MicroRNA-29a: a novel target for non-operative management of symptomatic lumbar spinal stenosis.

PURPOSE: Lumbar spinal stenosis (LSS) is the most common reason for spinal surgery in patients over the age of 65, and there are few effective non-surgical treatments. Therefore, the development of novel treatment or preventative modalities to decrease overall cost and morbidity associated with LSS is an urgent matter. The cause of LSS is multifactorial; however, a significant contributor is ligamentum flavum hypertrophy (LFH) which causes mechanical compression of the cauda equina or nerve roots. We assessed the role of a novel target, microRNA-29a (miR-29a), in LFH and investigated the potential for using miR-29a as a therapeutic means to combat LSS. METHODS: Ligamentum flavum (LF) tissue was collected from patients undergoing decompressive surgery for LSS and assessed for levels of miR-29a and pro-fibrotic protein expression. LF cell cultures were then transfected with either miR-29a over-expressor (agonist) or inhibitor (antagonist). The effects of over-expression and under-expression of miR-29a on expression of pro-fibrotic proteins was assessed. RESULTS: We demonstrated that LF at stenotic levels had a loss of miR-29a expression. This was associated with greater LF tissue thickness and higher mRNA levels of collagen I and III. We also demonstrated that miR29-a plays a direct role in the regulation of collagen gene expression in ligamentum flavum. Specifically, agents that increase miR-29a may attenuate LFH, while those that decrease miR-29a promote fibrosis and LFH. CONCLUSION: This study demonstrates that miR-29a may potentially be used to treat LFH and provides groundwork to initiate the development of a therapeutic product for LSS.

Immune exposure: how macrophages interact with the nucleus pulposus.

Intervertebral disc degeneration (IDD) is a primary contributor to low back pain. Immune cells play an extremely important role in modulating the progression of IDD by interacting with disc nucleus pulposus (NP) cells and extracellular matrix (ECM). Encased within the annulus fibrosus, healthy NP is an avascular and immune-privileged tissue that does not normally interact with macrophages. However, under pathological conditions in which neovascularization is established in the damaged disc, NP establishes extensive crosstalk with macrophages, leading to different outcomes depending on the different microenvironmental stimuli. M1 macrophages are a class of immune cells that are predominantly pro-inflammatory and promote inflammation and ECM degradation in the NP, creating a vicious cycle of matrix catabolism that drives IDD. In contrast, NP cells interacting with M2 macrophages promote disc tissue ECM remodeling and repair as M2 macrophages are primarily involved in anti-inflammatory cellular responses. Hence, depending on the crosstalk between NP and the type of immune cells (M1 vs. M2), the overall effects on IDD could be detrimental or regenerative. Drug or surgical treatment of IDD can modulate this crosstalk and hence the different treatment outcomes. This review comprehensively summarizes the interaction between macrophages and NP, aiming to highlight the important role of immunology in disc degeneration.

Ionizing Radiation Induces Disc Annulus Fibrosus Senescence and Matrix Catabolism via MMP-Mediated Pathways.

Previous research has identified an association between external radiation and disc degeneration, but the mechanism was poorly understood. This study explores the effects of ionizing radiation (IR) on inducing cellular senescence of annulus fibrosus (AF) in cell culture and in an in vivo mouse model. Exposure of AF cell culture to 10-15 Gy IR for 5 min followed by 5 days of culture incubation resulted in almost complete senescence induction as evidenced by SA-ßgal positive staining of cells and elevated mRNA expression of the p16 and p21 senescent markers. IR-induced senescent AF cells exhibited increased matrix catabolism, including elevated matrix metalloproteinase (MMP)-1 and -3 protein expression and aggrecanolysis. Analogous results were seen with whole body IR-exposed mice, demonstrating that genotoxic stress also drives disc cellular senescence and matrix catabolism in vivo. These results have important clinical implications in the potential adverse effects of ionizing radiation on spinal health.

ADAMTS5 Deficiency Protects Mice From Chronic Tobacco Smoking-induced Intervertebral Disc Degeneration.

STUDY DESIGN: ADAMTS5-deficient and wild type (WT) mice were chronically exposed to tobacco smoke to investigate effects on intervertebral disc degeneration (IDD). OBJECTIVE: The aim of this study was to demonstrate a role for ADAMTS5 in mediating tobacco smoking-induced IDD. SUMMARY OF BACKGROUND DATA: We previously demonstrated that chronic tobacco smoking causes IDD in mice because, in part, of proteolytic destruction of disc aggrecan. However, it was unknown which matrix proteinase(s) drive these detrimental effects. METHODS: Three-month-old WT (C57BL/6) and ADAMTS5 mice were chronically exposed to tobacco smoke (four cigarettes/day, 5 day/week for 6 months). ADAMTS-mediated cleavage of disc aggrecan was analyzed by Western blot. Disc total glycosaminoglycan (GAG) content was assessed by dimethyl methylene blue assay and safranin O/fast green histology. Vertebral osteoporosity was measured by microcomputed tomography. Human nucleus pulposus (hNP) cell cultures were also exposed directly to tobacco smoke extract (TSE), a condensate containing the water-soluble compounds inhaled by smokers, to measure ADAMTS5 expression and ADAMTS-mediated cleavage of aggrecan. Activation of nuclear factor (NF)-κB, a family of transcription factors essential for modulating the cellular response to stress, was measured by immunofluorescence assay. RESULTS: Genetic depletion of ADAMTS5 prevented vertebral bone loss, substantially reduced loss of disc GAG content, and completely obviated ADAMTS-mediated proteolysis of disc aggrecan within its interglobular domain (IGD) in mice following exposure to tobacco smoke. hNP cell cultures exposed to TSE also resulted in upregulation of ADAMTS5 protein expression and a concomitant increase in ADAMTS-mediated cleavage within aggrecan IGD. Activation of NF-κB, known to be required for ADAMTS5 gene expression, was observed in both TSE-treated hNP cell cultures and disc tissue of tobacco smoke-exposed mice. CONCLUSION: The findings demonstrate that ADAMTS5 is the primary aggrecanase mediating smoking-induced disc aggrecanolysis and IDD. Mouse models of chronic tobacco smoking are important and useful for probing the mechanisms of disc aggrecan catabolism and IDD. LEVEL OF EVIDENCE: N/A.

Investigating the role of DNA damage in tobacco smoking-induced spine degeneration.

BACKGROUND CONTEXT: Tobacco smoking is a key risk factor for spine degeneration. However, the underlying mechanism by which smoking induces degeneration is not known. Recent studies implicate DNA damage as a cause of spine and intervertebral disc degeneration. Because tobacco smoke contains many genotoxins, we hypothesized that tobacco smoking promotes spine degeneration by inducing cellular DNA damage. PURPOSE: To determine if DNA damage plays a causal role in smoking-induced spine degeneration. STUDY DESIGN: To compare the effect of chronic tobacco smoke inhalation on intervertebral disc and vertebral bone in normal and DNA repair-deficient mice to determine the contribution of DNA damage to degenerative changes. METHODS: Two-month-old wild-type (C57BL/6) and DNA repair-deficient Ercc1(-/Δ) mice were exposed to tobacco smoke by direct inhalation (4 cigarettes/day, 5 days/week for 7 weeks) to model first-hand smoking in humans. Total disc proteoglycan (PG) content (1,9-dimethylmethylene blue assay), PG synthesis ((35)S-sulfate incorporation assay), aggrecan proteolysis (immunoblotting analysis), and vertebral bone morphology (microcomputed tomography) were measured. RESULTS: Exposure of wild-type mice to tobacco smoke led to a 19% increase in vertebral porosity and a 61% decrease in trabecular bone volume. Intervertebral discs of smoke-exposed animals also showed a 2.6-fold decrease in GAG content and an 8.1-fold decrease in new PG synthesis. These smoking-induced degenerative changes were similar but not worse in Ercc1(-/Δ) mice. CONCLUSIONS: Short-term exposure to high levels of primary tobacco smoke inhalation promotes degeneration of vertebral bone and discs. Disc degeneration is primarily driven by reduced synthesis of proteoglycans needed for vertebral cushioning. Degeneration was not exacerbated in congenic DNA repair-deficient mice, indicating that DNA damage per se does not have a significant causal role in driving smoke-induced spine degeneration.

Injection of human umbilical tissue-derived cells into the nucleus pulposus alters the course of intervertebral disc degeneration in vivo.

BACKGROUND CONTEXT: Patients often present to spine clinic with evidence of intervertebral disc degeneration (IDD). If conservative management fails, a safe and effective injection directly into the disc might be preferable to the risks and morbidity of surgery. PURPOSE: To determine whether injecting human umbilical tissue-derived cells (hUTC) into the nucleus pulposus (NP) might improve the course of IDD. DESIGN: Prospective, randomized, blinded placebo-controlled in vivo study. PATIENT SAMPLE: Skeletally mature New Zealand white rabbits. OUTCOME MEASURES: Degree of IDD based on magnetic resonance imaging (MRI), biomechanics, and histology. METHODS: Thirty skeletally mature New Zealand white rabbits were used in a previously validated rabbit annulotomy model for IDD. Discs L2-L3, L3-L4, and L4-L5 were surgically exposed and punctured to induce degeneration and then 3 weeks later the same discs were injected with hUTC with or without a hydrogel carrier. Serial MRIs obtained at 0, 3, 6, and 12 weeks were analyzed for evidence of degeneration qualitatively and quantitatively via NP area and MRI Index. The rabbits were sacrificed at 12 weeks and discs L4-L5 were analyzed histologically. The L3-L4 discs were fixed to a robotic arm and subjected to uniaxial compression, and viscoelastic displacement curves were generated. RESULTS: Qualitatively, the MRIs demonstrated no evidence of degeneration in the control group over the course of 12 weeks. The punctured group yielded MRIs with the evidence of disc height loss and darkening, suggestive of degeneration. The three treatment groups (cells alone, carrier alone, or cells+carrier) generated MRIs with less qualitative evidence of degeneration than the punctured group. MRI Index and area for the cell and the cell+carrier groups were significantly distinct from the punctured group at 12 weeks. The carrier group generated MRI data that fell between control and punctured values but failed to reach a statistically significant difference from the punctured values. There were no statistically significant MRI differences among the three treatment groups. The treated groups also demonstrated viscoelastic properties that were distinct from the control and punctured values, with the cell curve more similar to the punctured curve and the carrier curve and carrier+cells curve more similar to the control curve (although no creep differences achieved statistical significance). There was some histological evidence of improved cellularity and disc architecture in the treated discs compared with the punctured discs. CONCLUSIONS: Treatment of degenerating rabbit intervertebral discs with hUTC in a hydrogel carrier solution might help restore the MRI, histological, and biomechanical properties toward those of nondegenerated controls. Treatment with cells in saline or a hydrogel carrier devoid of cells also might help restore some imaging, architectural, and physical properties to the degenerating disc. These data support the potential use of therapeutic cells in the treatment of disc degeneration.

Genotoxic stress accelerates age-associated degenerative changes in intervertebral discs.

Intervertebral disc degeneration (IDD) is the leading cause of debilitating spinal disorders such as chronic lower back pain. Aging is the greatest risk factor for IDD. Previously, we demonstrated IDD in a murine model of a progeroid syndrome caused by reduced expression of a key DNA repair enzyme. This led us to hypothesize that DNA damage promotes IDD. To test our hypothesis, we chronically exposed adult wild-type (Wt) and DNA repair-deficient Ercc1(-/Δ) mice to the cancer therapeutic agent mechlorethamine (MEC) or ionization radiation (IR) to induce DNA damage and measured the impact on disc structure. Proteoglycan, a major structural matrix constituent of the disc, was reduced 3-5× in the discs of MEC- and IR-exposed animals compared to untreated controls. Expression of the protease ADAMTS4 and aggrecan proteolytic fragments was significantly increased. Additionally, new PG synthesis was reduced 2-3× in MEC- and IR-treated discs compared to untreated controls. Both cellular senescence and apoptosis were increased in discs of treated animals. The effects were more severe in the DNA repair-deficient Ercc1(-/Δ) mice than in Wt littermates. Local irradiation of the vertebra in Wt mice elicited a similar reduction in PG. These data demonstrate that genotoxic stress drives degenerative changes associated with IDD.

ISSLS prize winner: inhibition of NF-κB activity ameliorates age-associated disc degeneration in a mouse model of accelerated aging.

STUDY DESIGN: NF-κB activity was pharmacologically and genetically blocked in an accelerated aging mouse model to mitigate age-related disc degenerative changes. OBJECTIVE: To study the mediatory role of NF-κB-signaling pathway in age-dependent intervertebral disc degeneration. SUMMARY OF BACKGROUND DATA: Aging is a major contributor to intervertebral disc degeneration (IDD), but the molecular mechanism behind this process is poorly understood. NF-κB is a family of transcription factors that play a central role in mediating cellular response to damage, stress, and inflammation. Growing evidence implicates chronic NF-κB activation as a culprit in many aging-related diseases, but its role in aging-related IDD has not been adequately explored. We studied the effects of NF-κB inhibition on IDD, using a DNA repair-deficient mouse model of accelerated aging (Ercc1 mice) previously been reported to exhibit age-related IDD. METHODS: Systemic inhibition of NF-κB activation was achieved either genetically by deletion of 1 allele of the NF-κB subunit p65 (Ercc1p65 mice) or pharmacologically by chronic intraperitoneal administration of the Nemo Binding Domain (8K-NBD) peptide to block the formation of the upstream activator of NF-κB, IκB Inducible Kinase (IKK), in Ercc1 mice. Disc cellularity, total proteoglycan content and proteoglycan synthesis of treated mice, and untreated controls were assessed. RESULTS.: Decreased disc matrix proteoglycan content, a hallmark feature of IDD, and elevated disc NF-κB activity were observed in discs of progeroid Ercc1 mice and naturally aged wild-type mice compared with young wild-type mice. Systemic inhibition of NF-κB by the 8K-NBD peptide in Ercc1 mice increased disc proteoglycan synthesis and ameriolated loss of disc cellularity and matrix proteoglycan. These results were confirmed genetically by using the p65 haploinsufficient Ercc1p65 mice. CONCLUSION: These findings demonstrate that the IKK/NF-κB signaling pathway is a key mediator of age-dependent IDD and represents a therapeutic target for mitigating disc degenerative diseases associated with aging.

Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model.

BACKGROUND CONTEXT: Intervertebral disc degeneration (IDD) is a common cause of back pain. Patients who fail conservative management may face the morbidity of surgery. Alternative treatment modalities could have a significant impact on disease progression and patients' quality of life. PURPOSE: To determine if the injection of a virus vector carrying a therapeutic gene directly into the nucleus pulposus improves the course of IDD. STUDY DESIGN: Prospective randomized controlled animal study. METHODS: Thirty-four skeletally mature New Zealand white rabbits were used. In the treatment group, L2-L3, L3-L4, and L4-L5 discs were punctured in accordance with a previously validated rabbit annulotomy model for IDD and then subsequently treated with adeno-associated virus serotype 2 (AAV2) vector carrying genes for either bone morphogenetic protein 2 (BMP2) or tissue inhibitor of metalloproteinase 1 (TIMP1). A nonoperative control group, nonpunctured sham surgical group, and punctured control group were also evaluated. Serial magnetic resonance imaging (MRI) studies at 0, 6, and 12 weeks were obtained, and a validated MRI analysis program was used to quantify degeneration. The rabbits were sacrificed at 12 weeks, and L4-L5 discs were analyzed histologically. Viscoelastic properties of the L3-L4 discs were analyzed using uniaxial load-normalized displacement testing. Creep curves were mathematically modeled according to a previously validated two-phase exponential model. Serum samples obtained at 0, 6, and 12 weeks were assayed for biochemical evidence of degeneration. RESULTS: The punctured group demonstrated MRI and histologic evidence of degeneration as expected. The treatment groups demonstrated less MRI and histologic evidence of degeneration than the punctured group. The serum biochemical marker C-telopeptide of collagen type II increased rapidly in the punctured group, but the treated groups returned to control values by 12 weeks. The treatment groups demonstrated several viscoelastic properties that were distinct from control and punctured values. CONCLUSIONS: Treatment of punctured rabbit intervertebral discs with AAV2-BMP2 or AAV2-TIMP1 helps delay degenerative changes, as seen on MRI, histologic sampling, serum biochemical analysis, and biomechanical testing. Although data from animal models should be extrapolated to the human condition with caution, this study supports the potential use of gene therapy for the treatment of IDD.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 1. RNA chain initiation, abortive initiation, and promoter escape at three bacteriophage promoters.

RNA chain initiation and promoter escape is the latter stage of transcription initiation. This stage is characterized by several well-defined biochemical events: synthesis and release of short RNA products ranging 2 to 15 nucleotides in length, release of the sigma subunit from the enzyme-promoter complex, and initial translocation of the polymerase away from the promoter. In this paper, we report the use of a steady-state transcription assay with [gamma-(32)P]ATP labeling to subject the RNA chain initiation-promoter escape reaction to quantitative analysis. The specific parameters we follow to describe the chain initiation-promoter escape process include the abortive and productive rates, the abortive probability, the abortive:productive ratio, and the maximal size of the abortive product. In this study, we measure these parameters for three bacteriophage promoters transcribed by Escherichia coli RNA polymerase: T7 A1, T5 N25, and T5 N25(antiDSR). Our studies show that all three promoters form substantial amounts of abortive products under all conditions we tested. However, each of the promoters shows distinct differences from the others when the various parameters are compared. At 100 microM NTP, in a 10 min reaction, the abortive and productive yields are 87 and 13%, respectively, for T7 A1; 97 and 3%, respectively, for T5 N25; and 99.4 and 0.6%, respectively, for T5 N25(antiDSR). These values correspond to approximately 7, 32, and 165 abortive transcripts per productive transcript for the three promoters, respectively. The yield of most of the abortive products is not affected by the elevated concentration of the NTP substrate corresponding to the next template-specified nucleotide; hence, abortive products are not normally formed through a simple process of "kinetic competition". Instead, formation of abortive products appears to be determined by intrinsic DNA signals embedded in the promoter recognition region and the initial transcribed sequence region of each promoter.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 2. Formation and characterization of two distinct classes of initial transcribing complexes.

By following the kinetics of abortive and productive synthesis in single-round transcription assays, we confirm the existence of two general classes of initial transcribing complexes (ITCs), which we term "productive ITC" and "unproductive ITC". The productive ITCs are able to escape from the promoter rapidly to produce full-length transcripts, but only after carrying out an obligate series of abortive initiation steps. The unproductive ITCs were found to synthesize mostly abortive transcripts of 2-3 nucleotides and escape from the promoter extremely slowly, if at all. Formation of the unproductive ITC is not due to the inactive RNA polymerase. Instead, RNA polymerase molecules recovered from both the productive and unproductive ITC fractions were shown to carry out abortive and productive synthesis with both the partitioning tendency and transcription kinetics similar to those of the original enzyme. Our results suggest that early transcription complexes are partitioned into the productive and unproductive ITCs most likely during the formation of open promoter complexes. The extent of partitioning varies with individual promoter sequences and is dependent on the nature and concentration of the initiating nucleotide. Thus, multiple classes of ITCs can be formed during promoter binding and transcript initiation.

In vitro studies of transcript initiation by Escherichia coli RNA polymerase. 3. Influences of individual DNA elements within the promoter recognition region on abortive initiation and promoter escape.

Abortive initiation and promoter escape are two principal biochemical reactions occurring in the latter stage of transcript initiation. We have analyzed the influences of individual DNA elements within the promoter recognition region (PRR) on these reactions by measuring the quantitative initiation parameters that describe abortive initiation and promoter escape; these parameters are the abortive rate, the productive rate, the abortive:productive ratio, the abortive probability, and the maximum size of abortive transcripts. Changes in the individual DNA elements within the PRR can have a substantial effect on each of these parameters. The discriminator region and the -10 element primarily influence the abortive probability at positions 2-5 and 6-10, respectively, while the -10 and -35 conserved hexamers and the spacer region affect the abortive probability at positions 11-15. Surprisingly, transcription of a consensus promoter invariably gives a higher abortive yield, a higher abortive probability, a longer abortive ladder, and a lower productive rate than promoter variants carrying even a single deviation in the consensus hexamers. These results suggest that strong RNA polymerase-PRR interactions stall the polymerase at the promoter, thereby reducing the rate of promoter escape and consequently enhancing the extent of abortive initiation.