The oxidized phospholipid PGPC impairs endothelial function by promoting endothelial cell ferroptosis via FABP3

Abstract

Ferroptosis is a novel cell death mechanism that is mediated by iron-dependent lipid peroxidation. It may be involved in atherosclerosis development. Products of phospholipid oxidation play a key role in atherosclerosis. 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) is a phospholipid oxidation product present in atherosclerotic lesions. It remains unclear whether PGPC causes atherosclerosis by inducing endothelial cell ferroptosis. In this study, human umbilical vein endothelial cells (HUVECs) were treated with PGPC. Intracellular levels of ferrous iron, lipid peroxidation, superoxide anions (O₂^•-), and glutathione were detected, and expression of fatty acid binding protein-3 (FABP3), glutathione peroxidase 4 (GPX4), and CD36 were measured. Additionally, the mitochondrial membrane potential (MMP) was determined. Aortas from C57BL6 mice were isolated for vasodilation testing. Results showed that PGPC increased ferrous iron levels, the production of lipid peroxidation and O₂^•-, and FABP3 expression. However, PGPC inhibited the expression of GPX4 and glutathione production and destroyed normal MMP. These effects were also blocked by ferrostatin-1, an inhibitor of ferroptosis. FABP3 silencing significantly reversed the effect of PGPC. Furthermore, PGPC stimulated CD36 expression. Conversely, CD36 silencing reversed the effects of PGPC, including PGPC-induced FABP3 expression. Importantly, E06, a direct inhibitor of the oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine IgM natural antibody, inhibited the effects of PGPC. Finally, PGPC impaired endothelium-dependent vasodilation, ferrostatin-1 or FABP3 inhibitors inhibited this impairment. Our data demonstrate that PGPC impairs endothelial function by inducing endothelial cell ferroptosis through the CD36 receptor to increase FABP3 expression. Our findings provide new insights into the mechanisms of atherosclerosis and a therapeutic target for atherosclerosis.

Keywords: Atherosclerosis; CD36; Endothelial function; Fatty acid binding protein-3; Oxidized lipids; PGPC.

Fig. 1

PGPC induced ferroptosis in human umbilical vein endothelial cells (HUVECs). A: CCK8 analysis of HUVECs treated with 12.5, 25, 50 μM PGPC for 24 h. (∗P < 0.05, ∗∗∗P < 0.001, n = 6). B: CCK8 analysis of HUVECs treated with 25 μM PGPC for 12, 24, 48 h. (∗∗∗P < 0.001, n = 7). C, D: Probe FerroOrange staining fluorescence (red) and bar chart showing the intracellular levels of ferrous iron (Fe²⁺) in cultured HUVECs after pretreatment with PGPC with or without Ferrostatin-1 (Fer-1) or erastin for 24 h. Erastin was used as a positive control. The nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. (∗∗∗P < 0.001, n = 8). E, F: Dihydrothidium staining fluorescence (red) and a bar chart showing the intracellular levels of superoxide anion (O₂^•−) in cultured HUVECs after pretreatment with or without N-acetylcysteine (NAC), which were then exposed to tumor necrosis factor alpha (TNF-α), erastin, and PGPC. TNF-α was used as a positive control. The scale bar represents 100 μm. (∗∗∗P < 0.001, n = 9). G: Representative transmission electron microscopy (TEM) images of mitochondria in HUVECs after erastin and PGPC treatment for 24 h. Scale bar represents 2 μm. H: Representative fluorescence-activated cell sorting (FACS) data for C11-BODIPY labeling of HUVECs following PGPC treatment with or without Fer-1 or erastin for 24 h. Unstained C11-BODIPY was not added. HUVEC count indicates the number of HUVECs. I: Statistical analysis of mean fluorescence intensity (MFI) of C11-BODIPY. (∗∗∗P < 0.001, n = 7). J: Relative glutathione (GSH) levels in HUVECs after PGPC treatment for 24 h. (∗∗P < 0.01, n = 8). K, L: Western blots and bar chart showing the expression levels of glutathione peroxidase 4 (GPX4) in HUVECs treated with PGPC with or without Fer-1 or erastin for 24 h (∗P < 0.05, ∗∗P < 0.01, n = 6).

Fig. 2

PGPC and ferroptosis promote fatty acid binding protein-3 (FABP3) expression in endothelial cells. A, B: Human umbilical vein endothelial cells (HUVECs) were pretreated with PGPC (25 μM) media for 24 h followed by mass spectrometry and enriched Kyoto Encyclopedia of Genes and Genomes analysis for regulated proteins. C: qRT-PCR showing the intracellular mRNA levels of ACSL3, ACSL4, FABP3, FABP4, and FABP5 in HUVECs after pretreatment with PGPC for 24 h (∗∗∗P < 0.001, n = 6). D, E: Western blots and bar charts showing the protein levels of ACSL4, FABP3, FABP4, and FABP5 in HUVECs after pretreatment with PGPC for 24 h (∗∗P < 0.01, ∗∗∗P < 0.001, n = 6). F: Immunofluorescence microscopy showing an increase in fluorescence intensity of FABP3 (green) after treatment of cultured HUVECs with PGPC for 24 h. F-actin was stained with phalloidin (red). The nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. G, H: Western blots and bar chart showing FABP3 expression levels in HUVECs treated with PGPC or erastin for 24 h (∗∗∗P < 0.001, n = 6). I, J: Western blots and bar chart showing FABP3 expression levels in HUVECs treated with PGPC with or without Fer-1 for 24 h (∗∗∗P < 0.001, n = 7).

Fig. 3

PGPC induces ferroptosis via upregulating fatty acid binding protein-3 (FABP3) in endothelial cells. A, B: Probe FerroOrange staining fluorescence (red) and bar chart showing the intracellular levels of ferrous iron (Fe²⁺) in cultured human umbilical vein endothelial cells (HUVECs) after knockdown of FABP3 followed with PGPC treatment with or without erastin for 24 h. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. (∗∗∗P < 0.001, n = 7). C, D: Dihydrothidium staining fluorescence (red) and bar chart showing the intracellular levels of superoxide anions (O₂^•−) in cultured HUVECs after pretreatment with or without N-acetylcysteine (NAC), which were then exposed to PGPC after knockdown of FABP3. Scale bar represents 100 μm. (∗∗∗P < 0.001, n = 6). E: The levels of C11-BODIPY in negative control and FABP3-knockdown HUVECs following PGPC treatment for 24 h were determined using fluorescence-activated cell sorting (FACS). HUVEC count indicates the number of HUVECs. F: Mean fluorescence intensity (MFI) values of C11-BODIPY in each group. (∗∗P < 0.01, ∗∗∗P < 0.001, n = 8). G: Relative glutathione (GSH) levels in negative control knockdown and FABP3-knockdown HUVECs following PGPC treatment for 24 h were determined. (∗∗P < 0.01, n = 6). H, I: Western blots and bar charts showing the levels of glutathione peroxidase 4 (GPX4) and FABP3 expression in negative control knockdown and FABP3-knockdown HUVECs after PGPC treatment for 24 h (∗∗P < 0.01, ∗∗∗P < 0.001, n = 6). si-FABP3, specific FABP3 siRNA; si-NC, negative control siRNA.

Fig. 4

Mitochondrial reactive oxygen species (MtROS) were involved in ferroptosis and mitochondrial dysfunction induced by PGPC via fatty acid binding protein-3 (FABP3) in endothelial cells. A: Representative transmission electron microscopy (TEM) images of mitochondria in negative control knockdown, FABP3-knockdown human umbilical vein endothelial cells (HUVECs) following PGPC treatment for 24 h. Scale bar represents 2 μm. B, C: MitoSOX Red staining fluorescence (red) and bar chart showing the intracellular levels of mitochondrial ROS in negative control knockdown, FABP3-knockdown following PGPC treatment for 24 h in cultured HUVECs. The nuclei were stained with Hoechst 33342 (blue). The scale bar represents 40 μm. (∗∗∗P < 0.001, n = 8). D, E: JC-1 staining fluorescence (red and green) and bar chart showing mitochondrial membrane potential (MMP) in negative control knockdown, FABP3-knockdown HUVECs following PGPC treatment for 24 h after pretreatment of cultured HUVECs with PGPC. Red represents aggregated JC-1 in the mitochondrial matrix, green represents JC-1 monomers in the cytoplasm of mitochondria. The ratio of red fluorescence to green fluorescence of the control was defined as 1. The nuclei were stained with Hoechst 33342 (blue). Scale bar represents 20 μm. (∗P < 0.05, n = 7). si-FABP3, specific FABP3 siRNA; si-NC, negative control siRNA.

Fig. 5

CD36 participates in PGPC-induced ferroptosis through its interaction with PGPC in endothelial cells. A, B: Probe FerroOrange staining fluorescence (red) and bar chart showing the levels of ferrous iron (Fe²⁺) in negative control knockdown, CD36-knockdown following PGPC treatment for 24 h in cultured human umbilical vein endothelial cells (HUVECs). Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. (∗∗∗P < 0.001, n = 7). C, D: Dihydroethidium staining fluorescence (red) and bar chart showing the intracellular levels of superoxide anion (O₂^•−) in negative control and CD36-knockdown HUVECs after pretreatment of cultured endothelial cells with PGPC and N-acetylcysteine (NAC). The scale bar represents 100 μm. (∗∗∗P < 0.001, n = 6). E, F: C11 BODIPY staining using followed fluorescence-activated cell sorting (FACS) analysis and bar chart showing the lipid peroxidation in negative control knockdown, CD36-knockdown HUVECs following PGPC treatment for 24 h. HUVEC count indicates the number of HUVECs. (∗P < 0.05, ∗∗∗P < 0.001, n = 8). G: Relative glutathione (GSH) levels in negative control knockdown, CD36-knockdown HUVECs following PGPC treatment for 24 h. (∗∗P < 0.01, n = 6). H, I: Western blots and bar charts showing the levels of CD36, FABP3, FABP4, FABP5, and glutathione peroxidase 4 (GPX4) expression in negative control and CD36-knockdown HUVECs following PGPC treatment for 24 h. (∗∗P < 0.01, ∗∗∗P < 0.001, n = 7). J: qRT-PCR showing the intracellular mRNA levels of FABP3, FABP4, FABP5 in HUVECs after in negative control and CD36-knockdown HUVECs following PGPC treatment for 24 h. (∗∗P < 0.01, ∗∗∗P < 0.001, n = 6). K, L: JC-1 staining fluorescence (red and green) and bar chart showing mitochondrial membrane potential (MMP) of PGPC-treated HUVECs for 24 h after negative control knockdown and CD36-knockdown in HUVECs. Red represents aggregated JC-1 in the mitochondrial matrix, green represents JC-1 monomers in the cytoplasm of mitochondria. The ratio of red fluorescence to green fluorescence of the control was defined as 1. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. (∗P < 0.05, n = 6). si-CD36, specific CD36 siRNA; si-NC, negative control siRNA.

Fig. 6

E06 rescues ferroptosis induced by PGPC in endothelial cells. A, B: Probe FerroOrange staining fluorescence (red) and bar chart showing the intracellular levels of ferrous iron (Fe²⁺) after pretreatment of cultured human umbilical vein endothelial cells (HUVECs) with erastin and PGPC with or without E06 (10 μg/ml) for 24 h. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. (∗∗∗P < 0.001, n = 6). C, D: C11 BODIPY staining using fluorescence-activated cell sorting (FACS) analysis and bar chart showing lipid peroxidation in HUVECs following erastin and PGPC with or without E06 treatment for 24 h. HUVEC count indicates the number of HUVECs. (∗∗∗P < 0.001, n = 7). E: Relative glutathione (GSH) levels in HUVECs after PGPC treatment with or without E06 for 24 h were determined. (∗P < 0.05, ∗∗P < 0.01, n = 6). F, G: Western blots and bar charts showing the protein levels of fatty acid binding protein-3 (FABP3) and glutathione peroxidase 4 (GPX4) after pretreatment of cultured HUVECs with PGPC with or without E06 treatment for 24 h. (∗∗P < 0.01, ∗∗∗P < 0.001, n = 4).

Fig. 7

PGPC impaired endothelium-dependent vasodilation by fatty acid binding protein-3 (FABP3)-mediated ferroptosis. A, B: Line chart showing the endothelium-dependent vasodilation of aortic rings ex vivo. Aortic rings isolated from C57BL6 were pretreated with PGPC or without Ferrostatin-1 (Fer-1), erastin, HTS01037 for 30 min. Subsequently, aortic rings were pre-constricted with 5-hydroxy tryptamine (5-HT). Endothelium-dependent vasodilation was detected using N-acetylcholine (Ach). (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, n = 6). C: Line chart showing the endothelium-dependent vasodilation of aortic rings ex vivo. Aortic rings isolated from high fed diet Apolipoprotein E^−/− mice intraperitoneally injected with or without 1 mg/kg of Fer-1 every day for 4 weeks. Subsequently, aortic rings were pre-constricted with 5-hydroxy tryptamine (5-HT). Endothelium-dependent vasodilation was detected using acetylcholine (Ach). (∗P < 0.05, n = 7).

Fig. 8

Ferroptosis occurs in endothelial cells of atherosclerotic vessels. A: Immunohistochemistry microscopy shows a decrease in the fluorescent intensity of CD31 (green) and in fluorescent intensity of GPX4 (red) in endothelial cells of atherosclerotic vessels compared with healthy individuals. Nuclei were stained with Hoechst 33342 (blue). Scale bar represents 50 μm. AS: atherosclerotic vessels tissues of patients. B: The ferrous iron levels in human aorta sections were measured by Ferrous Iron Colorimetric Assay Kit. (∗∗P < 0.01, n = 8). AS: atherosclerotic lesions tissues of patients. C: Representative images of mouse aorta sections stained with Perls Prussian blue, CD31 immunohistochemistry. Nuclei were stained with eosin (red) in Perls Prussian blue staining and hematoxylin (blue) in CD31 immunohistochemistry. Blue arrows indicate iron. Scale bar represents 40 μm. AS, atherosclerotic lesions tissues of the mouse.

Fig. 9

Schematic of the PGPC regulatory mechanism for ferroptosis in HUVECs.