Bromodomain-containing protein 4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity in NP cells.

An imbalance between matrix synthesis and degradation is the hallmark of intervertebral disc degeneration while inflammatory cytokines contribute to the imbalance. Bromodomain and extra-terminal domain (BET) family is associated with the pathogenesis of inflammation, and inhibition of BRD4, a vital member of BET family, plays an anti-inflammatory role in many diseases. However, it remains elusive whether BRD4 plays a similar role in nucleus pulposus (NP) cells and participates in the pathogenesis of intervertebral disc degeneration. The present study aims to observe whether BRD4 inhibition regulates matrix metabolism by controlling autophagy and NLRP3 inflammasome activity. Besides, the relationship was investigated among nuclear factor κB (NF-κB) signaling, autophagy and NLRP3 inflammasome in NP cells. Here, real-time polymerase chain reaction, western blot analysis and adenoviral GFP-LC3 vector transduction in vitro were used, and it was revealed that BRD4 inhibition alleviated the matrix degradation and increased autophagy in the presence or absence of tumor necrosis factor α. Moreover, p65 knockdown or treatment with JQ1 and Bay11-7082 demonstrated that BRD4 inhibition attenuated NLRP3 inflammasome activity through NF-κB signaling, while autophagy inhibition by bafilomycin A1 promoted matrix degradation and NLRP3 inflammasome activity in NP cells. In addition, analysis of BRD4 messenger RNA expression in human NP tissues further verified the destructive function of BRD4. Simply, BRD4 inhibition alleviates matrix degradation by enhancing autophagy and suppressing NLRP3 inflammasome activity through NF-κB signaling in NP cells.

Xylosyltransferase-1 expression is refractory to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1ß in nucleus pulposus cells: novel regulation by AP-1, Sp1, and Sp3.

We investigated whether expression of xylosyltransferase-1 (XT-1), a key enzyme in glycosaminoglycan biosynthesis, is responsive to disk degeneration and to inhibition by the inflammatory cytokines tumor necrosis factor α and IL-1ß in nucleus pulposus (NP) cells. Analysis of human NP tissues showed that XT-1 expression is unaffected by degeneration severity; XT-1 and Jun, Fos, and Sp1 mRNA were positively correlated. Cytokines failed to inhibit XT-1 promoter activity and expression. However, cytokines decreased activity of XT-1 promoters containing deletion and mutation of the -730/-723 bp AP-1 motif, prompting us to investigate the role of AP-1 and Sp1/Sp3 in the regulation of XT-1 in healthy NP cells. Overexpression and suppression of AP-1 modulated XT-1 promoter activity. Likewise, treatment with the Sp1 inhibitors WP631 and mithramycin A or cotransfection with the plasmid DN-Sp1 decreased XT-1 promoter activity. Inhibitors of AP-1 and Sp1 and stable knockdown of Sp1 and Sp3 resulted in decreased XT-1 expression in NP cells. Genomic chromatin immunoprecipitation analysis showed AP-1 binding to motifs located at -730/-723 bp and -684/-677 bp and Sp1 binding to -227/-217 bp and -124/-114 bp in XT-1 promoter. These results suggest that XT-1 expression is refractory to the disease process and to inhibition by inflammatory cytokines and that signaling through AP-1, Sp1, and Sp3 is important in the maintenance of XT-1 levels in NP cells.

Forelimb contractures and abnormal tendon collagen fibrillogenesis in fibulin-4 null mice.

Fibulin-4 is an extracellular matrix glycoprotein essential for elastic fiber formation. Mice deficient in fibulin-4 die perinatally because of severe pulmonary and vascular defects associated with the lack of intact elastic fibers. Patients with fibulin-4 mutations demonstrate similar defects, and a significant number die shortly after birth or in early childhood from cardiopulmonary failure. The patients also demonstrate skeletal and other systemic connective tissue abnormalities, including joint laxity and flexion contractures of the wrist. A fibulin-4 null mouse strain was generated and used to analyze the roles of fibulin-4 in tendon fibrillogenesis. This mouse model displayed bilateral forelimb contractures, in addition to pulmonary and cardiovascular defects. The forelimb and hindlimb tendons exhibited disruption in collagen fibrillogenesis in the absence of fibulin-4 as analyzed by transmission electron microscopy. Fewer fibrils were assembled, and fibrils were disorganized compared with wild-type controls. The organization of developing tenocytes and compartmentalization of the extracellular space was also disrupted. Fibulin-4 was co-localized with fibrillin-1 and fibrillin-2 in limb tendons by using immunofluorescence microscopy. Thus, fibulin-4 seems to play a role in regulating tendon collagen fibrillogenesis, in addition to its essential function in elastogenesis.

FIH-1-Mint3 axis does not control HIF-1 transcriptional activity in nucleus pulposus cells.

The objective of this study was to determine the role of FIH-1 in regulating HIF-1 activity in the nucleus pulposus (NP) cells and the control of this regulation by binding and sequestration of FIH-1 by Mint3. FIH-1 and Mint3 were both expressed in the NP and were shown to strongly co-localize within the cell nucleus. Although both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxiasensitive. Overexpression of FIH-1 was able to reduce HIF-1 function, as seen by changes in activities of hypoxia response element-luciferase reporter and HIF-1-C-TAD and HIF-2-TAD. Moreover, co-transfection of either full-length Mint3 or the N terminus of Mint3 abrogated FIH-1-dependent reduction in HIF-1 activity under both normoxia and hypoxia. Nuclear levels of FIH-1 and Mint3 decreased in hypoxia, and the use of specific nuclear import and export inhibitors clearly showed that cellular compartmentalization of overexpressed FIH-1 was critical for its regulation of HIF-1 activity in NP cells. Interestingly, microarray results after stable silencing of FIH-1 showed no significant changes in transcripts of classical HIF-1 target genes. However, expression of several other transcripts, including those of the Notch pathway, changed in FIH-1-silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF-1-TAD activity by exogenous FIH-1. Taken together, these results suggest that, possibly due to low endogenous levels and/or preferential association with substrates such as Notch, FIH-1 activity does not represent a major mechanism by which NP cells control HIF-1-dependent transcription, a testament to their adaptation to a unique hypoxic niche.

CCN2 suppresses catabolic effects of interleukin-1ß through α5ß1 and αVß3 integrins in nucleus pulposus cells: implications in intervertebral disc degeneration.

The objective of the study was to examine the regulation of CCN2 by inflammatory cytokines, IL-1ß, and TNF-α and to determine whether CCN2 modulates IL-1ß-dependent catabolic gene expression in nucleus pulposus (NP) cells. IL-1ß and TNF-α suppress CCN2 mRNA and protein expression in an NF-κB-dependent but MAPK-independent manner. The conserved κB sites located at -93/-86 and -546/-537 bp in the CCN2 promoter mediated this suppression. On the other hand, treatment of NP cells with IL-1ß in combination with CCN2 suppressed the inductive effect of IL-1ß on catabolic genes, including MMP-3, ADAMTS-5, syndecan 4, and prolyl hydroxylase 3. Likewise, silencing of CCN2 in human NP cells resulted in elevated basal expression of several catabolic genes and inflammatory cytokines like IL-6, IL-4, and IL-12 as measured by gene expression and cytokine protein array, respectively. Interestingly, the suppressive effect of CCN2 on IL-1ß was independent of modulation of NF-κB signaling. Using disintegrins, echistatin, and VLO4, peptide inhibitors to αvß3 and α5ß1 integrins, we showed that CCN2 binding to both integrins was required for the inhibition of IL-1ß-induced catabolic gene expression. It is noteworthy that analysis of human tissues showed a trend of altered expression of these integrins during degeneration. Taken together, these results suggest that CCN2 and inflammatory cytokines form a functional negative feedback loop in NP cells that may be important in the pathogenesis of disc disease.

COL6A3 protein deficiency in mice leads to muscle and tendon defects similar to human collagen VI congenital muscular dystrophy.

Collagen VI is a ubiquitously expressed extracellular microfibrillar protein. Its most common molecular form is composed of the α1(VI), α2(VI), and α3(VI) collagen α chains encoded by the COL6A1, COL6A2, and COL6A3 genes, respectively. Mutations in any of the three collagen VI genes cause congenital muscular dystrophy types Bethlem and Ullrich as well as intermediate phenotypes characterized by muscle weakness and connective tissue abnormalities. The α3(VI) collagen α chain has much larger N- and C-globular domains than the other two chains. Its most C-terminal domain can be cleaved off after assembly into microfibrils, and the cleavage product has been implicated in tumor angiogenesis and progression. Here we characterize a Col6a3 mutant mouse that expresses a very low level of a non-functional α3(VI) collagen chain. The mutant mice are deficient in extracellular collagen VI microfibrils and exhibit myopathic features, including decreased muscle mass and contractile force. Ultrastructurally abnormal collagen fibrils were observed in tendon, but not cornea, of the mutant mice, indicating a distinct tissue-specific effect of collagen VI on collagen I fibrillogenesis. Overall, the mice lacking normal α3(VI) collagen chains displayed mild musculoskeletal phenotypes similar to mice deficient in the α1(VI) collagen α chain, suggesting that the cleavage product of the α3(VI) collagen does not elicit essential functions in normal growth and development. The Col6a3 mouse mutant lacking functional α3(VI) collagen chains thus serves as an animal model for COL6A3-related muscular dystrophy.

The Inhibitory Effect of NSAIDs and Opioids on Spinal Fusion: An Animal Model.

STUDY DESIGN: Translational research. OBJECTIVE: To evaluate the relative effects of NSAIDs, opioids, and a combination of the two on spinal fusion inhibition in a rodent model. SUMMARY OF BACKGROUND DATA: Nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids are common postoperative analgesic agents. Since NSAIDs inhibit the cyclooxygenase (COX) pathway, they are seldom prescribed following spinal fusion. Opioids may be given instead, but recent evidence suggests opioids also adversely affect spinal fusion quality and success. METHODS: Eighty male Sprague-Dawley rats underwent L4-5 posterior lumbar fusion and were given one of the following analgesia regimens: saline, morphine (6 mg/kg), ketorolac (4 mg/kg), or morphine (3 mg/kg) and ketorolac (2 mg/kg). Serum samples were drawn to evaluate systemic pro-osteoblastic cytokines and vascular endothelial growth factor-A (VEGF-A) levels, which were measured through enzyme-linked immunosorbent assays (ELISA). After six weeks, the rats were sacrificed, and the operated spinal segments underwent manual palpation, microCT, and histologic analysis. RESULTS: Manual palpation scores were significantly diminished in the opioid, NSAID, and multimodal groups when compared with control ( P <0.001). MicroCT fusion scores ( P <0.001) and fusion rates (control: 75% vs . NSAID: 35% vs . opioid: 0% vs . combination: 15%, P <0.001) were significantly diminished in the treatment groups. The bone volume (BV) to tissue volume (TV) ratio (BV/TV) ( P <0.001) and bone mineral density (BMD) ( P <0.001) were all lower in the treatment groups, with the opioid and combined groups having the lowest BMD. Although statistically insignificant ( P <0.09), the concentration of VEGF-A was greater in the control group compared with opioids, NSAIDs, and the combined group. CONCLUSION: Opioids and NSAIDs, both independently and combined, inhibited spinal fusion and caused inferior bony callus. Administration of opioids resulted in the lowest rate of spinal fusion. We propose this may be due to the inhibition of VEGF-A, which limits angiogenesis to the burgeoning fusion mass.

Circulating microRNAs May Be Predictive of Degenerative Cervical Myelopathy.

STUDY DESIGN: Basic Science. OBJECTIVE: The objective of this study was to identify a unique serum profile of circulating miRNAs and inflammatory markers in patients with degenerative cervical myelopathy (DCM) compared to healthy controls (HC). SUMMARY OF BACKGROUND DATA: Currently, DCM is diagnosed with a combination of history, physical examination, and close correlation to advanced imaging. To date, no serum marker has been identified to be diagnostic of this condition. METHODS: Whole venous blood was collected from patients with DCM as well as healthy age- and sex-matched controls. miRNA was extracted from venous blood and a screening analysis was initially conducted to identify miRNA dysregulation in DCM patients. RT-qPCR was used to analyze the expression of 2 specific miRNAs based on screening analysis and literature review. Bioinformatics analysis was used to identify gene networks and potential targets of the miRNA. In addition, the serum inflammatory profile of DCM and HC groups was differentiated using a pro-inflammatory panel. RESULTS: Thirty-six patients were enrolled in the DCM group (36.1% male, 61.5±9.5 y) while 35 patients were enrolled in the HC group (31.4% male, 57.5±8.9 y). Of the 15 total miRNAs differentially expressed between DCM and HC groups, two were selected for further analysis: miR-223-3p (upregulated) and miR-451a (downregulated). Functional gene network analysis revealed the highest-ranking gene network was involved in neurological disease, while the most overexpressed miRNA in this network (miR-233-3p) was noted to have over 100 targets including CDKN1B and the insulin receptor. Serum cytokine analysis showed significant upregulation of several pro-inflammatory cytokines in the DCM cohort compared to the HC group. CONCLUSION: DCM patients demonstrated a set of unique circulating miRNAs in addition to a different serum inflammatory profile compared to HC. These miRNAs may potentially serve as targets for future therapeutic intervention or diagnostic/prognostic testing.

Expression of prolyl hydroxylases (PHDs) is selectively controlled by HIF-1 and HIF-2 proteins in nucleus pulposus cells of the intervertebral disc: distinct roles of PHD2 and PHD3 proteins in controlling HIF-1α activity in hypoxia.

Adaptive response to hypoxia in nucleus pulposus cells of the intervertebral disc is regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Moreover, oxygen-dependent turnover of HIF-1α in these cells is controlled by the prolyl-4-hydroxylase domain (PHD) family of proteins. Whether HIF homologues control expression of PHDs and whether PHDs control hypoxia-inducible factor (HIF) turnover and/or activity under hypoxia is not known. Here, we show that in nucleus pulposus cells, hypoxia robustly induces PHD3 expression and, to a lesser extent, of PHD2 and PHD1. Reporter analysis shows that the hypoxic induction of the PHD2 promoter is HIF-1α dependent, whereas PHD3 promoter/enhancer activity is dependent on both HIF-1α and HIF-2α. Lentiviral delivery of HIF-1α, ShHIF-1α, and ShHIF-1ß confirmed these observations. Noteworthy, HIF-1α maintains basal expression of PHD1 in hypoxia at the posttranscriptional level. Finally, loss of function studies using lentiviral transduction of ShPHDs clearly shows that even at 1% O(2), PHD2 selectively degrades HIF-1α. In contrast, in hypoxia, PHD3 enhances HIF-1α transcriptional activity without affecting protein levels. To correlate these observations with disc disease, a condition characterized by tissue vascularization, we analyzed human tissues. Increased PHD1 mRNA expression but decreased PHD2 and PHD3 expression is observed in degenerate tissues. Interestingly, the hypoxic responsiveness of all the PHDs is maintained in isolated nucleus pulposus cells regardless of the disease state. We propose that PHD2 and PHD3 can be used as a biomarker of tissue oxygenation in the disc and that, as such, it may have important clinical implications.

Varenicline mitigates the increased risk of pseudarthrosis associated with nicotine.

BACKGROUND CONTEXT: High serum nicotine levels increase the risk of nonunion after spinal fusion. Varenicline, a pharmaceutical adjunct for smoking cessation, is a partial agonist designed to displace and outcompete nicotine at its receptor binding site, thereby limiting downstream activation. Given its mechanism, varenicline may have therapeutic benefits in mitigating nonunion for active smokers undergoing spinal fusion. PURPOSE: To compare fusion rate and fusion mass characteristics between cohorts receiving nicotine, varenicline, or concurrent nicotine and varenicline after lumbar fusion. STUDY DESIGN: Rodent noninstrumented spinal fusion model. METHODS: Sixty eight-week-old male Sprague-Dawley rats weighing approximately 300 grams underwent L4-5 posterolateral fusion (PLF) surgery. Four experimental groups (control: C, nicotine: N, varenicline: V, and combined: NV [nicotine and varenicline]) were included for analysis. Treatment groups received nicotine, varenicline, or a combination of nicotine and varenicline delivered through subcutaneous osmotic pumps beginning two weeks before surgery until the time of sacrifice at age 14 weeks. Manual palpation testing, microCT imaging, bone histomorphometry, and biomechanical testing were performed on harvested spinal fusion segments. RESULTS: Control (p=0.016) and combined (p=0.032) groups, when compared directly to the nicotine group, demonstrated significantly greater manual palpation scores. The fusion rate in the control (93.3%) and combined (93.3%) groups were significantly greater than that of the nicotine group (33.3%) (p=0.007, both). Biomechanical testing demonstrated greater Young's modulus of the fusion segment in the control (17.1 MPa) and combined groups (34.5 MPa) compared to the nicotine group (8.07 MPa) (p<0.001, both). MicroCT analysis demonstrated greater bone volume fraction (C:0.35 vs N:0.26 vs NV:0.33) (p<0.001, all) and bone mineral density (C:335 vs N:262 vs NV:328 mg Ha/cm3) (p<0.001, all) in the control and combined groups compared to the nicotine group. Histomorphometry demonstrated a greater mineral apposition rate in the combined group compared to the nicotine group (0.34 vs 0.24 µm/day, p=0.025). CONCLUSION: In a rodent spinal fusion model, varenicline mitigates the adverse effects of high nicotine serum levels on the rate and quality of spinal fusion. CLINICAL SIGNIFICANCE: These findings have the potential to significantly impact clinical practice guidelines and the use of pharmacotherapy for active nicotine users undergoing fusion surgery.

Inhibition of Neurogenic Inflammatory Pathways Associated with the Reduction in Discogenic Back Pain.

STUDY DESIGN: Retrospective cohort study. PURPOSE: This study aimed to determine whether the initiation of anti-calcitonin gene-related peptide (CGRP inhibitor) medication therapy for migraines was also associated with improvements in back/neck pain, mobility, and function in a patient population with comorbid degenerative spinal disease and migraine. OVERVIEW OF LITERATURE: CGRP upregulates pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, brain-derived neurotrophic factor, and nerve growth factor in spinal spondylotic disease, which results in disc degeneration and sensitization of nociceptive neurons. Although CGRP inhibitors can quell neurogenic inflammation in migraines, their off-site efficacy as a therapeutic target for discogenic back/neck pain conditions remains unknown. METHODS: All adult patients diagnosed with spinal spondylosis and migraine treated with CGRP inhibitors at a single academic institution between 2017 and 2020 were retrospectively identified. Patient demographic and medical data, follow-up duration, migraine severity and frequency, spinal pain, functional status, and mobility before and after the administration of CGRP inhibitors were collected. Paired univariate analysis was conducted to determine significant changes in spinal pain, headache severity, and headache frequency before and after the administration of CGRP inhibitors. The correlation between changes in the spinal pain score and functional or mobility improvement was assessed with Spearman's rho. RESULTS: In total, 56 patients were included. The mean follow-up time after the administration of CGRP inhibitors was 123 days for spinal pain visits and 129 days for migraine visits. Back/neck pain decreased significantly (p <0.001) from 6.30 to 4.36 after starting CGRP inhibitor therapy for migraine control. As recorded in the spine follow-up notes, 25% of patients experienced a functional improvement in the activities of daily living, and 17.5% experienced mobility improvement while taking CGRP inhibitors. Change in back/ neck pain moderately correlated (ρ=-0.430) with functional improvement but was not correlated with mobility improvement (ρ=-0.052). CONCLUSIONS: Patients taking CGRP inhibitors for chronic migraines with comorbid degenerative spinal conditions experienced significant off-target reduction of back/neck pain.

Loss of fibulin-2 protects against progressive ventricular dysfunction after myocardial infarction.

Remodeling of the cardiac extracellular matrix (ECM) is an integral part of wound healing and ventricular adaptation after myocardial infarction (MI), but the underlying mechanisms remain incompletely understood. Fibulin-2 is an ECM protein upregulated during cardiac development and skin wound healing, yet mice lacking fibulin-2 do not display any identifiable phenotypic abnormalities. To investigate the effects of fibulin-2 deficiency on ECM remodeling after MI, we induced experimental MI by permanent coronary artery ligation in both fibulin-2 null and wild-type mice. Fibulin-2 expression was up-regulated at the infarct border zone of the wild-type mice. Acute myocardial tissue responses after MI, including inflammatory cell infiltration and ECM protein synthesis and deposition in the infarct border zone, were markedly attenuated in the fibulin-2 null mice. However, the fibulin-2 null mice had significantly better survival rate after MI compared to the wild-type mice as a result of less frequent cardiac rupture and preserved left ventricular function. Up-regulation of TGF-ß signaling and ECM remodeling after MI were attenuated in both ischemic and non-ischemic myocardium of the fibulin-2 null mice compared to the wild type counterparts. Increase in TGF-ß signaling in response to angiotensin II was also lessened in cardiac fibroblasts isolated from the fibulin-2 null mice. The studies provide the first evidence that absence of fibulin-2 results in decreased up-regulation of TGF-ß signaling after MI and protects against ventricular dysfunction, suggesting that fibulin-2 may be a potential therapeutic target for attenuating the progression of ventricular remodeling.

TNF-α and IL-1ß promote a disintegrin-like and metalloprotease with thrombospondin type I motif-5-mediated aggrecan degradation through syndecan-4 in intervertebral disc.

Elevated levels of TNF-α, IL-1ß and a resultant increase in ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type I motifs) expression is seen during disc degeneration. However, if these pro-inflammatory cytokines control ADAMTS activity is not definitively known. The goal of the investigation was to study if TNF-α and IL-1ß regulate syndecan-4 (SDC4) expression, and if SDC4 was responsible for promoting aggrecan degradation through controlling ADAMTS activity in nucleus pulposus cells of the intervertebral disc. Cytokine treatment increased SDC4 expression and promoter activity. Use of inhibitor, SM7368 and co-transfections with IκBα, RelA/p50 showed that NF-κΒ regulated both basal and cytokine-dependent SDC4 transcription. SDC4 promoter harboring RelA binding site mutation was unresponsive to the cytokines. Moreover, cytokines failed to increase SDC4 promoter activity in RelA-null cells. Cytokines increased ADAMTS-4/5 expression and aggrecan degradation and promoted SDC4 interaction with ADAMTS-5. Treatment with heparinase-III and p-nitrophenyl-ß-D-xylopyranoside (PNPX), an inhibitor of heparan sulfate synthesis and transfection with SDC4-shRNA partially blocked cytokine mediated aggrecan degradation. Analysis of human tissues showed increased aggrecan degradation with a concomitant increase in SDC4 and ADAMTS-5 protein expression with severity of disc disease. Likewise, SDC4, TNF-α, IL-1ß, ADAMTS-4, and ADAMTS-5 mRNA expression increased in degenerate tissues. We conclude that in nucleus pulposus, TNF-α and IL-1ß regulate SDC4 expression, which plays a key role in pathogenesis of degenerative disc disease by promoting aggrecan degradation by ADAMTS-5.

Hypoxia activates the notch signaling pathway in cells of the intervertebral disc: implications in degenerative disc disease.

OBJECTIVE: To investigate whether hypoxia regulates Notch signaling, and whether Notch plays a role in intervertebral disc cell proliferation. METHODS: Reverse transcription-polymerase chain reaction and Western blotting were used to measure expression of Notch signaling components in intervertebral disc tissue from mature rats and from human discs. Transfections were performed to determine the effects of hypoxia and Notch on target gene activity. RESULTS: Cells of the nucleus pulposus and annulus fibrosus of rat disc tissue expressed components of the Notch signaling pathway. Expression of Notch-2 was higher than that of the other Notch receptors in both the nucleus pulposus and annulus fibrosus. In both tissues, hypoxia increased Notch1 and Notch4 messenger RNA (mRNA) expression. In the annulus fibrosus, mRNA expression of the Notch ligand Jagged1 was induced by hypoxia, while Jagged2 mRNA expression was highly sensitive to hypoxia in both tissues. A Notch signaling inhibitor, L685458, blocked hypoxic induction of the activity of the Notch-responsive luciferase reporters 12xCSL and CBF1. Expression of the Notch target gene Hes1 was induced by hypoxia, while coexpression with the Notch-intracellular domain increased Hes1 promoter activity. Moreover, inhibition of Notch signaling blocked disc cell proliferation. Analysis of human disc tissue showed that there was increased expression of Notch signaling proteins in degenerated discs. CONCLUSION: In intervertebral disc cells, hypoxia promotes expression of Notch signaling proteins. Notch signaling is an important process in the maintenance of disc cell proliferation, and thus offers a therapeutic target for the restoration of cell numbers during degenerative disc disease.

Do Inflammatory Cytokines Affect Patient Outcomes After ACDF?

STUDY DESIGN: Prospective cohort study. OBJECTIVE: The aim was to determine the relationship between serum inflammatory mediators, preoperative cervical spine disease severity, and clinical outcomes after anterior cervical discectomy and fusion (ACDF). SUMMARY OF BACKGROUND DATA: Given the role of the inflammatory cascade in spinal degenerative disease, it has been hypothesized that inflammatory markers may serve as a predictor of patient outcomes after surgery. MATERIALS AND METHODS: All patients over age 18 who underwent ACDF for cervical spondylosis with associated radiculopathy and/or myelopathy between 2015 and 2017 from a single institution were prospectively recruited. Preoperative serum inflammatory markers including interleukin (IL)-6, IL-8, tumor necrosis factor-α, high-mobility group box-1 (HMGB1), and white blood cells were measured and correlated to patient demographics, surgical characteristics, duration of symptoms, previous opioid use, and preoperative and 1-year postoperative patient-reported outcomes measures (PROMs) including the neck disability index (NDI), visual analog scale neck pain, visual analog scale arm pain, and Physical and Mental Component Scores of the Short Form-12 (PCS and MCS, respectively) using spearman's rho coefficient. RESULTS: A total of 77 patients were enrolled with follow-up PROMs available for 62% (n=48) of patients at a minimum of 1-year after ACDF. The absolute concentrations of IL-6 and tumor necrosis factor-α were found to be weakly correlated with one another (ρ=0.479). Preoperative symptoms lasting <1-year were weakly correlated with elevation in HMGB1 (ρ=0.421). All other patient demographics exhibited negligible correlation with the preoperative inflammatory markers. Lower preoperative PCS (ρ=0.355) and higher preoperative NDI (ρ=0.336) were weakly correlated with elevated HMGB1. Lower MCS (ρ=0.395) and higher NDI (ρ=0.317) preoperatively were weakly correlated with elevated white blood cells. Postoperative improvement in MCS (ρ=0.306) and MCS recovery ratio (ρ=0.321) exhibited a weakly positive correlation with IL-6. CONCLUSION: Preoperative cytokine levels demonstrated minimal correlation with preoperative symptoms or clinical improvement, suggesting that profiling of patient cytokines has limited utility in predicting outcomes after ACDF. LEVEL OF EVIDENCE: Level III.

Recessive COL6A2 C-globular missense mutations in Ullrich congenital muscular dystrophy: role of the C2a splice variant.

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.

Hypoxia-inducible factor regulation of ANK expression in nucleus pulposus cells: possible implications in controlling dystrophic mineralization in the intervertebral disc.

OBJECTIVE: Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins. METHODS: Quantitative reverse transcription-polymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity. RESULTS: ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1α and HIF-2α resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1ß-null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1α and HIF-2α in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxia-responsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state. CONCLUSION: Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.

Regulation of CCN2/connective tissue growth factor expression in the nucleus pulposus of the intervertebral disc: role of Smad and activator protein 1 signaling.

OBJECTIVE: To investigate transforming growth factor beta (TGFbeta) regulation of connective tissue growth factor (CTGF) expression in cells of the nucleus pulposus of rats, mice, and humans. METHODS: Real-time reverse transcription-polymerase chain reaction and Western blot analyses were used to measure CTGF expression in the nucleus pulposus. Transfections were used to measure the effects of Smads 2, 3, and 7 and activator protein 1 (AP-1) on TGFbeta-mediated CTGF promoter activity. RESULTS: CTGF expression was lower in neonatal rat discs than in skeletally mature rat discs. An increase in CTGF expression and promoter activity was observed in rat nucleus pulposus cells after TGFbeta treatment. Deletion analysis indicated that promoter constructs lacking Smad and AP-1 motifs were unresponsive to treatment. Analysis showed that full-length Smad3 and the Smad3 MH-2 domain alone increased CTGF activity. Further evidence of Smad3 and AP-1 involvement was seen when DN-Smad3, SiRNA-Smad3, Smad7, and DN-AP-1 suppressed TGFbeta-mediated activation of the CTGF promoter. When either Smad3 or AP-1 sites were mutated, CTGF promoter induction by TGFbeta was suppressed. We also observed a decrease in the expression of CTGF in discs from Smad3-null mice as compared with those from wild-type mice. Analysis of human nucleus pulposus samples indicated a trend toward increasing CTGF and TGFbeta expression in the degenerated state. CONCLUSION: TGFbeta, through Smad3 and AP-1, serves as a positive regulator of CTGF expression in the nucleus pulposus. We propose that CTGF is a part of the limited reparative response of the degenerated disc.

Functional Deficits in Mice Expressing Human Interleukin 8.

We showed previously that inflammatory mediators, including IL8, in intervertebral disc tissues from patients with discogenic back pain may play a key role in back pain. To investigate the molecular mechanism of IL8 signaling in back pain, we generated a mouse model that conditionally expresses human (h) IL8. We hypothesized that hIL8 levels affect mouse activity and function. Briefly, hIL8 cDNA was inserted into the pCALL2 plasmid, linearized, and injected into mouse embryos. Resulting pCALL2-hIL8 mice were then bred with GDF5-Cre mice to express the transgene in cartilage and intervertebral disc (IVD) tissues. Functional capacities including nest-making and other natural behaviors were measured. Both male and female mice expressing hIL8 showed lower nesting scores than did littermates that did not express hIL8 (n = 14 to 16 per group). At 28 wk of age, mice expressing hIL8 (n = 35) spent more time immobile and eating during each night than littermate controls (n = 33). Furthermore, hIL8-expressing mice traveled shorter distances and at a lower average speed than littermate controls. Thus, in an initial effort to investigate the relationship between this chemokine and mouse behavior, we have documented changes in normal activities in mice conditionally expressing hIL8.

Correlation of Early Outcomes and Intradiscal Interleukin-6 Expression in Lumbar Fusion Patients.

OBJECTIVE: To determine if there is correlation between intradiscal levels of interleukin-6 (IL-6) and early outcome measures in patients undergoing lumbar fusion for painful disc degeneration. METHODS: Intervertebral disc tissue was separated into annulus fibrosus/nucleus pulposus and cultured separately in vitro in serum-free medium (Opti-MEM). Conditioned media was collected after 48 hours. The concentration of IL-6 was quantified using enzyme-linked immunosorbent assay. Pearson correlation coefficients quantified relationships between IL-6 levels and pre- and postoperative visual analogue scale (VAS) back pain and Oswestry Disability Index (ODI), as well as change in VAS/ODI. RESULTS: Sixteen discs were harvested from 9 patients undergoing anterior lumbar interbody fusion (mean age, 47.4 years; range, 21-70 years). Mean preoperative and 6-month postoperative VAS were 8.1 and 3.7, respectively. Mean preoperative and postoperative ODI were 56.2 and 25.6, respectively. There were significant positive correlations between IL-6 expression and postoperative VAS (ρ = 0.38, p = 0.048) and ODI (ρ = 0.44, p = 0.02). No significant correlations were found between intradiscal IL-6 expression and preoperative VAS (ρ = -0.12, p = 0.54). Trends were seen associating IL-6 expression and change in VAS/ODI (ρ = -0.35 p = 0.067; ρ = -0.34, p = 0.08, respectively). A trend associated IL-6 and preoperative ODI (ρ = 0.36, p = 0.063). CONCLUSION: The direct association between IL-6 expression and VAS/ODI suggests patients with elevated intradiscal cytokine expression may have worse early outcomes than those with lower expression of IL-6 after surgery for symptomatic disc degeneration.