Matrix Stiffness Activating YAP/TEAD1-Cyclin B1 in Nucleus Pulposus Cells Promotes Intervertebral Disc Degeneration
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PROVIDER: S-EPMC10529754 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:Intervertebral disc degeneration (IVDD) is the primary cause of low back pain; however, the molecular mechanisms involved in the pathogenesis of IVDD are not fully understood. Polo-like kinase 1 (PLK1) plays numerous roles in the cell cycle, including in cell proliferation and senescence. To investigate the involvement of PLK1 in IVDD, we used patient tissues and an animal model of IVDD. Samples were analyzed via immunoblotting, quantitative real-time polymerase chain reaction (qPCR), immunofluorescence, and immunohistochemistry. Our results demonstrated that PLK1 expression was decreased in nucleus pulposus cells (NPCs) of degenerative IVDs. The inhibition of PLK1 kinase activity in normal NPCs increased the expression of p53 protein, inhibited cell proliferation, and induced senescence. Our results suggest that PLK1 regulates the degeneration of the IVD through p53, revealing the function and mechanism of PLK1 in IVDD and providing a theoretical basis and experimental evidence for the potential treatment of low back pain.
| S-EPMC8964438 | biostudies-literature
Project description:Background/objectiveIntervertebral disc (IVD) degeneration (IVDD) that greatly affected by regional biomechanical environment is a major cause of low back pain. Injectable hydrogels have been commonly studied for treatment of IVDD due to their capability of mimicking extracellular matrix structure to support cellular behavior and clinical prospects in minimally invasive treatment. However, most hydrogels suffer from complicated chemistry, potential uncertainty and toxicity from in-situ gelation, and mismatch with IVD mechanical environment that limit their therapeutic effects or clinical translation in IVDD or intervertebral disc defect repair. For IVD lesion repair, the study aims to develop a novel hydrogel with shear-thinning enabled injectability, high bio-safety, and mechanical properties adaptable to the IVD environment, using a simple chemistry and method. And therapeutic efficacy of the novel hydrogel in the treatment of IVDD or intervertebral disc defect will be revealed.MethodsA glycerol cross-linked PVA gel (GPG) was synthesized based on multiple H-bonds formation between glycerol molecules and PVA chains. The rheological and mechanical properties were tested. The swelling ratio was measured. The micro-architecture was observed through scanning and transmission electron microscopes. Nucleus pulposus (NP) cells were cultured in GPG-coated plates or silicone chambers treated under hydrostatic or dynamic loading in vitro, and examined for proliferation, vitality, apoptosis, expression of catabolic and anabolic markers. GPG was injected in needle puncture (IDD) or NP discectomy (NPD) models in vivo, and examined through magnetic resonance imaging, micro-computed tomography scanning and histological staining.ResultsGPG had a highly porous structure consisting of interconnected pores. Meanwhile, the GPG had NP-like viscoelastic property, and was able to withstand the cyclic deformation while exhibiting a prominent energy-dissipating capability. In vitro cell tests demonstrated that, the hydrogel significantly down-regulated the expression of catabolic markers, maintained the level of anabolic markers, preserved cell proliferation and vitality, reduced apoptotic rate of NP cells under pathologically hydrostatic and dynamic loading environments compared to cells cultured on untreated plate or silicone chamber. In vivo animal studies revealed that injection of GPG efficiently maintained NP structural integrity, IVD height and relative water content in IDD models, and stimulated the fibrous repair in NPD models.ConclusionThis study showed that GPG, with high injectability, NP-like viscoelastic characteristics, good energy-dissipating properties and swelling capacities, preserved NP cells vitality against pathological loading, and had therapeutic effects on IVD repair in IDD and NPD models.The translational potential of this articleEffective clinical strategy for treatment of intervertebral disc degeneration (IVDD) is still lacking. This study demonstrates that injection of a hydrogel with nucleus pulposus-matched viscoelastic property could remarkably prevent the IVDD progress. Prepared with simple chemistry and procedure, the cell/drug-free GPG with high bio-safety and shear-thinning enabled injectability bears great translational potential for the clinical treatment of IVDD via a minimally invasive approach.
| S-EPMC8980713 | biostudies-literature
Project description:The nucleus pulposus (NP) plays a vital role in intervertebral disc degeneration (IVDD). Previous studies have revealed cellular heterogeneity in NP tissue during IVDD progression. However, the cellular and molecular alterations of diverse cell clusters during IVDD remain to be fully elucidated. NP tissues were isolated from patients with different grades of IVDD undergoing discectomy, and then subjected to single-cell RNA sequencing (scRNA-seq). Cell subsets were identified based on unbiased clustering of gene expression profiles. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed to determine the molecular features of diverse cell clusters. Monocle analysis was used to illustrate the differentiation trajectories of chondrocytes. Additionally, CellPhoneDB analysis revealed potential interactions between chondrocytes and other cells during IVDD. Based on the expression profiles of 47,610 individual cells, eight putative clusters including chondrocytes, endothelial cells, fibroblasts, macrophages, mural cells, osteoclasts, proliferating stromal cells and T cells were identified. The chondrocyte cluster was classified into three subsets, C1-C3, which were associated with stress-resistance, fibrosis and inflammatory responses, respectively. Pseudo-time trajectories suggested that chondrocytes gradually differentiated into fibroblasts during IVDD. Immune cells including cDC2s, macrophages and monocytes were identified. Further analysis showed that chondrocytes might communicate with immune cells via the MIF, TNFSF9, SPP1 and CCL4L2 signaling pathways. In addition, we found that invading endothelial cells might interact with chondrocytes through the COL4A1, CXCL12, VEGFA and SEMA3E signaling pathways. Our results reveal the cellular complexity and phenotypic characteristics of NP tissues at single-cell resolution, which will contribute to the in-depth investigation of preventative and regenerative strategies for IVDD.
| S-EPMC11549379 | biostudies-literature
Project description:Background contextLow back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear.PurposeTo study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling.Study designThe expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro.MethodsHuman disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells.ResultsIn vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM).ConclusionOur results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.
| S-EPMC9532572 | biostudies-literature
Project description:Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2+) and disialoganglioside 2 positive (GD2+), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2+ cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2+GD2+) can be induced from their precursor cells (Tie2+GD2-) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.
| S-EPMC3535337 | biostudies-literature
Project description:Lower back pain, a leading cause of disability worldwide, is associated with intervertebral disc degeneration (IDD) in approximately 40% of cases. Although nucleus pulposus (NP) cell senescence is a major contributor to IDD, the underlying mechanisms remain unclear. We collected NP samples from IDD patients who had undergone spinal surgery. Healthy and senescent NP tissues (n = 3) were screened using the Pfirrmann grading system combined with immunohistochemistry, as well as hematoxylin and eosin, Safranin O, Alcian blue, and Masson staining. Differentially expressed proteins (DEPs) were identified using quantitative TMT-based proteomics technology. Bioinformatics analyses included gene ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and protein-protein interaction (PPI) analyses. In addition, immunofluorescence was used to verify protein expression. In total, 301 DEPs were identified in senescent NP tissues, including 92 upregulated and 209 downregulated proteins. In GO, DEPs were primarily associated with NF-kappaB transcription factor, extracellular regions, cellular protein metabolic processes, and post-translational protein modification. The enriched KEGG pathways included TGF-β, Wnt, RAP1, interleukin-17, extracellular matrix-receptor adhesion, and PI3K/Akt signaling pathways. PPI analysis demonstrated interactions between multiple proteins. Finally, immunofluorescence verified the expressions of MMP3, LUM, TIMP1, and CDC42 in senescent NP cells. Our study provides valuable insights into the mechanisms underlying senescent NP tissues in IDD patients. DEPs provide a basis for further investigation of the effects of senescent factors on IDD.
| S-EPMC10488253 | biostudies-literature
Project description:Intervertebral Disc degeneration (IDD) is one of the leading causes of disability, and current therapies are ineffective. Phosphodiesterase 4B (PDE4B) plays an essential role in regulating the activation of nuclear factor E2-related factor 2 (Nrf2), while Nrf2 regulates ferroptosis. However, it is still unknown whether PDE4B is involved in the development of IDD. In this study, we explored the role of PDE4B on ferroptosis and Nrf2 in IDD pathogenesis by in vivo and in vitro experiments. The findings suggested that the expressions of PDE4B, ASCL4, and TRFC were significantly upregulated, and the expression of Nrf2 was significantly downregulated in nucleus pulposus (NP) tissues from human IDD patients dependent on IDD degeneration. Overexpression of PDE4B (PDE4B-OE) in NP cells upregulated the expression of ASCL4 and TRFC, and downregulated the expression of Nrf2. Meanwhile, the level of cytokine and oxidative stress were upregulated. Ferroptosis inhibitor Fer-1 or Nrf2 activator dimethyl fumarate (DMF) suppressed the effect of PDE4B-OE, while ferroptosis inducer elastin enhanced the effect of PDE4B-OE. In the IDD rat model, PDE4 inhibitor roflumilast, ferroptosis inhibitor Fer-1, or Nrf2 activator dimethyl fumarate (DMF) delayed IDD pathogenesis. While administration of ferroptosis inducer elastin enhanced IDD pathogenesis. Combination with PDE4B inhibitor and ferroptosis inhibitor Fer-1 significantly synergistic reversed IDD pathogenesis. While combination with PDE4B inhibitor or Nrf2 activator and elastin also decreased the degree of the IDD. The IHC suggested PDE4 inhibitor downregulated the expression of ASCL4 and TRFC. However, the combination effect of the Nrf2 activator was not obvious. Our study suggested that aberrant PDE4B activation in NP tissues induces pathological changes in IDD mediated by ferroptosis, and PDE4 inhibitor reveres the process of IDD by suppressing ferroptosis, and has a synergic effect with ferroptosis inhibitor. So PDE4B inhibition may be a potential therapeutic strategy for IDD.
| S-EPMC11787312 | biostudies-literature
Project description:Intervertebral disc degeneration (IDD) is considered one of the main causes of low back pain and lumbar disc herniation. Various studies have shown that disc cell senescence plays a critical role in this process. however, its role in IDD is yet unclear. In this study, we explored the role of senescence-related genes (SR-DEGs) and its underlying mechanism in IDD. A total of 1325 differentially expressed genes (DEGs) were identified using Gene Expression Omnibus (GEO) database GSE41883. 30 SR-DEGs were identified for further functional enrichment and pathway analysis, and two hub SR-DEGs (ERBB2 and PTGS2) were selected to construct transcription factor (TF)-gene interaction and TF-miRNA coregulatory networks, and 10 candidate drugs were screened for the treatment of IDD. Last but not least, in vitro experiments show that ERBB2 expression decreased and PTGS2 expression increased in human nucleus pulposus (NP) cell senescence model treated with TNF-α. After lentivirus-mediated overexpression of ERBB2, the expression of PTGS2 decreased and the senescence level of NP cells decreased. Overexpression of PTGS2 reversed the anti-senescence effects of ERBB2. The findings in this study suggested that ERBB2 overexpression further reduced NP cell senescence by inhibiting PTGS2 levels, which ultimately alleviated IDD. Taken together, our findings provide new insights into the roles of senescence-related genes in IDD and highlight a novel target of ERBB2-PTGS2 axis for therapeutic strategies.
| S-EPMC10280935 | biostudies-literature
Project description:Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non‑coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence‑associated β‑galactosidase (SA‑β‑gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis‑ and 2,170 trans‑acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon‑β production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans‑acting targets, C‑C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc‑ST8SIA5‑1:2 and lnc‑HRK‑2:1, were identified. Reverse transcription‑quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc‑HRK‑2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA‑β‑gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc‑HRK‑2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.
| S-EPMC7646984 | biostudies-literature
Project description:BackgroundIn recent years, intervertebral disc (IVD) degeneration (IDD) has increased in age. There is still a lack of effective treatment in clinics, which cannot improve the condition of IDD at the level of etiology.ObjectiveTo explore IDD pathogenesis at the cellular and gene levels and investigate lactotransferrin (LTF) expression in IDD patients and its possible mechanism.MethodsWe downloaded the IDD data set from the Gene Expression Omnibus (GEO) database, screened the differentially expressed genes (DEGs) and hub genes and performed Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis to construct a protein-protein interaction (PPI) network. Subsequently, we verified LTF's regulatory mechanism through cell experiments. IL-1β was used to intervene in nucleus pulposus cells (NPCs) to construct the IDD cell model, and LTF and Fas expression was detected by qRT-PCR. LTF inhibitor, Fas inhibitor, LTF mimic, and Fas mimic were used to intervene in each group. Western blotting was used to detect Fas, Caspase-3, Bax, and Bcl-2 expression.ResultsA total of 131 DEGs and 10 hub genes were screened. LTF mRNA in the IDD model was significantly higher than that in the control group, while Fas' mRNA was significantly lower. When LTF was upregulated or downregulated in NPCs, apoptosis marker expression showed the opposite trend. The rescue test showed that LTF and Fas' overexpression greatly enhanced NPC apoptosis.ConclusionLTF promotes IDD progression by regulating Fas in NPCs, and it may be an effective gene therapy target.
| S-EPMC9186764 | biostudies-literature