PEST domain
DMPE- DMPE-TMR
E- LDL and Ox-LDL loading
2.9. Analysis of proteins involved in cholesterol metabolism
2.9.1. Gene expression analysis
The analysis of genes involved in cholesterol metabolism by Microarray analysis during E-LDL and Ox-E-LDL loading and also during HDL3 deloading of macrophages revealed regulatory principles of the cholesterol metabolism during foam cell formation with the two different lipid loading agents E-LDL and Ox-LDL and cholesterol efflux with HDL3 (table 10).
lubrol-raft non-raft
MCSF
E-LDL
E-LDL/HDL3
Ox-LDL
Ox-LDL/HDL3 lubrol-raft non-raft
MCSF
E-LDL
E-LDL/HDL3
Ox-LDL
Ox-LDL/HDL3
Figure 35: Raft association of FcgRIIA/CD32
The LDL-receptor (LDLR), responsible for the binding and subsequent cellular uptake of apoB/E-containing lipoproteins plays a crucial role in cellular cholesterol homeostasis. The LDL-receptor is internalized via “clathrin coated pits” and following internalization of the LDL particles takes place. Cholesterol which is released into the cytoplasm suppresses cell surface expression of LDL receptors. This regulation corresponds with our chip data, which showed a -8.6 fold downregulation of the LDL receptor during E-LDL loading and a -4 fold downrgulation during Ox-LDL loading. During E-LDL deloading an upregulation of 3.2 fold and during Ox-LDL deloading an upregulation of 1.4 fold could be observed. The ABCA1 transporter responsible for cholesterol efflux was 2-fold upregulated during lipid loading indicating an enhanced amount of ABCA1 resulting in more effective cholesterol efflux due to an increase of cellular cholesterol.
2.9.2. Analysis of ABCA1
2.9.2.1. mRNA expression of ABCA1
The mRNA expression of ABCA1 determined with Taqman RT-PCR showed only a slight increase during E-LDL loading while upon Ox-LDL loading the mRNA expression was significantly induced. With HDL3 deloading a reduction of ABCA1 mRNA expression took place (Fig.36). The gene array data reflect these findings partly with a 2 fold increase of ABCA1 gene expression during E-LDL and Ox-LDL loading and a decrease (-1.4 and -1.2 fold) during HDL3 deloading (table 2).
2.9.2.2. Protein expression of ABCA1
Protein expression analysis of ABCA1 by Western Blot was performed to verify the Affymetrix gene array data with an additional method. Macrophages loaded with E-LDL and Ox-LDL showed in accordance with the gene array data an induction of ABCA1 total protein Figure 36: mRNA expression of ABCA1
0 5 10 15 20 25 30 35 40 45
d4 lipid loading lipid deloading
relative mRNA expression
Ox-LDL E-LDL
0 5 10 15 20 25 30 35 40 45
d4 lipid loading lipid deloading
relative mRNA expression
Ox-LDL E-LDL Ox-LDL E-LDL
expression compared to MCSF differentiated cells while the increase during Ox-LDL loading was stronger than the increase during E-LDL loading (Fig.37). This is in accordance to the mRNA expression of ABCA1 (Fig.36). The induction of ABCA1 protein expression was partially reversed during E-LDL deloading with HDL3 and there was a stronger reduction of ABCA1 protein expression during Ox-LDL deloading with HDL3 (Fig.37).
Figure 37: Protein expression of ABCA1
2.9.2.3. Analysis of membrane raft association of ABCA1
In a next step it should be analyzed whether the different lipid loading agents E-LDL and Ox-LDL or HDL3 deloading have influences on the raft association of ABCA1. Therefore raft fractions respectively non raft fractions were isolated from membrane preparations of cells from the different incubation states and the protein expressions of ABCA1 in these fractions were analyzed.
During MCSF differentiation no raft association of ABCA1 could be detected while during LDL and Ox-LDL loading a slight raft association could be observed (Fig.38). Also during E-LDL and Ox-E-LDL deloading with HDL3 a slight raft association was detectable. However the strongest raft association could be observed during Ox-LDL deloading (Fig.38).
MCSF Ox-LDL
E-LDL/
HDL3 E-LDL
Ox-LDL/
HDL3
250 kDA
MCSF Ox-LDL
E-LDL/
HDL3 E-LDL
Ox-LDL/
HDL3
250 kDA
MCSF
E-LDL
E-LDL/HDL3
Ox-LDL
Ox-LDL/HDL3 lubrol-raft non-raft
MCSF
E-LDL
E-LDL/HDL3
Ox-LDL
Ox-LDL/HDL3 lubrol-raft non-raft
Figure 38: Raft association of ABCA1
2.9.3. Analysis of apoE
2.9.3.1. Protein expression of apoE
Total protein expression of apoE was analyzed with western-blot. Protein expression of apoE was only slightly induced during E-LDL and Ox-LDL loading compared to MCSF differentiated macrophages (Fig.39). Deloading with HDL3 after E-LDL loading led to a stronger upregulation of apoE protein expression than after Ox-LDL loading compared to lipid loading (Fig.39).
Figure 39: Protein expression of apoE
2.9.3.2. Extracellular (surface) and intracellular expression of apoE
Extracellular (surface) and intracellular expression of apoE was measured by flow cytometry of MCSF differentiated, E-LDL and Ox-LDL loaded and HDL3 deloaded macrophages. In the differentiation phase with MCSF from day 1 to day 4 apoE surface expression was increased. From day 4 to day 6 no change and from day 6 to day 7 a further increase of apoE surface expression could be detected (Fig.40A). During lipid loading apoE surface expression increased with a stronger induction during E-LDL than during Ox-LDL loading (Fig.40A). This is in accordance with the protein expression of apoE (Fig.40). During HDL3 deloading apoE surface expression was decreased compared to lipid loading (Fig.40A).
Intracellular expression of apoE was increased during differentiation with MCSF from day 1 to day 6 and was decreased from day 6 to day 7. During E-LDL loading a slight increase and during Ox-LDL loading a decrease of apoE intracellular expression could be observed compared to MCSF differentiation (Fig.40B). HDL3 deloading decreased intracellular apoE expression compared to lipid loading (Fig.40B).
32 kDA
Figure 40A: ApoE surface expression
analyzed by flow cytometry Figure 40B: ApoE intracellular expression analyzed by flow cytometry
2.9.4. Analysis of apoC-I
2.9.4.1. Protein expression of apoC-I
Protein expression of apoC-I was analyzed with western-blot. Protein expression of apoC-I was slightly reduced during E-LDL while it was induced during Ox-LDL loading compared to MCSF differentiated macrophages (Fig.41). E-LDL and especially Ox-LDL deloading with HDL3 led to a stronger upregulation of apoC-I protein expression compared to lipid loading which is probably due to apoC-I as a constituent of HDL3 (Fig.41).
2.9.4.2. Extracellular (surface) and intracellular expression of apoC-I
Extracellular (surface) and intracellular expression of apoC-I was measured by flow cytometry of MCSF differentiated, E-LDL and Ox-LDL loaded and HDL3 deloaded macrophages. ApoC-I surface and intracellular expression was increased during differentiation with MCSF from day 1 to day 4, was slightly decreased from day 4 to day 6 and was increased from day 6 to day 7 (Fig.42A). ApoC-I intracellular expression was much higher compared to extracellular expression, which is obvious from the higher mean fluorescence intensity. During E-LDL loading apoC-I surface expression was only slightly increased while during Ox-LDL loading it was strongly increased compared to MCSF (Fig.42A). E-LDL deloading with HDL3 increased and Ox-LDL deloading slightly decreased apoC-I surface expression compared to lipid loading (Fig.42A).
Intracellular expression of apoC-I increased during lipid loading to a higher extent with Ox-LDL than with E-Ox-LDL loading compared to MCSF differentiated macrophages (Fig.42B).
During Ox-LDL deloading with HDL3 a slight decrease of apoC-I intracellular expression was observed while there was no change during E-LDL deloading compared to lipid loaded cells (Fig.42B).
Figure 42B: ApoC-I intracellular expression analyzed by flow cytometry
A. ApoCI extracellular B. ApoCI intracellular
0
A. ApoCI extracellular B. ApoCI intracellular
0
A. ApoCI extracellular B. ApoCI intracellular
0
Figure 42A: ApoC-I surface expression analyzed by flow cytometry
Figure 41: Protein expression of apoC-I
MCSF E-LDL/ Ox-LDL
2.9.5. Analysis of CETP
2.9.5.1. Extracellular (surface) and intracellular expression of cholesteryl ester transfer protein (CETP)
Extracellular (surface) and intracellular expression of CETP was measured by flow cytometry of MCSF differentiated, E-LDL and Ox-LDL loaded and HDL3 deloaded macrophages. During differentiation with MCSF from day 1 to day 7 CETP surface expression was increased (Fig.43A). During lipid loading CETP surface expression was increased with E-LDL loading while it was decreased with Ox-LDL loading compared to MCSF (Fig.43A). During E-LDL deloading with HDL3 CETP surface expression was slightly increased while during Ox-LDL deloading it was slightly decreased compared to lipid loading (Fig.43A).
Intracellular expression of CETP increased during differentiation with MCSF from day 1 to day 6 while no further change was observed from day 6 to day 7 (Fig.43B). During lipid loading intracellular expression of CETP was decreased compared to MCSF with a stronger decrease during Ox-LDL than during E-LDL loading (Fig.43B). HDL3 deloading of E-LDL loaded cells increased while deloading of Ox-LDL loaded cells slightly decreased intracellular CETP expression compared to lipid loading (Fig.43B).
2.9.6. Analysis of PLTP
2.9.6.1. Protein expression of PLTP
Total protein expression of PLTP was analyzed with western-blot. Protein expression of PLTP was increased during E-LDL and Ox-LDL loading compared to MCSF differentiated macrophages (Fig.44). E-LDL deloading with HDL3 led to an increase of PLTP protein expression while Ox-LDL deloading led to a decrease of PLTP protein expression compared Figure 43A: CETP surface expression
analyzed by flow cytometry
Figure 43B: CETP surface expression analyzed by flow cytometry
to lipid loading (Fig.44). Western Blot shows the two forms of PLTP with the molecular masses of 67kDa and 77kDa as described by Huuskonen et al. (388).
Figure 44: Protein expression of PLTP
2.9.7. Analysis of ATP-synthase (b-chain)
2.9.7.1. Protein expression of ATP-synthase (b-chain)
Total protein expression of ATP-synthase (b-chain) was analyzed with western-blot. Lipid loading with E-LDL and Ox-LDL revealed no change of ATP-synthase protein expression compared to MCSF (Fig.45). HDL3 deloading after E-LDL loading led to a slight increase of ATP-synthase protein expression compared to lipid loading (Fig.45).
Figure 45: Protein expression of ATP-synthase (b-chain)
MCSF E-LDL/ Ox-LDL
HDL3
E-LDL Ox-LDL/
HDL3
MCSF E-LDL/ Ox-LDL
HDL3
E-LDL Ox-LDL/
HDL3
MCSF E-LDL/ Ox-LDL
HDL3
E-LDL Ox-LDL/
HDL3 67 kDa
77 kDa
MCSF E-LDL/ Ox-LDL
HDL3
E-LDL Ox-LDL/
HDL3 67 kDa
77 kDa
MCSF E-LDL/ Ox-LDL
HDL3
E-LDL Ox-LDL/
HDL3
MCSF E-LDL/ Ox-LDL
HDL3
E-LDL Ox-LDL/
HDL3
3. Incubation of ApoE3/3 macrophages with LDL, E-LDL and Ox-LDL alone and as