differentiation. These findings identify CTSD as a potential therapeutic target for treating spinal stenosis associated with spinal ligament abnormalities.

Evaluation of differences in expression pattern of three isoforms of the transforming growth factor beta in patients with lumbosacral stenosis.

The study investigates molecular changes in the lumbosacral (L/S) spine's yellow ligamentum flavum during degenerative stenosis, focusing on the role of transforming growth factor beta 1-3 (TGF-ß-1-3). Sixty patients with degenerative stenosis and sixty control participants underwent molecular analysis using real-time quantitative reverse transcription reaction technique (RTqPCR), enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemical analysis (IHC). At the mRNA level, study samples showed reduced expression of TGF-ß-1 and TGF-ß-3, while TGF-ß-2 increased by only 4%. Conversely, at the protein level, the study group exhibited significantly higher concentrations of TGF-ß-1, TGF-ß-2, and TGF-ß-3 compared to controls. On the other hand, at the protein level, a statistically significant higher concentration of TGF-ß-1 was observed (2139.33 pg/mL ± 2593.72 pg/mL vs. 252.45 pg/mL ± 83.89 pg/mL; p < 0.0001), TGF-ß-2 (3104.34 pg/mL ± 1192.74 pg/mL vs. 258.86 pg/mL ± 82.98 pg/mL; p < 0.0001), TGF-ß-3 (512.75 pg/mL ± 107.36 pg/mL vs. 55.06 pg/mL ± 9.83 pg/mL, p < 0.0001) in yellow ligaments obtained from patients of the study group compared to control samples. The study did not establish a significant correlation between TGF-ß-1-3 concentrations and pain severity. The findings suggest that molecular therapy aimed at restoring the normal expression pattern of TGF-ß-1-3 could be a promising strategy for treating degenerative stenosis of the L/S spine. The study underscores the potential therapeutic significance of addressing molecular changes at the TGF-ß isoforms level for better understanding and managing degenerative spinal conditions.

Biomarkers related to hypertrophy of the ligamentum flavum: a systematic review of the literature.

OBJECTIVE: Spinal stenosis is one of the most common spinal disorders in the elderly. Hypertrophy of the ligamentum flavum (HLF) can contribute to spinal stenosis. The current literature suggests that various biomarkers may play important roles in the pathogenesis of HLF. However, the connection between these biomarkers and the development of HLF is still not well understood. This systematic review aims to explore the current literature on biomarkers related to the development of HLF. METHODS: A literature search was conducted using PubMed, Embase, Web of Science, and Cochrane Library. The search strategy looked for the titles, abstracts, and keywords of studies that contained a combination of the following phrases: "ligamentum flavum OR yellow ligament," "biomarkers," and "hypertrophy." Recorded data included study design, demographic characteristics (number of patients of each gender and mean age), study period, country where the study was conducted, biomarkers, and diagnostic modalities used. Risk of bias was assessed using the Newcastle-Ottawa Scale for case-control studies. RESULTS: The authors identified 39 studies. After screening, 26 full-text original articles assessing one or more biomarkers related to HLF were included. The included studies were conducted over a 22-year period. The most popular biomarkers studied, in order of frequency reported, were collagen types I and III (n = 10), transforming growth factor ß (TGF-ß) (n = 8), and interleukin (IL)-6 (n = 6). The authors found that mechanical stretching forces, tissue inhibitor of metalloproteinases 2 (TIMP-2) induction, and TGF-ß were associated with increased amounts of collagen I and III. IL-6 expression was increased by microRNA-21, as well as by leptin, through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. CONCLUSIONS: Biomarkers such as TGF-ß, IL-6, and collagen I and III have been consistently correlated with the development of HLF. However, the pathogenesis of HLF remains unclear due to the heterogeneity of the studies, patient populations, and research at the molecular level. Further studies are necessary to better characterize the pathogenesis of HLF and provide a more comprehensive understanding of how these biomarkers may aid in the diagnosis and treatment of HLF.

Increased matrix metalloproteinase-2 in ligamentum flavum hypertrophy and the regulation of MMP-2/TIMPs by elastin-derived peptides.

The study aimed to examine matrix metalloproteinase-2 (MMP-2) expression in a rat ligamentum flavum (LF) hypertrophy model in vivo, and the effect of elastin-derived peptides (EDPs) on MMP-2 and tissue inhibitors of metalloproteinases (TIMPs) in rat LF cells in vitro. Surgical destabilization was performed at the rat spinal L3/4 level to induce increased mechanical stress. Rats were killed at 6- and 12-weeks postsurgery for histological staining, immunohistochemical staining, RT-qPCR and western blot. 100 µg/mL EDPs were applied to isolated normal rat LF cells, with or without pretreatment of elastin receptor complex (ERC) inhibitors, to assess the expression of MMP-2, TIMP-1, and TIMP-2. Spinal destabilization led to LF hypertrophy, observed through increased LF thickness and area, along with histological changes of chondrometaplasia and elastic fiber degradation. LF was also stained positively for Col I and Col II, where elastic fiber has broken down. MMP-2 expression was notably elevated in the hypertrophied LF, accompanied by increased TIMP-2 and TIMP-3 levels. EDPs were found to suppress MMP-2 expression and reduce TIMP-1 and TIMP-2 levels in rat LF cells. Interestingly, exposure to EDPs led to a significant rise in MMP-2/TIMP-1 and MMP-2/TIMP-2 ratios, dependent on the ERC. Collectively, the study suggests that increased MMP-2 activity contributes to elastic fiber degradation in hypertrophied LF, generating EDPs that further enhance the MMP-2/TIMPs ratio in LF cells in an ERC-dependent manner. Further research is essential to delve into the mechanisms of EDPs in LF hypertrophy.

Comparative Gene-Expression Analysis of the Ligamentum Flavum of Patients with Lumbar Spinal Canal Stenosis: Comparison between the Dural and Dorsal Sides of the Thickened Ligamentum Flavum.

Thickening of the ligamentum flavum is the main factor in the development of lumbar spinal canal stenosis (LSCS). Although previous studies have reported factors related to ligamentum flavum thickening, its etiology has not been clarified. Furthermore, it is often difficult to set proper controls to investigate the pathologies of thickening due to differences in patient characteristics, such as age, sex, obesity, and comorbidities. This study aimed to elucidate the pathologies of ligamentum flavum thickening by comparing the dural and dorsal sides of the thickened ligamentum flavum in patients with LSCS. Ligamentum flavum samples were collected from 19 patients with LSCS. The samples were divided into the dural and dorsal sides. The dural side was used as a control to assess the pathologies occurring on the dorsal side. Elastic Masson staining was used to assess the elastic fibres. Gene expression levels were comprehensively assessed using quantitative reverse transcription polymerase chain reaction and DNA microarray analyses. Gene ontology analysis was used to identify biological processes associated with differentially expressed genes. The elastic fibres were significantly decreased on the dorsal side of the thickened ligamentum flavum. Genes related to fibrosis, inflammation, tissue repair, remodeling, and chondrometaplasia, such as COL1A2, COL3A1, COL5A1, TGFB1, VEGFA, TNFA, MMP2, COL10A1, and ADAMTS4, were highly expressed on the dorsal side of the thickened ligamentum flavum. The biological processes occurring on the dorsal side of the thickened ligamentum flavum were extracellular matrix organization, cell adhesion, extracellular matrix disassembly, and proteolysis.These are considered important pathologies of ligamentum flavum thickening.

Thrombospondin-1 promotes mechanical stress-mediated ligamentum flavum hypertrophy through the TGFß1/Smad3 signaling pathway.

Lumbar spinal canal stenosis is primarily caused by ligamentum flavum hypertrophy (LFH), which is a significant pathological factor. Nevertheless, the precise molecular basis for the development of LFH remains uncertain. The current investigation observed a notable increase in thrombospondin-1 (THBS1) expression in LFH through proteomics analysis and single-cell RNA-sequencing analysis of clinical ligamentum flavum specimens. In laboratory experiments, it was demonstrated that THBS1 triggered the activation of Smad3 signaling induced by transforming growth factor ß1 (TGFß1), leading to the subsequent enhancement of COL1A2 and α-SMA, which are fibrosis markers. Furthermore, experiments conducted on a bipedal standing mouse model revealed that THBS1 played a crucial role in the development of LFH. Sestrin2 (SESN2) acted as a stress-responsive protein that suppressed the expression of THBS1, thus averting the progression of fibrosis in ligamentum flavum (LF) cells. To summarize, these results indicate that mechanical overloading causes an increase in THBS1 production, which triggers the TGFß1/Smad3 signaling pathway and ultimately results in the development of LFH. Targeting the suppression of THBS1 expression may present a novel approach for the treatment of LFH.

PTGR1 is involved in cell proliferation in thoracic ossification of the ligamentum flavum.

Thoracic ossification of the ligamentum flavum (TOLF) is a heterotopic ossification of spinal ligaments, leading to serious myelopathy. TOLF underlying mechanisms are not well understood. Our iTRAQ analysis have identified ten inflammatory factors related to TOLF, including l. We found that PTGR1 expressions increased in TOLF by RT-PCR and western blot in this study. Both cell proliferation and differentiation are important for the process of bone formation. In our previous study, we demonstrated that TOLF primary cells grew faster than control cells. It was reported that knockdown of PTGR1 inhibited cell proliferation. We hypothesize that PTGR1 may participate in cell proliferation in TOLF. To test this hypothesis, TOLF primary cells were treated for 24h with PTGR1. We observed that PTGR1 increased cell proliferation. The effect of PTGR1 on cell proliferation related genes was examined in TOLF primary cells. Our results showed that PTGR1 was able to activate expressions of c-Myc and CyclinD1. Moreover, blocking JNK pathway by selective JNK inhibitor SP600125 eliminated the positive effect of PTGR1 on c-Myc expression, indicating that PTGR1 activated the expression of c-Myc via JNK pathway. Our new findings suggest that PTGR1 is involved in cell proliferation of TOLF.

ACSM5 inhibits ligamentum flavum hypertrophy by regulating lipid accumulation mediated by FABP4/PPAR signaling pathway.

BACKGROUND: Ligamentum flavum (LF) hypertrophy is the main cause of lumbar spinal canal stenosis (LSCS). Previous studies have shown that LF hypertrophy tissue exhibits abnormal lipid accumulation, but the regulatory mechanism remains unclear. The objective of this study was to explore the function and potential mechanism of ACSM5 in LF lipid accumulation. METHODS: To assess the ACSM5 expression levels, lipid accumulation and triglyceride (TG) level in LF hypertrophy and normal tissue, we utilized RT-qPCR, western blot, oil red O staining, and TG assay kit. The pearson correlation coefficient assay was used to analyze the correlation between ACSM5 levels and lipid accumulation or TG levels in LF hypertrophy tissue. The role of ACSM5 in free fatty acids (FFA)-induced lipid accumulation in LF cells was assessed in vitro, and the role of ACSM5 in LF hypertrophy in mice was verified in vivo. To investigate the underlying mechanisms of ACSM5 regulating lipid accumulation in LF, we conducted the mRNA sequencing, bioinformatics analysis, and rescue experiments. RESULTS: In this study, we found that ACSM5, which was significantly down-regulated in LF tissues, correlated with lipid accumulation. In vitro cell experiments demonstrated that overexpression of ACSM5 significantly inhibited FFA-induced lipid accumulation and fibrosis in LF cells. In vivo animal experiments further confirmed that overexpression of ACSM5 inhibited LF thickening, lipid accumulation, and fibrosis. Mechanistically, ACSM5 inhibited lipid accumulation of LF cells by inhibiting FABP4-mediated PPARγ signaling pathway, thereby improving hypertrophy and fibrosis of LF. CONCLUSIONS: our findings elucidated the important role of ACSM5 in the regulation of LF lipid accumulation and provide insight into potential therapeutic interventions for the treatment of LF hypertrophy. This study further suggested that therapeutic strategies targeting lipid deposition may be an effective potential approach to treat LF hypertrophy-induced LSCS.

Transthyretin amyloid deposition in ligamentum flavum (LF) is significantly correlated with LF and epidural fat hypertrophy in patients with lumbar spinal stenosis.

Lumbar spinal stenosis (LSS) is a degenerative disease characterized by intermittent claudication and numbness in the lower extremities. These symptoms are caused by the compression of nerve tissue in the lumbar spinal canal. Ligamentum flavum (LF) hypertrophy and spinal epidural lipomatosis in the spinal canal are known to contribute to stenosis of the spinal canal: however, detailed mechanisms underlying LSS are still not fully understood. Here, we show that surgically harvested LFs from LSS patients exhibited significantly increased thickness when transthyretin (TTR), the protein responsible for amyloidosis, was deposited in LFs, compared to those without TTR deposition. Multiple regression analysis, which considered age and BMI, revealed a significant association between LF hypertrophy and TTR deposition in LFs. Moreover, TTR deposition in LF was also significantly correlated with epidural fat (EF) thickness based on multiple regression analyses. Mesenchymal cell differentiation into adipocytes was significantly stimulated by TTR in vitro. These results suggest that TTR deposition in LFs is significantly associated with increased LF hypertrophy and EF thickness, and that TTR promotes adipogenesis of mesenchymal cells. Therapeutic agents to prevent TTR deposition in tissues are currently available or under development, and targeting TTR could be a potential therapeutic approach to inhibit LSS development and progression.

Interleukin-17A Promotes Proliferation and Osteogenic Differentiation of Human Ligamentum Flavum Cells Through Regulation of ß-Catenin Signaling.

STUDY DESIGN: A basic experimental study. OBJECTIVE: To elucidate the role and mechanism of interleukin (IL)-17A in thoracic ossification of the ligamentum flavum (TOLF). SUMMARY OF BACKGROUND DATA: TOLF is characterized by the replacement of the thoracic ligamentum flavum with ossified tissue and is one of the leading causes of thoracic spinal stenosis. IL-17A is an important member of the IL-17 family that has received widespread attention for its key contributions to the regulation of bone metabolism and heterotopic ossification. However, it is unclear whether IL-17A is involved in TOLF. MATERIALS AND METHODS: Cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed to assess the proliferation of ligamentum flavum cells (LFCs). Alkaline phosphatase activity assay, Alizarin red staining, and protein level expression of osteogenic-related genes were used to evaluate the osteogenic differentiation potential of LFCs. The effect of IL-17A on the proliferation and osteogenic differentiation of LFCs was further assessed after silencing ß-catenin by transfection with small interfering RNA. In addition, the possible source of IL-17A was further demonstrated by coculture assays of T helper 17 (Th17) cells with LFCs. Student t test was used for comparisons between groups, and the one-way analysis of variance, followed by the Tukey post hoc test, was used for comparison of more than two groups. RESULTS: IL-17A was elevated in TOLF tissue compared with normal ligamentum flavum. IL-17A stimulation promoted the proliferation and osteogenic differentiation of LFCs derived from patients with TOLF. We found that IL-17A promoted the proliferation and osteogenic differentiation of LFCs by regulating the ß-catenin signaling. Coculture of Th17 cells with LFCs enhanced ß-catenin signaling-mediated proliferation and osteogenic differentiation of LFCs. However, these effects were markedly attenuated after the neutralization of IL-17A. CONCLUSIONS: This is the first work we are aware of to highlight the importance of IL-17A in TOLF. IL-17A secreted by Th17 cells in the ligamentum flavum may be involved in the ossification of the microenvironment by regulating ß-catenin signaling to promote the proliferation and osteogenic differentiation of LFCs.

Decorin: a potential therapeutic candidate for ligamentum flavum hypertrophy by antagonizing TGF-ß1.

Ligamentum flavum hypertrophy (LFH) is the main physiological and pathological mechanism of lumbar spinal canal stenosis (LSCS). The specific mechanism for LFH has not been completely clarified. In this study, bioinformatic analysis, human ligamentum flavum (LF) tissues collection and analysis, and in vitro and in vivo experiments were conducted to explore the effect of decorin (DCN) on LFH pathogenesis. Here, we found that TGF-ß1, collagen I, collagen III, α-SMA and fibronectin were significantly upregulated in hypertrophic LF samples. The DCN protein expression in hypertrophic LF samples was higher than that in non-LFH samples, but the difference was not significant. DCN inhibited the expression of TGF-ß1-induced fibrosis-associated proteins in human LF cells, including collagen I, collagen III, α-SMA, and fibronectin. ELISAs showed that TGF-ß1 can upregulate PINP and PIIINP in the cell supernatant, and this effect was inhibited after DCN administration. Mechanistic studies revealed that DCN suppressed TGF-ß1-induced fibrosis by blocking the TGF-ß1/SMAD3 signaling pathway. In addition, DCN ameliorated mechanical stress-induced LFH in vivo. In summary, our findings indicated that DCN ameliorated mechanical stress-induced LFH by antagonizing the TGF-ß1/SMAD3 signaling pathway in vitro and in vivo. These findings imply that DCN is a potential therapeutic candidate for ligamentum flavum hypertrophy.

Smurf1 Facilitates Oxidative Stress and Fibrosis of Ligamentum Flavum by Promoting Nrf2 Ubiquitination and Degradation.

Lumbar spinal stenosis (LSS), which can lead to irreversible neurologic damage and functional disability, is characterized by hypertrophy and fibrosis in the ligamentum flavum (LF). However, the underlying mechanism is still unclear. In the current study, the effect of Smurf1, a kind of E3 ubiquitin ligase, in promoting the fibrosis and oxidative stress of LF was investigated, and its underlying mechanism was explored. The expression of oxidative stress and fibrosis-related markers was assessed in the tissue of lumbar spinal stenosis (LSS) and lumbar disc herniation (LDH). Next, the expression of the top 10 E3 ubiquitin ligases, obtained from Gene Expression Omnibus (GEO) dataset GSE113212, was assessed in LDH and LSS, and confirmed that Smurf1 expression was markedly upregulated in the LSS group. Furthermore, Smurf1 overexpression promotes the fibrosis and oxidative stress of LF cells. Subsequently, NRF2, an important transcription factor for oxidative stress and fibrosis, was predicted to be a target of Smurf1. Mechanistically, Smurf1 directly interacts with Nrf2 and accelerates Nrf2 ubiquitination and degradation. In conclusion, the current study suggests that Smurf1 facilitated the fibrosis and oxidative stress of LF and induced the development of LSS by promoting Nrf2 ubiquitination and degradation.

Circular RNA PDK1 targets miR-4731-5p to enhance TNXB expression in ligamentum flavum hypertrophy.

Hypertrophic ligamentum flavum (LF) is a main factor responsible for lumbar spinal stenosis (LSS); however, the exact mechanisms of the pathogenesis of these processes remain unknown. This study aimed to elucidate whether circular RNAs and microRNAs regulate the pathogenesis of LF and LSS, especially focusing on circPDK1 (hsa_circ_0057105), a circRNA targeting pyruvate dehydrogenase kinase 1 and differentially expressed in LF tissues between lumbar disk herniation and LSS patients. The circPDK1/miR-4731 and miR-4731/TNXB (Tenascin XB) interactions were predicted and validated by luciferase reporter assay. Colony formation, wound-healing, and MTT assays were used for estimating cell proliferation and migration. Protein expression levels were evaluated using Western blotting. TNXB expression was verified using immunohistochemistry (IHC). Overexpressing circPDK1 promoted the proliferation, migration, and expression of fibrosis-related protein (alpha smooth muscle actin (α-SMA), lysyl oxidase like 2 (LOXL2), Collagen I, matrix metalloproteinase-2 (MMP-2) and TNXB) in LF whereas miR-4731-5p showed opposite effects. The expression of TNXB was promoted by circPDK1; contrary results were observed with miR-4731-5p. Co-overexpression of miR-4731-5p partially reversed the proliferative and fibrosis-prompting effects of circPDK1 or TNXB. The circPDK1-miR-4731-TNXB pathway may be proposed as a regulatory axis in LF hypertrophy, which might shed light on in-depth research of LSS, as well as providing a novel therapeutic target for LF hypertrophy-induced LSS.

Piezo1 involves in intracellular osteogenic transformation signal to promote the ossification of ligamentum flavum.

Ectopic osteogenesis refers to the occurrence of osteoblasts in soft tissues other than bone tissue and the formation of bone tissue. The ligamentum flavum is an essential connecting structure between adjacent vertebral lamina, which participates in the formation of the vertebral canal's posterior wall and maintains the vertebral body's stability. Ossification of the ligamentum flavum (OLF) is one of the manifestations of systemic ossification of the spinal ligaments and one of the degenerative diseases related to the spine. However, there is a lack of research on the expression and biological function of Piezo1 in ligamentum flavum. Whether Piezo1 participates in the development of OLF is still unclear. The FX-5000C cell or tissue pressure culture and real-time observation and analysis system was applied to stretch ligamentum flavum cells to detect the expression of mechanical stress channel and osteogenic markers after the effect of different stretching durations. The results showed elevated expression of mechanical stress channel Piezo1 and osteogenic markers with the effect of tensile time duration. In conclusion, Piezo1 involves in intracellular osteogenic transformation signal to promote the ossification of ligamentum flavum. An approved explanatory model and further research will be required in the future.

Periostin increased by mechanical stress upregulates interleukin-6 expression in the ligamentum flavum.

Ligamentum flavum (LF) hypertrophy is a major cause of lumbar spinal canal stenosis. Although mechanical stress is thought to be a major factor involved in LF hypertrophy, the exact mechanism by which it causes hypertrophy has not yet been fully elucidated. Here, changes in gene expression due to long-term mechanical stress were analyzed using RNA-seq in a rabbit LF hypertrophy model. In combination with previously reported analysis results, periostin was identified as a molecule whose expression fluctuates due to mechanical stress. The expression and function of periostin were further investigated using human LF tissues and primary LF cell cultures. Periostin was abundantly expressed in human hypertrophied LF tissues, and periostin gene expression was significantly correlated with LF thickness. In vitro, mechanical stress increased gene expressions of periostin, transforming growth factor-ß1, α-smooth muscle actin, collagen type 1 alpha 1, and interleukin-6 (IL-6) in LF cells. Periostin blockade suppressed the mechanical stress-induced gene expression of IL-6 while periostin treatment increased IL-6 gene expression. Our results suggest that periostin is upregulated by mechanical stress and promotes inflammation by upregulating IL-6 expression, which leads to LF degeneration and hypertrophy. Periostin may be a pivotal molecule for LF hypertrophy and a promising therapeutic target for lumbar spinal stenosis.

Administration of N-Acetylcysteine to Regress the Fibrogenic and Proinflammatory Effects of Oxidative Stress in Hypertrophic Ligamentum Flavum Cells.

Ligamentum flavum hypertrophy (LFH) is a major cause of lumbar spinal stenosis (LSS). In hypertrophic ligamentum flavum (LF) cells, oxidative stress activates intracellular signaling and induces the expression of inflammatory and fibrotic markers. This study explored whether healthy and hypertrophic LF cells respond differently to oxidative stress, via examining the levels of phosphorylated p38 (p-p38), inducible nitric oxide synthase (iNOS), and α-smooth muscle actin (α-SMA). Furthermore, the efficacy of N-acetylcysteine (NAC), an antioxidant, in reversing the fibrogenic and proinflammatory effects of oxidative stress in hypertrophic LF cells was investigated by assessing the expression levels of p-p38, p-p65, iNOS, TGF-ß, α-SMA, vimentin, and collagen I under H2O2 treatment with or without NAC. Under oxidative stress, p-p38 increased significantly in both hypertrophic and healthy LF cells, and iNOS was elevated in only the hypertrophic LF cells. This revealed that oxidative stress negatively affected both hypertrophic and healthy LF cells, with the hypertrophic LF cells exhibiting more active inflammation than did the healthy cells. After H2O2 treatment, p-p38, p-p65, iNOS, TGF-ß, vimentin, and collagen I increased significantly, and NAC administration reversed the effects of oxidative stress. These results can form the basis of a novel therapeutic treatment for LFH using antioxidants.

EGF Contributes to Hypertrophy of Human Ligamentum Flavum via the TGF-ß1/Smad3 Signaling Pathway.

Background: The most common spinal disorder in elderly is lumbar spinal canal stenosis (LSCS). Previous studies showed that ligamentum flavum hypertrophy (LFH) with fibrosis as the main pathological change is one of the pathogenic factors leading to LSCS. Epidermal Growth Factor (EGF) is known to have an intimate relationship with fibrosis in various tissues. Nevertheless, currently, there are few studies regarding EGF in LFH. The effect of EGF on the development of LFH is unknown, and the underlying pathomechanism remains unclear. In this study, we investigated the role of EGF in LFH and its potential molecular mechanism. Methods: First, the expression levels of EGF, phosphorylation of EGF receptor (pEGFR), Transforming growth factor-ß1 (TGF-ß1), Phosphorylated Smad3 (pSmad3), collagen I and collagen III were examined via immunohistochemistry and Western blot in LF tissues from patients with LSCS or Non-LSCS. Second, primary LF cells were isolated from adults with normal LF thickness and were cultured with different concentrations of exogenous EGF with or without erlotinib/TGF-ß1-neutralizing antibody. Results: The results showed that EGF, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression in the LSCS group was significantly higher than that in the Non-LSCS group. Meanwhile, pEGFR, TGF-ß1, pSmad3, collagen I and collagen III protein expression was significantly enhanced in LF cells after exogenous EGF exposure, which can be notably blocked by erlotinib. In addition, pSmad3, collagen I and collagen III protein expression was blocked by TGF-ß1-neutralizing antibody. Conclusions: EGF promotes the synthesis of collagen I and collagen III via the TGF-ß1/Smad3 signaling pathway, which eventually contributes to LFH.

Revealing the novel autophagy-related genes for ligamentum flavum hypertrophy in patients and mice model.

Background: Fibrosis is a core pathological factor of ligamentum flavum hypertrophy (LFH) resulting in degenerative lumbar spinal stenosis. Autophagy plays a vital role in multi-organ fibrosis. However, autophagy has not been reported to be involved in the pathogenesis of LFH. Methods: The LFH microarray data set GSE113212, derived from Gene Expression Omnibus, was analyzed to obtain differentially expressed genes (DEGs). Potential autophagy-related genes (ARGs) were obtained with the human autophagy regulator database. Functional analyses including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Gene Set Enrichment Analysis (GSEA), and Gene Set Variation Analysis (GSVA) were conducted to elucidate the underlying biological pathways of autophagy regulating LFH. Protein-protein interaction (PPI) network analyses was used to obtain hub ARGs. Using transmission electron microscopy, quantitative RT-PCR, Western blotting, and immunohistochemistry, we identified six hub ARGs in clinical specimens and bipedal standing (BS) mouse model. Results: A total of 70 potential differentially expressed ARGs were screened, including 50 up-regulated and 20 down-regulated genes. According to GO enrichment and KEGG analyses, differentially expressed ARGs were mainly enriched in autophagy-related enrichment terms and signaling pathways related to autophagy. GSEA and GSVA results revealed the potential mechanisms by demonstrating the signaling pathways and biological processes closely related to LFH. Based on PPI network analysis, 14 hub ARGs were identified. Using transmission electron microscopy, we observed the autophagy process in LF tissues for the first time. Quantitative RT-PCR, Western blotting, and immunohistochemistry results indicated that the mRNA and protein expression levels of FN1, TGFß1, NGF, and HMOX1 significantly higher both in human and mouse with LFH, while the mRNA and protein expression levels of CAT and SIRT1 were significantly decreased. Conclusion: Based on bioinformatics analysis and further experimental validation in clinical specimens and the BS mouse model, six potential ARGs including FN1, TGFß1, NGF, HMOX1, CAT, and SIRT1 were found to participate in the fibrosis process of LFH through autophagy and play an essential role in its molecular mechanism. These potential genes may serve as specific therapeutic molecular targets in the treatment of LFH.

Relationship between Severity of Lumbar Spinal Stenosis and Ligamentum Flavum Hypertrophy and Serum Inflammatory Factors.

Objective: This study is aimed at investigating the correlation between lumbar spinal stenosis (LSS) severity, ligamentum flavum hypertrophy, and the upregulation of inflammatory markers. Methods: From March 2019 and May 2022, eighty-five inpatients with LSS were enlisted as the study's research group, while sixty-five patients hospitalized for lumbar intervertebral disc herniation over the same time period served as the study's control group. Moreover, mild, moderate, and severe subgroups of patients were created within the research population based on their LSS severity. The ligamentum flavum thickness and the positive expression rates of TNF-α, TGF-ß1, and IL-1α were compared between the study group and the control group. The levels of TNF-α, TGF-ß1, and IL-1α that were found to be positively expressed were compared between the mild, moderate, and severe groups. Patients with LSS had their ligamentum flavum thickness and their positive expression rates of TNF-α, TGF-ß1, and IL-1α analyzed using Spearman correlation analysis. We evaluated the diagnostic utility of the positive expression rates of IL-α1, TGF-ß1, and TNF-α and ligamentum flavum thickness in distinguishing the severity of LSS using a receiver operating characteristic (ROC) curve. Results: The rates of both lower limb pain (40.00%) and intermittent claudication (80.00%) in the LSS group were higher than those in the lumbar disc herniation group (15.38%, 12.31%), with statistical significance (P < 0.05). However, no substantial disparity was observed in left lower limb pain, right lower limb pain, low back pain, lower limb sensation, muscle strength, and reflex abnormalities between the two groups (P > 0.05). Positive expressions of TGF-ß1, TNF-α, and IL-1α and thicker ligamentum flavum were more prevalent in the LSS group than in the lumbar intervertebral disc herniation group. All indexes were significantly (P < 0.05) higher in the moderate stenosis group than in the severe stenosis group. Additionally, the thickness of the ligamentum flavum and the positive expression rates of TNF-α, TGF-ß1, and IL-1α were higher in the mild and moderate stenosis groups than in the severe stenosis group. The expression levels of TNF-α, TGF-ß1, and IL-1α were favorably linked with ligamentum flavum thickness (P < 0.05). ROC curve analysis showed that the thickness of ligamentum flavum, the expression of IL-1α, the expression of TGF-ß1, and the expression of TNF-α could effectively diagnose mild, moderate, and severe LSS (P < 0.05). Conclusion: Ligamentum flavum hypertrophy and positive expression rates of IL-1α, TGF-ß1, and TNF-α are closely linked to LSS, which can effectively identify mild, moderate, and severe LSS.

Elucidating the effect of mechanical stretch stress on the mechanism of ligamentum flavum hypertrophy: Development of a novel in vitro multi-torsional stretch loading device.

OBJECTIVE: We developed a novel multi-torsional mechanical stretch stress loading device for ligamentum flavum cells and evaluated its influence on the development of ligamentum flavum hypertrophy, a common cause of lumbar spinal canal stenosis. MATERIALS AND METHODS: Stretch strength of the device was optimized by applying 5% and 15% MSS loads for 24, 48, and 72 h. A cytotoxicity assay of human ligamentum flavum cells was performed and the results were compared to control (0% stress). Inflammatory markers (interleukin [IL]-6, IL-8), vascular endothelial growth factor [VEGF], and extracellular matrix (ECM)-regulating cytokines (matrix metalloproteinase [MMP]-1, MMP-3 and MMP-9, and tissue inhibitor of metalloproteinase [TIMP]-1 and TIMP-2) were quantified via enzyme-linked immunosorbent assay. RESULTS: Using our multi-torsional mechanical stretch stress loading device, 5% stress for 24 hour was optimal for ligamentum flavum cells. Under this condition, the IL-6 and IL-8 levels, VEGF level, and MMP-1, MMP-3, and TIMP-2 were significantly increased, compared to the control. CONCLUSION: Using the novel multi-torsional mechanical stretch stress loading device we confirmed that, mechanical stress enhances the production of inflammatory cytokines and angiogenic factors, and altered the expression of ECM-regulating enzymes, possibly triggering ligamentum flavum hypertrophy.