ALDH4A1 knockdown inhibits in vitro atherosclerosis model by modulating Trim28-mediated P53 ubiquitination to suppress ferroptosis of vascular endothelial cells

Abstract

Atherosclerosis (AS) is a primary contributor to cardiovascular disease (CVD), resulting in high mortality. Ferroptosis, triggered by lipid peroxidation, contribute to AS development. This study aimed to explore the regulatory relationships of Trim28, ALDH4A1, P53, and ferroptosis in the pathogenesis of AS. The AS cell model was constructed by treating HUVECs with oxidized low-density lipoprotein (ox-LDL). The roles of Trim28 overexpression in regulating AS development, P53 ubiquitination, and ferroptosis of vascular endothelial cells were investigated. Moreover, the interaction between Trim28 and ALDH4A1 was explored, followed by analyzing the effect of ALDH4A1 knockdown on P53 ubiquitination. Additionally, the impact of ALDH4A1 knockdown and P53 overexpression on AS development and ferroptosis of vascular endothelial cells was explored. Reduced Trim28 expression and increased ALDH4A1 and P53 expression were observed in HUVECs after treatment with ox-LDL. Overexpression of Trim28 mitigated AS development, promoted P53 ubiquitination, and suppressed ferroptosis of vascular endothelial cells. Additionally, ALDH4A1 could interact with Trim28, and ALDH4A1 knockdown enhanced P53 ubiquitination. Moreover, P53 overexpression reversed the inhibitory effects of ALDH4A1 knockdown on AS development and ferroptosis of vascular endothelial cells. Our findings indicate that Trim28, ALDH4A1, and P53 may be key regulators in AS development. Silencing of ALDH4A1 may alleviate AS development through regulating Trim28-mediated P53 ubiquitination to inhibit ferroptosis of vascular endothelial cells. These molecules may by promising therapeutic targets for AS and related CVD.

Keywords: ALDH4A1; Atherosclerosis; Ferroptosis; P53; Trim28; Ubiquitination.

Figure 1.

Verification of ALDH4A1, Trim28, and P53 expression in vascular endothelial cells treated with ox-LDL. (A): The viability of HUVECs after ox-LDL treatment. (B): Oil red O staining observed intracellular lipid accumulation after ox-LDL treatment. Scale bar: 50 µm. (C): TG and TC concentrations after ox-LDL treatment. (D): The ALDH4A1, Trim28, and P53 expression after ox-LDL treatment. Data are the means ± SD (n = 3). Student’s t-test was used. ** P < 0.01 compared to control group. HUVECs: human umbilical vein endothelial cells; ox-LDL: oxidized low-density lipoprotein; TC: total cholesterol; TG: total triglyceride.

Figure 2.

Trim28 overexpression alleviated atherosclerosis by mediating the ubiquitination of P53 to inhibit ferroptosis of vascular endothelial cells. (A): The protein expression of P53 and Trim28 after treatments. (B): The viability of HUVECs after treatments. (C): Oil red O staining observed intracellular lipid accumulation after treatments. Scale bar: 50 µm. (D): TG and TC concentrations after treatments. (E): Ubiquitination assays showed the ubiquitinated P53 levels after treatments. (F): The Fe²⁺ levels after treatments. (G): The protein expression of SLC7A11 and GPX4 after treatments. Data are the means ± SD (n = 3). One-way analysis of variance assay was used. * P < 0.05 and ** P < 0.01 compared to control group; # P < 0.05 and ## P < 0.01 compared to ox-LDL + oe-NC group. HUVECs: human umbilical vein endothelial cells; ox-LDL: oxidized low-density lipoprotein; TC: total cholesterol; TG: total triglyceride.

Figure 3.

Molecular docking results of Trim28 and ALDH4A1 (input the numbered amino acids).

Figure 4.

ALDH4A1 knockdown promoted Trim28-mediated P53 ubiquitination. (A): The co-IP assays revealed the interaction of Trim28 and ALDH4A1 in HUVECs. (B): Ubiquitination assay showed the ubiquitinated P53 protein level after treatments. (C): The protein expression of ALDH4A1, Trim28, and P53 expression after treatments. Data are the means ± SD (n = 3). One-way analysis of variance assay was used. * P < 0.05 and ** P < 0.01 compared to control group; # P < 0.05 and ## P < 0.01 compared to ox-LDL + sh-NC group. HUVECs: human umbilical vein endothelial cells; ox-LDL: oxidized low-density lipoprotein; co-IP: co-immunoprecipitation; TC: total cholesterol; TG: total triglyceride.

Figure 5.

Overexpression of P53 attenuated the inhibitory effect of ALDH4A1 knockdown on AS by promoting ferroptosis of vascular endothelial cells. (A): The protein expression of Trim28, ALDH4A1 and P53 expression after treatments. (B): The viability of HUVECs after treatments. (C): Oil red O staining observed intracellular lipid accumulation after treatments. Scale bar: 50 µm. (D): TG and TC concentrations after treatments. (E): The Fe²⁺ levels after treatments. (F): The protein expression of SLC7A11 and GPX4 after treatments. Data are the means ± SD (n = 3). One-way analysis of variance assay was used. * P < 0.05 and ** P < 0.01 compared to ox-LDL group; # P < 0.05 and ## P < 0.01 compared to ox-LDL + sh-ALDH4A1 + oe-NC group. HUVECs: human umbilical vein endothelial cells; ox-LDL: oxidized low-density lipoprotein; TC: total cholesterol; TG: total triglyceride.

Figure 6.

Mechanism diagram.