Calcium Nodule Formation Research Articles

Mg2+ in β-TCP/Mg-Zn composite enhances the differentiation of human bone marrow stromal cells into osteoblasts through MAPK-regulated Runx2/Osx.

Inducing the osteogenic differentiation from bone marrow stromal cells (BMSCs) might be a potent strategy for treating bone loss and nonunion during fracture and improving fracture healing. Among several signaling pathways involved, mitogen-activated protein kinases (MAPKs) have been reported to play a critical role. Magnesium (Mg)-based alloys, including Mg-Zn alloy, have been used clinically as implants in the musculoskeletal field and could promote BMSC osteogenic differentiation. However, the underlying mechanisms remain unclear. In this study, we produced Mg-Zn alloy consists of Mg and low concentrations of Zn, calcium carbonate, and β-tricalcium phosphate (β-TCP; manifesting process not shown), prepared Mg, Zn, and Mg-Zn extracts, and investigated the specific effects of these extracts on human BMSC (hBMSC) osteogenic differentiation and MAPK signaling. Mg extracts and Mg-Zn extracts could significantly promote the osteogenic differentiation of hBMSCs as manifested as increased alkaline phosphatase levels, enhanced calcium nodules formation, and increased messenger RNA expression and protein levels of osteogenesis markers, including BMPs, Col-I, Runx2, and Osx; in the meantime, Mg culture medium (CM) and Mg-Zn CM both significantly enhanced the activation of MAPK signaling in hBMSCs. By adding ERK1/2 signaling, p38 signaling, or JNK signaling inhibitor to Mg-Zn CM, or conducting p38 MAPK silence in hBMSCs, we revealed that these extracts might promote hBMSC osteogenic differentiation via p38 MAPK signaling and MAPK-regulated Runx2/Osx. In conclusion, Mg2+ in β-TCP/Mg-Zn extract promotes the osteogenic differentiation of hBMSCs via MAPK-regulated Runx2/Osx interaction.

Role of nanoparticles in osteogenic differentiation of bone marrow mesenchymal stem cells.

The present study aimed to investigate the osteoinductive potentiality of some selected nanostructures; Hydroxyapatite (HA-NPs), Gold (Au-NPs), Chitosan (C-NPs), Gold/hydroxyapatite (Au/HA-NPs) and Chitosan/hydroxyapatite (CH-NPs) on bone marrow- derived mesenchymal stem cells (BM-MSCs). These nanostructures were characterized using transmission electron microscope and Zetasizer. MSCs were isolated from bone marrow of rat femur bones and their identity was documented by morphology, flow cytometry and multi-potency capacity. The influence of the selected nanostructures on the viability, osteogenic differentiation and subsequent matrix mineralization of BM-MSCs was determined by MTT assay, molecular genetic analysis and alizarin red S staining, respectively. MTT analysis revealed insignificant toxicity of the tested nanostructures on BM-MSCs at concentrations ranged from 2 to 25µg/ml over 48h and 72h incubation period. Notably, the tested nanostructures potentiate the osteogenic differentiation of BM-MSCs as evidenced by a prominent over-expression of runt-related transcription factor 2 (Runx-2) and bone morphogenetic protein 2 (BMP-2) genes after 7days incubation. Moreover, the tested nanostructures induced matrix mineralization of BM-MSCs after 21days as manifested by the formation of calcium nodules stained with alizarin red S. Conclusively, these data provide a compelling evidence for the functionality of the studied nanostructures as osteoinductive materials motivating the differentiation of BM-MSCs into osteoblasts with the most prominent effect observed with Au-NPs and Au/HA-NPs, followed by CH-NPs.

Effect of 25-Hydroxycholesterol on the Differentiation of Bone Marrow Mesenchymal Stem Cells Through Wnt5/TGF-β Signaling Pathway

Bone marrow mesenchymal stem cells (BMSCs) can be multi-directionally differentiated and are widely used in tissue engineering. 25-hydroxycholesterol (25-HC) can induce osteogenesis and is involved in osteogenic formation. However, the role of 25-hydroxycholesterol in BMSCs is unclear. Rat BMSCs were isolated and divided into control group and 25-HC treatment (2 and 4 μM) group. Cell proliferation was detected by MTT assay. Caspase-3 and ALP activity was analyzed. Real time PCR was done to analyze Runx2, OPN, FABP4 and PPARγ2 expression. Red staining detects the calcified nodule formation. Wnt5 level was detected by western blot and TGF-β secretion was analyzed by ELISA. 25-HC treatment significantly inhibited cell proliferation, increased Caspase 3 activity, decreased ALP activity and the expression of Runx2 and OPN, increased expression of FABP4 and PPARγ2, decreased formation of calcified nodules, secretion of TGF-β and reduced expression of Wnt5 compared to control group (P < 0.05), and the above changes were significant with the increase of the concentration of 25-HC (P < 0.05). 25-hydroxycholesterol regulates the proliferation and apoptosis of BMSCs by regulating Wnt5/TGF-β signaling pathway, inhibiting the differentiation of BMSCs into osteogenic direction and promoting its adipogenic differentiation.

Uncarboxylated osteocalcin promotes osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells by activating the Erk-Smad/β-catenin signalling pathways.

Uncarboxylated osteocalcin (unOc) is an osteoblast-derived hormone with multiple regulatory functions. Osteocalcin knockdown delays the maturation of mineral species and downregulates the expression of osteogenic-specific genes in human mesenchymal stromal cells. However, the underlying mechanisms remain unclear. Here, we investigated the effects of unOc on the osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells (BMSCs) and discovered that unOc promoted osteogenic differentiation of BMSCs, which was characterized by increases in alkaline phosphatase (ALP) activity, type I collagen (COLI) production, calcified nodule formation, and expression of osteogenic-specific genes including the osterix, runt-related transcription factor 2 (Runx2), ALP, and COLI genes. Further experiments indicated that unOc promoted the osteogenic differentiation of BMSCs via activation of the Erk-Smad/β-catenin signalling pathways. SIGNIFICANCE OF THE STUDY: Osteoporosis is associated with the osteogenic differentiation of BMSCs. In recent years, the role of unOc function as an endocrine hormone has received much attention. In this study, we reported for the first time that unOc promoted the osteogenic differentiation of mouse BMSCs through Erk-Smad/β-catenin signalling pathway. Our results highlight the importance of unOc as a hormone in promoting the osteogenic differentiation of BMSCs, indicating that this hormone may be beneficial in treatments for osteoporosis and fracture healing.

Transferrin promotes calcified nodule formation in rat bone marrow cell culture with dexamethasone

Some chemicals can promote the differentiation of stem cells and the formation of hard tissues. In this study, the effects of transferrin on calcified nodule formation were evaluated using bone marrow cells obtained from the femora of Fischer 344 rats. Transferrin was added to the culture medium to promote the proliferation and differentiation of stem cells among the bone marrow cells into osteoblasts or chondroblasts. Calcified nodule formation was confirmed macroscopically in the culture medium and evaluated quantitatively by measuring Ca2+ in the solution after demineralization using formic acid. The cells were cultured in 2 ml of minimum essential medium with 20 μl of solution containing 100, 200 or 400 ng of transferrin. Dexamethasone was also added to the medium at 10 nmol. Subculturing was performed for 2 weeks. The concentration of Ca2+ after decalcification of the calcified nodule formed in the bone marrow cell culture with dexamethasone was approximately 10 mg/ml. The addition of 100~200 ng of transferrin to the bone marrow cell culture resulted in the maximum concentration of Ca2+ and size of the formed calcified nodule in the medium. For calcified nodule formation by bone marrow cells, the optimal concentration of transferrin in the culture medium was suggested to be 200 ng in 2 ml. This study suggested that transferrin is beneficial together with dexamethasone. It may play an important role in bone formation in vivo.

Transferrin promotes calcified nodule formation in rat bone marrow cell culture with dexamethasone

Transferrin promotes calcified nodule formation in rat bone marrow cell culture with dexamethasone

Improvement of osteogenic differentiation of human mesenchymal stem cells on composite poly l-lactic acid/nano-hydroxyapatite scaffolds for bone defect repair

Tissue engineering offers new approaches to repair bone defects, which cannot be repaired physiologically, developing scaffolds that mimic bone tissue architecture. Furthermore, biomechanical stimulation induced by bioreactor, provides biomechanical cues that regulate a wide range of cellular events especially required for cellular differentiation and function. The improvement of human mesenchymal stem cells (hMSCs) colonization in poly-l-lactic-acid (PLLA)/nano-hydroxyapatite (nHA) composite scaffold was evaluated in terms of cell proliferation (dsDNA content), bone differentiation (gene expression and protein synthesis) and ultrastructural analysis by comparing static (s3D) and dynamic (d3D) 3D culture conditions at 7 and 21 days. The colonization rate of hMSCs and osteogenic differentiation were amplified by d3D when physical stimulation was provided by a perfusion bioreactor. Increase in dsDNA content (p < 0.0005), up-regulation of RUNX2, ALPL, SPP1 (p < 0.0005) and SOX9 (p < 0.005) gene expression, and more calcium nodule formation (p < 0.0005) were observed in d3D cultures in comparison to s3D ones over time. Dynamic 3D culture, mimicking the mechanical signals of bone environment, improved significantly osteogenic differentiation of hMSCs on PLLA/nHA scaffold, without the addition of growth factors, confirming this composite scaffold suitable for bone regeneration.

Effect of SIRT1 on Bone Marrow Stromal Stem Cells in High Glucose Environment and Related Mechanisms

Silencing information regulator (SIRT1) involves in endocrine diseases. However, whether SIRT1 participates in BMSCs under high glucose environment remains unclear. Rat BMSCs were isolated and divided into control group; high glucose group; and SIRT1 group, in which SIRT1 agonist (Resveratrol) was added to high glucose BMSCs followed by analysis of SIRT1, Bax and Bcl-2 expression by real time PCR and ELISA, cell proliferation by MTT assay, Caspase3 activity, ALP activity, calcification nodule formation by alizarin red staining, changes of ROS and SOD activities, PI3K signaling pathway by Western blot. SIRT1 expression was significantly decreased in BMSCs in high glucose group, with inhibited cell proliferation, increased Caspase 3 activity, Bax expression, and ROS content, decreased Bcl-2 expression, ALP activity, SOD activity, as well as reduced formation of calcified nodules and phosphorylation of PI3K compared to control (P &lt; 0.05). Addition of Resveratrol significantly promoted SIRT1 expression and cell proliferation of BMSCs in high glucose group, decreased Caspase 3 activity, Bax expression, and ROS content, increased Bcl-2 expression, ALP activity, SOD activity, as well as increased formation of calcified nodules and phosphorylation of PI3K (P &lt; 0.05). SIRT1 expression is decreased in BMSCs in high glucose group. Increasing SIRT1 expression in BMSCs under high glucose environment promoted proliferation of BMSCs and the formation of calcified nodules by regulating oxidative stress and activating PI3K signaling pathway.

Effect of GGCX on the differentiation function of osteoporosis bone marrow mesenchymal stem cells through regulating TGFβ/smad signaling pathway.

Osteoporosis (OP) has a high incidence and can be found in multiple age groups. The bone marrow mesenchymal stem cells (BMSCs) have the potential for self-renewal and multi-directional differentiation, which are often used for investigating the differentiation function of osteoporosis bone marrow mesenchymal stem cells. γ-glutamyl carboxylase (GGCX) is a carboxylase-related carboxylase and was observed to be abnormally expressed in osteoarthritis. However, the role and related mechanisms of GGCX in OP have not been fully elucidated. This work aimed to evaluate the effect of GGCX on the differentiation function of BMSCs. Sprague-Dawley rats were randomly divided into the OP group prepared by ovariectomy and sham group. GGCX expression was tested by enzyme-linked immunosorbent assay (ELISA). BMSCs were isolated from OP rats and transfected with pcDNA-GGCX plasmids. BMSC proliferation was detected by tetrazolium salt colorimetry (MTT) assay. The osteogenic and adipogenic differentiation of BMSCs was analyzed by alizarin red staining and oil red O staining. The ALP activity was determined by alkaline phosphatase (ALP) activity colorimetric assay. Real time-PCR was used to test the expressions of osteogenesis-related genes RUNX2 and OPN mRNA. Western blot was adopted to assess the TGFβ/smad signaling pathway activity. GGCX expression was significantly decreased in the serum of OP rats compared with the sham group (p < 0.05). The transfection of pcDNA-GGCX plasmid significantly promoted BMSC cell proliferation, increased calcified nodule formation, inhibited adipogenic differentiation, enhanced ALP activity, elevated RUNX2, and OPN mRNA expressions, and upregulated TGFβ1, Smad2, and Smad7 expressions (p < 0.05). GGCX secretion is reduced in osteoporosis. GGCX can regulate osteoporosis via promoting the TGFβ/smad signaling pathway, facilitating BMSCs osteogenic differentiation, and inhibiting BMSCs adipogenic differentiation.

Bioinformatic Analysis of Transcriptomic Data Reveals Novel Key Genes Regulating Osteogenic Differentiation of Human Adipose Stem Cells.

Adipose stem cells (ASCs) are an attractive cell source for treating many human diseases including osteoporosis. However, the molecular mechanisms accounting for ASC osteogenesis are poorly known. In this study, ASCs were first isolated from the fat tissues from the patients with osteoporosis. The global transcriptome profile between osteogenic differentiated ASCs and undifferentiated ASCs was compared using RNA sequencing (RNA-seq). Then, bioinformatic analysis was performed to reveal the central genes and pathways that regulated the osteogenic differentiation of ASCs. One of the interested genes C5AR1 was chosen for further investigation. A total of 1521 upregulated and 3020 downregulated genes were identified between the ASCs with osteogenic induction and controls. Functional gene ontology analysis revealed that these significantly differentially expressed genes (DEGs) were associated with cell cycle, protein binding, and nucleotide binding. Pathway analysis showed that many canonical pathways, such as the MAPK signaling pathway and the PI3K-AKT pathway, might actively be involved in regulating osteogenic differentiation of ASCs. A total of three subnetworks and 20 central nodes were identified by the protein-protein interaction analysis. In addition, the expression level of C5AR1 was significantly increased during osteogenic differentiation of ASCs. The downregulation of C5AR1 dramatically reduced the expression levels of osteogenic differentiation biomarkers and calcium nodule formation capacity. Collectively, we have provided a number of novel genes and pathways that might be indispensable for ASC osteogenic differentiation. Manipulating the levels of this candidate gene might contribute to the osteoporosis therapy.

Heterogeneous multi-laminar tissue constructs as a platform to evaluate aortic valve matrix-dependent pathogenicity

Designing and constructing controlled in vitro cell culture platforms is imperative toward pinpointing factors that contribute to the development of calcific aortic valve disease. A 3D, laminar, filter paper-based cell culture system that was previously established as a method of analyzing valvular interstitial cell migration and protein expression was adapted here for studying the impact of specific extracellular matrix proteins on cellular viability and calcification proclivity. Hydrogels incorporating hyaluronan and collagen I, two prevalent valvular extracellular matrix proteins with altered pathological production, were designed with similar mechanics to parse out effects of the individual proteins on cell behavior. Laminar constructs containing varying combinations of discrete layers of collagen and hyaluronan were assembled to mimic native and pathological valve compositions. Proteinaceous and genetic expression patterns pertaining to cell viability and calcific potential were quantified via fluorescent imaging. A significant dose-dependency was observed, with increased collagen content associated with decreased viability and increased calcific phenotype. These results suggest that extracellular composition is influential in calcific aortic valve disease progression and will be key toward development of future tissue-engineered or pharmaceutical calcific aortic valve treatments. Statement of SignificanceCalcific aortic valve disease (CAVD), a widespread heart valve disorder, is characterized by fibrotic leaflet thickening and calcific nodule formation. This pathological remodeling is an active process mediated by the valvular interstitial cells (VICs). Currently, the only treatment available is surgical replacement of the valve – a procedure associated with significant long-term risk and morbidity. Development of effective alternate therapies is hindered by our poor understanding of CAVD etiology. Previous work has implicated the composition and mechanics of the extracellular matrix in the progression of CAVD. These individual factors and their magnitude of influence have not been extensively explored - particularly in 3D systems. Here, we have bridged this gap in understanding through the employment of a heterogeneous 3D filter-paper culture system.

The effect of external lateral stabilization on the control of mediolateral stability in walking and running.

Development of scaffolds from naturally available biomaterials for bone tissue engineering is an interesting research area. Among the different scaffolds established for this purpose, biogenic silica nanostructure (BSN) serves as a promising naturally available inorganic and biocompatible material. A few studies have demonstrated the intrinsic biological activity of synthetic silica nanoparticles. The virtually infinite promising applications of these structures rely on their erratic physicochemical properties. We have derived BSNs from Sorghum bicolor seed head using a progressive approach. The intrinsic biological activities were analyzed using human mesenchymal stem cells (hMSCs) as an in vitro model with MTT assay and acridine orange/ethidium bromide staining. We also studied the role of BSNs in the osteogenic differentiation of hMSCs using alkaline phosphatase staining, alizarin red staining, and gene expression analysis. BSNs increased the formation of calcium nodules and stimulated alkaline phosphatase (ALP) activity. Significant changes and/or upregulation in the expression of osteogenic prominent markers such as ALP, bone morphogenetic protein-2 (BMP-2), BMP-4, bone sialoprotein (BSP), collagen-1 (Col-1), and Runt-related transcription factor 2 (RUNX2) genes were observed. Taken together, these results suggest that BSNs exhibited biocompatibility and induced osteogenic differentiation of hMSCs, indicative of their potential applications for bone tissue engineering.

Effects of EZH2 inhibitor DZNeP on osteogenic differentiation of periodontal ligament stem cells

Objective To observe the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and to investigate the epigenetic regulation of EZH2 inhibitor DZNeP on osteogenic differentiation of hPDLSCs. Methods The hPDLSCs were isolated and cultured, and their proliferation under different concentrations of DZNeP (0, 1, 2, 5 and 10 μmol/L) was detected by MTT. The effects of DZNeP on osteogenic differentiation of hPDLSCs were observed by alkaline phosphatase (ALP) staining and alizarin red staining. The effect of DZNeP on the trimethylation of histone H3K27 in hPDLSCs was detected by immunofluorescence staining. Results Compared with the control group, the proliferation of hPDLSCs after 1, 2, 5 and 10 μmol/L DZNeP treatment for 48 h was significantly decreased, respectively (all P<0.05), and it was concentration-dependent. The result of ALP staining and alizarin red staining showed that DZNeP could promote the expression of early osteogenic markers ALP and the formation of advanced calcified nodules of hPDLSCs. The immunofluorescence staining result showed that the trimethylation fluorescence intensity of histone H3K27 was significantly decreased in the DZNeP group compared with the control group. Conclusions As an EZH2 inhibitor, DZNeP can inhibit the proliferation of hPDLSCs and promote the differentiation of hPDLSCs into osteoblasts in vitro, suggesting that DZNeP can be used as a potential small molecule drug for the treatment of periodontitis. Key words: Epigenetic regulation; EZH2; DZNeP; Human periodontal ligament stem cells; Osteogenic differentiation

MicroRNA-96 promotes osteoblast differentiation and bone formation in ankylosing spondylitis mice through activating the Wnt signaling pathway by binding to SOST.

Ankylosing spondylitis (AS) refers to a type of arthritis manifested with chronic inflammation of spine joints. microRNAs (MiRNAs) have been identified as new therapeutic targets for inflammatory diseases. In this study, we evaluated the influence of microRNA-96 (miR-96) on osteoblast differentiation together with bone formation in a murine model of AS. The speculated relationship that miR-96 could bind to sclerostin (SOST) was verified by dual luciferase reporter assay. After successful model establishment, the mice with AS and osteoblasts isolated from mice with AS were treated with mimics or inhibitors of miR-96, or DKK-1 (a Wnt signaling inhibitor). The effects of gain- or loss-of-function of miR-96 on the inflammatory cytokine release (IL-6, IL-10, and TNF-α), alkaline phosphatase (ALP) activity, calcium nodule formation, along with the viability of osteoblasts were determined. It was observed that miR-96 might target and regulate SOST. Besides, miR-96 was expressed at a high level in AS mice while SOST expressed at a low level. TOP/FOP-Flash luciferase reporter assay confirmed that miR-96 activated the Wnt signaling pathway. Moreover, AS mice overexpressing miR-96 exhibited increased contents of IL-6, IL-10 and TNF-α, ALP activity, calcium nodule numbers, and viability of osteoblasts. In contrast, inhibition of miR-96 resulted in suppression of the osteoblast differentiation and bone formation. In conclusion, the study implicates that overexpressing miR-96 could improve osteoblast differentiation and bone formation in AS mice via Wnt signaling pathway activation, highlighting a potential new target for AS treatment.

Correlational study on altered epicardial adipose tissue as a stratification risk factor for valve disease progression through IL-13 signaling

AimsGenetic and environmental factors all interact in the risk of progression of valvular dysfunctions. Previous studies reported a relation between valve diseases and epicardial adipose tissue (EAT) thickness. The aim of this study was to verify the possible relationship between the molecular pattern of EAT related to IL-13 fibrogenic cytokine expression and valve dysfunction. Methods and resultsA valvular heart disease (VHD) population was stratified according to their median EAT thickness (7 mm). The molecular expression of IL-13 in EAT is directly related to the molecular expression of genes associated with extracellular matrix (ECM) turnover, macrophage infiltration and promotion of the formation of ectopic calcific nodules involved in aorta coarctation and calcification. ConclusionIL-13 gene expression in altered EAT is directly related to the expression of genes involved in ECM turnover and the formation of ectopic calcific nodules, suggesting measurements of EAT as a stratification risk factor for valve instability in the VHD patients.

Preparation, Characterization and In Vitro Biological Evaluation of a Novel Pearl Powder/Poly-Amino Acid Composite as a Potential Substitute for Bone Repair and Reconstruction.

Many studies about fabricating organic-inorganic composite materials have been carried out in order to mimic the natural structure of bone. Pearl, which has a special block-and-mortar hierarchical structure, is a superior bone repair material with high osteogenic activity, but it shows few applications in the clinical bone repair and reconstruction because of its brittle and uneasily shaped properties. In this work, pearl powder (P)/poly (amino acid) (PAA) composites were successfully prepared by a method of in situ melting polycondensation to combine the high osteogenic activity of the pearl and the pliability of the PAA. The mechanical properties, in vitro bioactivity and biocompatibility as well as osteogenic activity of the composites were investigated. The results showed that P/PAA composites have both good mechanical properties and bioactivity. The compressive strength, bending strength and tensile strength of the composites reached a maximum of 161 MPa, 50 MPa and 42 MPa, respectively; in addition, apatite particles successfully deposited on the composites surface after immersion in simulated body fluid (SBF) for 7 days indicated that P/PAA composites showed an enhanced mineralization capacity and bioactivity due to incorporation of pearl powder and PAA. The cell culture results revealed that higher cell proliferation and better adhesion morphology of mouse bone marrow mesenchymal stem cells (MSCs) appeared on the composite surface. Moreover, cells growing on the surface of the composites exhibited higher alkaline phosphatase (ALP) activity, more calcium nodule-formation, and higher expression levels of osteogenic differentiation-related genes (COL 1, RunX2, OCN, and OPN) than cells grown on PAA surface. The P/PAA composites exhibited both superior mechanical properties to the pearl powder, higher bioactivity and osteogenic capability compared with those of PAA.

Dipeptidyl peptidase-4 promotes calcification of human vascular smooth muscle cells by inhibiting LincRNA ENST00000540293 expression

Objective To explore the molecular mechanism of dipeptidyl peptidase-4(DPP4)in the calcification of human vascular smooth muscle cells(HVSMCs). Methods The osteogenic differentiation of HVSMCs was induced by 200 ng/ml DPP4 as calcification model. The differentially expressed long non-coding RNAs(lncRNAs)between DPP4 group and control group were analyzed by microarray, and the microarray results of LincRNA ENS0540293 were validated by real-time PCR. After HVSMCs were incubated with LincRNA ENS0540293 silencing positive reagent for 48 h, the expressions of calcification-related proteins osteoprotegerin(OPG)and bone morphogenetic protein 2(BMP-2)were detected by Western blotting and the formation of calcified nodules was observed by Alizarin red staining. Results The protein expressions of OPG and BMP-2 in HVSMCs were significantly increased after DPP4 intervention(P<0.05), with the increased formation of calcified nodules. RT-qPCR showed that LincRNA ENS0540293 expression was significantly decreased in DPP4 group as compared with the control group(P<0.05). The expressions of calcification-related proteins OPG and BMP-2 were significantly increased after LincRNA ENS0540293 silence(P<0.05). Conclusion DPP4 may promote the calcification of HVSMC through inhibiting LincRNA ENS0540293 expression. Key words: Dipeptidyl peptidase-4; Vascular calcification; Microarray; Long non-coding RNA

Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α Inhibits Vascular Calcification Through Sirtuin 3-Mediated Reduction of Mitochondrial Oxidative Stress.

Aims: Vascular calcification is associated with cardiovascular death in patients with chronic kidney disease (CKD). Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) plays an important role in various cardiovascular diseases. However, its role in vascular calcification remains unknown. Results: Adenine-induced rat CKD model was used to induce arterial medial calcification. The level of PGC-1α decreased in abdominal aorta of CKD rats. Overexpression of PGC-1α significantly ameliorated calcium deposition in rat abdominal aorta, isolated carotid rings, and cultured vascular smooth muscle cells (VSMCs). Mitochondrial reactive oxygen species (mtROS) increased in calcifying aorta and VSMCs. Upregulation of PGC-1α inhibited, whereas PGC-1α depletion promoted β-glycerophosphate-induced mtROS production and calcium deposition. Moreover, PGC-1α increased superoxide dismutase 1 (SOD1) and SOD2 contents in vivo and in vitro, whereas SOD2 deletion eliminated PGC-1α-mediated mtROS change and promoted calcium deposition. Mechanistically, sirtuin 3 (SIRT3) expression declined in calcifying aorta and VSMCs, while PGC-1α overexpression restored SIRT3 expression. Inhibition of SIRT3 by 3-TYP or siRNA (small interfering RNA) reduced PGC-1α-induced upregulation of SOD1 and SOD2, and abolished the protective effect of PGC-1α on calcification of VSMCs. Importantly, PGC-1α was reduced in calcified femoral arteries in CKD patients. In phosphate-induced human umbilical arterial calcification, upregulation of PGC-1α attenuated calcium nodule formation, while this protective effect was abolished by SIRT3 inhibitor. Innovation: We showed for the first time that PGC-1α is an important endogenous regulator against vascular calcification. Induction of PGC-1α could be a potential strategy to treat vascular calcification in CKD patients. Conclusions: PGC-1α protected against vascular calcification by SIRT3-mediated mtROS reduction.

Modulation of a new pathway prevent valvular interstitial cells calcification, a potential innovative therapeutic target in aortic valve stenosis

Aortic valve stenosis (AVS) is the narrowing of the aortic valve (AV) opening caused by fibrosis and calcification. Despite this paradigm, no targeted medical therapy has proven to prevent, slow down or treat AVS. Using transcriptomic approach on human valvular interstitial cells (VIC) isolated from fibrocalcific and normal valves, our team has identified an enzyme X downregulated in fibro-calcific VIC. This enzyme X and its product Y have documented anti-calcification effects in other cell types than VIC. Our goal is to decipher the implication of this pathway on an in vitro model of human primary cultures of VIC in ostegenic conditions. Normal AV unsuitable for grafting and fibrocalcific AV from valve replacement (ATHERAO protocol) were collected. Levels of enzyme X in AV was assessing by histology and by Western Blot on tissue homogenate. VIC isolation was realized by enzymatic digestion of normal AV. Primary cultures were exposed to ostegenic media (OM) or control media with or without the product Y of the enzyme (1–1000 nM) during 7 days. Evaluation of calcification was realized by alizarin red staining, mRNA levels of calcification markers. We confirmed a differential expression of enzyme X on tissue by histology and by western blot (− 5.2 fold, P < 0.05) from fibro-calcific AV compared to those from control AV. In OM, enzyme X is decreased at the mRNA (− 5.2 fold, P < 0.05) and protein level (− 5.2 fold, P < 0.05) compared to control media. VIC treatments with product Y in presence of OM decrease the formation of calcium nodules in a dose dependent manner (− 5.2 fold, P < 0.05, vs OM) and mRNA levels for RUNX2 (− 2.4 fold, P < 0.05 vs OM), attenuating osteoblastic VIC differentiation. Our results demonstrate the involvement of this new pathway in an in vitro model of AVS by decreasing human VIC calcification and associated markers. Enzyme X is a potential innovative pharmacological therapeutic target in AVS.

Effects of graphene oxide and graphene oxide quantum dots on the osteogenic differentiation of stem cells from human exfoliated deciduous teeth

Graphene and its derivatives, graphene oxide (GO) and graphene oxide quantum dots (GOQDs), have recently attracted much attention as bioactive factors in differentiating stem cells towards osteoblastic lineage. The stem cells from human exfoliated deciduous teeth (SHEDs) possess the properties of self-renewal, extensive proliferation, and multiple differentiation potential, and have gradually become one of the most promising mesenchymal stem cells (MSCs) in bone tissue engineering. The purpose of this study was to explore the effects of GO and GOQDs on the osteogenic differentiation of SHEDs. In this study, GO and GOQDs facilitated SHED proliferation up to 7 days in vitro at the concentration of 1 μg/ml. Because of their excellent fluorescent properties, GOQD uptake by SHEDs was confirmed and distributed in the SHED cytoplasm. Calcium nodules formation, alkaline phosphatase (ALP) activity, and RNA and protein expression increased significantly in SHEDs treated with osteogenic induction medium containing GOQDs but decreased with osteogenic induction medium containing GO. Interestingly, the Wnt/β-catenin signaling pathway appeared to be involved in osteogenic differentiation of SHEDs induced with GOQDs. In summary, GO and GOQDs at the concentration of 1 μg/ml promoted SHED proliferation. GOQDs induced the osteogenic differentiation of SHEDs, whilst GO slightly inhibited it.