Grant acordat de Autoritatea Nationala Pentru Cercetare Stiintifica si Inovare, CNCS-UEFISCDI, Program Resurse umane, Proiecte de cercetare pentru stimularea tinerelor echipe independente (TE)

Cod Proiect: PN-III-P1-1.1-TE-2019-0811/ Nr. Contract: TE 97 /2020

TITLU PROIECT: Modularea imuna a celulelor T de catre plachete si microvezicule plachetare in ateroscleroza indusa experimental; rolul microRNA-142-3p

DIRECTOR DE PROIECT: Dr. CSII Nicoleta Alexandru-Moise

Perioada de desfasurare: 15. 09.2020-14.09.2022

Buget proiect: 431.900 lei

Echipa de cercetare: Ph.D. Habil, CSI Adriana Georgescu, Ph.D. CSIII Alina Constantin, Ph.D. CSIII Miruna Nemecz, Ph.D. AC Alexandru Filippi, AC Ioana Karla Comarita, AC Alexandra Vilcu, AC Anastasia Procopciuc.

Rezumat

 Ateroscleroza reprezinta principala cauza a bolilor cardiovasculare ischemice, aflate pe primul loc privind mortalitatea la nivel mondial. Celulele imune, atat ale sistemului imun innascut, cat si adaptativ, si plachetele sunt prezente in toate etapele dezvoltarii leziunii aterosclerotice. Desi, exista dovezi care sugereaza faptul ca plachetele regleaza mai multe functii ale celulelor imune, sunt necesare studii suplimentare pentru a intelege mai bine modul in care acestea moduleaza raspunsul imun. Scop: Acest proiect isi propune sa exploreze interactia dintre celulele T (CD4⁺ si CD8⁺) si plachete, intr-un model animal de ateroscleroza indusa experimental, sa investigheze, in vitro, efectele plachetelor si a microveziculelor plachetare (PMVs) de origine sanatoasa, asupra functiilor celulelor T si sa descifreze mecanismelor moleculare implicate, cu o atentie speciala asupra transportului miR-142-3p de catre PMVs. Modele experimentale: Soarecii vor fi impartiti in doua grupe: (1) soareci ApoE-/-, hraniti cu o dieta bogata in grasimi si colesterol, grupul HFHC; si (2) soareci C57BL/6J, grupul control (C).

Obiective specifice: (1.) izolarea, caracterizarea si evaluarea functiilor celulelor T; (2.) evaluarea efectului direct al plachetelor asupra functiilor celulelor T; (3) explorarea mecanismelor moleculare implicate in modularea functiilor celulelor T de catre PMVs.

IPOTEZA

Se cunoaste ca, leucocitele și plachetele au un rol deosebit de important în dezvoltarea procesului de ateroscleroza. Posibilul rol al interactiei dintre leucocite si plachete, respectiv dintre leucocite si microveziculele eliberate de plachete, in procesul complex al aterosclerozei a atras atentia in ultimii ani, insa cunostintele cu privire la acest aspect sunt limitate si necesita o cercetare profunda (Figura 1).

Figura 1. Actiunea plachetelor si a leucocitelor in procesul de ateroscleroza (Coenen et al., 2021).

REZULTATE PROIECT

Etapa I - 2020

In cadrul acestei etape a proiectului au fost realizate si caracterizate din punct de vedere al parametrilor plasmatici, modelele animale experimentale: soarecii HFHC (animale cu ateroscleroza accelerata)  si  C (control, animale sanatoase). Astfel, rezultatele au aratat ca dieta hiperlipidemica (îmbogățită cu unt si colesterol) administrata pe parcursul celor 3 luni a avut un impact major atat asupra parametrilor plasmatici (concentrațiile de glucoză, colesterol, LDL-colesterol, HDL-colesterol si de trigliceride), cat si asupra structurii peretelui vascular la nivelul aortei, la soarecii din grupul HFHC.

Etapa a II-a - 2021

Dupa ce in prima etapa a proiectului a fost disponibil modelulexperimental animal de ateroscleroza HFHC (soarecele ApoE-/-cu dieta hiperlipidemică - dieta standard suplimentată cu 1% colesterol și 15% unt), in cea de-a doua etapa cele doua obiective propuse, obiectivul 1 - Izolarea, caracterizarea si evaluarea funcțiilor celulelor imune T, si obiectivul 2 - Evaluarea efectului direct al plachetelor asupra functiilor celulelor imune T, au avut ca finalitate urmatoarele rezultate:

- au fost izolate si caracterizate celulele imune T (CD4+/CD8+) de la modelul animal experimental de ateroscleroza (grupul HFHC)

- au fost evaluate activarea plachetara si a celulelor imune T (CD4+/CD8+) asociate bolii aterosclerotice

- au fost cuantificate nivelurile de microparticule plachetare (PMVs) in modelul animal de ateroscleroza

- au fost urmarite efectele plachetelor de origine sanatoasa asupra activarii si functiilor celulelor T aterogene

Etapa a III-a - 2022

Cel de-al treilea obiectiv propus, Obiectiv 3-Explorarea mecanismelor moleculare implicate in modularea funcţiilor celulelor imune T de catre PMVs (sau PMPs= microveziculele plachetare), a avut ca finalitate urmatoarele rezultate:

- prezenta PMVs in mediul de cultura al celulelor T provenite de la soarecii din grupul HFHC (soareci ApoE cu hrana “high fat-high cholesterol”=animale cu ateroscleroza accelerata) a redus expresia markerilor de activare CD25 si CD154 la nivelul acestor celule, expresia markerului CD69 fiind nemodificata

- prezenta PMVs nu modifica proliferarea, apoptoza si necroza celulelor T de la grupul de animale HFHC

- s-a arătat că după 3 zile de incubare, PMVs de origine sanatoasa au fost preluate de celulele T aterogene intr-un procent foarte mare

- citokinele IL-6, IL-12, IL-17, CD40L si factorii de crestere TGF-β, PDGF si VEGF au prezentat nivele semnificativ crescute in mediul de cultura provenit de la celulele T obtinute din splina soarecilor HFHC comparativ cu mediul provenit de la celulele T obtinute din splina soarecilor control. Incubarea cu PMVs a celulelor T provenite de la soarecii HFHC timp de 3 zile a redus semnificativ nivelele acestor citokine si factori de crestere.

- in urma incubarii cu plachete sau PMVs nivelele de miR-142-3p au fost semnificativ crescute in celulele T, cresteri mai mari inregistrandu-se in prezenta PMVs. Rezultatele dovedesc capacitatea PMVs si a plachetelor de origine sanatoasa de a transfera miR-142-3p catre celulele T.

IMPACTUL estimat al rezultatelor obţinute

Studiul prezent ofera noi oportunitati de cercetare pentru avansarea cunostintelor  in domeniul aterosclerozei, subliniind influenta plachetelor si a microveziculelor plachetare (PMVs) asupra homeostazei celulelor T.

Rezultatele obtinute in acest studiu ofera noi informatii asupra mecanismelor moleculare implicate in interactia dintre celulele T si plachete in ateroscleroza si demonstreaza ca PMVs provenite de la plachetele sanatoase pot controla functiile celulelor T prin transportul de miR-142-3p. Ca urmare, miR-142-3p si PMVs ar putea fi noi tinte terapeutice pentru mentinerea homeostazei celulelor T in ateroscleroza.

In plus, rezultatele noastre vor aduce o noua perspectiva asupra reglarii functiilor celulelor T, care ar putea ajuta la manipularea sistemului imunitar adaptativ si la elaborarea de noi abordări pentru tratarea altor boli vasculare si inflamatorii.

INDICATORI DE REZULTAT

Articole ISI

1. Adriana Georgescu, Maya Simionescu. Extracellular Vesicles: Versatile nanomediators, potential biomarkers and therapeutic agents in atherosclerosis and COVID-19-related thrombosis. International Journal of Molecular Sciences 22(11): 5967-5994, 2021. Q1, IF-5.92 

2. Nicoleta Alexandru, Anastasia Procopciuc, Alexandra Vîlcu, Ioana Karla Comariţa, Elisabeta Bӑdilӑ, Adriana Georgescu. Extracellular vesicles—incorporated microRNA signature as biomarker and diagnosis of prediabetes state and its complications. Rev Endocr Metab Disord, 1-24, 2021 Jun 18. doi: 10.1007/s11154-021-09664-y. Q1, IF=6.51

3. Ioana Karla Comarița, Alexandra Vîlcu, Alina Constantin, Anastasia Procopciuc,  Florentina Safciuc, Nicoleta Alexandru,  Emanuel Dragan, Miruna Nemecz, Alexandru Filippi, Leona Chitoiu, Mihaela Gherghiceanu, Adriana Georgescu.Therapeutic potential of stem cell-derived extracellular vesicles on atherosclerosis-induced vascular dysfunction and its key molecular players. Frontiers in Cell and Developmental Biology, vol. 10 Article 817180:1-30, 2022. Q1, IF=6.684

4. Alina Constantin, Nicoleta Alexandru, Ioana Karla Comarița, Alexandru Filippi, Florina Bojin, Mihaela Gherghiceanu, Alexandra Vîlcu, Miruna Nemecz, Loredan Niculescu, Virgil Paunescu, Adriana Georgescu. Stem cell - derived extracellular vesicles reduce the expression of molecules involved in cardiac hypertrophy - in a model of human-induced pluripotent stem cell-derived cardiomyocytes. Frontiers in Pharmacology, 2022.Q1, IF-5.988 articol acceptat spre publicare in septembrie 2022.

Conferinte Internationale

1. Alina Constantin, Nicoleta Alexandru, Miruna Nemecz, Alexandra Vîlcu, Anastasia Procopciuc, Ioana Karla Comarița, Maya Simionescu, Adriana Georgescu. Stem cell - derived extracellular vesicles reduce the expression of molecules involved in cardiac hypertrophy-in a model of human-induced pluripotent stem cell-derived cardiomyocytes. The 42nd Anniversary Symposium Of The Institute Of Cellular Biology And Pathology “Nicolae Simionescu” Held Jointly With 38th Annual Scientific Session Of The Romanian Society For Cell Biology, November 4-6, 2021, Bucharest, Romania.

2.Miruna Nemecz, Diana Simona Stefan, Alina Constantin, Anastasia Procopciuc, Ioana Karla Comarita, Gabriela Tanko, Maya Simionescu, Adriana Georgescu. The profiles of microRNAs detected in the plasma and circulating microvesicles are possible biomarkers for diagnosis of diabetic dyslipidemia. The 42nd Anniversary Symposium Of The Institute Of Cellular Biology And Pathology “Nicolae Simionescu” Held Jointly With 38th Annual Scientific Session Of The Romanian Society For Cell Biology, November 4-6, 2021, Bucharest, Romania.

3. Ioana Karla Comarița, Alina Constantin, Alexandra Vîlcu, Anastasia Procopciuc, Florentina Safciuc, Nicoleta Alexandru, Emanuel Dragan, Miruna Nemecz, Alexandru Filippi, Adriana Georgescu. Inflammation-induced arterial dysfunction in atherosclerosis; the modulating action of mesenchymal stem cell-derived extracellular vesicles. ‘Frontiers in CardioVascular Biomedicine,’ 29 April - 1 May 2022, Budapest – Hungary. Abstract No. 70108 in Cardiovascular Research 118 (Supplement_1), cvac066. 187, 2022. Q1, IF=10,787

4. Miruna Nemecz, Diana Simona Stefan, Alina Constantin, Anastasia Procopciuc, Ioana Karla Comarita, Gabriela Tanko, Adriana Georgescu. MicroRNAs in circulating microvesicles and plasma as biomarkers that complement the clinical diagnosis of diabetic dyslipidemia and its complications. ‘Frontiers in CardioVascular Biomedicine’, 29 April - 1 May 2022, Budapest – Hungary. Abstract No. 70246 in Cardiovascular Research 118 (Supplement_1), cvac066. 224, 2022, Q1, IF=10,787

5. Ioana Karla Comarița, Alina Constantin, Alexandra Vîlcu, Anastasia Procopciuc, Florentina Safciuc, Nicoleta Alexandru, Emanuel Dragan, Miruna Nemecz, Alexandru Filippi, Adriana Georgescu. Vascular wall damage in atherosclerotic cardiovascular disease; positive effect of extracellular vesicle-based nanotherapeutics on endothelial dysfunction and its key molecular players. ‘90th European Atherosclerosis Society Congress (90th EAS Congress)’, 22-25 May, 2022, Milan, Italy. Abstract in Atherosclerosis, Volume 355:29, DOI: 10.1016/j.atherosclerosis.2022.06.315.  Q1, IF=6,847

6. Alina Constantin, Nicoleta Alexandru, Miruna Nemecz, Alexandra Vîlcu, Anastasia Procopciuc, Ioana Karla Comarița, Adriana Georgescu. Mesenchimal stem cell-derived extracellular vesicles attenuate cardiac hypertrophy in a cellular model of human-induced pluripotent stem cell-derived cardiomyocytes. ‘90th European Atherosclerosis Society Congress (90th EAS Congress)’, 22-25 May, 2022, Milan, Italy. Abstract in Atherosclerosis, Volume 355:233, DOI: 10.1016/j.atherosclerosis.2022.06.902. Q1, IF=6,847

Premii

1. ‘Scientific Achievements – Original Article’ Award offered by Ministry for Education and Research and Uefiscdi, Subprogram 1.1 - Human Resources - Awarding research results - Articles, Competition 2020, Evaluation results List 2 - Award applications submitted for articles published in 2021/18.11.2021 – for the paper ‘Extracellular Vesicles: Versatile nanomediators, potential biomarkers and therapeutic agents in atherosclerosis and COVID-19-related thrombosis. International Journal of Molecular Sciences 22(11): 5967-5994, 2021’. (Adriana Georgescu, Maya Simionescu).

2. ‘Scientific Achievements – Original Article’ Award offered by Ministry for Education and Research and Uefiscdi, Subprogram 1.1 - Human Resources - Awarding research results - Articles, Competition 2020, Evaluation results List 2 - Award applications submitted for articles published in 2021/18.11.2021 – for the paper „Extracellular vesicles—incorporated microRNA signature as biomarker and diagnosis of prediabetes state and its complications. Rev Endocr Metab Disord, 1-24, 2021 Jun 18. doi: 10.1007/s11154-021-09664-y”. (Nicoleta Alexandru, Anastasia Procopciuc, Alexandra Vîlcu, Ioana Karla Comariţa, Elisabeta Bӑdilӑ, Adriana Georgescu).

Grant awarded by the Romanian National Authority for Scientific Research and Innovation, CNCS –UEFISCDI, Program Human Resources/ Research projects to stimulate young independent teams (TE).

Project no. PN-III-P1-1.1-TE-2019-0811/ Grant no. 97 /2020

PROJECT TITLE: Immune modulation of T-cells by platelets and platelet-derived microvesicles in experimental induced atherosclerosis; the role of microRNA-142-3p

Project leader: Ph.D. CSII Nicoleta Alexandru-Moise

Duration: 15.09.2020-14.09.2022

Budget: 431.900 lei

Research Team members: Ph.D. Habil, CSI Adriana Georgescu, Ph.D. CSIII Alina Constantin, Ph.D. CSIII Miruna Nemecz, Ph.D. AC Alexandru Filippi, AC Ioana Karla Comarita, AC Alexandra Vilcu, AC Anastasia Procopciuc.

Abstract

Atherosclerosis is the primary cause for the ischaemic cardiovascular diseases, which are the leading cause of human mortality worldwide. Immune cells, both from innate and adaptive immunity, and likewise, platelets, are present throughout all stages of atherosclerotic lesion development. Although there are evidences suggesting that platelets assist and regulate several functions of immune cells, supplementary studies are necessary to better understand how platelets modulate the immune response. Aims: The purposes of this project are to explore the interaction between T-cells (CD4⁺ and CD8⁺) and platelets, in an experimental induced atherosclerosis animal model, to investigate the in vitro effects of healthy platelets and healthy platelet-derived microvesicles (PMVs) on T-cell functions, and to disclose the involved molecular mechanisms, with special attention on the miR-142-3p delivery via PMVs. Experimental models: Mice will be divided in two groups: (1) ApoE-/- mice feed with a high-fat, high-cholesterol diet, HFHC group; and (2) C57BL/6J mice, as control (C) group.

The specific objectives: (1.) the isolation, characterization and function evaluation of the immune T-cells; (2.) the evaluation of the direct effect of platelets on immune T-cell functions; (3.) the exploration of the molecular mechanisms involved in modulation of immune T-cell functions by PMVs.