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1.  Contribution of Endothelial Cells to Human Bone-Derived Cells Expansion in Coculture 
Tissue Engineering. Part A  2012;19(3-4):393-402.
Creating a functional vascularized bone tissue remains one of the main goals of bone tissue engineering. Recently, a growing interest in the crosstalk between endothelial cells (EC) and osteoblasts (OB), the two main players in a new bone formation, has been observed. However, only a few reports have addressed a mutual influence of OB and EC on cell proliferation. Our study focuses on this issue by investigating cocultures of human bone-derived cells (HBDC) and human umbilical vein endothelial cells (HUVEC). Three various proportions of cells have been used that is, HBDC:HUVEC 1:1, 1:4, and 4:1 and the cocultures were investigated on day 1, 4, and 7, while HUVEC and HBDC monocultures served as reference. We have detected enhanced alkaline phosphatase (ALP) activity in a direct HBDC–HUVEC coculture. This effect was not observed when cells were separated by an insert, which is consistent with other reports on various OB–EC lineages. The appearance of gap-junctions in coculture was confirmed by a positive staining for connexin 43. The number of cells of both phenotypes has been determined by flow cytometry: CD-31-positive cells have been considered EC, while CD-31-negative have been counted as OB. We have observed an over 14-fold increase in OB number after a week in the 1:4 HBDC:HUVEC coculture as compared with less than fourfold in monoculture. The increase in HBDC number in 1:1 coculture has been less pronounced and has reached the value of about sevenfold. These results correspond well with the cell proliferation rate, which has been measured by 5-bromo-2′-deoxyuridine incorporation. Moreover, at day 7 EC have been still present in the coculture, which is inconsistent with some other reports. Real-time polymerase chain reaction analysis has revealed the upregulation of ALP and collagen type I genes, but not osteocalcin gene, in all the cocultures grown without pro-osteogenic additives. Our study indicates that HUVEC significantly promote HBDC expansion and upregulate collagen I gene expression in these cells. We believe that these findings have application potency in bone tissue engineering.
PMCID: PMC3542902  PMID: 22924666
2.  Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells 
PLoS ONE  2012;7(12):e51547.
Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-α. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that β-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.
PMCID: PMC3519707  PMID: 23251572
3.  Statins Impair Glucose Uptake in Tumor Cells1 
Neoplasia (New York, N.Y.)  2012;14(4):311-323.
Statins, HMG-CoA reductase inhibitors, are used in the prevention and treatment of cardiovascular diseases owing to their lipid-lowering effects. Previous studies revealed that, by modulating membrane cholesterol content, statins could induce conformational changes in cluster of differentiation 20 (CD20) tetraspanin. The aim of the presented study was to investigate the influence of statins on glucose transporter 1 (GLUT1)-mediated glucose uptake in tumor cells. We observed a significant concentration- and time-dependent decrease in glucose analogs' uptake in several tumor cell lines incubated with statins. This effect was reversible with restitution of cholesterol synthesis pathway with mevalonic acid as well as with supplementation of plasma membrane with exogenous cholesterol. Statins did not change overall GLUT1 expression at either transcriptional or protein levels. An exploratory clinical trial revealed that statin treatment decreased glucose uptake in peripheral blood leukocytes and lowered 18F-fluorodeoxyglucose (18F-FDG) uptake by tumor masses in a mantle cell lymphoma patient. A bioinformatics analysis was used to predict the structure of human GLUT1 and to identify putative cholesterol-binding motifs in its juxtamembrane fragment. Altogether, the influence of statins on glucose uptake seems to be of clinical significance. By inhibiting 18F-FDG uptake, statins can negatively affect the sensitivity of positron emission tomography, a diagnostic procedure frequently used in oncology.
PMCID: PMC3349257  PMID: 22577346
4.  Modulation of endothelial cell migration by extracellular nucleotides. Involvement of focal adhesion kinase and phosphatidylinositol 3-kinase - mediated pathways 
Thrombosis and haemostasis  2005;93(4):735-742.
Extracellular nucleotides bind to type-2 purinergic/pyrimidinergic (P2) receptors that mediate various responses, such as cell activation, proliferation and apoptosis, implicated in inflammatory processes. The role of P2 receptors and their associated signal transduction pathways in endothelial cell responses has not been fully investigated. Here, it is shown that stimulation of human umbilical vein endothelial cells (HUVEC) with extracellular ATP or UTP increased intracellular free calcium ion concentrations ([Ca2+]i), induced phosphorylation of focal adhesion kinase (FAK), p130cas and paxillin, and caused cytoskeletal rearrangements with consequent cell migration. Furthermore, UTP increased migration of HUVEC in a phosphatidylinositol 3-kinase (PI3-K)-dependent manner. BAPTA or thapsigargin inhibited the extracellular nucleotide-induced increase in [Ca2+]i, a response crucial for both FAK phosphorylation and cell migration. Furthermore, long-term exposure of HUVEC to ATP and UTP, agonists of the G protein-coupled P2Y2 and P2Y4 receptor subtypes, caused upregulation of αv integrin expression, a cell adhesion molecule known to directly interact with P2Y2 receptors. Our results suggest that extracellular nucleotides modulate signaling pathways in HUVEC influencing cell functions, such as cytoskeletal changes, cellular adhesion and motility, typically associated with integrin-activation and the action of growth factors. We propose that P2Y2 and possibly P2Y4 receptors mediate those responses that are important in vascular inflammation, atherosclerosis and angiogenesis.
PMCID: PMC2830093  PMID: 15841322
endothelial cells; extracellular nucleotides; migration; P2 receptors; FAK
5.  Loss of ATP Diphosphohydrolase Activity with Endothelial Cell Activation 
Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.
PMCID: PMC2196106  PMID: 8996251

Results 1-5 (5)