Georgia State University, Atlanta, GA
A NOVEL ROLE OF METHYLTRANSFERASE SMYD2 IN ATHEROSCLEROTIC PLAQUE CALCIFICATION nBackground: Atherosclerotic plaque calcification involves complex signaling pathways, including the differentiation of vascular smooth muscle cells (VSMCs) into osteo/chondrogenic-like cells, associated with an increased risk of cardiovascular mortality. Expression of Runx2, RANKL, and SOX9 by osteoblast-like VSMCs stimulates plaque mineralization and remodeling. The study aims to explore the role of methyltransferase SMYD2 in atherosclerotic plaque calcification. Methods: VSMC-specific SMYD2 knockout (ApoE-/-SMYD2?SMC) were generated in ApoE-/- background mice. Age and body weight-matched mice were put on Western diet (TD88137) for 26 weeks. Aortas collected from mice were analyzed by Oil-red-O Staining (ORO) for plaque formation, von Kossa for calcification, Masson's trichrome for collagen distribution, and mRNA analysis for calcification associated genes (MGP1, ACC, SPARC, OPG, ALP, RANKL, SOST, TNF? and transcription factors SOX9, MSX2, BMP2, and RUNX2). The effect of SMYD2 NOTCH signaling pathway (NOTCH1-4, Hey, Hes1, PSEN2) was also examined. Results: VSMC specific loss of SMYD2 protected mice from atherosclerotic plaque calcification. SMYD2 increased total plaque area and promoted formation of necrotic core while decreasing total fibrous cap, lipid core area and total ORO positive areas, without affecting the distribution of collagen fibers. von Kossa staining showed that micro- and macro-calcification depots were significantly higher in ApoE-/- group than ApoE-/-SMYD2?SMC mice, and subsequent mRNA analysis indicated significant upregulation of SOX9, RANKL, TNF? with decreasing trend of MGP1 and AGG. Analysis of mRNA expression of molecules in NOTCH signaling showed that downregulation of VSMC specific SMYD2 significantly enhanced PSEN2, an endoprotease which catalyzes intramembranous cleavage of NOTCH, suggesting that NOTCH signaling may also be involved. Further, analysis of NOTCH genes (NOTCH1-4) and their downstream molecules (Heyl and Hes1) revealed that increased PSEN2 led to an increase in NOTCH4 expression, though NOTCH 1-3, Hey, Hes1 mRNA remained unchanged or even decreased. Conclusion: SMYD2 promotes plaque calcification in atherosclerosis through upregulation of SOX9, RANKL, TNF? and inhibition of MGP1 and AGG. The endoprotease PSEN2 mediates S3 cleavage of NOTCH to release NICD, and nuclear translocation of NICD induces expression of Hey and Hes1, suggesting that SMYD2 is essential for NICD functional activity and could serve as a novel therapeutic target in atherosclerotic plaque calcification.