[Show abstract][Hide abstract] ABSTRACT: The ECM of the intervertebral disc and articular cartilage contain a highly organised network of collagens and proteoglycans which resist compressive forces applied to these tissues. A pathological hallmark of the intervertebral disc is the imbalance between production of anabolic and catabolic factors by the resident cells. This process is thought to be mediated by pro-inflammatory cytokines, predominantly TNF-α and IL-1β, which upregulate expression of matrix degrading enzymes such as MMPs and ADAMTSs. This imbalance ultimately results in tissue degeneration causing failure of the biomechanical function of the tissues. A similar cascade of events is thought to occur in articular cartilage during development of osteoarthritis. Within these skeletal tissues a small, cell surface heparan sulphate proteoglycan; syndecan-4 (SDC4) has been implicated in maintaining physiological functions. However in the degenerating niche of the intervertebral disc and cartilage, dysregulated activities of this molecule may exacerbate pathological changes. Studies in recent years have elucidated a role for SDC4 in mediating matrix degradation in both intervertebral discs and cartilage by controlling ADAMTS-5 function and MMP3 expression. Discourse presented in this review highlights the potential of SDC4 as possible therapeutic target in slowing the progression of ECM degradation in both degenerative disc disease and osteoarthritis.
Full-text · Article · Jan 2016 · Matrix biology: journal of the International Society for Matrix Biology
[Show abstract][Hide abstract] ABSTRACT: The objective of this study was to determine the role of histone deacetylases (HDACs) in regulating HIF-1α protein stability and activity in nucleus pulposus (NP) cells. Treatment of NP cells with pan-HDAC inhibitor TSA resulted in decreased HIF-1α levels under both normoxia and hypoxia in a dose-dependent fashion. TSA-mediated HIF-1α degradation was rescued by concomitant inhibition of not only the 26S proteasome but also PHD2 function. Moreover, TSA treatment of PHD2(-/-) cells had little effect on HIF-1α levels, supporting the notion that inhibition of PHD2 function by HDACs contributed to HIF-1α stabilization. Surprisingly, class-specific HDAC-inhibitors did not affect HIF-1α protein stability, indicating that multiple HDACs controlled HIF-1α stability by regulating HIF-1α-PHD2 interaction in NP cells. Interestingly, lower-dose TSA that did not affect HIF-1α stability decreased its activity and target gene expression. Likewise, rescue of TSA-mediated HIF-1α protein degradation by blocking proteasomal or PHD activity did not restore HIF-1 activity, suggesting that HDACs independently regulate HIF-1α stability and activity. Noteworthy, selective inhibition of HDAC6 and not of class I and IIa HDACs decreased HIF-1-mediated transcription under hypoxia, to a similar extent as lower-dose TSA, contrasting the reported role of HDAC6 as a transcriptional repressor in other cell types. Moreover, HDAC6 inhibition completely blocked TSA effects on HIF-1 activity. HDAC6 associated with and deacetylated HSP90, an important cofactor for HIF-1 function in NP cells, and HDAC6 inhibition decreased p300 transactivation in NP cells. Taken together, these results suggest that while multiple Class I and Class IIa HDACs control HIF-1 stability, HDAC6, a class IIb HDAC, is a novel mediator of HIF-1 activity in NP cells possibly through promoting action of critical HIF-1 cofactors. This article is protected by copyright. All rights reserved.
No preview · Article · Jan 2016 · Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research
[Show abstract][Hide abstract] ABSTRACT: The intervertebral disc is a unique avascular organ that supports axial skeleton flexion and rotation. The high proteoglycan content of the nucleus pulposus tissue, present at the center of the disc, is pivotal for its mechanical function, distribution of compressive loads. Chronic low back pain, a prevalent and costly condition, is strongly associated with disc degeneration. Degenerated discs exhibit high levels of inflammatory cytokines, matrix catabolizing enzymes, and an overall reduction in proteoglycan content. Although the cytokine profile of diseased discs has been widely studied, little is known of what initiates and drives inflammation and subsequent low back pain. Recent studies have shown that anaerobic bacteria are present in a high percentage of painful, herniated discs and long-term treatment with antibiotics resolves symptoms associated with chronic low back pain. It is thought that these anaerobic bacteria in the disc may stimulate inflammation through toll-like receptors to further exacerbate disc degeneration. Despite the promise and novelty of this theory, there are other possible inflammatory mediators that need careful consideration. The metabolic environment associated with diabetes and atypical matrix degradation products also have the ability to activate many of the same inflammatory pathways as seen during microbial infection. It is therefore imperative that the research community must investigate the contribution of all possible drivers of inflammation to address the wide spread problem of discogenic chronic low back pain.
No preview · Article · Nov 2015 · Discovery medicine
[Show abstract][Hide abstract] ABSTRACT: Galectin-3 is highly expressed in notochordal nucleus pulposus (NP) and thought to play important physiological roles; however, regulation of its expression remains largely unexplored. The aim of the study was to investigate if TGFβ regulates Galectin-3 expression in NP cells. TGFβ treatment resulted in decreased galectin-3 expression. Bioinformatic analysis using JASPAR and MatInspector databases cross-referenced with published ChIP-Seq data showed nine locations of highly probable Smad3 binding in the LGALS3 proximal promoter. In NP cells, TGFβ treatment resulted in decreased activity of reporters harboring several 5' deletions of the proximal Galectin-3 promoter. While transfection of NP cells with constitutively active (CA)-ALK5 resulted in decreased promoter activity, DN-ALK5 blocked the suppressive effect of TGFβ on the promoter. The suppressive effect of Smad3 on the Galectin-3 promoter was confirmed using gain- and loss-of-function studies. Transfection with DN-Smad3 or Smad7 blocked TGFβ mediated suppression of promoter activity. We also measured Galectin-3 promoter activity in Smad3 null and wild type cells. Noteworthy, promoter activity was suppressed by TGFβ only in wild type cells. Likewise, stable silencing of Smad3 in NP cells using sh-Smad3 significantly blocked TGFβ-dependent decrease in Galectin-3 expression. Treatment of human NP cells isolated from tissues with different grades of degeneration showed that Galectin-3 expression was responsive to TGF-β-mediated suppression. Importantly, Galectin-3 synergized effects of TNF-α on inflammatory gene expression by NP cells. Together these studies suggest that TGFβ, through Smad3 controls Galectin-3 expression in NP cells and may have implications in the intervertebral disc degeneration.
No preview · Article · Nov 2015 · Matrix biology: journal of the International Society for Matrix Biology
[Show abstract][Hide abstract] ABSTRACT: The intervertebral disc is an important mechanical structure that allows range of motion of the spinal column. Degeneration of the intervertebral disc - incited by aging, traumatic insult, genetic predisposition, or other factors - is often defined by functional and structural changes in the tissue, including excessive breakdown of the extracellular matrix, increased disc cell senescence and death, as well as compromised biomechanical function of the tissue. Intervertebral disc degeneration is strongly correlated with low back pain, which is a highly prevalent and costly condition, significantly contributing to loss in productivity and health care costs. Disc degeneration is a chronic, progressive condition, and current therapies are limited and often focused on symptomatic pain relief rather than curtailing the progression of the disease. Inflammatory processes exacerbated by cytokines tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) are believed to be key mediators of disc degeneration and low back pain. In this review, we describe the contributions of TNF-α and IL-1β to changes seen during disc degeneration at both cellular and tissue level, as well as new evidence suggesting a link between infection of the spine and low back pain, and the emerging therapeutic modalities aimed at combating these processes.
No preview · Article · Sep 2015 · European cells & materials
[Show abstract][Hide abstract] ABSTRACT: The intervertebral disc is an important mechanical structure that allows range of motion of the spinal column. Degeneration of the intervertebral disc - incited by aging, traumatic insult, genetic predisposition, or other factors - is often defined by functional and structural changes in the tissue, including excessive breakdown of the extracellular matrix, increased disc cell senescence and death, as well as compromised biomechanical function of the tissue. Intervertebral disc degeneration is strongly correlated with low back pain, which is a highly prevalent and costly condition, significantly contributing to loss in productivity and health care costs. Disc degeneration is a chronic, progressive condition, and current therapies are limited and often focused on symptomatic pain relief rather than curtailing the progression of the disease. Inflammatory processes exacerbated by cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) are believed to be key mediators of disc degeneration and low back pain. In this review, we describe the contributions of TNF-alpha and IL-1 beta to changes seen during disc degeneration at both cellular and tissue level, as well as new evidence suggesting a link between infection of the spine and low back pain, and the emerging therapeutic modalities aimed at combating these processes.
No preview · Article · Jul 2015 · European cells & materials
[Show abstract][Hide abstract] ABSTRACT: Allograft bone is commonly used to augment bone stock. Unfortunately, allograft is prone to bacterial contamination and current antimicrobial therapies are inadequate. Photoactivated porphyrins combat bacterial growth by production of reactive oxygen species (ROS); however, to our knowledge, they have not been tested in the setting of allograft bone.
We asked: (1) Does 5,10,15,20-tetrakis-(4-aminophenyl)-porphyrin (TAPP) stably adsorb to morselized, mineralized allograft? (2) Does Staphylococcus aureus acquire TAPP from TAPP-allograft? (3) Is TAPP-allograft antibacterial to S. aureus? (4) Is ROS production critical for antimicrobial activity? (5) Does illuminated TAPP-allograft dislodge biofilm? (6) Could other photoactive dyes (TAPP, TMPyP, TSP, THP, and methylene blue) confer antimicrobial properties to allograft?
TAPP adsorption to allograft (TAPP-allograft), its localization in S. aureus, and TAPP-allograft long-term stability were determined spectrophotometrically. Antimicrobial activity was measured while activated with light or in the dark during incubation with S. aureus or after allograft biofilm formation. Glutathione was added to illuminated TAPP-allograft to quench ROS and antimicrobial activity was determined. Light-dependent antimicrobial activity of other photoactive dyes (TMPyP, TSP, THP, and methylene blue) adsorbed to allograft was also tested.
We found (1) porphyrins strongly adhere to bone allograft; and (2) the bacteria are not able to sequester TAPP from the TAPP-allograft; (3) when illuminated, TAPP-allograft is resistant to bacterial adherence; (4) the effects of TAPP are inhibited by the radical scavenger glutathione, indicating ROS-dependent antimicrobial activity; (5) illumination of TAPP-allograft disrupts biofilms; and, (6) other photoactive dyes impede biofilm formation on allograft bone in the presence of light.
Porphyrins stably associate with allograft and are inactive until illuminated. Illuminated TAPP-allograft markedly reduces bacterial colonization, which is restored in the presence of radical scavengers. Finally, illuminated TAPP-allograft disrupts biofilms.
The findings of this in vitro study suggest that loading bone allograft with biocompatible porphyrins before surgery might allow increased sterility of the allograft during implantation. Future testing in an animal model will determine if these in vitro activities can be used to prevent allograft-based infection in an establishing osteomyelitis.
No preview · Article · Apr 2015 · Clinical Orthopaedics and Related Research
[Show abstract][Hide abstract] ABSTRACT: Periprosthetic joint infection (PJI) is one of the most challenging complications compromising the outcome of an otherwise successful operation. Considerable efforts have been invested in the recent years to address paradigm shifts in our understanding of the complex microbiological phenomena that contribute to the pathophysiology of PJI, such as microbial adherence, biofilm formation, and resistance to antibiotics. This article is an introduction to some of the recent advancements in the prevention, diagnosis, and treatment of PJI. It describes how industry, academic researchers, and government are increasing collaboration to address PJI through development of novel technologies, therapeutic strategies, and regulatory science that specifically target the unique biofilm-associated aspects of its pathogenesis.
Copyright 2015 by the American Academy of Orthopaedic Surgeons.
Full-text · Article · Apr 2015 · The Journal of the American Academy of Orthopaedic Surgeons
[Show abstract][Hide abstract] ABSTRACT: Greater documentation of patient history and clinical course is crucial for identifying factors that can influence surgical outcomes. The Centers for Medicare and Medicaid Services have already begun public reporting of hospital data on readmission, complication, and infection rates and will soon launch a website to make physician-specific outcomes data public. The orthopaedic community has the opportunity to lead the way in ensuring that adequate and accurate data is collected to facilitate appropriate comparisons that are based on patients' true risk of complications and the complexity of treatment. Several studies have reported a link between oral pathogens and periprosthetic infection, although it remains unclear whether organisms unique to dental tissues are also present in osteoarthritic joints and tissues affected by periprosthetic joint infection. The American Academy of Orthopaedic Surgeons and the American Dental Association are aware of these concerns and have created guidelines for antibiotic prophylaxis in patients who have undergone total hip or knee arthroplasty and require high-risk dental procedures. Because these guidelines have received considerable criticism, recommendations that are based on scientific and case-controlled clinical studies and provide effective guidance on this important subject are needed.
Copyright 2015 by the American Academy of Orthopaedic Surgeons.
Preview · Article · Apr 2015 · The Journal of the American Academy of Orthopaedic Surgeons
[Show abstract][Hide abstract] ABSTRACT: Objectives of this study were to investigate whether AQP1 and AQP5 expression is altered during intervertebral disc degeneration and if hypoxia and HIF-1 regulate their expression in NP cells. AQP expression was measured in human tissues from different degenerative grades; regulation by hypoxia and HIF-1 was studied using promoter analysis and gain- and loss-of-function experiments. We show that both AQPs are expressed in the disc and that mRNA and protein levels decline with human disease severity. Bioinformatic analyses of AQP promoters showed multiple evolutionarily conserved HREs. Surprisingly, hypoxia failed to induce promoter activity or expression of either AQP. While genomic chromatin immunoprecipitation showed limited binding of HIF-1α to conserved HREs, their mutation did not suppress promoter activities. Stable HIF-1α suppression significantly decreased mRNA and protein levels of both AQPs, but HIF-1α failed to induce AQP levels following accumulation. Together, our results demonstrate that AQP1 and AQP5 expression is sensitive to human disc degeneration and that HIF-1α uniquely maintains basal expression of both AQPs in NP cells, independent of oxemic tension and HIF-1 binding to promoter HREs. Diminished HIF-1 activity during degeneration may suppress AQP levels in NP cells, compromising their ability to respond to extracellular osmolarity changes.
[Show abstract][Hide abstract] ABSTRACT: Degeneration of the intervertebral disc is characterized by changes in proteoglycan status, loss of bound water molecules, decreased tissue osmotic pressure and a resulting mechanical failure of the disc. A similar spectrum of changes is evident in osteoarthritic articular cartilage. When healthy, resident cells in these skeletal tissues respond to applied mechanical loads by regulating their own osmotic state and the hydration of the extracellular matrix. The transcription factor Tonicity-Responsive Enhancer Binding Protein (TonEBP or NFAT5) is known to mediate the osmoadaptive response in these and other tissues. While the molecular basis of how osmotic loading controls matrix homeostasis is not completely understood, TonEBP regulates the expression of aggrecan and β1,3-glucoronosyltransferase in nucleus pulposus cells, in addition to targets that allow for survival under hypertonic stress. Moreover, in chondrocytes, TonEBP controls expression of several collagen subtypes and Sox9, a master regulator of aggrecan and collagen II expression. Thus, TonEBP-mediated regulation of the matrix composition allows disc cells and chondrocytes to modify the extracellular osmotic state itself. On the other hand, TonEBP in immune cells induces expression of TNF-α, ΙL-6 and MCP-1, pro-inflammatory molecules closely linked to matrix catabolism and pathogenesis of both disc degeneration and osteoarthritis, warranting investigations of this aspect of TonEBP function in skeletal cells. In summary, the TonEBP system, through its effects on extracellular matrix and osmoregulatory genes can be viewed primarily as a protective or homeostatic response to physiological loading.
[Show abstract][Hide abstract] ABSTRACT: The intervertebral disc (IVD) is one of the largest avascular organs in vertebrates. The nucleus pulposus (NP), a highly hydrated and proteoglycan-enriched tissue, forms the inner portion of the IVD. The NP is surrounded by a multi-lamellar fibrocartilaginous structure, the annulus fibrosus (AF). This structure is covered superior and inferior side by cartilaginous endplates (CEP). The NP is a unique tissue within the IVD as it results from the differentiation of notochordal cells, whereas, AF and CEP derive from the sclerotome. The hypoxia inducible factor-1α (HIF-1α) is expressed in NP cells but its function in NP development and homeostasis is largely unknown. We thus conditionally deleted HIF-1α in notochordal cells and investigated how loss of this transcription factor impacts NP formation and homeostasis at E15.5, birth, 1 and 4 months of age, respectively. Histological analysis, cell lineage studies, and TUNEL assay were performed. Morphologic changes of the mutant NP cells were identified as early as E15.5, followed, postnatally, by the progressive disappearance and replacement of the NP with a novel tissue that resembles fibrocartilage. Notably, lineage studies and TUNEL assay unequivocally proved that NP cells did not transdifferentiate into chondrocyte-like cells but they rather underwent massive cell death, and were completely replaced by a cell population belonging to a lineage distinct from the notochordal one. Finally, to evaluate the functional consequences of HIF-1α deletion in the NP, biomechanical testing of mutant IVD was performed. Loss of the NP in mutant mice significantly reduced the IVD biomechanical properties by decreasing its ability to absorb mechanical stress. These findings are similar to the changes usually observed during human IVD degeneration. Our study thus demonstrates that HIF-1α is essential for NP development and homeostasis, and it raises the intriguing possibility that this transcription factor could be involved in IVD degeneration in humans.
[Show abstract][Hide abstract] ABSTRACT: The pathogenesis of joint infections is not well understood. In particular, we do not know why these infections respond poorly
to antibiotic treatment. Here we show that methicillin-resistant Staphylococcus aureus, a major cause of joint infections, forms exceptionally strong biofilmlike aggregates in human synovial fluid (SF), to an
extent significantly exceeding biofilm formation observed in growth medium or serum. Screening a transposon bank identified
bacterial fibronectin- and fibrinogen-binding proteins as important for the formation of macroscopic clumps in SF, suggesting
an important role of fibrin-containing clots in the formation of bacterial aggregates during joint infection. Pretreatment
of SF with plasmin led to a strongly reduced formation of aggregates and increased susceptibility to antibiotics. These results
give important insight into the pathogenesis of staphylococcal joint infection and the mechanisms underlying resistance to
treatment. Furthermore, they point toward a potential novel approach for treating joint infections.
Full-text · Article · Sep 2014 · The Journal of Infectious Diseases
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinase-3 (MMP-3) plays an important role in intervertebral disc degeneration, a ubiquitous condition closely linked to low back pain and disability. Elevated expression of syndecan 4, a cell surface heparan sulfate proteoglycan, actively controls disc matrix catabolism. However, the relationship between MMP-3 expression and syndecan 4 in the context of inflammatory disc disease has not been clearly defined. We investigated the mechanisms by which cytokines control MMP-3 expression in rat and human nucleus pulposus cells. Cytokine treatment increased MMP-3 expression and promoter activity. Stable silencing of syndecan 4 blocked cytokine-mediated MMP-3 expression; more important, syndecan 4 did not mediate its effects through NF-κB or mitogen-activated protein kinase (MAPK) pathways. However, treatment with MAPK and NF-κB inhibitors resulted in partial blocking of the inductive effect of cytokines on MMP-3 expression. Loss-of-function studies confirmed that NF-κB, p38α/β2/γ/δ, and extracellular signal-regulated kinase (ERK) 2, but not ERK1, contributed to cytokine-dependent induction of MMP3 promoter activity. Similarly, inhibitor treatments, lentiviral short hairpin-p65, and short hairpin-I κ B kinase β significantly decreased cytokine-dependent up-regulation in MMP-3 expression. Finally, we show that transforming growth factor-β can block the up-regulation of MMP-3 induced by tumor necrosis factor (TNF)-α by counteracting the NF-κB pathway and syndecan 4 expression. Taken together, our results suggest that cooperative signaling through syndecan 4 and the TNF receptor 1-MAPK-NF-κB axis is required for TNF-α-dependent expression of MMP-3 in nucleus pulposus cells. Controlling these pathways may slow the progression of intervertebral disc degeneration and matrix catabolism.
No preview · Article · Jul 2014 · American Journal Of Pathology