Abstract
Purpose: To study the effect of lysine demethylase 1A (LSD1) on inflammatory responses of vascular smooth muscle cells (VSMCs), and investigate the mechanism.
 Methods: VSMCs were treated with lipopolysaccharide (LPS). Overexpression and knockdown of LSD1 in VSMCs were performed by transfecting with LSD1 overexpression plasmid and small interfering RNAs (siRNAs), respectively. Western blot and quantitative real-time polymerase chain reaction (qRTPCR) were used to measure protein and mRNA levels. Enzyme-linked immunosorbent (ELISA) assay was used to determine the levels of inflammatory cytokines.
 Results: Phosphorylation of LSD1 (p-LSD1) was significantly increased in LPS-induced VSMCs. Monocyte chemoattractant protein-1 and IL-6 levels were also increased by LPS, but attenuated by LSD1 knockdown in VSMCs. Activation of NF-κB was increased by LPS, but was also decreased by LSD1 knockdown. Level of methylated p65 (p65-me) in VSMCs was increased by treatment with SET7/9 (p65 methyltransferase), but this effect was attenuated by overexpression of LSD1. Besides, the increased levels of MCP-1 and IL-6 induced by overexpression of LSD1 were reversed by NF-κB signaling inhibitor, PDTC.
 Conclusion: LSD1 exacerbates LPS-induced inflammation of VSMCs through NF-κB activation via p65 demethylation, which indicates that LSD1 might be a potential target for the treatment of cardiovascular diseases.
 Keywords: Vascular smooth muscle cells, Lysine demethylase 1A, Phosphorylation, NF-κB, p65, Demethylation
Highlights
Vascular smooth muscle cells (VSMCs) are main components of the vascular wall, contributing primarily to maintain the stability of blood flow and pressure [1,2]
To investigate the role of lysine demethylase 1A (LSD1) in the LPSinduced inflammatory response of VSMCs, the phosphorylation level of LSD1 (p- LSD1) in VSMCs that treated with LPS was determined
The protein level ofpLSD1 was significantly higher in VSMCs treated with 100 or 1000 ng/mL LPS, while Phosphorylation of LSD1 (p-LSD1) protein expression was not affected by 10 ng/mL LPS as compared to that without LPS treatment
Summary
Vascular smooth muscle cells (VSMCs) are main components of the vascular wall, contributing primarily to maintain the stability of blood flow and pressure [1,2]. Protein kinase Cα (PKCα)LSD1-nuclear factor-κB (NF-κB) signaling was a critical for LPS-induced inflammatory response in mice and bone marrow-derived macrophages (Raw264.7 cells) [9]. LSD1 might be a novel target for regulating the inflammatory response by NF-κB signaling. Small interfering RNA (siRNA) (siNC), siRNA for LSD1 (siLSD1), and LSD1 overexpression plasmid were synthesized at Shanghai GenePharma Co., Ltd. For cell transfection, 4 × 105 VSMCs were seeded into each well of a 6well plate and cultured in DMEM for 24 h. The VSMCs were transfected with siNC, siLSD1, or LSD1 overexpression plasmid using Lipofectamine 3000 reagent (ThermoFisher Scientific, Waltham, MA). VSMCs were collected after 48 h of transfection These results indicated that high concentrations of LPS (100 and 1000 ng/mL) and long treatment time significantly increased p-LSD1 protein expression.
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