Pro-ferroptotic signaling promotes arterial aging via vascular smooth muscle cell senescence.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging-related cardiovascular diseases by promoting arterial remodelling and stiffness. Ferroptosis is a novel type of regulated cell death associated with lipid oxidation. Here, we show that pro-ferroptosis signaling drives VSMCs senescence to accelerate vascular NAD+ loss, remodelling and aging. Pro-ferroptotic signaling is triggered in senescent VSMCs and arteries of aged mice. Furthermore, the activation of pro-ferroptotic signaling in VSMCs not only induces NAD+ loss and senescence but also promotes the release of a pro-senescent secretome. Pharmacological or genetic inhibition of pro-ferroptosis signaling, ameliorates VSMCs senescence, reduces vascular stiffness and retards the progression of abdominal aortic aneurysm in mice. Mechanistically, we revealed that inhibition of pro-ferroptotic signaling facilitates the nuclear-cytoplasmic shuttling of proliferator-activated receptor-γ and, thereby impeding nuclear receptor coactivator 4-ferrtin complex-centric ferritinophagy. Finally, the activated pro-ferroptotic signaling correlates with arterial stiffness in a human proof-of-concept study. These findings have significant implications for future therapeutic strategies aiming to eliminate vascular ferroptosis in senescence- or aging-associated cardiovascular diseases.

The Effect of the NFκB-USP9X-Cx43 Axis on the Dynamic Balance of Bone Formation/Degradation during Ossification of the Posterior Longitudinal Ligament of the Cervical Spine.

Connexin 43- (Cx43-) mediated nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling has been found involved in the ossification of the posterior longitudinal ligament (OPLL). However, the underlying mechanism how OPLL is regulated has not been elucidated. In the present study, primary ligament fibroblast were isolated; immunoprecipitation (IP) and liquid chromatography-mass spectrometry (LC-MS) assays were applied to identify potential binding proteins of Cx43. Protein interaction was then confirmed by co-IP assay. Alkaline phosphatase (ALP) activity and alizarin red staining were used to evaluate ossification. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were employed to assess the binding between NF-κB p65 and target gene. Lipoxygenase inhibitor (5,8,11-eicosatriynoic acid, EPA) was applied to induce endoplasmic reticulum (ER) stress, and 4-phenylbutyrate (4-PBA) was used as an ER-stress inhibitor. Expression of USP9X, Cx43, and nuclei p65 in ligaments from patients and controls was detected by Western blotting. The results showed that ubiquitin-specific protease 9 X-chromosome (USP9X), a deubiquitylating enzyme, was a candidate of Cx43 binding proteins, and USP9X inhibited Cx43 ubiquitination. In vitro experiments showed that USP9X promoted ossification of primary ligament fibroblasts and nuclear translocation of NF-κB p65 by regulating Cx43 expression. Moreover, NF-κB can bind to the USP9X promoter to promote its transcription. When ER stress was inhibited by 4-PBA, USP9X levels, NF-κB nuclei translocation, and ALP activity were decreased. Reverse results were obtained when ER stress was induced by EPA. PDTC, an NF-κB inhibitor, could abolish the effects of EPA. Furthermore, USP9X, Cx43, and nuclei p65 were significantly upregulated in ligaments from OPLL patients than non-OPLL controls. USP9X was positively correlated with CX43 and nuclei p65 in OPLL samples. Overall, the findings suggest that the ER stress-NFκB-USP9X-Cx43 signaling pathway plays an important role in OPLL progression.

Human umbilical cord mesenchymal stem cells deliver exogenous miR-26a-5p via exosomes to inhibit nucleus pulposus cell pyroptosis through METTL14/NLRP3.

BACKGROUND: Intervertebral disc degeneration (IVDD) is the breakdown of the discs supporting the vertebrae. It is one of the most frequent causes of back pain worldwide. Currently, the clinical interventions for IVDD are mainly focused on symptom releases. Thus, new therapeutic options are needed. METHODS: Nucleus pulposus (NP) samples were obtained from 20 patients experiencing IVDD and 10 healthy volunteers compared for mRNA N6-methyladenosine (m6A) mRNA modification as well as methyltransferase (METT) like METTL3, METTL14, and Wilms' tumor 1-associated protein mRNA and protein abundance following exosomes exposure from mesenchymal stem cells. In addition, microRNA expressions were also compared. The correlation between the NLR family pyrin domain containing 3 (NLRP3) and METTL14 was measured by luciferase reporter assay. Cytokines were evaluated using an enzyme-linked immunosorbent assay. METTL14, NLRP3, and insulin-like growth factor 2 mRNA-binding protein 2 mRNAs were measured via a quantitative reverse transcription-polymerase chain reaction. Protein was assayed using western blots. Cell death was assessed by propidium iodide staining, lactate dehydrogenase release, gasdermin-N domain abundance, and caspase-1 activation. RESULTS: The human umbilical cord mesenchymal stem cell (hucMSC) exosomes were found to effectively improve the viability of NP cells and protect them from pyroptosis through targeting METTL14, with a methyltransferase catalyzing m6A modification. METTL14 was highly present in NP cells from IVDD patients, which stabilize NLRP3 mRNA in an IGFBP2-dependent manner. The elevated NLRP3 levels result in the increase of interleukin 1ß (IL-1ß) and IL-18 levels and trigger pyroptotic NP cell death. Such pathogenic axis could be blocked by hucMSC exosomes, which directly degrade METTL14 through exosomal miR-26a-5p. CONCLUSIONS: The results of the current study revealed the beneficial effects of hucMSC exosomes on NP cells and determined a potential mechanism inducing IVDD.

METTL3 Regulates Ossification of the Posterior Longitudinal Ligament via the lncRNA XIST/miR-302a-3p/USP8 Axis.

The prevalence of ossification of the posterior longitudinal ligament (OPLL) is increasing, and currently there is no effective medical treatment for OPLL. Methyltransferase like 3 (METTL3), one of the components of the N 6-methyladenosine (m6A) methyltransferase complex, regulates gene expression via modification of mRNA. Although METTL3 has been implicated in a variety of diseases, its role in OPLL remains to be elucidated. Primary ligament fibroblasts were used in this study. To investigate the role of METTL3 in OPLL, METTL3 was silenced or overexpressed. m6A RNA methylation was measured by commercially available kits. Luciferase reporter assay was performed to investigate the binding of miR-302a-3p and METTL3, and the binding of miR-302a-3p and USP8. Quantitative RT-PCR and western blots were used to evaluate mRNA and protein expression, respectively. OPLL increases METTL3 and its m6A modification. Overexpressing METTL3 significantly promoted osteogenic differentiation of primary ligament fibroblasts. Mechanism study showed that METTL3 increased m6A methylation of long non-coding RNA (lncRNA) X-inactive specific transcript (XIST). Further study showed that lncRNA XIST regulates osteogenic differentiation of primary ligament fibroblasts via miR-302a-3p, which targets ubiquitin-specific protease 8 (USP8). METTL3 enhanced osteogenic differentiation of primary ligament fibroblasts via the lncRNA XIST/miR-302a-3p/USP8 axis. The findings highlight the importance of METTL3-mediated m6A methylation of XIST in OPLL and provide new insights into therapeutic strategies for OPLL.

The endoplasmic reticulum stress response participates in connexin 43-mediated ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) manifests as ectopic bone formation in spinal ligament tissue. As revealed by in vitro studies, fibroblasts from patients with OPLL or healthy ligament fibroblasts undergo mechanical stress (MS). We previously demonstrated that a cell-cell junction protein, connexin 43 (Cx43), is significantly up-regulated in OPLL cells and previous data indicated that some proteins related to the endoplasmic reticulum (ER) stress response are elevated during the development of OPLL. The present study utilized gain- and loss-of-function tools to delineate the contribution of the ER stress response within ligament fibroblasts under OPLL-inducing stimuli and the crosstalk between Cx43 signaling and the ER stress response. The ER stress process was augmented by the induction of Cx43 expression in OPLL cells or cells under MS. Cx43 over-expression also promoted ER stress and ossification in OPLL cells. Moreover, the activation of ER stress was accompanied with increased oxidative stress, which was inhibited by Cx43 gene silencing. Cx43 knockdown also improved ER stress-related ossification in OPLL cells. The blockage of ER stress using a chemical compound or small interfering RNA was sufficient to overcome MS-induced ossification in OPLL cells. These findings were further validated in patients with OPLL, as the mRNA levels of Cx43 and PKR-like endoplasmic reticulum kinase (a single-pass type I ER membrane protein kinase), a major transducer of ER stress, were significantly increased compared with non-OPLL subjects. In conclusion, this study demonstrates that ER stress participates in Cx43-related OPLL.

Endoplasmic reticulum stress regulates mechanical stress-induced ossification of posterior longitudinal ligament.

PURPOSE: The pathogenesis of ossification of posterior longitudinal ligament (OPLL) is not completely clear. Previous study has confirmed a single-pass type I endoplasmic reticulum (ER) membrane protein kinase (PERK), which is a major transducer of the ER stress, participates in the process of OPLL in vitro. This study aimed to demonstrate the role of ER stress in mechanical stress (MS)-induced OPLL. METHODS: The posterior longitudinal ligaments were collected intraoperatively. The expression of ER stress markers in ligament tissue samples was compared between OPLL and non-OPLL patients in vivo. Ligament fibroblasts were isolated and cultured. Loaded by MS, the expression of ER stress markers in fibroblasts deriving from non-ossified areas of the ligament tissues from OPLL patients was detected. The influence of inhibition of ER stress on MS-induced OPLL and activation of mitogen-activated protein kinase (MAPK) pathways by MS was also investigated. RESULTS: We confirmed the ER stress markers were highly expressed in non-ossified areas of the ligament tissues from OPLL patients but could barely be detected in the ligaments from non-OPLL patients in vivo. We also found ER stress could be activated by MS during the process of OPLL in vitro. Moreover, inhibition of ER stress could hinder MS-induced OPLL and activation of MAPK signaling pathways by MS in vitro. CONCLUSION: Activated ER stress was observed in OPLL patients both in vitro and in vivo. Mechanical stress could activate ER stress response in posterior longitudinal ligament fibroblasts and further promote OPLL in vitro. In this process, ER stress might work through the MAPK signaling pathways. These slides can be retrieved under Electronic Supplementary Material.

Long non-coding RNA MALAT1 functions as miR-1 sponge to regulate Connexin 43-mediated ossification of the posterior longitudinal ligament.

Ossification of the posterior longitudinal ligament (OPLL) is the major cause for several deteriorate bone and joint diseases. Its development is a highly organized dynamic process as modulated by various physiological and pathophysiological factors. Both long non-coding RNAs (lncRNAs) and small non-coding RNAs (miRNAs) have been postulated to involve into almost all the biological conditions. Here, we applied high through-put transcriptome screening to unveil lncRNAs highly regulated under OPLL condition. siRNA assay in combination with western blot and quantitative PCR deciphered the lncRNA and miRNA functions in OPLL and their underlying mechanism. Here we identified an lncRNA, named Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1) engaged into the development of OPLL by indirectly targeting Connexin 43 (Cx43) gene. As previously reported, Cx43 is one of the main proteins contributing to OPLL partially through enhancing inflammatory signaling. On top of that, we provided another regulatory layer that MALAT1 served as the upstream effector governing the transcription of Cx43 gene. Perturbation of MALAT1 significantly inhibited Cx43 expression, inflammation, and osteogenesis. Mechanistically, in silico analysis and experimental validation both confirmed that microRNA-1 (miR-1) was the mediator connecting MALAT1-Cx43 axis: overexpression of miR-1 diminished Cx43 expression and OPLL process; meanwhile, MALAT1 acted as miR-1 sponge to inhibit its suppressive transcription effect on downstream ossification related genes. Knock-down of MALAT1 released sequestered miR-1, which repressed Cx43 expression and associated OPLL. Likewise, induced OPLL caused by overexpression of MALAT1 can be ameliorated by enhanced miR-1 function, knock-down of Cx43 or inhibition of inflammation. More importantly, further validation using patient ligament samples from non-OPLL and OPLL individuals identified MALAT1-miR-1-Cx43 regulatory axis. Collectively, we found a novel mechanism through lncRNA-miRNA interaction that provides more insights into understanding the development of OPLL.

Clinical Features and Long-Term Surgical Outcomes of Patients with Cervical Spondylotic Amyotrophy.

BACKGROUND: Cervical spondylotic amyotrophy (CSA) is not common. The clinical features and long-term surgical outcomes of patients with CSA are also unclear. We sought to summarize clinical features, assess long-term surgical outcomes, and determine the prognostic factors relevant for patients with CSA. METHODS: A total of 136 patients with CSA who underwent anterior or posterior decompression during January 2001 to December 2012 were included. Their clinical and radiologic data were collected. The surgical outcome was evaluated using manual muscle test and improvements in the muscle strength. Correlations between the surgical outcome and various factors also were analyzed. RESULTS: In total, 128 patients underwent anterior decompression and 8 patients underwent posterior decompression. At the final follow-up, the surgical outcome was significantly better after anterior decompression compared with that after posterior decompression. Statistical analyses showed the type of CSA, duration of symptoms, and association with ossification of the posterior longitudinal ligament were associated with a poor outcome after anterior surgery (P < 0.05). CONCLUSIONS: Besides significant muscular atrophy in one upper extremity, CSA also occasionally presents with mild atrophy in the other upper extremity, sensory disturbance in the upper extremities, or hyperflexia in the lower extremities. Anterior decompression is generally effective in the treatment of patients with CSA. Preoperative duration of symptoms, type of CSA, and ossification of the posterior longitudinal ligament are important predictors for the surgical outcome.

Reduction of Slippage Influences Surgical Outcomes of Grade II and III Lumbar Isthmic Spondylolisthesis.

OBJECTIVE: To explore influence of reduction of slippage on radiologic parameters, clinical outcomes, and perioperative complications in treatment of grade II/III lumbar isthmic spondylolisthesis. METHODS: We divided 156 patients with grade II/III spondylolisthesis into 2 groups with preoperative balanced or unbalanced pelvis. We further divided each group into group A with postoperative grade I or less slippage and group B with persistent grade II/III slippage postoperatively. Outcome scores were measured for clinical evaluation. Radiologic parameters included pelvic incidence, sacral slope, pelvic tilt, and lumbar lordosis. RESULTS: In group A patients with preoperative balanced pelvis, lumbar lordosis significantly decreased from 60.2° ± 10.6° to 50.9° ± 9.8° after operation (P < 0.05). In group A patients with preoperative unbalanced pelvis, pelvic tilt decreased from 29.1° ± 8.6° to 24.1° ± 9.1°, and sacral slope increased from 36.1° ± 9.0° to 41.3° ± 8.4°, significantly (P < 0.05). There were significant differences (P < 0.05) between group A and B in postoperative visual analog scale for low back pain (1.5 ± 0.8 vs. 2.1 ± 0.9), Oswestry Disability Index (13.8 ± 8.7 vs. 18.1 ± 7.6), and EuroQol-5 dimensions (0.75 ± 0.14 vs. 0.68 ± 0.11) scores in patients with preoperative unbalanced pelvis. CONCLUSIONS: In patients with grade II/III lumbar isthmic spondylolisthesis, if postoperative slippage was grade I or less, pelvic tilt and sacral slope could be corrected more effectively, and better clinical outcomes would be obtained for cases with preoperative unbalanced pelvis. In cases with balanced pelvis, lumbar lordosis could be better corrected by the same degree of reduction, although clinical outcomes would not be influenced significantly. Perioperative complications would not be influenced by reduction of slippage.

Surgical Treatment for Odontoid Fractures in Patients with Long-Standing Ankylosing Spondylitis: A Report of 3 Cases and Review of the Literature.

BACKGROUND: Ankylosing spondylitis (AS) is classified as a chronic inflammatory seronegative rheumatic arthritis. Patients with AS are more likely to sustain a fracture of the cervical spine compared with the general population. Most fractures occur in the lower cervical spine and manifest at the level of the intervertebral disc. There have been few reports about the surgical treatment for upper cervical spine fractures in patients with AS, especially odontoid fractures. We present 3 cases of odontoid fracture in patients with long-standing AS. METHODS: Odontoid fracture with atlantoaxial displacement was identified on radiologic imaging in 3 patients. In 1 patient, fracture was a missed diagnosis after initial trauma, and the fracture and displacement were discovered 3 months later owing to aggravation of symptoms. Posterior occipitocervical fusion with iliac autograft was performed under general anesthesia in all cases. RESULTS: All 3 patients recovered postoperatively without any complications related to surgery. Cervical radiographs obtained at 12-month follow-up demonstrated healed fracture and replacement of the atlantoaxial joint. CONCLUSIONS: Odontoid fracture with atlantoaxial dislocation in patients with long-standing AS is uncommon. Clinicians must be cautious in assessing such patients with any episode of trauma. Computed tomography and magnetic resonance imaging can be helpful in demonstrating occult odontoid fractures. Posterior occipitocervical fusion with internal fixation may benefit these patients, although at the cost of sacrificing the last motion segment.

A Novel Anterior Revision Surgery for the Treatment of Cervical Ossification of Posterior Longitudinal Ligament: Case Report and Review of the Literature.

PURPOSE: A 62-year-old blind man with severe ossification of the posterior longitudinal ligament was presented. The patient underwent posterior laminectomy and fixation. After surgery, the patient was not satisfied with the recovery of his upper limbs although his Japanese Orthopedic Association (JOA) score had increased from 9 to 12 points. Because the tactile sensation of his hands was especially important to his daily life, the patient asked for further treatment after 6 months. This report describes the novel revision surgery we invented and the outcome in the patient after the surgery. METHODS: We performed the revision surgery using an anterior approach from C3 to C7 with a novel technique, anterior controllable antedisplacement and fusion (ACAF). The patient was followed up for 6 months. The clinical data, including JOA score and radiologic images, were collected and analyzed. RESULTS: After the revision surgery, the patient had improved sensation in both hands, and his JOA score increased to 14 points. Satisfactory decompression was assessed by magnetic resonance imaging after operation. Bone fusion was confirmed by computed tomography 3 months after operation. No specific complications related to this surgery were observed. CONCLUSIONS: The application of such an operative procedure in revision surgery for OPLL has not been reported earlier and might be an alternative choice for patients with an unsatisfactory outcome from previous posterior surgery.

Cervical Artificial Disc Replacement With Discover Prosthesis Does Not Reduce the Midterm Risk of Heterotopic Ossification: Results of a Cohort Study.

STUDY DESIGN: A prospective cohort study with 79 patients. OBJECTIVE: To analyze the midterm incidence of heterotopic ossification (HO) after cervical artificial disc replacement with Discover prosthesis in treatment of cervical degenerative disc disease. MATERIALS AND METHODS: A cohort of 79 patients with 102 Discover prostheses (56 monosegment and 23 bisegments) was evaluated. Clinical and radiographic follow-up was performed. The performance of HO of the operative segments was assessed annually after the operation for 4 years. RESULTS: Early follow-up at 2 years postsurgical treatment showed satisfactory clinical outcomes. No significant changes on the mean Japanese Orthopaedic Association and Visual Analog Scale scores were detected at 3 and 4 years postoperation. The occurrence rate of HO was 5.9% in 1-year follow-up and 17.6% in 2-year follow-up, and then the percentages increased to 49.2% in 3-year follow-up and 65.6% in 4-year follow-up, respectively. No association between the HO grades and the type of the surgical segment was detected by the Fisher exact test (P-value=0.56). No prosthesis subsidence or excursion was identified during the whole follow-up period. CONCLUSIONS: The use of Discover prosthesis resulted in satisfactory clinical outcomes after operation. However, the new prosthesis did not reduce the midterm risk of HO, despite the fact that the observed incidence was relatively low in the early follow-up.

Roles of the Endoplasmic Reticulum Stress Transducer OASIS in Ossification of the Posterior Longitudinal Ligament.

STUDY DESIGN: In vitro molecular research on the posterior longitudinal ligament fibroblasts. OBJECTIVE: To investigate different expression of old astrocyte specifically induced substance (OASIS) between spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament (OPLL) and from non-OPLL patients and demonstrate knockdown of OASIS protein expression by RNA interference inhibiting expression of type I collagen (COL I) in OPLL cells. SUMMARY OF BACKGROUND DATA: OPLL is characterized by ectopic bone formation in spinal ligaments. Some evidence indicates that ligament fibroblasts from OPLL patients have osteogenic characteristics. However, the relevant intracellular signaling pathways remain unclear. METHODS: Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of osteocalcin, alkaline phosphatase, and COL I were detected in OPLL and non-OPLL cells by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of OASIS was detected by Western blotting. And then, after 72 hours, when RNA interference against OASIS was performed in OPLL cells, expression of the osteoblast-specific genes was compared again between the transfection group and the nontransfection group. RESULTS: Spinal ligament fibroblasts were observed 7 to 10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of osteocalcin, alkaline phosphatase, and COL I and protein expressions of OASIS from OPLL cells were significantly greater than those from non-OPLL cells. In addition, knockdown of OASIS protein expression inhibited the mRNA expressions of COL I remarkably in the transfection group compared with the nontransfection group, at 72 hours after RNA interference targeting OASIS was performed in OPLL cells. CONCLUSIONS: The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and OASIS expression plays an important role in the development of OPLL through the expression of COL I.

A meta-analysis of the fusion rate from surgical treatment for odontoid factures: anterior odontoid screw versus posterior C1-C2 arthrodesis.

PURPOSE: Surgical treatment for odontoid fractures is widely performed in practice. However, the choice of different surgical procedures remains controversial. Regardless of the surgical technique, the fusion situation is one of the important factors that affect the clinical efficacy. A discrepancy in fusion rate between the anterior odontoid screw fixation approach and the posterior C1-C2 arthrodesis approach has been suggested in clinical research, yet no consensus has been reached. This meta-analysis aims to synthesize the currently available evidence on the topic. METHODS: Most published comparative studies have limited statistical power to reach a solid conclusion due to the sample size constraint. In this condensed meta-analysis, we focused on the analysis of the reported fusion rates among selected comparative studies. The targeted comparative study design was chosen to control for potential confounding factors. However, the inclusion criteria of comparative studies limited our sample size and we were not able to obtain statistically meaningful sample size for other endpoints. On the other hand, fusion rate serves as an important clinical outcome and is the most commonly reported one from odontoid fracture studies. RESULTS: Results show that the overall fusion rate is lower in anterior group than in posterior group in both fixed effect model (RR = 0.90, 95 % CI 0.82-0.99) and random effect model (RR = 0.90, 95 % CI 0.83-0.97). There was no significant heterogeneity between these studies (p value = 0.5718), and no evident publication bias was detected by the Egger's test (t = -0.3541, df = 6, p value = 0.7354) and funnel plots. In general, age is not statistically associated with the choice of surgical approach (χ(2) = 0.29, df = 1, p value = 0.59) but appears to affect the clinical efficacy. The protective effect of posterior C1-C2 arthrodesis treatment on fusion remains significant in the elderly (≥60 years), but loses its significance in the younger age population (<60 years). CONCLUSIONS: In light of these findings, we concluded that significant higher fusion rates were observed in patients who underwent posterior C1-C2 arthrodesis surgeries compared to those treated with anterior odontoid screw fixation.

Knockdown of galectin-1 suppresses the growth and invasion of osteosarcoma cells through inhibition of the MAPK/ERK pathway.

Galectin-1 (GAL1), a widely expressed ß­galacto-side-binding protein, exerts pleiotropic biological functions. GAL1 has been found to be upregulated in many malignancies; yet the role of GAL1 in the pathophysiology of human osteosarcoma (OS) remains uncertain. The present study was carried out to investigate the expression of GAL1 in human OS tissues and to explore its effects on the growth and invasion of OS cells. OS and corresponding adjacent non-cancerous tissues (ANCT) were collected from 30 consecutive cases. The expression of GAL1 was detected by immunohistochemical assay through tissue microarray procedure. Using small hairpin RNA (shRNA)-mediated GAL1 knockdown (Lv-shGAL1) in OS (MG-63 and U-2 OS) cells, we observed the changes in the malignant phenotype in OS cells in vitro and in vivo. As a consequence, the positive expression of GAL1 was significantly higher in OS tissues than that in the ANCT (63.3 vs. 36.7%, P=0.029), and was positively correlated with distant metastasis in the OS patients (P=0.022). Knockdown of GAL1 suppressed cell proliferative activities and invasive potential and induced apoptosis in OS cells with decreased expression of p38MAPK, p-ERK, Ki-67 and matrix metallopeptidase-9 (MMP-9) and increased expression of caspase-3. In addition, the tumor volume in the MG-63 subcutaneous tumor models treated with Lv-shGAL1 was significantly smaller than that in the negative control (NC) group (P<0.01). Altogether, our findings indicate that high expression of GAL1 is associated with distant metastasis of OS patients, and knockdown of GAL1 inhibits growth and invasion of OS cells possibly through inhibition of the MAPK/ERK pathway, suggesting that GAL1 may represent a potential target for the treatment of cancer.

Clinical and radiographic outcomes of cervical disc replacement with a new prosthesis.

BACKGROUND CONTEXT: Anterior cervical discectomy and interbody fusion was a classical treatment for cervical degenerative disc disease (CDDD). However, the rigid fusion also leads to a reduction in normal cervical spine motion and to increased biomechanical stress at adjacent levels, which in turn accelerates degenerative changes of the discs at these levels. Cervical disc replacement (CDR) is a new technology with the aim of addressing the limitations of fusion procession and preserving motion at the treated level. Discover prosthesis (DePuy Spine, Raynham, MA, USA) is a new type artificial disc and there are few reports about it. PURPOSE: The purpose of this study was to analyze the primary clinical and radiographic outcomes of CDR with Discover prosthesis to treat mono- or bi-segment CDDD in a Chinese population. STUDY DESIGN: The study design was prospective and single-center clinical trial of the Discover prosthesis in the treatment of patients with mono- or bi-segment CDDD. PATIENTS SAMPLE: Seventy-nine patients with 102 Discover prosthesis arthroplasty performed (56 mono-segment and 23 bi-segment) were evaluated. OUTCOME MEASURES: Clinical outcomes based on Japanese Orthopaedic Association (JOA), visual analog scale (VAS) pain score, and Odom's scale and radiographic outcomes including the anterior disc heights (ADH), posterior disc heights (PDH), range of motion, and performance of heterotopic ossification (HO) of the operative segment were assessed. METHODS: Clinical and radiographic follow-up was performed. Preoperative and postoperative ADH, PDH, and range of motion were measured from lateral and flexion-extension radiographs. The paired t test was used to assess the difference of clinical and radiographic outcomes before and after operation. The performance of HO was observed by two independent MD. RESULTS: The mean follow-up time for all the patients was 31.6 months, ranging from 24 to 43 months. Mean preoperative JOA score was 9.5, and VAS overall pain score was 7.2. At 2-, 6-, 12-, and 24-month follow-up, the mean JOA score was 14.1, 14.7, 15.3, and 14.9, whereas the mean VAS overall pain score was 1.9, 1.7, 1.8, and 1.4, respectively. Mean JOA and VAS scores showed statistical improvements in the postoperative period. Seven patients had mild dysphagia within the first month after operation. According to Odom's scale, 52 patients had excellent outcomes, 25 patients had good outcomes, and 2 patients had fair outcomes at 2-year follow-up. The Mean preoperative ADH and PDH of the operative segment were 4.9 mm and 3.1 mm. Compared with preoperative, there were significantly increased and maintenance well at 2- (7.5 mm, 5.1 mm), 6- (7.5 mm, 5.0 mm), 12- (7.4 mm, 4.9 mm) and 24-month (7.2 mm, 5.0 mm) follow-up. Range of motion of the operative segment in the postoperative follow-up was slightly increased than the preoperative follow-up but not statistically significant. Heterotopic ossification was presented in six replaced levels at 1-year follow-up including 4 Grade I and 2 Grade II and 18 replaced levels at the follow-up more than 2 years including 8 grade I and 10 grade II. No prosthesis subsidence or excursion was identified. CONCLUSIONS: The use of Discover prostheses in our study resulted in satisfactory clinical and radiographic outcomes. The prostheses can restore and maintain interbody height, while preserve the motion of the treated segment. Although the results of this study demonstrate initial safety and effectiveness in a Chinese population, we need further studies to know more about the impact of CDR with Discover prosthesis, especially on HO and adjacent segment degeneration.

Upregulated expression of PERK in spinal ligament fibroblasts from the patients with ossification of the posterior longitudinal ligament.

PURPOSE: Molecular mechanism of ossification of the posterior longitudinal ligament (OPLL) remains unclear. This study was to investigate different expressions of PERK between the spinal ligament fibroblasts from OPLL patients and non-OPLL patients, and demonstrate knockdown of PERK protein expression by RNA interference inhibiting expression of osteocalcin (OCN), alkaline phosphatase (ALP), and type I collagen (COL I) in the cells from OPLL patients. METHODS: Spinal ligament cells were cultured using tissue fragment cell culture and identified by immunocytochemistry and immunofluorescence. The mRNA expression of osteoblast-specific genes of OCN, ALP and COL I was detected in the cells from OPLL and non-OPLL patients by semiquantitative reverse transcription-polymerase chain reaction. The protein expression of PERK was detected by Western blotting. And then, after 72 h, when RNA interference against PERK was performed on the cells from OPLL patients, expression of the osteoblast-specific genes was compared again between the transfection group and non-transfection group. RESULTS: Spinal ligament fibroblasts were observed 7-10 days after cell culture. Immunocytochemistry and immunofluorescence exhibited positive results of vimentin staining. The mRNA expressions of OCN, ALP and COL I and protein expression of PERK in the cells from OPLL patients were significantly greater than those from non-OPLL patients. In addition, knockdown of PERK protein expression inhibited the mRNA expressions of OCN, ALP and COL I remarkably in the transfection group compared with the non-transfection group, at 72 h after RNA interference targeting PERK was performed on the cells from OPLL patients. CONCLUSIONS: The cultured fibroblasts from OPLL patients exhibited osteogenic characteristics, and PERK-mediated ER stress might be involved in development of OPLL.

Association between osteoprotegerin genetic variants and osteoporosis in Chinese postmenopausal women.

The objective of this study was to evaluate the association of single nucleotide polymorphisms (SNPs) of osteoprotegerin gene (OPG) with bone mineral density (BMD) and osteoporosis. A total of 338 Chinese postmenopausal women with primary osteoporosis and 367 healthy controls were enrolled. The lumbar spine (L2₋4), total hip and femoral neck hip of BMD were assessed by dual-energy X-ray absorptiometry (DEXA). OPG genetic variants were genotyped through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), created restriction site-PCR (CRS-PCR) and DNA sequencing methods. In this study, the g.18861A>G and g.25548C>T SNPs were detected and our data suggested that the significant differences of spine BMD, femoral neck hip BMD and total hip BMD were found among different g.18861A>G genotype, subjects with the AA genotype were significantly higher than those of AG and GG genotypes (p < 0.05). The g.25548C>T variant was not significantly associated with spine BMD, femoral neck hip BMD and total hip BMD (p > 0.05), while almost reached at the significant level in total hip BMD (p = 0.061). These findings suggeste that OPG gene variants are related to BMD and osteoporosis in Chinese postmenopausal women.

Significant association between cytotoxic T lymphocyte antigen 4 +49G>A polymorphism and risk of malignant bone tumors.

Cytotoxic T lymphocyte antigen 4 (CTLA-4) gene +49G>A polymorphism was implicated to be associated with risk of malignant bone tumors, but the finding was inconclusive owing to the limited sample of a single study. The objective of the current study was to conduct a pooled analysis of four previously published studies to investigate the association between CTLA-4 +49G>A polymorphism and the risk of malignant bone tumors. Data were extracted, and the pooled odds ratio (OR) with the corresponding 95% confidence interval (95% CI) was calculated to assess the association. Those four published studies included a total of 2,165 subjects. The pooled results indicated that CTLA-4 +49G>A polymorphism was significantly associated with risk of malignant bone tumors (AA versus GG: OR = 2.24, 95% CI 1.67-2.99, P < 0.001; AA/GA versus GG: OR = 1.35, 95% CI 1.14-1.61, P = 0.001; AA versus GG/GA: OR = 2.00, 95% CI 1.53-2.62, P < 0.001). Stratified analyses by tumor type showed that CTLA-4 +49G>A polymorphism was associated with risks of both osteosarcoma (AA versus GG: OR = 2.23, 95% CI 1.45-3.43, P < 0.001; AA/GA versus GG: OR = 1.35, 95% CI 1.04-1.75, P = 0.024; AA versus GG/GA: OR = 2.00, 95% CI 1.34-2.98, P = 0.001) and Ewing's sarcoma (AA versus GG: OR = 2.24, 95% CI 1.51-3.31, P < 0.001; AA/GA versus GG: OR = 1.36, 95% CI 1.07-1.72, P = 0.011; AA versus GG/GA: OR = 2.01, 95 % CI 1.39-2.89, P < 0.001). Therefore, results from the current pooled analysis suggest that CTLA-4 +49G>A polymorphism is associated with risk of malignant bone tumors, including osteosarcoma and Ewing's sarcoma.