Changes in cellular lipid content determined by mass spectrometry

ESI-tandem mass spectrometry was used to measure cellular content of total, esterified and unesterified cholesterol, sphingomyelin, ceramide and sphingosine in four independent experiments and the results are shown in Fig.28. Total, esterified and unesterified cholesterol did not change during MCSF-induced differentiation (Fig.28A-C). However, monocyte derived macrophages loaded with E-LDL (40mg/ml) showed a 4-fold higher content of total cholesterol and a 99-fold increase of esterified cholesterol while Ox-LDL loaded cells (80mg/ml) showed only a 2.3-fold increase of total cholesterol and an 18-fold enhancement of esterified cholesterol (Fig.28A,B). Unesterified cholesterol increased with E-LDL and Ox-LDL loading to the same extent (Fig.28C). Cellular sphingomyelin content increased during monocyte to macrophage differentiation up to 1.7-fold and no further change could be observed during E-LDL and Ox-LDL loading (Fig.28D). The ceramide content during MCSF-induced differentiation showed no significant change (Fig.28E). Ox-LDL incubation however, led to a significant 3.6-fold increase in cellular ceramide content while E-LDL showed only a 1.4-fold increase (Fig.28E). As shown before, this effect was not due to higher ceramide content in Ox-LDL itself (Fig.25C). These data allow the conclusion that E-LDL loading predominantly increases cellular cholesterol content while Ox-LDL loading preferentially increases cellular ceramide content. Sphingosine content of the cells revealed a 1.6-fold upregulation during MCSF-dependent differentiation, an additional 1.2-/1.3-fold increase occurred with E-LDL and Ox-LDL respectively (Fig.28F). HDL₃-dependent deloading reversed the increase of cholesterol mass of both lipid loading agents (Fig.28A-C). Deloading of E-LDL loaded cells with HDL3 diminished total cholesterol content up to 40% (Fig.28A), mostly (up to 140%) at the expense of esterified cholesterol (Fig.28B) while unesterified cholesterol decreased only by 2% (Fig.28C). HDL3 deloading of Ox-LDL loaded cells led to a 20% decrease of total cholesterol (Fig.28A), a 10% decrease of esterified (Fig.28B) and a neglegible decrease of unesterifed cholesterol (Fig.28C). Sphingomyelin content however increased during HDL₃ deloading for both LDL-modifications up to 24% (Fig.28D) and cellular ceramide increased slightly during E-LDL deloading up to 13% (Fig.28E) while it decreased during Ox-LDL deloading up to 50%. Along with the decrease in ceramide, sphingosine increased with HDL₃ deloading of Ox-LDL loaded cells up to 130%. In E-LDL loaded cells however, HDL3 did not affect sphingosine levels (Fig.28F).

Figure 28: Cellular content of total, esterified and unesterified cholesterol, sphingomyelin, ceramide and sphingosine

Mass spectrometry was used to determine the cellular lipid content of human monocyte derived macrophages at the status of differentiation with MCSF (day 0 and day 4), during E-LDL and Ox-LDL loading after 48 hours and during HDL3 deloading after 24 hours. The intracellular content of total cholesterol (A), esterified cholesterol (B), unesterified cholesterol (C), sphingomyelin (D), ceramide (E) and sphingosine (F) is shown.

2.2. RT-PCR analysis of enzymes involved in ceramide generation and degradation To elucidate the reason for the Cer elevation during Ox-LDL loading, TaqMan realtime RT-PCR was performed for the enzymes involved in Cer generation and degradation of MCSF differentiated, E-LDL and Ox-LDL loaded and HDL₃ deloaded human monocyte-derived macrophages. During MCSF-dependent monocyte/macrophage differentiation, mRNA expressions of serine palmitoyltransferases 1+2 (SPTLC1+2), which are involved in Cer synthesis as well as the transcriptional levels of sphingomyelin synthases, 1 and 2 which convert Cer into sphingomyelin were downregulated (Fig.29A,B,C,D). Analysis of the sphingomyelinases, which convert sphingomyelin to Cer, revealed an upregulation of mRNA expression of acid sphingomyelinase (SMPD1) (2.8fold) and acid sphingomyelinase like phosphodiesterase (ASML3B) (3.5fold) (Fig.29E,F) whereas the mRNA expression of neutral sphingomyelinase (SMPD2) and neutral sphingomyelinase activation associated factor (NSMAF) was downregulated -1,5 fold during differentiation (Fig.29G,H). mRNA expression of glucosidase, beta; acid (GBA) which includes glucosylceramidase involved in the

0

degradation of glucosylceramide into Cer showed no change during differentiation (Fig.29I).

UDP-glucose ceramide glucosyltransferase (UGCG) the enzyme which converts Cer to glucosylceramide and N-acylsphingosine amidohydrolase 1 (ASAH1) (acid ceramidase) degrading Cer to sphingosine showed an upregulation of mRNA expression (2 and 3.9 fold) during differentiation (Fig.29J,K).

During E-LDL loading SPTLC1 and SPTLC2 were 1.7 and 1.5 fold and during Ox-LDL loading 2.1 and 1.3 fold upregulated (Fig.29A,B). Sphingomyelin synthase 1 was 1.1 fold and sphingomyelin synthase 2 was 1.6 fold upregulated during both lipid loading agents (Fig.29C,D). SMPD1 and ASML3B mRNA expression was -1.7 and -1.8 fold downregulated during E-LDL loading. Ox-LDL loading however revealed a 3.1 fold upregulation of SMPD1 and a -2.3 fold downregulation of ASML3B compared to MCSF. mRNA expression of SMPD2 and NSMAF showed a stronger upregulation during Ox-LDL loading (5.2 and 2.2 fold) than during E-LDL loading (2.7 and 1.3 fold) (Fig.29G,H). The stronger upregulation of the sphingomyelinases SMPD1, SMPD2 and the activation factor NSMAF during Ox-LDL loading compared with E-LDL loading can be considered as causal for the Ox-LDL induced Cer elevation.

mRNA expression of GBA, was stronger upregulated during Ox-LDL than during E-LDL loading (3.7 vs 1.8 fold) (Fig.29I) while UGCG mRNA expression showed a higher upregulation during E-LDL loading (3.2 vs 2.2 fold) (Fig.29J). ASAH1 mRNA expression was -1.5 fold downregulated with E-LDL and Ox-LDL loading (Fig.29K).

E-LDL deloading with HDL₃ revealed almost no change of the mRNA expression of SPTLC1 and 2 (Fig.29A,B). With Ox-LDL deloading SPTLC1 decreased up to 1 fold (Fig.29A) while SPTLC2 levels remained unchanged (Fig.29B). Both sphingomyelinases, 1 and 2 were upregulated with HDL₃ after E-LDL deloading up to 2 fold (Fig.29C,D) With Ox-LDL deloading sphingomyelin synthase 1 increased 1.7 fold while sphingomyelin synthase 2 decreased 1.2 fold (Fig.29C,D). SMPD1, ASML3B as well as SMPD2 and NSMAF expression decreased with HDL₃ deloading compared to lipid loading, however from a higher level in Ox-LDL loaded cells (Fig.29E,F,G,H). HDL₃ deloading led also to a downregulation of GBA (Fig.29I), UGCG (Fig.29J) and ASHA1 (Fig.29K). These results show that all tested sphingomyelinases were strongly downregulated with HDL₃ deloading which reflects an inversion of the lipid loading effects of E- and Ox-LDL and a reduction of the enzymatic functions in converting sphingomyelin to ceramide to provide sphingomyelin for cholesterol efflux.

Figure 29. Taqman analysis of enzymes involved in ceramide synthesis and degradation Using real time Taqman RT-PCR standardized to 18s rRNA as a reference, gene expression was monitored of 4 day differentiated macrophages, 48 hours E- and Ox-LDL loaded macrophages (day 4 to day 6) and 24 hours HDL3 (day 6 to day 7) deloaded macrophages. Serine palmitoyltransferase 1 (SPTLC1) (A), serine palmitoyltransferase 2 (SPTLC2) (B), sphingomyelin synthase 1 (MOB) (C), sphingomyelin synthase 2 (D), acid sphingomyelinase (SMPD1) (E), acid sphingomyelinase like phosphodiesterase (ASML3B) (F), neutral sphingomyelinase (SMPD2) (G), neutral sphingomyelinase (N-SMase) activation associated factor (NSMAF) (H), glucosylceramidase (glucosidase beta acid) (GBA) (I), UDP-glucose ceramide glucosyltransferase (UGCG) (J), acid ceramidase (ASAH1)

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A. serine palmitoyltransferase 1 (SPTLC1) B. serine palmitoyltransferase 2 (SPTLC2)

D. sphingomyelin synthase 2

2,5 C. sphingomyelin synthase 1 (MOB) E-LDL

A. serine palmitoyltransferase 1 (SPTLC1) B. serine palmitoyltransferase 2 (SPTLC2)

D. sphingomyelin synthase 2

2,5 C. sphingomyelin synthase 1 (MOB) E-LDL

2,5 C. sphingomyelin synthase 1 (MOB) E-LDL

relative mRNA expression I. glucosylceramidase (GBA)

E-LDL

relative mRNA expression K. acid ceramidase (ASAH1)

E-LDL

relative mRNA expression J. UDP-glucose ceramide glucosyltransferase

relative mRNA expression I. glucosylceramidase (GBA)

E-LDL

relative mRNA expression K. acid ceramidase (ASAH1)

E-LDL

relative mRNA expression J. UDP-glucose ceramide glucosyltransferase