Kindlin-2 inhibits Nlrp3 inflammasome activation in nucleus pulposus to maintain homeostasis of the intervertebral disc.
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SUBMITTER: Chen S
PROVIDER: S-EPMC8748798 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundAccumulating evidence indicates that intervertebral disc degeneration (IDD) is associated with diabetes mellitus (DM), while the underlying mechanisms still remain elusive. Herein, the current study sought to explore the potential molecular mechanism of IDD in diabetic rats based on transcriptome sequencing data.MethodsStreptozotocin (STZ)-induced diabetes mellitus type 1 (T1DM) rats were used to obtain the nucleus pulposus tissues for transcriptome sequencing. Next, differentially expressed genes (DEGs) in transcriptome sequencing data and GSE34000 microarray dataset were obtained and intersected to acquire the candidate genes. Moreover, GO and KEGG enrichment analyses were performed to analyze the cellular functions and molecular signaling pathways primarily regulated by candidate DEGs.ResultsA total of 35 key genes involved in IDD of T1DM rats were mainly enriched in the extracellular matrix (ECM) and cytokine adhesion binding-related pathways. NLRP3 inflammasome activation promoted the pyroptosis of nucleus pulposus cells (NPCs). Besides, BMP7 could affect the IDD of T1DM rats by regulating the inflammatory responses. Additionally, NPCs were isolated from STZ-induced T1DM rats to illustrate the effects of BMP7 on IDD of T1DM rats using the ectopic expression method. Both in vitro and in vivo experiments validated that BMP7 alleviated IDD of T1DM rats by inhibiting NLRP3 inflammasome activation and pyroptosis of NPCs.ConclusionCollectively, our findings provided novel mechanistic insights for understanding of the role of BMP7 in IDD of T1DM, and further highlighted BMP7 as a potential therapeutic target for preventing IDD in T1DM.
| S-EPMC9979491 | biostudies-literature
Project description:Intervertebral disc degeneration (IDD) results from the dysfunction of nucleus pulposus (NP) cells and the exhaustion of NP progenitors (ProNPs). The cellular applications of NP cells during IDD are currently limited due to the lack of in vivo studies showing whether NP cells are heterogeneous and contain ProNPs throughout postnatal stages. In this study, single-cell RNA sequencing of purified NP cells is used to map four molecularly defined populations and urotensin II receptor (UTS2R)-expressing postnatal ProNPs is identified, which are markedly exhausted during IDD, in mouse and human specimens. The lineage tracing shows that UTS2R+ ProNPs preferentially resides in the NP periphery with its niche factor tenascin-C and give rise to functional NP cells. It is also demonstrated that transplanting UTS2R+ ProNPs with tenascin-C into injured intervertebral discs attenuate the progression of IDD. The study provides a novel NP cell atlas, identified resident ProNPs with regenerative potential, and revealed promising diagnostic and therapeutic targets for IDD.
| S-EPMC9069184 | biostudies-literature
Project description:STUDY DESIGN:Profiling proteins expressed in the nucleus pulposus of fetal intervertebral disc (IVD). PURPOSE:To evaluate the molecular complexity of fetal IVDs not exposed to mechanical, traumatic, inflammatory, or infective insults to generate improved knowledge on disc homeostasis. OVERVIEW OF LITERATURE:Low back pain is the most common musculoskeletal disorder, causing a significant reduction in the quality of life, and degenerative disc disorders mainly contribute to the increasing socioeconomic burden. Despite extensive research, the causative pathomechanisms behind degenerative disc disorders are poorly understood. Precise molecular studies on the intricate biological processes involved in maintaining normal disc homeostasis are needed. METHODS:IVDs of nine fetal specimens obtained from medical abortions were used to dissect out the annulus fibrosus and nucleus pulposus under sterile operating conditions. Dissected tissues were transferred to sterile Cryovials and snap frozen in liquid nitrogen before transporting to the research laboratory for protein extraction and further liquid chromatography tandem mass spectrometry (LC-MS/ MS) analysis. Collected data were further analyzed using Gene Functional Classification Tool in DAVID and STRING databases. RESULTS:A total of 1,316 proteins were identified through LC-MS/MS analysis of nine fetal IVD tissues. Approximately 247 proteins present in at least four fetal discs were subjected to further bioinformatic analysis. The following 10 clusters of proteins were identified: collagens, ribosomal proteins, small leucine-rich proteins, matrilin and thrombospondin, annexins, protein disulfide isomerase family proteins and peroxiredoxins, tubulins, histones, hemoglobin, and prolyl 4-hydroxylase family proteins. CONCLUSIONS:This study provides fundamental information on the proteome networks involved in the growth and development of healthy fetal discs in humans. Systematic cataloging of proteins involved in various structural and regulatory processes has been performed. Proteins expressed most abundantly (collagen type XIV alpha 1 chain, biglycan, matrilin 1, and thrombospondin 1) in their respective clusters also elucidate the possibility of utilizing these proteins for potential regenerative therapies.
| S-EPMC7435318 | biostudies-literature
Project description:Intervertebral Disc (IVD) degeneration leading to Low back pain (LBP) is the most common musculoskeletal disorder. Lack of knowledge on the intricate homeostatic mechanisms necessitates proteomic characterisation of a normal human IVD to understand the biological process and unravel the pathomechanisms of degenerative disc disease (DDD). In this study, we employed proteomic approach coupled with tandem mass-spectrometry to derive the comprehensive list of proteins expressed in true biologically normal control discs. This would serve as the basis for identifying the interacting molecules participating in significant biological processes and pathways disrupted during aging and degeneration.
2021-09-09 | PXD016560 | Pride
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:Intervertebral disc (IVD) disorder and age-related degeneration are believed to contribute to low back pain. Cell-based therapies represent a promising strategy to treat disc degeneration; however, the cellular and molecular characteristics of disc cells during IVD maturation and aging still remain poorly defined. This study investigated novel molecular markers and their age-related changes in the rat IVD. Affymetrix cDNA microarray analysis was conducted to identify a new set of genes characterizing immature nucleus pulposus (NP) cells. Among these markers, select neuronal-related proteins (Basp1, Ncdn and Nrp-1), transcriptional factor (Brachyury T), and cell surface receptors (CD24, CD90, CD155 and CD221) were confirmed by real-time PCR and immunohistochemical (IHC) staining for differential expression between IVD tissue regions and among various ages (1, 12 and 21 months). NP cells generally possessed higher levels of mRNA or protein expression for all aforementioned markers, with the exception of CD90 in anulus fibrosus (AF) cells. In addition, CD protein (CD24 and CD90) and Brachyury (T) expression in immature disc cells were also confirmed via flow cytometry. Similar to IHC staining, results revealed a higher percentage of immature NP cells expressing CD24 and Brachyury, while higher percentage of immature AF cells was stained positively for CD90. Altogether, this study identifies that tissue-specific gene expression and age-related differential expression of the above markers do exist in immature and aged disc cells. These age-related phenotype changes provide a new insight for a molecular profile that may be used to characterize NP cells for developing cell-based regenerative therapy for IVD regeneration.
| S-EPMC3526492 | biostudies-literature
Project description:Study designThe diversity of microflora inhabiting endplate (EP) and nucleus pulposus (NP) tissues of human intervertebral disc (IVD) was profiled through NGS-supported 16S rRNA amplicon sequencing. Sixteen EP and their corresponding NP were excised from the brain-dead voluntary organ donors with no clinical history of low back pain, and 12 herniated and 8 degenerated NP tissues isolated from the patients undergoing spinal surgery were subjected to study the alteration in the microbial diversity.ObjectivesTo understand in normal IVD, whether the colonization of bacteria to the NP is through the EP in discs with intact annulus fibrosus. To identify significantly differing microbial population(s) between normal and diseased IVD (NP).Background of the studyThere is increasing evidence for subclinical infection by fastidious low, growing bacteria to be a cause of disc degeneration. Although the presence of bacteria in NP has been reported well in literature, the source of bacteria is not clearly proved as the disc is avascular in healthy condition. Documentation of similar bacterial populations in the EP and NP may add proof that bacterial inoculation of NP occurs via the EP.Materials and methodsSixteen EP and their corresponding NP excised from brain-dead voluntary organ donors with no history of back pain and 20 diseased discs collected from patients undergoing microdiscectomy/fusion surgery were used for profiling microbiome through 16S rRNA amplicon sequencing using primers specific for V1-V9 hypervariable regions. Changes in bacterial diversity and abundance were analysed to identify the key microbial populations in normal IVD NP and EP tissues and those significantly altered in diseased IVD (NP).ResultsNP and EP shared a similar spectrum of microbiome but with varying abundance. The five dominant phyla identified were Proteobacteria, Firmicutes, Actinobacteria, OD1, and Bacteroidetes. Proteobacteria was found to be the most abundant phyla in both NP (62%) and EP (53%) of the normal IVD. This was followed by Firmicutes (16%), Actinobacteriota (11%), OD1 (Parcubacteria) (7.6%), and Bacteroidetes (2%) in NP and Firmicutes (23.4%), OD1 (Parcubacteria) (17.6%), Actinobacteriota (2.8%), and Bacteroidetes (2.6%) in EP, respectively. Under diseased conditions, Proteobacteria (68%) was dominant when compared with other phyla. However, there was no significant difference in the abundance of Proteobacteria between the normal and diseased discs. Interestingly, the other dominant phyla such as Firmicutes (Normal-NP: 16.2%; Diseased-NP: 4.02%) and Actinobacteria (Normal-NP: 11%; Diseased-NP: 0.99%) showed a significant reduction in degenerated discs. To understand the key microbial populations that are significantly altered during disease, correlation analysis was performed among the three phyla, which revealed a negative correlation in the ratio of Actinobacteria + Firmicutes vs. Proteobacteria (p = 0.001) in DD.ConclusionResults of our study clearly demonstrated a similar bacterial diversity but with varying abundance between the EP and NP, suggesting the existence of the endplate-nucleus pulposus axis in the normal IVD microbiome. Further, our results have indicated that the changes in the abundance of Actinobacteria + Firmicutes vs. Proteobacteria during DDD need further investigation.
| S-EPMC9554234 | 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:BackgroundRecently, Tie2/TEK receptor tyrosine kinase (Tie2 or syn. angiopoietin-1 receptor) positive nucleus pulposus progenitor cells were detected in human, cattle, and mouse. These cells show remarkable multilineage differentiation capacity and direct correlation with intervertebral disc (IVD) degeneration and are therefore an interesting target for regenerative strategies. Nevertheless, there remains controversy over the presence and function of these Tie2+ nucleus pulposus cells (NPCs), in part due to the difficulty of identification and isolation.PurposeHere, we present a comprehensive protocol for sorting of Tie2+ NPCs from human, canine, bovine, and murine IVD tissue. We describe enhanced conditions for expansion and an optimized fluorescence-activated cell sorting-based methodology to sort and analyze Tie2+ NPCs.MethodsWe present flow cytometry protocols to isolate the Tie2+ cell population for the aforementioned species. Moreover, we describe crucial pitfalls to prevent loss of Tie2+ NPCs from the IVD cell population during the isolation process. A cross-species phylogenetic analysis of Tie2 across species is presented.ResultsOur protocols are efficient towards labeling and isolation of Tie2+ NPCs. The total flow cytometry procedure requires approximately 9 hours, cell isolation 4 to 16 hours, cell expansion can take up to multiple weeks, dependent on the application, age, disease state, and species. Phylogenetic analysis of the TEK gene revealed a strong homology among species.ConclusionsCurrent identification of Tie2+ cells could be confirmed in bovine, canine, mouse, and human specimens. The presented flow cytometry protocol can successfully sort these multipotent cells. The biological function of isolated cells based on Tie2+ expression needs to be confirmed by functional assays such as in vitro differentiation. in vitro culture conditions to maintain and their possible proliferation of the Tie2+ fraction is the subject of future research.
| S-EPMC6686801 | biostudies-literature
Project description:Intervertebral disc degeneration (IVDD) is a common cause of lower back pain. Programmed cell death (PCD) including apoptosis and autophagy is known to play key mechanistic roles in the development of IVDD. We hypothesized that the nucleus pulposus cells that make up the center of the IVD can be affected by aging and environmental oxygen concentration, thus affecting the development of IVDD. Here, we evaluated the phenotype changes and PCD signaling in nucleus pulposus cells in two different oxygen percentages (5% (hypoxia) and 20% (normoxia)) up to serial passage 20. NP cells were isolated from the lumbar discs of rats, and the chondrogenic, autophagic, and apoptotic gene expressions were analyzed during cell culture up to serial passage 20. Hypoxia significantly increased the number of autophagosomes, as determined by monodansylcadaverine staining and transmission electron microscopy. Furthermore, hypoxia triggered the activation of autophagic flux (beclin-1, LC3-II/LC3-I ratio, and SIRT1) with a concomitant decrease in the expression of apoptotic proteins (Bax and caspase-3). Despite injury and age differences, no significant differences were observed between the ex vivo lumbar disc cultures of groups incubated in the hypoxic chamber. Our study provides a better understanding of autophagy- and apoptosis-related senescence in NP cells. These results also provide insight into the effects of aging on NP cells and their PCD levels during aging.
| S-EPMC7528147 | biostudies-literature