Kevin Ngo

Mitochondrial-derived reactive oxygen species (ROS) play a causal role in aging-related intervertebral disc degeneration

Oxidative damage is a well‐established driver of aging. Evidence of oxidative stress exists in aged and degenerated discs, but it is unclear how it affects disc metabolism. In this study, we first determined whether oxidative stress negatively impacts disc matrix metabolism using disc organotypic and cell cultures. Mouse disc organotypic culture grown at atmospheric oxygen (20% O2) exhibited perturbed disc matrix homeostasis, including reduced proteoglycan synthesis and enhanced expression of matrix metalloproteinases, compared to discs grown at low oxygen levels (5% O2). Human disc cells grown at 20% O2 showed increased levels of mitochondrial‐derived superoxide anions and perturbed matrix homeostasis. Treatment of disc cells with the mitochondria‐targeted reactive oxygen species (ROS) scavenger XJB‐5‐131 blunted the adverse effects caused by 20% O2. Importantly, we demonstrated that treatment of accelerated aging Ercc1−/Δ mice, previously established to be a useful in vivo model to study age‐related intervertebral disc degeneration (IDD), also resulted in improved disc total glycosaminoglycan content and proteoglycan synthesis. This demonstrates that mitochondrial‐derived ROS contributes to age‐associated IDD in Ercc1−/Δ mice. Collectively, these data provide strong experimental evidence that mitochondrial‐derived ROS play a causal role in driving changes linked to aging‐related IDD and a potentially important role for radical scavengers in preventing IDD.

Bupivacaine decreases cell viability and matrix protein synthesis in an intervertebral disc organ model system

BACKGROUND CONTEXT Bupivacaine is a local anesthetic commonly used for back pain management in interventional procedures. Cytotoxic effects of bupivacaine have been reported in articular cartilage and, recently, in intervertebral disc cell culture. However, the relevance of these effects to discs in vivo remains unclear. This study examines the effect of bupivacaine on disc cell metabolism using an organotypic culture model system that mimics the in vivo environment. PURPOSE To assess the effect of bupivacaine on disc cell viability and matrix protein synthesis using an organotypic model system and to determine whether this anesthetic has toxic effects. STUDY DESIGN Mouse intervertebral discs were isolated and maintained ex vivo in an organotypic culture then exposed to clinically relevant concentrations of bupivacaine, and the impact on disc cell viability and matrix proteoglycan (PG) and collagen syntheses were measured in the presence and absence of the drug. SUBJECTS Mouse functional spine units (FSUs) were isolated from the lumbar spines of 10-week-old mice. OUTCOME MEASURES Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Total PG and collagen syntheses were determined by measuring the incorporation of radioactive (35)S-sulfate and (3)H-l-proline into PG and collagen, respectively. METHODS Organotypic cultures of mouse FSUs were exposed to different concentrations (0%-0.5%) of bupivacaine for variable amounts of time (0-2 hours). Cell viability within disc tissue was quantified by MTT staining and histologic assay. Matrix protein synthesis was measured by incorporation of radioactive (35)S-sulfate (for PG synthesis) and (3)H-l-proline (for collagen synthesis). RESULTS Untreated mouse disc organs were maintained in culture for up to 1 month with minimal changes in tissue histology, cell viability, and matrix protein synthesis. Exposure to bupivacaine decreased cell viability in a dose- and time-dependent manner. Exposure to bupivacaine at concentrations less than or equal to 0.25% did not significantly affect matrix protein synthesis. However, at 0.5% bupivacaine, collagen synthesis was reduced by fourfold and PG synthesis by threefold. CONCLUSIONS Mouse discs can be successfully maintained ex vivo for upward of 4 weeks with little cell death, change in histologic structure, or matrix protein synthesis. This organotypic model system closely mimics the in vivo environment of the disc. Exposure of these cultures to bupivacaine dramatically decreased cell viability and matrix protein synthesis in a dose- and time-dependent manner. These findings corroborate those previously reported by Lee et al. using disc cell culture and demonstrate that this anesthetic at clinically relevant doses is toxic to intervertebral discs in both cell culture and disc organ models representative of the native architectural context.

ISSLS prize winner: inhibition of NF-κB activity ameliorates age-associated disc degeneration in a mouse model of accelerated aging.

Study Design. NF-&kgr;B activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. Objective. To study the mediatory role of NF-&kgr;B-signaling pathway in age-dependent intervertebral disc degeneration. Summary of Background Data. Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-&kgr;B is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-&kgr;B activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-&kgr;B inhibition on IDD, using a DNA repair-deficient mouse model of accelerated aging (Ercc1−/&Dgr; mice) previously been reported to exhibit age-related IDD. Methods. Systemic inhibition of NF-&kgr;B activation was achieved either genetically by deletion of 1 allele of the NF-&kgr;B subunit p65 (Ercc1−/&Dgr;p65+− mice) or pharmacologically by chronic intraperitoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-&kgr;B, I&kgr;B Inducible Kinase (IKK), in Ercc1−/&Dgr; mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice, and untreated controls were assessed. Results. Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-&kgr;B activity were observed in discs of progeroid Ercc1−/&Dgr; mice and naturally aged wild-type mice compared with young wild-type mice. Systemic inhibition of NF-&kgr;B by the 8K-NBD peptide in Ercc1−/&Dgr; mice increased disc proteoglycan synthesis and ameriolated loss of disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1−/&Dgr;p65+/− mice. Conclusion. These findings demonstrate that the IKK/NF-&kgr;B signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.

Differential effects of nicotine and tobacco smoke condensate on human annulus fibrosus cell metabolism

Tobacco smoking increases the risk of intervertebral disc degeneration (IDD) and back pain, but the mechanisms underlying the adverse effects of smoking are largely unknown. Current hypotheses predict that smoking contributes to IDD indirectly through nicotine‐mediated vasoconstriction which limits the exchange of nutrients between the discs and their surroundings. We alternatively hypothesize that direct contact of disc cells, that is, cells in the outermost annulus and those present along fissures in degenerating discs, with the vascular system containing soluble tobacco smoking constituents could perturb normal metabolic activities resulting in IDD. In this study, we tested our hypothesis by comparing the effects of direct exposure of human disc cells to tobacco smoke condensate and nicotine on cell viability and metabolic activity. We showed that smoke condensate, which contains all of the water‐soluble compounds inhaled by smokers, exerts greater detrimental effects on human disc cell viability and metabolism than nicotine. Smoke condensate greatly induced an inflammatory response and gene expression of metalloproteinases while reduced active matrix synthesis and expression of matrix structural genes. Therefore, we have demonstrated that disc cell exposure to the constituents of tobacco smoke has negative consequences which have the potential to alter disc matrix homeostasis.

Investigating the role of DNA damage in tobacco smoking-induced spine degeneration

BACKGROUND CONTEXT Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage. PURPOSE To determine if DNA damage plays a causal role in smoking-induced spine degeneration. STUDY DESIGN To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes. METHODS Two-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1(-/Δ) mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis ((35)S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured. RESULTS Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1(-/Δ) mice. CONCLUSIONS Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.

Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs.

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

Biological responses to flexion/extension in spinal segments ex‐vivo

Mechanical loading is a salient factor in the progression of spinal disorders that contribute to back pain. Biological responses to loading modes like flexion/extension (F/E) in relevant spinal tissues remain unstudied. A novel, multi‐axial experimental system was developed to subject viable functional spinal units (FSUs) to complex, in‐situ loading. The objective was to determine biological effects of F/E in multiple spinal tissues—annulus fibrosus, nucleus pulposus, facet cartilage, and ligamentum flavum. Rabbit lumbar FSUs were mounted in a bioreactor within a robotic testing system. FSUs underwent small (0.17/0.05 Nm) and large (0.5/0.15 Nm) range‐of‐motion F/E for 1 or 2 h of cycling. Outcomes in each tissue, compared to unloaded FSUs, included (i) relative mRNA expression of catabolic (MMP‐1, 3 and ADAMTS‐5), pro‐inflammatory (COX‐2), and anabolic (ACAN) genes and (ii) immunoblotting of aggrecan degradation. Total energy applied to FSUs increased in groups subject to large range‐of‐motion and 2‐h cycling, and moment relaxation was higher with large range‐of‐motion. F/E significantly modulated MMP1,‐3 and COX‐2 in facet cartilage and MMP‐3 and ACAN in annulus fibrosus. Large range‐of‐motion loading increased MMP‐mediated aggrecan fragmentation in annulus fibrosus. Biological responses to complex loading ex vivo showed variation among spinal tissues that reflect tissue structure and mechanical loading in F/E.

Catabolic effects of endothelial cell-derived microparticles on disc cells: Implications in intervertebral disc neovascularization and degeneration.

Neovascularization of intervertebral discs, a phenomenon considered pathological since normal discs are primarily avascular structures, occurs most frequently in annulus fibrosus (AF) of degenerated discs. Endothelial cells (ECs) are involved in this process, but the mechanism of the interaction between AF and endothelial cells is unclear. In this study, we evaluated the effects on matrix catabolic activity of AF cells by the extracellular endothelial microparticles (EMPs) and soluble protein factors (SUP fraction) produced from ECs. Passage 1 human AF cells grown in monolayer cultures were treated for 72 h with 250 µg of EMPs or SUP fraction isolated from culture of the microvascular endothelial cell line, HEMC‐I. Live‐cell imaging revealed uptake of EMPs by AF cells. RT‐PCR analysis demonstrated increased mRNA expression of MMP‐1 (50.3‐fold), MMP‐3 (4.5‐fold) and MMP‐13 (5.5‐fold) in AF cell cultures treated with EMPs compared to untreated control. Western analysis also demonstrated increased MMP protein expression in EMP‐treated AF cells. AF cells treated with the SUP fraction also exhibited a dramatic increase in MMP mRNA and protein expression. Increased MMP expression is primarily due to EMP or SUP stimulation of AF cells since EMPs or SUP fraction alone contained negligible amount of MMPs. Interestingly, MMP activity was elevated in AF cell cultures treated with EMPs but not with SUP. This study revealed enhanced matrix catabolism as a molecular consequence of action of ECs on AF cells via EMPs, which might be expected during neo‐angiogenesis of degenerating disc.

Adamts5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration

Study Design. ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). Objective. The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. Summary of Background Data. We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. Methods. Three-month-old WT (C57BL/6) and ADAMTS5−/− mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-&kgr;B, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. Results. Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-&kgr;B, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. Conclusion. The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. Level of Evidence: N/A

Senescent intervertebral disc cells exhibit perturbed matrix homeostasis phenotype

Aging greatly increases the risk for intervertebral disc degeneration (IDD) as a result of proteoglycan loss due to reduced synthesis and enhanced degradation of the disc matrix proteoglycan (PG). How disc matrix PG homeostasis becomes perturbed with age is not known. The goal of this study is to determine whether cellular senescence is a source of this perturbation. We demonstrated that disc cellular senescence is dramatically increased in the DNA repair-deficient Ercc1-/Δ mouse model of human progeria. In these accelerated aging mice, increased disc cellular senescence is closely associated with the rapid loss of disc PG. We also directly examine PG homeostasis in oxidative damage-induced senescent human cells using an in vitro cell culture model system. Senescence of human disc cells treated with hydrogen peroxide was confirmed by growth arrest, senescence-associated β-galactosidase activity, γH2AX foci, and acquisition of senescence-associated secretory phenotype. Senescent human disc cells also exhibited perturbed matrix PG homeostasis as evidenced by their decreased capacity to synthesize new matrix PG and enhanced degradation of aggrecan, a major matrix PG. of the disc. Our in vivo and in vitro findings altogether suggest that disc cellular senescence is an important driver of PG matrix homeostatic perturbation and PG loss.