Effect of Oxidized Low Density Lipoprotein on the Expression of Runx2 and SPARC Genes in Vascular Smooth Muscle Cells.

BACKGROUND Vascular calcification is an important stage in atherosclerosis. During this stage, vascular smooth muscle cells (VSMC) synthesize many osteogenic factors such as osteonectin (encoded by SPARC). Oxidative stress plays a critical role in atherosclerosis progression, and its accumulation in the vascular wall stimulates the development of atherosclerosis and vascular calcification. The osteonectin overexpression has been observed in the arterial wall during the course of atherosclerosis. However, the regulatory mechanism of oxidized low density lipoprotein (oxLDL)-mediated vascular calcification remains to be clarified. The aim of this study was to investigate the effect of oxLDL on the osteonectin gene expression through the Runx2 transcription factor. METHODS In this experimental study, VSMC were cultured in F-12K media and then treated with oxLDL. The expression of Runx2 and osteonectin genes was determined by real-time PCR method. Protein levels were investigated by the western blotting technique. The Runx2 gene was knocked down using siRNA in order to determine whether Runx2 regulates the osteonectin expression in VSMC induced by oxLDL. Then transfected cells were treated with oxLDL, and the expression levels of Runx2 and osteonectin were determined again. RESULTS oxLDL was found to increase Runx2 and osteonectin gene expression (4.8±0.47- and 9.2±1.96-fold, respectively) after 48 h. Western blotting analysis confirmed the induced levels of Runx2 and osteonectin proteins. However, oxLDL-induced osteonectin expression was not observed to be blocked by Runx2 knockdown. CONCLUSION The up-regulation of osteonectin by oxLDL is independent of Runx2, and it may be mediated by other transcription factors

Caspase-independent programmed cell death triggers Ca2PO4 deposition in an in vitro model of nephrocalcinosis

We provide evidence of caspase-independent cell death triggering the calcification process in GDNF-silenced HK-2 cells

The Effect of Adiponectin on Osteonectin Gene Expression by Oxidized Low Density Lipoprotein-Treated Vascular Smooth Muscle Cells

Osteonectin is a bone-associated protein involved in vascular calcification. Adiponectin may protect against cardiovascular disease but possible effects on vascular calcification have been poorly studied. The aim of this study was to investigate the modulatory effect of adiponectin on oxidized low density lipoprotein (oxLDL)-induced expression of osteonectin in human aorta vascular smooth muscle cells (HA/VSMCs). HA/VSMCs were cultured in F12K media and then treated with oxLDL (100 mu g/mL) in the presence or absence of adoponectin (5 mu g/mL) for 24 and 48 hours. mRNA expression and protein level of osteonectin were determined by quantitative real-time PCR and western blot analysis, respectively. After exposure to oxLDL, osteonectin expression increased 1.62 +/- 0.23- and 6.62 +/- 0.48-fold after 24 and 48 hours respectively compared to the control. Adiponectin increased oxLDL-induced osteonectin expression in a time-dependent manner after 24 and 48 hours (3.24 +/- 0.39- and 24.93 +/- 2.15-fold, respectively). Western blotting confirmed that osteonectin protein was upregulated by adiponectin. Our data suggest that OxLDL might cause the increase of osteonectin expression both at mRNA and protein level. This upregulation is intensified by adiponectin

Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices

Journal of Osseointegration Volume 7, Issue 1, 1 March 2015, Pages 2-7 Open Access Genetical stability and osteogenic ability of mesenchimal stem cells on demineralized bone matrices (Article) Pozzuoli, A.a, Gardin, C.b, Aldegheri, R.a, Bressan, E.c, Isola, M.d, Calvo-Guirado, J.L.e, Biz, C.a, Arrigoni, P.a, Feroni, L.b, Zavan, B.b a Department of Surgical,Oncological and Gastroenterological Sciences, University of Padua, Padua, Italy b Department of Biomedical Sciences, University of Padua, Padua, Italy c Department of Neurosciences, University of Padua, Padua, Italy d Department of Animal Medicine, Production and Health (MAPS), Italy e Department of General Dentistry, Faculty of Medicine and Dentistry, University of Murcia, Murcia, Spain Hide additional affiliations View references (44) Abstract Aim: Tissue engineering is a rapidly expanding field with regard to the use of biomaterials and stem cells in the orthopedic surgery. Many experimental studies have been done to understand the best characteristics of cells, materials and laboratory methods for safe clinical applications. The aim of this study was to compare the ability of 2 different human demineralized bone matrices (DBMs), the one enriched and the other not enriched with hyaluronic acid, to stimulate in vitro the proliferation and the osteogenic differentiation of human adipose-derived stem cells (ADSCs) seeded onto an osteoconductive scaffold. Materials and Methods: ADSCs were isolated, by enzymatic digestion, from abdominal adipose tissue of 5 patients undergoing cosmetic lipoaspiration surgery. ADSCs were then seeded onto a 3D scaffold in the presence of the two different osteoinductive matrices of human demineralized bone and evaluated for proliferation and osteogenic differentiation. The safety of the methods was verified using array-Comparative Genomic Hybridization (array-CGH). Results: ADSCs were able to differentiate in osteogenic sense. Both DBMs showed the ability to induce osteogenic differentiation of the cells. Conclusion: array-CGH showed no changes at genome level, thus confirming the safety of materials and method

Histomorphometric evaluation of bone regeneration induced by biodegradable scaffolds as carriers for dental pulp stem cells in a rat model of calvarial "critical size" defect

Objective: The aim of this study was to test specific stem cells that could enhance bone formation in combination with specific scaffolds. Methods: Dental Pulp Stem Cells (DPSCs) were seeded with Granular Deproteinized Bovine Bone (GDPB) or Beta-Tricalcium Phosphate (ß-TCP) in a rat model of calvarial "critical size" defect. DPSCs were isolated from permanent human teeth, obtained and characterized using specific stem cells markers (Nanog and Oct-4) by real time-PCR and immunofluorescence. Cells were differentiated for 10-15 days towards the osteoblastic phenotype with 100μM L-ascorbic acid, added every day in culture medium and 20 vol. percentage of FBS in α-MEM medium. Osteogenic commitment was evaluated with real time-PCR by measuring the expression of specific markers (osteonectin and runx2). When a sufficient cell number was obtained, DPSCs were trypsinized, washed in culture medium and seeded onto the GDPB and ß-TCP scaffold sat a density of 0.5-1×106 cells/scaffold. Two bilateral critical-size circular defects (5 mm diameter; 1 mm thickness) were created from the parietal bone of the 8 athymic T-cell deficient nude rats. One cranial defect for each rat was filled with the scaffold alone and the other defect with the scaffold seeded with stem cells. After 12 weeks post-surgery animals were euthanized and histomorphometric analysis was performed. Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's Protected Least Significant Difference (PLSD) post-hoc test. A p-value <0.05 was considered statistically significant. Results: GDPB group presented higher percentage of lamellar bone than that of GDPB/DPSC, ß-TCP alone had lower levels as compared to ß-TCP/DPSC. The addition of stem cells significantly increased woven bone formation in both scaffold-based implants, although still higher in GDPB based implants. Conclusion: Our findings indicate that GDPB and ß-TCP used as scaffold to induce bone regeneration may benefit from adding DPSC to tissue-engineered constructs

Porcine bone scaffolds adsorb growth factors secreted by MSCs and improve bone tissue repair

An ideal tissue-engineered bone graft should have both excellent pro-osteogenesis and pro-angiogenesis properties to rapidly realize the bone regeneration in vivo . To meet this goal, in this work a porcine bone scaffold was successfully used as a Trojan horse to store growth factors produced by mesenchymal stem cells (MSCs). This new scaffold showed a time-dependent release of bioactive growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in vitro . The biological effect of the growth factors-adsorbed scaffold on the in vitro commitment of MSCs into osteogenic and endothelial cell phenotypes has been evaluated. In addition, we have investigated the activity of growth factor-impregnated granules in the repair of critical-size defects in rat calvaria by means of histological, immunohistochemical, and molecular biology analyses. Based on the results of our work bone tissue formation and markers for bone and vascularization were significantly increased by the growth factor-enriched bone granules after implantation. This suggests that the controlled release of active growth factors from porcine bone granules can enhance and promote bone regeneratio

Characterization of Sparus aurata osteonectin cDNA and in silico analysis of protein conserved features: Evidence for more than one osteonectin in Salmonidae

Osteonectin is a matricellular protein involved in various cellular mechanisms but its exact function remains unclear despite numerous studies. We present here the cloning of Sparus aurata partial osteonectin cDNA and the reconstruction of 15 other sequences from both vertebrates and invertebrates, almost doubling the set of available sequences (a total of 35 sequences is now available). Taking advantage of the resulting large amount of data, we have created multiple sequence alignments and identified osteonectin putative conserved features (intra- and inter-disulfide bonds, collagen- and calcium-binding domains and phosphorylation sites) likely to be important for protein structure and function. This work also provides the first evidence for the presence of more than one osteonectin in some species. Finally, S. aurata osteonectin gene expression has been shown to initiate during larval development shortly after gastrulation, and to be high in bone-derived cell lines while down-regulated during extracellular matrix mineralization, further emphasizing the important role of osteonectin in skeletal development and bone formation

Oxidized Low-Density Lipoprotein and Upregulated Expression of Osteonectin and Bone Sialoprotein in Vascular Smooth Muscle Cells

Background: Oxidative stress has been associated with the progression of atherosclerosis and activation of genes that lead to increased deposition of proteins in the extracellular matrix. Bone sialoprotein (BSP) and osteonectin are proteins involved in the initiation and progression of vascular calcification. Objective: To investigate the effect of oxidized low-density lipoprotein on osteonectin and BSP expression in human aorta vascular smooth muscle cells (HA/VSMCs). Methods: We treated HA/VSMCs with oxidized low-density lipoprotein (oxLDL) and measured the relative expression of osteonectin and BSP genes using the real-time polymerase chain reaction (PCR) method. We investigated the protein levels produced by each gene using the western blotting technique. Results: oxLDL increased osteonectin and BSP levels (mean SD], 9.1 2.1]-fold and 4.2 0.75]-fold, respectively) after 48 hours. The western blotting results also confirmed the increased levels of osteonectin and BSP. Conclusion: oxLDL may enhance vascular calcification by promoting the expression of osteonectin and BSP

Bioactive sphene-based ceramic coatings on cpTi substrates for dental implants: An in vitro study

Titanium implant surface modifications have been widely investigated to favor the process of osseointegration. The present work aimed to evaluate the effect of sphene (CaTiSiO5) biocoating, on titanium substrates, on the in vitro osteogenic differentiation of Human Adipose-Derived Stem Cells (hADSCs). Sphene bioceramic coatings were prepared using preceramic polymers and nano-sized active fillers and deposited by spray coating. Scanning Electron Microscopy (SEM) analysis, surface roughness measurements and X-ray diffraction analysis were performed. The chemical stability of the coatings in Tris-HCl solution was investigated. In vitro studies were performed by means of proliferation test of hADSCs seeded on coated and uncoated samples after 21 days. Methyl Thiazolyl-Tetrazolium (MTT) test and immunofluorescent staining with phalloidin confirmed the in vitro biocompatibility of both substrates. In vitro osteogenic differentiation of the cells was evaluated using Alizarin Red S staining and quantification assay and real-time PCR (Polymerase Chain Reaction). When hADSCs were cultured in the presence of Osteogenic Differentiation Medium, a significantly higher accumulation of calcium deposits onto the sphene-coated surfaces than on uncoated controls was detected. Osteogenic differentiation on both samples was confirmed by PCR. The proposed coating seems to be promising for dental and orthopedic implants, in terms of composition and deposition technology

Overexpression of SPARC obliterates the in vivo tumorigenicity of human hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third leading cause of cancer-related death worldwide. Current treatments are extremely disappointing. SPARC (Secreted protein, acidic and rich in cysteine) is a matricellular glycoprotein with differential expression in several tumors, including HCC, which significance remains unclear. We infected HCC cells (HepG2, Hep3B and Huh7) with an adenovirus expressing SPARC (AdsSPARC) to examine the role of SPARC expression on HCC cells and its effect on tumor aggressiveness. The in vitro HCC cells substrate-dependent proliferation and cell cycle profile were unaffected; however, SPARC overexpression reduced HCC proliferation when cells were grown in spheroids. A mild induction of cellular apoptosis was observed upon SPARC overexpression. SPARC overexpression resulted in spheroid growth inhibition in vitro while no effects were found when recombinant SPARC was exogenously applied. Moreover, the clonogenic and migratory capabilities were largely decreased in SPARC-overexpressing HCC cells, altogether suggesting a less aggressive HCC cell phenotype. Consistently, AdsSPARC-transduced cells showed increased E-cadherin expression and a concomitant decrease in N-cadherin expression. Furthermore, SPARC overexpression was found to reduce HCC cell viability in response to 5-FU-based chemotherapy in vitro, partially through induction of apoptosis. In vivo experiments revealed that SPARC overexpression in HCC cells inhibited their tumorigenic capacity and increased animal survival through a mechanism that partially involves host macrophages. Our data suggest that SPARC overexpression in HCC cells results in a reduced tumorigenicity partially through the induction of mesenchymal-to-epithelial transition (MET). These evidences point to SPARC as a novel target for HCC treatment.Fil: Atorrasagasti, María Catalina. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Malvicini, Mariana. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Aquino, Jorge Benjamin. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Alaniz, Laura Daniela. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: García, Mariana Gabriela. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bolontrade, Marcela Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rizzo, Manglio Miguel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Podhajcer, Osvaldo Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Mazzolini Rizzo, Guillermo Daniel. Universidad Austral. Facultad de Ciencias Biomédicas. Laboratorio de Terapia Genética; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin