Wnt5a deficiency in osteocalcin-expressing cells could not alleviate the osteoarthritic phenotype in a mouse model of post-traumatic osteoarthritis.

Objectives: Wnt5a, which regulates the activities of osteoblasts and osteoclasts, is reportedly overexpressed in osteoarthritis (OA) tissues. The purpose of this study was to elucidate its role in the development of OA by deleting Wnt5a in osteocalcin (OCN)-expressing cells. Materials and Methods: Knee OA was induced by anterior cruciate ligament transection (ACLT) in OCN-Cre;Wnt5afl/fl knockout (Wnt5a-cKO) mice and control littermates. Eight weeks after surgery, histological changes, cell apoptosis, and matrix metabolism of cartilage were evaluated by toluidine blue, TUNEL staining, and im-immunohistochemistry analyses, respectively. In addition, the subchondral bone microarchitecture of mice was examined by micro-computed tomography (micro-CT). Results: Histological scores show substantial cartilage degeneration occurred in ACLT knees, coupled with decreased collagen type II expression and enhanced matrix metalloproteinase 13 expression, as well as higher proportions of apoptotic cells. Micro-CT results show that ACLT resulted in decreased bone mineral density, bone volume/trabecular volume, trabecular number, and structure model index of subchondral bones in both Wnt5a-cKO and control littermates; although Wnt5a-cKO mice display lower BMD and BV/TV values, no significant difference was observed between Wnt5a-cKO and control mice for any of these values. Conclusion: Our findings indicate that Wnt5a deficiency in OCN-expressing cells could not prevent an osteoarthritic phenotype in a mouse model of post-traumatic OA.

Vitexin inhibits APEX1 to counteract the flow-induced endothelial inflammation.

Vascular endothelial cells are exposed to shear stresses with disturbed vs. laminar flow patterns, which lead to proinflammatory vs. antiinflammatory phenotypes, respectively. Effective treatment against endothelial inflammation and the consequent atherogenesis requires the identification of new therapeutic molecules and the development of drugs targeting these molecules. Using Connectivity Map, we have identified vitexin, a natural flavonoid, as a compound that evokes the gene-expression changes caused by pulsatile shear, which mimics laminar flow with a clear direction, vs. oscillatory shear (OS), which mimics disturbed flow without a clear direction. Treatment with vitexin suppressed the endothelial inflammation induced by OS or tumor necrosis factor-α. Administration of vitexin to mice subjected to carotid partial ligation blocked the disturbed flow-induced endothelial inflammation and neointimal formation. In hyperlipidemic mice, treatment with vitexin ameliorated atherosclerosis. Using SuperPred, we predicted that apurinic/apyrimidinic endonuclease1 (APEX1) may directly interact with vitexin, and we experimentally verified their physical interactions. OS induced APEX1 nuclear translocation, which was inhibited by vitexin. OS promoted the binding of acetyltransferase p300 to APEX1, leading to its acetylation and nuclear translocation. Functionally, knocking down APEX1 with siRNA reversed the OS-induced proinflammatory phenotype, suggesting that APEX1 promotes inflammation by orchestrating the NF-κB pathway. Animal experiments with the partial ligation model indicated that overexpression of APEX1 negated the action of vitexin against endothelial inflammation, and that endothelial-specific deletion of APEX1 ameliorated atherogenesis. We thus propose targeting APEX1 with vitexin as a potential therapeutic strategy to alleviate atherosclerosis.

MiR-1908/EXO1 and MiR-203a/FOS, regulated by scd1, are associated with fracture risk and bone health in postmenopausal diabetic women.

BACKGROUND: Stearoyl-coenzyme A desaturase-1 (SCD1) can inhibit the development of diabetic bone disease by promoting osteogenesis. In this study, we examined whether this regulation by SCD1 is achieved by regulating the expression of related miRNAs. METHODS: SCD1 expression levels were observed in human bone-marrow mesenchymal stem cells (BM-MSCs) of patients with type 2 diabetes mellitus (T2DM), and the effect of SCD1 on osteogenesis was observed in human adipose-derived MSCs transfected with the SCD1 lentiviral system. We designed a bioinformatics prediction model to select important differentially expressed miRNAs, and established protein-protein interaction and miRNA-mRNA networks. miRNAs and mRNAs were extracted and their differential expression was detected. The SCD1-miRNA-mRNA network was validated. FINDINGS: SCD1 expression in bone marrow was downregulated in patients with T2DM and low-energy fracture, and SCD1 expression promotes BM-MSC osteogenic differentiation. The predictors in the nomogram were seven microRNAs, including hsa-miR-1908 and hsa-miR-203a. SCD1 inhibited the expression of CDKN1A and FOS, but promoted the expression of EXO1 and PLS1. miR-1908 was a regulator of EXO1 expression, and miR-203a was a regulator of FOS expression. INTERPRETATION: The regulation of BM-MSCs by SCD1 is a necessary condition for osteogenesis through the miR-203a/FOS and miR-1908/EXO1 regulatory pathways.

Predicting the risk of sarcopenia in elderly patients with patellar fracture: development and assessment of a new predictive nomogram.

PURPOSE: To develop a risk prediction model for postoperative sarcopenia in elderly patients with patellar fractures in China. PATIENTS AND METHODS: We conducted a community survey of patients aged ≥55 years who underwent surgery for patellar fractures between January 2013 and October 2018, through telephone interviews, community visits, and outpatient follow-up. We established a predictive model for assessing the risk of sarcopenia after patellar fractures. We developed the prediction model by combining multivariate logistic regression analysis with the least absolute shrinkage model and selection operator regression (lasso analysis) as well as the Support Vector Machine (SVM) algorithm. The predictive quality and clinical utility of the predictive model were determined using C-index, calibration plots, and decision curve analysis. We also conducted internal sampling methods for qualitative assessment. RESULT: We recruited 137 participants (53 male; mean age, 65.7 years). Various risk factors were assessed, and low body mass index and advanced age were identified as the most important risk factor (P < 0.05). The prediction rate of the model was good (C-index: 0.88; 95% CI [0.80552-0.95448]), with a satisfactory correction effect. The C index is 0.97 in the validation queue and 0.894 in the entire cohort. Decision curve analysis suggested good clinical practicability. CONCLUSION: Our prediction model shows promise as a cost-effective tool for predicting the risk of postoperative sarcopenia in elderly patients based on the following: advanced age, low body mass index, diabetes, less outdoor exercise, no postoperative rehabilitation, different surgical methods, diabetes, open fracture, and removal of internal fixation.

DNA methyltransferase 1 and Krüppel-like factor 4 axis regulates macrophage inflammation and atherosclerosis.

Macrophage-mediated inflammatory responses occur throughout all stages of atherosclerosis. DNA methylation is one of the critical epigenetic mechanisms and is associated with the development of atherosclerosis. The underlying mechanism of epigenetic regulation of macrophage inflammation (M1 activation) remains unclear. Here we aim to study the role of DNA methyltransferase 1 (DNMT1) in modulating macrophage inflammation and atherosclerosis. DNMT1 expression is up-regulated in THP-1-derived macrophages upon treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Overexpression of DNMT1 promotes the LPS- and IFN-γ-induced M1 activation whereas inhibition of DNMT1 attenuates it. Consistently, DNMT1 expression is elevated in macrophages in atherosclerotic plaques from human and mouse specimens; compared with the Dnmt1wild-type, myeloid Dnmt1 deficiency in mice in an Apolipoprotein E (ApoE) knockout background or receiving AAV-PSCK9 injection and carotid partial ligation results in ameliorated atheroma formation and suppressed plaque inflammation. The promoter regions of atheroprotective Krüppel-like factor 4 (KLF4) are hypermethylated in M1- activated macrophages. DNMT1 down-regulates the expression of KLF4, probably through catalyzing DNA methylation of the promoter regions of KLF4. Gain- and loss-of function study of KLF4 indicates that the DNMT1-mediated macrophage M1 activation is dependent on KLF4. Our data demonstrate a proatherogenic role for DNMT1 as a defining factor in macrophage inflammation both in vitro and in vivo. DNMT1 promotes macrophage M1 activation by suppressing KLF4 expression. Thus macrophage-specific DNMT1 inhibition may provide an attractive therapeutic potential to prevent or reduce atherosclerosis.

Accuracy of Freehand Pedicle Screw Placement in Surgical Correction of Thoracolumbar Kyphosis Secondary to Ankylosing Spondylitis: A Computed Tomography Investigation of 2314 Consecutive Screws.

OBJECTIVE: To evaluate the accuracy and safety of freehand pedicle screw placement in surgical correction for thoracolumbar kyphosis caused by ankylosing spondylitis (AS). METHODS: We retrospectively reviewed 266 consecutive patients with AS who underwent osteotomy for kyphosis correction with freehand screw insertion from January 1998 to April 2015 at our institution. A total of 2314 pedicle screws in 158 patients with AS with postoperative computed tomography scans were included in the study. Postoperative computed tomography was performed to classify accuracy of screws, using the established Gertbein classification (grade 0: no perforation, grade 1: perforation <2 mm, grade 2: perforation between 2 and 4 mm, and grade 3: perforation >4 mm). Patients were divided into 2 groups according to coronal Cobb angle: group A (n = 21, Cobb angle ≥10°), group B (n = 137, Cobb angle <10°). RESULTS: Among the 2314 pedicle screws, 2168 pedicle screw placements were categorized as grade 0, 71 were grade 1, 51 were grade 2, and 24 were grade 3. Breaches occurred more frequently in L1-S1 than the thoracic spine (7.1% and 5.4%, respectively). T5 (25.0%) and S1 (17.7%) experienced the greatest breach rate, whereas T8, L1, and L3 had the lowest breach rate. The breach rate of group A was greater than that of group B (7.9% vs. 6.1%). None of the breaches resulted in either neurologic deficits or vascular complications. CONCLUSIONS: Freehand pedicle screw placement can be performed safely with acceptable breach rate in patients with AS and thoracolumbar kyphosis.

Analogous ß-Carboline Alkaloids Harmaline and Harmine Ameliorate Scopolamine-Induced Cognition Dysfunction by Attenuating Acetylcholinesterase Activity, Oxidative Stress, and Inflammation in Mice.

The analogous ß-carboline alkaloids, harmaline (HAL) and harmine (HAR), possess a variety of biological properties, including acetylcholinesterase (AChE) inhibitory activity, antioxidant, anti-inflammatory, and many others, and have great potential for treating Alzheimer's disease (AD). However, studies have showed that the two compounds have similar structures and in vitro AChE inhibitory activities but with significant difference in bioavailability. The objective of this study was to comparatively investigate the effects of HAL and HAR in memory deficits of scopolamine-induced mice. In the present study, mice were pretreated with HAL (2, 5, and 10 mg/kg), HAR (10, 20, and 30 mg/kg) and donepezil (5 mg/kg) by intragastrically for 7 days, and were daily intraperitoneal injected with scopolamine (1 mg/kg) to induce memory deficits and then subjected to behavioral evaluation by Morris water maze. To further elucidate the underlying mechanisms of HAL and HAR in improving learning and memory, the levels of various biochemical factors and protein expressions related to cholinergic function, oxidative stress, and inflammation were examined. The results showed that HAL and HAR could effectively ameliorate memory deficits in scopolamine-induced mice. Both of them exhibited an enhancement in cholinergic function by inhibiting AChE and inducing choline acetyltransferase (ChAT) activities, and antioxidant defense via increasing the antioxidant enzymes activities of superoxide dismutase and glutathione peroxidase, and reducing maleic diadehyde production, and anti-inflammatory effects through suppressing myeloperoxidase, tumor necrosis factor α, and nitric oxide as well as modulation of critical neurotransmitters such as acetylcholine (ACh), choline (Ch), L-tryptophan (L-Trp), 5-hydroxytryptamine (5-HT), γ-aminobutyric acid (γ-GABA), and L-glutamic acid (L-Glu). Furthermore, the regulations of HAL on cholinergic function, inflammation, and neurotransmitters were more striking than those of HAR, and HAL manifested a comparable antioxidant capacity to HAR. Remarkably, the effective dosage of HAL (2 mg/kg) was far lower than that of HAR (20 mg/kg), which probably due to the evidently differences in the bioavailability and metabolic stability of the two analogs. Taken together, all these results revealed that HAL may be a promising candidate compound with better anti-amnesic effects and pharmacokinetic characteristics for the treatments of AD and related diseases.

The Mammalian Target of Rapamycin and DNA methyltransferase 1 axis mediates vascular endothelial dysfunction in response to disturbed flow.

The earliest atherosclerotic lesions preferentially develop in arterial regions experienced disturbed blood flow, which induces endothelial expression of pro-atherogenic genes and the subsequent endothelial dysfunction. Our previous study has demonstrated an up-regulation of DNA methyltransferase 1 (DNMT1) and a global hypermethylation in vascular endothelium subjected to disturbed flow. Here, we determined that DNMT1-specific inhibition in arterial wall ameliorates the disturbed flow-induced atherosclerosis through, at least in part, targeting cell cycle regulator cyclin A and connective tissue growth factor (CTGF). We identified the signaling pathways mediating the flow-induction of DNMT1. Inhibition of the mammalian target of rapamycin (mTOR) suppressed the DNMT1 up-regulation both in vitro and in vivo. Together, our results demonstrate that disturbed flow influences endothelial function and induces atherosclerosis in an mTOR/DNMT1-dependent manner. The conclusions obtained from this study might facilitate further evaluation of the epigenetic regulation of endothelial function during the pathological development of atherosclerosis and offer novel prevention and therapeutic targets of this disease.

sPAGM: inferring subpathway activity by integrating gene and miRNA expression-robust functional signature identification for melanoma prognoses.

MicroRNAs (miRNAs) regulate biological pathways by inhibiting gene expression. However, most current analytical methods fail to consider miRNAs, when inferring functional or pathway activities. In this study, we developed a model called sPAGM to infer subpathway activities by integrating gene and miRNA expressions. In this model, we reconstructed subpathway graphs by embedding miRNA components, and characterized subpathway activity (sPA) scores by simultaneously considering the expression levels of miRNAs and genes. The results showed that the sPA scores could distinguish different samples across tumor types, as well as samples between tumor and normal conditions. Moreover, the sPAGM model displayed more specificities than the entire pathway-based analyses. This model was applied to melanoma tumors to perform a prognosis analysis, which identified a robust 55-subpathway signature. By using The Cancer Genome Atlas and independently verified data sets, the subpathway-based signature significantly predicted the patients' prognoses, which were independent of clinical variables. In the prognostic performance comparison, the sPAGM model was superior to the gene-only and miRNA-only methods. Finally, we dissected the functional roles and interactions of components within the subpathway signature. Taken together, the sPAGM model provided a framework for inferring subpathway activities and identifying functional signatures for clinical applications.

Can acetabular orientation be restored by lumbar pedicle subtraction osteotomy in ankylosing spondylitis patients with thoracolumbar kyphosis?

PURPOSE: To evaluate whether acetabular orientation (abduction and anteversion) can be restored by lumbar pedicle subtraction osteotomy (PSO) in ankylosing spondylitis (AS) patients with thoracolumbar kyphosis. MATERIALS AND METHODS: A total of 33 consecutive AS patients with thoracolumbar kyphosis undergoing one-level lumbar PSO were retrospectively reviewed. Radiographical measurements included sagittal vertical axis, global kyphosis, thoracic kyphosis, local kyphosis, lumbar lordosis, pelvic incidence, sacral slope, and pelvic tilt. Acetabular abduction and anteversion were measured on CT scans of the pelvis before and after lumbar PSO. The preoperative and postoperative parameters were compared by the paired samples t test. Pearson's correlation analysis was conducted to determine the correlations between the changes in acetabular abduction and anteversion and the changes in sagittal spinopelvic parameters. RESULTS: After lumbar PSO, sagittal vertical axis, global kyphosis, and pelvic tilt were corrected from 15.7 ± 6.7 cm, 66.8° ± 17.5°, and 38.6° ± 9.0° to 2.9 ± 4.9 cm, 21.3° ± 8.2°, and 23.2° ± 8.2°, respectively (p < 0.001). Of note, acetabular abduction and anteversion decreased from 59.6° ± 4.6° to 31.4° ± 6.5° before surgery to 51.4° ± 6.5° and 20.2° ± 4.4° after surgery, respectively (p < 0.001). Moreover, the changes in acetabular abduction and anteversion were observed significantly correlated with the change in pelvic tilt (r = 0.527, p = 0.002; r = 0.586, p < 0.001). CONCLUSION: Abnormal acetabular abduction and anteversion could be corrected by lumbar PSO in AS patients with thoracolumbar kyphosis. Consequently, a relatively normal acetabular orientation could be achieved after lumbar PSO, which might decrease the potential risk of dislocation in AS patients with spine and hip deformities requiring subsequent THR surgery.

Sagittal Vertical Axias, Spinosacral Angle, Spinopelvic Angle, and T1 Pelvic Angle: Which Parameters May Effectively Predict the Quality of Life in Ankylosing Spondylitis Patients With Thoracolumbar Kyphosis?

STUDY DESIGN: This is a retrospective study. OBJECTIVE: To identify the relationship between global sagittal alignment and health-related quality of life (HRQoL) in ankylosing spondylitis (AS) patients with thoracolumbar kyphosis. SUMMARY OF BACKGROUND DATA: Little data are available on correlation between global sagittal alignment and HRQoL in AS. MATERIALS AND METHODS: A total of 107 AS patients were included in this study. The radiographic parameters were measured on lateral radiographs of the whole spine, including sagittal vertical axias (SVA), spinosacral angle (SSA), spinopelvic angle (SPA), and T1 pelvic angle (TPA). HRQoL was assessed using the oswestry disability index questionnaire, the bath ankylosing spondylitis disease activity index, the bath ankylosing spondylitis functional index, and short form-36 questionnaire. The patients were divided into 2 groups: group A (n=76, global kyphosis≤70 degrees), group B (n=31, global kyphosis>70 degrees). Statistical analysis was performed to identify significant differences between these 2 groups. In addition, correlation analysis and multiple regression analysis between radiologic parameters and clinical questionnaires were conducted. RESULTS: With respect to SVA, SSA, SPA, TPA, and HRQoL scores, significant differences were observed between 2 groups (P<0.05). Also, SVA, SSA, SPA, and TPA were significantly related to HRQoL. Multiple regression analysis revealed that SVA, SSA, SPA, and TPA were significant parameters in the prediction of HRQoL in AS patients with thoracolumbar kyphosis. Of note, HRQoL related much more to SSA and SPA than SVA and TPA. CONCLUSIONS: AS patients with moderate and severe deformity were demonstrated to be significantly different in terms of SVA, SSA, SPA, TPA, and HRQoL. Moreover, SVA, SSA, SPA, and TPA correlated with HRQoL significantly. In particular, SSA and SPA could better predict HRQoL than SVA and TPA in AS patients with thoracolumbar kyphosis.

Synthesis of CdTe quantum dot-conjugated CC49 and their application for in vitro imaging of gastric adenocarcinoma cells.

The purpose of this experiment was to investigate the visible imaging of gastric adenocarcinoma cells in vitro by targeting tumor-associated glycoprotein 72 (TAG-72) with near-infrared quantum dots (QDs). QDs with an emission wavelength of about 550 to 780 nm were conjugated to CC49 monoclonal antibodies against TAG-72, resulting in a probe named as CC49-QDs. A gastric adenocarcinoma cell line (MGC80-3) expressing high levels of TAG-72 was cultured for fluorescence imaging, and a gastric epithelial cell line (GES-1) was used for the negative control group. Transmission electron microscopy indicated that the average diameter of CC49-QDs was 0.2 nm higher compared with that of the primary QDs. Also, fluorescence spectrum analysis indicated that the CC49-QDs did not have different optical properties compared to the primary QDs. Immunohistochemical examination and in vitro fluorescence imaging of the tumors showed that the CC49-QDs probe could bind TAG-72 expressed on MGC80-3 cells.

In vitro gastric cancer cell imaging using near-infrared quantum dot-conjugated CC49.

In this experiment, we developed a bioprobe label for immunofluorescence using gastric tumor-specific quantum dots (QDs) to detect gastric tumor cells in vitro. The fluorescent probe, which is capable of specifically labeling gastric tumor cells, was constructed by taking advantage of the unique and superior properties of QDs. We grafted primary QDs onto the tumor-associated glycoprotein 72 (TAG-72) monoclonal antibody CC49 to produce CC49-QDs that specifically label tumor cells. Following a series of tests on the diameter and emission spectrum of CC49-QDs, they were employed in immunofluorescence analysis. Transmission electron microscopy and fluorescence spectrum analyses indicated that CC49-QDs had a 0.25 nm higher average diameter than the primary QDs, and that the grafted CC49 had no difference in optical properties compared to the primary QDs. In cell imaging, the cells labeled with CC49-QDs generated brighter fluorescence compared with the cells of the primary QD group. The results of immunofluorescence analysis demonstrated that antibody grafting reinforced the specific binding of QDs to tumor cells. This probe may also be further applied to live gastric cancer animal models to track lymphatic metastasis. In addition, it may potentially offer theoretical support for lymphadenectomy in the treatment of gastric cancer.