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

Atherosclerosis is an inflammatory disease, generated and/or aggravated by lipid metabolism disorders. The discovery of microRNAs (miRNAs) and their role in the regulation of the gene expression is one of the most exciting scientific breakthroughs of the last period. Identifying functional miRNA target genes is crucial to understand cellular function of specific miRNAs. Our preliminary results show that miR-486 and miR-92a are significantly associated with coronary artery disease (CAD) vulnerability and are associated primarily with high density lipoproteins (HDL), specifically with HDL₂ and HDL₃. Existing data do not refer to the existence of lipid metabolism-related genes that are functionally targeted by miR-486 and miR-92a, although these miRNAs individually target over 250 genes. The main aim of the project is to evaluate molecular therapy for dyslipidaemia by in vivo modulation of microRNAs expression. Our working hypothesis is that dyslipidemia may be treated by using specific inhibition of miRNAs. Accordingly, the first objective is to identify by bioinformatics analysis and to validate lipid metabolism-related genes targeted by miR-486 and miR-92a. We will further explore blood and tissue-specific microarray distribution of miRNAs in hyperlipidemic animals. Furthermore, we will evaluate the effects of in vivo inhibition of miRNAs in atherosclerosis development. Finally, we will assess the HDL-mediated transfer of specific microRNAs to cultured cells.

Rezumat

Ateroscleroza este o boala inflamatorie, generata si/sau agravata de dereglari ale metabolismului lipidic. Descoperirea microARN (miRNA) si a rolului lor in reglarea expresiei genice este una dintre cele mai importante realizari stiintifice din ultima perioada. Identificarea genelor tinta functionale ale miRNA este cruciala pentru intelegerea functiilor celulare ale miRNA specifice. Rezultatele noastre preliminare arata ca miR-486 si miR-92a se coreleaza semnificativ cu vulnerabilitatea bolii coronariene si sunt asociate in special cu lipoproteinele de inalta densitate (HDL), specific cu HDL₂ si HDL₃. Datele din literatura nu fac referire la genele implicate in metabolismul lipidic care sunt functional modulate de miR-486 si miR-92a, desi aceste miRNA tintesc individual peste 250 de gene. Scopul principal al acestui proiect este evaluarea terapiilor moleculare pentru dislipidemie prin modularea in vivo a expresiei miRNA. Ipoteza noastra de lucru este aceea ca dislipidemia poate fi tratata folosind inhibitia specifica a miRNA. Astfel, primul obiectiv este identificarea prin analiza bioinformatica si validarea genelor metabolismului lipidic tintite de catre miR-486 si miR-92a. Mai departe, ne propunem explorarea distributiei miRNA in sangele si tesuturile animalelor hiperlipidemice. Apoi, vom evalua efectele inhibitiei in vivo a miRNA asupra evolutiei aterosclerozei. In final, vom evalua transferul mediat de HDL al unor miRNA specifice catre celule in cultura.

General objectives and workplan

WP1. Functional analysis of the cardiovascular-related microRNAs, miR-486 and miR-92a:in silico and in vitro approaches (months 1-6)

Objective 1: To identify and validate in vitro the target genes of miR-486 and miR-92a

WP2. In vivo functional analysis of the cardiovascular-related microRNAs, miR-486 and miR-92a (months 5-11)

Objective 2: To investigate in vivo the functional roles of miR-486 and miR-92a

WP3. In vivo molecular therapy against miR-486 and miR-92a (months 9-15)

Objective 3: To treat hyperlipidemia by in vivo inhibition of miR-486 and miR-92a

WP4. High density lipoprotein-mediated miRNA communication (months 16-23)

Objective 4: To investigate the specific mechanisms of cellular miRNA communication mediated by high density lipoproteins [WP4]

WP5. Dissemination and training (months 1-24)

Objective 5: To widely disseminate the results and to develop the technical knowledge of team members

WP6. Management of the project (months 1-24)

Objective 6: To manage the overall project tasks, deliverables and milestones.

2015 WORKPLAN

WP1. Functional analysis of the cardiovascular-related microRNAs, miR-486 and miR-92a: in silico approaches (months 1-3)

Task 1.1. Bioinformatics analysis of the predicted genes targeted by human miR-486 and miR-92a.

Task 1.2. In silico analysis by KEGG signalling pathways and functional protein association networks; selection of maximum five target genes involved in human lipid metabolism that are most likely to be modulated by miR-486 and miR-92a.

Task 1.3. Design of luciferase reporter plasmids containing the 3’UTR sequence of the selectedtarget genes.

WP5. Dissemination and training (months 1-3)

Task 5.1. Contribution to doctoral studies by integrating scientific programs of Ph.D. students.

Task 5.2. Design and build of a website dedicated to the project’s results.

WP6. Management of the project (months 1-3)

Task 6.1. Monitoring the project’s progress, defining the specific role and working protocol of each team member, timing and synchronizing tasks.

Task 6.2. Organizing periodic meetings between team members.

Task 6.3. Communication management: generating and storing data, documentation and deliverables generated by the project; periodic contact with the Contracting Authority; managing the dedicated website.

Task 6.4. Preparation of periodic deliverables and reports, including achievement of financial management and delivery of financial and scientific reports to the Contracting Authority.

2015 Deliverables:

Technical-scientific report (month 3)

Possible target genes for miR-486 and miR-92a

Project’s website

2016 WORKPLAN

WP1. Functional analysis of the cardiovascular-related microRNAs, miR-486 and miR-92a: in vitro approach (months 4-6)

Objective: To identify and validate in vitro the target genes of miR-486 and miR-92a.

Task 1.3. Design, cloning and validation of luciferase reporter plasmids containing the 3’UTR sequence of the selected target genes.

Task 1.4. In vitro testing of the selected target genes inhibition by miR-486 and miR-92a using 3’UTR plasmids and miRNAs mimics; evaluation of the target gene expression and associated protein levels.

WP2. In vivo functional analysis of the cardiovascular-related microRNAs, miR-486 and miR-92a (months 5-11)

Objective: To investigate in vivo the functional roles of miR-486 and miR-92a.

Task 2.1. Obtaining of experimental animal models of diet-induced; establishing the animal groups and starting the hyperlipidemic diet administration.

Task 2.2. Monitoring blood and tissue miR-486 and miR-92a levels during hyperlipidemic diet; correlation with serum lipids profile, lipoprotein-related enzymes activity.

WP3. In vivo molecular therapy against miR-486 and miR-92a (months 9-15)

Objective: To treat hyperlipidemia by in vivo inhibition of miR-486 and miR-92a.

Task 3.1. Establishing the hyperlipidemic animal groups and starting the administration of in vivo LNA microRNA inhibitors against miR-486 and miR-92a.

Task 3.2. In vivo molecular therapy targeting the hyperlipidemia using LNA microRNA inhibitors against miR-486 and miR-92a: follow-up of the serum lipids profile and lipoprotein-related enzyme activity.

Task 3.3. In vivo molecular therapy targeting the hyperlipidemia using LNA microRNA inhibitors against miR-486 and miR-92a: organ-specific distribution of miRNAs and of the selected and validated targets.

Task 3.4. In vivo molecular therapy targeting the hyperlipidemia using LNA microRNA inhibitors against miR-486 and miR-92a: tissue-specific miRNAs machinery regulation (Dicer, Drosha and DGCR8).

WP5. Dissemination and training (months 4-24)

Objective: To widely disseminate the results and to develop the technical knowledge of team members.

Task 5.1. Attending national and international scientific events with communications of the obtained results.

Task 5.2. Preparing and publishing the results in ISI-Thomson ranked and/or BDI journals.

Task 5.3. Performing training and research stages for team members (Ph.D. students, postdocs).

Task 5.4. Contribution to doctoral studies by integrating scientific programs of Ph.D. students.

Task 5.5. Design, build and periodic update of a website dedicated to the project’s results.

WP6. Management of the project (months 4-24)

Objective: To manage the overall project tasks, deliverables and milestones.

Task 6.1. Monitoring the project’s progress, defining the specific role and working protocol of each team member, timing and synchronizing tasks.

Task 6.2. Organizing periodic meetings between team members.

Task 6.3. Communication management: generating and storing data, documentation and deliverables generated by the project; periodic contact with the Contracting Authority; managing the dedicated website.

Task 6.4. Preparation of periodic deliverables and reports, including achievement of financial management and delivery of financial and scientific reports to the Contracting Authority.

2016 Deliverables

Technical-scientific report (month 15)

Validated target genes for miR-486 and miR-92a

Anti-miRNA therapy in hyperlipidemic animals

Communications at scientific events and research stages

2017 WORKPLAN

WP2. In vivo functional analysis of the cardiovascular-related microRNAs, miR-486 and miR-92a (months 16-24)

Objective: To investigate in vivo the functional roles of miR-486 and miR-92a.

Task 2.3. Tissue-specific miRNAs microarray profiling of hyperlipidemic animals’ liver, gut and myocardium (valves, ventricles, apex); correlation with circulating miR-486 and miR-92a levels.

Task 2.4. Circulating miRNAs microarray profiling of hyperlipidemic animals’ serum and circulating monocytes; correlation with circulating miR-486 and miR-92a levels.

Task 2.5. Tissue-specific miRNAs machinery regulation: expression of Dicer, Drosha and DGCR8 in hyperlipidemic animals’ liver, gut and myocardium (valves, ventricles, apex).

WP4. High density lipoprotein-mediated miRNA communication (months 16-26).

Objective: To investigate the specific mechanisms of cellular miRNA communication mediated by HDL.

Task 4.1. Isolation of human LDL and HDL (HDL₂ and HDL₃ subpopulations) from healthy donors’ plasma and preparation of in vitro oxidized LDL.

Task 4.2. Isolation of circulating human CD14⁺ monocytes from healthy donors’ blood.

Task 4.3. Obtaining lipid-loaded macrophages by differentiating monocytes into macrophages and subsequent lipid loading by incubation with in vitro oxidized LDL.

Task 4.4. miRNAs microarray profiling of human monocytes and lipid-loaded macrophages; correlation with miRNAs profile of circulating monocytes from hyperlipidemic animals (obtained in WP2); evaluation of the cell-specific distribution of miR-486 and miR-92a and their selected target genes.

Task 4.5. Assessment of miR-486 and miR-92a and their selected target genes expression in lipid-loaded macrophages after exposure to HDL₂ or HDL₃.

Task 4.6. Enrichment of human HDL with exogenous non-human (cel-miR-39) miRNA; measurement of exogenous miRNAs in the lipid-loaded macrophages exposed to HDL enriched with cel-miR-39.

Task 4.7. Enrichment of human HDL with miR-486 or miR-92a; evaluation of selected target genes expression in lipid-loaded macrophages incubated with HDL enriched with miR-486 or miR-92a.

WP5. Dissemination and training (months 4-26)

Objective: To widely disseminate the results and to develop the technical knowledge of team members.

Task 5.1. Attending national and international scientific events with communications of the obtained results.

Task 5.2. Preparing and publishing the results in ISI-Thomson ranked and/or BDI journals.

Task 5.3. Performing training and research stages for team members (Ph.D. students, postdocs).

Task 5.4. Contribution to doctoral studies by integrating scientific programs of Ph.D. students.

Task 5.5. Design, build and periodic update of a website dedicated to the project’s results.

Task 5.6. Uploading the microarray data to specific online databases.

WP6. Management of the project (months 4-26)

Objective: To manage the overall project tasks, deliverables and milestones.

Task 6.1. Monitoring the project’s progress, defining the specific role and working protocol of each team member, timing and synchronizing tasks.

Task 6.2. Organizing periodic meetings between team members.

Task 6.4. Preparation of periodic deliverables and reports, including achievement of financial management and delivery of financial and scientific reports to the Contracting Authority.

2017 Deliverables

Technical-scientific report (month 26)

miRNA microarray profile in hyperlipidemic animals

miRNA distribution in human monocytes and macrophages

Communications at scientific events and research stages

Article in ISI-Thomson and/or BDI journals

Update of the project’s website

DISSEMINATION

Articles published in ISI-ranked journals

Simionescu Natalia, Loredan S. Niculescu, Gabriela M. Sanda, Mihaela G. Cărnuţă, Camelia S. Stancu, Andreea C. Popescu, Mihaela R. Popescu, Adelina Vlad, Doina R. Dimulescu, Maya Simionescu, Anca V. Sima. Hyperglycemia Determines Increased Specific MicroRNAs Levels in Sera and HDL of Acute Coronary Syndrome Patients and Stimulates MicroRNAs Production in Human Macrophages. PLoS ONE 11(8): e0161201, 2016; doi: 10.1371/journal.pone.0161201.

Loredan S. Niculescu, Natalia Simionescu, Elena V. Fuior, Camelia S. Stancu, Mihaela G. Cărnuţă, Mădălina D. Dulceanu, Mina Răileanu, Emanuel Drăgan, Anca V. Sima. Targeting lipid metabolism-related genes by in vivo inhibition of miR-486 and miR-92a to decrease plasma and liver cholesterol levels of hyperlipidemic hamsters. Molecular Biology Reports 45(4):497-509, 2018; doi: 10.1007/s11033-018-4186-8.

Communications at congresses

2016

Niculescu L.S., N. Simionescu, G.M. Sanda, M.G. CRNAuta, C.S. Stancu, A.C. Popescu, M.R. Popescu, A. Vlad, D.R. Dimulescu, A.V. Sima. Hyperglycemia increases microRNAs in circulating HDL from acute coronary syndrome patients and modulates miRNA processing machinery in human macrophages. 84^th Congress of European Atherosclerosis Society, Innsbruck, Austria, 29 May - 2 June 2016, Atherosclerosis Supplements 252: e82, 2016. DOI: 10.1016/j.atherosclerosis.2016.07.504.

Niculescu L.S., N. Simionescu, G.M. Sanda, M.G. CRNAuta, C.S. Stancu, A.C. Popescu, M.R. Popescu, A. Vlad, D.R. Dimulescu, A.V. Sima. Specific circulating microRNAs levels associate with hypertension, hyperglycemia and dysfunctional HDL in acute coronary syndrome patients. Poster presentation at 4^th Frontiers in Cardiovascular Biology, Florence, Italy, 8–10 July 2016, Cardiovascular Research, 111(Suppl 1): S56-S81, 2016; DOI: http://dx.doi.org/ 10.1093/cvr/cvw149.

Niculescu L.S., N. Simionescu, G.M. Sanda, A.C. Popescu, M.R. Popescu, A. Vlad, D.R. Dimulescu, M. Simionescu, A.V. Sima. Hyperglycemia is Associated with Increased Circulating Micrornas Levels in Acute Coronary Syndrome Patients’ Sera and Determines the Upregulation of microRNA Production in Human Macrophages. Moderated poster at 55^th National Congress of the Romanian Society of Cardiology, Sinaia, 21-24 September 2016, Romanian Journal of Cardiology Supplements 2016; awarded by the Romanian Society Cardiology Program - Excellence in Research.

Niculescu L.S., N. Simionescu, G.M. Sanda, M.G. Cărnuță, C.S. Stancu, A.C. Popescu, M.R. Popescu, A. Vlad, D.R. Dimulescu, A.V. Sima. Hyperglycemia Increases Micro-RNAs in Sera and HDL From Acute Coronary Syndrome Patients; in Search for the Processing Machinery of Micro-RNAs in Human Macrophages. Poster presentation at 8^th National Congress with international participation and 34^th Annual scientific session of the Romanian Society for Cell Biology, 8-12 June 2016, Oradea, Bulletin of RSCB 44: 63, 2016.

2017

Simionescu N, Cărnuţă MG, Fuior E, Stancu CS, Răileanu M, Dulceanu MD, Drăgan E, Sima AV, Niculescu LS. Targeting lipid metabolism-related genes by in vivo inhibition of miR-486 and miR-92a lowers plasma cholesterol levels in hyperlipidemic hamsters; autori Science at a Glance Session at The 85^th Congress of European Atherosclerosis Society, Prague, Czech Republic, 23 – 26 April 2017. Atherosclerosis Supplements, 263: e34–e35, 2017. DOI: 10.1016/ j.atherosclerosis.2016.07.504.

Simionescu N, Cărnuţă MG, Fuior E, Stancu CS, Răileanu M, Dulceanu MD, Drăgan E, Sima AV, Niculescu LS. Molecular therapy regulates dyslipidemia in experimental atherosclerosis; in vivo targeting of miR-486 and miR-92a. International Symposium Acad. Nicolae Cajal of the Academy for Medical Sciences: Translational research - Actualities in virusology, Bucharest, 30 March - 1 April 2017.

Simionescu N., Cărnuţă MG, Fuior E, Stancu CS, Răileanu M, Dulceanu MD, Drăgan E, Sima AV, Niculescu LS. MiR-486 and miR-92a modulate SOAT2, SREBF1, ABCG4 and NPC1 expression in hyperlipidemic hamsters to lower plasma cholesterol levels. The 9th National Congress with international participation and The 35th Annual scientific session of Romanian Society for Cell Biology, Iasi, Romania, 7 - 11 June 2017, Bulletin of RSCB 45: 10, 2017.

Awards obtained by the team members

EAS Young Investigator Fellowship for Natalia Simionescu to attend The 85^th Congress of European Atherosclerosis Society, Prague, Czech Republic, 23 – 26 April 2017 with the communication Targeting lipid metabolism-related genes by in vivo inhibition of miR-486 and miR-92a lowers plasma cholesterol levels in hyperlipidemic hamsters, authors Simionescu N, Cărnuţă MG, Fuior E, Stancu CS, Răileanu M, Dulceanu MD, Drăgan E, Sima AV, Niculescu LS.

Award „Constantin Velican” of the Romanian Society for Cell Biology, for outstanding research in the field of cardiovascular disease biopathology - Loredan Niculescu, 8 June 2017.