Dataset Information

Proinflammatory intervertebral disc cell and organ culture models induced by tumor necrosis factor alpha

ABSTRACT: Abstract Inflammation plays an important role in the pathogenesis of intervertebral disc (IVD) degeneration. The proinflammatory cytokine tumor necrosis factor alpha (TNF??) has shown markedly higher expression in degenerated human disc tissue compared with healthy controls. Anti?inflammatory treatment targeting TNF?? has shown to alleviate discogenic pain in patients with low back pain. Therefore, in vitro and ex vivo inflammatory models utilizing TNF?? provide relevant experimental conditions for drug development in disc degeneration research. The current method article addressed several specific questions related to the model establishment. (a) The effects of bovine and human recombinant TNF?? on bovine nucleus pulposus (NP) cells were compared. (b) The required dose for an inflammatory IVD organ culture model with intradiscal TNF?? injection was studied. (c) The effect of TNF?? blocking at different stages of inflammation was evaluated. Outcomes revealed that bovine and human recombinant TNF?? induced equivalent inflammatory effects in bovine NP cells. A bovine whole IVD inflammatory model was established by intradiscal injection of 100?ng TNF??/ cm3 disc volume, as indicated by increased nitric oxide, glycosaminoglycan, interleukin 6 (IL?6), and interleukin 8 (IL?8) release in culture media, and upregulation of MMP3, ADAMTS4, IL?8, IL?6, and cyclooxygenase (COX)?2 expression in NP tissue. However, results in human NP cells showed that the time point of anti?inflammatory treatment was crucial to achieve significant effects. Furthermore, anticatabolic therapy in conjunction with TNF?? inhibition would be required to slow down the pathologic cascade of disc degeneration. This article describes the methods for establishment of proinflammatory intervertebral disc cell and organ culture models induced by TNF?a.

SUBMITTER: Du J

PROVIDER: S-EPMC7524256 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:IntroductionThe goal of this study is to characterize transcriptome changes and gene regulation networks in an organ culture system that mimics early post-traumatic intervertebral disc (IVD) degeneration.MethodsTo mimic a traumatic insult, bovine caudal IVDs underwent one strike loading. The control group was cultured under physiological loading. At 24 hours after one strike or physiological loading, RNA was extracted from nucleus pulposus (NP) and annulus fibrosus (AF) tissue. High throughput next generation RNA sequencing was performed to identify differentially expressed genes (DEGs) between the one strike loading group and the control group. Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes analyses were performed to analyze DEGs and pathways. Protein-protein interaction (PPI) network was analyzed with cytoscape software. DEGs were verified using qRT-PCR. Degenerated human IVD tissue was collected for immunofluorescence staining to verify the expression of DEGs in human disc tissue.ResultsOne strike loading resulted in significant gene expression changes compared with physiological loading. In total 253 DEGs were found in NP tissue and 208 DEGs in AF tissue. Many of the highly dysregulated genes have known functions in disc degeneration and extracellular matrix (ECM) homeostasis. ACTB, ACTG, PFN1, MYL12B in NP tissue and FGF1, SPP1 in AF tissue were verified by qRT-PCR and immunofluorescence imaging. The identified DEGs were involved in focal adhesion, ECM-receptor interaction, PI3K-AKT, and cytokine-cytokine receptor interaction pathways. Three clusters of PPI networks were identified. GO enrichment revealed that these DEGs were mainly involved in inflammatory response, the ECM and growth factor signaling and protein folding biological process.ConclusionOur study revealed different DEGs, pathways, biological process and PPI networks involved in post-traumatic IVD degeneration. These findings will advance the understanding of the pathogenesis of IVD degeneration, and help to identify novel biomarkers for the disease diagnosis.

Project description:ObjectiveIn this work, we examine the molecular basis for capillary tube regression and identify key proregressive factors, signaling pathways, and pharmacological antagonists of this process. Approach and Results: We demonstrate that the proinflammatory mediators, IL (interleukin)-1β, TNF (tumor necrosis factor) α, and thrombin, singly and in combination, are potent regulators of capillary tube regression in vitro. These proregressive factors, when added to endothelial cell-pericyte cocultures, led to selective loss of endothelial cell-lined tube networks, with retention and proliferation of pericytes despite the marked destruction of adjacent capillary tubes. Moreover, treatment of macrophages with the TLR (toll-like receptor) agonists Pam3CSK4 and lipopolysaccharide generates conditioned media with marked proregressive activity, that is completely blocked by a combination of neutralizing antibodies directed to IL-1β and TNFα but not to other factors. The same combination of blocking antibodies, as well as the anti-inflammatory cytokine IL-10, interfere with macrophage-dependent hyaloid vasculature regression in mice suggesting that proinflammatory cytokine signaling regulates capillary regression in vivo. In addition, we identified a capillary regression signaling signature in endothelial cells downstream of these proregressive agents that is characterized by increased levels of ICAM-1 (intercellular adhesion molecule-1), phospho-p38, and phospho-MLC2 (myosin light chain-2) and decreased levels of phospho-Pak2, acetylated tubulin, phospho-cofilin, and pro-caspase3. Finally, we identified combinations of pharmacological agents (ie, FIST and FISTSB) that markedly rescue the proregressive activities of IL-1β, TNFα, and thrombin, individually and in combination.ConclusionsOverall, these new studies demonstrate that the major proinflammatory mediators, IL-1β, TNFα, and thrombin, are key regulators of capillary tube regression-a critical pathological process regulating human disease.

Dataset Information

Proinflammatory intervertebral disc cell and organ culture models induced by tumor necrosis factor alpha

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets