Abbie L.A. Binch

Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION

Background: The regulation of NOTCH signaling under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of select NOTCH pathway genes, including NOTCH2 and NOTCH signaling, is regulated by IL-1β and TNF-α. Conclusion: Inflammatory cytokines promote NOTCH signaling in disc. Significance: NOTCH signaling may play a role in pathogenesis of disc disease. The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

Expression and regulation of neurotrophic and angiogenic factors during human intervertebral disc degeneration

IntroductionThe degenerate intervertebral disc (IVD) becomes innervated by sensory nerve fibres, and vascularised by blood vessels. This study aimed to identify neurotrophins, neuropeptides and angiogenic factors within native IVD tissue and to further investigate whether pro-inflammatory cytokines are involved in the regulation of expression levels within nucleus pulposus (NP) cells, nerve and endothelial cells.MethodsQuantitative real-time PCR (qRT-PCR) was performed on 53 human IVDs from 52 individuals to investigate native gene expression of neurotrophic factors and their receptors, neuropeptides and angiogenic factors. The regulation of these factors by cytokines was investigated in NP cells in alginate culture, and nerve and endothelial cells in monolayer using RT-PCR and substance P (SP) protein expression in interleukin-1 (IL-1β) stimulated NP cells.ResultsInitial investigation on uncultured NP cells identified expression of all neurotrophins by native NP cells, whilst the nerve growth factor (NGF) receptor was only identified in severely degenerate and infiltrated discs, and brain derived neurotrophic factor (BDNF) receptor expressed by more degenerate discs. BDNF expression was significantly increased in infiltrated and degenerate samples. SP and vascular endothelial growth factor (VEGF) were higher in infiltrated samples. In vitro stimulation by IL-1β induced NGF in NP cells. Neurotropin-3 was induced by tumour necrosis factor alpha in human dermal microvascular endothelial cells (HDMECs). SP gene and protein expression was increased in NP cells by IL-1β. Calcitonin gene related peptide was increased in SH-SY5Y cells upon cytokine stimulation. VEGF was induced by IL-1β and interleukin-6 in NP cells, whilst pleiotrophin was decreased by IL-1β. VEGF and pleiotrophin were expressed by SH-SY5Y cells, and VEGF by HDMECs, but were not modulated by cytokines.ConclusionsThe release of cytokines, in particular IL-1β during IVD degeneration, induced significant increases in NGF and VEGF which could promote neuronal and vascular ingrowth. SP which is released into the matrix could potentially up regulate the production of matrix degrading enzymes and also sensitise nerves, resulting in nociceptive transmission and chronic low back pain. This suggests that IL-1β is a key regulatory cytokine, involved in the up regulation of factors involved in innervation and vascularisation of tissues.

Nucleus pulposus phenotypic markers to determine stem cell differentiation : fact or fiction?

Progress in mesenchymal stem cell (MSC) based therapies for nucleus pulposus (NP) regeneration are hampered by a lack of understanding and consensus of the normal NP cell phenotype. Despite the recent consensus paper on NP markers, there is still a need to further validate proposed markers. This study aimed to determine whether an NP phenotypic profile could be identified within a large population of mature NP samples. qRT-PCR was conducted to assess mRNA expression of 13 genes within human non-degenerate articular chondrocytes (AC) (n=10) and NP cells extracted from patients across a spectrum of histological degeneration grades (n=71). qRT-PCR results were used to select NP marker candidates for protein expression analysis. Differential expression at mRNA between AC and non-degenerate NP cells was only observed for Paired Box Protein 1 (PAX1) and Forkhead box F1 (FOXF1). In contrast no other previously suggested markers displayed differential expression between non-degenerate NP and AC at mRNA level. PAX1 and FOXF1 protein expression was significantly higher in the NP compared to annulus fibrosus (AF), cartilaginous endplate (CEP) and AC. In contrast Laminin-5 (LAM-332), Keratin-19 (KRT-19) and Hypoxia Inducible Factor 1 alpha (HIF1α) showed no differential expression in NP cells compared with AC cells. A marker which exclusively differentiates NP cells from AF and AC cells remains to be identified, raising the question: is the NP a heterogeneous population of cells? Or does the natural biological variation during IVD development, degeneration state and even the life cycle of cells make finding one definitive marker impossible?

Integrative epigenomics, transcriptomics and proteomics of patient chondrocytes reveal genes and pathways involved in osteoarthritis.

Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood. We investigated genes and pathways that mark OA progression in isolated primary chondrocytes taken from paired intact versus degraded articular cartilage samples across 38 patients undergoing joint replacement surgery (discovery cohort: 12 knee OA, replication cohorts: 17 knee OA, 9 hip OA patients). We combined genome-wide DNA methylation, RNA sequencing, and quantitative proteomics data. We identified 49 genes differentially regulated between intact and degraded cartilage in at least two –omics levels, 16 of which have not previously been implicated in OA progression. Integrated pathway analysis implicated the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. Using independent replication datasets, we showed that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. AQP1, COL1A1 and CLEC3B were significantly differentially regulated across all three –omics levels, confirming their differential expression in human disease. Through integration of genome-wide methylation, gene and protein expression data in human primary chondrocytes, we identified consistent molecular players in OA progression that replicated across independent datasets and that have translational potential.

Nerves are more abundant than blood vessels in the degenerate human intervertebral disc.

BackgroundChronic low back pain (LBP) is the most common cause of disability worldwide. New ideas surrounding LBP are emerging that are based on interactions between mechanical, biological and chemical influences on the human IVD. The degenerate IVD is proposed to be innervated by sensory nerve fibres and vascularised by blood vessels, and it is speculated to contribute to pain sensation. However, the incidence of nerve and blood vessel ingrowth, as well as whether these features are always associated, is unknown. We investigated the presence of nerves and blood vessels in the nucleus pulposus (NP) of the IVD in a large population of human discs.MethodsImmunohistochemistry was performed with 61 human IVD samples, to identify and localise nerves (neurofilament 200 [NF200]/protein gene product 9.5) and blood vessels (CD31) within different regions of the IVD.ResultsImmunopositivity for NF200 was identified within all regions of the IVD within post-mortem tissues. Nerves were seen to protrude across lamellar ridges and through matrix towards NP cells. Nerves were identified deep within the NP and were in many cases, but not always, seen in close proximity to fissures or in areas where decreased matrix was seen. Fifteen percent of samples were degenerate and negative for nerves and blood vessels, whilst 16 % of all samples were degenerate with nerves and blood vessels. We identified 52 % of samples that were degenerate with nerves but no blood vessels. Interestingly, only 4 % ofall samples were degenerate with no nerves but positive for blood vessels. Of the 85 samples investigated, only 6 % of samples were non-degenerate without nerves and blood vessels and 7 % had nerves but no blood vessels.ConclusionsThis study addresses the controversial topic of nerve and blood vessel ingrowth into the IVD in a large number of human samples. Our findings demonstrate that nerves are present within a large proportion of NP samples from degenerate IVDs. This study shows a possible link between nerve ingrowth and degeneration of the IVD and suggests that nerves can migrate in the absence of blood vessels.

Syndecan-4 in intervertebral disc and cartilage: Saint or synner?

The ECM of the intervertebral disc and articular cartilage contains 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 a possible therapeutic target in slowing the progression of ECM degradation in both degenerative disc disease and osteoarthritis.

Integrated molecular phenotyping identifies genes and pathways disrupted in osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease with substantial global health economic burden and no curative therapy. Here we investigate genes and pathways underpinning disease progression, combining methylation typing, RNA sequencing and quantitative proteomics in chondrocytes from matched damaged and healthy articular cartilage samples from OA patients undergoing knee replacement surgery. Our data highlight 49 genes differentially regulated at multiple levels, identifying 19 novel genes with a potential role in OA progression. An integrated pathway analysis identifies established and emerging biological processes. We perform an in silico search for drugs predicted to target the differentially regulated factors and identify several established therapeutic compounds which now warrant further investigation in OA. Overall this work provides a first integrated view of the molecular landscape of human primary chondrocytes and offers insights into the mechanisms of cartilage degeneration. The results point to new therapeutic avenues, highlighting the translational potential of integrated functional genomics. All data from these experiments are freely available for access in the appropriate repositories.Background Osteoarthritis (OA) is a common disease characterized by cartilage degeneration and joint remodeling. The underlying molecular changes underpinning disease progression are incompletely understood, but can be characterized using recent advances in genomics technologies, as the relevant tissue is readily accessible at joint replacement surgery. Here we investigate genes and pathways that mark OA progression, combining genome-wide DNA methylation, RNA sequencing and quantitative proteomics in isolated primary chondrocytes from matched intact and degraded articular cartilage samples across twelve patients with OA undergoing knee replacement surgery. Results We identify 49 genes differentially regulated between intact and degraded cartilage at multiple omics levels, 16 of which have not previously been implicated in OA progression. Using independent replication datasets, we replicate statistically significant signals and show that the direction of change is consistent for over 90% of differentially expressed genes and differentially methylated CpG probes. Three genes are differentially regulated across all 3 omics levels: AQP1, COL1A1 and CLEC3B, and all three have evidence implicating them in OA through animal or cellular model studies. Integrated pathway analysis implicates the involvement of extracellular matrix degradation, collagen catabolism and angiogenesis in disease progression. All data from these experiments are freely available as a resource for the scientific community. Conclusions This work provides a first integrated view of the molecular landscape of human primary chondrocytes and identifies key molecular players in OA progression that replicate across independent datasets, with evidence for translational potential.

Unique Regulation of AQP1 and AQP5 Expression by HIF-1 in Nucleus Pulposus Cells

Introduction Aquaporins are a family of transmembrane water channels that aid in osmoregulation, a critical function in the proteoglycan-rich nucleus pulposus. Previous reports have demonstrated expression of aquaporins in the intervertebral disc.1,2 Hypoxia, a defining feature of the nucleus pulposus environment has been shown to regulate expression of aquaporin1 (AQP1) and aquaporin 5 (AQP5).3-6 In some cases, this regulation has been shown to occur through HIF-1α and to depend on hypoxia responsive elements (HREs). The goal of this study was to examine if hypoxia and HIF-1α play a role in regulation and function of aquaporin1 (AQP1) and aquaporin5 (AQP5) in nucleus pulposus (NP) cells of the intervertebral disc. Materials and Methods Quantitative reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry were used to measure AQP1 and AQP5 expression in nucleus pulposus (NP) and annulus fibrosus (AF) cells and tissues. Transfections were used to determine the role of HIF-1α on AQP1 and AQP5 promoter activity in normoxia and hypoxia. The JASPAR database was used to identify two putative hypoxia response elements (HREs) in the promoter of each aquaporin. HIF-1α levels were modulated with treatment by DMOG or shRNAs. Expression of aquaporins in human tissue samples was evaluated using immunohistochemistry and polymerase chain reaction. Results We found that both AQP1 and AQP5 were expressed in tissues and cells of human and rat discs. To determine whether expression of the aquaporins was regulated by hypoxia, we treated NP cells with hypoxia (1% O2) for 8 to 72 hours. Treatment had no effect on promoter activity, mRNA expression, or protein expression of either aquaporin. Using the JASPAR database, we identified two HREs in the promoter of each aquaporin. Mutation of the HREs in either AQP1 or AQO5 had no suppressive effect on the basal activity of the promoters. We then investigated whether AQP1 and AQP5 were regulated in a HIF1α-dependent manner. Accumulation of HIF-1α by DMOG did not affect expression of either aquaporin. However, suppression of HIF-1α by lentiviral delivery of shRNA significantly decreased both mRNA and protein expression of AQP1 and AQP5. In addition, we have found changes in expression of these aquaporins in human tissue samples from patients with varying degrees of intervertebral disc degeneration. Conclusion These results indicate that, under hypoxic conditions, HIF-1α maintains basal expression of both AQP1 and AQP5 in the NP; however, this regulation is independent of HIF binding to the identified HREs in their promoters. Analysis of human tissue samples suggests that aquaporin expression may be linked to health of the intervertebral disc. Disclosure of Interest None declared References Richardson SM, Knowles R, Marples D, Hoyland JA, Mobasheri A. Aquaporin expression in the human intervertebral disc. J Mol Histol 2008;39(3):303–309 Gajghate S, Hiyama A, Shah M, et al. Osmolarity and intracellular calcium regulate aquaporin2 expression through TonEBP in nucleus pulposus cells of the intervertebral disc. J Bone Miner Res 2009;24(6):992–1001 Tanaka A, Sakurai K, Kaneko K, et al. The role of the hypoxia-inducible factor 1 binding site in the induction of aquaporin-1 mRNA expression by hypoxia. DNA Cell Biol 2011;30(8):539–544 Tie L, Lu N, Pan XY, et al. Hypoxia-induced up-regulation of aquaporin-1 protein in prostate cancer cells in a p38-dependent manner. Cell Physiol Biochem 2012;29(1-2):269–280 Zhang J, Xiong Y, Lu LX, et al. AQP1 expression alterations affect morphology and water transport in Schwann cells and hypoxia-induced up-regulation of AQP1 occurs in a HIF-1α-dependent manner. Neuroscience 2013;252(252):68–79 Kawedia JD, Yang F, Sartor MA, Gozal D, Czyzyk-Krzeska M, Menon AG. Hypoxia and hypoxia mimetics decrease aquaporin 5 (AQP5) expression through both hypoxia inducible factor-1α and proteasome-mediated pathways. PLoS ONE 2013;8(3):e57541