A Novel Role of Histone Methyltransferase SMYD2 in Atherosclerosis and Plaque Calcification

Introduction: Clinical manifestation of atherosclerosis is marked by plaque buildup in the brachiocephalic artery (BCA) and vasculature, accompanied by upregulation of mineralization regulating genes (Sox9 and Runx2) by vascular smooth muscle cells (VSMCs) and macrophages (MΦ). Pathogenic remodeling of VSMCs and MΦ into osteochondrogenic phenotypes is accompanied by inflammation, proliferation, migration, and secretion of extracellular matrix proteins. This study aimed to determine the role of SMYD2 (VSMC and MΦ-specific) in atherosclerotic progression and plaque calcification. Methods: SMC- and MΦ- specific SMYD2 knockout was generated in ApoE-/- mice (SMYD2ΔSMCApoE-/- andSMYD2ΔMΦApoE-/-). 10-26 weeks Western diet (WD) fed mice aortic tissue were examined for atherosclerotic plaque distribution using En-face Oil-Red-O staining (ORO). Plaque burden was determined by Hematoxylin & Eosin (H&E) staining, followed by staining for collagen, and calcification of plaque using Masson\u27s trichrome, and von Kossa (VKs)/Alizarin Red S (ARs) respectively. Calcification-regulatory gene Sox9 and Runx2 in aortas were determined by RT-qPCR. The molecular mechanism of calcification was elucidated in 10T1/2, primary VSMCs, and RAW 264.7 cells. Results: SMYD2 expression and function differ profoundly among cell/tissue types (VSMC & MΦ) involved in atherosclerotic plaque progression and calcification. SMC-specific depletion of SMYD2 protected mice from atherosclerotic plaque burden and calcification. En-face ORO and H&E analysis of BCA demonstrated significant decrease in ORO+ve area, total plaque size, and necrotic core area in SMYD2ΔSMCApoE-/- mice as compared to ApoE-/- mice. VKs staining showed microcalcification depots at medial lining of the vasculature in ApoE-/- mice fed with 18-week WD, while a smaller macrocalcification depots was observed in SMYD2ΔSMCApoE-/- mice compared with ApoE-/- mice when fed with 26-week WD. mRNA expression analysis of key calcification transcription factors showed significant suppression of Sox9 gene in aortas of SMYD2ΔSMCApoE-/- mice compared with ApoE-/- mice. Contrary to SMCs, MΦ-specific SMYD2 deletion promotes vascular pathogenesis including plaque burden and plaque /vascular calcification, confirmed En-face ORO+ve, H&E, ARS and VKs stanning’s, and RT-qPCR analysis. Conclusion: Our study unveiled a novel role of SMYD2 in Vascular homeostasis, with VSMC-SMYD2, not MΦ-SMYD2, driving atherosclerosis and vascular calcification. Therefore, assessing the pleiotropic effects of SMYD2 may improve therapeutic strategies for atherosclerotic complications and CVD