Jianmei Li

Effect of microRNA‐145 on IL‐1β‐induced cartilage degradation in human chondrocytes

MicroRNA‐145 has been shown to regulate chondrocyte homeostasis. It seems that miR‐145 is implicated in cartilage dysfunction in Osteoarthritis (OA). However, the functional role of miR‐145 in interleukin‐1 beta (IL‐1β)‐induced extracellular matrix (ECM) degradation of OA cartilage has never been clarified. Here, we show that miR‐145 expression increased in OA chondrocytes and in response to IL‐1β stimulation. We confirm that mothers against decapentaplegic homolog 3 (Smad3), a key factor in maintaining chondrocyte homeostasis, is directly regulated by miR‐145. Modulation of miR‐145 affects the expression of Smad3 causing a change of its downstream target gene expression as well as IL‐1β‐induced ECM degradation in OA chondrocytes. This indicates that miR‐145 contributes to impaired ECM in OA cartilage probably in part via targeting Smad3.

Changing expression profiles of lncRNAs, mRNAs, circRNAs and miRNAs during osteoclastogenesis

Bone is a dynamic organ continuously undergoing shaping, repairing and remodeling. The homeostasis of bone is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts (OCs) are specialized multinucleated cells derived from hematopoietic stem cells (HSCs) or monocytes/macrophage progenitor cells. There are different stages during osteoclastogenesis, and one of the most important steps to form functional osteoclasts is realized by cell-cell fusion. In our study, microarray was performed to detect the expression profiles of lncRNA, mRNA, circRNA and miRNA at different stages during osteoclastogenesis of RAW264.7 cells. Often changed RNAs were selected and clustered among the four groups with Venn analysis. The results revealed that expressions of 518 lncRNAs, 207 mRNAs, 24 circRNAs and 37 miRNAs were often altered at each stage during OC differentiation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis were performed to predict the functions of differentially expressed lncRNAs and co-expressed potential targeting genes. Co-expression networks of lncRNA-mRNA and circRNA-miRNA were constructed based on the correlation analysis between the differentially expressed RNAs. The present study provided a systematic perspective on the potential function of non-coding RNAs (ncRNAs) during osteoclastogenesis.

Dual Effect of Cyanidin on RANKL-Induced Differentiation and Fusion of Osteoclasts.

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. Osteoclasts are multinucleated cells derived from hematopoietic stem cells (HSCs) or monocyte/macrophage progenitor cells and formed by osteoclasts precursors (OCPs) fusion. Cyanidin is an anthocyanin widely distributed in food diet with novel antioxidant activity. However, the effect of cyanidin on osteoclasts is still unknown. We investigated the effect of cyanidin on RANKL‐induced osteoclasts differentiation and cell fusion. The results showed that cyanidin had a dual effect on RANKL‐induced osteoclastogenesis. Lower dosage of cyanidin (< 1µg/ml) has a promoting effect on osteoclastogenesis while higher dosage of cyanidin (> 10µg/ml) has an inhibitory effect. Fusogenic genes like CD9, ATP6v0d2, DC‐STAMP, OC‐STAMP, and osteoclasts related genes like NFATc1, mitf, and c‐fos were all regulated by cyanidin consistent to its dual effect. Further exploration showed that low concentration of cyanidin could increase osteoclasts fusion whereas higher dosage of cyanidin lead to the increase of LXR‐β expression and activation which is suppressive to osteoclasts differentiaton. All these results showed that cyanidin exhibits therapeutic potential in prevention of osteoclasts related bone disorders. J. Cell. Physiol. 231: 558–567, 2016.

Cerium Oxide Nanoparticle Modified Scaffold Interface Enhances Vascularization of Bone Grafts by Activating Calcium Channel of Mesenchymal Stem Cells

Insufficient blood perfusion is one of the critical problems that hamper the clinical application of tissue engineering bone (TEB). Current methods for improving blood vessel distribution in TEB mainly rely on delivering exogenous angiogenic factors to promote the proliferation, migration, differentiation, and vessel formation of endothelial cells (ECs) and/or endothelial progenitor cells (EPCs). However, obstacles including limited activity preservation, difficulty in controlled release, and high cost obstructed the practical application of this strategy. In this study, TEB scaffold were modified with cerium oxide nanoparticles (CNPs) and the effects of CNPs existed at the scaffold surface on the growth and paracrine behavior of mesenchymal stem cells (MSCs) were investigated. The CNPs could improve the proliferation and inhibit the apoptosis of MSCs. Meanwhile, the interaction between the cell membrane and the nanoparticle surface could activate the calcium channel of MSCs leading to the rise of intracellular free Ca(2+) level, which subsequently augments the stability of HIF-1α. These chain reactions finally resulted in high expression of angiogenic factor VEGF. The improved paracrine of VEGF could thereby promote the proliferation, differentiation, and tube formation ability of EPCs. Most importantly, in vivo ectopic bone formation experiment demonstrated this method could significantly improve the blood vessel distribution inside of TEB.

Long noncoding RNA expression profiles in chondrogenic and hypertrophic differentiation of mouse mesenchymal stem cells

Long noncoding RNAs (lncRNAs) are important regulators for a variety of biological processes. Chondrogenic differentiation of mesenchymal stem cells (MSCs) is a crucial stage in chondrogenesis while chondrocyte hypertrophy is related to endochondral ossification and osteoarthritis. However, the effects of lncRNAs on chondrogenic and hypertrophic differentiation of mouse MSCs are unclear. To explore the potential mechanisms of lncRNAs during chondrogenesis and chondrocyte hypertrophy, microarray was performed to investigate the expression profiles of lncRNA and mRNA in MSCs, pre-chondrocytes, and hypertrophic chondrocytes. Then, we validated microarray data by RT-PCR and screened three lncRNAs from upregulating groups during chondrogenesis and chondrocyte hypertrophy respectively. After downregulating any of the above lncRNAs, we found that the expression of chondrogenesis-related genes such as Sox9 and Col2a1 and hypertrophy-related genes including Runx2 and Col10a1 was inhibited, respectively. Furthermore, the target genes of above lncRNAs were predicted by bioinformatics approaches. Gene ontology and Kyoto encyclopedia of genes and genome biological pathway analysis were also made to speculate the functions of above lncRNAs. In conclusion, the study first revealed the expression profile of lncRNAs in chondrogenic and hypertrophic differentiations of mouse MSCs and presented a new prospect for the underlying mechanisms of chondrogenesis and endochondral ossification.

LncRNA-AK131850 Sponges MiR-93-5p in Newborn and Mature Osteoclasts to Enhance the Secretion of Vascular Endothelial Growth Factor a Promoting Vasculogenesis of Endothelial Progenitor Cells

Background/Aims: In the process of bone development and remodeling, the vasculature is regarded as the communicative network between the bone and neighboring tissues. Recently, it has been reported that the processes of angiogenesis and osteogenesis are coupled temporally and spatially. However, few studies reported the relationship and relevant mechanism between osteoclastogenesis and vasculogenesis. Methods: Arraystar Mouse lncRNA microarray V3.0 was firstly used to analyze the differentially expressed lncRNA genes in osteoclast different stages during osteoclastogenesis. Cell counting kit 8 (CCK-8) analysis, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, migration and tube formation assays were used to detect impact of osteoclast different stages on the proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs), respectively. Finally, transfection of AK131850 shRNA, miR-93-5p mimic and miR-93-5p inhibitor, qRT-PCR, western blotting, enzyme-linked immunosorbent assay (ELISA), fluorescence in situ hybridization (FISH) and luciferase reporter assay were carried out to dissect molecular mechanisms. Results: In this study, we found that newborn OCs (N-OC) and mature OCs (M-OC) during osteoclastogenesis significantly promoted proliferation, differentiation, migration and tube formation of endothelial progenitor cells (EPCs). Through lncRNA microarray and GO&pathway analysis, we found that AK131850 and co-expressed gene, vascular endothelial growth factor a (VEGFa), were significantly up-regulated in N-OC and M-OC. After inhibition of AK131850 the promoting effect of N-OC and M-OC on EPCs was reversed. Furthermore, we found that AK131850 directly competed miR-93-5p in N-OC and M-OC through sponge, thereby increasing VEGFa transcription, expression and secretion through derepressing of miR-93-5p on VEGFa. Conclusion: Our results provided the first finding that lncRNA-AK131850 sponged miR-93-5p in N-OC and M-OC during osteoclastogenesis to enhance the secretion of VEGFa, thus promoting vasculogenesis of EPCs.

Hypertrophic differentiation of mesenchymal stem cells is suppressed by xanthotoxin via the p38‑MAPK/HDAC4 pathway

Chondrocyte hypertrophy is a physiological process in endochondral ossification. However, the hypertrophic-like alterations of chondrocytes at the articular surface may result in osteoarthritis (OA). In addition, the generation of fibrocartilage with a decreased biological function in tissue engineered cartilage, has been attributed to chondrocyte hypertrophy. Therefore, suppressing chondrocyte hypertrophy in OA and the associated regeneration of non-active cartilage is of primary concern. The present study examined the effects of xanthotoxin (XAT), which is classified as a furanocoumarin, on chondrocyte hypertrophic differentiation of mesenchymal stem cells. Following XAT treatment, the expression levels of genes associated with chondrocyte hypertrophy were detected via immunohistochemistry, western blotting and reverse transcription-quantitative polymerase chain reaction. The results revealed that XAT inhibited the expression of various chondrocyte hypertrophic markers, including runt related transcription factor 2 (Runx2), matrix metalloproteinase 13 and collagen type X α1 chain. Further exploration indicated that XAT reduced the activation of p38-mitogen activated protein kinase and then increased the expression of histone deacetylase 4 to suppress Runx2. The findings indicated that XAT maintained the chondrocyte phenotype in regenerated cartilage and therefore may exhibit promise as a potential drug for the treatment of OA in the future.

Cordycepin inhibits chondrocyte hypertrophy of mesenchymal stem cells through PI3K/Bapx1 and Notch signaling pathway.

Mesenchymal stem cells (MSCs) are widely used in cartilage tissue engineering to repair articular cartilage defects. However, hypertrophy of chondrocytes derived from MSCs might hinder the stabilization of hyaline cartilage. Thus, it is very important to find a suitable way to maintain the chondrogenic phenotype of chondrocytes. It has been reported that cordycepin has anti-inflammatory and anti-tumor functions. However, the role of cordycepin in chondrocyte hypertrophy remains unclear. Therefore, the objective of this study was to determine the effect of cordycepin on chondrogenesis and chondrocyte hypertrophy in MSCs and ATDC5 cells. Cordycepin upregulated chondrogenic markers including Sox9 and collagen type II while down-regulated hypertrophic markers including Runx2 and collagen type X. Further exploration showed that cordycepin promoted chondrogenesis through inhibiting Nrf2 while activating BMP signaling. Besides, cordycepin suppressed chondrocyte hypertrophy through PI3K/Bapx1 pathway and Notch signaling. Our results indicated cordycepin had the potential to maintain chondrocyte phenotype and reconstruct engineered cartilage. [BMB Reports 2016; 49(10): 548-553]

Mangiferin enhances endochondral ossification-based bone repair in massive bone defect by inducing autophagy through activating AMP-activated protein kinase signaling pathway

Endochondral ossification is crucial for bone formation in both adult bone repair process and embryo long‐bone development. In endochondral ossification, bone marrow‐derived mesenchymal stem cells (BMSCs) first differentiate to chondrocytes, then BMSC‐derived chondrocytes endure a hypertrophic process to generate new bone. Endochondral ossification‐based bone repair is a promising strategy to cure massive bone defect, which is a major clinical issue in orthopedics. However, challenges still remain for this novel strategy. One challenge is to ensure the sufficient hypertrophic differentiation. Another is to maintain the survival of the above hypertrophic chondrocytes under the hypoxic environment of massive bone defect. To solve this issue, mangiferin (MAG) was introduced to endochondral ossification‐based bone repair. In this report, we proved MAG to be a novel autophagy inducer, which promoted BMSC‐derived hypertrophic chondrocyte survival against hypoxia‐induced injury through inducing autophagy. Furthermore, MAG enhances hypertrophic differentiation of BMSC‐derived chondrocytes via upregu‐lating key hypertrophic markers. Mechanistically, MAG induced autophagy in BMSC‐derived chondrocytes by promoting AMPKa phosphorylation. Additionally, MAG balanced the expression of sex‐determining region Y‐box 9 and runt‐related transcription factor 2 to facilitate hypertrophic differentiation. These results indicated that MAG was a potential drug to improve the efficacy of endochondral ossification‐based bone repair in massive bone defects.—Bai, Y., Liu, C., Fu, L., Gong, X., Dou, C., Cao, Z., Quan, H., Li, J., Kang, F., Dai, J., Zhao, C., Dong, S. Mangiferin enhances endochondral ossification‐based bone repair in massive bone defect by inducing autophagy through activating AMP‐activated protein kinase signaling pathway. FASEB J. 32, 4573–4584 (2018). www.fasebj.org

Inhibitory effect of vanillin on RANKL-induced osteoclast formation and function through activating mitochondrial-dependent apoptosis signaling pathway

&NA; Bone matrix homeostasis associated diseases such as osteoporosis and erosive arthritis were caused by the imbalance of osteoclast‐mediated bone‐resorption and osteoblast‐mediated bone‐formation. Suppressing the fusion and differentiation of osteoclast from osteoclast precursors are an essential way to maintain the dynamic balance of resorption and formation. Recently, some natural products were discovered to inhibit osteoclast formation and function for potential treatment of osteoporosis. Vanillin was previously reported to have anti‐tumor and anti‐oxidant activities; however, its effect on bone health has not been elucidated. In this study, we found that the inhibitory effect of vanillin on RANKL‐induced multinucleated osteoclast formation and bone resorption (concentration of 0.25 mM–2.5 mM). Morphologically, the number of mature osteoclasts was decreased after treating with vanillin in a dose‐dependent manner, which was evaluated by TRAP staining and FAK staining. Vanillin could significantly inhibit bone resorption and promote the early apoptosis rate during RANKL‐induced osteoclastogenesis. Furthermore, vanillin could activate the mitochondrial‐dependent apoptosis via inducing the expression of cytochrome c, cleaved caspease‐3, BAX and Apaf‐1 both on mRNA and protein level. Otherwise, the expression of Bcl‐2 was inhibited. Thereby, these data provide the clue that vanillin could be a candidate to treat bone matrix metabolic diseases.