Kenichiro Kakutani

Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy.

Study Design. In vivo studies using a rat model were performed to determine the feasibility of microbubble-enhanced ultrasound gene transfer technique to the intervertebral disc. Objectives. 1) To establish this microbubble-enhanced ultrasound gene therapy technique for intervertebral disc cells in vivo without using viral vectors and 2) to estimate the duration of transgene expression in vivo. Summary of Background Data. Intervertebral disc degeneration and associated spinal disorders remain a formidable problem. Although gene therapy approaches have been reported as having promising therapeutic potential to regenerate the disc, concerns over safety issues using recombinant viral vectors limits its application. Successful gene transfer using ultrasound has been reported in muscle and cardiovascular systems in vivo. Materials and Methods. Two different reporter plasmid DNA encoding green fluorescent protein (GFP) and firefly luciferase were used. Plasmid DNA was mixed with ultrasonography contrast agent (microbubbles) and injected into coccygeal intervertebral discs of Sprague-Dawley rats. The therapeutic ultrasound was irradiated on the surface of injected discs. Rats were killed 1, 3, 6, 12, and 24 weeks after gene transduction. Harvested nucleus pulposus tissues were used for evaluation of transgene expression. The intact discs were used as a control. Results. Seven days after gene transfection, considerable numbers of GFP-positive cells were observed in nucleus pulposus from the GFP-transfected group. Luciferase assay revealed that the ultrasound group demonstrated approximately an 11-fold increase in luciferase activity over the plasmid DNA-only group. Furthermore, transgene expression mediated by this method was observed, at least up to 24 weeks. Conclusions. Our study indicated that ultrasound transfection method with microbubbles significantly enhanced transfection efficiency of plasmid DNA into the nucleus pulposus cells in vivo. Furthermore, the sustained transgene expression in vivo was possible up to 24 weeks. The long-term gene expression mediated by simple and safe procedure has important clinical applications, including the treatment of chronic types of disease such as degenerative disc diseases.

Rat tail static compression model mimics extracellular matrix metabolic imbalances of matrix metalloproteinases, aggrecanases, and tissue inhibitors of metalloproteinases in intervertebral disc degeneration

IntroductionThe longitudinal degradation mechanism of extracellular matrix (ECM) in the interbertebral disc remains unclear. Our objective was to elucidate catabolic and anabolic gene expression profiles and their balances in intervertebral disc degeneration using a static compression model.MethodsForty-eight 12-week-old male Sprague-Dawley rat tails were instrumented with an Ilizarov-type device with springs and loaded statically at 1.3 MPa for up to 56 days. Experimental loaded and distal-unloaded control discs were harvested and analyzed by real-time reverse transcription-polymerase chain reaction (PCR) messenger RNA quantification for catabolic genes [matrix metalloproteinase (MMP)-1a, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, and ADAMTS-5], anti-catabolic genes [tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, and TIMP-3], ECM genes [aggrecan-1, collagen type 1-α1, and collagen type 2-α1], and pro-inflammatory cytokine genes [tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, and IL-6]. Immunohistochemistry for MMP-3, ADAMTS-4, ADAMTS-5, TIMP-1, TIMP-2, and TIMP-3 was performed to assess their protein expression level and distribution. The presence of MMP- and aggrecanase-cleaved aggrecan neoepitopes was similarly investigated to evaluate aggrecanolytic activity.ResultsQuantitative PCR demonstrated up-regulation of all MMPs and ADAMTS-4 but not ADAMTS-5. TIMP-1 and TIMP-2 were almost unchanged while TIMP-3 was down-regulated. Down-regulation of aggrecan-1 and collagen type 2-α1 and up-regulation of collagen type 1-α1 were observed. Despite TNF-α elevation, ILs developed little to no up-regulation. Immunohistochemistry showed, in the nucleus pulposus, the percentage of immunopositive cells of MMP-cleaved aggrecan neoepitope increased from 7 through 56 days with increased MMP-3 and decreased TIMP-1 and TIMP-2 immunopositivity. The percentage of immunopositive cells of aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with decreased TIMP-3 immunopositivity. In the annulus fibrosus, MMP-cleaved aggrecan neoepitope presented much the same expression pattern. Aggrecanase-cleaved aggrecan neoepitope increased at 7 and 28 days only with increased ADAMTS-4 and ADAMTS-5 immunopositivity.ConclusionsThis rat tail sustained static compression model mimics ECM metabolic imbalances of MMPs, aggrecanases, and TIMPs in human degenerative discs. A dominant imbalance of MMP-3/TIMP-1 and TIMP-2 relative to ADAMTS-4 and ADAMTS-5/TIMP-3 signifies an advanced stage of intervertebral disc degeneration.

Gene therapy approach for disc degeneration and associated spinal disorders

Disc degeneration is deeply associated with many spinal disorders and thus has a significant clinical impact on society. The currently available surgical treatment often necessitates removing a pathological disc and spinal fusion. However, it is also well known that these surgical treatments have many potential problems including invasion and cost. Therefore, biological approaches for regenerating these pathological discs have received much attention. Gene therapy is one of these biological approaches. Gene therapy involves the transfer of genes to cells so the recipient cells express these genes and thereby synthesize the RNA and protein they encode in a continuous fashion. One of the significant advantages of gene therapy is that we can expect a lasting duration of biological effect which is potentially beneficial for most disc degeneration associated disorders, as they are, by nature, chronic conditions. Originally, gene therapy was mediated by viral vectors, but recent technological progress has enabled us to opt for non-virus-mediated gene therapy for the disc. Furthermore, the development of the RNA interference technique has enabled us to down-regulate a specific gene expression in the disc opening the door for a new generation of intradiscal gene therapy.

Progression of Cervical Spine Instabilities in Rheumatoid Arthritis : A Prospective Cohort Study of Outpatients over 5 Years

Study Design. A 5-year prospective cohort study of cervical spine instabilities in rheumatoid arthritis (RA). Objective. To clarify the natural course of cervical instabilities in RA patients and to determine predictors for the prognosis of RA cervical spine. Summary of Background Data. Although several previous studies investigating the natural history of RA cervical spine have been reported, few of them have described radiological predictive factors for the aggravation of these instabilities. Methods. Two hundred sixty-seven outpatients with “definite” or “classical” RA initially assigned were prospectively followed for over 5 years. Radiographic cervical findings were classified into three representative instabilities: atlantoaxial subluxation (AAS), vertical subluxation (VS), and subaxial subluxation (SAS). The aggravations of these instabilities were identified in the cases with a decrease of at least 2 mm in the Ranawat value of VS, an increase of at least 1 mm in translation of SAS, or a new development of respective instabilities. RA stages and mutilating changes were assessed in the hand radiograms. Results. Fifty-two point four percent of 267 patients, without any cervical instability at the beginning of follow-up, decreased to 29.6% at the end (P < 0.01), whereas VS and SAS increased significantly (P < 0.01). The aggravation of VS was observed at statistically higher rates in patients with pre-existing instabilities as follows; 25.7% of AAS (P = 0.01), 49.1% of VS (P < 0.01), and 41.2% of SAS (P = 0.06). The aggravation of SAS was also detected in 47.2% of VS and 64.7% of SAS (P < 0.01). Patients with pre-existing mutilating changes exhibited the aggravations of VS and SAS in significantly higher incidences (P < 0.01). Furthermore, the cases with development into mutilating changes during the follow-up showed significantly higher tendencies for the aggravations of these instabilities (P < 0.01). Conclusion. The incidences of VS and SAS significantly increased during the minimum 5-year follow-up. Prognostic factors of these instabilities were revealed to be the initial radiological findings of VS, SAS, and mutilating changes.

Fas ligand plays an important role for the production of pro‐inflammatory cytokines in intervertebral disc nucleus pulposus cells

It is suggested that pro‐inflammatory cytokines, which are produced by interaction of the intervertebral nucleus pulposus cells and macrophages, may be linked to the cause of pain of the intervertebral disc herniation. This study carries out the in vitro experiments to examine the mechanism, with the use of the co‐culture of an immortalized cell line of nucleus pulposus of the human intervertebral disc and the macrophage cell line. As a result, it is found that the production of pro‐inflammatory cytokines is significantly larger at the co‐culture group than at the independent culture group. Also, at the co‐culture group of macrophages and intervertebral nucleus pulposus cells with over‐expression of fas ligand (FasL), the production of pro‐inflammatory cytokines is found to be far larger. Furthermore, it is found that these pro‐inflammatory cytokines are produced mainly by the intervertebral nucleus pulposus cells with over‐expression of FasL, and that the expression of a disintegrin and metalloproteinase (ADAM) 10, which controls the expression of FasL and activates reverse signaling inside cells, also increases. From these findings, it is suggested that FasL and ADAM10 play an important role in the production of pro‐inflammatory cytokines coming from interaction of the intervertebral nucleus pulposus cells and macrophages.

Intervertebral disc and macrophage interaction induces mechanical hyperalgesia and cytokine production in a herniated disc model in rats

OBJECTIVE The expression of proinflammatory factors such as tumor necrosis factor α (TNFα), interleukin-6 (IL-6), IL-8, and prostaglandin E(2) (PGE(2) ) is significantly correlated with the symptoms of herniated disc disease. Among the different types of immune cells, macrophages are frequently noted in the herniated disc tissue. We undertook this study to clarify the interaction of the intervertebral disc (IVD) and macrophages with regard to the production of TNFα, IL-6, IL-8, and PGE(2) . METHODS We developed 2 animal models to assess the interactions of IVDs with macrophages in terms of TNFα, IL-6, IL-8, and PGE(2) production and pain-related behavior. We also cocultured IVDs and macrophages to assess the role of TNFα in IL-6, IL-8, and PGE(2) production. RESULTS IVD autografts induced TNFα, IL-6, IL-8, and cyclooxygenase 2 (COX-2) messenger RNA (mRNA) up-regulation; macrophage infiltration was seen shortly after the autograft was implanted. A significant decrease was noted in the mechanical threshold of the ipsilateral paw following the up-regulation of TNFα, IL-6, IL-8, and COX-2 mRNA. Only IVD and macrophage cocultures resulted in IL-8 and PGE(2) up-regulation. TNFα up-regulation was maximized before that of IL-6 and IL-8. TNFα neutralization attenuated production of IL-6 and PGE(2) , but not that of IL-8. Neutralization of TNFα and IL-8 significantly increased the paw withdrawal mechanical threshold in the IVD autograft and spinal nerve ligation model. CONCLUSION IVD-macrophage interaction plays a major role in sciatica and in the production of TNFα, IL-6, IL-8, and PGE(2) . TNFα is required for IL-6 and PGE(2) production, but not for IL-8 production, during IVD-macrophage interaction. Neutralization of TNFα and IL-8 can be a valuable therapy for herniated disc disease.

Matrix metalloproteinase (MMP)-3 gene up-regulation in a rat tail compression loading-induced disc degeneration model.

The rodent static compression loading‐induced disc degeneration model still has important gaps among the radiographic, magnetic resonance imaging (MRI), and histological schemes and the acute and chronic expression of catabolic genes such as matrix metalloproteinase (MMP)‐3. Our objectives were to assess the validity of a rat tail two‐disc static compression model and to elucidate a representative catabolic marker, MMP‐3 gene alterations, throughout the degenerative process. Static compression at 1.3 MPa for up to 56 days produced progressive disc height loss in radiographs, lower nucleus intensity on T2‐weighted MRIs, and histomorphological degeneration. Real‐time RT‐PCR mRNA quantification showed significant MMP‐3 up‐regulation in nucleus pulposus cells from 7 days and a significantly progressive increase as the loading duration lengthened, with high correlations to radiological degenerative scores. Immunohistochemistry demonstrated progressively increased positive staining for MMP‐3. These results validate this animal model for disc degeneration research. Progressive mRNA and protein‐distributional up‐regulations indicate the significant role of MMP‐3 and its feasibility as a disc degenerative marker. This model should prove useful for investigating the pathomechanism and for evaluating molecular therapies for degenerative disc disease.

A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype

The intervertebral disc nucleus pulposus (NP) has two phenotypically distinct cell types—notochordal cells (NCs) and non‐notochordal chondrocyte‐like cells. In human discs, NCs are lost during adolescence, which is also when discs begin to show degenerative signs. However, little evidence exists regarding the link between NC disappearance and the pathogenesis of disc degeneration. To clarify this, a rat tail disc degeneration model induced by static compression at 1.3 MPa for 0, 1, or 7 days was designed and assessed for up to 56 postoperative days. Radiography, MRI, and histomorphology showed degenerative disc findings in response to the compression period. Immunofluorescence displayed that the number of DAPI‐positive NP cells decreased with compression; particularly, the decrease was notable in larger, vacuolated, cytokeratin‐8‐ and galectin‐3‐co‐positive cells, identified as NCs. The proportion of TUNEL‐positive cells, which predominantly comprised non‐NCs, increased with compression. Quantitative PCR demonstrated isolated mRNA up‐regulation of ADAMTS‐5 in the 1‐day loaded group and MMP‐3 in the 7‐day loaded group. Aggrecan‐1 and collagen type 2α‐1 mRNA levels were down‐regulated in both groups. This rat tail temporary static compression model, which exhibits decreased NC phenotype, increased apoptotic cell death, and imbalanced catabolic and anabolic gene expression, reproduces different stages of intervertebral disc degeneration.

Modified house-keeping gene expression in a rat tail compression loading-induced disc degeneration model.

House‐keeping genes (HKGs) are generally used as endogenous controls for molecular normalization in quantitative PCR analysis. However, whether all the so‐called HKGs are useful for intervertebral disc research is controversial. Our objective was, using a prevalidated rat tail static compression loading‐induced disc degeneration model, to clarify the feasibility of common HKGs for gene‐quantification in the nucleus pulposus cells. In real‐time RT‐PCR for five HKGs [β‐actin, β‐glucuronidase, β‐2 microglobulin, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA)], static compression at 1.3 MPa for up to 56 days demonstrated messenger RNA (mRNA) expression levels of consistent β‐2 microglobulin and GAPDH, slightly up‐regulated β‐glucuronidase, and fairly down‐regulated β‐actin and LDHA. Especially, β‐actin had a drastic suppression to 0.15‐fold in the loaded relative to unloaded discs at 7 days. In immunofluorescence, β‐actin showed a significant down‐regulation to almost undetectable levels from 28 days, while GAPDH was constantly detected throughout. β‐Actin mRNA and protein‐distribution are thought to be affected by the loading treatment, however, GAPDH mRNA and protein‐distribution can retain relatively stable expressions. Under prolonged static compression, β‐actin and probably LDHA are inappropriate, and GAPDH is a feasible HKG as internal references in the disc cells.

Expression of silent mating type information regulator 2 homolog 1 and its role in human intervertebral disc cell homeostasis

IntroductionIntervertebral disc tissue homeostasis is modulated by a variety of molecules. Silent mating type information regulator 2 homolog 1 (SIRT1) plays a key role in various physiological processes. The aim of the present study was to verify the expression of SIRT1 and determine SIRT1 function in human intervertebral disc cell homeostasis.MethodsHuman nucleus pulposus (NP) cells were obtained from 24 surgical patients (mean age: 39.4 years) and monolayer-cultured. SIRT1 expression was investigated using RT-PCR analysis and immunohistochemical staining. Quantitative real-time RT-PCR was performed to detect mRNA expression of SIRT1 and other genes: aggrecan, collagen type 2 and Sox9. The effect of SIRT1 on the extracellular matrix metabolism of NP cells was examined using recombinant human SIRT1 protein and a protein delivery reagent. Cell number and proliferation activity were measured following SIRT1 treatment. To reveal the deacetylation potential of transfected recombinant human SIRT1, western blotting for acetylated p53 was utilized. R-phycoerythrin was used for the negative control.ResultsSIRT1 expression was confirmed at both mRNA and protein levels in almost all NP cells. Real-time RT-PCR analysis showed SIRT1 mRNA expression significantly increased with donor age (P <0.05, ρ = 0.492). Pfirrmann grade 3 discs showed significantly higher SIRT1 mRNA expression than other grades. SIRT1 treatment significantly reduced aggrecan, Sox9 and collagen type 2 mRNA expression in a dose-dependent manner in all disease classes and disc degeneration grades. Proliferation activity was decreased by SIRT1 treatment in lumbar spinal stenosis and lumbar disc herniation, Pfirrmann grade 3 and grade 4 discs. In contrast, it was significantly upregulated in idiopathic scoliosis, Pfirrmann grade 2 discs. The negative control protein did not affect extracellular matrix metabolism or proliferation activity.ConclusionsWe demonstrate for the first time that SIRT1 is expressed by human NP cells. SIRT1 expression was significantly elevated in an early degeneration stage. SIRT1 affected both extracellular matrix metabolism and proliferation activity; the effect of SIRT1 was altered according to disease class and disc degeneration grade. SIRT1 appears to play a key role in homeostasis during the human intervertebral disc degeneration process.