BackgroundOsteosarcoma (OS) is a malignant tumor mainly occurring in young people. Due to the limited effective therapeutic strategies, OS patients cannot achieve further survival improvement. G-protein-coupled receptors (GPCRs) constitute the largest family of cell membrane receptors and consequently hold the significant promise for tumor imaging and targeted therapy. We aimed to explore the biological functions of Sphingosine 1-phosphate receptor 3 (S1PR3), one of the members of GPCRs family, in OS and the possibility of S1PR3 as an effective target for the treatment of osteosarcoma.MethodsThe quantitative real time PCR (qRT-PCR) and western blotting were used to analyze the mRNA and protein expressions. Cell counting kit-8 (CCK8), colony formation assay and cell apoptosis assay were performed to test the cellular proliferation in vitro. Subcutaneous xenograft mouse model was generated to evaluate the functions of S1PR3 in vivo. RNA sequencing was used to compare gene expression patterns between S1PR3-knockdown and control MNNG-HOS cells. In addition, metabolic alternations in OS cells were monitored by XF96 metabolic flux analyzer. Co-immunoprecipitation (Co-IP) assay was used to explore the interaction between Yes-associated protein (YAP) and c-MYC. Chromatin immunoprecipitation was used to investigate the binding capability of PGAM1 and YAP or c-MYC. Moreover, the activities of promoter were determined by the luciferase reporter assay.FindingsS1PR3 and its specific ligand Sphingosine 1-phosphate (S1P) were found elevated in OS, and the higher expression of S1PR3 was correlated with the poor survival rate. Moreover, our study has proved that the S1P/S1PR3 axis play roles in proliferation promotion, apoptosis inhibition, and aerobic glycolysis promotion of osteosarcoma cells. Mechanistically, the S1P/S1PR3 axis inhibited the phosphorylation of YAP and promoted the nuclear translocation of YAP, which contributed to the formation of the YAP–c-MYC complex and enhanced transcription of the important glycolysis enzyme PGAM1. Moreover, the S1PR3 antagonist TY52156 exhibited in vitro and in vivo synergistic inhibitory effects with methotrexate on OS cell growth.InterpretationOur study unveiled a role of S1P, a bioactive phospholipid, in glucose metabolism reprogram through interaction with its receptor S1PR3. Targeting S1P/S1PR3 axis might serve as a potential therapeutic target for patients with OS.FundThis research was supported by National Natural Science Foundation of China (81472445 and 81672587).

Rationale: Peripheral nerves are unique in their remarkable elasticity. Schwann cells (SCs), important components of the peripheral nervous system (PNS), are constantly subjected to physiological and mechanical stresses from dynamic stretching and compression forces during movement. So far, it is not clear if SCs sense and respond to mechanical signals. It is also unknown whether mechanical stimuli can interfere with the intercellular communications between neurons and SCs, and what role extracellular vesicles (EVs) play in this process. The present study aimed to examine the effect of mechanical stimuli on the EV-mediated intercellular communication between neurons and SCs, explore their effect on axonal regeneration, and investigate the underlying mechanism. Methods: Purified SCs were stimulated using a magnetic force-based mechanical stimulation (MS) system and EVs were purified from mechanically stimulated SCs (MS-SCs-EVs) and non-stimulated SCs (SCs-EVs). The effect of MS-SCs-EVs on axonal elongation was examined in vitro and in vivo . High throughput miRNA sequencing was performed to compare the differential miRNA profiles between MS-SCs-EVs and SCs-EVs. The functional role of differentially expressed miRNAs on neurite extension in MS-SCs-EVs was examined. Also, the putative target genes of differentially expressed miRNAs in MS-SCs-EVs were predicted by bioinformatics tools, and the regulatory effect of those miRNAs on putative target genes was validated both in vitro and in vivo . Results: The MS-SCs-EVs showed an average size of 137.52±1.77 nm, and could be internalized by dorsal root ganglion (DRG) neurons. Compared to SCs-EVs, MS-SCs-EVs showed a stronger ability to enhance neurite outgrowth in vitro and nerve regeneration in vivo . High throughput miRNA sequencing identified a number of differentially expressed miRNAs in MS-SCs-EVs. Further analysis of those EV-miRNAs demonstrated that miR-23b-3p played a predominant role in MS-SCs-EVs since its deprivation abolished their enhanced axonal elongation. Furthermore, we identified neuropilin 1 (Nrp1) in neurons as the target gene of miR-23b-3p in MS-SCs-EVs. This observation was supported by the evidence that miR-23b-3p could decrease Nrp1-3'-UTR-WT luciferase activity in vitro and down-regulate Nrp1 expression in neurons. Conclusion: Our findings suggested that mechanical stimuli are capable of modulating the intercellular communication between neurons and SCs by altering miRNA composition in MS-SCs-EVs. Transfer of miR-23b-3p by MS-SCs-EVs from mechanically stimulated SCs to neurons decreased neuronal Nrp1 expression, which was responsible, at least in part, for the beneficial effect of MS-SCs-EVs on axonal regeneration. Our results highlighted the potential therapeutic value of MS-SCs-EVs and miR-23b-3p-en...

Rationale: Local hypoxia is a challenge for fabrication of cellular grafts and treatment of peripheral nerve injury. In our previous studies, we demonstrated that perfluorotributylamine (PFTBA) could provide short term oxygen supply to Schwann cells (SCs) and counteract the detrimental effects of hypoxia on SCs during the early stages of nerve injury. However, the quick release of oxygen in PFTBA compromised its ability to counteract hypoxia over an extended time, limiting its performance in peripheral nerve injury. Methods: In this study, PFTBA-based oxygen carrier systems were prepared through coaxial electrospinning to prolong the time course of oxygen release. Core-shell structures were fabricated, optimized, and the oxygen kinetics of PFTBA-enriched core-shell fibers evaluated. The effect of core-shells on the survival and function of SCs was examined in both 2D and 3D systems as well as in vivo . The system was used to bridge large sciatic nerve defects in rats. Results: PFTBA core-shell fibers provided high levels of oxygen to SCs in vitro , enhancing their survival, and increasing NGF, BDNF, and VEGF expression in 2D and 3D culture systems under hypoxic condition. In vivo analysis showed that the majority of GFP-expressing SCs in the PFTBA conduit remained viable 14 days post-implantation. We found that axons in PFTBA oxygen carrier scaffold improved axonal regeneration, remyelination, and recovery. Conclusion: A synthetic oxygen carrier in core-shell fibers was fabricated by the coaxial electrospinning technique and was capable of enhancing SC survival and nerve regeneration by prolonged oxygen supply. These findings provide a new strategy for fabricating cellular scaffolds to achieve regeneration in peripheral nerve injury treatment and other aerobic tissue injuries.

In the present study, we investigated whether mesenchymal stem cells (MSCs) overexpressing integrin-linked kinase (ILK) might regulate ventricular remodeling and cardiac function in a porcine myocardial infarction model. ILK-modified MSCs (ILK-MSCs) (n = 8), MSCs (n = 8) or placebo (n = 8) were injected into peri-infarct myocardium 7 days after ligation of the left anterior descending coronary artery. ILK expression was confirmed by immunofluorescence, real-time PCR, Western blot analysis, and flow cytometry. In vitro assays indicated increased proliferation and reduced apoptosis of MSCs due to overexpression of ILK. Echocardiographic, single-photon emission computed tomography and positron emission tomography analyses demonstrated preserved cardiac function and myocardial perfusion. Reduced fibrosis, increased cardiomyocyte proliferation, and enhanced angiogenesis were observed in the ILK-MSC group. Reduced apoptosis, as demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling analysis, was also noted. In conclusion, ILK promotes MSC proliferation and suppresses apoptosis. ILK-MSC transplantation improves ventricular remodeling and cardiac function in pigs after MI. It is associated with increased angiogenesis, reduced apoptosis, and increased cardiomyocyte proliferation. This may represent a new approach to the treatment of post-infarct remodeling and subsequent heart failure.

Biochemical and physical guidance cues are both pivotal for axonal guidance and nerve regeneration. However, fabrication of a platform that can integrate biochemical gradients and topographical guidance cues remains challenging, especially in a three-dimensional (3D) scaffold that closely mimics in vivo axonal outgrowth conditions and ready to be used for in vivo nerve repair. In this study, a new method was introduced to construct 3D scaffolds displaying continuous biochemical gradients along longitudinally oriented microchannels by combining the modified 3D printing and directional freezing techniques. Fluorescence analysis and ELISA results demonstrated that a continuous biochemical gradient was formed, and scanning electron microscopy revealed a longitudinally oriented microstructure. Dorsal root ganglia explants seeded on the longitudinal sections of the newly developed scaffold (scaffold with nerve growth factor gradient along oriented microstructure, G-NGF + OS) showed that 81.3 ± 4.5% of neurites oriented within ±10°, 0.3 ± 0.1 of guidance ratio, and 1.5-fold of the average length of neurites on the high-nerve growth factor (NGF) concentration side compared to that on the low-NGF concentration side, which were significantly higher than those in the other groups. In addition, the G-NGF + OS scaffold was used to repair a 15 mm sciatic nerve defect in rats. Immunofluorescence staining, Fluoro-Gold retrograde tracing, and transmission electron microscopy results confirmed that the G-NGF + OS scaffold enhanced nerve regeneration to the distal target and promoted myelination of regenerated axons. More importantly, the sciatic functional index and the von Frey test demonstrated that the G-NGF + OS scaffold accelerated sensory and motor functional recovery. These results provide new insights into the importance of combining topographical guidance cues with bioactive molecule gradient cues for neural tissue engineering. The 3D scaffold displaying biochemical gradients along longitudinally oriented microchannels represents a promising platform for the development of advanced devices for severe nervous system injuries.

Delivery of multiple neurotrophic factors (NTFs), especially with time‐restricted release kinetics, holds great potential for nerve repair. In this study, we utilized the tetracycline‐regulatable Tet‐On 3G system to control the expression of c‐Jun, which is a common regulator of multiple NTFs in Schwann cells (SCs). In vitro, Tet‐On/c‐Jun‐modified SCs showed a tightly controllable secretion of multiple NTFs, including glial cell line‐derived NTF, nerve growth factor, brain‐derived NTF, and artemin, by the addition or removal of doxycycline (Dox). When Tet‐On/c‐Jun‐transduced SCs were grafted in vivo, the expression of NTFs could also be regulated by oral administration or removal of Dox. Fluoro‐Gold retrograde tracing results indicated that a biphasic NTF expression scheme (Dox+3/−9, NTFs were up‐regulated for 3 wk and declined to physiologic levels for another 9 wk) achieved more axonal regeneration than continuous up‐regulation of NTFs (Dox+12) or no NTF induction (Dox−12). More importantly, the Dox+3/−9–group animals showed much better functional recovery than the animals in the Dox+12 and Dox−12 groups. Our findings, for the first time, demonstrated drug‐controllable expression of multiple NTFs in nerve repair cells both in vitro and in vivo. These findings provide new hope for developing an optimal therapeutic alternative for nerve repair through the time‐restricted release of multiple NTFs using Tet‐On/c‐Jun‐modified SCs.—Huang, L., Xia, B., Shi, X., Gao, J., Yang, Y., Xu, F., Qi, F., Liang, C., Huang, J., Luo, Z. Time‐restricted release of multiple neurotrophic factors promotes axonal regeneration and functional recovery after peripheral nerve injury. FASEB J. 33, 8600–8613 (2019). http://www.fasebj.org

Studies of the effects of low glycemic index (LGI) diets on gestational diabetes mellitus (GDM) have reported conflicting findings.The aim of the study was to evaluate the results of randomized controlled trials (RCTs) that investigated the effects of LGI diets with and without added dietary fiber (DF) on maternal and neonatal outcomes in GDM patients.We searched the MEDLINE, EMBASE, EBSCO, Springer, Ovid, and Cochrane Library databases for studies of the effects of LGI diets in GDM patients. We performed a meta-analysis of the effects of the LGI diets with and without added dietary fiber (DF) on GDM outcomes. Risk ratios (RR) and 95% confidence intervals (CIs) were calculated using random- and fixed-effects models.Five RCTs involving 302 participants were included in our meta-analysis. No statistically significant differences in the risks of cesarean section delivery, large for gestational age, and small for gestational age were observed. The risk of macrosomia in the LGI groups was significantly lower (RR = 0.27; 95% CI: 0.10–0.71; P = 0.008) than that in the control groups. Our subgroup analysis of the effects of DF showed that LGI diets with an increased level of DF, relative to the control diet, reduced the risk of macrosomia beyond that of the LGI diets alone (RR: 0.17 vs 0.47, respectively). The subgroup analysis also showed that LGI diets in which the level of DF was approximately equivalent to that in the control diets significantly reduced the risk of insulin usage (RR = 0.69; 95% CI: 0.52–0.92; P = 0.01).The LGI diets reduced the risk of macrosomia in GDM patients, and LGI diets with added DF reduced the risk of macrosomia further. The LGI diets with levels of DF approximately equivalent to that in the control diets reduced the risk of insulin usage in GDM patients.

Background/Aims: Microbes reside in a number of body sites, including the oral cavity, and are associated with the progression of many systemic diseases. In this study, we aimed to investigate the effects of gout and hyperuricemia (HUA) on the composition of oral microbiomes. Methods: Analysis of the oral microbiota from 12 gout patients, 11 HUA patients, and 19 healthy control subjects was performed using a deep sequencing approach, and validation of significant changes in Prevotella intermedia and Serratia marcescens in new patient cohorts was performed using quantitative PCR (qPCR). Results: Our analysis indicated that both gout and HUA significantly altered the composition of the oral microbiome in patients. Patients with gout or HUA had significantly greater levels of salivary Prevotella intermedia but significantly lower levels of Serratia marcescens than healthy control subjects. Conclusion: We demonstrated the association between the oral microbiome and gout and HUA for the first time. In particular, 16S sequencing and qPCR analysis revealed significantly higher levels of oral Prevotella intermedia in gout/HUA patients, which suggests that these patients might be at risk for the development of periodontitis.