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Contracting authority: Ministry of National Education - Executive Agency for Higher Education, Research, Development and Innovation(MEN-UEFISCDI)

Contractor:Institute of Cellular Biology and Pathology “Nicolae Simionescu”

Project director: Acad. Maya Simionescu, Ph.D., Director, Institute of Cellular Biology and Pathology "N. Simionescu", Bucharest, Romania (e-mail: maya.simionescu@icbp.ro)

Research team:

Adrian Manea, Ph.D.

Monica Raicu, Ph.D.

Simona-Adriana Manea, Ph.D.

Ioana Madalina Fenyo, Ph.D Student

Mihaela-Loredana Antonescu, Ph.D. Student

Summary of the project:

Convincing evidence, including ours reveal that oxidative stress (OS) and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) play a key role in all stages of atherosclerosis. Uncovering the molecular mechanisms of Nox regulation is a prerequisite of an effective anti-oxidative stress therapy. We hypothesize that a complex interplay of epigenetic/non-epigenetic factors, transcription factors, co-activators, and co-repressors are coordinately involved in the up-regulation of Nox activity in atherogenesis. Our goals are (a) to uncover the mechanisms involved in the regulation of Nox expression/function in vascular cells in atherogenesis; (b) to investigate the contribution of OS on vascular epigenetic status, and (c) to employ the data to reduce ROS by pharmacological targeting of the newly identified up-stream regulators of Nox and counteract the OS-induced deleterious effects in atherosclerosis. Our objectives aim to search for the molecular alterations induced by ROS in endothelial and smooth muscle cells, to analyze the cooperation among the transcription factors, co-activators, and/or co-repressors that control Nox expression, to investigate the epigenetic control mechanisms involved in Nox regulation in vitro and in vivo and to develop novel pharmacological tactic based on biodegradable nanocarriers for drug/siRNA delivery to defeat Nox effects. The outcome will be the design of new nanotherapeutic strategies for the reduction of oxidative stress in atherosclerosis.

Project goals:

In atherogenesis, excessive Nox-dependent ROS formation induces deregulation of the redox control systems and promotes oxidative injury and inflammation of the vascular cells. Changes in the gene expression of the Nox subtypes are critical for their function. Recently we have found that in human aortic SMC, the pro-inflammatory transcription factors NF-kB, AP-1, and STAT1/3 are important regulators of the genes coding for p22phox, Nox1, and Nox4 transcription as well as Nox-derived O₂^•⁻ production under pro-inflammatory conditions. Since these transcription factors are essential transducers of many cardiovascular risk factors, modulation of the upstream regulators of Nox could represent a novel and efficient pharmacological strategy to attenuate the pathological effects of oxidative stress. Despite of the numerous existing data, the precise mechanisms of Nox regulation in atherosclerosis is poorly understood. We hypothesize that a complex interplay of epigenetic and non-epigenetic factors, transcription factors, co-activators, and/or co-repressors are coordinately involved in the up-regulation of Nox activity in atherogenesis. To test this hypothesis, we plan to elucidate the complex interactions among different protein kinases/phosphateses, pro-inflammatory transcription factors, and epigenetic mechanisms that control Nox subtypes expression and activity. An open area in the field is the ‘fine tuning’ mechanisms implicated in the regulation of Nox-derived ROS; this knowledge will permit to find novel approaches for the therapeutic management of atherosclerosis. Thus, the goals of this proposal are: (a) to uncover the molecular mechanisms involved in the regulation of Nox expression and function in vascular cells in atherogenesis, (b) to investigate the contribution of oxidative stress on vascular epigenetic status, and(c) to use the datato reduce Nox-derived ROS by pharmacological targeting of the newly identified up-stream regulators of Nox so as to counteract the oxidative stress-induced deleterious effects in atherosclerosis. The general concept and the major goals of the proposal are shown below (see figure).

Figure legend: Schematic depiction of the main concept and the major goals of the project. In response to cardiovascular risk factors, vascular cells through their receptors activate a range of signaling pathways that up-regulate Nox expression, activity, and the ensuing ROS production. This triggers a chain of critical events that generally amplify the initial response to vascular insults (i.e., activation of other cellular sources of ROS and redox-sensitive signaling effectors). Persistent Nox activation leads to oxidative stress that is a major contributor to the initiation and the development of atherosclerotic lesions. The diagram highlights (by question marks) that we intend to uncover the epigenetic or non-epigenetic mechanisms linked to Nox up-regulation and hyperactivity in atherosclerosis and to use the data to target and control pharmacologically the Nox-derived oxidative stress (green text).

Specific objectives:

1. Investigation of cellular and molecular alterations induced by Nox-dependent ROS in EC and SMC exposed to pro-inflammatory conditions.

2. Analysis of the potential cooperation among multiple transcription factors, co-activators, and/or co-repressors to control Nox expression and cellular redox state.

3. Investigation of epigenetic control mechanisms involved in the regulation of Nox enzymes and oxidative stress in vitro and in vivo.

4. Development and testing of novel pharmacological approaches: biodegradable nanocarriers for drug and siRNA delivery to control Nox expression/activity and oxidative stress.

Expected results

Expected results 2011-2012:

• At least one study (oral presentation/poster) presented at a national/international scientific events.

• At least one original paper published in a journal indexed by ISI.

Expected results 2013:

• An original article submitted for publication in a journal indexed by ISI.

Expected results 2014:

• At least one study (oral presentation/poster) presented at a national/international scientific events.

• An accepted original article in a journal indexed by ISI.

• An original article submitted for publication in a journal indexed by ISI.

Expected results 2015:

• At least one study (oral presentation/poster) presented at a national/international scientific meeting.

• An original article accepted for publication in a journal indexed by ISI.

Expected results 2016:

• At least one study (oral presentation/poster) presented at a national/international scientific meeting.

• An original article accepted for publication in a journal indexed by ISI.

Scientific report – brief description of the main achievements

Results

1. Positive regulation of NADPH oxidase 5 by proinflammatory-related mechanisms in human aortic smooth muscle cells. NADPH oxidase Nox5 subtype expression is significantly increased in vascular smooth muscle cells (SMCs) underlying fibro-lipid atherosclerotic lesions. The mechanisms that up-regulate Nox5 are not understood. Consequently, we characterized the promoter of the human Nox5 gene and investigated the role of various proinflammatory transcription factors in the regulation of Nox5 in human aortic SMCs. The Nox5 promoter was cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5′ deletion mutants to identify the sequences necessary to effect high levels of expression in SMCs. Transcriptional initiation site was detected by rapid amplification of the 5′-cDNA ends. In silico analysis indicated the existence of typical NF-kB, AP-1, and STAT1/STAT3 sites. Transient overexpression of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 increased significantly the Nox5 promoter activity. Chromatin immunoprecipitation demonstrated the physical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the Nox5 promoter. Lucigenin-enhanced chemiluminescence, real-time PCR, and Western blot assays showed that pharmacological inhibition and the silencing of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 reduced significantly the interferon γ-induced Ca2+-dependent Nox activity and Nox5 expression. Up-regulated Nox5 correlated with increases in intracellular Ca²⁺, an essential condition for Nox5 activity. NF-kB, AP-1, and STAT1/STAT3 are important regulators of Nox5 in SMCs by either direct or indirect mechanisms. Overexpressed Nox5 may generate free radicals in excess, further contributing to SMCs dysfunction in atherosclerosis. (published inFree Radical Biology and Medicine, 52(9), 1497-1507, 2012, Impact Factor: 5.271)

2. CCAAT/enhancer-binding proteins regulate Nox transcription in human vascular smooth muscle cells. In atherosclerosis, oxidative stress-induced vascular smooth muscle cells (SMCs) dysfunction is partially mediated by upregulated NADPH oxidase (Nox); the mechanisms of enzyme regulation are not entirely defined. CCAAT/enhancer-binding proteins (C/EBP) regulate cellular proliferation and differentiation, and the expression of many inflammatory and immune genes.We aimed at elucidating the role of C/EBP in the regulation of Nox in SMCs exposed to proinflammatory conditions.Human aortic SMCs were treated with interferon gamma (IFNγ) for up to 24h. Lucigenin-enhanced chemiluminescence, real-time PCR, Western blot, promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate Nox regulation. IFNγ dose-dependently induced Nox activity and expression, nuclear translocation and upregulation of C/EBPα, C/EBPβ, and C/EBPδ protein expression levels. Silencing of C/EBPα, C/EBPβ or C/EBPδ reduced significantly but differentially the IFNγ-induced upregulation of Nox activity, gene, and protein expression. In silico analysis indicated the existence of typical C/EBP sites within Nox1, Nox4, and Nox5 promoters. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ enhanced the luciferase level directed by the promoters of the Nox subtypes. Chromatin immunoprecipitation demonstrated the physical interaction of C/EBPα, C/EBPβ, and C/EBPδ proteins with the Nox1/4/5 promoters. C/EBP transcription factors are important regulators of Nox enzymes in IFNγ-exposed SMCs. Activation of C/EBP may induce excessive Nox-derived reactive oxygen species formation, further contributing to SMCs dysfunction and atherosclerotic plaque development. Pharmacological targeting of C/EBP-related signalling pathways may be used to counteract the adverse effects of oxidative stress (published in Journal of Cellular and Molecular Medicine 18(7), pg. 1467-1477, 2014, Impact Factor: 4.014).).

3. Epigenetic regulation of NADPH oxidase by histone acetylation in human aortic smooth muscle cells. Members of the vascular NADPH oxidase (Nox) family are key regulators of cell physiology. Produced in excess, Nox-derived reactive oxygen species (ROS) are highly detrimental in numerous cardiovascular pathologies such as atherosclerosis. The mechanisms of Nox regulation and the specific function of each Nox subtype are yet to be discovered. Post-translational modifications of histones of conserved lysine residues, is accomplished by specialized enzymes and results in chromatin conformational changes that influence the DNA accessibility for transcription factors. Yet, the precise role of epigenetic mechanisms of Nox regulation by histone modification/histone-modifying enzymes is scantly elucidated. In this study we aimed at investigating the implication of histone acetylation in mediating Nox regulation in human aortic smooth muscle cells (SMCs) exposed to pro-inflammatory conditions. Human aortic SMCs were treated with CTPB, a potent activator of histone acetyltransferases (HAT) or interferon gamma (IFNγ) for up to 24h. Lucigenin-enhanced chemiluminescence, dichlorofluorescein assay, real-time PCR, Western blot, and chromatin immunoprecipitation assays were employed to investigate Nox regulation. CTBP-activated HAT dose-dependently induced up-regulation of intracellular ROS formation, Nox activity, and the mRNA and protein expression levels of the Nox1, Nox4, and Nox5 isoforms. IFNγ treatment mimicked the effect of HAT agonist. It also induced significant increases in HAT1 protein expression level and acetylation of H3K27 (H3K27ac). Pharmacological inhibition as well as silencing of HAT1 reduced significantly but differentially the IFNγ-induced Nox activity and expression. Specific H3K27ac (a marker of positive-acting regulatory regions in the genome) and HAT1 – Nox1/4/5 promoters interactions were identified in various locations by chromatin immunoprecipitation assays. Immunofluorescence microscopyindicated an increased expression of HAT1 and H3K27ac in SMCs underlying fibro-lipid and unstable atherosclerotic lesions in human carotid arteries.The data provide evidence that histone acetylation play a role in mediating Nox expression and function in IFNγ - exposed SMCs. Understanding the complex networking among transcription factors and epigenetic mechanismsconverging to Nox regulation may contribute to developing novel pharmacological strategies to reduce the adverse effects of oxidative stress in atherosclerosis.

4. Pharmacological inhibition of histone deacetylase reduces oxidative stress and inflammation in the aorta of diabetic mice. Hyperglycaemia-induced functional and structural alterations of the vascular wall in diabetes are partially mediated by oxidative stress generated by the activated NADPH oxidase (Nox). Hitherto, the molecular mechanisms that accounts for Nox upregulation are not entirely elucidated. In this study we hypothesize that histone acetylation has a role in the regulation of Nox and that histone deacetylase (HDAC) inhibition may have the potential to counteract the vascular oxidative stress and inflammation in diabetes. Male C57BL/6J mice were rendered diabetic with streptozotocin. The animals were distributed into four experimental groups to receive vehicle or suberoylanilide hydroxamic acid (SAHA), a selective HDAC inhibitor, every other day for four weeks: i) non-diabetic + vehicle, ii) non-diabetic + SAHA, iii) diabetic + vehicle, and iv) diabetic + SAHA. Lucigenin-enhanced chemiluminescence assay, real-time PCR, and Western blot analysis were employed to investigate vascular epigenetic changes, Nox regulation, and the expression of pro-inflammatory markers. Western blot analysis revealed that the HDAC1 and HDAC2 protein expression levels were significantly elevated in the aorta of diabetic mice compared to non-diabetic control animals. Treatment of diabetic mice with SAHA greatly reduced the augmented Nox activity and the gene and protein expression of the Nox1, Nox2, and Nox4 subtypes. Pharmacological targeting of HDAC generated vascular anti-inflammatory activities as illustrated by the inhibitory effects of SAHA on intercellular adhesion molecule 1, vascular cell adhesion molecule
1, and monocyte chemoattractant protein-1 gene expression in the aorta of diabetic mice. Pharmacological inhibition of HDAC reduced Nox expression and the ensuing reactive oxygen species formation in the aorta of diabetic mice. These data indicate the existence of epigenetic-based mechanisms whereby changes in chromatin conformation leads to Nox upregulation and inflammation in vascular cells in diabetes.

5. Systemic deliveryof HDAC1 siRNA reduces vascular oxidative stress and inflammation in the aorta of hypercholesterolemic apolipoprotein E deficient mice. Evidence is accumulating that epigenetic regulation of gene expression by changes in chromatin conformation due to histone acetylation play a major role in all major cardiovascular diseases. Histone acetylation is regulated by the activities of two major enzyme families, namely histone acetyltransferases (HAT) and histone deacetylases (HDAC). As general principle, histone acetylation induces chromatin relaxation, a condition that facilitates the accessibility of transcription factors to their cognate DNA elements in the genome. Thus, histone acetylation is associated with active gene expression. Yet, recent evidence indicates that not only nucleosomal histones are the substrates of HAT/HDAC but also other non-histone proteins such as transcription factors. Consequently, the activity of several transcription factors can be directly influenced via lysine acetylation by HAT/HDAC system. The beneficial effects of several pan-HDAC inhibitors have been demonstrated in various experimental models of heart failure and hypertension. However, the role of HDAC in atherosclerosis and the rationale of using HDAC inhibitors as therapeutic cues are not established. In this study we have investigated the expression pattern of HDAC1 and HDAC2 essential isoforms in both human and experimental atherosclerosis (ApoE-/- mice). We found that HDAC1 and HDAC2 are significantly up-regulated in atherosclerotic plaques derived from human carotid artery compared to non-atherosclerotic arterial samples (superior thyroid arteries). A similar expression pattern of HDAC1 and HDAC2 proteins was found in the aorta of atherosclerotic ApoE-/- mice. Interestingly, the increased expression of HDAC1 and HDAC2 correlated with the severity of atherosclerotic lesions and plasma cholesterol level in mice. In order to investigate the specific contribution HDAC1 and HDAC2 isoforms in mediating oxidative stress and inflammation in atherosclerosis a nanotechnology-based procedure for in vivo siRNA delivery was employed. The results showed that systemic delivery of HDAC1 and HDAC2 siRNA pools resulted in a significant decrease in HDAC1 and HDAC2 protein expression levels in the aorta of atherosclerotic ApoE-/- mice. In vivo silencing of HDAC1 but not HDAC2 significantly reduced the expression of reactive oxygen species-generating enzymes NADPH oxidase 1 (Nox1), Nox2, and Nox4. In addition, silencing of HDAC1 correlated with significant decreases in vascular inflammation (e.g., NOS2, MMP9, CD45), matrix deposition and remodeling (e.g., fibronectin, MMP9), and cell proliferation (e.g., PCNA) markers. Our preclinical studies indicate that selective targeting of a specific HDAC isoform (e.g., HDAC1) rather than pan-HDAC inhibition may be a novel therapeutic strategy to counteract vascular oxidative stress and inflammation in atherosclerosis.

Publications

Peer-review ISI articles:

1. Adrian Manea, Simona-Adriana Manea, Irina Cristina Florea, Catalina Maria Luca, Monica Raicu. Positive regulation of NADPH oxidase 5 by proinflammatory-related mechanisms in human aortic smooth muscle cells. Free Radical Biology and Medicine, 52(9), 1497-1507, 2012. Impact Factor: 5.271.

2. Simona-Adriana Manea, Andra Todirita, Monica Raicu, Adrian Manea. C/EBP transcription factors regulate NADPH oxidase in human aortic smooth muscle cells, Journal of Cellular and Molecular Medicine, 18(7), 1467-1477, 2014. Impact Factor: 4.014.

3. Adrian Manea, Simona-Adriana Manea, Andra Todirita, Irina Cristina Albulescu, Monica Raicu, Shlomo Sasson, Maya Simionescu. Cell and Tissue Research, 361(2), 593-604, 2015. Impact Factor: 3.565.

4. Adrian Manea, Simona-Adriana Manea, Ana Maria Gan, Alina Constantin, Ioana Madalina Fenyo, Monica Raicu, Horia Muresian, Maya Simionescu. Human monocytes and macrophages express NADPH oxidase 5; a potential source of reactive oxygen species in atherosclerosis. Biochemical and Biophysical Research Communications, 461(1), 172-179, 2015. Impact Factor: 2.297.

5. Simona-Adriana Manea, Alina Constantin, Gina Manda, Shlomo Sasson, Adrian Manea. Regulation of Nox enzymes expression in vascular pathophysiology: Focusing on transcription factors and epigenetic mechanisms. Redox Biology, 5, 358-366, 2015. Impact Factor: 6.235.

6. Simona-Adriana Manea, Ioana Madalina Fenyo, Adrian Manea. c-Src tyrosine kinase mediates high glucose-induced endothelin-1 expression. The International Journal of Biochemistry & Cell Biology, 75, 123-130, 2016. Impact Factor: 3.905.

Book chapter:

1. Manuela Calin, Elena Butoi, Simona-Adriana Manea, Maya Simionescu, Adrian Manea. Lessons from Experimental-Induced Atherosclerosis: Valuable for the Precision Medicine of Tomorrow. In: Arterial Revascularization of the Head and Neck - Text Atlas for Prevention and Management of Stroke, 2016; Chapter 17, 341-365, ISBN: 978-3-319-34191-0. Book edited by: Horia Mureasian MD, PhD, Bucharest University Hospital. Publishing house: Springer, New York, USA.

Oral communications:

1.Adrian Manea. CCAAT/enhancer-binding proteins mediate interferon gamma-induced upregulation of NADPH oxidase in human aortic smooth muscle cells. European Society of Cardiology Congress, August 31- September 3, 2013. European Heart Journal (2013) 34 (Abstract Supplement), pg. 509.

2. Adrian Manea. Histone deacetylase inhibition reduces NADPH oxidase activity and expression in the aorta of diabetic mice. “Reactive oxygen species in cellular biology and pathology” European Cooperation in Science and Technology - Biomedicine and Molecular Biosciences COST Action BM1203 EU-ROS, 21-24 October 2015, Bucharest, Romania.

3. Adrian Manea, Simona-Adriana Manea, Ioana Madalina Fenyo, Mihaela Loredana Antonescu, Monica Raicu, Maya Simionescu.Pharmacological inhibition of histone deacetylase reduces oxidative stress and inflammation in the aorta of diabetic mice. The 4th International Symposium on Adipobiology and Adipopharmacology (ISAA), 28-31 October 2015, Bucharest. Romanian Journal of Diabetes, Nutrition and Metabolic Diseases, Volume 22 (2015)/Supplement 2, pg. 29.

Posters:

1. Manea A, Todirita A, Manea SA, Florea IC, Raicu M, Sasson S, Simionescu M. Peroxisome proliferator-activated receptor isoforms α and β/δ mediate 4-hydroxynonenal-induced upregulation of NADPH oxidase in human aortic smooth muscle cells. The 4th EMBO Meeting, 2012.

2. Raicu M, Manea SA, Florea IC, Luca C, Manea A. Up-regulation NADPH oxidase 5 is mediated via pro-inflammatory mechanisms in human aortic smooth muscle cells. The 4th EMBO Meeting, 2012.

3. Raicu M, Manea SA, Florea IC, Luca C, Manea A. NADPH oxidase 5 is up-regulated by proinflammatory-associated processes in human aortic smooth muscle cells. 17th Annual Meeting of the Israeli Society for Research, Prevention and Treatment of Atherosclerosis, 2012.

4. Todirita A, Florea IC, Manea SA, Raicu M, Sasson S, Manea A, Simionescu M. PPARα and PPARβ/δ control 4-hydroxynonenal-induced upregulation of NADPH oxidase in human aortic smooth muscle cells. Al IV-lea Congres International si A XXX-a Sesiune Anuala a Societatii Romane de Biologie Celulara, 2012.

5. Todirita A, Manea SA, Raicu M, Manea A. CCAAT/enhancer-binding proteins mediate interferon gamma-induced upregulation of NADPH oxidase in human aortic smooth muscle cells. 5th International Congress and the 31st Annual Session of the Romanian Society for Cell Biology, 2013. Bulletin of Romanian Society for Cell Biology, no. 41, pg. 183.

6. Manea SA, Todirita A, Manea A. High glucose-induced increased expression of endothelin-1 in human endothelial cells is mediated by activated CCAAT/enhancer-binding proteins. 5th International Congress and the 31st Annual Session of the Romanian Society for Cell Biology, 2013. Bulletin of Romanian Society for Cell Biology, no. 41, pg. 145.

7.Manea A, Manea SA, Gan AM, Constantin A, Fenyo IM, Todirita A, Raicu M, Muresian H, Simionescu M. Human monocytes and macrophages express functional NADPH oxidase 5; a novel source of reactive oxygen species in atherosclerosis. Congresul National al Societatii Romane de Biologie Celulara cu Participare Internationala si A XXXII-A Sesiune Stiintifica Anuala, 2014. Bulletin of Romanian Society for Cell Biology, no. 42, pg. 96(First prize for poster presentation).

8.Manea SA, Todirita A, Fenyo IM, Constantin A, Manea A. c-Src tyrosine kinase mediates high glucose-induced endothelin-1 expression in diabetes. European Society of Cardiology Congress, 2014. European Heart Journal - Abstract Supplement, 2014, pg. 788-789.

9.Manea A, Todirita A, Constantin A, Manea SA. Epigenetic regulation of NADPH oxidase by histone acetylation in human aortic smooth muscle cells. European Society of Cardiology Congress, 2014. European Heart Journal - Abstract Supplement, 2014, pg. 613.

10. Manea SA, Fenyo IM, Manea A.Epigenetic regulation of endothelin-1 expression by histone acetylation/deacetylation in diabetes. 7th National Congress with International Participation and 33rd Annual Scientific Session of the RSCB, 2015. Bulletin of Romanian Society for Cell Biology, no. 43, pg. 71(First prize for poster presentation).

11. Manea A, Manea SA, Fenyo IM, Raicu M, Simionescu M. High glucose-induced NADPH oxidase expression and activity is mediated by epigenetic mechanisms in vascular smooth muscle cells. 7th National Congress with International Participation and 33rd Annual Scientific Session of the RSCB, 2015. Bulletin of Romanian Society for Cell Biology, no. 43, pg. 70.

12. Manea A, Fenyo IM,Manea SA. High glucose-induced NADPH oxidase expression and activity is mediated by epigenetic mechanisms in vascular smooth muscle cells. 40^th FEBS Congress, 2015.FEBS Journal 282(Suppl. 1) (2015) 55, pg. 65.

13.Manea SA, Fenyo IM, Manea A. Histone deacetylase mediates high glucose-induced endothelin-1 expression via direct and indirect mechanisms. 8th National Congress with International Participation and 34rd Annual Scientific Session of the RSCB, 2016. Bulletin of Romanian Society for Cell Biology, No. 44, June 2016, pg. 67.

14. Antonescu ML, Manea SA, Simionescu M, Manea A.Epigenetic regulation of NADPH oxidase 5 expression by p300/histone acetyltransferase in human macrophages. 8th National Congress with International Participation and 34rd Annual Scientific Session of the RSCB, 2016. Bulletin of Romanian Society for Cell Biology, No. 44, June 2016, pg. 74.

15. Manea A, Manea SA, Antonescu ML, Fenyo IM, Raicu M, Simionescu M. Pharmacological inhibition of histone deacetylase reduces vascular NADPH oxidase expression and reactive oxygen species formation in experimental diabetes. 8th National Congress with International Participation and 34rd Annual Scientific Session of the RSCB, 2016. Bulletin of Romanian Society for Cell Biology, No. 44, June 2016, pg 80.

Awards:

Adrian Manea: “Nicolae Simionescu Award” of the Romanian Academy for outstanding contribution in the field of vascular biology of reactive oxygen species, 2012.
First prize for poster presentation: Human monocytes and macrophages express functional NADPH oxidase 5; a novel source of reactive oxygen species in atherosclerosis. Authors: Manea A, Manea SA, Gan AM, Constantin A, Fenyo IM, Todirita A, Raicu M, Muresian H, Simionescu M. National Congress with International Participation and 32nd Annual Scientific Session of the Romanian Society for Cell Biology ”. Targu Mures, June 4-7, 2014.
First prize for poster presentation: Epigenetic regulation of endothelin-1 expression by histone acetylation/deacetylation in diabetes. Authors: Simona-Adriana Manea, Ioana Madalina Fenyo, Adrian Manea. 7th National Congress with International Participation and 33rd Annual Scientific Session of the Romanian Society for Cell Biology”, Baia Mare, June 10 – 14, 2015.