scholarly journals Expression and activity of hyaluronidases HYAL-1, HYAL-2 and HYAL-3 in the human intervertebral disc

2019 ◽  
Vol 29 (3) ◽  
pp. 605-615 ◽  
Author(s):  
Olga Krupkova ◽  
Helen Greutert ◽  
Norbert Boos ◽  
Johannes Lemcke ◽  
Thomas Liebscher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hyaluronic Acid ◽  
Intervertebral Disc ◽  
Protein Expression ◽  
Severe Group ◽  
Health And Disease ◽  
Catabolic Response ◽  
Donor Variation ◽  
Ivd Degeneration ◽  
Expression And Activity

Abstract Purpose Hyaluronic acid plays an essential role in water retention of the intervertebral disc (IVD) and thus provides flexibility and shock absorbance in the spine. Hyaluronic acid gets degraded by hyaluronidases (HYALs), and some of the resulting fragments were previously shown to induce an inflammatory and catabolic response in human IVD cells. However, no data currently exist on the expression and activity of HYALs in IVD health and disease. Methods Gene expression, protein expression and activity of HYALs were determined in human IVD biopsies with different degrees of degeneration (n = 50 total). Furthermore, freshly isolated human IVD cells (n = 23 total) were stimulated with IL-1β, TNF-α or H2O2, followed by analysis of HYAL-1, HYAL-2 and HYAL-3 gene expression. Results Gene expression of HYAL-1 and protein expression of HYAL-2 significantly increased in moderate/severe disc samples when compared to samples with no or low IVD degeneration. HYAL activity was not significantly increased due to high donor–donor variation, but seemed overall higher in the moderate/severe group. An inflammatory environment, as seen during IVD disease, did not affect HYAL-1, HYAL-2 or HYAL-3 expression, whereas exposure to oxidative stress (100 µM H2O2) upregulated HYAL-2 expression relative to untreated controls. Conclusion Although HYAL-1, HYAL-2 and HYAL-3 are all expressed in the IVD, HYAL-2 seems to have the highest pathophysiological relevance. Nonetheless, further studies will be needed to comprehensively elucidate its significance and to determine its potential as a therapeutic target. Graphic abstract These slides can be retrieved under Electronic Supplementary Material.

scholarly journals Differential regulation of TRP channel gene and protein expression by intervertebral disc degeneration and back pain

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
A. Sadowska ◽  
W. Hitzl ◽  
A. Karol ◽  
P. Jaszczuk ◽  
H. Cherif ◽  
...  
Keyword(s):  
Gene Expression ◽  
Back Pain ◽  
Intervertebral Disc ◽  
Protein Expression ◽  
Pain Intensity ◽  
Trp Channels ◽  
Gene Array ◽  
Exact Function ◽  
Gene And Protein Expression ◽  
Ivd Degeneration

AbstractIntervertebral disc (IVD) degeneration and consequent low back pain (LBP) are common and costly pathological processes that require improved treatment strategies. Transient Receptor Potential (TRP) channels constitute a family of multimodal ion channels that have recently emerged as contributors to disc pathologies and were thus proposed as potential therapeutic targets, although limited data on their presence and function in the IVD exist. The purpose of this study was to determine the mRNA and protein expression of TRP channels in non-degenerated and degenerated human IVD tissue (with different pain intensity and chronicity) using gene array, conventional qPCR and immunohistochemistry. We could demonstrate that 26 out of 28 currently known TRP channels are expressed in the IVD on the mRNA level, thereby revealing novel therapeutic candidates from the TRPC, TRPM and TRPML subfamilies. TRPC6, TRPM2 and TRPML1 displayed enhanced gene and protein expression in degenerated IVDs as compared to non-degenerated IVDs. Additionally, the gene expression of TRPC6 and TRPML1 was influenced by the IVD degeneration grade. Pain intensity and/or chronicity influenced the gene and/or protein expression of TRPC6, TRPM2 and TRML1. Interestingly, decreased gene expression of TRPM2 was observed in patients treated with steroids. This study supports the importance of TRP channels in IVD homeostasis and pathology and their possible application as pharmacological targets for the treatment of IVD degeneration and LBP. However, the exact function and activation of the highlighted TRP channels will have to be determined in future studies.

scholarly journals Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

2021 ◽  
Vol 11 (15) ◽  
pp. 7144
Author(s):  
Muriel C. Bischof ◽  
Sonja Häckel ◽  
Andrea Oberli ◽  
Andreas S. Croft ◽  
Katharina A. C. Oswald ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Metabolic Activity ◽  
Cell Metabolism ◽  
Trauma Patients ◽  
Low Back ◽  
Relative Gene Expression ◽  
Progenitor Cell Population ◽  
Ivd Degeneration ◽  
Relative Gene

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2- or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤ 5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration.

scholarly journals Unravelling the role of Mesenchymal Stem/Stromal Cells Secretome in intervertebral disc degeneration in a pro-inflammatory/degenerative ex vivo model

2020 ◽  
Author(s):  
JR Ferreira ◽  
GQ Teixeira ◽  
E Neto ◽  
C Ribeiro-Machado ◽  
AM Silva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Stromal Cells ◽  
Ex Vivo ◽  
Low Oxygen ◽  
Ex Vivo Model ◽  
A Cell ◽  
New Perspective ◽  
Regulated Gene Expression ◽  
Ivd Degeneration

Abstract Background: Mesenchymal stem/stromal cells (MSCs) have been increasingly used in clinical trials for intervertebral disc (IVD) degeneration. Here, we aimed to evaluate the potential of a cell-free approach to degenerated IVD, testing if MSCs secretome can stimulate a regenerative response by modulating the IVD inflammatory cascade. Methods: Human bone marrow-derived MSCs were pre-conditioned with IL-1β (10 ng/mL) and low oxygen (6% O2). The secretome of MSCs (MSCsec) was collected after 48h. Bovine IVD tissue explants cultured in pro-inflammatory/degenerative conditions (needle puncture + IL-1β) were treated with MSCsec or co-cultured with MSCs. Results: MSCsec obtained upon IL-1β-pre-conditioning, as well as MSCs co-culture, down-regulated gene expression of pro-inflammatory cytokines, bIL-6 and bIL-8 after 48h, in IVD. IVD matrix degrading enzymes, bMMP1 and bMMP3, were downregulated and upregulated, respectively, in the presence of MSCsec, but not MSCs. After 14 days, MSCsec-treated IVDs revealed increased aggrecan content at the protein level, contrarily to MSCs/IVD co-cultures. Interestingly, IL-1β-preconditioning only, but not IL-1β-IVD, increased gene expression of hADAMTS5 and hTIMP-1in MSCs. Additionally, conditioned medium from MSCsec-treated IVDs did not promote angiogenesis or neurogenesis. In MSCsec-treated IVD, an increase in MCP-3 and GCP-2 was observed, while SDF-1α, TNF-α, IGF-1, Eotaxin 3, FGF-9, MIP-1δ, IFN-γ, IL-5, TNF-β, IL-4, TGF-β1, IL-16, IGFBP-3 and IGFBP-4 were decreased, compared with MSCs/IVD co-cultures. Conclusions: MSCsec obtained upon IL1β-preconditioning, present an immunomodulatory role in degenerated IVD, as well as MSCs. Nevertheless, MSCsec but not MSCs, potentiate aggrecan deposition in IVD in pro-inflammatory/degenerative conditions. This finding can open new perspective on the use of MSCsec as a cell-based/cell-free approach to LBP.

scholarly journals Effect of Static Load on the Nucleus Pulposus of Rabbit Intervertebral Disc Motion Segment in an Organ Culture

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Jia-Wen Zhan ◽  
Min-Shan Feng ◽  
Li-Guo Zhu ◽  
Ping Zhang ◽  
Jie Yu
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Static Load ◽  
Ex Vivo ◽  
Compressive Load ◽  
Type Ii ◽  
Matrix Gene ◽  
Motion Segment ◽  
Custom Made ◽  
Ivd Degeneration

The development of mechanically active culture systems helps in understanding of the role of mechanical stress in intervertebral disc (IVD) degeneration. Motion segment cultures facilitate the application and control of mechanical loads. The purpose of this study was to establish a culturing method for rabbit IVD motion segments to observe the effect of static load on the whole disc organ. Segments were cultured in custom-made apparatuses under a constant, compressive load (3 kg) for 2 weeks. Tissue integrity, matrix synthesis, and matrix gene expression profile were assessed and compared with fresh one. The results showedex vivoculturing of samples gradually destroyed the morphology. Proteoglycan contents and gene expression were decreased and downregulated obviously. However, immunohistochemical staining intensity and collagen type II gene expression were significantly enhanced and upregulated. In contrast, these trends were reversed under constant compression. These results indicated short-term static load stimulated the synthesis of type II collagen; however, constant compression led to progressive degeneration and specifically to proteoglycan. Through this study a loading and organ-culturing system forex vivorabbit IVD motion segments was developed, which can be used to study the effects of mechanical stimulation on the biology of IVDs and the pathomechanics of IVD degeneration.

NO regulates LPS-stimulated cyclooxygenase gene expression and activity in pulmonary artery endothelium

2001 ◽  
Vol 280 (3) ◽  
pp. L450-L457 ◽  
Author(s):  
Jian-Xiong Chen ◽  
Leonard C. Berry ◽  
Brian W. Christman ◽  
Miles Tanner ◽  
Paul R. Myers ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pulmonary Artery ◽  
Protein Expression ◽  
Gas Chromatography Mass Spectrometry ◽  
No Donors ◽  
Gene And Protein Expression ◽  
Cox 2 ◽  
Spermine Nonoate ◽  
Cox 1 ◽  
Expression And Activity

We examined whether nitric oxide (NO) inhibits prostanoid synthesis through actions on cyclooxygenase (COX) gene expression and activity. Bovine pulmonary artery endothelial cells were pretreated for 30 min with the NO donors 1 mM S-nitroso- N-acetylpenicillamine (SNAP), 0.5 mM sodium nitroprusside (SNP), or 0.2 μM spermine NONOate; controls included cells pretreated with either 1 mM N-acetyl-d-penicillamine or the NO synthase (NOS) inhibitor 1 mM N G-nitro-l-arginine methyl ester with and without addition of lipopolysaccharide (LPS; 0.1 μg/ml) for 8 h. COX-1 and COX-2 gene and protein expression were examined by RT-PCR and Western analysis, respectively; prostanoid measurements were made by gas chromatography-mass spectrometry, and COX activity was studied after a 30-min incubation with 30 μM arachidonic acid. LPS induced COX-2 gene and protein expression and caused an increase in COX activity and an eightfold increase in 6-keto-PGF1αrelease. LPS-stimulated COX-2 gene expression was decreased by ∼50% by the NO donors. In contrast, LPS caused a significant reduction in COX-1 gene expression and treatment with NO donors had little effect. SNAP, SNP, and NONOate significantly suppressed LPS-stimulated COX activity and 6-keto-PGF1α release. Our data indicate that increased generation of NO attenuates LPS-stimulated COX-2 gene expression and activity, whereas inhibition of endogenous NOS has little effect.

scholarly journals TonEBP regulates the hyperosmotic expression of aquaporin 1 and 5 in the intervertebral disc

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
J. W. Snuggs ◽  
S. Tessier ◽  
R. A. B. Bunning ◽  
I. M. Shapiro ◽  
M. V. Risbud ◽  
...  
Keyword(s):  
Gene Expression ◽  
Intervertebral Disc ◽  
Nucleus Pulposus ◽  
Central Region ◽  
Aquaporin 1 ◽  
Enhancer Binding Protein ◽  
Ivd Degeneration ◽  
Hyperosmotic Environment

AbstractThe central region of the intervertebral disc (IVD) is rich in proteoglycans, leading to a hyperosmotic environment, which fluctuates with daily loading. The cells of the nucleus pulposus (NP cells) have adapted to this environment via the function of tonicity enhancer binding protein (TonEBP), and NP cells have been shown to express several water channels known as aquaporins (AQP). We have previously shown that AQP1 and 5 decrease during IVD degeneration. Here, the regulation of AQP1 and 5 by hyperosmotic conditions and the role of TonEBP in this regulation was investigated. AQP1 and 5 gene expression was upregulated by hyperosmotic conditions mimicking the osmolality of the healthy IVD, which was abrogated by TonEBP knockdown. Furthermore, AQP1 and 5 immunopositivity was significantly reduced in TonEBPΔ/Δ E17.5 mice when compared with wildtype controls, indicating in vivo expression of AQP1 and 5 is controlled at least in part by TonEBP. This hyperosmotic regulation of AQP1 and 5 could help to explain the decreased AQP1 and 5 expression during degeneration, when the osmolality of the NP decreases. Together this data suggests that TonEBP-regulated osmo-adaptation may be disrupted during IVD degeneration when the expression of both AQPs is reduced.

scholarly journals Multiscale Regulation of the Intervertebral Disc: Achievements in Experimental, In Silico, and Regenerative Research

2021 ◽  
Vol 22 (2) ◽  
pp. 703
Author(s):  
Laura Baumgartner ◽  
Karin Wuertz-Kozak ◽  
Christine L. Le Maitre ◽  
Francis Wignall ◽  
Stephen M. Richardson ◽  
...  
Keyword(s):  
Intervertebral Disc ◽  
In Silico ◽  
Current Knowledge ◽  
Treatment Options ◽  
Cell Tissue ◽  
Advanced Therapies ◽  
Holistic Understanding ◽  
Health And Disease ◽  
In Silico Models ◽  
Ivd Degeneration

Intervertebral disc (IVD) degeneration is a major risk factor of low back pain. It is defined by a progressive loss of the IVD structure and functionality, leading to severe impairments with restricted treatment options due to the highly demanding mechanical exposure of the IVD. Degenerative changes in the IVD usually increase with age but at an accelerated rate in some individuals. To understand the initiation and progression of this disease, it is crucial to identify key top-down and bottom-up regulations’ processes, across the cell, tissue, and organ levels, in health and disease. Owing to unremitting investigation of experimental research, the comprehension of detailed cell signaling pathways and their effect on matrix turnover significantly rose. Likewise, in silico research substantially contributed to a holistic understanding of spatiotemporal effects and complex, multifactorial interactions within the IVD. Together with important achievements in the research of biomaterials, manifold promising approaches for regenerative treatment options were presented over the last years. This review provides an integrative analysis of the current knowledge about (1) the multiscale function and regulation of the IVD in health and disease, (2) the possible regenerative strategies, and (3) the in silico models that shall eventually support the development of advanced therapies.

Abstract 16396: Phosphodiesterase-5 Expression and Activity is Increased in Children With Single Ventricle Heart Failure

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Shelley D Miyamoto ◽  
Penny Nelson ◽  
Rebecca Sobus ◽  
Karin Nunley ◽  
Valencia Peterson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heart Failure ◽  
Protein Expression ◽  
Single Ventricle ◽  
Specific Activity ◽  
Pde5 Inhibitor ◽  
Cardiovascular Death ◽  
Promising Therapeutic Target ◽  
Phosphodiesterase 5 ◽  
Expression And Activity

Introduction: Single ventricle congenital heart disease (SV) is the leading cause of cardiovascular death and indication for heart transplantation in infancy. There are no proven therapies for SV heart failure (HF). Human and animal models of HF demonstrate that myocardial phosphodiesterase-5 (PDE5) is increased with cardiac stress and treatment with a PDE5 inhibitor (PDE5i) results in enhanced cardiac function and prevents remodeling. Sildenafil, a PDE5i, is increasingly utilized for the treatment of patients suffering from failing SV. The objective of this study was to determine myocardial PDE5 expression and activity in children transplanted for failing SV. Methods: At the time of cardiac transplantation, explanted pediatric hearts were immediately cooled in ice cold oxygenated Tyrodes in the operating room. The tissue is rapidly dissected, flash frozen and stored at -80 0 C until further use. RNA, protein and cytosolic fractions were isolated from explanted right ventricle (RV) tissue from SV and non-failing (NF) donors. RTqPCR for PDE5, Western blot (normalized to calnexin loading control) for PDE5 and PDE5 activity assays were performed. For PDE 5 activity, cGMP hydrolysis was measured using [ 3 H]cGMP as the substrate. Sildenafil was added to measure PDE5 specific activity, and activity was calculated using nonlinear regression. Results: All patients used in the SV analysis had a failing morphologic RV and were selected from a cohort of 17 SV (median age 0.5, range 0.05-10 yrs) and 8 NF controls (median age 7, range 1.3-13 yrs). PDE5 gene expression was higher in SV myocardium compared to NF (1.9±0.7, n=17 SV vs 1.1±0.5 ct fold change, n=8 NF, p=0.02). There was a trend towards higher PDE5 protein expression in SV myocardium compared to NF (1.5±0.7, n=4 SV vs 1.0±0.4 protein expression normalized to NF, n=3 NF; p=ns). There was increased PDE5 activity in SV compared to NF (22.3±1.2, n=3 SV vs 11.9±4.2 pmol/mg/min, n=2 NF; p=0.02). Conclusions: There is increasing evidence that PDE5i has beneficial direct myocardial effects. There is increased PDE5 gene expression and activity in failing SV myocardium compared to NF control suggesting that PDE5 may represent a promising therapeutic target in this challenging population.

scholarly journals Glucose Oxidase Induces Cellular Senescence in Immortal Renal Cells through ILK by DownregulatingKlothoGene Expression

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Nuria Troyano-Suárez ◽  
María del Nogal-Avila ◽  
Inés Mora ◽  
Patricia Sosa ◽  
Susana López-Ongil ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Protein Expression ◽  
Glucose Oxidase ◽  
Cellular Senescence ◽  
Renal Cells ◽  
Long Time ◽  
P16 Expression ◽  
Expression And Activity ◽  
In Cells

Cellular senescence can be prematurely induced by oxidative stress involved in aging. In this work, we were searching for novel intermediaries in oxidative stress-induced senescence, focusing our interest on integrin-linked kinase (ILK), a scaffold protein at cell-extracellular matrix (ECM) adhesion sites, and on theKlothogene. Cultured renal cells were treated with glucose oxidase (GOx) for long time periods. GOx induced senescence, increasing senescence associatedβ-galactosidase activity and the expression of p16. In parallel, GOx increased ILK protein expression and activity. Ectopic overexpression of ILK in cells increased p16 expression, even in the absence of GOx, whereas downregulation of ILK inhibited the increase in p16 due to oxidative stress. Additionally, GOx reducedKlothogene expression and cells overexpressing Klotho protein did not undergo senescence after GOx addition. We demonstrated a direct link between ILK andKlothosince silencing ILK expression in cells and mice increasesKlothoexpression and reduces p53 and p16 expression in renal cortex. In conclusion, oxidative stress induces cellular senescence in kidney cells by increasing ILK protein expression and activity, which in turn reducesKlothoexpression. We hereby present ILK as a novel downregulator ofKlothogene expression.

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