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Titlul proiectului: Strategie terapeutică în nefropatia diabetică bazată pe țintirea unor mecanisme epigenetice implicate în insuficiența renală și memoria metabolică

Project title: Advanced therapeutic strategy in diabetic nephropathy based on the targeting of epigenetic mechanisms underlying renal failure and metabolic memory

Acronimul proiectului/Acronym: NEPHRODIAB

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: 12/05/2022 - 11/04/2024

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

Rezumatul proiectului:
Boala renală diabetică (DKD) este o tulburare degenerativă care afectează structura și funcția glomerulilor, determinând în cele din urmă insuficiență renală. Strategiile terapeutice actuale pot întârzia progresia DKD. Hemodializa intensivă și transplantul renal reprezintă opțiunile terapeutice pentru pacienții în stadiile finale ale DKD. Astfel, există o nevoie stringentă pentru dezvoltarea unor strategii terapeutice avansate pentru a reduce eficient impactul DKD. Date recente, inclusiv ale noastre, demonstrează că modificările histonelor promovează instabilitatea epigenetică și, în consecință, alterări transcriptomice care inițiază și amplifică inflamația și stresul oxidativ în bolile cardiovasculare. Metilarea histonelor ar putea sta la baza mecanismului imprimării unei memorii epigenetice „toxice” în diabet. Ipoteza noastră este că dereglarea indusă de diabet a unor enzime implicate in metilarea histonelor (KMT, KDM) și modificările epigenomice asociate sunt implicate în mod coordonat în debutul și progresia DKD. Scopul acestui proiect este de a descoperi și caracteriza funcțional izoformele KMT/KDM care stau la baza proceselor patologice relevante pentru DKD. Strategia propusă include metode și instrumente de ultimă generație și modele experimentale in vitro și in vivo de diabet. Rezultatul așteptat: Strategie pentru reversarea/ atenuarea DKD prin intervenții orientate către KMT/KDM prin reducerea proceselor pro-oxidante, pro-inflamatorii și pro-fibrotice.

Project summary:
Diabetic kidney disease (DKD) is a degenerative disorder affecting the structure and function of the glomeruli, ultimately leading to kidney failure. Current therapeutic strategies can only delay the progression of DKD. Hemodialysis and eventually kidney transplantation represent the major therapeutic options in end-stage DKD patients. Thus, there is a stringent need for the development of advanced therapeutic strategies to efficiently reduce the burden of DKD. Cutting-edge evidence, including ours, emphasizes that specific histone-based epigenetic mechanisms promote epigenetic instability and consequently, transcriptomic changes that initiates and amplifies the inflammation and oxidative stress in cardiovascular diseases. Histone methylation could provide the underlying mechanistic support for an imprinted “toxic” epigenetic memory in diabetes. We hypothesize that the diabetes-induced dysregulation of selective histone methylation enzymes (KMT, KDM) and the ensuing epigenomic alterations are coordinately implicated in the onset and progression of DKD. The aim of this project is to uncover and functionally characterize KMT/KDM isoforms underlying DKD-relevant pathological processes. The proposed strategy entails state-of-the-art methods and tools, and in vitro and in vivo experimental models of diabetes. Expected outcome: Strategy to reverse/attenuate DKD by KMT/KDM-oriented interventions through reduction of pro-oxidant, pro-inflammatory, and pro-fibrotic processes.

Rezultate semnificative:
• În condiţii de diabet experimental se manifestă la nivel renal/vascular modificări transcriptomice asociate unor enzime epigenetice reprezentative implicate în metilarea (histon-metiltransferaze)/demetilarea (histon-demetilaze) histonelor nucleozomale.
• Rezultatele experimentale sugerează existenţa unor mecanisme de auto-reglare/compensare la nivelul sistemului enzimatic implicat în modularea statusului de metilare al histonelor nucleozomale.
• În condiţii diabetice, histon-demetilaza KDM1A/LSD1 şi histon-metiltransferaza SET7 mediază la nivel renal activarea unor mecanisme moleculare cu rol important în instalarea stresului oxidativ (activarea/supraexprimarea complexului NADPH oxidaza), inflamaţiei (molecule de adeziune, citokine, chemokine) şi fibrozei (colagen IV, fibronectina, laminină), procese caracteristice bolii renale diabetice.

Significant results:
• Transcriptomic changes and alterations in gene and protein expression levels of selected epigenetic enzymes involved in the methylation of nucleosomal histones (histone-methyltransferases)/demethylation (histone-demethylases) have been identified at both the renal and vascular (aorta) level in diabetic mice.
• The experimental evidence suggests the existence of negative feed-back mechanisms at the level of the enzyme system involved in modulating the methylation status of nucleosomal histones.
• In diabetic conditions, histone-demethylase KDM1A/LSD1 and histone-methyltransferase SET7 mediate at renal level the activation of molecular mechanisms with an important role in the installation of oxidative stress (activation/overexpression of the NADPH oxidase complex), inflammation (adhesion molecules, cytokines, chemokines) and fibrosis (collagen IV, fibronectin, laminin), processes characteristic of diabetic kidney disease.

Publicaţii/Publications

Peer-review ISI articles:

1. 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).
2. Lazar AG, Vlad ML, Manea A, Manea SA. Epigenetic regulation of NADPH oxidase expression, inflammation and fibrosis in diabetic kidney by KDM1A/LSD1-related signaling pathways (Manuscript in preparation).
3. Vlad ML, Lazar AG, Manea A, Manea SA. The SET7 lysine methyltransferase regulates the reactive oxygen species overproduction, inflammation, and fibrosis in the kidney of diabetic mice (Manuscript in preparation).
4. 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).

Comunicări orale (Oral communications):

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. 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.
9. 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.
10. 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.