Anca V. Gafencu
Romanian Academy
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Featured researches published by Anca V. Gafencu.
Arteriosclerosis, Thrombosis, and Vascular Biology | 2008
Adrian Manea; Simona A. Manea; Anca V. Gafencu; Monica Raicu; Maya Simionescu
Objective—NADPH oxidase (NADPHox) is the major source of reactive oxygen species in vascular diseases; the mechanisms of enzyme activation are not completely elucidated. AP-1 controls the expression of many genes linked to vascular smooth muscle cells (SMCs) dysfunction. In this study we searched for the role of AP-1 in the regulation of NADPHox expression and function in human aortic SMCs exposed to proinflammatory conditions. Methods and Results—Cultured SMCs were exposed to either angiotensin II (Ang II) or tumor necrosis factor (TNF)-&agr;. The lucigenin-enhanced chemiluminescence assay and real-time polymerase chain reaction analysis revealed that AP-1 and mitogen-activated protein kinase inhibitors reduced both Ang II or TNF-&agr;-dependent upregulation of NADPHox activity and mRNA expression (NOX1, NOX4, p67phox, p47phox, p22phox). Inhibitors of AP-1 significantly diminished the Ang II or TNF-&agr;-stimulated p22phox promoter activity and protein level. Transient overexpression of c-Jun/c-Fos upregulated p22phox promoter activity. Transcription factor pull-down assay and chromatin immunoprecipitation demonstrated the physical interaction of c-Jun protein with predicted AP-1–binding sites in the p22phox gene promoter. Conclusions—In SMCs exposed to Ang II or TNF-&agr;, inhibition of AP-1–related pathways reduces NADPHox expression and the O2− production. The physical interaction of AP-1 with p22phox gene promoter facilitates NADPHox regulation.
Immunobiology | 2013
Ioana Madalina Fenyo; Anca V. Gafencu
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.
Journal of Biological Chemistry | 2007
Anca V. Gafencu; Marius R. Robciuc; Elena Valeria Fuior; Vassilis I. Zannis; Dimitris Kardassis; Maya Simionescu
The atheroprotective role of apolipoprotein E (apoE) is well established. During inflammation, expression of apoE in macrophages is reduced leading to enhanced atheromatous plaque development. In the present study, we investigated the signaling pathways involved in the repression of apoE gene expression in response to lipopolysaccharide (LPS) treatment, a condition that mimics the inflammatory stress, in mouse macrophages RAW 264.7. We identified Tpl-2 and MEKK1 as the kinases that are primarily responsible for the down-regulation of apoE promoter activity by LPS. Using a dominant negative form of IκB, we established that Tpl-2 and MEKK1 signaling pathways converge to NF-κB acting on the apoE core promoter –55/+73. In addition to NF-κB activation, LPS also activated c-Jun via its phosphorylation by JNK. The activity of the apoE promoter was repressed by c-Jun, whereas small interference RNA-mediated inhibition of endogenous c-Jun expression reversed the inhibitory effect of Tpl-2 on the apoE promoter. Transfection experiments and DNA binding assays showed that the binding site for c-Jun is in the –55/+73 region of the apoE promoter. Finally, we showed that LPS inhibited apoE gene expression via activation of the Tpl-2/MEK/ERK pathway acting on a different apoE promoter region. In summary, LPS represses apoE gene expression in macrophages via signaling pathways that involve the upstream kinases Tpl-2 and MEKK1, the intermediate mitogen-activated protein kinases ERK and JNK, and the downstream transcription factors AP-1 and NF-κB that inhibit the apoE promoter activity via distinct regions.
Journal of Cellular and Molecular Medicine | 2005
Constantina Heltianu; Gabriela Costache; Anca V. Gafencu; Mihaela Diaconu; Mihaela Bodeanu; Carmen Cristea; Kemal Azibi; Livia Poenaru; Maya Simionescu
The endothelial cell (EC) dysfunction is a common characteristic of various pathologies that include atherosclerosis, hypertension, and Fabrys disease. Aware of the role of eNO and ACE in EC dysfunction, we questioned whether polymorphism of eNOS and/or ACE gene may be a common denominator in these pathologies. Patients with CHD (108), HT (109), Fabrys disease (37) and healthy subjects (control, 141) were genotyped for the eNOSG894T by RFLP‐PCR technique and for eNOS4b/a, and ACEI/D polymorphisms by PCR amplification. The results of these studies were statistically evaluated. Compared to controls, the frequency of the eNOSG894T (T allele) was higher in CHD (P=0.03) and Fabry (P=0.01), while the eNOS4b/a (a allele) in CHD (P=0.01) and HT patients (P=0.01). The proportion of the ACEI/D was similar in all subjects. In CHD patients at “low risk” of atherogenic factors, the frequency of the T and a alleles of eNOS gene was high (P=0.03 and 0.02, respectively). Carriers of the T allele of eNOSG894T were over‐represented (P=0.04) in Fabry subgroup with renal failure. Compared to women, the eNOS894T alleles were more frequent (P=0.03) in men with CHD and HT, whereas ACE I/D in men (P=0.03) with HT. These findings suggest: (i) the frequency of eNOSG894T and/or eNOS4b/a is significantly associated with coronary dysfunction; (ii) eNOS4b/a confers a relatively high risk of hypertension in subjects with atherogenic risk factors; (iii) the frequency of eNOSG894T is high in Fabry hemizygotes with renal complications. Therefore, eNOS gene polymorphism represent a frequent risk factor for vascular abnormalities in CHD, HT and Fabrys disease, afflictions which have in common, the endothelial dysfunction.
Cell and Tissue Research | 1998
Anca V. Gafencu; Mihaela Stanescu; Aurel Mircia Toderici; Constantina Heltianu; Maya Simionescu
Abstract In endothelial cells (EC), caveolae or plasmalemmal vesicles (PVs) represent a structurally and biochemically specialized membrane microdomain. Since few data are available on the biochemical composition of PVs of large vessel endothelium, we have designed experiments to isolate this domain and to analyze its chemical components. A highly purified apical membrane fraction was obtained from cultured bovine aortic EC by using cationic colloidal silica (silica-ap), or the EC were surface-radioiodinated and a cell homogenate was prepared. Detergent treatment (Triton X-100; TX) and mechanical disruption of both the silica-ap fraction and cell homogenate followed by ultracentrifugation on a sucrose gradient gave detergent-soluble and detergent-insoluble membranous fractions. The lowest density TX-insoluble fraction appeared morphologically as distinct vesicles (caveolae; 60 nm average diameter; PVs fraction). Biochemical characterization of the PVs fraction (by comparison with the soluble fraction) revealed the presence, at high concentration, of specific caveolar markers, viz., caveolin (both isoforms, the 24-kDa form being conspicuously more abundant) and Ca2+-ATPase. By contrast, angiotensin-converting enzyme and alkaline phosphodiesterase were present almost exclusively in the TX-soluble fraction. The glycoproteins in the PVs fraction were of apparent molecular weights 52, 68, 95, and 114 kDa. Analysis of the fatty acid composition revealed more palmitoleic and stearic acid in the PVs fraction then in the TX-soluble fraction. Thus, in comparison with the plasmalemma proper, the PVs fraction (1) is detergent-insoluble; (2) contains caveolin in two isoforms; (3) contains Ca2+-ATPase at high concentration; (4) contains a set of specific glycoproteins; and (5) is enriched in palmitoleic and stearic acids.
Journal of Biological Chemistry | 2011
Violeta Georgeta Trusca; Elena Valeria Fuior; Irina C. Florea; Dimitris Kardassis; Maya Simionescu; Anca V. Gafencu
In atherogenesis, macrophage-derived apolipoprotein E (apoE) has an athero-protective role by a mechanism that is not fully understood. We investigated the regulatory mechanisms involved in the modulation of apoE expression in macrophages. The experiments showed that the promoters of all genes of the apoE/apoCI/apoCIV/apoCII gene cluster are enhanced by multienhancer 2 (ME.2), a regulatory region that is located 15.9 kb downstream of the apoE gene. ME.2 interacts with the apoE promoter in a macrophage-specific manner. Transient transfections in RAW 264.7 macrophages showed that the activity of ME.2 was strongly decreased by deletion of either 87 bp from the 5′ end or 131 bp from the 3′ end. We determined that the minimal fragment of this promoter that can be activated by ME.2 is the proximal −100/+73 region. The analysis of the deletion mutants of ME.2 revealed the importance of the 5′ end of ME.2 in apoE promoter transactivation. Chromatin conformational capture assays demonstrated that both ME.2 and ME.1 physically interacted with the apoE promoter in macrophages. Our data showed that phorbol 12-myristate 13-acetate-induced differentiation of macrophages is accompanied by a robust induction of apoE and STAT1 expression. In macrophages (but not in hepatocytes), STAT1 up-regulated apoE gene expression via ME.2. The STAT1 binding site was located in the 174–182 region of ME.2. In conclusion, the specificity of the interactions between the two multienhancers (ME.1 and ME.2) and the apoE promoter indicates that these distal regulatory elements play an important role in the modulation of apoE gene expression in a cell-specific manner.
Computational and structural biotechnology journal | 2017
Irina Florina Tudorache; Violeta Georgeta Trusca; Anca V. Gafencu
Apolipoprotein E (apoE), a 34 kDa glycoprotein, mediates hepatic and extrahepatic uptake of plasma lipoproteins and cholesterol efflux from lipid-laden macrophages. In humans, three structural different apoE isoforms occur, with subsequent functional changes and pathological consequences. Here, we review data supporting the involvement of apoE structural domains and isoforms in normal and altered lipid metabolism, cardiovascular and neurodegenerative diseases, as well as stress-related pathological states. Studies using truncated apoE forms provided valuable information regarding the regions and residues responsible for its properties. ApoE3 renders protection against cardiovascular diseases by maintaining lipid homeostasis, while apoE2 is associated with dysbetalipoproteinemia. ApoE4 is a recognized risk factor for Alzheimers disease, although the exact mechanism of the disease initiation and progression is not entirely elucidated. ApoE is also implicated in infections with herpes simplex type-1, hepatitis C and human immunodeficiency viruses. Interacting with both viral and host molecules, apoE isoforms differently interfere with the viral life cycle. ApoE exerts anti-inflammatory effects, switching macrophage phenotype from the proinflammatory M1 to the anti-inflammatory M2, suppressing CD4+ and CD8+ lymphocytes, and reducing IL-2 production. The anti-oxidative properties of apoE are isoform-dependent, modulating the levels of various molecules (Nrf2 target genes, metallothioneins, paraoxonase). Mimetic peptides were designed to exploit apoE beneficial properties. The “structure correctors” which convert apoE4 into apoE3-like molecules have pharmacological potential. Despite no successful strategy is yet available for apoE-related disorders, several promising candidates deserve further improvement and exploitation.
Handbook of experimental pharmacology | 2015
Dimitris Kardassis; Anca V. Gafencu; Vassilis I. Zannis; Alberto Dávalos
HDL regulation is exerted at multiple levels including regulation at the level of transcription initiation by transcription factors and signal transduction cascades; regulation at the posttranscriptional level by microRNAs and other noncoding RNAs which bind to the coding or noncoding regions of HDL genes regulating mRNA stability and translation; as well as regulation at the posttranslational level by protein modifications, intracellular trafficking, and degradation. The above mechanisms have drastic effects on several HDL-mediated processes including HDL biogenesis, remodeling, cholesterol efflux and uptake, as well as atheroprotective functions on the cells of the arterial wall. The emphasis is on mechanisms that operate in physiologically relevant tissues such as the liver (which accounts for 80% of the total HDL-C levels in the plasma), the macrophages, the adrenals, and the endothelium. Transcription factors that have a significant impact on HDL regulation such as hormone nuclear receptors and hepatocyte nuclear factors are extensively discussed both in terms of gene promoter recognition and regulation but also in terms of their impact on plasma HDL levels as was revealed by knockout studies. Understanding the different modes of regulation of this complex lipoprotein may provide useful insights for the development of novel HDL-raising therapies that could be used to fight against atherosclerosis which is the underlying cause of coronary heart disease.
PLOS ONE | 2012
Violeta Georgeta Trusca; Irina C. Florea; Dimitris Kardassis; Anca V. Gafencu
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.
PLOS ONE | 2017
Violeta Georgeta Trusca; Elena Valeria Fuior; Ioana Madalina Fenyo; Dimitris Kardassis; Maya Simionescu; Anca V. Gafencu
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.