Untargeted Lipidomic Profiling Reveals Lysophosphatidylcholine and Ceramide as Atherosclerotic Risk Factors in apolipoprotein E Knockout Mice

Abstract

Despite the availability and use of numerous cholesterol-lowering drugs, atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of mortality globally. Many researchers have focused their effort on identifying modified lipoproteins. However, lipid moieties such as lysophosphatidylcholine (LPC) and ceramide (CER) contribute to atherogenic events. LPC and CER both cause endothelial mitochondrial dysfunction, leading to fatty acid and triglyceride (TG) accumulation. In addition, they cause immune cells to differentiate into proinflammatory phenotypes. To uncover alternative therapeutic approaches other than cholesterol- and TG-lowering medications, we conducted untargeted lipidomic investigations to assess the alteration of lipid profiles in apolipoprotein E knockout (apoE^-/-) mouse model, with or without feeding a high-fat diet (HFD). Results indicated that, in addition to hypercholesterolemia and hyperlipidemia, LPC levels were two to four times higher in apoE^-/- mice compared to wild-type mice in C57BL/6 background, regardless of whether they were 8 or 16 weeks old. Sphingomyelin (SM) and CER were elevated three- to five-fold in apoE^-/- mice both at the basal level and after 16 weeks when compared to wild-type mice. After HFD treatment, the difference in CER levels elevated more than ten-fold. Considering the atherogenic properties of LPC and CER, they may also contribute to the early onset of atherosclerosis in apoE^-/- mice. In summary, the HFD-fed apoE^-/- mouse shows elevated LPC and CER contents and is a suitable model for developing LPC- and CER-lowering therapies.

Keywords: apolipoprotein E (apoE) knockout; atherosclerosis; atherosclerotic cardiovascular disease (ASCVD); ceramide (CER); cholesteryl ester (CE); lipidomic; lysophosphatidylcholine (LPC); mass spectrometry; triglyceride (TG).

Figure A1

ApoE^−/− mice had altered plasma lipid patterns at 8 weeks of age, with higher levels of lysophosphatidylcholine (LPC), sphingomyelin (SM), and ceramide (CER). Data was presented in (A) a heatmap and (B) bar plots with dots. Similar to Figure 2, but the bar plots here combined data from the two apoE^−/− groups and was in signal intensity. The LPC(16:0) and LPC(18:0) were the most abundant LPC species, whereas the SM(d18:1/22:0) was the most dominant sphingolipid species in the 8-week-old mice. The data are presented in signal intensity. **** p < 0.0001. Abbreviations: apoE^−/−: apolipoprotein E knockout; LPC: lysophosphatidylcholine; SM: sphingomyelin; CER: ceramide.

Figure A2

High-fat diet-fed apoE^−/− mice show enhanced levels of sphingomyelin and ceramide. Data was presented in (A) a heatmap and (B) bar plots with dots in signal intensity. Similar to Figure 3, the 16-week-old mice showed that LPC(16:0), LPC(18:0) and SM(d18:1/22:0) remain the most dominant LPC and sphingolipid species in the plasma, respectively. *** p < 0.001, ** p < 0.01, * p < 0.05, ns: not significant between groups. Abbreviations: apoE^−/−: apolipoprotein E knockout; LPC: lysophosphatidylcholine; SM: sphingomyelin; CER: ceramide; HFD: high-fat diet.

Figure A3

ApoE^−/− mice fed with a high-fat diet showed higher SM and CER levels. Data was displayed in heatmaps (A) individually and (B) average of n = 7. We accessed the disease model’s trajectory using all of the lipid profile data from mice plasma. Our findings showed that apoE^−/− mice had increased lysophospholipid levels regardless of whether they were fed NCD or HFD. LPC levels in apoE^−/− mice were two to four times greater than in C57BL/6 mice, whether they were 8 or 16 weeks old. The results demonstrated that apoE^−/− mice exhibited higher lysophospholipid levels regardless of whether they were administered NCD or HFD. LPC levels were two to four times higher in apoE^−/− mice compared to C57BL/6 mice, regardless of whether they were 8 or 16 weeks old. Sphingolipids such as SM and CER were determined to be significantly higher in apoE^−/− mice when administered HFD. At the baseline or 16-week-old, SM and CER were elevated three- to five-fold in apoE^−/− mice that compared to C57BL/6. After HFD treatment, the difference could be more than ten times elevated in comparison to C57BL/6. Abbreviations: apoE^−/−: apolipoprotein E knockout; HFD: high-fat diet; NCD: normal-chow diet; LPC: lysophosphatidylcholine; SM: sphingomyelin; CER: ceramide.

Figure A4

High-fat diet-fed apoE^−/− mice showed decreased cholesteryl ester levels. Data was presented in (A) a heatmap and (B) bar plots with dots in signal intensity. Similar to Figure 4 but presented with signal intensity, the 16-week-old mice showed that CE(18:2) and CE(20:4) were the most dominant CE species in the plasma of apoE^−/− mice. All CE species were down-regulated in NCD- or HFD-fed apoE^−/− mice. *** p < 0.001, ** p < 0.01. Abbreviations: apoE^−/−: apolipoprotein E knockout; HFD: high-fat diet; NCD: normal-chow diet; CE: cholesteryl ester.

Figure 1

Lipidomic profiling of mouse plasma using UPLC/MS^E. (A) Total lipids were extracted from the plasma from the respective groups (n = 7, each). MarkerLynx and Progenesis QI software were used to analyze and quantify the MS data. Heatmaps were used generated to display the results. (B) Using a CSH™ C18 column, total lipids were separated into lysophospholipids, glycerophospholipids, sphingolipids, CEs, and glycerolipids (DG, TG). The results were acquired in the positive mode. (C) The representative lipidomic profiling in plasma from the respective groups are demonstrated based on retention time and signal intensity. (D) Pattern of parent and daughter ion of LPC from the respective groups with their signal intensities. Abbreviations: apoE^−/−: apolipoprotein E knockout; HFD: high-fat diet; UPLC: ultra-performance liquid chromatography; MS^E: data-independent collection mode mass spectrometry; MS: mass spectrometry; CSH: charged surface hybrid; CE: cholesteryl ester; DG: diacylglycerol; TG: triacylglycerol.

Figure 2

Altered lipid profile in the respective animal groups at eight weeks of age. We chose m/z signals with (1) higher abundance, (2) a fold change greater than 2, and (3) p values less than 0.001 to be the selecting markers of untargeted plasma lipidomic profiling of all the three groups of mice at the 8-week-old baseline. (A) A total of 27 markers were chosen to represent various lipid components between groups in a heatmap. By the exact mass and retention time, some of the 27 markers were identified. (B) LPC(16:0), LPC(18:0), LPC(20:0), SM(d18:1/16:0), SM(d18:1/18:0), SM(d18:1/22:0), CER(d18:1/16:0), CER(d18:1/22:0), and CER(d18:1/24:0) were compared among groups. N = 7 in each group. The data are presented in the fold change. *** p < 0.001, ns: not significant between groups. Abbreviations: apoE^−/−: apolipoprotein E knockout; LPC: lysophosphatidylcholine; SM: sphingomyelin; CER: ceramide.

Figure 3

Elevated levels of sphingomyelin and ceramide in high-fat diet-fed apoE^−/− mice. The analysis of untargeted lipidomic data was carried on as described earlier. (A) A total of 30 markers were picked from each category and displayed in a heatmap. Of the 30 markers, 27 were the same as those identified at the baseline. (B) LPC(16:0), LPC(18:0), LPC(20:0), SM(d18:1/16:0), SM(d18:1/18:0), SM(d18:1/22:0), CER(d18:1/16:0), CER(d18:1/22:0), and CER(d18:1/24:0) were the most abundant lipid species in each category. Levels of each lipid species were compared among groups (N = 7 each). The information is provided in the fold change format. *** p < 0.001, ** p < 0.01, * p < 0.05, ns: not significant between groups. Abbreviations: apoE^−/−: apolipoprotein E knockout; HFD: high-fat diet; NCD: normal-chow diet; LPC: lysophosphatidylcholine; SM: sphingomyelin; CER: ceramide.

Figure 4

Analysis of cholesteryl ester levels in high-fat diet-fed apoE^−/− mice. (A) By the exact mass and retention time, CEs were analyzed similarly to other lipid parameters. CE(18:2), CE(20:2), CE(20:4), CE(22:2), CE(22:4), and CE(22:6) were the most abundant CE species in mice. CE levels were compared among three groups. Data were presented in a heatmap. (B) CE(18:2), CE(20:2), CE(20:4), CE(22:2), CE(22:4), and CE(22:6) were all reduced significantly in apoE^−/− mice given NCD or HFD. Notably, the CE levels were lowest in apoE^−/− mice on a high-fat diet. The data are presented in the fold change. *** p < 0.001, ** p < 0.01. Abbreviations: apoE^−/−: apolipoprotein E knockout; HFD: high-fat diet; NCD: normal chow diet; CE: cholesteryl ester.