Nam Vo

NF-κB inhibition delays DNA damage–induced senescence and aging in mice

The accumulation of cellular damage, including DNA damage, is thought to contribute to aging-related degenerative changes, but how damage drives aging is unknown. XFE progeroid syndrome is a disease of accelerated aging caused by a defect in DNA repair. NF-κB, a transcription factor activated by cellular damage and stress, has increased activity with aging and aging-related chronic diseases. To determine whether NF-κB drives aging in response to the accumulation of spontaneous, endogenous DNA damage, we measured the activation of NF-κB in WT and progeroid model mice. As both WT and progeroid mice aged, NF-κB was activated stochastically in a variety of cell types. Genetic depletion of one allele of the p65 subunit of NF-κB or treatment with a pharmacological inhibitor of the NF-κB-activating kinase, IKK, delayed the age-related symptoms and pathologies of progeroid mice. Additionally, inhibition of NF-κB reduced oxidative DNA damage and stress and delayed cellular senescence. These results indicate that the mechanism by which DNA damage drives aging is due in part to NF-κB activation. IKK/NF-κB inhibitors are sufficient to attenuate this damage and could provide clinical benefit for degenerative changes associated with accelerated aging disorders and normal aging.

Expression and regulation of metalloproteinases and their inhibitors in intervertebral disc aging and degeneration

BACKGROUND CONTEXT Destruction of extracellular matrix (ECM) leads to intervertebral disc degeneration (IDD), which underlies many spine-related disorders. Matrix metalloproteinases (MMPs), and disintegrins and metalloproteinases with thrombospondin motifs (ADAMTSs) are believed to be the major proteolytic enzymes responsible for ECM degradation in the intervertebral disc (IVD). PURPOSE To summarize the current literature on gene expression and regulation of MMPs, ADAMTSs, and tissue inhibitors of metalloproteinases (TIMPs) in IVD aging and IDD. METHODS A comprehensive literature review of gene expression of MMP, ADAMTS, and TIMP in human IDD and reported studies on regulatory factors controlling their expressions and activities in both human and animal model systems. RESULTS Upregulation of specific MMPs (MMP-1, -2, -3, -7, -8, -10, and -13) and ADAMTS (ADAMTS-1, -4, and -15) were reported in human degenerated IVDs. However, it is still unclear from conflicting published studies whether the expression of ADAMTS-5, the predominant aggrecanase, is increased with IDD. Tissue inhibitors of metalloproteinase-3 is downregulated, whereas TIMP-1 is upregulated in human degenerated IVDs relative to nondegenerated IVDs. Numerous studies indicate that the expression levels of MMP and ADAMTS are modulated by a combination of many factors, including mechanical, inflammatory, and oxidative stress, some of which are mediated in part through the p38 mitogen-activated protein kinase pathway. Genetic predisposition also plays an important role in determining gene expression of MMP-1, -2, -3, and -9. CONCLUSIONS Upregulation of MMP and ADAMTS expression and enzymatic activity is implicated in disc ECM destruction, leading to the development of IDD. Future IDD therapeutics depends on identifying specific MMPs and ADAMTSs whose dysregulation result in pathological proteolysis of disc ECM.

p38 MAPK Inhibition Modulates Rabbit Nucleus Pulposus Cell Response to IL-1

Analysis of disc gene expression implicated IL‐1 in the development of intervertebral disc degeneration (IDD) in a rabbit stab model. The purpose of these studies is to determine the role of p38 Mitogen Activated Protein Kinase (p38 MAPK) signaling in nucleus pulposus cell response to IL‐1, and to compare rabbit nucleus pulposus (rNP) cell responses to IL‐1 activation with those in a stab model of disc degeneration. NP cells maintained in alginate bead culture were exposed to IL‐1, with or without p38 MAPK inhibition. RNA was isolated for reverse transcription polymerase chain reaction (RT‐PCR) analysis of gene expression, conditioned media analyzed for accumulation of nitric oxide (NO) and prostaglandin E‐2 (PGE‐2), and proteoglycan synthesis measured after 10 days. IL‐1 upregulation of mRNA for cycloxygenase‐2 (COX‐2), matrix metalloproteinase‐3 (MMP‐3), IL‐1, and IL‐6, was blunted by p38 inhibition while downregulation of matrix proteins (collagen I, collagen II, aggrecan) and insulin‐like‐growth‐factor I (IFG‐1) was also reversed. mRNA for tissue inhibitor of matrixmetalloproteinase‐1 (TIMP‐1) was modestly increased by IL‐1, while those for Transforming Growth Factor‐β (TGF‐β) SOX‐9, and versican remained unchanged. Blocking p38 MAPK reduced IL‐1 induced NO and PGE‐2 accumulation and partially restored proteoglycan synthesis. p38 MAPK inhibition in control cells increased mRNA for matrix proteins (aggrecan, collagen II, versican, collagen I) and anabolic factors (IGF‐1, TGF, and SOX‐9) from 50% to 120%, decreased basal PGE‐2 accumulation, but had no effect on message for TIMP‐1, MMP‐3, or COX‐2. Inhibition of p38 MAPK in cytokine‐activated disc cells blunts gene expression and production of factors associated with inflammation, pain, and disc matrix catabolism while reversing IL‐1 downregulation of matrix protein gene expression and proteoglycan synthesis. The results support the hypothesis that IL‐1 could be responsible for many of the mRNA changes seen in rabbit NP in the stab model of disc degeneration, and uphold the concept that development of molecular techniques to block p38 MAPK could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

p38 MAPK inhibition in nucleus pulposus cells: a potential target for treating intervertebral disc degeneration.

Study Design. Human nucleus pulposus cells were cultured in alginate beads and activated with IL-1&bgr; or TNF-&agr;, with and without inhibition of p38 mitogen activated protein kinase (p38 MAPK) activity. Cell production of factors modulating the anabolic/catabolic balance of the disc was determined. Objective. To determine the role of signaling through p38 MAPK in nucleus pulposus cells response to inflammatory cytokines and whether it might be a valid target for the development of molecular therapies for disc degeneration. Summary of Background Data. Multiple factors contribute to intervertebral disc degeneration (IDD), and development of effective therapies depends on understanding the underlying cellular pathophysiology. Interleukin-1&bgr; and tumor necrosis factor-&agr; are implicated in the development of IDD, and p38 MAPK is part of cytokine and mechanical stress signal pathways in other cells. These studies determine whether inhibiting p38 MAPK can decrease factors that negatively affect the metabolic balance and viability of nucleus pulposus cells. Materials and Methods. Degenerated intervertebral disc tissue was obtained from patients undergoing elective surgical procedures. Nucleus pulposus cells in alginate bead culture were exposed to IL-1 or TNF-&agr;, with or without p38 MAPK inhibition, and conditioned media analyzed for accumulation of nitric oxide (NO), prostaglandin E2 (PGE2), IL-6, matrix metalloproteinase-3 (MMP-3), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) through 10 days. Results. Inhibition of p38 MAPK decreased PGE2 in conditioned medium of control, unstimulated cells while not affecting TIMP-1 accumulation. Blocking cytokine activation of p38 MAPK reduced IL-1 and TNF-&agr; induced PGE2 and IL-6 accumulation. p38 MAPK inhibition increased the ratio of TIMP-1 to MMP-3 in conditioned medium of cells activated by IL-1 or TNF-&agr;. Conclusion. Inhibition of p38 MAPK in cytokine-activated disc cells blunts production of factors associated with inflammation, pain, and disc matrix catabolism. The data support further analysis of these effects on the anabolic/catabolic balance of nucleus pulposus cells and suggest that molecular techniques blocking this signal could provide a therapeutic approach to slow the course of intervertebral disc degeneration.

Human Nucleus Pulposus Cells React to IL-6 : Independent Actions and Amplification of Response to IL-1 and TNF-α

Study Design. Human nucleus pulposus cells were activated with IL-6 plus IL-6 soluble receptor (sR) in the presence or absence of IL-1&bgr; or TNF-&agr;. Cell production of factors modulating the anabolic/catabolic balance of the disc and proteoglycan synthesis were determined. Objective. To evaluate NP cell response to exogenous IL-6, and how IL-6 modulates IL-1 and TNF-&agr; actions in these cells. Summary of Background Data. Interleukin-6 (IL-6) is produced by cervical and lumbar herniated discs and is associated with neurological symptoms of intervertebral disc degeneration. It upregulates catabolic gene expression and downregulates matrix protein gene expression in chondrocytes. However, no studies have evaluated the effects of IL-6 on disc nucleus pulposus (NP) cells. Methods. NP cells from degenerated human discs were expanded in monolayer, maintained in alginate bead culture, and activated with IL-6 plus IL-6 soluble receptor (sR), in the presence or absence of IL-1&bgr; or TNF-&agr;. Conditioned media was collected and analyzed for nitrite, PGE-2, TIMP-1, MMP-3, VEGF, and IL-8. Proteoglycan synthesis was assayed as 35S-sulfate incorporation normalized to DNA content and relative gene expression measured by rtPCR. Results. IL-6 + sR decreased collagen and aggrecan message, proteoglycan synthesis, and exacerbated the downregulation of proteoglycan synthesis effected by IL-1. PGE-2 synthesis was increased by IL-6 + sR, as was the induction of COX-2 mRNA. IL-6 + sR also enhanced IL-1 and TNF-&agr; stimulated synthesis of PGE-2. IL-6 + sR induced MMP-3 approximately twofold and increased gene expression and synthesis in cells exposed to IL-1 and TNF-&agr;. MMP-13 induction by TNF-&agr; was also potentiated by IL-6 + sR. IL-6 + sR induced IL-6 gene expression and increased that stimulated by TNF-&agr; fourfold. Conclusion. The results suggest maneuvers to diminish IL-6 production in the disc could provide some protection against the adverse effects of IL-1 and TNF-&agr;, thus, helping preserve disc composition, structure, and function.

Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro

Study Design. We demonstrated the differentiation of notochordal cells by direct observation using a live automated cell imaging system. We also hypothesized that notochordal cells have characteristics of chondrocyte-like cells. Objective. To determine characteristics of notochordal cells by matrix protein expression and their differentiation using a live automated cell imager. Summary of Background Data. Although notochordal cells are critical to homeostasis of intervertebral disc, their fate has not been extensively studied and there is little evidence of notochordal cells as progenitors. Methods. Notochordal cells purified from rabbit nucleus pulposus were isolated after serial filtration. Notochordal cells in 3-dimensional culture were compared to chondrocyte-like cells by 35S sulfate incorporation into proteoglycan and reverse transcription polymerase chain reaction for gene expression(collagen II and aggrecan). Notochordal cells in 2-D culture were used for immunocytochemical staining (collagen II, aggrecan, and SOX9) and time-lapsed cell tracking study. Results. Notochordal cells were capable of proteoglycan production at a rate comparable to chondrocyte-like cells (108% ± 22.6% to chondrocyte-like cells) and expressed collagen II, aggrecan, and SOX9. In time-lapsed cell tracking analysis, notochordal cells were slower in population doubling time than chondrocyte-like cells and differentiated into 3 morphologically distinct cell types: vacuolated cells (area: 2392 ± 507.1 &mgr;m2, velocity: 0.09 ± 0.01 &mgr;m/min); giant cells (area: 12678 ± 1637.0 &mgr;m2, velocity: 0.08 ± 0.01 &mgr;m/min) which grew rapidly without cell division; polygonal cells (area: 3053 ± 751.2 &mgr;m2, 0.14 ± 0.01 &mgr;m/min) morphologically similar to typical differentiation type of chondrocyte-like cells (area: 2671 ± 235.6 &mgr;m2, 0.19 ± 0.01 &mgr;m/min). Rarely, notochordal cells formed clusters analogous to that observed in vivo. Conclusion. These studies demonstrate a chondrocyte phenotype of notochordal cells and are the first direct evidence of notochordal cell differentiation, suggesting that they may act as progenitor cells, which has the potential to lead to their use in novel approaches to regeneration of degenerative intervertebral disc.

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.

Spine degeneration in a murine model of chronic human tobacco smokers

OBJECTIVE To investigate the mechanisms by which chronic tobacco smoking promotes intervertebral disc degeneration (IDD) and vertebral degeneration in mice. METHODS Three month old C57BL/6 mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 6 months) to model long-term smoking in humans. Total disc proteoglycan (PG) content [1,9-dimethylmethylene blue (DMMB) assay], aggrecan proteolysis (immunobloting analysis), and cellular senescence (p16INK4a immunohistochemistry) were analyzed. PG and collagen syntheses ((35)S-sulfate and (3)H-proline incorporation, respectively) were measured using disc organotypic culture. Vertebral osteoporosity was measured by micro-computed tomography. RESULTS Disc PG content of smoke-exposed mice was 63% of unexposed control, while new PG and collagen syntheses were 59% and 41% of those of untreated mice, respectively. Exposure to tobacco smoke dramatically increased metalloproteinase-mediated proteolysis of disc aggrecan within its interglobular domain (IGD). Cellular senescence was elevated two-fold in discs of smoke-exposed mice. Smoke exposure increased vertebral endplate porosity, which closely correlates with IDD in humans. CONCLUSIONS These findings further support tobacco smoke as a contributor to spinal degeneration. Furthermore, the data provide a novel mechanistic insight, indicating that smoking-induced IDD is a result of both reduced PG synthesis and increased degradation of a key disc extracellular matrix protein, aggrecan. Cleavage of aggrecan IGD is extremely detrimental as this results in the loss of the entire glycosaminoglycan-attachment region of aggrecan, which is vital for attracting water necessary to counteract compressive forces. Our results suggest identification and inhibition of specific metalloproteinases responsible for smoke-induced aggrecanolysis as a potential therapeutic strategy to treat IDD.

An overview of underlying causes and animal models for the study of age-related degenerative disorders of the spine and synovial joints

As human lifespan increases so does the incidence of age‐associated degenerative joint diseases, resulting in significant negative socioeconomic consequences. Osteoarthritis (OA) and intervertebral disc degeneration (IDD) are the most common underlying causes of joint‐related chronic disability and debilitating pain in the elderly. Current treatment methods are generally not effective and involve either symptomatic relief with non‐steroidal anti‐inflammatory drugs and physical therapy or surgery when conservative treatments fail. The limitation in treatment options is due to our incomplete knowledge of the molecular mechanism of degeneration of articular cartilage and disc tissue. Basic understanding of the age‐related changes in joint tissue is thus needed to combat the adverse effects of aging on joint health. Aging is caused at least in part by time‐dependent accumulation of damaged organelles and macromolecules, leading to cell death and senescence and the eventual loss of multipotent stem cells and tissue regenerative capacity. Studies over the past decades have uncovered a number of important molecular and cellular changes in joint tissues with age. However, the precise causes of damage, cellular targets of damage, and cellular responses to damage remain poorly understood. The objectives of this review are to provide an overview of the current knowledge about the sources of endogenous and exogenous damaging agents and how they contribute to age‐dependent degenerative joint disease, and highlight animal models of accelerated aging that could potentially be useful for identifying causes of and therapies for degenerative joint diseases.

Effect of bupivacaine on intervertebral disc cell viability

BACKGROUND CONTEXT Bupivacaine is a local anesthetic commonly used to relieve or control pain in interventional spine procedures. Bupivacaine has been shown to be toxic to articular cartilage, which has similarities to intervertebral disc (IVD) cartilage, raising concern over a potentially negative effect of bupivacaine on the disc. PURPOSE To determine bupivacaines effect on cell viability of IVD cells in vitro and to elucidate whether this is through apoptosis or necrosis. STUDY DESIGN In vitro controlled study of bupivacaine effect on cell viability in human and rabbit IVD cells. SUBJECTS Rabbit annulus fibrosus (AF) tissue, nucleus pulposus (NP) cells, and knee articular chondrocytes were isolated from New Zealand white rabbits. Human AF and NP cells were isolated from stage 3 to 4 degenerative disc surgical specimens. OUTCOME MEASURES Cell viability was assessed after exposure to bupivacaine via trypan blue staining or flow cytometry. METHODS Annulus fibrosus and NP cells were grown in monolayer and alginate beads, respectively, to simulate their physiologic environment. The cells were then exposed to bupivacaine or saline control at 60 and 120 minutes and examined for cell viability. RESULTS Rabbit NP cell death demonstrated a time and dose dependence in response to bupivacaine. In addition, cell death was greater than that observed for articular chondrocytes. Rabbit AF tissue also demonstrated increased cell death in response to bupivacaine exposure. Human NP cells demonstrated time-dependent cell death, with greater necrosis than apoptosis. Annulus fibrosus cells grown in monolayers also resulted in similar effects, with greater necrosis rather than apoptosis. CONCLUSIONS Despite its pain relieving properties, bupivacaine decreases cell viability in rabbit and human disc cells in a time-dependent manner. In addition, the changes observed are greater than that seen for articular chondrocytes. This increase in cell death appears to be related to an increase in necrosis rather than apoptosis. Whether bupivacaine exerts similar effects in vivo or how this relates to overall clinical outcome remains to be explored.