Author ORCID Identifier

https://orcid.org/0009-0008-6758-5089

Date of Award

5-1-2024

Degree Type

Thesis

Degree Name

Master of Interdisciplinary Studies (MIS)

Department

Biomedical Sciences

First Advisor

Dr. Chunying Li

Second Advisor

Dr. Ping Song

Abstract

Introduction: Oxidative stress induced vascular remodeling promotes development and progression of atherosclerosis. By scavenging excessive Lipid ROS, GPX4 reduces atherosclerotic plaque severity and VSMC sensitivity, whereas BRD4 promotes plaque progression by facilitating lipid uptake associated with atherosclerosis. This study aimed to explore smooth muscle cell (SMC)-specific role of SMYD2 in modulating oxidative stress in vitro through regulation of GPX4/BRD4

Methods: To evaluate the effect of ferroptosis on SMYD2 and BRD4, C3H/10T1/2 (10T1/2 cells) mouse embryonic fibroblasts were treated with Ferrostatin-1 (Potent and selective inhibitor of ferroptosis), and 1S,3R-RSL3 (Ferroptosis inducer), PAz-PC (oxLDL), in dose and time dependent manner. Next, to evaluate if SMYD2’s mediated ferroptosis is dependent on BRD4, control and Smyd2-depelted 10T1/2 cells were treated with Ferrostatin-1/1S,3R-RSL3 and (+)-JQ1. Lipid hydroperoxides, and Glutathione assay along with Immunofluorescence C11-BODIPY581/591 staining was used to measure oxidative stress in the cellular in cultured cells. Primary VSMCs isolated from control and SMYD2 depleted mice and cultured C3H/10T1/2 cells were used for in vitro studies.

Results: Smyd2 depletion in C3H/10T1/2 and primary cells protected against oxidative stress by regulating GPX4 and BRD4 axis. Pharmacological inhibition (Ferrostatin-1, 2µM, 24hr) or activation (3R-RSL3, 0.8nM, 8hr) of GPX4 activity either by resulted in a significant upregulation and suppression of SMYD2 respectively. OxLDL (PAz-PC, 15ug, 48r) treatment resulted in elevated GPX4 expression in SMYD2-depleted cells compared to control, suggesting that SMYD2 deficiency mitigating ferroptosis by upregulating GPX4. Moreover, inhibiting BRD4 with (+)-JQ1 treatment in C3H/10T1/2 cells, regardless of SMYD2 presence, led to an increase in ferroptosis. Immunofluorescence and C11-BODIPY581/591 staining of these cells revealed higher GPX4 expression and lower BRD4 expression in SMYD2-depleted cells compared to controls. MDA assay results from both cells and culture media corroborated these findings. qPCR analysis and the re-examination of our RNA-seq data demonstrated that either depletion of SMYD2, or (+)-JQ1 treatment significantly downregulated the expression of ferroptosis-associated genes, such as Hmox1, Acsl3, Nox2, GPX4, and Slc40a1. This suggests that SMYD2 may govern lipid peroxidation by modulating the expression of ferroptosis-associated genes regulated by BRD4.

Conclusion: SMYD2 depletion protects against the severity of oxidative stress by regulating the GPX4/BRD4 axis, suggesting its therapeutic potential against lipid peroxidation and cardiometabolic disease.

DOI

https://doi.org/10.57709/36899966

Download

Available for download on Friday, April 11, 2025

Share

COinS