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Trusca V.G.,Nicolae Simionescu of the Romanian Academy | Fuior E.V.,Nicolae Simionescu of the Romanian Academy | Fenyo I.M.,Nicolae Simionescu of the Romanian Academy | Kardassis D.,University of Crete | And 3 more authors.
PLoS ONE | Year: 2017

Apolipoprotein E (apoE) has anti-atherosclerotic properties, being involved in the transport and clearance of cholesterol-rich lipoproteins as well as in cholesterol efflux from cells. We hypothesized that glucocorticoids may exert anti-inflammatory properties by increasing the level of macrophage-derived apoE. Our data showed that glucocorticoids increased apoE expression in macrophages in vitro as well as in vivo. Dexamethasone increased ~6 fold apoE mRNA levels in cultured peritoneal macrophages and RAW 264.7 cells. Administered to C57BL/6J mice, dexamethasone induced a two-fold increase in apoE expression in peritoneal macrophages. By contrast, glucocorticoids did not influence apoE expression in hepatocytes, in vitro and in vivo. Moreover, dexamethasone enhanced apoE promoter transcriptional activity in RAW 264.7 macrophages, but not in HepG2 cells, as tested by transient transfections. Analysis of apoE proximal promoter deletion mutants, complemented by protein-DNA interaction assays demonstrated the functionality of a putative glucocorticoid receptors (GR) binding site predicted by in silico analysis in the -111/-104 region of the human apoE promoter. In hepatocytes, GR can bind to their specific site within apoE promoter but are not able to modulate the gene expression. The modulatory blockade in hepatocytes is a consequence of partial involvement of transcription factors and other signaling molecules activated through MEK1/2 and PLA2/PLC pathways. In conclusion, our study indicates that glucocorticoids (1) differentially target apoE gene expression; (2) induce a significant increase in apoE level specifically in macrophages. The local increase of apoE gene expression in macrophages at the level of the atheromatous plaque may have therapeutic implications in atherosclerosis. © 2017 Trusca et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Preda M.B.,Nicolae Simionescu of the Romanian Academy | Preda M.B.,University of Oslo | Valen G.,University of Oslo
Current Stem Cell Research and Therapy | Year: 2013

Although the treatment of acute myocardial infarction has improved considerably and the mortality rate is reduced, patients who survive may develop loss of cardiomyocytes, scar formation, ventricular remodeling, and ultimately heart failure. The treatment of the most severe types of heart failure is heart transplantation, but this therapeutic intervention is not available for a large number of patients due to a shortage of donor hearts. Since current pharmacological and interventional approaches are unsuccessful to regenerate infarcted myocardium, new approaches like gene-or cell-based therapies are tested to prevent loss of cardiac tissue, enhance angiogenesis, and to reduce left ventricular remodeling. Exciting and promising data on laboratory animals have moved the field rapidly into clinical trials. Although several clinical trials proved the safety and feasibility of using gene-and cell-based therapies, many challenges remain before large-scale novel treatment modules will be available. The purpose of this review is to summarize the key findings of larger, randomized clinical trials in cardiovascular medicine using both gene and cell-based therapy, and to emphasize the most significant questions that emerged from the clinical experience so far, such as the optimal gene or cell type to be used, the ideal delivery route, and for DNA the ideal delivery system. Understanding the mechanisms of gene-and cell-based therapies is essential for designing the next phase clinical studies in the field of regenerative medicine. © 2013 Bentham Science Publishers.


Rosca A.-M.,Nicolae Simionescu of the Romanian Academy | Matei C.,Nicolae Simionescu of the Romanian Academy | Dragan E.,Nicolae Simionescu of the Romanian Academy | Burlacu A.,Nicolae Simionescu of the Romanian Academy | Burlacu A.,Petru Poni Institute of Macromolecular Chemistry
Cell Biology International | Year: 2013

Various studies performed on different models have demonstrated that apoptosis occurs in ischaemic-reperfused myocardium in vivo; however, the individual contribution of ischaemia and reperfusion to CMC (cardiomyocyte) apoptosis remains uncertain. We have determined the main inducer of CMC apoptosis in ischaemia-reperfusion by exposing CMCs to either 30 min ischaemia followed by reperfusion or to 25-OH-cholesterol (25-hydroxycholesterol) for 1-3 days. Both ischaemia-reperfusion and exogenous oxidants increased the Bax/Bcl-2 ratio, a favourable effect for the apoptotic process. However, apoptosis was not observed in ischaemic CMCs in the absence of reperfusion. Moreover, reperfusion after 30 min ischaemia did not make an important contribution to CMC apoptosis in culture in terms of caspase 3 activation. In contrast, 25-OH-cholesterol promoted CMC apoptosis by a caspase 3-dependent mechanism that involved the transcriptional activation of the pro-apoptotic protein, Bax and post-translational degradation of the anti-apoptotic protein, Bcl-2. From these results, we conclude that CMC apoptosis is not induced by ischaemia per se, but by the oxidants from the surrounding environment at the time of reperfusion. These exogenous oxidants exacerbate the alterations induced by ischaemia and complete the apoptotic process at the time when ATP and glucose levels are restored. © The Author(s) Journal compilation. © 2012 International Federation for Cell Biology.


Alexandru N.,Petru Poni Institute of Macromolecular Chemistry | Alexandru N.,Nicolae Simionescu of the Romanian Academy | Popov D.,Nicolae Simionescu of the Romanian Academy | Dragan E.,Nicolae Simionescu of the Romanian Academy | And 3 more authors.
PLoS ONE | Year: 2013

Aim: The purpose of this project was to evaluate the influence of circulating endothelial progenitor cells (EPCs) and platelet microparticles (PMPs) on blood platelet function in experimental hypertension associated with hypercholesterolemia. Methods: Golden Syrian hamsters were divided in six groups: (i) control, C; (ii) hypertensive-hypercholesterolemic, HH; (iii) 'prevention', HHin-EPCs, HH animals fed a HH diet and treated with EPCs; (iv) 'regression', HHfin-EPCs, HH treated with EPCs after HH feeding; (v) HH treated with PMPs, HH-PMPs, and (vi) HH treated with EPCs and PMPs, HH-EPCs-PMPs. Results: Compared to HH group, the platelets from HHin-EPCs and HHfin-EPCs groups showed a reduction of: (i) activation, reflected by decreased integrin 3β, FAK, PI3K, src protein expression; (ii) secreted molecules as: SDF-1, MCP-1, RANTES, VEGF, PF4, PDGF and (iii) expression of pro-inflammatory molecules as: SDF-1, MCP-1, RANTES, IL-6, IL-1β; TFPI secretion was increased. Compared to HH group, platelets of HH-PMPs group showed increased activation, molecules release and proteins expression. Compared to HH-PMPs group the combination EPCs with PMPs treatment induced a decrease of all investigated platelet molecules, however not comparable with that recorded when EPC individual treatment was applied. Conclusion: EPCs have the ability to reduce platelet activation and to modulate their pro-inflammatory and anti-thrombogenic properties in hypertension associated with hypercholesterolemia. Although, PMPs have several beneficial effects in combination with EPCs, these did not improve the EPC effects. These findings reveal a new biological role of circulating EPCs in platelet function regulation, and may contribute to understand their cross talk, and the mechanisms of atherosclerosis. © 2013 Alexandru et al.


Burlacu A.,Petru Poni Institute of Macromolecular Chemistry | Burlacu A.,Nicolae Simionescu of the Romanian Academy | Grigorescu G.,Nicolae Simionescu of the Romanian Academy | Rosca A.-M.,Nicolae Simionescu of the Romanian Academy | And 2 more authors.
Stem Cells and Development | Year: 2013

Stem cell-based therapy for myocardial regeneration has reported several functional improvements that are attributed mostly to the paracrine effects stimulating angiogenesis and cell survival. This study was conducted to comparatively evaluate the potential of factors secreted by mesenchymal stem cells (MSCs) in normoxic and hypoxic conditions to promote tissue repair by sustaining endothelial cell (EC) adhesion and proliferation and conferring protection against apoptosis. To this aim, a conditioned medium (CM) was generated from MSCs after 24-h incubation in a serum-free normal or hypoxic environment. MSCs exhibited resistance to hypoxia, which induced increased secretion of vascular endothelial growth factor (VEGF) and decreased levels of other cytokines, including stromal-derived factor-1 (SDF). The CM derived from normal (nMSC-CM) and hypoxic cells (hypMSC-CM) induced similar protective effects on H9c2 cells in hypoxia. Minor differences were noticed in the potential of normal versus hypoxic CM to promote angiogenesis, which were likely connected to SDFα and VEGF levels: the nMSC-CM was more effective in stimulating EC migration, whereas the hypMSC-CM had an enhanced effect on EC adhesion. However, the factors secreted by MSCs in normoxic or hypoxic conditions supported adhesion, but not proliferation, of ECs in vitro, as revealed by impedance-based dynamic assessments. Surprisingly, factors secreted by other stem/progenitor cells, such as endothelial progenitor cells (EPCs), had complementary effects to the MSC-CM. Thus, the EPC-CM, in either a normal or hypoxic environment, supported EC proliferation, but did not sustain EC adhesion. Combined use of the MSC-CM and EPC-CM promoted both EC adhesion and proliferation, suggesting that the local angiogenesis at the site of ischemic injury might be better stimulated by simultaneous releasing of factors secreted by multiple stem/progenitor cell populations. © Copyright 2013, Mary Ann Liebert, Inc. 2013.


Toma L.,Nicolae Simionescu of the Romanian academy | Sanda G.M.,Nicolae Simionescu of the Romanian academy | Stancu C.S.,Nicolae Simionescu of the Romanian academy | Rogoz D.,Nicolae Simionescu of the Romanian academy | Sima A.V.,Nicolae Simionescu of the Romanian academy
Annals of the Romanian Society for Cell Biology | Year: 2012

Data from literature show that endothelial dysfunction is a key factor in atherogenesis; it is characterized by a reduction of the bioavailability of nitric oxide (NO), whereas endothelium-derived contracting factors are increased leading to an impairment of endothelium-dependent vasodilation. The main causes of NO reduced bioavailability are its interaction with reactive oxygen species leading to reactive nitrogen species (RNS) formation, and the decreased capacity of endothelial nitric oxide synthase (eNOS) to produce NO. Inflammation is another end-result of endothelial dysfunction. Our aim was to evaluate the effect of C-reactive protein (CRP), known marker of inflammation, on NO bioavailability and endothelial-derived inflammatory molecules. To this purpose, we prepared pure CRP and incubated it with human umbilical vein endothelial cells (HEC) in DMEM with 5 mM or 25 mM glucose. We determined the expression of eNOS, the levels of NO and intracellular RNS. Results showed that CRP inhibits the gene and protein expression of eNOS and NO production, determines intracellular accumulation of RNS and up regulation of MCP-1 expression. We demonstrated that the signalling pathways activated by CRP in HEC were p38MAPK and NF-kB dependent. Addition of 25 mM glucose in the culture medium aggravates this process. In conclusion, CRP combined with hyperglycemia may contribute to accelerated atherosclerosis in diabetes by inducing endothelial cell dysfunction.


Trusca V.G.,Nicolae Simionescu of the Romanian Academy | Florea I.C.,Nicolae Simionescu of the Romanian Academy | Kardassis D.,Foundation for Research and Technology Hellas | Gafencu A.V.,Nicolae Simionescu of the Romanian Academy
PLoS ONE | Year: 2012

Apolipoprotein CII (apoCII) is a specific activator of lipoprotein lipase and plays an important role in triglyceride metabolism. The aim of our work was to elucidate the regulatory mechanisms involved in apoCII gene modulation in macrophages. Using Chromosome Conformation Capture we demonstrated that multienhancer 2 (ME.2) physically interacts with the apoCII promoter and this interaction facilitates the transcriptional enhancement of the apoCII promoter by the transcription factors bound on ME.2. We revealed that the transcription factor STAT1, previously shown to bind to its specific site on ME.2, is functional for apoCII gene upregulation. We found that siRNA-mediated inhibition of STAT1 gene expression significantly decreased the apoCII levels, while STAT1 overexpression in RAW 264.7 macrophages increased apoCII gene expression. Using transient transfections, DNA pull down and chromatin immunoprecipitation assays, we revealed a novel STAT1 binding site in the -500/-493 region of the apoCII promoter, which mediates apoCII promoter upregulation by STAT1. Interestingly, STAT1 could not exert its upregulatory effect when the RXRα/T3Rβ binding site located on the apoCII promoter was mutated, suggesting physical and functional interactions between these factors. Using GST pull-down and co-immunoprecipitation assays, we demonstrated that STAT1 physically interacts with RXRα. Taken together, these data revealed that STAT1 bound on ME.2 cooperates with RXRα located on apoCII promoter and upregulates apoCII expression only in macrophages, due to the specificity of the long-range interactions between the proximal and distal regulatory elements. Moreover, we showed for the first time that STAT1 and RXRα physically interact to exert their regulatory function. © 2012 Trusca et al.


Manea S.-A.,Nicolae Simionescu of the Romanian Academy | Fenyo I.M.,Nicolae Simionescu of the Romanian Academy | Manea A.,Nicolae Simionescu of the Romanian Academy
International Journal of Biochemistry and Cell Biology | Year: 2016

Endothelin-1 (ET-1) plays an important role in the pathophysiology of diabetes-associated cardiovascular disorders. The molecular mechanisms leading to ET-1 upregulation in diabetes are not entirely defined. c-Src tyrosine kinase regulates important pathophysiological aspects of vascular response to insults. In this study, we aimed to elucidate whether high glucose-activated c-Src signaling plays a role in the regulation of ET-1 expression. Human endothelial cells EAhy926 (ECs) were exposed to normal or high levels of glucose for 24 h. Male C57BL/6J mice were rendered diabetic with streptozotocin and then treated with a specific c-Src inhibitor (Src I1) or c-Src siRNA. Real-time PCR, Western blot, and ELISA, were used to investigate ET-1 regulation. The c-Src activity and expression were selectively downregulated by pharmacological inhibition and siRNA-mediated gene silencing, respectively. High glucose dose-dependently up-regulated c-Src phosphorylation and ET-1 gene and protein expression levels in human ECs. Chemical inhibition or silencing of c-Src significantly decreased the high-glucose augmented ET-1 expression in cultured ECs. In vivo studies showed significant elevations in the aortic ET-1 mRNA expression and plasma ET-1 concentration in diabetic mice compared to non-diabetic animals. Treatment with Src I1, as well as in vivo silencing of c-Src, significantly reduced the upregulated ET-1 expression in diabetic mice. These data provide new insights into the regulation of ET-1 expression in endothelial cells in diabetes. Pharmacological targeting of c-Src activity and/or expression may represent a potential therapeutic strategy to reduce ET-1 level and to counteract diabetes-induced deleterious vascular effects. © 2016 Elsevier Ltd. All rights reserved.


Titorencu I.,Nicolae Simionescu of the Romanian Academy | Pruna V.,Nicolae Simionescu of the Romanian Academy | Jinga V.V.,Nicolae Simionescu of the Romanian Academy | Simionescu M.,Nicolae Simionescu of the Romanian Academy
Cell and Tissue Research | Year: 2014

Osteoblasts are specialized mesenchyme-derived cells accountable for bone synthesis, remodelling and healing. Differentiation of osteoblasts from mesenchymal stem cells (MSC) towards osteocytes is a multi-step process strictly controlled by various genes, transcription factors and signalling proteins. The aim of this review is to provide an update on the nature of bone-forming osteoblastic cells, highlighting recent data on MSC - osteoblast - osteocyte transformation from a molecular perspective and to discuss osteoblast malfunctions in various bone diseases. We present here the consecutive stages occurring in the differentiation of osteoblasts from MSC, the transcription factors involved and the role of miRNAs in the process. Recent data concerning the pathogenic mechanisms underlying the loss of bone mass and architecture caused by malfunctions in the synthetic activity and metabolism of osteoblasts in osteoporosis, osteogenesis imperfecta, osteoarthritis and rheumatoid arthritis are discussed. The newly acquired knowledge of the ontogeny of osteoblasts will assist in unravelling the abnormalities taking place during their differentiation and will facilitate the prevention and/or treatment of bone diseases by therapy directed against altered molecules and mechanisms. © 2013 Springer-Verlag Berlin Heidelberg.


Fenyo I.M.,Nicolae Simionescu of the Romanian Academy | Gafencu A.V.,Nicolae Simionescu of the Romanian Academy
Immunobiology | Year: 2013

Atherosclerosis is a progressive chronic disease of large and medium arteries, characterized by the formation of atherosclerotic plaques. Monocytes and macrophages are key factors in lesion development, participating to the processes that mediate the progression of the atherosclerotic plaque (lipid accumulation, secretion of pro-inflammatory and cytotoxic factors, extracellular matrix remodeling). The recruitment of the monocytes in the vascular wall represents a hallmark in the pathology of the atherosclerotic lesion. Monocyte adhesion and transmigration are dependent on the complementary adhesion molecules expressed on the endothelial surface, whose expression is modulated by chemical mediators. The atherosclerotic plaque is characterized by a heterogeneous population of macrophages reflecting the complexity and diversity of the micro-environment to which cells are exposed after entering the arterial wall. Within the atherosclerotic lesions, macrophages differentiate, proliferate and undergo apoptosis. Taking into account that their behavior has a direct and critical influence on all lesional stages, the development of therapeutic approaches to target monocytes/macrophages in the atherosclerotic plaque became a focal interest point for researchers in the field. © 2013 Elsevier GmbH.

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