Mesenchymal stem cells deliver exogenous miR‐21 via exosomes to inhibit nucleus pulposus cell apoptosis and reduce intervertebral disc degeneration

Although mesenchymal stem cells (MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells (NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC‐derived exosomes (MSC‐exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of miRNAs in MSC‐exosomes and associated mechanisms for NPC apoptosis. MSC‐exosomes were isolated from MSC medium, and its anti‐apoptotic effect was assessed in a cell and rat model. The down‐regulated miRNAs in apoptotic NPCs were identified, and their contents in MSC‐exosomes were detected. The target genes of eligible miRNAs and possible downstream pathway were investigated. Purified MSC‐exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR‐21 were down‐regulated in apoptotic NPCs while MSC‐exosomes were enriched in miR‐21. The exosomal miR‐21 could be transferred into NPCs and alleviated TNF‐α induced NPC apoptosis by targeting phosphatase and tensin homolog (PTEN) through phosphatidylinositol 3‐kinase (PI3K)‐Akt pathway. Intradiscal injection of MSC‐exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC‐derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR‐21 contained in exosomes. Exosomal miR‐21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration.     

Characterization of Krt19CreERT allele for targeting the nucleus pulposus cells in the postnatal mouse intervertebral disc

Intervertebral disc degeneration and associated back pain are relatively common but sparsely understood conditions, affecting over 70% of the population during some point of life. Disc degeneration is often associated with a loss of nucleus pulposus (NP) cells. Genetic mouse models offer convenient avenues to understand the cellular and molecular regulation of the disc during its formation, growth, maintenance, and aging. However, due to the lack of inducible driver lines to precisely target NP cells in the postnatal mouse disc, progress in this area of research has been moderate. NP cells are known to express cytokeratin 19 (Krt19), and tamoxifen (Tam)-inducible Krt19^CreERT allele is available. The current study describes the characterization of Krt19^CreERT allele to specifically and efficiently target NP cells in neonatal, skeletally mature, middle-aged, and aged mice using two independent fluorescent reporter lines. The efficiency of recombination at all ages was validated by immunostaining for KRT19. Results show that following Tam induction, Krt19^CreERT specifically drives recombination of NP cells in the spine of neonatal and aged mice, while no recombination was detected in the surrounding tissues. Knee joints from skeletally mature Tam-treated Krt19^CreERT/+; R26^tdTOM mouse show the absence of recombination in all tissues and cells of the knee joint. Thus, this study provides evidence for the use of Krt19^CreERT allele for genetic characterization of NP cells at different stages of the mouse life.     

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.     

Down‐regulation of insulin‐like growth factor binding protein 5 is involved in intervertebral disc degeneration via the ERK signalling pathway

It is obvious that epigenetic processes influence the evolution of intervertebral disc degeneration (IDD). However, its molecular mechanisms are poorly understood. Therefore, we tested the hypothesis that IGFBP5, a potential regulator of IDD, modulates IDD via the ERK signalling pathway. We showed that IGFBP5 mRNA was significantly down‐regulated in degenerative nucleus pulposus (NP) tissues. IGFBP5 was shown to significantly promote NP cell proliferation and inhibit apoptosis in vitro, which was confirmed by MTT, flow cytometry and colony formation assays. Furthermore, IGFBP5 was shown to exert its effects by inhibiting the ERK signalling pathway. The effects induced by IGFBP5 overexpression on NP cells were similar to those induced by treatment with an ERK pathway inhibitor (PD98059). Moreover, qRT‐PCR and Western blot analyses were performed to examine the levels of apoptosis‐related factors, including Bax, caspase‐3 and Bcl2. The silencing of IGFBP5 up‐regulated the levels of Bax and caspase‐3 and down‐regulated the level of Bcl2, thereby contributing to the development of human IDD. Furthermore, these results were confirmed in vivo using an IDD rat model, which showed that the induction of Igfbp5 mRNA expression abrogated the effects of IGFBP5 silencing on intervertebral discs. Overall, our findings elucidate the role of IGFBP5 in the pathogenesis of IDD and provide a potential novel therapeutic target for IDD.     

Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by Transforming Growth Factor-beta(1)and insulin like growth factor-I.

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-beta 1 (TGF-beta 1) and Insulin-like Growth Factor-1 (beta IGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24 h, then stimulated with TGF-beta 1 (0.1 or 10 ng/ml) or IGF-I (2 or 50 ng/ml) +/-Concanavalin A (20 microg/ml) for an additional 48 h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72 kDa) and active (59 kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-beta 1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-beta 1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-beta 1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.     

Retracted: HO-1 overexpression alleviates senescence by inducing autophagy via the mitochondrial route in human nucleus pulposus cells

Intervertebral disc degeneration (IDD) is closely associated with aging. Our previous studies have confirmed that heme oxygenase-1 (HO-1) can inhibit nucleus pulposus (NP) cell apoptosis. However, whether or not HO-1 is involved in NP cell senescence and autophagy is unclear. Our results indicated that HO-1 expression was reduced in IDD tissues and replicative senescent NP cells. HO-1 overexpression using a lentiviral vector reduced the NP cell senescence level, protected mitochondrial function, and promoted NP cell autophagy through the mitochondrial pathway. Autophagy inhibitor 3-MA pretreatment reversed the anti-senescent and protective effects on the mitochondrial function of HO-1, which promoted the degradation of the extracellular matrix (ECM) in the intervertebral disc. In vivo, HO-1 overexpression inhibited IDD and enhanced autophagy. In summary, these results suggested that HO-1 overexpression alleviates NP cell senescence by inducing autophagy via the mitochondrial route.     

Carbohydrate sulfotransferase 3 (CHST3) overexpression promotes cartilage endplate-derived stem cells (CESCs) to regulate molecular mechanisms related to repair of intervertebral disc degeneration by rat nucleus pulposus

To investigate the regulatory effect of carbohydrate sulfotransferase 3 (CHST3) in cartilage endplate-derived stem cells (CESCs) on the molecular mechanism of intervertebral disc degeneration after nucleus pulposus repair in rats. We performed GO and KEGG analysis of GSE15227 database to select the differential genes CHST3 and CSPG4 in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration, IHC and WB to detect the protein profile of CHST3 and CSPG4, Co-IP for the interaction between CHST3 and CSPG4. Then, immunofluorescence was applied to measure the level of CD90 and CD105, and flow cytometry indicated the level of CD73, CD90 and CD105 in CESCs. Next, Alizarin red staining, Alcian blue staining and TEM were performed to evaluate the effects of CESCs into osteoblasts and chondroblasts, respectively, CCK8 for the cell proliferation of osteoblasts and chondroblasts after induction for different times; cell cycle of osteoblasts or chondroblasts was measured by flow cytometry after induction, and WB for the measurement of specific biomarkers of OC and RUNX in osteoblasts and aggrecan, collagen II in chondroblasts. Finally, colony formation was applied to measure the cell proliferation of CESCs transfected with ov-CHST3 or sh-CHST3 when cocultured with bone marrow cells, WB for the protein expression of CHST3, CSPG4 and ELAVL1 in CSECs, transwell assay for the migration of CESCs to bone marrow cells, TEM image for the cellular characteristics of bone marrow cells, and WB for the protein profile of VCAN, VASP, NCAN and OFD1 in bone marrow cells. CHST3 and CSPG4 were differentially expressed and interacted in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration; CD73, CD90 and CD105 were lowly expressed in CESCs, osteogenic or chondroblastic induction changed the characteristics, proliferation, cell cycle and specific biomarkers of osteoblasts and chondroblasts after 14 or 21 days,; CHST3 affected the cell proliferation, protein profile, migration and cellular features of cocultured CESCs or bone marrow cells. CHST3 overexpression promoted CESCs to regulate bone marrow cells through interaction with CSPG4 to repair the grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration.     

Downregulation of microRNA-129-5p increases the risk of intervertebral disc degeneration by promoting the apoptosis of nucleus pulposus cells via targeting BMP2

miR-129-5p is implicated in many diseases, such as laryngeal cancer and breast cancer. In this study, we studied the mechanism underlying the role of BMP2 in intervertebral disc degeneration (IDD). We used a luciferase assay system to determine the relationship between BMP2 and miR-129-5 expression. In addition, Western blot and real-time PCR were used to confirm the regulatory relationship between miR-129-5p and its targets, while flow cytometry was used to evaluate the effect of miR-129-5p on the apoptosis of neural progenitor cells (NPCs). BMP2 was confirmed as a direct target of miR-129-5p. Furthermore, the expression of miR-129 was downregulated along with upregulated BMP2 expression in IDD patients. Meanwhile, BMP2 was validated as the target of miR-129-5p in cells transfected with miR-129-5p and BMP2 siRNA. Also, compared with NPCs transfected with blank/scramble controls or miR-129-5p inhibitors, the NPCs treated with miR-129-5p mimics or BMP2 siRNA exhibited evidently elevated viability and inhibited apoptosis. The data demonstrated that miR-129-5p was poorly expressed in IDD patients, and the dysregulation of miR-129-5p might contribute to the development of IDD by targeting BMP2 expression.     

Therapeutic potential of melatonin in the intervertebral disc degeneration through inhibiting the ferroptosis of nucleus pulpous cells

Ferroptosis, a novel type of cell death mediated by the iron-dependent lipid peroxidation, contributes to the pathogenesis of the intervertebral disc degeneration (IDD). Increasing evidence demonstrated that melatonin (MLT) displayed the therapeutic potential to prevent the development of IDD. Current mechanistic study aims to explore whether the downregulation of ferroptosis contributes to the therapeutic capability of MLT in IDD. Current studies demonstrated that conditioned medium (CM) from the lipopolysaccharide (LPS)-stimulated macrophages caused a series of changes about IDD, including increased intracellular oxidative stress (increased reactive oxygen species and malondialdehyde levels, but decreased glutathione levels), upregulated expression of inflammation-associated factors (IL-1β, COX-2 and iNOS), increased expression of key matrix catabolic molecules (MMP-13, ADAMTS4 and ADAMTS5), reduced the expression of major matrix anabolic molecules (COL2A1 and ACAN), and increased ferroptosis (downregulated GPX4 and SLC7A11 levels, but upregulated ACSL4 and LPCAT3 levels) in nucleus pulposus (NP) cells. MLT could alleviate CM-induced NP cell injury in a dose-dependent manner. Moreover, the data substantiated that intercellular iron overload was involved in CM-induced ferroptosis in NP cells, and MLT treatment alleviated intercellular iron overload and protected NP cells against ferroptosis, and those protective effects of MLT in NP cells further attenuated with erastin and enhanced with ferrostatin-1(Fer-1). This study demonstrated that CM from the LPS-stimulated RAW264.7 macrophages promoted the NP cell injury. MLT alleviated the CM-induced NP cell injury partly through inhibiting ferroptosis. The findings support the role of ferroptosis in the pathogenesis of IDD, and suggest that MLT may serve as a potential therapeutic approach for clinical treatment of IDD.     

Astrocyte‐like cells differentiated from a novel population of CD45‐positive cells in adult human peripheral blood

We have previously reported a novel CD45‐positive cell population called peripheral blood insulin‐producing cells (PB‐IPCs) and its unique potential for releasing insulin in vitro. Despite the CD45‐positive phenotype and self‐renewal ability, PB‐IPCs are distinguished from hemopoietic and endothelial progenitor cells (EPCs) by some characteristics, such as a CD34‐negative phenotype and different culture conditions. We have further identified the gene profiles of the embryonic and neural stem cells, and these profiles include Sox2, Nanog, c‐Myc, Klf4, Notch1 and Mash1. After treatment with all‐trans retinoic acid (ATRA) in vitro, most PB‐IPCs exhibited morphological changes that included the development of elongated and branched cell processes. In the process of induction, the mRNA expression of Hes1 was robustly upregulated, and a majority of cells acquired some astrocyte‐associated specific phenotypes including anti‐glial fibrillary acidic protein (GFAP), CD44, Glutamate‐aspartate transporter (GLAST) and S100β. In spite of the deficiency of glutamate uptaking, the differentiated cells significantly relaxed the regulation of the expression of brain‐derived neurotrophic factor (BDNF) mRNA. This finding demonstrates that PB‐IPCs could be induced into a population of astrocyte‐like cells and enhanced the neurotrophic potential when the state of proliferation was limited by ATRA, which implies that this unique CD45+ cell pool may have a protective role in some degenerative diseases of the central nervous system (CNS).