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Autoritatea finanţatoare: Ministerul Cercetării, Inovării şi Digitalizării, Unitatea Executivă pentru Finanțarea Învățământului Superior, a Cercetării, Dezvoltării și Inovării (UEFISCDI)

Funding authority: Ministry of Research, Innovation and Digitization, Executive Agency for Higher Education, Research, Development and Innovation (UEFISCDI)

Project duration: 2021 - 2023

Director de proiect/Project director: Adrian Manea, Ph.D., Institute of Cellular Biology and Pathology "N. Simionescu", Bucharest, Romania (e-mail: adrian.manea@icbp.ro)

Rezumatul proiectului:
Ateroscleroza, cea mai frecventă boală cardiovasculară (BCV), reprezintă principala cauză de morbiditate și mortalitate la nivel mondial. Evenimentele asociate BCV pot fi prevenite la mai puțin de jumătate dintre pacienți prin implementarea strategiilor terapeutice convenționale. Dezvoltarea unor noi algoritmi terapeutici reprezintă o condiție esențială pentru reducerea impactului BCV. Epigenetica are un rol important in ateroscleroză. Metilarea histonelor reprezintă un marker epigenetic care poate fi transmisă prin mai multe cicluri celulare pentru a induce o memorie epigenetică toxică care ar putea explica eficiența slabă a terapiilor actuale la un număr mare de pacienți. Ipoteza noastră este că reprogramarea epigenetică prin țintirea farmacologică selectivă a enzimelor implicate în metilarea histonelor (KMT/KDM) ar putea reprezenta o strategie terapeutică importantă în ateroscleroză. Implicarea funcțională a KMT/KDM în ateroscleroză rămâne slab înțeleasă. În acest proiect ne propunem să descoperim rolul KMT/KDM și efectorilor lor moleculari cu scopul de a reduce progresia aterosclerozei mai eficient decât cu terapiile convenționale. Strategia integrează experimente pe probe biologice umane, modele in vitro și in vivo de ateroscleroză și tehnici avansate. Rezultate: 1) Panel validat al subtipurilor KMT și KDM ca biomarkeri și potențiale ținte terapeutice în ateroscleroză; 2) Strategie terapeutică validată preclinic bazată pe reprogramare epigenetică în ateroscleroză.

Project summary:
Atherosclerosis-related cardiovascular diseases (CVD) are the leading cause of morbidity and mortality worldwide. The outcomes of CVD can be improved in less than a half of patients under conventional standards of care. The development of novel and comprehensive treatment algorithms is a prerequisite to limit the burden of CVD. Epigenetics has been mechanistically connected to atherosclerosis. Histone methylation marks can be transmitted through multiple cell cycles to induce the imprinting of a toxic epigenetic memory that could explain the poor effectiveness of the standard drugs in a large number of patients. We hypothesize that epigenetic reprogramming via pharmacological targeting of selective histone methylation enzymes (KMTs/KDMs) could represent an important therapeutic strategy in atherosclerosis. The functional implication of KMTs/KDMs in the pathoetiology of atherosclerosis remains elusive. In this project we aim to uncover the role of KMTs/KDMs and their down-stream molecular effectors in order to reduce atherosclerosis more effectively than conventional therapies. The proposed strategy entails experiments on human biological samples, in vitro and in vivo experimental models of atherosclerosis, and state-of-the-art techniques. Expected outcomes: 1) Validated panel of KMT and KDM subtypes as potential biomarkers and therapeutic targets in atherosclerosis; 2) Preclinical validated histone methylation reprogramming-based therapeutic strategy in atherosclerosis.

Rezultate semnificative obtinute in cadrul proiectului: Mecanisme, modulatori şi efectori epigenetici în bolile cardiovasculare: noi oportunităţi pentru diagnostic, prognostic şi tratament
Context: Histon-metiltransferazele şi histon-demetilazele reprezintă sisteme enzimatice epigenetice cu rol important în modularea exprimării genelor, insă rolul acestor enzime în procesul de formare a leziunlor aterosclerotice nu este complet cunoscut.
Obiectivul major al proiectului a constat în dezvoltarea la nivel preclinic a unei strategii terapeutice avansate în ateroscleroză bazată pe reprogramare epigenetică, dependentă de metilarea histonelor. Obiectivele stiintifice propuse în cadrul acestui proiect au fost realizate în totalitate şi au constat în: (1) Identificarea și predicția funcțională a potențialelor căi de semnalizare dependente de histon-metiltransferaze (KMT) și histon-demetilaze (KDM) cu rol important în ateroscleroză - studii in vitro, in vivo și in silico. (2) Modularea farmacologică a funcției unor subtipuri KMT și KDM specifice pentru a reduce progresia aterosclerozei - studii în vitro și in vivo. Activităţile specifice au vizat: (1) Determinarea nivelurilor de exprimare a unor izoforme KMT/KDM și a unor markeri specifici de metilare a histonelor în țesuturile arteriale non-/aterosclerotice. (2) Analiza modificărilor epigenetice dependente de metilarea histonelor specifice tipurilor celulare existente în plăcile aterosclerotice umane. (3) Determinarea nivelelor de exprimare KMT/KDM și a statusului de metilare al histonelor în modele experimentale de ateroscleroză. (4) Evaluarea potențialelor modificări epigenetice dependente de metilarea histonelor în aortele aterosclerotice la șoareci. (5) Determinarea potențialelor ținte terapeutice dependente de KMT și KDM. (6) Validarea preclinică a implicării funcționale a diferitelor subtipuri KMT (SET7, DOT1L) și KDM (LSD1) în inițierea și progresia aterosclerozei la șoarecii ApoE-/-. (7) Evaluarea potențialelor efecte adverse ale intervențiilor farmacologice la șoareci. Livrabile: (1) Panel validat de subtipuri KMT și KDM ca potențiali biomarkeri și ținte terapeutice în ateroscleroză. (2) Efectori moleculari validați preclinic ai unor subtipuri KMT și KDM cu expresie/funcție modificată care pot contribui la formarea și progresia ateromului. (3) Strategie terapeutică în ateroscleroză, validată preclinic, bazată pe reprogramarea metilării histonice. Diseminare: Articole: 2 articole publicate într-o revista ISI cu factor de impact ridicat [Antioxidants (2021, 2022), Q1, Factor de impact: 7.675] şi 2 manuscrise in pregătire. Comunicări orale la manifestări ştiinţifice internaţionale: 8. Postere prezentate la manifestări ştiinţifice naţionale şi internaţionale: 21.

Rezultate semnificative:
• Histon-demetilaza LSD1 şi histon-metiltransferazele SET7 şi DOT1L contribuie la modularea unor procese moleculare cu rol important ȋn creşterea producţiei de specii reactive de oxigen (complexul NADPH oxidază) şi a unor mediatori pro-inflamatori (inflamazomul NLRP3, IL18) ȋn ateroscleroza experimentală (studii in vitro şi in vivo).
• Rezultatele acestor studii sugerează posibilitatea utilizării inhibitorilor farmacologici specifici pentru lizin-demetilaza LSD1 şi respectiv, lizin-metiltransferazelor SET7 sau DOT1L drept suport terapeutic în ateroscleroză.
Rezultatele ştiinţifice obţinute în cadrul acestui proiect au fost publicate (2 articole originale) într-o revistă (Q1) cu factor de impact ridicat şi incluse în două manuscrise în pregatire, prezentate în cadrul unor manifestări ştiinţifice internaţionale ale unor societăţi profesionale de prestigiu sub formă de prezentări orale (Societatea Europeană de Ateroscleroză; Societatea Europeană de Cardiologie, Societatea Europeană de Patologie) sau postere (Congresul Societăţii Europene de Cardiologie, Congresul Societăţii Europene de Ateroscleroză, Congresul Societăţilor Europene de Biochimie; Congresul Anual al Societăţii Române de Biologie Celulară). Prin rezultatele obţinute, proiectul furnizează cunoştinţe avansate pentru transferul la etapele următoare de dezvoltare și validare preclinică, o condiție esențială pentru utilizarea în clinică. Prin aplicarea in practica medicală, această strategie terapeutică ar putea îmbunătăţi semnificativ tratamentul pacienţilor cu ateroscleroză.

Significant results obtained within the project: Epigenetic mechanisms, modulators and effectors in cardiovascular diseases: new opportunities for diagnosis, prognosis and treatment.
Background: Histone-methyltransferases and histone-demethylases represent epigenetic enzyme systems with an important role in the modulation of gene expression, but the role of these enzymes in the formation process of atherosclerotic lesions is not completely elucidated.
The main goal of the project consisted in the preclinical development of an advanced therapeutic strategy in atherosclerosis based on histone methylation-related epigenetic reprogramming of key genes associated with atherogenesis. The scientific objectives proposed within this project were fully achieved and consisted of: (1) Identification and functional prediction of potential signalling pathways dependent on histone-methyltransferases (KMT) and histone-demethylases (KDM) with an important role in atherosclerosis - in vitro, in vivo and in silico studies. (2) Pharmacological modulation of the function of specific KMT and KDM subtypes to reduce the progression of atherosclerosis - in vitro and in vivo studies. The specific activities aimed at: (1) Determination of the expression levels of some KMT/KDM isoforms and of specific histone methylation markers in non-/atherosclerotic arterial tissues. (2) Analysis of histone methylation-dependent epigenetic changes specific to cell types existing in human atherosclerotic plaques. (3) Determination of KMT/KDM expression levels and histone methylation status in experimental atherosclerosis models. (4) Evaluation of potential histone methylation-dependent epigenetic changes in mouse atherosclerotic aortas. (5) Determination of potential KMT- and KDM-dependent therapeutic targets. (6) Preclinical validation of the functional involvement of different KMT (SET7, DOT1L) and KDM (LSD1) subtypes in the initiation and progression of atherosclerosis in ApoE−/− mice. (7) Evaluation of potential adverse effects of pharmacological interventions in mice. Deliverables: (1) Validated panel of KMT and KDM subtypes as potential biomarkers and therapeutic targets in atherosclerosis. (2) Preclinical validated molecular effectors of KMT and KDM subtypes with altered expression/function that may contribute to atheroma formation and progression. (3) Preclinical validated therapeutic strategy in atherosclerosis based on histone methylation reprogramming. Dissemination: Articles: 2 articles published in a high impact factor journal [Antioxidants (2021, 2022), Q1, Impact Factor: 7.675] and 2 manuscripts in preparation. Oral communications at international scientific meetings: 8. Posters presented at national and international scientific at international scientific meetings: 21.
Significant results:
• Histone-demethylase LSD1 and histone-methyltransferases SET7 and DOT1L contribute to the modulation of key molecular processes with an important role in reactive oxygen species overproduction of and oxidative stress (NADPH oxidase complex up-regulation) and important pro-inflammatory mediators (NLRP3 inflammasome, IL18) in experimental atherosclerosis (in vitro and in vivo studies).
• The results of these studies suggest the possibility of using specific pharmacological inhibitors for lysine-demethylase LSD1 and, respectively, lysine-methyltransferases SET7 or DOT1L as therapeutic support in atherosclerosis-related cardiovascular disorders.
The scientific results obtained within this project were published (2 original articles) in a high impact journal (Q1) and included in two manuscripts in preparation, presented at international scientific meetings of prestigious professional societies as oral communications (European Society of Atherosclerosis; European Society of Cardiology, European Society of Pathology) or poster presentations (Congress of the European Society of Cardiology, Congress of the European Atherosclerosis Society, Congress of European Societies of Biochemistry; Annual Congress of the Romanian Society of Cell Biology). Collectively, the project provides advanced knowledge ready to be translated to the next stages of development and preclinical validation, an essential condition for clinical implementation. By applying it in medical practice, this therapeutic strategy could significantly improve the treatment of patients with atherosclerosis.

Publicaţii/Publications

Peer-review ISI articles:

1. Lazar AG, Vlad ML, Manea A, Simionescu M, Manea SA. Activated histone acetyltransferase p300/CBP-related signalling pathways mediate up-regulation of NADPH oxidase, inflammation, and fibrosis in diabetic kidney. Antioxidants, 2021; 10(9):1356. doi: 10.3390/antiox10091356. Impact factor: 7.675 (Q1).
2. Manea SA, Vlad ML, Lazar AG, Muresian H, Simionescu M, Manea A. Pharmacological inhibition of lysine-specific demethylase 1A reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice by a mechanism involving decreased oxidative stress and inflammation; potential implications in human atherosclerosis. Antioxidants, 2022; 11: 2382. https://doi.org/10.3390/antiox11122382. Impact factor: 7.675 (Q1).
3. Manea SA, Vlad ML, Lazar AG, Muresian H, Simionescu M, Manea A. The epigenetic enzyme SET7 lysine methyltransferase mediates the up-regulation of NADPH oxidase expression and oxidative stress in atherosclerotic apolipoprotein E knockout mice (Manuscript in preparation).
4. Vlad ML, Manea SA, Lazar AG, Muresian H, Simionescu M, Manea A. Histone methyltransferase DOT1L mediates NADPH oxidase up-regulation, oxidative stress and NLRP3 inflammasome activation in atherosclerotic apolipoprotein E-deficient mice; potential functional implication in human atherosclerosis (Manuscript in preparation).

Comunicări orale (Oral communications):

1. Manea A. Epigenetic mechanisms in cardiometabolic disorders: potential biomarkers and therapeutic targets. 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. 2021.
2. Manea A, Vlad ML, Lazar AG, Manea SA. Focused ultrasound-induced targeted delivery of miR-155-5p inhibitor employing biomimetic microbubbles reduces NADPH oxidase expression and inflammation in atherosclerotic ApoE-/- mice. Cardiovascular Research, 2022, Vol. 118, Supplement 1, pg. cvac066. 197.
3. Manea A, Vlad ML, Lazar AG, Manea SA. SET7 methyltransferase mediates the up-regulation of NADPH oxidase expression and oxidative stress in the atherosclerotic aorta of apolipoprotein E-deficient mice. Virchows Archiv (2022) 481 (Suppl 1):S157.
4. Manea A. Pathophysiological role and potential therapeutic implications of histone methylation-related epigenetic pathways in atherosclerosis. The 43nd Anniversary Symposium of the Institute of Cellular Biology and Pathology “Nicolae Simionescu”, 2022.
5. Lazar AG, Vlad ML, Manea A, Manea SA, Simionescu M. Ursolic acid reduces atherosclerotic lesion progression in hypercholesterolemic apolipoprotein E-deficient mice by a mechanism involving decreased oxidative stress and inflammation. The 43nd Anniversary Symposium of the Institute of Cellular Biology and Pathology “Nicolae Simionescu”, 2022.
6. Vlad ML, Lazar AG, Manea SA, Manea A. Histone methyltransferase SET7 mediates the inflammatory response in atherosclerotic apolipoprotein E-deficient mice. 91th EAS Congress, 2023, Science at a Glance Section, Atherosclerosis 379, S15, Mannheim, Germany.
7. Manea A, Vlad ML, Lazar AG, Manea SA. Histone methyltransferase DOT1L mediates the up-regulation of NADPH oxidase expression and oxidative stress in atherosclerotic ApoE knockout mice; potential implication in human atherosclerosis. European Journal of Heart Failure (2023) 25 (Suppl. S2) S455. Heart Failure Congress 2023, Prague, Czechia.
8. Manea SA, Vlad ML, Lazar AG, Manea A. Pharmacological inhibition of histone demethylase 1A reduces oxidative stress and inflammation in the aorta of diabetic mice. European Journal of Heart Failure (2023) 25 (Suppl. S2) S455. Heart Failure Congress 2023, Prague, Czechia.

Postere prezentate la manifestări stiinţifice naţionale sau internaţionale (Posters presented at national and international meetings):

1. Manea A, Manea SA, Vlad LM, Lazar AG, Muresian H, Simionescu M. Identification of potential lysine-specific histone demethylase-dependent mechanisms underlying oxidative stress and inflammation in human and experimental atherosclerosis: a transcriptomics approach. The 45th FEBS Congress 2021. FEBS OPEN BIO. 2021, 11, 142.
2. Lazar AG, Vlad LM, Manea A, Manea SA. Activation of histone acetyltransferase p300/CBP -dependent signaling pathways induces oxidative stress, inflammation, and fibrosis in the kidney of diabetic mice. The 38th Annual Scientific Session of the Romanian Society for Cell Biology. 2021.
3. Vlad LM, Lazar AG, Manea SA, Manea A. Histone demethylase KDM5B mediates the up-regulation of pro-oxidant and pro-inflammatory genes in M1-type macrophages. The 38th Annual Scientific Session of the Romanian Society for Cell Biology. 2021.
4. Manea SA, Lazar AG, Vlad ML, Manea A. Ursolic acid reduces inflammation and fibrosis in the kidney of diabetic mice. Cardiovascular Research, 2022, Vol. 118, Supplement 1, pg. cvac066. 225.
5. Lazar AG, Vlad ML, Manea A, Olariu L, Manea SA. Ursolic acid reduces NADPH oxidase expression and ensuing oxidative stress in diabetic kidney. Atherosclerosis, 2022, Vol. 355, pg. 206.
6. Vlad ML, Mares RG, Lazar AG, Manea SA, Preda BM, Simionescu M, Schiopu A, Manea A. Monocyte-derived macrophages mediate S100a8/A9-induced oxidative stress and inflammation in the ischemic myocardium. Atherosclerosis, 2022, Vol 355, pg. 268.
7. Manea SA, Vlad ML, Lazar AG, Manea A. miR-210-3p mediates the up-regulation of NADPH oxidase expression in the atherosclerotic aorta of hypercholesterolemic apolipoprotein E-deficient mice; potential implications for human atherosclerosis. Atherosclerosis, 2022, Vol. 355, pg. 49-50.
8. Manea A, Manea SA, Vlad ML, Lazar AG, Muresian H, Simionescu M. Lysine-specific histone demethylase 1A mediates oxidative stress, inflammation, and atherogenesis in apolipoprotein E-deficient mice; prospective implications for human atherosclerosis. Atherosclerosis, 2022, Vol. 355, pg. 50.
9. Manea A, Vlad ML, Lazar AG, Manea SA. MicroRNA-210-3p contributes to inflammatory response in the atherosclerotic aorta of apolipoprotein E-deficient mice: potential role in human atherosclerosis. FEBS OPEN BIO, 2022, Vol. 12, pg. 152.
10. Manea SA, Lazar AG, Vlad ML, Manea A. Ursolic acid reduces NADPH oxidase expression and oxidative stress in the atherosclerotic aorta of apolipoprotein E-deficient mice by inhibiting NF-kB and STAT1/3 signaling. FEBS OPEN BIO, 2022, 12, pg. 151.
11. Manea SA, Lazar AG, Vlad ML, Manea A. Ursolic acid prevents the dysregulation in the expression of histone methylation-related epigenetic enzymes in diabetic kidney. Virchows Archiv (2022) 481 (Suppl 1): S86-S87.
12. Lazar AG, Vlad ML, Manea A, Manea SA. Ursolic acid reduces inflammation and the development of atherosclerotic lesions in hypercholesterolemic apolipoprotein E-deficient mice. International Conference and XXXIX Scientific Session of the Romanian Society for Cell Biology, 2022.
13. Lazar AG, Vlad ML, Manea A, Olariu L, Manea SA. Ursolic acid reduces inflammation and oxidative stress in the kidney of diabetic mice. The 43nd Anniversary Symposium of the Institute of Cellular Biology and Pathology “Nicolae Simionescu”, 2022.
14. Vlad ML, Mares RG, Lazar AG, Manea SA, Preda BM, Simionescu M, Schiopu A, Manea A. Short-term pharmacological inhibition of alarmin S100A9 reduces oxidative stress and inflammation after myocardial infarction in mice. The 43nd Anniversary Symposium of the Institute of Cellular Biology and Pathology “Nicolae Simionescu”, 2022.
15. Lazar AG, Vlad ML, Manea A, Manea SA. Lysine-specific histone demethylase 1A mediates the up-regulation of NADPH oxidase expression in the kidney of diabetic mice. 91th EAS Congress, 2023, Atherosclerosis 379, S161, Mannheim, Germany.
16. Manea SA, Lazar AG, Vlad ML, Manea A. Pharmacological inhibition of lysine specific demethylase 1A reduces inflammation and fibrosis in the kidney of diabetic mice. The 47th FEBS Congress, 2023, FEBS Open Bio 13 (Suppl. S2) (2023), 105, Tours, France.
17. Manea A, Vlad ML, Lazar AG, Manea SA. Histone methyltransferase DOT1L-dependent signaling pathways mediate the inflammatory response in the atherosclerotic aorta of ApoE knockout mice; potential role in human atherosclerosis. The 47th FEBS Congress, 2023, FEBS Open Bio 13 (Suppl. S2) (2023), 243, Tours, France.
18. Manea SA, Lazar AG, Vlad ML, Manea A. Pharmacological inhibition of histone methyltransferase SET7 reduces inflammation and fibrosis in the kidney of diabetic mice. 35th European Congress of Pathology, 2023, Virchows Archiv (2023) 483 (Suppl 1): S126, Dublin, Ireland.
19. Manea A, Vlad ML, Lazar AG, Manea SA. Histone methyltransferase DOT1L mediates NLRP3 inflammasome priming and activation in atherosclerotic apolipoprotein E knockout mice; potential functional implication in human atherosclerosis. 35th European Congress of Pathology, 2023, Virchows Archiv (2023) 483 (Suppl 1): S126, Dublin, Ireland.
20. Vlad ML, Lazar AG, Manea SA, Manea A. Activation of histone methyltransferase SET7 induces inflammatory response in the atherosclerotic aorta of apolipoprotein E knockout mice. 40th Annual Scientific Session of the Romanian Society for Cell Biology, 2023, Bucharest, Romania.
21. Lazar AG, Vlad ML, Manea A, Manea SA. The epigenetic enzyme LSD1 mediates the up-regulation of NADPH oxidase expression and oxidative stress in diabetic kidney. 40th Annual Scientific Session of the Romanian Society for Cell Biology, 2023, Bucharest, Romania.