Preosteoblast-enriched lnc-Evf2 facilitates osteogenic differentiation by targeting Notch

Ossification of ligaments(OL)and osteoporosis(OP)are multifactorial disorders without definitive clinical biomarkers.Long non-coding RNAs(lncRNAs)are known to involve in regulating pathogenesis.Here,we have identified a preosteoblast-enriched lnc-Evf2 that was overexpressed in ossified ligamentum flavum(OLF)and down-expressed in OP.lnc-Evf2 is gradually upregulated during osteogenic induction,correlating with the enhanced expression of osteogenic marker genes and matrix mineralization.Moreover,knockdown of lnc-Evf2 significantly inhibits the expression of osteogenic differentiation markers and delays the osteoblastic mineralization process,indicating that this molecule is involved in osteogenesis.Mechanistically,we demonstrated that silencing of lnc-Evf2 decreases the protein level but not the mRNA levels of Notch2,Notch3,and Hes1,all of which correlate with osteogenesis.Taken together,our data demonstrate that lnc-Evf2 promotes osteogenic differentiation and bone formation through the Notch signaling,revealing that lnc-Evf2 may serve as a novel potential clinical target of OL and OP.     

Perspectives of gene combinations in phenotype presentation

Cells exhibit a variety of phenotypes in different stages and diseases. Although several markers for cellular phenotypes have been identified, gene combinations denoting cellular phenotypes have not been completely elucidated. Recent advances in gene analysis have revealed that various gene expression patterns are observed in each cell species and status. In this review, the perspectives of gene combinations in cellular phenotype presentation are discussed. Gene expression profiles change during cellular processes, such as cell proliferation, cell differentiation, and cell death. In addition, epigenetic regulation increases the complexity of the gene expression profile. The role of gene combinations and panels of gene combinations in each cellular condition are also discussed.     

Protective effects of resveratrol on postmenopausal osteoporosis： regulation of SIRT1-NF-κB signaling pathway

Postmenopausal osteoporosis severely jeopardizes human health. Seeking for therapeutic drugs without side effects is of great necessity. Our study was designed to investigate whether resveratrol, an agonist of SIRT1, could have favor- able effect on osteoporosis and to explore the underlying mechanisms. Rat osteoporosis model （ovariectomy group, OVX） was established by bilateral ovariectomy. Three dif- ferent doses of resveratrol were used： 5 mg/kg/d （low- dosed, RESCD）, 25 mg/kg/d （medium-dosed, RESMD）, and 45 mg/kg/d （high-dosed, RESUD）. Results showed that RESLD did not show any significant effect on OVX altera- tions, while RESMD and RESXD significantly elevated the decreased bone mineral density induced by osteoporosis （RESMD 0.205±0.023, RESm） 0.214 ± 0.053 vs. OVX 0.165 ± 0.050 g/cm2 respectively; P 〈 0.05）. Serum markers alkaline phosphatase （ALP） and osteocalcin were moderately restored by resveratrol. Moreover, resveratrol improved bone structure in OVX rats, demonstrated by hematoxylin-eosin staining and micro-computed tomo- graphic results. In vitro results revealed that resveratrol promoted osteoblast differentiation of bone marrow mesen- chymal stromal cells, evidenced by the increase of ALP generation and mRNA expression of collagen 1 （P 〈 0.05; RESMD, RESm） vs. control group）. SIRT1 gene silencing by siRNA transfection blocked these beneficial effects of resveratrol （P〈0.05; RES ＋ SIRT1^KD vs RES^HD）. Western blot results showed that resveratrol activated SIRT1 and subsequently suppressed the activity of NF-κB with decreased expression level of p-IκBa and NF-κB p65 （P〈 0.05）. Our findings verified the effects of specific dosed resveratrol on postmenopausal osteoporosis through osteoblast differentiation via SIRT1-NF-κB signaling pathway. This study suggested the therapeutic potential of resveratroi against osteoporosis and stressed the importance of effective doses.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-κB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expression of IL-12 p40 and p35 mRNA, and the degradation of κBα induced by LPS or LPS/IFN-γ. EMSA showed that LPS could augment the NF-κB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-κB activity nor promote the degradation of kBa. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided wit     

Sphingosine 1-phosphate induces epicardial progenitor cell differentiation into smooth muscle-like cells

Epicardial progenitor cells (EpiCs) which are derived from the proepicardium have the potential to differentiate into coronary vascular smooth muscle cells during development. Whether sphingosine 1-phosphate (S1P), a highly hydrophobic zwitterionic lysophospholipid in signal transduction, in duces the differe ntiati on of EpiCs is unk nown. In the present study, we dem on strated that S1P significantly induced the expression of smooth muscle cell specific markers a-smooth muscle actin and myosin heavy chain 11 in the EpiCs. And the smooth muscle cells differentiated from the EpiCs stimulated by S1P were further evaluated by gel contraction assay. To further confirm the major subtype of sphingosine 1-phosphate receptors (S1 PRs) involved in the differentiation of EpiCs, we used the agonists and antagonists of different S1PRs. The results showed that the S1 Pq/ S1P3 antagonist VPC23019 and the S1P2 antagonist JTE013 significantly attenuated EpiCs differentiation, while the S1 P-i agonist SEW2871 and antagonist W146 did not affect EpiCs differentiation. These results collectively suggested that S1P, principally through its receptor S1P3, in creases EpiCs differentiation into VSMCs and thus indicated the importance of S1P signaling in the embryonic coronary vasculature, while S1P2 plays a sec on dary role.     

Application of cell penetrating peptide in magnetic resonance imaging of bone marrow mesenchymal stem cells

Tracking the distribution and differentiation of stem cells by high-resolution imaging techniqueswould have significant clinical and research implications.In this study,a model cell-penetrating peptide wasused to carry gadolinium particles for magnetic resonance imaging (MRI) of mesenchymal stem cells (MSCs).MSCs were isolated from rat bone marrow and identified by osteogenic differentiation in vitro.The cell-penetrating peptide labeled with fluorescein-5-isothiocyanate (FITC) and gadolinium was synthesized by asolid-phase peptide synthesis method.Fluorescein imaging analysis confirmed that this new peptide couldinternalize into the cytoplasm and nucleus at room temperature,4℃ and 37℃.Gadolinium were efficientlyinternalized into mesenchymal stem cells by the peptide in a time or concentration-dependent manner,resulting in intercellular shortening of longitudinal relaxation enhancements,which were obviously detectedby 1.5 Tesla Magnetic Resonance Imaging.Cytotoxicity assay and flow cytometric analysis showed thatthe intercellular contrast medium incorporation did not affect cell viability at the tested concentrations.Thein vitro experiment results suggested that the new constructed peptides could be a vector for trackingMSCs.     

Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells

BACKGROUND A major problem in the healing of bone defects is insufficient or absent blood supply within the defect.To overcome this challenging problem,a plethora of approaches within bone tissue engineering have been developed recently.Bearing in mind that the interplay of various diffusible factors released by endothelial cells(ECs)and osteoblasts(OBs)have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions,we set the focus on the simultaneous application of these cell types together with platelet-rich plasma(PRP)as a growth factor reservoir within ectopic bone tissue engineering constructs.AIM To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells(ASCs)induced into ECs and OBs.METHODS ASCs isolated from adipose tissue,induced in vitro into ECs,OBs or just expanded were used for implant construction as followed:BPEO,endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix;BPUI,uninduced ASCs with PRP and bone mineral matrix;BC(control),only bone mineral matrix.At 1,2,4 and 8 wk after subcutaneous implantation in mice,implants were extracted and endothelial-related and bone-related gene expression were analyzed,while histological analyses were performed after 2 and 8 wk.RESULTS The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation.BC had the lowest endothelial-related gene expression,weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO.Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO.BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs.Except Vwf,endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk.Tissue regression was noticed in BPEO constructs after 8 wk.CONCLUSION Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis,but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.