:: En > En > Networking Grants > Grants > National Grants

Title: Molecules and mechanisms involved in cytokine and chemokine-dependent vascular inflammation as targets for novel nanotherapeutic strategies

Funds

Year	Funds
2011	200.000
2012	515.000
2013	189.626,16
2014	182.500
2015	133.215
2016	279.658,84
TOTAL	1.500.000

Abstract. Atherosclerosis is considered as the end result of dyslipidemia and a strong inflammatory process. Extensive evidence revealed that inflammation is a key contributor to all stages of this disease, from the initial lesion to the ruptured plaque. However, there are many instances in which inflammatory reactions induced by putative antigens that stimulate immune cells induce atherosclerotic plaque formation in the absence of systemic dyslipidemia. Moreover recent clinical studies indicate thatthe vascular inflammation might be a factor that promotes atherosclerosis and the formation of aneurysms. The mechanism of vascular inflammation and the reason for accelerated atherosclerosis in patients with inflammatory rheumatic disease remain unclear. Although it was reported that cytokines and chemokines participate in this process, their mechanism of action and their effect on the cellular constituents of the atheroma is not very well known.Resistin is a 12. 5- kD cysteine-rich protein belonging to the family of proteins named FIZZ (found in inflammatory zone) or RELM (resistin-like molecules). It is a cytokine with an established function in mice as a molecular link between obesity and diabetes and with an unclear function in humans, where it appears as a pro-inflammatory molecule associated with both acute and chronic inflammation (neutrophils and monocytes are important sources of resistin). In the atherosclerotic lesions increased resistin presence was associated with macrophages and very recent data suggested that resistin, when present at high levels locally, in the arterial vessel wall significantly contributes to an enhanced accumulation of macrophages by increasing monocytes chemotaxis and survival,bymechanisms that need to be further clarified. The resistin receptor is not known in vascular cells. Recently, Toll-like receptor 4 (TLR 4) was proposed to be a receptor for the proinflammatory effects of resistin in monocytes and Gi proteins signaling suggested to be involved in resistin chemotactic effects in human. Fractalkine (CX3CL1) is an atypical chemokine that functions as chemoattractant or as adhesion molecule, facilitating monocyte and T cell transmigration in atherosclerotic lesion prone areas. Recently, fractalkine was even proposed as an early endothelial marker of atherosclerosis by a complex study of global gene expression profile from human arterial endothelial cells in different stages of atherosclerosis. Fractalkine recognize a Pertussis toxin-sensitive G-protein-coupled receptor, CX3CR1. The presence of fractalkine and its cognate receptor was demonstrated in human coronary artery atherosclerotic plaque, in the sub-endothelium and not in normal artery, but the role of fractalkine presence in this location was not well characterized. Previous reports have shown that within the atherosclerotic plaque smooth muscle cells (SMC) and monocytes interact via the CX3CL1/CX3CR1 axis and that their interaction regulates monocyte survival and differentiation. Both resistin and fractalkine were found at increased levels in the human atherosclerotic lesions resistin being associated with CD68 monocytes/macrophages in carotid arteries and aortic aneurisms, and fractalkine predominantly associated with the intimal SMC and with monocytes/macrophages. Thus we questioned whether the interaction between resistin and fractalkine, enhances the inflammatory process and if so, one could find ways to reduce the process. Our recent datauncovered fractalkine (Fk) as a new molecular link between resistin and inflammation and showedthat resistin increases monocyte adhesion by mechanisms involving endothelial cell adhesion molecules P-selectin and Fk. Moreover, our preliminary data indicate that resistin may use toll-like receptor 4 (TLR4), Gi proteins and JAK/STAT pathways in its pro-inflammatory effects in endothelial cells and that resistin up-regulates fractalkine expression in smooth muscle cells (SMC).

Our goals are to search for the mechanisms by which Fk and resistin (R) modulate the inflammation in endothelial cells (EC), smooth muscle cells (SMC) and monocytes/macrophages (Mac), to determine if they contribute to SMC-Mac cross-talk, to get insights in the signalling mechanisms and gene regulation involved and to develop new targeted nanotechnology-based therapeutic strategies to reduce vascular wall inflammation. The use of nanotechnology in cardiovascular disease treatment may have unprecedented benefits in preventing atherosclerosis progression. The concept of targeted drug delivery using nanoparticles (NP) is an appealing therapeutic strategy because of its advantages such as the ability to target specific and restricted locations in the body, to deliver effective concentration of drugs to the diseased sites and to reduce the drug concentrations at non-target sites resulting in fewer side effects.

Working hypothesis. In inflammation, resistin (R), that is present in high concentration in the inflamed vessel wall, activates endothelial cells (EC) and intimal smooth muscle cells (SMC) determining enhanced monocytes (M) infiltration by specific mechanisms involving up-regulation of fraktalkine, MCP-1, TLR4 and Gi protein activation (Figure, panel A). Subsequently, within the intima, modulation of monocytes phenotype, their proliferation and differentiation in activated macrophages and the cross-talk between the monocyte-derived macrophages and SMC induces changes in the signaling pathways, augments expression of pro-atherogenic molecules that accelerate inflammation-induced atheroma formation (Figure, panel B). The relevant molecules found to be instrumental in the EC and M activation could be used as targets for a novel anti-inflammatory nanotherapy employing drugs-carrying nanoparticles (NP). This hypothesis (based on our preliminary data) will be tested by the objectives of this proposal.

Objectives:

Objective 1: To explore the role of subendothelial resistin on monocytes transmigration.

Objective 2: To analyze the resistin and fractalkine-induced monocytes and smooth muscle cells phenotype modulation and the molecular mechanisms involved in macrophage-SMC cross-talk.

Objective 3: To develop new targeted therapeutic strategies to reduce vascular wall inflammation based on nanotechnology.

Collectively, the molecular mechanism revealed by our study will help to get further insight into the complexity of the signaling mechanisms and gene regulation of some key molecules involved and to develop new targeted therapeutic strategies based on nanotechnology to reduce vascular wall inflammation. Our project will identify novel drug targets and would provide important knowledge for developing strategies for better management of vascular disease. The data will be applicable to the numerous diseases in which inflammation is involved, such as such as atherosclerosis, chronic rheumatic diseases, diabetes, cancer and neurodegenerative diseases.

Impact and relevance. Our study is relevant for cardiovascular diseases (CVD) that represents more than 55% of the death rate in the world and the World Health Organization predicts that by 2020 CVD will be the leading cause of the global disease burden. However the results will go well beyond the CVD because inflammation is implicated in numerous pathologies such as diabetes, cancer, neurodegenerative diseases and chronic rheumatic diseases. Thus the results will have an impact in all these pathologies. The molecular mechanism revealed will help to get further insight into the complexity of the signaling mechanisms and gene regulation of some key molecules involved and to develop new targeted therapeutic strategies based on nanotechnology to reduce vascular wall inflammation. Our project will identify novel drug targets and would provide important knowledge for developing strategies for better management of vascular disease. The outcome of this project is highly relevant for biomedical science because: it uses cellular and molecular biology tools to help to better understand clinical data, and by our ongoing collaboration with rheumatologists and cardiologists we will correlate the obtained data with clinical data facilitating a continuous two way transfer from bench to bedside. Altogether, the continuous close collaboration we have with a rheumatology and a cardiology clinic will increase the professional competency of researchers, clinicians, medical residents, masters and PhD students and by dissemination of the results the visibility of biomedical Romanian science.

Project implementation

Stage 1

During the period between October and December 2011, the project implementation was performed by executing Task 1.1: Analysis of the EC expression of relevant chemokines for monocyte transmigration: Fk and MCP-1.

A scientific report was delivered at the end of the period and the results obtained were communicated at The 4^th EMBO Meeting (European Molecular Biology Organization), Advancing the Life Sciences in Nice, France, in September 2012 with the title: “Resistin activates TLR4, Gi proteins and JAK/STAT signaling pathways in human endothelial cells”, authors: Ileana Manduteanu, Monica Pirvulescu, Cristina Remes, Elena Butoi, Anca Gan, Viorel Simion, Daniela Stan, Manuela Calin, Maya Simionescu The abstract was published in The EMBO Meeting 2012 Abstract Book, C 014, p. 172:

Abstract: Background: Resistin is a significant modulator of local and systemic inflammation. In the human atherosclerotic plaque resistin accumulates within the subendothelial space and enhances leukocyte transmigration by mechanisms that are not well known. Receptors for resistin have not been identified in vascular cells. The aim of our study is to uncover the signaling pathways involved in the production of the chemokines fractalkine and MCP-1 by high subendothelial resistin. Observations: Human endothelial cells (HEC) were cultured on membrane inserts in two compartment systems and resistin was added in the lower chamber for 6,18 and 24 hours. Conditioning medium was collected and analyzed for soluble Fk and MCP-1, by sandwich ELISA. The role of JAK/STAT in Fk and MCP-1 regulation was examined by using pharmacologic inhibitors for JAK/STAT pathway or specific si RNA for STAT1, STAT3, STAT5 and analysis of FK and MCP-1 expression by WB and ELISA. Resistin effects on TLR4 expression was examined by WB and flow cytometry. To analyze the role of TLR4 and Gi proteins in resistin-induced chemokines up-regulation, TLR4 function was blocked in HEC using mAb or specific siRNA, and Gi proteins signaling by preincubation with Pertussis toxin, next resistin-induced production of fractalkine and MCP-1 in HECs was assessed by WB and ELISA kits. Our data showed that in HEC subendothelial resistin enhanced Fk secretion after 6 hours activation by JAK-STAT, and Gi proteins signaling pathways and increased the secretion of MCP-1 after 24 hours stimulation by activation of TLR4 receptor. Conclusions: The novel findings of our study are that resistin may activate JAK/STAT, TLR4 and Gi proteins signaling pathways in HEC and enhance the secretion MCP-1 and Fk. Our data will help to develop new targeted anti-inflammatory therapy employing drugs-carrying nano-particles.

Stage 2

Project implementation during January to December 2012 was carried out by performing Stage 2: Signaling pathways involved in up-regulating fractalkine and MCP-1 expression by subendothelial resistin in vascular cells.

In the period mentioned above, tasks of the first objective (To explore the role of subendothelial resistin on monocytes transmigration) were performed:

Task 1.2. Revealing of new intracellular signaling pathways involved in chemokines (Fk, MCP-1) expression in response to high resistin in subendothelium.

Task 1.3. Investigation of the possible involvement of endothelial TLR4 and Gi proteins in subendothelial resistin-induced chemokines (Fk and MCP-1) expression

1.3.1. Resistin effect on TLR4 expression.

1.3.2. Resistin effect on TLR4 and MyD88 association.

1.3.3. TLR4 implication in FK and MCP-1 expression induced by resistin in human EC

1.3.4. Investigation of Gi proteins role in FK and MCP-1 induced by resistin in human EC

The results obtained in task 1.2 and 1.3 were communicated at The 81^st EAS Congress (European Atherosclerosis Society) in Lyon, France in June 2013 with the title “Molecular mechanisms involved in resistin dependent enhanced monocytes infiltration in the vessel wall”, authors: M Pirvulescu, AM Gan, E Butoi, D Stan, V Simion, M Calin, I Manduteanu. The abstract was selected for the Moderated Poster Session (assigned as M37) and the no. 1400 was attributed in the Abstract Book published online:

Abstract. Aim: It has been reported that within the human plaque, resistin secreted by the recruited leukocytes accumulates within the subendothelial space. We hypothesize that in this location resistin activates endothelial cells (EC) and smooth muscle cells (SMC) and enhance monocyte transmigration, by mechanisms that we plan to uncover. Methods: We have used an in vitro system, a co-culture in which SMC were grown on the lower chamber and the EC were grown on membrane insert (upper chamber). Resistin was added luminal or subendothelial for 2 and 24h and calcein-labeled human monocytes isolated with CD14 microbeads were added on the EC layer. The transmigrated monocytes were counted under the microscope or by measure of calcein fluorescence. The subendothelial and luminal expression of the chemokines fractalkine (Fk) and MCP1 was determined by ELISA. To evaluate the role of the chemokines in monocyte transmigration in the EC-SMC co-culture system, the function of Fk and MCP1 was blocked with specific antibodies and with Fk or MCP1 siRNAs. The role of TLR4 and Gi proteins activation in resistin-induced monocyte transmigration was evaluated by incubation with specific antibodies or inhibitors for these pathways. Results: Our data showed that: (i) subendothelial resistin induced enhanced monocyte transmigration compared to luminal resistin and (ii) subendothelial resistin induced an increased monocyte transmigration in co-cultured EC-SMC compared to monocyte transmigration through only the EC monolayer. Blocking the function of chemokines Fk and MCP1 and blocking TLR4 signaling or Gi proteins activation decreased monocyte transmigration through EC towards SMC. Conclusion: Resistin enhances monocyte transmigration by mechanisms involving fractalkine and MCP1 and the activation of TLR4 and Gi proteins signaling. Investigation of the molecular mechanisms instrumental in resistin induced monocyte transmigration may be important for using the involved molecules as targets for novel anti-inflammatory nanotherapy employing drugs-carrying nanoparticles.

Task 1.4. Assessment ofthe resistin-induced gene and protein expression of chemokines Fk and MCP-1 in SMC

1.4.1. The effect of resistin on FK expression in SMC

1.4.2. The effect of resistin on MCP-1 expression in SMC

Task 1.5.Investigation ofthe possible involvement of TLR 4 and Gi proteins in resistin-induced chemokines (Fk and MCP-1) expression in SMC

1.5.1. Investigation of resistin role on TLR4 expression on the cell surface

1.5.2. Analysis of TLR4 implication in FK and MCP-1 expression induced by resistin in vascular SMC

1.5.3. Evaluation of Gi proteins role in FK and MCP-1 expression induced by resistin in vascular SMC

The results obtained in task 1.4 and 1.5 were communicated in a scientific paper in a ISI Journal: Inflammatory effects of resistin on human smooth muscle cells: up-regulation of fractalkine and its receptor, CX3CR1 expression by TLR4 and Gi-protein pathways. Gan AM, Butoi ED, Manea A, Simion V, Stan D, Parvulescu MM, Calin M, Manduteanu I, Simionescu M. Cell Tissue Res. 2013 Jan;351(1):161-74. doi: 10.1007/s00441-012-1510-9. Epub 2012 Oct 20.

Abstract: In the atherosclerotic plaque, smooth muscle cells (SMC) acquire an inflammatory phenotype. Resistin and fractalkine (CX3CL1) are found in human atheroma and not in normal arteries. CX3CL1 and CX3CR1 are predominately associated with SMC. We have questioned whether resistin has a role in the expression of CX3CL1 and CX3CR1 in SMC thus contributing to the pro-inflammatory status of these cells. Cultured human aortic SMC were stimulated with 100 ng/ml resistin for 4, 6, 12, and 24 h, and then CX3CL1 and CX3CR1 expression was assessed by quantitative reverse transcription with the polymerase chain reaction and Western blot. We found that resistin up-regulated CX3CL1 and CX3CR1 in SMC and induced the phosphorylation of p38MAPK and STAT3. Inhibitors of p38MAPK, JAK JAKSTAT, NF-kB, and AP-1 significantly reduced CX3CL1 and CX3CR1 expression. Knockdown of STAT1 and STAT3 with decoy oligodeoxinucleotides and the silencing of p65 and cJun with short interfering RNA decreased CX3CL1 and CX3CR1 expression. Anti-TLR4 antibody and pertussis toxin also reduced CX3CL1 and CX3CR1 protein expression. xCELLigence experiments revealed that resistin probably uses Gi proteins for its effect on SMC. The CX3CL1 induced by resistin exhibited a chemotactic effect on monocyte transmigration. Thus, (1) resistin contributes to the pro-inflammatory state of SMC by the up-regulation of CX3CL1 and CX3CR1 expression via a mechanism involving NF-kB, AP-1, and STAT1/3 transcription factors, (2) resistin employs TLR4 and Gi-protein signaling for its effect on SMC, (3) CX3CL1 induced by resistin is functional in monocyte chemotaxis. The data reveal new mechanisms by which resistin promotes the inflammatory phenotype of SMC.

Task 1.6. Investigationof the resistin-dependent signaling pathways involved in regulation of Fk expression in SMC

1.6.1. In silico analysis of human fractalkine promoter

1.6.2. Cloning the human fractalkine promoter

1.6.3. Functional activity evaluation of fractalkine promoter in vascular SMC

1.6.4. Physical interactions of transcription factors in fractalkine promoter – canonic and non-canonic protein-DNA interactions

Task 1.7.Investigation the role of oxidative stress in resistin-induced Fk expression in SMC

1.7.1. Intracellular reactive oxygen species (ROS) quantification induced by resistin in vascular SMC

1.7.2. Evaluation of resistin role in NADPH oxidase activity

The results obtained in task 1.6 and 1.7 were communicated at The 5^th International Congress and The 31th Annual Scientific Session of Romanian Society for Cell Biology in Timisoara, Romania in June 2013 with the title “Molecular mechanism regulating fractalkine gene expression in human aortic smooth muscle cells”, authors: Ana-Maria Gan, Adrian Manea, Monica Pirvulescu, Stan Daniela, Viorel Simion, Manuela Calin, Ileana Manduteanu, Elena Butoi. The abstract was published in the Bulletin of Romanian Society for Cell Biology, no. 41, june 2013, p. 136:

Abstract: Introduction. Fractalkine (Fk, CX3CL1) and its receptor CX3CR1 participate in the atherosclerotic process mediating the recruitment of leukocytes and facilitating the interactions between monocytes/macrophages and smooth muscle cells (SMC) by mechanisms not very well known. In order to uncover transcriptional regulation of Fk expression, in this study, we characterized the promoter of the human Fk gene and investigated the role of various transcription factors in its regulation in human aortic SMC. Materials and Methods.The Fk promoter was cloned in the pGL3 basic reporter vector. Functional analysis was done employing 5′ deletion mutants to identify the functionally active sequences that modulate Fk in SMCs (luciferase assay). The FK gene and protein expression was evaluated by Real Time-PCR and Western blot, respectively. Identification of specific transcription factors that are involved in Fk regulation was determined by in silico analysis and chromatin immunoprecipitation (ChIP) methods. Results and Discussions. Exposure of SMCs to IFN-γ for 24h over-expressed Fk, which further induced monocyte chemotaxis. Pharmacological inhibition and silencing of STAT1/STAT3 transcription factors decreased the Fk induced by IFN-γ. In silico analysis indicated the existence of typical sites for NF-kB, AP-1, and STAT1/STAT3 transcription factors binding. Transient overexpression of p65/NF-kB, c-Jun/AP-1, or STAT1/STAT3 increased the Fk promoter activity. ChIP demonstrated the physical interaction of c-Jun/AP-1 and STAT1/STAT3 proteins with the predicted elements of the Fk promoter. Conclusions. NF-kB, AP-1, and STAT1/STAT3 transcription factors are important regulators of Fk expression in SMCs by either canonical or/and noncanonical mechanisms. Overexpressed Fk promote monocyte chemotaxis dependent by JAK-STAT pathway, which may further contribute to atherosclerosis progression.

Task 2 conclusion: High concentration of resistin activates human vascular cells (endothelial and smooth muscle cells) by specific mechanisms involving up-regulation of FK and MCP-1 expression, increasing oxidative stress and activation of Gi proteins and TLR4 signaling pathways. All these mechanisms could be involved in monocyte infiltration in the vessel wall induced by resistin and could be proposed as therapeutic targets in reducing vessel wall inflammation.

Stage 3

Project implementation during January to December 2013 was carried out by performing Stage 3: Investigation of the role of subendothelial resistin in monocyte transmigration; analysis of monocyte and smooth muscle cells (SMC) phenotype induced by resistin and fractalkine and analysis of molecular mechanisms involved in macrophage-SMC cross-talk.

In the period mentioned above the first objective of the project was finalized with the execution of Task 1.8.: Validation of the functional role of resistin-induced chemokines (Fk and MCP-1) in monocyte transmigration. Moreover, in the same period, were accomplished tasks 2.1.: Analysis of the effect of resistin and fractalkine on monocytes proliferation, 2.2.: Analysis of monocytes differentiation towards macrophages induced by local resistin and fractalkine and 2.3.: Investigation of inflammatory mediator’s expression in macrophages induced by macrophages-SMC cross-talk of Objective 2 (To analyze the resistin and fractalkine-induced monocytes and smooth muscle cells phenotype modulation and the molecular mechanisms involved in macrophage-SMC cross-talk.)

Task 1.8. Validation of the functional role of resistin-induced chemokines (Fk and MCP-1) in monocyte transmigration

The results obtained in task 1.8 were communicated in a scientific paper in a ISI Journal: “Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine, MCP-1 and activation of TLR4 and Gi/o proteins signaling”, Pirvulescu MM, Gan AM, Stan D, Simion V, Calin M, Butoi E, Manduteanu I. Int J Biochem Cell Biol. 2014 May;50:29-37. doi: 10.1016/j.biocel.2014.01.022. Epub 2014 Feb 6.

Abstract. „The cytokine resistin and the chemokine fractalkine (FKN) were found at increased levels in human atherosclerotic plaque, in the subendothelium, but their role in this location still needs to be characterized. Recently, high local resistin in the arterial vessel wall was shown to contribute to an enhanced accumulation of macrophages by mechanisms that need to be clarified. Our recent data showed that resistin activated smooth muscle cells (SMC) by up-regulating FKN and MCP-1 expression and monocyte chemotaxis by activating toll-like receptor 4 (TLR4) and Gi/o proteins. Since in the vessel wall both endothelial cells (EC) and SMC respond to cytokines and promote atherosclerosis, we questioned whether subendothelial resistin (sR) has a role in vascular cells cross-talk leading to enhanced monocyte transmigration and we investigated the mechanisms involved. To this purpose we used an in vitro system of co-cultured SMC and EC activated by sR and we analyzed monocyte transmigration. Our results indicated that: (1) sR enhanced monocyte transmigration in EC/SMC system compared to EC cultured alone; (2) sR activated TLR4 and Gi/o signaling in EC/SMC system and induced the secretion of more FKN and MCP-1 compared to EC cultured alone and used both chemokines to specifically recruit monocytes by CX3CR1 and CCR2 receptors. Moreover, FKN produced by resistin in EC/SMC system, by acting on CX3CR1 on EC/SMC specifically contributes to MCP-1 secretion in the system and to the enhanced monocyte transmigration. Our study indicates new possible targets for therapy to reduce resistin-dependent enhanced macrophage infiltration in the atherosclerotic arterial wall.”

Task 3 conclusion: The results indicate the important role of macrophage – SMC interaction, either in absence or presence of resistin, in modulation of citokines, chemokines, citokine receptors or chemokine receptors expression in macrophages.

Stage 4: “The role of resistin and fractalkine on SMC and macrophage cross-talk and the design, preparation and characterization of different types of nanoparticles to target resistin activated endothelium”.

During January to December 2014 the Objective 2 of the project was implemented with the execution of Task 2.4 and 2.5 and one activity of the Objective 3 was performed with the execution of Task 3.1, as described below.

Objective 2: “To analyze the resistin and fractalkine-induced monocytes and smooth muscle cells phenotype modulation and the molecular mechanisms involved in macrophage-SMC cross-talk.”

Materials and methods

Experimental design

In this objective we performed in vitro experiments using human aortic smooth muscle cells (SMC) line and human macrophages (derived from THP-1 monocytes). SMC were co-cultured with macrophages in presence or absence of resistin (R) or fractalkine (FKN) in a double chamber culture system for 1-3 days. The control cells are macrophages or SMCs grown separately (no co-culture). In this co-culture system, the macrophages (obtained by differentiation of THP-1 with PMA 100nM for 3 days) are cultured on the bottom of culture plate and the SMCs on the insert filter (pore diameter 0.4µm); each cell type is maintained separately until ready for co-culture. Co-culture is achieved by placing the insert into the culture plate. After co-culture, the gene and protein expression of different inflammatory molecules are determined by quantitative Real Time PCR (qRT-PCR) and Proteome Profiler array.

Real-time PCR (qPCR)

Total mRNA was isolated from macrophages or SMC after interaction and activation with resistin or fractalkine by using a commercial extraction kit (Sigma Aldrich) and transcribed to cDNA utilizing 1µg of total ARN and MMLV revers-transcriptase. cDNA amplification was performed by using specific primers for the following molecules: collagen I, collagen III, MMP2, MMP9, elastin, fibronectin (FN), IL-1β, TNFα, IFNγ, TGF β, IL-5, IL-10, IL-8, IL-1α, Midkine, CD40L, MCP-1, TLR4, fractalkin (FKN), CX3CR1, CXCL16, SDF-1, Serpin E1, GROα, CCL1, NAP2, PARC, PF4, RANTES, VCC-1, TARC, GM-CSF, G-CSF, MIP-1β, MIP-1δ, MIP-3β and β-actin. Gene expression was quantified by the ΔΔCt algorithm.

ProteomeProfiler

In order to investigate the proteomic profile of interacted macrophages and SMC, we used a ProteomeProfiler Array (R&D Systems): Human Chemokine Proteome and Human Cytokine Proteome. Principle of the assay, according to the manufacturer’s protocol: selected capture antibodies have beeen spotted in duplicate on nitrocellulose membranes. Cell culture lysates are diluted and mixed with a cocktail of biotinylated detection antibodies. The sapmle/antibody mixture is then incubated with the membrane. Any cytokine or chemokine/detection antibody complex present is bound by its cognate immobilized capture antibody on the membrane. Following a wash to remove unbound material, Streptavidin-HRP and chemiluminescent detection reagents are added sequentially. Light is produced at each spot in proportion to the amount of cytokine/chemokine bound.

The activities are described in the scientific report of stage 4.

Task 2.4:”Analysis of SMC and macrophage activation induced by macrophage and SMC cross-talk (the role of resistin or fractalkine) on macrophage and SMC interactions”.

Background and scientific results

Resistin is a cytokine that in humans is secreted by peripheral blood mononuclear cells and macrophages. Serum resistin levels are elevated in patients with obesity-associated diabetes or cardiovascular disease. Resistin was found in human atherosclerotic lesions and was suggested to be an inflammatory marker of atherosclerosis and to promote atherogenesis. Fractalkine (FKN) has dual function acting as a cell adhesion molecule and chemokine mediating direct capture, firm adhesion and transmigration of leukocytes and its expression is enhanced in human atherosclerotic plaques. Both resistin and fractalkine were found at increased levels in the human atherosclerotic lesions, resistin being associated with CD68 monocytes/macrophages in carotid arteries and aortic aneurisms, and fractalkine predominantly associated with intimal smooth muscle cells (SMC) and with monocytes/macrophages but their role in this location is not very clear yet. Our recent data showed that resistin has pro-inflammatory effects on SMC and that fractalkine has proatherogenic effects in human monocytes/SMC cross-talk. The objective of this study was to explore the role of resistin and FKN in macrophage-SMC cross-talk and to uncover the molecular mechanisms involved. To this purpose, THP-1 cells (a monocytic cell line) were differentiated to macrophages with phorbol myristate acetate (PMA) and then interacted with SMC cultured on membrane inserts in the presence or absence of resistin or FKN. After 24 hours, the gene and protein expression of inflammatory mediators induced in macrophages by macrophages-SMC cross-talk was determined by Q-PCR and Proteome Profiler™ Array (R&D Systems). Macrophages alone or macrophages activated with resistin or FKN were used as controls. Protein array experiments revealed that macrophages exhibit after their interaction with SMC an increased expression of chemokines ( CCL1, CCL4, CCL5, CCL19, CXCL1, CXCL5, CXCL7) and cytokines (IFNγ, TNFα, IL-1α, IL-10, IL-2, IL-23, IL-27, IL-5, IL-6 and IL-8) and PAI-1 (plasminogen activator inhibitor-1). Moreover, resistin enhanced the inflammatory macrophage phenotype induced by their interaction with SMC, by increasing the level of the chemokines CCL1, CCL4, CCL5, CXCL1 and of the cytokines: IFNγ, IL-1α, IL-5, IL-6, IL-23, IL-27, IL-8 and PAI-1. In addition, resistin induced in macrophages interacted with SMC an increased expression of the chemokines: CCL10, CCL17, CCL3, CXCL11 and cyokines IL-13, IL-17, IL-17E, IL-ra, IL-1β and ICAM-1, MIF (Macrophage migration inhibitory factor), CD40L. Our preliminary data indicate that FKN also modulate macrophage-SMC interaction by increasing the expression of mediators involved in inflammation and fibrosis.

In conclusion, the results of Task 2.4 suggest that resistin and fractalkine may exert pro-inflammatory effects in macrophages-SMC cross-talk and the molecular mechanisms involved may reveal targets for novel anti-inflammatory therapies.

Task 2.5: “Role of CX3CR1 in macrophage-SMC cross talk. Regulation of CX3CR1 by AP-1 in macrophages exposed to fractalkine and resistin.”

Background and scientific results

Macrophages, smooth muscle cells (SMCs) and their interactions have key roles in the pathogenesis of atherosclerotic vascular diseases. Along atheroma development, the phenotype of macrophages and SMCs changes and may influence the disease progression. The accumulating data on the phenotypes exhibited by these cells within atherosclerotic lesions raise many questions, regarding the mechanisms and factors that might control the transition of cell phenotype. SMCs often reside in vascular lesions in close proximity to macrophage clusters and are most likely influenced by factors released from these pro-inflammatory phagocytes. Moreover, macrophages may be influenced by direct contact with smooth muscle cells or soluble factors released by these cells. Particularly, macrophages may promote activation and induce pro-atherogenic functions of SMCs and smooth muscle cells may modulate macrophage phenotype. Addressing the mechanisms involved in SMC-macrophages cross-talk that lead to phenotypic modulation of both cell types may provide new insight into atherogenesis and new targets for therapies of various vascular diseases.

The results of Task 2.5 show that fractalkine-CX3CR1 axis mediates the interaction between macrophages and SMC, but resistin does not affect CX3CR1 expression in macrophages.

Some of the results of Task 2.4 together with results from task 2.3 were communicated at the 6th National Congress with international Participation and 32nd Annual Scientific Session of Romanian Society for Cell Biology, 4-7 June 2014, at Targu Mures, Romania and at the FEBS EMBO Conference, 30 August – 4 September 2014, in Paris, France. The title of the communication:” The effect of resistin and fractalkine on macrophage-smooth muscle cells cross-talk”, authors: Pirvulescu M, Butoi E, Gan AM, Stan D, Simion V, Calin M, Manduteanu I.

Results of the activities of REFINATER project were presented in the review:

“Molecular and functional interactions among monocytes/macrophages and smooth muscle cells and their relevance for atherosclerosis”, authors: Elena Butoi, Ana Maria Gan, Ileana Manduteanu, review published in Crit Rev Eukaryot Gene Expr. 2014; 24(4):341-55.

Objective 3: “To develop new targeted therapeutic strategies to reduce vascular wall inflammation based on nanotechnology”,

Task 3.1: “Design, preparation and characterization of different NP for targeted delivery to resistin-activated endothelium”.

This activity is described in the scientific report of stage 4.

Materials and methods

Preparation of lipid nanoemulsions (LN)

Lipid nanoemulsions (LN) were prepared using the ultrasonication method. Briefly, the organic and the aqueous phase were prepared separately. The organic phase composed of EPC, phospholipidic derivates of PEG (PEG-DSPE and Maleimide-PEG-DSPE) and soybean oil dissolved in chloroform was evaporated using a vacuum rotary evaporator. The aqueous phase containing 3 ml of double distilled water, Tween 80 and/or glycerine at different concentrations was added to the organic phase and sonicated for 10 min, at 40V intensity using a UP200H probe-type sonicator from Heidolph. The obtained nanoemulsion was further vacuum evaporated, in order to eliminate the organic solvent traces. The formulation of nanoemulsions has been optimized by varying the concentration of surfactants.

Coupling of P-seectinl peptide to CmLN

The P-selectin (Psel) peptide with high affinity for the cell adhesion molecule P-selectin has been coupled to the maleimide group at the distal end of PEG (Mal-PEG-DSPE) by sulfhydryl-maleimide. Briefly, the Psel peptide was activated by adding a reducing agent to break the disulfide bonds (TCEP Buffer) and mixed for 2 hours at room temperature. The excess of TCEP was removed by dialysis (using cellulose ester membrane with a cut-off of 500-1000 Da), overnight at 4°C against coupling buffer (10mM Na₂HPO₄, 10mM NaH₂PO₄, 2 mM EDTA, 30 mM, pH:6.7). Then, the Psel peptide was added (molar ratio 1:1, peptide:maleimide-PEG-DSPE) to LN,suspension and mixed together at room temperature for 6 hours. At the end, to saturate the uncoupled maleimide groups, L-cysteine (1mM) was added for 30 min. As a purification step, we performed centrifugation using Amicon ultracentrifugal filter units of 100 kDa (Millipore, Billerica, Massachusetts, USA) in order to separate the uncoupled Psel peptide and L-cysteine. By this step, the excess of Tween-80 and glycerine are either filtrated or retained by the ultra-filtration membrane.

Background and scientific results

Inflammation is a common occurrence associated, involved and/or responsible for many pathologies. P-selectin is a cell adhesion molecule highly expressed by endothelium in inflammation, and therefore a potential target for nanotherapy. We hypothesize that functionalization of nanoparticles with P-selectin high affinity peptide will increase their binding to activated endothelium in inflamed tissues. Thus, the aim of this study was to develop nanoparticles loaded with curcumin (Cm, a hydrophobic anti-inflammatory polyphenol) that are able to target the inflammatory process in both, acute and chronic inflammation. Curcumin-loaded lipid nanoparticles (CmLN) functionalized with a P-selectin affinity peptide (P-sel_CmLN) have been developed. The CmLN were characterized for size and structure (by DLS and TEM), entrapment efficiency and curcumin release capacity (by HPLC). In vitro studies using human endothelial cells (HEC) were performed to investigate the cytotoxicity of CmLN (by MTT), their cell binding and internalization (by flow cytometry and confocal microscopy), the signaling pathways involved (Western Blot) and the effect on monocytes adhesion to HEC (fluorescence microscopy). In vivo studies were performed using IVIS Caliper live imaging system on an acute [C57BL mice injected with lipopolysaccharide (LPS) for 5 h] and chronic inflammation mice models [atherosclerotic ApoE-/- mice fed a high cholesterol diet for 4 months].

The results of Task 2.5 show that CmLN have: (1) low cytotoxicity; (2) anti-inflammatory effect by down-regulating ERK1/2 and p38 MAPK signaling pathways and (3) impaired adhesion of monocytes to activated endothelial cells. Functionalization of CmLN with P-sel peptide induced (4) an increased binding to activated HEC. In vivo studies (after 1h of nanoparticles administration) revealed (6) an increased accumulation of Psel_CmLN in the lungs of LPS-injected C57BL mice compared to controls (i.e. non-targeted CmLN and PBS-injected C57BL mice) and (7) an increased binding of Psel-CmLN to the aorta (in atherosclerotic plaque-prone areas) of ApoE-/- mice, compared to non-targeted CmLN binding.

In conclusion, P-selectin coupled CmLN efficiently bind to activated endothelial cells in vitro and in vivo in acute and chronic experimental inflammation. Therefore, P-selectin exposed on activated HEC surface represents a reliable target for delivery of nanoparticles-carrying drugs.

Some of the results of the Task 2.5 were presented at 2 important international scientific meetings: at the 6^th National Congress with international Participation and 32nd Annual Scientific Session of Romanian Society for Cell Biology, 4-7 June 2014, Targu Mures, Romania and at FEBS EMBO Conference, 30 August – 4 September 2014, Paris, France. The title of the communication: “P-selectin directed lipid nanoparticles specifically target activated endothelium.“, the authors: Simion V, Stan D, Constantinescu C, Dragan E, Pirvulescu M, Gan AM, Butoi E, Manduteanu I, Simionescu M, Calin M.

Stage 5

Project implementation during January to December 2015 was carried out by performing Stage 5, accomplishing tasks of the Objective 2: “To analyze the resistin and fractalkine-induced monocytes and smooth muscle cells phenotype modulation and the molecular mechanisms involved in macrophage-SMC cross-talk.” and Objective 3: “To develop new targeted therapeutic strategies to reduce vascular wall inflammation based on nanotechnology”,

In the period mentioned above, were accomplished: Task 2.4. - Analysis of macrophages and smooth muscle cells activation induced by their interaction (role of concomitant activation with resistin and fractalkine on macrophage-smooth muscle cells cross-talk) and Task 3.2. - Assessment of the therapeutic effect of targeted lipid NP-mediated anti-inflammatory agents delivery to activated EC by detecting if they reduce monocytes adhesion and migration. These activities are described in the scientific report of stage 5.

The results of Stage 5 were presented in two ISI articles and at an important international scientific meeting:

ISI articles:

1. VCAM-1 directed target-sensitive liposomes carrying CCR2 antagonists bind to activated endothelium and reduce adhesion and transmigration of monocytes. Manuela Calin, Daniela Stan, Martin Schlesinger, Viorel Simion, Mariana Deleanu, Cristina Ana Constantinescu, Ana-Maria Gan, Monica Madalina Pirvulescu, Elena Butoi, Ileana Manduteanu, Marian Bota, Marius Enachescu, Lubor Borsig, Gerd Bendas, Maya Simionescu. European Journal of Pharmaceutics and Biopharmaceutics 89 (2015) 18–29.

Abstract:

Chemokines are critically involved in the development of chronic inflammatory-associated diseases such as atherosclerosis. We hypothesized that targeted delivery of compounds to the surface of activated endothelial cells (EC) interferes with chemokine/receptor interaction and thereby efficiently blocks inflammation. We developed PEGylated target-sensitive liposomes (TSL) encapsulating a CCR2 antagonist (Teijin compound 1) coupled with a specific peptide recognized by endothelial VCAM-1 (Vp-TSL-Tj). TSL were characterized for size (by dynamic light scattering), the amount of peptide coupled at the liposomal surface and Teijin release (by HPLC). We report that Vp-TSL-Tj binds specifically to activated EC in vitro and in situ, release the entrapped Teijin and prevent the transmigration of monocytes through activated EC. This is the first evidence that nanocarriers which transport and release chemokine inhibitors at specific pathological sites can reduce chemokine-dependent inflammatory processes.

2. Conjugation of curcumin-loaded lipid nanoemulsions with cell penetrating peptides increases their cellular uptake and enhances the anti-inflammatory effects in endothelial cells. Simion Viorel, Stan Daniela, Constantinescu Cristina Ana, Deleanu Mariana, Dragan Emanuel, Tucureanu Monica Madalina, Gan Ana-Maria, Butoi Elena, Constantin Alina, Manduteanu Ileana, Simionescu Maya, Calin Manuela. Journal of Pharmacy and Pharmacology, accepted on 29 November 2015.

Abstract:

Objectives. To prepare and characterize in vitro and in vivo lipid nanoemulsions loaded with curcumin (Cm) and functionalized with a cell penetrating peptide.

Methods. Cm-loaded lipid nanoemulsions (CmLN) functionalized with a nona-arginine peptide (R9-CmLN) have been obtained, characterized and optimized for size, entrapment efficiency and in vitro Cm release. The interaction of R9-CmLN with human endothelial cells (HEC) was investigated using cultured EA.hy926 cells and in vivo biodistribution studies were performed using C57BL6 mice.

Key findings. When used in therapeutically relevant concentration, R9-CmLN have low haemolytic activity, low cytotoxicity on HEC, and show anti-inflammatory effects by reducing the monocytes adhesion to TNF-α activated HEC. Moreover, HEC uptake and internalization of R9-CmLN was significantly higher compared to the non-functionalized CmLN. In vivo biodistribution studies in mice revealed a higher accumulation of R9-CmLN in the liver and the lungs compared to CmLN and the body clearance of the both nanoformulations after 72 hours.

Conclusions. Cell penetrating peptides-functionalized CmLN have superior characteristics compared to their non-functionalized counterparts: are more efficiently internalized by the cells, produces anti-inflammatory effects in human endothelial cells and when administrated intravenously in mice exhibit increased accumulation in the liver and the lungs, suggesting their potential therapeutic applications in different inflammatory pathologies localized in the liver or the lungs.

International Conference: The 6th EMBO Meeting 2015, Birmingham, United Kingdom, 4-8 september 2015: Communication between macrophages and smooth muscle cells promotes extracellular matrix alterations. Elena Butoi, Ana Maria Gan, Monica Madalina Pirvulescu, Daniela Stan, Cristina Ana Constantinescu, Manuela Calin, Ileana Manduteanu.

Abstract:

Background: Coronary atherosclerosis complicated by plaque rupture and thrombosis is one of the leading causes of cardiovascular death. Although reduced matrix synthesis as well as increased matrix degradation predisposes vulnerable plaques to rupture, the effect of communication between cells of plaque on this process, is not well known. The aim of our study was to investigate the effects of smooth muscle cells (SMCs) – macrophage communication on extracellular matrix alterations and neo-angiogenesis.

Observations: Cell communication was achieved using trans-well chambers, where the aortic SMCs (isolated from the media of fetal thoracic aorta) were grown on the filter and, at confluence were placed in the wells, above differentiated macrophages (THP1 monocytes differentiated with PMA). SMCs and macrophages which were not co-cultured, were used as controls. The results show that co-culture between macrophages and SMCs: i) significantly decreased expression of the extracellular matrix proteins (collagen I, III and elastin) in SMCs; ii) increased the metalloprotease MMP-9 and MMP-1 in both macrophages and SMCs; iii) augmented the VEGF levels in conditioned media (CM) from cell co-culture, and VEGF gene in both cell types. In addition, inflammatory IL-1β and TLR2 were increased in both SMCs and macrophages,and could be responsible for triggering the signaling mechanism in co-culture conditions. Moreover, CM isolated from macrophage-SMC co-culture had an increased angiogenic effect on endothelial cells, promoting tube formation. Interestingly, when MMP-9, IL-1β or TLR2 were blocked with specific antibody, the CM from co-culture produced a lessen number of tube-like structures.

Conclusions: These data extend the previous results that highlighted the role of macrophage-SMCs communication in atherosclerotic plaque evolution, showing that this cell cross-talk may be involved in the vulnerable plaque rupture by promoting extracellular matrix alteration and neo-angiogenesis.

Stage 6

Effect of AP-1 on CX3CR1 regulation in resistin and fractalkine-activated macrophages; development and characterization of nanoparticles to target activated monocytes and macrophages.

Project implementation during January to October 2016 was carried out by performing Stage 6, accomplishing tasks of Objective 2 (Task 2.5) and Objective 3 (Tasks 3.3 and 3.4).

In the period mentioned above, were accomplished:

Task2.5. Role of CX3CR1 in macrophage-SMC cross talk. Regulation of CX3CR1 by AP-1 in macrophages exposed to fractalkine and resistin

Task 3.3. Development and characterization of different NP for targeted delivery to activated monocytes/macrophages

Task 3.4. Assessment of the therapeutic effect of targeted NP-mediated Gi protein inhibitors delivery to activated monocytes/macrophages.

Results

Objective 2: To analyze the resistin and fractalkine-induced monocytes and smooth muscle cells phenotype modulation and the molecular mechanisms involved in macrophage-SMC cross-talk

Task 2.5. Role of CX3CR1 in macrophage-SMC cross talk. Regulation of CX3CR1 by AP-1 in macrophages exposed to fractalkine and resistin

CX3CR1 expression induced by fractalkine – Our data showed that fractalkine induced CX3CR1 receptor expression after 1h activation, this expression being decreased at the control level after 2h activation.

Role of AP-1 transcription factor in CX3CR1 regulation – Our results showed that CX3CR1 protein expression was induced by fractalkine and IFNγ, but not by resistin; also, CX3CR1 expression was not influenced by cJun siRNA transfection of macrophages.

CX3CR1 promoter cloning – In order to identify and characterize the regulatory elements in the promoter region of CX3CR1 gene, we amplified using specific primers a site of ~2000 nucleotides of the promoter. Using specific methods for molecular cloning, we obtained 12 regions of the CX3CR1 promoter, that were used in transfection reactions.

Functional analysis of CX3CR1 promoter activity in smooth muscle cells (SMC) – SMC were transfected using the plasmid DNA obtained in the previous step. After transfection, the activity of CX3CR1 promoter was calculated by measuring the values of β-galacosidase.

In conlcusion, our data regarding the protein and gene expression of CX3CR1 in macrophages showed that fractalkine-CX3CR1 axis is modulate in macrophages by different activators, but further experiments are needed to determine the optimal activation period, in order to evaluate the role of AP-1 transcription factor in CX3CR1 modulation. By cloning CX3CR1 promoter, we could observe that the 87bp and 253bp regions contain multiple positive regulation sites of CX3CR1 receptor, and the longer regions contain negative regulation sites of CX3CR1 promoter activity.

Objective 3: To develop new targeted therapeutic strategies to reduce vascular wall inflammation based on nanotechnology

Task 3.3. Development and characterization of different NP for targeted delivery to activated monocytes/macrophages

Nanoparticles design and functionalization with specific peptides to recognize fractalkine receptor (CX3CR1) – In this stage were optimized the methods of preparation for two types of nanoparticles: lipidic nanoemulsions (LN), suitable for effective incorporation of hydrophobic therapeutic agents and liposomes (Lipo), suitable for hydrophilic therapeutic agents incorporation.

CX3CR1 expression in THP-1 human monocytes – Using flow cytometry techniques, we investigated the presence of CX3CR1 receptor on THP-1 monocytes surface. Monocytes were activated for different periods with resistin or LPS. Our results showed that monocytes express in a constitutive fashion the CX3CR1 expression, and the activation with resistin and LPS determined an increase in protein expression after 48h.

Specific association of CX3CR1-targeted nanoparticles with THP-1 monocytes – To evaluate the binding capacity of nanoparticles functionalized with a specific peptide to target CX3CR1 receptor, the nanoparticles were labeled with a fluorescent compound by inserting in its composition a phospholipid labeled with rodhamine. These nanoparticles were verified using flow cytometry and confocal microscopy.

Task 3.4: Assessment of the therapeutic effect of targeted NP-mediated Gi protein inhibitors delivery to activated monocytes/macrophages

The effect of liposomes loaded with Gi proteins inhibitors and targeted to CX3CR1 on monocytes/macrophages proliferation - To evaluate the effect of CX3CR1-targeted liposomes loaded with GOT (a specific inhibitor for Gi proteins) on the proliferation of monocytes and macrophages, first of all we performed MTT test. Our experiments showed that CX3CR1-targeted liposomes loaded with GOT had no effect on monocytes and macrophages proliferation.

The effect of CX3CR1-targeted liposomes loaded with GOT on signaling pathways activated by resistin or LPS in monocytes/macrophages – Western Blot experiments revealed that resistin and LPS induce the phosphorylation of p38 MAPK protein kinase in monocytes and macrophages. In resistin-activated monocytes, the treatment with GOT-loaded liposomes determined a decrease of p38 MAPK phosphorylation, compared with monocytes treated with resistin only. Investigating JNK signaling pathway, we observed that in monocytes, resistin induced a slight decrease of JNK phosphorylation compared with control cells. Treatment of monocytes with GOT only did not influenced JNK phosphorylation, but the treatment with GOT-loaded liposomes inhibited the level of pJNK. Stimulation with resistin did not activate ERK signaling pathway in monocytes or macrophages, and neither in monocytes activated with LPS. The level of ERK phosphorylation in monocytes and macrophages activated by resistin and LPS and treated with CX3CR1-targeted liposomes loaded with GOT is significantly decreased compared with monocytes activated only by resistin or LPS.

The effect of CX3CR1-targeted liposomes loaded with GOT on cytokine production in monocytes/macrophages activated with resistin or LPS –In this stage we determined the effect of CX3CR1-targeted liposomes loaded with GOT on the expression of inflammatory cytokines like IL-1β, IL-6, TNF-α and MCP-1 chemokine in monocytes/macrophages. ELISA results showed that the targeted liposomes are internalized by monocytes and macrophages and are capable of efficient intracellular transport of GOT inhibitor that is progressively released in cells, determining a significant decrease of pro-inflammatory cytokine production, induced by resistin and LPS.

The effect of CX3CR1-targeted liposomes loaded with GOT on monocyte adhesion to endothelial cells – Cell adhesion experiments showed that the number of adhered monocytes to resistin/LPS-activated endothelium was increased when the monocytes are also treated with these activators, compared to the number of adhered non-stimulated monocytes to non-activated endothelium. The treatment of monocytes with CX3CR1-targeted liposomes loaded with GOT showed that the adhesion to endothelial cells is significantly decreased, regardless the activation status of the endothelium.

In conclusion, the results obtained in Objective 3 showed that Gi protein inhibitor (GOT - guanosine-5'-O-2-thiodiphosphate trilithium salt) can be incorporated with good efficiency in CX3CR1-targeted liposomes. This incorporation determines an efficient intracellular transport of GOT in monocytes/macrophages, that further diminishes the activation of signaling pathways like p38, JNK and ERK MAPK and the production of inflammatory cytokines and chemokines (IL-1β, TNF-α, IL-6, MCP-1). The function of resistin/LPS-activated monocytes is specifically modulated by the treatment with CX3CR1-targeted liposomes loaded with GOT, so that their adhesion to endothelial cells is significantly decreased. These nanoparticles that specifically recognize fractalkine receptor (CX3CR1) could be used to transport different therapeutic agents to monocytes/macrophages and to modulate the cells functions in different diseases associated with inflammatory conditions, such as atherosclerosis.

The results of Stage 6 were presented in four ISI articles and at two important international scientific meetings:

ISI Papers

1. Amendment of the cytokine profile in macrophages subsequent to their interaction with smooth muscle cells: Differential modulation by fractalkine and resistin. Tucureanu MM, Butoi E, Gan AM, Stan D, Constantinescu CA, Calin M, Simionescu M, Manduteanu I. Cytokine. 2016 Jul;83:250-61. doi: 10.1016/j.cyto.2016.04.019. Epub 2016 May 12.

Abstract:

In atherosclerotic plaques, macrophages (MAC) and smooth muscle cells (SMC) frequently reside in close proximity and resistin (Rs) and fractalkine (Fk) are present at increased levels, resistin being associated with CD68 macrophages and fractalkine predominantly associated with intimal SMC; however, their role in this location is not clear, yet. The objective of this study was to determine whether the cross-talk between MAC-SMC induces changes in MAC cytokine phenotype and if Fk and Rs have a role in the process. To this purpose, macrophages (THP-1 monocytes differentiated with phorbol myristate acetate) were interacted with SMC cultured on the membrane inserts in the presence or absence of Rs or Fk. After 24h, MAC were removed from the co-culture and the gene and protein expression of 57 cytokines was assessed by QPCR and Proteome Profiler™ Array. Fk secreted in the culture medium following MAC-SMC interaction was determined (ELISA assay) and the role of Fk in MAC cytokine gene expression was assessed by silencing the Fk receptor in both cell types. The results showed that subsequent to the interaction with SMC, MAC exhibit: (1) a general increased expression of chemokines (the highest fold increase: VCC-1 and GRO-α) and of some interleukins, such as interleukins IL-5 (∼8-fold) and IL-6; (2) an increased Fk expression that in turn induces expression of: CXCL17, CCL19, CCL2, CXCL10, CXCL12, CXCL4, CXCL7, CCL4, CCL18, CXCL16, CXCL1 and IL-27; (3) in the presence of Rs, a predominant increased expression of interleukins (the highest fold increase: IL-6, IL-27, IL-23 and IL-5) and an augmented expression of some chemokines such as MIP-1β, GRO-α and CCL1. In addition, the secretome collected from the SMC-MAC co-culture increased human monocytes chemotaxis. DAVID analysis of the data revealed that the switch of MAC to a pro-inflammatory phenotype, prime the cells to intervene in the immune response, chemotaxis and inflammatory response. In conclusion, MAC cytokines expression is considerable augmented upon their interaction with SMC and Fk and Rs have distinct immunomodulatory roles: Fk predominantly increases the pro-angiogenic and inflammatory chemokines expression and Rs mostly the pro-inflammatory interleukins with consequences on monocyte chemotaxis. The novel data could help to develop targeted nanotherapies to reduce leukocyte chemotaxis and the ensuing inflammatory process associated with atherosclerosis.

2. Cross-talk between macrophages and smooth muscle cells impairs collagen and metalloprotease synthesis and promotes angiogenesis. Butoi E, Gan AM, Tucureanu MM, Stan D, Macarie RD, Constantinescu C, Calin M, Simionescu M, Manduteanu I. Biochim Biophys Acta. 2016 Jul;1863(7 Pt A):1568-78. doi: 10.1016/j.bbamcr.2016.04.001. Epub 2016 Apr 7.

Abstract:

Coronary atherosclerosis complicated by plaque disruption and thrombosis is a critical event in myocardial infarction and stroke, the major causes of cardiovascular death. In atherogenesis, macrophages (MAC) and smooth muscle cells (SMC) are key actors; they synthesize matrix components and numerous factors involved in the process. Here, we design experiments to investigate whether SMC-MAC communication induces changes in ECM protein composition and/or neo-angiogenesis. Cell to cell communication was achieved using trans-well chambers, where SMCs were grown in the upper chamber and differentiated MAC in the bottom chamber for 24 or 72h. We found that cross-talk between MAC and SMC during co-culture: (i) significantly decreased the expression of ECM proteins (collagen I, III, elastin) in SMC; (ii) increased the expression and activity of metalloprotease MMP-9 and expression of collagenase MMP-1, in both MAC and SMC; (iii) augmented the secretion of soluble VEGF in the conditioned media of cell co-culture and VEGF gene expression in both cell types, compared with control cells. Moreover, the conditioned media collected from MAC-SMC co-culture promoted endothelial cell tube formation in Matrigel, signifying an increased angiogenic effect. In addition, the MAC-SMC communication led to an increase in inflammatory IL-1β and TLR-2, which could be responsible for cellular signaling. In conclusion, MAC-SMC communication affects factors and molecules that could alter ECM composition and neo-angiogenesis, features that could directly dictate the progression of atheroma towards the vulnerable plaque. Targeting the MAC-SMC cross-talk may represent a novel therapeutic strategy to slow-down or retard the plaque progression.

3. Conjugation of curcumin-loaded lipid nanoemulsions with cell-penetrating peptides increases their cellular uptake and enhances the anti-inflammatory effects in endothelial cells. Simion V, Stan D, Constantinescu CA, Deleanu M, Dragan E, Tucureanu MM, Gan AM, Butoi E, Constantin A, Manduteanu I, Simionescu M, Calin M. J Pharm Pharmacol. 2016 Feb;68(2):195-207. doi: 10.1111/jphp.12513. Epub 2016 Jan 8.

Abstract:

Objectives: To prepare and characterize in vitro and in vivo lipid nanoemulsions (LN) loaded with curcumin (Cm) and functionalized with a cell-penetrating peptide. Methods: Curcumin-loaded lipid nanoemulsions (CmLN) functionalized with a nona-arginine peptide (R9-CmLN) have been obtained, characterized and optimized for size, entrapment efficiency and in vitro Cm release. The interaction of R9-CmLN with human endothelial cells (HEC) was investigated using cultured EA.hy926 cells, and in vivo biodistribution studies were performed using C57BL6 mice. Key findings: When used in therapeutically relevant concentration, R9-CmLN have low haemolytic activity, low cytotoxicity on HEC, and show anti-inflammatory effects by reducing the monocytes adhesion to TNF-α activated HEC. Moreover, HEC uptake and internalization of R9-CmLN was significantly higher compared to the non-functionalized CmLN. In vivo biodistribution studies in mice revealed a higher accumulation of R9-CmLN in the liver and the lungs compared to CmLN and the body clearance of the both nanoformulations after 72 h. Conclusions: Cell-penetrating peptides-functionalized CmLN have superior characteristics compared to their non-functionalized counterparts: are more efficiently internalized by the cells, produces anti-inflammatory effects in HEC and when administrated intravenously in mice exhibit increased accumulation in the liver and the lungs, suggesting their potential therapeutic applications in different inflammatory pathologies localized in the liver or the lungs.

4. P-Selectin Targeted Dexamethasone-Loaded Lipid Nanoemulsions: A Novel Therapy to Reduce Vascular Inflammation. Viorel Simion, Cristina Ana Constantinescu, Daniela Stan, Mariana Deleanu, Monica Madalina Tucureanu, Elena Butoi, Ileana Manduteanu, Maya Simionescu, and Manuela Calin. Mediators of Inflammation, Volume 2016, Article ID 1625149, 15 pages, http://dx.doi.org/10.1155/2016/1625149.

Abstract:

Inflammation is a common process associated with numerous vascular pathologies. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. We developed and characterized dexamethasone-loaded lipid nanoemulsions directed towards P-selectin (PLN-Dex) and monitored their anti-inflammatory effects in vitro using cultured EC (EA.hy926 cells) and in vivo using a mouse model of acute inflammation [lipopolysaccharides (LPS) intravenously administered in C57BL/6 mice]. We found that PLN-Dex bound specifically to the surface of activated EC are efficiently internalized by EC and reduced the expression of proinflammatory genes, thus preventing the monocyte adhesion and transmigration to/through activated EC. Given intravenously in mice with acute inflammation, PLN-Dex accumulated at a significant high level in the lungs (compared to nontargeted nanoemulsions) and significantly reduced mRNA expression level of key proinflammatory cytokines such as IL-1β, IL-6, and MCP-1. In conclusion, the newly developed nanoformulation, PLN-Dex, is functional in vitro and in vivo, reducing selectively the endothelium activation and the consequent monocyte infiltration and diminishing significantly the lungs’ inflammation, in a mouse model of acute inflammation.

Communications at International meetings

1. P-selectin targeted dexamethasone-loaded lipid nanoemulsions reduce endothelium inflammation. Viorel Simion, Cristina Ana Constantinescu, Daniela Stan, Mariana Deleanu, Monica Madalina Tucureanu, Elena Butoi,Ileana Manduteanu, Maya Simionescu, Manuela Calin. Poster presented at The 8th National Congress with International Participation and 34th Annual Scientific Session of the Romanian Society for Cell Biology, June 08-12 2016, Oradea, Romania.

Abstract:

Introduction. P-selectin is a cell adhesion molecule highly expressed specifically by endothelium in inflammatory disorders, and therefore a potential target for nanotherapy. We hypothesized that targeting the inflamed endothelium by coupling a peptide with high affinity for P-selectin to the surface of dexamethasone-loaded lipid nanoemulsions will highly increase their specific binding to activated endothelial cells (EC) and reduce the cell activation. Materials and methods. Dexamethasone-loaded lipid nanoemulsions (LN-Dex) have been prepared using the ultrasonication method. Targeted LN-Dex were obtained by coupling a peptide with high affinity for P-selectin to the distal end of PEGylated phospholipid in the lipid monolayer of LN via a thioether bond (PLN-Dex). The nanoemulsions were characterized for size by dynamic light scattering (DLS), the amount of peptide coupled to the surface and for entrapment efficiency of dexamethasone by HPLC. The binding and internalization of fluorescently labeled PLN-Dex was determined in TNF-α activated endothelial cells (EC) using fluorescence microscopy and flow cytometry. The anti-inflammatory effects of PLN-Dex were investigated by assessing in EC the gene expression of several pro-inflammatory molecules using qRT-PCR and by evaluating the monocytes adhesion and transmigration to/through EC monolayer using a Boyden chambers. Results. 1) the hydrodynamic diameter of LN was 143±2.6 nm with a polydispersity index of 0.083±0.004; 2) the amount of P-selectin recognizing peptide to the LN surface was 9.25 μg peptide/μmol lipids; 3) the entrapment efficiency of dexamethasone into LN was high (95%); 4) P-selectin targeted LN bound specifically to the surface of activated EC and were efficiently internalized by the cells; 5) dexPLN reduced the gene expression of pro-inflammatory cytokines TNF-α, IL-1β and chemokines IL-8 and MCP-1 in activated EC and, 6) dexPLN is functional since could inhibit monocyte adhesion and transmigration to/through activated EC. Conclusions. (1) Targeted delivery of dexamethasone to TNF-α-activated endothelium can be accomplished by lipid nanoemulsions covered with P-selectin specific peptide. (2) Our newly developed formulation is functional being able to reduce selectively the endothelium activation and the consequent monocyte infiltration.

2. Dexamethasone-encapsulated lipid nanoemulsions targeted to P-selectin reduce endothelium inflammation. Viorel Simion, Daniela Stan, Cristina Constantinescu, Monica Tucureanu, Mariana Deleanu, Elena Butoi, Ileana Manduteanu, Maya Simionescu, Manuela Calin. Poster presented at European Society of Cardiology, August 27-31 2016, Rome, Italy.

Abstract:

Background. Inflammation is a common process associated with many pathologic conditions. P-selectin is a cell adhesion molecule highly expressed specifically by endothelium in inflammatory disorders, and therefore a potential target for nanotherapy. We hypothesize that the coupling of a peptide with high affinity for P-selectin to the surface of nanoparticles will increase the binding to activated endothelium in inflamed tissues. Thus, the aim of this study was to develop nanoparticles loaded with the potent anti-inflammatory drug, dexamethasone and able to target the inflamed endothelium and reduce its activation. Materials and methods. Dexamethasone-loaded lipid nanoemulsions (dexLN) have been prepared using the ultrasonication method. Targeted dexLN were obtained by coupling a peptide with high affinity for P-selectin to the distal end of PEGylated phospholipid in the lipid monolayer of LN via a thioether bond (dexPLN). The nanoemulsions were characterized for size by dynamic light scattering (DLS), the amount of peptide coupled to the surface and for entrapment efficiency of dexamethasone by HPLC. The binding and internalization of fluorescently labeled dexPLN was determined in TNF-α activated endothelial cells (EC) using fluorescence microscopy and flow cytometry. The anti-inflammatory effects of dexPLN were investigated by assessing in EC the gene expression of several pro-inflammatory molecules using qRT-PCR and by evaluating the monocytes adhesion and transmigration to/through EC monolayer using a Boyden chambers. Results. 1) the hydrodynamic diameter of LN was 143±2.6 nm with a polydispersity index of 0.083±0.004; 2) the amount of P-selectin recognizing peptide to the LN surface was 9.25 μg peptide/μmol lipids; 3) the entrapment efficiency of dexamethasone into LN was high (95%); 4) P-selectin targeted LN bound specifically to the surface of activated EC and were efficiently internalized by the cells; 5) dexPLN reduced the gene expression of pro-inflammatory cytokines TNF-α, IL-1β and chemokines IL-8 and MCP-1 in activated EC and, 6) dexPLN is functional since could inhibit monocyte adhesion and transmigration to/through activated EC. Conclusions. (1) Targeted delivery of dexamethasone to TNF-α-activated endothelium can be accomplished by lipid nanoemulsions covered with P-selectin specific peptide. (2) Our newly developed formulation is functional being able to reduce selectively the endothelium activation and the consequent monocyte infiltration.

Contact: Project Manager: Ph.D. Ileana Manduteanu

Head of the Department of Biopathology and Therapy of Inflammation

Deputy Director

e-mail: ileana.manduteanu@icbp.ro

Address: B.P.HASDEU Street, No. 8, 5^th District, Bucharest

Code: 050568, PO Box 35-14

Phone: (+4021)319.45.18

Fax: (+4021)319.45.19

Proiect de Cercetare exploratorie Refinater

Program IDEI - Competitia PCE 2011

Contract nr.108/28.10 2011

Director proiect: dr. Ileana Manduteanu

Echipa:

dr. Ileana Manduteanu (CS I)

dr. Manuela Calin (CS I)

dr. Adrian Manea (CS I)

drd. Daniela Stan (CS)

drd. Viorel Simion (CS)

dr. Elena Butoi (CS II)

dr. Ana-Maria Gan (CS)

drd. Monica-Madalina Pirvulescu (CS)

Gabriela Mesca (tehnician)

Titlu: Molecule si mecanisme implicate in inflamatia vasculara dependenta de citokine si chemokine, posibile tinte pentru noi strategii nanoterapeutice

Fonduri

AN	Fonduri RON
2011	200.000
2012	515.000
2013	189.626,16
2014	182.500
2015	133.215
2016	279.658,84
TOTAL	1.500.000

Rezumat. Inflamatia reprezinta o cauza a multor patologii incluzind ateroscleroza. Mediatorii inflamatiei (citokinele si chemokinele) afecteaza functia celulelor vasculare rezidente si a leucocitelor infiltrate precum si dialogul lor cu consecinte pentru formarea ateromului. Noi am gasit ca rezistina (o citokina) si fractalkina (o chemokina), actioneaza ca legaturi moleculare in inflamatia vasculara prin mecanisme care nu sunt inca cunoscute. Scopurile noastre sunt de a evidentia mecanismele prin care fractalkina si rezistina moduleaza inflamatia in celulele endoteliale, celulele musculare netede, monocite, macrofage, daca ele contribuie la dialogul intre celulele musculare netede si macrofage, evidentierea cailor de semnalizare si reglarea genica implicate si de a dezvolta noi strategii terapeutice tintite bazate pe nanotehnologii pentru a reduce inflamatia peretelui vascular. Obiectivele sunt: (a) Explorarea rolului rezistinei subendoteliale asupra transmigrarii monocitelor; (b) Analiza modularii de catre fractalkina si rezistina a fenotipului macrofagelor si al celulelor musculare netede si elucidarea mecanismelor moleculare implicate in dialogul intre macrofage si celulele musculare netede; (c) Folosirea moleculelor relevante implicate in activarea celulelor endoteliale si monocitelor ca tinte pentru noi terapii antiinflamatorii folosind nanoparticule purtatoare de medicamente. Impact si aplicatii. Proiectul nostru va identifica noi tinte terapeutice si va furniza cunostinte importante pentru dezvoltarea de strategii pentru un mai bun management al bolilor vasculare. Rezultatele vor fi aplicabile la numeroasele boli in care e implicata inflamatia, precum ateroscleroza, boli cronice reumatismale, diabet, cancer si boli neurodegenerative. Efectele acestui proiect vor avea impact pentru stiintele biomedicale deoarece: se folosesc unelte ale biologiei celulare si moleculare pentru a ajuta intelegerea datelor clinice si prin colaborarea in desfasurare cu reumatologi si cardiologi vom corela datele noastre cu date clinice facilitind un transfer continuu in ambele sensuri, de la masa de lucru la patul bolnavului. In plus, prin diseminarea rezulatatelor vom contrubui la cresterea vizibilitatii stiintei biomedicale romanesti.

Proiect

Rezistina si fractalkina au fost evidentiate la nivele crescute in leziuni aterosclerotice umane asociate cu monocite/macrofage in artere carotide si anevrism aortic, iar FK predominant asociata cu celule musculare netede (SMC) intimale si cu monocite/macrofage. Ne-am intrebat daca interactia dintre rezistina si fractalkina intensifica procesul inflamator si daca da, ce mijloace putem gasi pentru a reduce intensitatea procesului.

Ipoteza de lucru:

In inflamatie resistina (R), care este prezenta in concentratii mari in peretele vascular inflamat, activeaza CE si celulele musculare netede intimale (SMC), determinind amplificarea infiltrarii monocitelor (M) prin cresterea expresiei de fraktalkina, MCP-1, TLR4 si activarea proteinelor Gi. (A). Ulterior, in intima, modularea fenotipului monocitelor, proliferarea lor, diferentierea in macrofage activate si dialogul intre macrofagele derivate din monocite si SMC induce modificari in caile de semnalizare, creste expresia de molecule pro-aterogene care accelereaza formarea ateromului indusa de inflamatie. (B). Moleculele relevante gasite a fi instrumentale in activarea CE si M vor fi folosite ca tinte pentru o noua nanoterapie anti-inflamatorie folosind nanoparticule purtatoare de medicamente (NP).

Obiective:

Obiectiv 1: Explorarea rolului rezistinei subendoteliale asupra transmigrarii monocitelor

Obiectiv 2: Analiza modularii fenotipului celulelor musculare netede si a monocitelor indus de rezistina si fractalkina si mecanisme moleculare implicate in dialogul dintre macrofage si celule musculare.

Obiectiv 3: Dezvoltarea de noi strategii terapeutice tintite bazate pe nanotehnologii pentru a reduce inflamatia peretelui vascular.

Impreuna, aceste mecanisme moleculare relevate de studiile noastre vor ajuta la intelegerea complexitatii mecanismelor de semnalizare si de reglare genica a unor molecule cheie implicate si dezvoltarea de noi terapii tintite bazate pe nanotehnologii pentru a reduce inflamatia vasculara. Proiectul nostru va identifica noi tinte pentru medicamente si va furniza o cunoastere importanta pentru dezvoltarea de strategii pentru un tratament mai bun al bolilor vasculare. Datele vor fi aplicabile la numeroase boli in care e implicata inflamatia, cum ar fi ateroscleroza, bolile cronice reumatismale, diabetul, cancerul si bolile neurodegenerative.

Implementare

I. Etapa 1

Implementarea proiectului in perioada octombrie - decembrie 2011 s-a facut prin realizarea Etapei 1, si anume: Analiza expresiei de catre celulele endoteliale a chemokinelor relevante pentru transmigrarea monocitelor: fractalkina si MCP-1.

S-a predat un raport stiintific in care au fost prezentate rezultatele inceputului implementarii obiectivului 1 al proiectului prin evidentierea chemokinelor induse de concentratiile crescute de rezistina subendoteliala in CE umane.

Concluzii Etapa 1: Rezistina subendoteliala induce expresia de chemokine MCP-1 si fractalkina in celule endoteliale umane, chemokine care pot avea un rol important in transmigrarea monocitelor.

II. Etapa 2

Implementarea proiectului in perioada ianuarie-decembrie 2012 s-a facut prin realizarea Etapei 2, si anume: Cai de semnalizare pentru expresia de Fk si MCP-1 induse de rezistina subendoteliala in celulele vasculare

In perioada ianuarie-decembrie 2012 au fost realizate activitati ale obiectivului 1 (Explorarea rolului rezistinei subendoteliale asupra transmigrarii monocitelor) si anume:

Activitatea 1.2. Evidentierea unor noi cai de semnalizare implicate in expresia chemokinelor FK si MCP-1 induse de rezistina in celule endoteliale umane

Activitatea 1.3. Implicarea TLR4 si proteinelor Gi in expresia chemokinelor FK si MCP-1 induse de rezistina in CEU

1.3.1. Efectul rezistinei asupra expresiei de TLR4.

1.3.2. Efectul rezistinei asupra asocierii receptorului TLR 4 cu proteina MyD88.

1.3.3. Analiza implicarii TLR4 in expresia FK si MCP-1 indusa de rezistina in CEU.

1.3.4. Rolul proteinelor Gi in expresia FK si MCP-1 induse de rezistina in CEU

Activitatea 1.4. Expresia genica si proteica de FK si MCP-1 induse de rezistina in celulele musculare netede (CMN) vasculare

1.4.1. Efectul rezistinei asupra expresiei de Fk in CMN

1.4.2. Efectul rezistinei asupra expresiei MCP-1 in CMN

Activitatea 1.5. Implicarea TLR4 si a proteinelor Gi in inducerea de FK si MCP-1 de catre rezistina in celulele musculare netede (CMN)

1.5.1. Efectul rezistinei asupra expresiei de suprafata a receptorului TLR4

1.5.2. Analiza rolului receptorului TLR4 in expresia FK si MCP-1 indusa de rezistina in CMN vasculare

1.5.3. Rolul proteinelor Gi in expresia FK si MCP-1 indusa de rezistina in CMN vasculare.

Activitatea 1.6. Investigarea cailor de semnalizare dependente de rezistina implicate in reglarea expresiei Fk in CMN

1.6.1. Analiza in silico a promotorului uman de fractalkina (CX3CL1)

1.6.2. Clonarea promotorului uman de fractalkina

1.6.3. Analiza functionala a activitatii promotorului CX3CL1 in CMN vasculare:

1.6.4. Interactii fizice ale factorilor de transcriptie la nivelul promotorului CX3CL1- interactii proteina - ADN canonice sau non-canonice

Activitatea 1.7. Rolul stresului oxidativ in expresia FK indusa de rezistina in CMN

1.7.1.Cuantificarea speciilor reactive de oxigen intracelulare induse de rezistina in CMN vasculare

1.7.2. Efectul rezistinei asupra activitatii NADPH oxidazei

Concluzii Etapa 2: Concentratiile crescute de rezistina activeaza celulele vasculare umane (endoteliale si celulele musculare netede) prin mecanisme specifice implicind cresterea productiei de chemoatractanti, fractalkina si MCP-1 , cresterea stresului oxidativ si activarea semnalizarii proteinelor Gi si a receptorului TLR 4. Aceste mecanisme pot fi implicate in cresterea infiltrarii monocitelor induse de rezistina in peretele vascular in conditii inflamatorii si pot constituii tinte terapeutice pentru reducerea inflamatiei peretelui vascular

III. Etapa 3

Implementarea proiectului in perioada ianuarie-decembrie 2013 s-a facut prin realizarea Etapei 3, si anume: Explorarea rolului rezistinei subendoteliale in transmigrarea monocitelor; analiza fenotipului monocitelor si celulelor musculare netede (CMN) indus de rezistina si fractalkina si analiza mecanismelor moleculare implicate in interactia macrofagelor cu CMN

In perioada ianuarie-decembrie 2013 a fost finalizat obiectivul 1 al proiectului de explorare a rolului rezistinei subendoteliale in transmigrarea monocitelor cu efectuarea activitatii 1.8.: Validarea rolului functional al chemokinelor induse de rezistina (fractalkina si MCP-1) in transmigrarea monocitelor. De asemenea au fost realizate activitatile 2.1.: Analiza efectului rezistinei si fractalkinei asupra proliferarii monocitelor, 2.2.: Analiza diferentierii monocitelor in macrofage indusa de rezistina si fractalkine si 2.3.: Investigarea in macrofage a expresiei mediatorilor inflamatori indusi de interactia macrofagelor cu celule musculare netede ale obiectivului 2 (Analiza modularii fenotipului celulelor musculare netede si a monocitelor indus de rezistina si fractalkina si mecanisme moleculare implicate in dialogul dintre macrofage si celule musculare).

Activitatea 1.8. Validarea rolului functional al chemokinelor induse de rezistina (fractalkina si MCP-1) in transmigrarea monocitelor

Rezultatele obtinute in Activitatea 1.8 au fost comunicate intr-o lucare stiintifica publicata intr-o revista cotata ISI: „Subendothelial resistin enhances monocyte transmigration in a co-culture of human endothelial and smooth muscle cells by mechanisms involving fractalkine, MCP-1 and activation of TLR4 and Gi/o proteins signaling”, Pirvulescu MM, Gan AM, Stan D, Simion V, Calin M, Butoi E, Manduteanu I. Int J Biochem Cell Biol. 2014 May;50:29-37. doi: 10.1016/j.biocel.2014.01.022. Epub 2014 Feb 6.

Concluzii etapa 3: Rezultatele obtinute indica rolul important al interactiei dintre macrofage si celulele musculare netede atit in absenta cit si in prezenta rezistinei in modularea expresiei de citokine, chemokine si receptori citokinici si chemokinici in macrofage.

Etapa 4 : “Rolul rezistinei si fractalkinei in cross-talk-ul macrofagelor cu CMN si design-ul, preparearea si caracterizarea mai multor tipuri de nanoparticule pentru tintirea endoteliului activat de rezistina”.

In perioada Ianuarie-Decembrie 2014, a fost implementat Obiectivul 2 al proiectului prin executarea Activitatilor 2.4 si 2.5 si a inceput implementarea e Obiectivului 3 prin executarea Activitatii 3.1, asa cum este descris mai jos.

Obiectivul 2: Analiza fenotipului monocitelor si CMN indus de rezistina si fractalkina si analiza mecanismelor moleculare implicate in interactia macrofagelor cu CMN

Activitatea 2.4. “Analiza activarii CMN si macrofagelor indusa de cross-talk-ul macrofagelor cu CMN (rolul rezistinei sau fractalkinei asupra interactiei macrofagelor cu CMN).”

Activitatea 2.5. “Rolul CX3CR1 in interactia macrofagelor cu CMN; efectul rezistinei si fractalkinei asupra CX3CR1 in macrofage.”

Activitatile si rezultatele obtinute sunt descrise in raportul stiintific al etapei 4.

Rezultate din Activitatea 2.4 impreuna cu rezultate din Activitatea 2.3 au fost comunicate la congresul cu participare internationala “6th National Congress with international Participation and 32nd Annual Scientific Session of Romanian Society for Cell Biology” ce a avut loc la Targu Mures, Romania in perioada 4-7 iunie 2014 si la Conferinta FEBS EMBO ce a avut loc la Paris, Franta in perioada 30 august-4 septembrie 2014. Titlul comunicarii: ”The effect of resistin and fractalkine on macrophage-smooth muscle cells cross-talk”, autori: Pirvulescu M, Butoi E, Gan AM, Stan D, Simion V, Calin M, Manduteanu I.

Rezumatul comunicarii“Resistin is a cytokine that in humans is secreted by peripheral blood mononuclear cells and macrophages. Serum resistin levels are elevated in patients with obesity-associated diabetes or cardiovascular disease. Resistin was found in human atherosclerotic lesions and was suggested to be an inflammatory marker of atherosclerosis and to promote atherogenesis. Fractalkine (FKN) has dual function acting as a cell adhesion molecule and chemokine mediating direct capture, firm adhesion and transmigration of leukocytes and its expression is enhanced in human atherosclerotic plaques. Both resistin and fractalkine were found at increased levels in the human atherosclerotic lesions, resistin being associated with CD68 monocytes/macrophages in carotid arteries and aortic aneurisms, and fractalkine predominantly associated with intimal smooth muscle cells (SMC) and with monocytes/macrophages but their role in this location is not very clear yet. Our recent data showed that resistin has pro-inflammatory effects on SMC and that fractalkine has proatherogenic effects in human monocytes/SMC cross-talk. The objective of this study was to explore the role of resistin and FKN in macrophage-SMC cross-talk and to uncover the molecular mechanisms involved. To this purpose, THP-1 cells (a monocytic cell line) were differentiated to macrophages with phorbol myristate acetate (PMA) and then interacted with SMC cultured on membrane inserts in the presence or absence of resistin or FKN. After 24 hours, the gene and protein expression of inflammatory mediators induced in macrophages by macrophages-SMC cross-talk was determined by Q-PCR and Proteome Profiler™ Array (R&D Systems). Macrophages alone or macrophages activated with resistin or FKN were used as controls.

Protein array experiments revealed that macrophages exhibit after their interaction with SMC an increased expression of chemokines ( CCL1, CCL4, CCL5, CCL19, CXCL1, CXCL5, CXCL7) and cytokines (IFNγ, TNFα, IL-1α, IL-10, IL-2, IL-23, IL-27, IL-5, IL-6 and IL-8) and PAI-1 (plasminogen activator inhibitor-1). Moreover, resistin enhanced the inflammatory macrophage phenotype induced by their interaction with SMC, by increasing the level of the chemokines CCL1, CCL4, CCL5, CXCL1 and of the cytokines: IFNγ, IL-1α, IL-5, IL-6, IL-23, IL-27, IL-8 and PAI-1. In addition, resistin induced in macrophages interacted with SMC an increased expression of the chemokines: CCL10, CCL17, CCL3, CXCL11 and cyokines IL-13, IL-17, IL-17E, IL-ra, IL-1β and ICAM-1, MIF (Macrophage migration inhibitory factor), CD40L. Our preliminary data indicate that FKN also modulate macrophage-SMC interaction by increasing the expression of mediators involved in inflammation and fibrosis. In conclusion, our results suggest that resistin and fractalkine may exert pro-inflammatory effects in macrophages-SMC cross-talk and the molecular mechanisms involved may reveal targets for novel anti-inflammatory therapies.”

Rezultate ale activitatilor din cadrul proiectului REFINATER au fost de asemenea prezentate in review-ul: “Molecular and functional interactions among monocytes/macrophages and smooth muscle cells and their relevance for atherosclerosis”, autori: Elena Butoi, Ana Maria Gan, Ileana Manduteanu, review publicat in Crit Rev Eukaryot Gene Expr. 2014; 24(4):341-55.

Rezumatal reviewului:“Macrophages, smooth muscle cells (SMCs) and their interactions have key roles in the pathogenesis of atherosclerotic vascular diseases. Along atheroma development, the phenotype of macrophages and SMCs changes and may influence the disease progression. The accumulating data on the phenotypes exhibited by these cells within atherosclerotic lesions raise many questions, regarding the mechanisms and factors that might control the transition of cell phenotype. SMCs often reside in vascular lesions in close proximity to macrophage clusters and are most likely influenced by factors released from these pro-inflammatory phagocytes. Moreover, macrophages may be influenced by direct contact with smooth muscle cells or soluble factors released by these cells. Particularly, macrophages may promote activation and induce pro-atherogenic functions of SMCs and smooth muscle cells may modulate macrophage phenotype. Addressing the mechanisms involved in SMC-macrophages cross-talk that lead to phenotypic modulation of both cell types may provide new insight into atherogenesis and new targets for therapies of various vascular diseases.”

Obiectivul 3: Dezvoltarea unor noi strategii terapeutice tintite bazate pe nanotehnologii pentru a reduce inflamatia peretelui vascular, Activitatea 3.1: “Design-ul, prepararea si caracterizarea mai multor tipuri de nanoparticule pentru tintirea endoteliului activat de rezistina”.

Activitatea si rezultatele ei sunt descrise in raportul stiintific al etapei 4.

Rezultate ale acestei activitati au fost prezentate la doua conferinte internationale importante: “6th National Congress with international Participation and 32nd Annual Scientific Session of Romanian Society for Cell Biology” ce a avut loc la Targu Mures, Romania in perioada 4-7 iunie 2014, titlul comunicarii: “P-selectin directed lipid nanoparticles specifically target activated endothelium”, autori: Simion V, Stan D, Constantinescu C, Dragan E, Pirvulescu M, Gan AM, Butoi E, Manduteanu I, Simionescu M, Calin M. si la Conferinta FEBS EMBO ce a avut loc la Paris, Franta in perioada 30 august-4 septembrie 2014, titlul comunicarii: “P-selectin functionalized lipid nanoparticles specifficaly target activated endothelium in acute and chronic inflammation mice models”, autori: Simion V, Stan D, Constantinescu C, Pirvulescu M, Gan AM, Butoi E, Manduteanu I, Simionescu M, Calin M.

Rezumat al comunicarii: “Background. Inflammation is a common occurrence associated, involved and/or responsible for many pathologies. P-selectin is a cell adhesion molecule highly expressed by endothelium in inflammation, and therefore a potential target for nanotherapy. We hypothesize that functionalization of nanoparticles with P-selectin high affinity peptide will increase their binding to activated endothelium in inflamed tissues. Thus, the aim of this study was to develop nanoparticles loaded with curcumin (Cm, a hydrophobic anti-inflammatory polyphenol) that are able to target the inflammatory process in both, acute and chronic inflammation. Materials and methods. Curcumin-loaded lipid nanoparticles (CmLN) functionalized with a P-selectin affinity peptide (P-sel_CmLN) have been developed. The CmLN were characterized for size and structure (by DLS and TEM), entrapment efficiency and curcumin release capacity (by HPLC). In vitro studies using human endothelial cells (HEC) were performed to investigate the cytotoxicity of CmLN (by MTT), their cell binding and internalization (by flow cytometry and confocal microscopy), the signaling pathways involved (Western Blot) and the effect on monocytes adhesion to HEC (fluorescence microscopy). In vivo studies were performed using IVIS Caliper live imaging system on an acute [C57BL mice injected with lipopolysaccharide (LPS) for 5 h] and chronic inflammation mice models [atherosclerotic ApoE-/- mice fed a high cholesterol diet for 4 months]. Results. In vitro studies on HEC revealed that CmLN have: (1) low cytotoxicity; (2) anti-inflammatory effect by down-regulating ERK1/2 and p38 MAPK signaling pathways and (3) impaired adhesion of monocytes to activated endothelial cells. Functionalization of CmLN with P-sel peptide induced (4) an increased binding to activated HEC. In vivo studies (after 1h of nanoparticles administration) revealed (6) an increased accumulation of Psel_CmLN in the lungs of LPS-injected C57BL mice compared to controls (i.e. non-targeted CmLN and PBS-injected C57BL mice) and (7) an increased binding of Psel-CmLN to the aorta (in atherosclerotic plaque-prone areas) of ApoE-/- mice, compared to non-targeted CmLN binding. Conclusion. P-selectin coupled CmLN efficiently bind to activated endothelial cells in vitro and in vivo in acute and chronic experimental inflammation. Therefore, P-selectin exposed on activated HEC surface represents a reliable target for delivery of nanoparticles-carrying drugs.”

Etapa 5

In perioada Ianuarie-Decembrie 2015, a fost implementat Obiectivul 2 al proiectului prin executarea Activitatii 2.4 si Obiectivul 3 prin executarea Activitatii 3.2, asa cum este descris mai jos.

Obiectivul 2. Analiza fenotipului monocitelor si CMN indus de rezistina si fractalkina si analiza mecanismelor moleculare implicate in interactia macrofagelor cu CMN: Activitatea 2.4 - Analiza activarii CMN si macrofagelor indusa de cross-talk-ul macrofagelor cu CMN (rolul activarii simultane cu rezistina si fractalkina asupra interactiei macrofagelor cu CMN).

Obiectivul 3. Dezvoltarea unor noi strategii terapeutice tintite bazate pe nanotehnologii pentru a reduce inflamatia peretelui vascular: Activitatea 3.2 - Determinarea efectelor terapeutice ale unor compusi anti-inflamatori livrati prin intermediul nanoparticulelor la endoteliul activat prin analiza adeziunii si migrarii monocitelor.

Rezultatele acestor activitati au fost descrise in raportul stiintific aferent Etapei 5 si au fost prezentate in doua articole publicate in reviste cotate ISI si comunicate la un congres cu participare internationala:

Articole ISI:

Rezumat:

Objectives. To prepare and characterize in vitro and in vivo lipid nanoemulsions loaded with curcumin (Cm) and functionalized with a cell penetrating peptide.

Conferinta internationala The 6th EMBO Meeting 2015, care a avut loc in Birmingham, Marea Britanie, intre 4-8 septembrie 2015 in cadrul careia a fost prezentat posterul „Communication between macrophages and smooth muscle cells promotes extracellular matrix alterations.” Elena Butoi, Ana Maria Gan, Monica Madalina Pirvulescu, Daniela Stan, Cristina Ana Constantinescu, Manuela Calin, Ileana Manduteanu.

Rezumat:

Etapa 6

Reglarea CX3CR1 de catre AP-1 in macrofagele activate cu rezistina si fractalkina, si dezvoltarea si caracterizarea diferitelor nanoparticule pentru tintirea monocitelor activate/macrofagelor. Determinarea efectelor terapeutice ale inhibitorilor specifici proteinelor Gi livrati monocitelor activate/macrofagelor prin intermediul nanoparticulelor

In perioada ianuarie-octombrie 2016 au fost realizate activitati ale Obiectivului 2 (Activitatea 2.5) si ale Obiectivului 3 (Activitatea 3.3 si 3.4) si anume:

Activitatea 2.5. Rolul CX3CR1 in interactia macrofagelor cu CMN. Reglarea CX3CR1 de catre AP-1 in macrofagele activate cu rezistina si fractalkina

Activitatea 3.3. Dezvoltarea si caracterizarea diferitelor nanoparticule pentru tintirea monocitelor activate/macrofagelor

Activitatea 3.4. Determinarea efectelor terapeutice ale inhibitorilor specifici proteinelor Gi livrati monocitelor activate/macrofagelor prin intermediul nanoparticulelor

Rezultate

Obiectivul 2: Analiza fenotipului monocitelor si CMN indus de rezistina si fractalkina si analiza mecanismelor moleculare implicate in interactia macrofagelor cu CMN

Activitatea 2.5. Reglarea CX3CR1 de catre AP-1 in macrofagele activate cu rezistina si fractalkina

Expresia CX3CR1 indusa de fractalkina - Datele noastre au aratat ca fractalkina induce expresia receptorului CX3CR1 dupa 1h de activare cu fractalkina, aceasta expresie scazand la nivelul controlului dupa 2h de activare.

Rolul factorului de transcriptie AP-1 in reglarea receptorului CX3CR1 - Rezultatele au aratat ca expresia proteica a CX3CR1 a fost indusa de fractalkina si de IFNγ, dar nu si de rezistina, insa nu a fost influentata de transfectia macrofagelor cu cJun siRNA.

Clonarea promotorului genei CX3CR1 - Pentru a identifica si caracteriza elementele reglatoare din regiunea promotor a genei CX3CR1, am amplificat utilizand primeri specifici, o regiune a promotorului de ~2000 nucleotide. Primerii utilizati in toate reactiile de amplificare, au fost proiectati sa aiba incorporat in capatul terminal 5’ situsuri ale enzimelor de restrictie KpnI pentru primerii sens si XhoI pentru primerul antisens.

Analiza functionala a activitatii promotorului CX3CR1 in CMN vasculare - Pentru analiza functionala a activitatii promotorului CX3CR1, CMN au fost transfectate cu ADN-ul plasmidial obtinut prin clonare. Dupa transfectie, activitatea promotorului CX3CR1 a fost calculata prin raportarea valorilor de luciferaza la β-galactozidaza si exprimata ca valori arbitrare.

In concluzie, datele obtinute privind atat expresia genica, cat si proteica a CX3CR1 in macrofage, arata ca axa fractalkina-CX3CR1 este modulata in macrofage de diferiti agonisti, insa sunt necesare experimente suplimentare pentru a stabili timpul optim de activare al macrofagelor astfel incat sa fie posibila evaluarea rolul factorului de transcriptie cJun in modularea expresiei CX3CR1. De asemenea, prin clonarea promotorului CX3CR1, s-a observat ca fragmentele de 87pb si 253 pb contin multiple situsuri de reglare pozitiva a activitatii receptorului CX3CR1, in timp ce fragmentele mai mari contin situsuri de inhibare a activitatii promotorului CX3CR1.

Obiectivul 3: Dezvoltarea unor noi strategii terapeutice tintite bazate pe nanotehnologii pentru a reduce inflamatia peretelui vascular

Activitatea: 3.3. Dezvoltarea si caracterizarea diferitelor nanoparticule pentru tintirea monocitelor activate/macrofagelor

Prepararea nanoparticulelor si functionalizarea acestora cu peptidele specifice de recunoasterea a receptorului pentru fractalkina (CX3CR1) - Au fost optimizate metodele de preparare pentru doua tipuri de nanoparticule: nanoemulsiile lipidice (LN), potrivite pentru incorporarea eficienta a agentilor terapeutici hidrofobi si liposomii (Lipo), utili pentru incroporarea agentilor terapeutici hidrofili.

Evaluarea exprimarii receptorului CX3CR1 de catre monocitele umane THP-1 - Cu ajutorul citometriei de flux s-a investigat prezenta receptorului CX3CR1 pe suprafata monocitelor THP-1 ne-activate sau la diferite intervale de timp dupa activare cu rezistina sau LPS. Rezultatele obtinute au aratat ca monocitele THP-1 exprima constitutiv receptorul CX3CR1, iar activarea cu rezistina si LPS determina o crestere a exprimarii acestuia dupa 48 ore de activare.

Asocierea specifica a nanoparticulelor tintite catre CX3CR1 cu monocitele THP-1 - Pentru a evalua capacitatea de legare a nanoparticulelor functionalizate cu peptidul cu afinitate specifica pentru receptorul CX3CR1 de pe monocite, nanoparticulele au fost etichetate fluorescent prin inserarea fosfolipidului DSPE marcat fluorescent cu Rhodamina si au fost utilizate metodele de citometrie de flux si microscopie de fluorescenta confocala.

Activitatea 3.4.: Determinarea efectelor terapeutice ale inhibitorilor proteinelor Gi livrati monocitelor activate/macrofagelor prin intermediul nanoparticulelor

Efectul liposomilor incarcati cu inhibitori de proteine Gi si directionati catre CX3CR1 asupra proliferarii monocitelor/macrofagelor - Pentru a evalua efectul liposomilor incarcati cu GOT (inhibitor al proteinelor Gi) si directionati catre CX3CR1 asupra proliferarii monocitelor si macrofagelor, in primul rand s-a efectuat testul de viabilitate si proliferare celulara MTT. Experimentele au aratat ca liposomii incarcati cu GOT si directionati catre CX3CR1 nu au avut nici un efect asupra proliferarii monocitelor sau macrofagelor.

Efectul tratamentului cu liposomi directionati catre CX3CR1, transportori de inhibitori de proteine Gi asupra cailor de semnalizare activate de rezistina sau LPS in monocite/macrofage -Atat rezistina cat si LPS induc fosforilarea protein kinazei p38 MAPK in monocite si in macrofage. In monocitele activate cu rezistina, tratamentul cu liposomi transportori de GOT determina o inhibare a fosforilarii p38 MAPK fata de monocitele activate cu rezistina. Investigarea caii de semnalizare JNK a relevat faptul ca, in monocite, rezistina nu induce, ci determina o mica scadere a fosforilarii JNK comparativ cu celulele ne-activate. Tratarea monocitelor cu GOT liber nu a avut influenta asupra nivelului de JNK fosforilat, in schimb tratamentul cu GOT incorporat in liposomi a inhibat nivelul de pJNK. Stimularea monocitelor cu rezistina nu a determinat activarea caii ERK in monocite si macrofage activate cu rezistina si nici in monocite activate cu LPS. Nivelul de fosforilare a ERK1/2 in monocitele si macrofagele activate atat cu rezistina cat si cu LPS si incubate cu liposomi directionati catre CX3CR1, este semnificativ redus in comparatie cu pERK1/2 in monocite activate cu rezistina.

Efectul tratamentului cu liposomi directionati catre CX3CR1, transportori de inhibitori de proteine Gi asupra productiei de citokine in monocite/macrofage activate cu rezistina sau LPS - A fost determinat efectul tratamentului monocitelelor/macrofagelor umane THP-1, activate cu rezistina sau LPS, cu inhibitorul de proteine Gi (GOT), liber sau incorporat in liposomi ne-directionati sau directionati catre CX3CR1 asupra productiei de citokine inflamatorii IL-1β, IL-6, TNF-α si a chemokinei MCP-1. Rezultatele obtinute sugereaza ca liposomii, atat cei ne-tintiti cat si cei tintiti sunt internalizati de catre monocite/macrofage si sunt capabili sa realizeze un transport intracelular eficient de inhibitori de proteine Gi, pe care ii elibereaza in celula treptat determinand scaderea seminificativa si sustinuta a productiei de citokine pro-inflamatorii indusa de activarea monocitelor si macrofagelor cu rezistina sau LPS.

Efectul tratamentului monocitelor activate cu rezistina sau LPS cu liposomi directionati catre CX3CR1, transportori de inhibitori de proteine Gi asupra adeziunii acestora la celulele endoteliale si asupra capacitatii lor de transmigrare - Rezultatele arata ca numarul monocitelor aderate la endoteliul activat cu rezistina/LPS creste semnificativ in cazul in care acestea sunt stimulate cu rezistina/LPS si tratate in diferite conditii, comparativ cu numarul monocitelor ne-tratate aderate la endoteliul ne-stimulat. In cazul monocitelor tratate cu GOT incapsulat in liposomi tintiti sau ne-tintiti, acestea adera semnificativ mai putin la endoteliul activat sau ne-activat cu rezistina/LPS, comparativ cu monocitele tratate cu liposomi ce nu contin GOT incapsulat.

In concluzie, rezultatele obtinute au aratat ca inhibitorul de proteine Gi, guanosine-5'-O-2-thiodiphosphate trilithium salt (GOT) poate fi incorporat cu o eficienta buna in liposomi PEGylati directionati catre receptorul chemokinic CX3CR1 exprimat pe suprafata monocitelor/macrofagelor. Aceasta incorporare in liposomi determina un transport intracelular eficient de GOT in monocite/macrofage, fapt ce duce la o diminuare semnificativa a activarii cailor de semnalizare intracelulara de catre rezistina sau LPS (p38MAPK, JNK, ERK 1/2) si a productiei de citokine/chemokine pro-inflamatorii (IL-1β, TNF-α, IL-6, MCP-1). Functia monocitelor activate cu rezistina sau LPS este specific modulata de tratamentul cu IC13-Lipo/GOT, astfel incat adeziunea acestora la celule endoteliale si capacitatea lor de transmigarea este semnificativ redusa comparativ cu tratamentul cu GOT liber sau incorporat in liposomi ne-directionati. Aceste nanoparticulele functionalizate cu peptide ce recunosc specific receptorul pentru fractalkina CX3CR1 pot fi utilizate pentru transportul tintit de agenti terapeutici la monocite/macrofage in scopul modularii functiei acestora in anumite conditii inflamatorii asociate unor boli cum ar fi ateroscleroza.

Rezultatele obtinute la finalizarea obiectivelor 2 si 3 au fost prezentate in patru articole publicate in reviste cotate ISI si comunicate la doua congrese cu participare internationala:

Articole ISI

Rezumat:

Comunicari

1. P-selectin targeted dexamethasone-loaded lipid nanoemulsions reduce endothelium inflammation. Viorel Simion, Cristina Ana Constantinescu, Daniela Stan, Mariana Deleanu, Monica Madalina Tucureanu, Elena Butoi,Ileana Manduteanu, Maya Simionescu, Manuela Calin. Poster prezentat la al 8-lea Congres cu Participare Internationala si a 34-a Sesiune Stiintifica Anuala a Societatii Romane de Biologie Celulara, 08-12 Iunie 2014, Oradea, Romania.

Rezumat:

2. Dexamethasone-encapsulated lipid nanoemulsions targeted to P-selectin reduce endothelium inflammation. Viorel Simion, Daniela Stan, Cristina Constantinescu, Monica Tucureanu, Mariana Deleanu, Elena Butoi, Ileana Manduteanu, Maya Simionescu, Manuela Calin. Poster prezentat at Conferinta Societatii Europene de Cardiologie, 27-31 August 2016, Roma, Italia.

Rezumat:

Contact: director proiect dr. Ileana Manduteanu

Sef Department Biopatologia si Terapia Inflamatiei

Director adjunct

email: ileana.manduteanu@icbp.ro

Adresa: Strada B.P.HASDEU Nr. 8, sector 5, Bucuresti

Cod: 050568, PO Box 35-14

Telefon: (+4021)319.45.18

Fax: (+4021)319.45.19

ABSTRACT

IMPLEMENTATION

DISSEMINATION

DISEMINARE

CONTACT

Funds

Fonduri

Proiect

Implementare

Contact: director proiect dr. Ileana Manduteanu

email: ileana.manduteanu@icbp.ro