High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain

Introduction

Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.

Methods

Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20% cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor α (TNFα) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.

Results

HMS caused upregulation of TLR2, TLR4, NGF and TNFα gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by γ interferon, transforming growth factor β1, TNFα and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.

Conclusions

HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

Clustered gene expression changes flank targeted gene loci in knockout mice

Background

Gene expression profiling using microarrays is a powerful technology widely used to study regulatory networks. Profiling of mRNA levels in mutant organisms has the potential to identify genes regulated by the mutated protein.

Methodology/Principle Findings

Using tissues from multiple lines of knockout mice we have examined genome-wide changes in gene expression. We report that a significant proportion of changed genes were found near the targeted gene.

Conclusions/Significance

The apparent clustering of these genes was explained by the presence of flanking DNA from the parental ES cell. We provide recommendations for the analysis and reporting of microarray data from knockout mice     

miRNA-mediated functional changes through co-regulating function related genes

Background

microRNAs play important roles in various biological processes involving fairly complex mechanism. Analysis of genome-wide miRNA microarray demonstrate that a single miRNA can regulate hundreds of genes, but the regulative extent on most individual genes is surprisingly mild so that it is difficult to understand how a miRNA provokes detectable functional changes with such mild regulation.

Results

To explore the internal mechanism of miRNA-mediated regulation, we re-analyzed the data collected from genome-wide miRNA microarray with bioinformatics assay, and found that the transfection of miR-181b and miR-34a in Hela and HCT-116 tumor cells regulated large numbers of genes, among which, the genes related to cell growth and cell death demonstrated high Enrichment scores, suggesting that these miRNAs may be important in cell growth and cell death. MiR-181b induced changes in protein expression of most genes that were seemingly related to enhancing cell growth and decreasing cell death, while miR-34a mediated contrary changes of gene expression. Cell growth assays further confirmed this finding. In further study on miR-20b-mediated osteogenesis in hMSCs, miR-20b was found to enhance osteogenesis by activating BMPs/Runx2 signaling pathway in several stages by co-repressing of PPARγ, Bambi and Crim1.

Conclusions

With its multi-target characteristics, miR-181b, miR-34a and miR-20b provoked detectable functional changes by co-regulating functionally-related gene groups or several genes in the same signaling pathway, and thus mild regulation from individual miRNA targeting genes could have contributed to an additive effect. This might also be one of the modes of miRNA-mediated gene regulation.     

Identification and interpretation of longitudinal gene expression changes in trauma

Rationale

The relationship between leukocyte gene expression and recovery of respiratory function after injury may provide information on the etiology of multiple organ dysfunction.

Objectives

To find a list of genes for which expression after injury predicts respiratory recovery, and to identify which networks and pathways characterize these genes.

Methods

Blood was sampled at 12 hours and at 1, 4, 7, 21 and 28 days from 147 patients who had been admitted to the hospital after blunt trauma. Leukocyte gene expression was measured using Affymetrix oligonucleotide arrays. A linear model, fit to each probe-set expression value, was used to impute the gene expression trajectory over the entire follow-up period. The proportional hazards model score test was used to calculate the statistical significance of each probe-set trajectory in predicting respiratory recovery. A list of genes was determined such that the expected proportion of false positive results was less than 10%. These genes were compared to the Gene Ontology for ‘response to stimulus’ and, using Ingenuity software, were mapped into networks and pathways.

Measurements and Main Results

The median time to respiratory recovery was 6 days. There were 170 probe-sets representing 135 genes that were found to be related to respiratory recovery. These genes could be mapped to nine networks. Two known pathways that were activated were antigen processing and presentation and JAK- signaling.

Conclusions

The examination of the relationship of gene expression over time with a patient''s clinical course can provide information which may be useful in determining the mechanism of recovery or lack of recovery after severe injury.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

Prenatal Polycyclic Aromatic Hydrocarbon,Adiposity, Peroxisome Proliferator-Activated Receptor (PPAR) γ Methylation in Offspring,Grand-Offspring Mice

Rationale

Greater levels of prenatal exposure to polycyclic aromatic hydrocarbon (PAH) have been associated with childhood obesity in epidemiological studies. However, the underlying mechanisms are unclear.

Objectives

We hypothesized that prenatal PAH over-exposure during gestation would lead to weight gain and increased fat mass in offspring and grand-offspring mice. Further, we hypothesized that altered adipose gene expression and DNA methylation in genes important to adipocyte differentiation would be affected.

Materials and Methods

Pregnant dams were exposed to a nebulized PAH mixture versus negative control aerosol 5 days a week, for 3 weeks. Body weight was recorded from postnatal day (PND) 21 through PND60. Body composition, adipose cell size, gene expression of peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding proteins (C/EBP) α, cyclooxygenase (Cox)-2, fatty acid synthase (FAS) and adiponectin, and DNA methylation of PPAR γ, were assayed in both the offspring and grand-offspring adipose tissue.

Findings

Offspring of dams exposed to greater PAH during gestation had increased weight, fat mass, as well as higher gene expression of PPAR γ, C/EBP α, Cox2, FAS and adiponectin and lower DNA methylation of PPAR γ. Similar differences in phenotype and DNA methylation extended through the grand-offspring mice.

Conclusions

Greater prenatal PAH exposure was associated with increased weight, fat mass, adipose gene expression and epigenetic changes in progeny.     

IL-1ra delivered from poly(lactic-co-glycolic acid) microspheres attenuates IL-1β-mediated degradation of nucleus pulposus in vitro

Introduction

Inflammation plays a key role in the progression of intervertebral disc degeneration, a condition strongly implicated as a cause of lower back pain. The objective of this study was to investigate the therapeutic potential of poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with interleukin-1 receptor antagonist (IL-1ra) for sustained attenuation of interleukin-1 beta (IL-1β) mediated degradative changes in the nucleus pulposus (NP), using an in vitro model.

Methods

IL-1ra was encapsulated in PLGA microspheres and release kinetics were determined over 35 days. NP agarose constructs were cultured to functional maturity and treated with combinations of IL-1β and media conditioned with IL-1ra released from microspheres at intervals for up to 20 days. Construct mechanical properties, glycosaminoglycan content, nitrite production and mRNA expression of catabolic mediators were compared to properties for untreated constructs using unpaired Student''s t-tests.

Results

IL-1ra release kinetics were characterized by an initial burst release reducing to a linear release over the first 10 days. IL-1ra released from microspheres attenuated the degradative effects of IL-1β as defined by mechanical properties, glycosaminoglycans (GAG) content, nitric oxide production and mRNA expression of inflammatory mediators for 7 days, and continued to limit functional degradation for up to 20 days.

Conclusions

In this study, we successfully demonstrated that IL-1ra microspheres can attenuate the degradative effects of IL-1β on the NP for extended periods. This therapeutic strategy may be appropriate for treating early-stage, cytokine-mediated disc degeneration. Ongoing studies are focusing on testing IL-1ra microspheres in an in vivo model of disc degeneration, as a prelude to clinical translation.     

Novel Immortal Cell Lines Support Cellular Heterogeneity in the Human Annulus Fibrosus

Introduction

Loss of annulus fibrosus (AF) integrity predisposes to disc herniation and is associated with IVD degeneration. Successful implementation of biomedical intervention therapy requires in-depth knowledge of IVD cell biology. We recently generated unique clonal human nucleus pulposus (NP) cell lines. Recurring functional cellular phenotypes from independent donors provided pivotal evidence for cell heterogeneity in the mature human NP. In this study we aimed to generate and characterize immortal cell lines for the human AF from matched donors.

Methods

Non-degenerate healthy disc material was obtained as surplus surgical material. AF cells were immortalized by simian virus Large T antigen (SV40LTAg) and human telomerase (hTERT) expression. Early passage cells and immortalized cell clones were characterized based on marker gene expression under standardized culturing and in the presence of Transforming Growth factor β (TGFβ).

Results

The AF-specific expression signature included COL1A1, COL5A1, COL12A1, SFRP2 and was largely maintained in immortal AF cell lines. Remarkably, TGFβ induced rapid 3D sheet formation in a subgroup of AF clones. This phenotype was associated with inherent differences in Procollagen type I processing and maturation, and correlated with differential mRNA expression of Prolyl 4-hydroxylase alpha polypeptide 1 and 3 (P4HA1,3) and Lysyl oxidase (LOX) between clones and differential P4HA3 protein expression between AF cells in histological sections.

Conclusion

We report for the first time the generation of representative human AF cell lines. Gene expression profile analysis and functional comparison of AF clones revealed variation between immortalized cells and suggests phenotypic heterogeneity in the human AF. Future characterization of AF cellular (sub-)populations aims to combine identification of additional specific AF marker genes and their biological relevance. Ultimately this knowledge will contribute to clinical application of cell-based technology in IVD repair.     

Growth differentiation factor 6 and transforming growth factor-beta differentially mediate mesenchymal stem cell differentiation,composition, and micromechanical properties of nucleus pulposus constructs

Introduction

Currently, there is huge research focus on the development of novel cell-based regeneration and tissue-engineering therapies for the treatment of intervertebral disc degeneration and the associated back pain. Both bone marrow-derived (BM) mesenchymal stem cells (MSCs) and adipose-derived MSCs (AD-MSCs) are proposed as suitable cells for such therapies. However, currently no consensus exists as to the optimum growth factor needed to drive differentiation to a nucleus pulposus (NP)-like phenotype. The aim of this study was to investigate the effect of growth differentiation factor-6 (GDF6), compared with other transforming growth factor (TGF) superfamily members, on discogenic differentiation of MSCs, the matrix composition, and micromechanics of engineered NP tissue constructs.

Methods

Patient-matched human AD-MSCs and BM-MSCs were seeded into type I collagen hydrogels and cultured in differentiating media supplemented with TGF-β3, GDF5, or GDF6. After 14 days, quantitative polymerase chain reaction analysis of chondrogenic and novel NP marker genes and sulfated glycosaminoglycan (sGAG) content of the construct and media components were measured. Additionally, construct micromechanics were analyzed by using scanning acoustic microscopy (SAM).

Results

GDF6 stimulation of BM-MSCs and AD-MSCs resulted in a significant increase in expression of novel NP marker genes, a higher aggrecan-to-type II collagen gene expression ratio, and higher sGAG production compared with TGF-β or GDF5 stimulation. These effects were greater in AD-MSCs than in BM-MSCs. Furthermore, the acoustic-wave speed measured by using SAM, and therefore tissue stiffness, was lowest in GDF6-stiumlated AD-MSC constructs.

Conclusions

The data suggest that GDF6 stimulation of AD-MSCs induces differentiation to an NP-like phenotype and results in a more proteoglycan-rich matrix. Micromechanical analysis shows that the GDF6-treated AD-MSCs have a less-stiff matrix composition, suggesting that the growth factor is inducing a matrix that is more akin to the native NP-like tissue. Thus, this cell and growth-factor combination may be the ideal choice for cell-based intervertebral disc (IVD)-regeneration therapies.     

VEGF Gene Expression in Adult Human Thymus Fat: A Correlative Study with Hypoxic Induced Factor and Cyclooxigenase-2

It is well known that the adult human thymus degenerates into fat tissue; however, it has never been considered as a potential source of angiogenic factors. Recently, we have described that this fat (TAT) produces angiogenic factors and induces human endothelial cell proliferation and migration, indicating its potential angiogenic properties.

Design

Adult thymus fat and subcutaneous adipose tissue specimens were obtained from 28 patients undergoing cardiac surgery, making this tissue readily available as a prime source of adipose tissue. We focused our investigation on determining VEGF gene expression and characterizing the different genes, mediators of inflammation and adipogenesis, and which are known to play a relevant role in angiogenesis regulation.

Results

We found that VEGF-A was the isoform most expressed in TAT. This expression was accompanied by an upregulation of HIF-1α, COX-2 and HO-1 proteins, and by increased HIF-1 DNA binding activity, compared to SAT. Furthermore, we observed that TAT contains a high percentage of mature adipocytes, 0.25% of macrophage cells, 15% of endothelial cells and a very low percentage of thymocyte cells, suggesting the cellular variability of TAT, which could explain the differences in gene expression observed in TAT. Subsequently, we showed that the expression of genes known as adipogenic mediators, including PPARγ1/γ2, FABP-4 and adiponectin was similar in both TAT and SAT. Moreover the expression of these latter genes presented a significantly positive correlation with VEGF, suggesting the potential association between VEGF and the generation of adipose tissue in adult thymus.

Conclusion

Here we suggest that this fat has a potential angiogenic function related to ongoing adipogenesis, which substitutes immune functions within the adult thymus. The expression of VEGF seems to be associated with COX-2, HO-1 and adipogenesis related genes, suggesting the importance that this new fat has acquired in research in relation to adipogenesis and angiogenesis.     

The notch and TGF-β signaling pathways contribute to the aggressiveness of clear cell renal cell carcinoma

Background

Despite recent progress, therapy for metastatic clear cell renal cell carcinoma (CCRCC) is still inadequate. Dysregulated Notch signaling in CCRCC contributes to tumor growth, but the full spectrum of downstream processes regulated by Notch in this tumor form is unknown.

Methodology/Principal Findings

We show that inhibition of endogenous Notch signaling modulates TGF-β dependent gene regulation in CCRCC cells. Analysis of gene expression data representing 176 CCRCCs showed that elevated TGF-β pathway activity correlated significantly with shortened disease specific survival (log-rank test, p = 0.006) and patients with metastatic disease showed a significantly elevated TGF-β signaling activity (two-sided Student''s t-test, p = 0.044). Inhibition of Notch signaling led to attenuation of both basal and TGF-β1 induced TGF-β signaling in CCRCC cells, including an extensive set of genes known to be involved in migration and invasion. Functional analyses revealed that Notch inhibition decreased the migratory and invasive capacity of CCRCC cells.

Conclusion

An extensive cross-talk between the Notch and TGF-β signaling cascades is present in CCRCC and the functional properties of these two pathways are associated with the aggressiveness of this disease.     

Polymorphism in the Hypoxia-Inducible Factor 1alpha Gene May Confer Susceptibility to LDD in Chinese Cohort

Objective

This study aimed to investigate whether or not hypoxia-inducible factor-1α (HIF-1α) gene variants are associated with the susceptibility and clinical characteristics of lumbar disc degeneration (LDD).

Methods

We examined 320 patients with LDD and 447 gender- and age-matched control subjects. We also determined the HIF-1α gene variants, including C1772T (P582S) and G1790A (A588T) polymorphisms.

Results

Significant differences were observed in allelic and genotypic distributions of 1790 A > G polymorphisms between LDD cases and control subjects. Logistic regression revealed that 1790 AA genotypes indicated a protective effect against the development of LDD. The HIF-1α 1790 A > G polymorphisms also affected the severity of LDD as evaluated based on the modified Japanese Orthopedic Association (mJOA) scores. The 1790 AA genotype carriers exhibited significantly lower mJOA scores than AG and GG carriers. C1772T did not show any association with the risk and severity of LDD.

Conclusion

Our study suggested that HIF-1α 1790 A > G polymorphisms may be used as a molecular marker to determine the susceptibility and severity of LDD.