Recombinant human SIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human intervertebral disc nucleus pulposus cells

IntroductionNutrient deprivation is a likely contributor to intervertebral disc (IVD) degeneration. Silent mating type information regulator 2 homolog 1 (SIRT1) protects cells against limited nutrition by modulation of apoptosis and autophagy. However, little evidence exists regarding the extent to which SIRT1 affects IVD cells. Therefore, we conducted an in vitro study using human IVD nucleus pulposus (NP) cells.MethodsThirty-two IVD specimens were obtained from patients who underwent surgical intervention and were categorized based on Pfirrmann IVD degeneration grades. Cells were isolated from the NP and cultured in the presence of recombinant human SIRT1 (rhSIRT1) under different serum conditions, including 10 % (v/v) fetal bovine serum (FBS) as normal nutrition (N) and 1 % (v/v) FBS as low nutrition (LN). 3-Methyladenine (3-MA) was used to inhibit autophagy. Autophagic activity was assessed by measuring the absorbance of monodansylcadaverine and immunostaining and Western blotting for light chain 3 and p62/SQSTM1. Apoptosis and pathway analyses were performed by flow cytometry and Western blotting.ResultsCells cultured under LN conditions decreased in number and exhibited enhanced autophagy compared with the N condition. Medium supplementation with rhSIRT1 inhibited this decrease in cell number and induced an additional increase in autophagic activity (P < 0.05), whereas the combined use of rhSIRT1 and 3-MA resulted in drastic decreases in cell number and autophagy (P < 0.05). The incidence of apoptotic cell death increased under the LN condition, which was decreased by rhSIRT1 (P < 0.05) but increased further by a combination of rhSIRT1 and 3-MA (P < 0.05). Under LN conditions, NP cells showed a decrease in antiapoptotic Bcl-2 and an increase in proapoptotic Bax, cleaved caspase 3, and cleaved caspase 9, indicating apoptosis induction via the mitochondrial pathway. These changes were suppressed by rhSIRT1 but elevated further by rhSIRT1 with 3-MA, suggesting an effect of rhSIRT1-induced autophagy on apoptosis inhibition. Furthermore, the observed autophagy and apoptosis were more remarkable in cells from IVDs of Pfirrmann grade IV than in those from IVDs of Pfirrmann grade II.ConclusionsSIRT1 protects against nutrient deprivation-induced mitochondrial apoptosis through autophagy induction in human IVD NP cells, suggesting that rhSIRT1 may be a potent treatment agent for human degenerative IVD disease.     

Circular RNAs in nucleus pulposus cell function and intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is a common cause of low back pain, which inflicts more global disability than any other condition. Although IDD was deemed to be a natural process that comes with ageing, a growing body of evidence suggested that both genetic and environmental factors could modify the development of IDD. In this connection, aberrant function of nucleus pulposus cells has been implicated in IDD pathogenesis. Circular RNAs are a novel class of endogenous non‐coding RNAs that play crucial regulatory roles in diverse cellular processes. Recently, deregulation of circRNAs in nucleus pulposus cells was found to functionally participate in IDD development. In this review, we summarize the current knowledge regarding the deregulation of circRNAs in IDD in relation to their actions on nucleus pulposus cell functions, including cell proliferation, apoptosis and extracellular matrix synthesis/degradation. The potential clinical utilities of circRNAs as therapeutic targets for the management of IDD are also discussed.     

The role of the nucleus pulposus in neutral zone human lumbar intervertebral disc mechanics

To study the effect of denucleation on the mechanical behavior of the human lumbar intervertebral disc through a 2mm incision, two groups of six human cadaver lumbar spinal units were tested in axial compression, axial rotation, lateral bending and flexion/extension after incremental steps of "partial" denucleation. Neutral zone, range of motion, stiffness, intradiscal pressure and energy dissipation were measured; the results showed that the contribution of the nucleus pulposus to the mechanical behavior of the intervertebral disc was more dominant through the neutral zone than at the farther limits of applied loads and moments.     

PTEN promotes intervertebral disc degeneration by regulating nucleus pulposus cell behaviors

Intervertebral disc degeneration (IDD) is induced by multiple factors including increased apoptosis, decreased survival, and reduced extracellular matrix (ECM) synthesis in the nucleus pulposus (NP) cells. The tumor suppressor phosphatase and tensin homolog deleted from chromosome 10 (PTEN) is the only known lipid phosphatase counteracting the PI3K/AKT pathway. Loss of PTEN leads to activated PI3K/AKT signaling, which plays a key role in a variety of cancers. However, the role of PTEN/PI3K/AKT signaling nexus in IDD remains unknown. Here, we report that PTEN is overexpressed in degenerative NP, which correlates with inactivated AKT. Using the PTEN knockdown approach by lentivirus‐mediated short interfering RNA gene transfer technique, we report that PTEN decreases survival but induces apoptosis and senescence of NP cells. PTEN also inhibits expression and production of ECM components including collagen II, aggrecan, and proteoglycan. Furthermore, PTEN modulates the expression of ECM regulatory molecules SOX‐9 and matrix metalloproteinase‐3 (MMP‐3). Using small‐molecule AKT inhibitor GDC‐0068, we confirm that PTEN regulates NP cell behaviors through its direct targeting of PI3K/AKT. These findings demonstrate for the first time that PTEN/PI3K/AKT signaling axis plays an important role in the pathogenesis of IDD. Targeting PTEN using gene therapy may represent a promising therapeutic approach against disc degenerative diseases.     

Reduced nucleus pulposus glycosaminoglycan content alters intervertebral disc dynamic viscoelastic mechanics

The intervertebral disc functions over a range of dynamic loading regimes including axial loads applied across a spectrum of frequencies at varying compressive loads. Biochemical changes occurring in early degeneration, including reduced nucleus pulposus glycosaminoglycan content, may alter disc mechanical behavior and thus may contribute to the progression of degeneration. The objective of this study was to determine disc dynamic viscoelastic properties under several equilibrium loads and loading frequencies, and further, to determine how reduced nucleus glycosaminoglycan content alters dynamic mechanics. We hypothesized that (1) dynamic stiffness would be elevated with increasing equilibrium load and increasing frequency, (2) the disc would behave more elastically at higher frequencies, and finally, (3) dynamic stiffness would be reduced at low equilibrium loads under all frequencies due to nucleus glycosaminoglycan loss. We mechanically tested control and chondroitinase ABC injected rat lumbar motion segments at several equilibrium loads using oscillatory loading at frequencies ranging from 0.05 to 5 Hz. The rat lumbar disc behaved non-linearly with higher dynamic stiffness at elevated compressive loads irrespective of frequency. Phase angle was not affected by equilibrium load, although it decreased as frequency was increased. Reduced glycosaminoglycan decreased dynamic stiffness at low loads but not at high equilibrium loads and led to increased phase angle at all loads and frequencies. The findings of this study demonstrate the effect of equilibrium load and loading frequencies on dynamic disc mechanics and indicate possible mechanical mechanisms through which disc degeneration can progress.     

Enhancing intervertebral disc repair and regeneration through biology: platelet-rich plasma as an alternative strategy

Intervertebral disc degeneration (IDD) is a common orthopedic disease associated with mechanical changes that may result in significant pain. Current treatments for IDD mainly depend on conservative therapies and spinal surgeries that are only able to relieve the symptoms but do not address the cause of the degeneration and even accelerate the degeneration of adjacent segments. This has prompted research to improve our understanding of the biology of intervertebral disc healing and into methods to enhance the regenerative process. Recently, biological therapies, including active substances, gene therapy and tissue engineering based on certain cells, have been attracting more attention in the field of intervertebral disc repair and regeneration. Early selection of suitable biological treatment is an ideal way to prevent or even reverse the progressive trend of IDD. Growth factors have been enjoying more popularity in the field of regeneration of IDD and many have been proved to be effective in reversing the degenerative trend of the intervertebral disc. Identification of these growth factors has led to strategies to deliver platelet-derived factors to the intervertebral disc for regeneration. Platelet-rich plasma (PRP) is the latest technique to be evaluated for promoting intervertebral disc healing. Activation of the PRP leads to the release of growth factors from the α-granules in the platelet cytoplasm. These growth factors have been associated with the initiation of a healing cascade that leads to cellular chemotaxis, angiogenesis, synthesis of collagen matrix, and cell proliferation. This review describes the current understanding of IDD and related biological therapeutic strategies, especially the promising prospects of PRP treatment. Future limitations and perspectives of PRP therapy for IDD are also discussed.     

Upregulation of P53 promotes nucleus pulposus cell apoptosis in intervertebral disc degeneration through upregulating NDRG2

P53 is an apoptosis marker which is involved in determining nucleus pulposus (NP) cell fate. Little is known about P53 interaction with N-Myc downstream-regulated gene 2 (NDRG2) in intervertebral disc degeneration (IVDD). Here, we studied the role of the P53-NDRG2 axis in IVDD. We found that NDRG2 was expressed in NP tissue obtained from patients with IVDD. The level of NDRG2 was positively related to the severity of IVDD, as determined by Pfirrmann grading. Subsequently, we overexpressed NDRG2 in human NP cells by adenoviral transfection and studied the effects of increased levels of NDRG2 on the viability and apoptosis of these cells. NDRG2 overexpression induced NP cell apoptosis and reduced viability in NP cells obtained from patient with IVDD. We also found that the level of P53 was elevated in NP cells from patients with IVDD and treatment with exogenous P53 upregulated NDRG2 in NP cells. Last, IVDD model was established in P53 knockout mice and the pathological changes in the intervertebral discs and NDRG2 expression were examined. P53 knockout can reduce the damage of NP tissues after IVDD surgery to some extent. Restoration of NDRG2 antagonized the effect of P53 knockout on IVDD. Collectively, this study suggests that elevated P53 in NP cells stimulates apoptosis of the cells by upregulating NDRG2 expression, thereby exacerbating IVDD.     

Secretory cells in the nucleus pulposus of the adult human intervertebral disc. A preliminary report

A light microscopical study was conducted to ascertain the type of cells in the nucleus pulposus of the adult human intervertrebral disc. Three lumbar intervertebral discs were removed from each of 15 male and female adults at autopsy (ages ranged from 19 to 62 years). The tissue was fixed in formalin, decalcified in formic acid, dehydrated in a graded series of ethanol, embedded in paraffin, and serially sectioned at 7-10 micron. Tissue sections were affixed to albuminized glass slides and stained either by hematoxylin and eosin or hematoxylin and Van Gieson's stain. The cells of the bulk of the nucleus pulposus consisted of chondrocytes and a few fibroblasts; however, the subchondral matrix of the nucleus pulposos contained numerous stellate cells with (from 1 to 8) unusually long (up to 80 micron) primary cytoplasmic processes that often branch into secondary processes. The cell processes contained cytoplasmic varicosities at various intervals along their lengths; and their endings often expanded into bulbous, vesicle-filled process terminals. The surrounding extracellular matrix usually contained numerous, vesicle-filled, eosinophil matrix bodies. Morphological similarities of cytoplasmic varicosities, process terminals, and matrix bodies, as well as the apparent budding of process terminals, suggest that these previously unidentified cells are secreting an unknown matrix component into the subchondral matrix of the nucleus pulposus of the adult human.     

A new non-enzymatic method for isolating human intervertebral disc cells preserves the phenotype of nucleus pulposus cells

Cells isolated from intervertebral disc (IVD) tissues of human surgical samples are one of potential sources for the IVD cellular therapy. The purpose of this study was to develop a new non-enzymatic method, “tissue incubation”, for isolating human IVD cells. The IVD tissues of annulus fibrosus (AF) and nucleus pulposus (NP) were incubated separately in tissue culture flasks with culture medium. After 7–10 days incubation, cells were able to migrate out of IVD tissues and proliferate in vitro. After 3–4 weeks culture, expanded cells were harvested by trypsinization, and the remaining tissues were transferred to a new flask for another round of incubation. The molecular phenotype of IVD cells from juvenile and adult human samples was evaluated by both flow cytometry analysis and immunocytochemical staining for the expression of protein markers of NP cells (CD24, CD54, CD239, integrin α6 and laminin α5). Flow cytometry confirmed that both AF and NP cells of all ages positively expressed CD54 and integrin α6, with higher expression levels in NP cells than in AF cells for the juvenile group sample. However, CD24 expression was only found in juvenile NP cells, and not in AF or older disc cells. Similar expression patterns for NP markers were also confirmed by immunocytochemistry. In summary, this new non-enzymatic tissue incubation method for cell isolation preserves molecular phenotypic markers of NP cells and may provide a valuable cell source for the study of NP regeneration strategies.     

TIGAR impedes compression-induced intervertebral disc degeneration by suppressing nucleus pulposus cell apoptosis and autophagy

To investigate whether TP53-induced glycolysis and apoptosis regulator (TIGAR) participates in compression-induced intervertebral disc (IVD) degeneration, and to determine the regulatory effect of TIGAR on nucleus pulposus (NP) cell autophagy and apoptosis following compression-induced injuries. IVD tissues were collected from human patients undergoing surgery (n = 20) and skeletally mature Sprague-Dawley rats (n = 15). Initially, the effect of compression on the expression of TIGAR was evaluated with in vivo and in vitro models. In addition, TIGAR was silenced to investigate the regulatory effect of TIGAR on compression-induced intracellular reactive oxygen species (ROS) levels, autophagy, and apoptosis in rat NP cells. Furthermore, the P53 inhibitor pifithrin-α (PFTα) and SP1 inhibitor mithramycin A were employed to detect expression level changes of TIGAR and autophagy-associated target molecules. TIGAR expression of NP cells increased gradually in human degenerative IVDs and in rat NP cells under compression both in vivo and in vitro. TIGAR knockdown enhanced compression-induced intracellular ROS generation and the NADPH/NADP⁺ and GSH/GSSG ratios. Moreover, TIGAR knockdown amplified the compression-induced caspase-3 activation and the apoptosis rate of rat NP cells. Likewise, knockdown of TIGAR significantly accelerated LC3B expression and autophagosome formation in rat NP cells during compression-induced injuries. The results also established that mithramycin A could inhibit TIGAR expression and autophagy levels in NP cells under compression conditions, while PFTα had no similar effect. Our data demonstrated that TIGAR acted as an important endogenous negative regulator of ROS levels, which might inhibit compression-induced apoptosis and autophagy through SP1-dependent mechanisms.     

Long non‐coding RNAs in nucleus pulposus cell function and intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is the major cause of low back pain which incurs a significant public‐health and economic burden. The aetiology of IDD is complex, with developmental, genetic, biomechanical and biochemical factors contributing to the disease development. Deregulated phenotypes of nucleus pulposus cells, including aberrant differentiation, apoptosis, proliferation and extracellular matrix deposition, are involved in the initiation and progression of IDD. Non‐coding RNAs, including long non‐coding RNAs (lncRNAs), have recently been identified as important regulators of gene expression. Research into their roles in IDD has been very active over the past 5 years. Our review summarizes current research regarding the roles of deregulated lncRNAs (eg, RP11‐296A18.3, TUG1, HCG18) in modulating nucleus pulposus cell functions in IDD. These exciting findings suggest that specific modulation of lncRNAs or their downstream signalling pathways might be an attractive approach for developing novel therapeutics for IDD.     

Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus.

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.     

Expression of acid-sensing ion channels in nucleus pulposus cells of the human intervertebral disk is regulated by non-steroid anti-inflammatory drugs

Non-steroid anti-inflammatory drugs （NSAIDs） are general- ly used in the treatment of inflammation and pain through cyclooxygenase （COX） inhibition. Mounting evidence has indicated additional COX-independent targets for NSAIDs including acid-sensing ion channels （ASICs） la and 3. However, detailed function and mechanism of ASICs still remain largely elusive. In this study, the impact of NSAIDs on ASICs in nucleus puiposus cells of the human interverte- bral disk was investigated. Nucleus pulposus cells were iso- lated and cultured from protruded disk tissues of 40 patients. It was shown that ASICla and ASIC3 were expressed and functional in these cells by analyzing proton- gated currents after ASIC inhibition. We further investi- gated the neuroprotective capacity of ibuprofen （a COX in- hibitor）, psaimotoxin-1 （PcTX1, a tarantula toxin specific for homomeric ASICla）, and amiloride （a classic inhibitor of the epithelial sodium channel ENaC/DEG family to which ASICs belong）. PcTXl-containing venom has been shown to be comparable with amiloride in its neuroprotective features in rodent models of ischemia. Taken together, our data showed that amiloride, PcTX1, and ibuprofen decreased ASIC protein expression and thereby exerted protective effects from ASIC inhibition-mediated cell damage.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

Differential response of nucleus pulposus intervertebral disc cells to high salt, sorbitol, and urea

Nucleus pulposus intervertebral disc cells are routinely confronted with high osmolality in their microenvironment and respond to this stress in vitro by regulating cell cycle progression and by activating a DNA repair machinery in order to counteract its genotoxic effect. In the present study, we attempted to identify the origin of this osmo-regulatory response, by using an ionic NaCl/KCl solution, the compatible osmolyte sorbitol, and the readily permeant urea. High salt and sorbitol were found to activate similar molecular pathways, including the p38 MAPK and the p53-p21(WAF1)-pRb axis, that were not stimulated by high urea. On the other hand, only high urea led to the phosphorylation of ERKs and JNKs. Furthermore, salt- and sorbitol-treated cells were able to phosphorylate histone H2A.X on Ser139, in contrast to cells exposed to urea, indicating a common mechanism for DNA repair, which was achieved by a p53-dependent activation of the G1 checkpoint by both solutes. DNA repair, as directly measured by a host cell reactivation assay, occurred under conditions of hyperosmolar salt and sorbitol, although to a lesser extent in sorbitol-treated cells than in cells exposed to high salinity. Taken as a whole, our findings suggest that the hyperosmolality-provoked DNA damage and the responses of nucleus pulposus cells induced by this genotoxic stress most probably originate from cell volume alterations mediated by hypertonicity and not from increased intracellular ionic concentration.     

TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.     

Intradiscal transplantation of synovial mesenchymal stem cells prevents intervertebral disc degeneration through suppression of matrix metalloproteinase-related genes in nucleus pulposus cells in rabbits

Introduction  

Synovial mesenchymal stem cells (MSCs) have high proliferative and chondrogenic potentials, and MSCs transplanted into the articular cartilage defect produce abundant extracellular matrix. Because of similarities between the articular cartilage and the intervertebral disc cartilage, synovial MSCs are a potential cell source for disc regeneration. Here, we examined the effect of intradiscal transplantation of synovial MSCs after aspiration of nucleus pulposus in rabbits.