The authors have declared that no competing interests exist.
Conceived and designed the experiments: SZB JH PR JDS. Performed the experiments: SZB JH PR. Analyzed the data: SZB JH PR JDS. Contributed reagents/materials/analysis tools: JH JDS. Wrote the paper: SZB JH PR JDS. Conceived of the study, obtained funding, participated in its design and coordination, and helped assemble the final manuscript: JDS. Read and approved the final manuscript: SZB JH PR JDS .
Cholesterol loaded macrophages in the arterial intima are the earliest histological evidence of atherosclerosis. Studies of mouse models of atherosclerosis have shown that the strain background can have a significant effect on lesion development. We have previously shown that DBA/2
Atherosclerosis is a complex and progressive pathology of arteries that can be initiated by the accumulation and entrapment of lipoproteins in the extracellular matrix of the sub-endothelial intima layer
The genetic background strain has been shown to effect atherosclerotic lesion area in hyperlipidemic
Age matched AKR and DBA/2
All experiments were performed in accordance with protocols approved by the Cleveland Clinic Institutional Animal Care and Use Committee.
Bone marrow derived macrophages (BMM) from AKR and DBA/2
Differentiated BMM in p100 dishes from AKR and DBA/2
Total RNA was isolated from cell pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA), following the manufacturer's protocol. On-column digestion with RNase-free DNase (Qiagen, Valencia, CA) was performed to remove genomic DNA. DNase was removed in the subsequent washing steps. RNA integrity was tested by overnight incubation of 200–500 ng of total RNA at 37°C and observation of the 18S and 28S ribosomal RNA bands on a 1.2% agarose/ethidium bromide gel.
An aliquot of total RNA for each sample (∼ 2 µg) was submitted to the Genomic Core at Lerner Research Institute. Total RNA was used as template to synthesize single stranded cDNA following the Illumina protocol. Single stranded cDNA is then converted into double stranded cDNA, purified and
Expression data were quantile normalized and log2 transformed using the R-package
Gene set enrichment analysis was performed for all expressed transcripts to identify possible pathways altered by strain, loading and strain-loading interactions. The
To validate the findings of gene transcript measurements by microarray, we assessed the expression of selected gene transcripts by using qPCR approach. The expression levels of tribbles homolog 3 (
Unloaded and cholesterol loaded BMM from AKR and DBA/2 were lysed with 0.5 mL of M-PER mammalian protein extraction reagent (Pierce Biotechnology). Cell lysates (45 µg protein/lane) were loaded, separated on a 4–15% gradient polyacrylamide gel and transferred to PVDF membranes by semi-dry electroblotting. After blocking with rapid blocking buffer (Amresco, Cat # M325) for 1 hour, the membrane was incubated overnight with rabbit antibody to TRIB3 (Proteintech, Cat # 13300) at 4°C. The membrane was washed and further incubated with HRP goat anti-rabbit IgG (Amresco, Cat # N791) secondary antibody for 1 hour. The membrane was then exposed to an enhanced chemiluminescent system, and bands were visualized by exposure to X-ray film. After stripping, GAPDH protein was visualized as loading control using goat antibody to GAPDH (Abcam, Cat # ab9483), followed by HRP rabbit anti-goat IgG as described above. TRIB3 band density was quantified using Total Lab TL120 software (Nonlinear Dynamics) and normalized to the respective GAPDH band density.
Bone marrow derived macrophages of the two strains were incubated with 50 µg/ml AcLDL for 48 hours. We observed that macrophages from the atherosclerosis susceptible DBA/2 strain had significantly higher levels of total cholesterol (p-value<0.0001) and cholesterol esters (p-value <0.0001) compared to atherosclerosis resistant AKR macrophages (
All data are mean±SD, N = 4 per group using the average of triplicate assays per sample. P-values were calculated by ANOVA with Newman-Keuls posttest, showing only the strain differences after loading. There were no significant strain differences in unloaded cells.
Bone marrow derived macrophages from mice of both strains were incubated with or without AcLDL for 1 day (n = 4 per group, total of 8 groups) in two independent experiments. Total RNA was applied to Illumina expression arrays and analyzed as described in
3,059 transcripts were identified as differentially expressed between AKR and DBA/2 unloaded macrophages at a stringent false discovery rate (FDR) adjusted p-value<0.01 in experiment 1 samples.
Gene Symbol | Log2Fold Change |
P-Value | Adjusted P-Value |
Description | |
Experiment 1 | H2-D1 | 7.7 | 2.7E-23 | 2.5E-19 | Histocompatibility 2, D region locus 1 |
H2-Q6 | 5.6 | 1.1E-20 | 5.1E-17 | Histocompatibility 2, Q region locus 6 | |
Gpr137b | 4.2 | 1.5E-19 | 4.6E-16 | G protein-coupled receptor 137B | |
Ncf2 | 6.1 | 5.0E-19 | 1.2E-15 | Neutrophil cytosolic factor 2 | |
Sfi1 | 3.7 | 7.0E-19 | 1.3E-15 | Sfi1 homolog, spindle assembly associated (yeast) | |
Psmb6 | −3.7 | 9.4E-19 | 1.3E-15 | Proteasome (prosome, macropain) subunit, beta type 6 | |
Ccl5 | −4.5 | 9.2E-19 | 1.3E-15 | Chemokine (C-C motif) ligand 5 | |
Baat1 | 3.2 | 1.8E-18 | 2.1E-15 | BRCA1-associated ATM activator 1 | |
Gpnmb | −3.7 | 2.8E-18 | 2.9E-15 | Glycoprotein (transmembrane) nmb | |
Prcp | 3.3 | 5.7E-18 | 5.3E-15 | Prolylcarboxypeptidase (angiotensinase C) | |
Experiment 2 | H2-K1 | 4.3 | 9.4E-14 | 1.1E-09 | Histocompatibility 2, K region locus 1 |
Pilrb1 | 5.2 | 5.2E-13 | 1.4E-09 | Paired immunoglobin-like type 2 receptor beta 1 | |
H2-Q5 | 4.7 | 7.1E-13 | 1.4E-09 | Histocompatibility 2, Q region locus 5 | |
H2-Q8 | 4.6 | 2.8E-12 | 1.4E-09 | Histocompatibility 2, Q region locus 8 | |
Eif2s3y | 4.4 | 3.9E-13 | 1.4E-09 | Eukaryotic translation initiation factor 2, subunit 3, structural gene Y-linked | |
Prcp | 3.3 | 6.5E-13 | 1.4E-09 | Prolylcarboxypeptidase (angiotensinase C) | |
Lrrc57 | 3.6 | 5.0E-12 | 8.5E-09 | Leucine rich repeat containing 57 | |
Ogfrl1 | 3.1 | 6.3E-12 | 9.3E-09 | Opioid growth factor receptor-like 1 | |
Napsa | −3.1 | 7.2E-12 | 9.5E-09 | Napsin A aspartic peptidase | |
Baat1 | 3.3 | 8.1E-12 | 9.6E-09 | BRCA1-associated ATM activator 1 |
Positive log2 fold change, transcripts expressed higher in DBA/2; negative log2 fold change, transcript expressed higher in AKR macrophages.
FDR adjusted p-value based upon permutation.
A similar analysis was performed on the experiment 2 samples and 1,703 transcripts were found to be differentially expressed between AKR and DBA/2 macrophages (FDR adjusted p-value<0.01;
522 differentially expressed transcripts overlapped among the 3,059 and 1,703 strain significant differences identified in the experiment 1 and 2, respectively (significance threshold set at 0.01 FDR,
To identify the common biological pathways most relevant to the genes that differ in expression between AKR and DBA/2 BMM, transcriptomes from both experiments were subjected to Gene Set Enrichment Analysis (GSEA) using KEGG pathways, and we report here only the pathways identified as significantly enriched in both. Strain effects on geneset enrichment were found for the hematopoietic cell lineage, chemokine signaling, toll like receptor signaling and aldosterone regulated sodium reabsorption pathways (permutation test p-value<0.0001,
In conclusion, significant basal gene expression differences were observed between the two strains in both experiments, which need to be considered in the following analysis of gene expression changes in the AKR and DBA/2 macrophages in response to cholesterol loading.
To identify the differentially regulated transcript in response to cholesterol loading, the expression data were fitted in a linear model with strain as an additive variable and strain-loading interaction as an interactive variable. This model identified transcripts whose expression was either up-regulated or down-regulated in one or both strains upon cholesterol loading.
3,758 transcripts were identified as differentially expressed in response to cholesterol loading in AKR and DBA/2 macrophages at an FDR adjusted p-value<0.01 (
Gene Symbol | Log2Fold Change |
P-Value | Adjusted P-Value |
Description | |||
Experiment 1 | P2ry13 | −2.8 | 7.8E-20 | 2.7E-16 | Purinergic receptor P2Y, G-protein coupled 13 | ||
Clec4a3 | −2.5 | 8.7E-20 | 2.7E-16 | C-type lectin domain family 4, member A3 | |||
Npy | 2.1 | 6.1E-20 | 2.7E-16 | Neuropeptide Y | |||
Ms4a6c | −2.3 | 4.6E-19 | 5.9E-16 | Membrane-spanning 4-domains, subfamily A, member 6C | |||
H2-Ab1 | −2.3 | 3.7E-19 | 5.9E-16 | Histocompatibility 2, class II antigen A, beta 1 | |||
Hyal1 | 2.3 | 5.7E-19 | 5.9E-16 | Hyaluronoglucosaminidase 1 | |||
Trib3 | 2.6 | 5.1E-19 | 5.9E-16 | Tribbles homolog 3 (Drosophila) | |||
Ppap2b | 2.7 | 4.9E-19 | 5.9E-16 | Phosphatidic acid phosphatase type 2B | |||
Chac1 | 2.9 | 3.6E-19 | 5.9E-16 | ChaC, cation transport regulator-like 1 (E. coli) | |||
Ly6a | −2.2 | 6.7E-19 | 6.2E-16 | Lymphocyte antigen 6 complex, locus A | |||
Experiment 2 | Ccr5 | −2.9 | 3.8E-16 | 2.8E-12 | Chemokine (C-C motif) receptor 5 | ||
Ccr5 | −3.0 | 6.0E-16 | 2.8E-12 | Chemokine (C-C motif) receptor 5 | |||
Ifit3 | −3.3 | 7.1E-16 | 2.8E-12 | Interferon-induced protein with tetratricopeptide repeats 3 | |||
Fcgr1 | −2.0 | 1.2E-15 | 3.7E-12 | Fc receptor, IgG, high affinity I | |||
Ccr5 | −2.9 | 1.8E-15 | 4.4E-12 | Chemokine (C-C motif) receptor 5 | |||
P2ry14 | −1.9 | 3.2E-15 | 6.4E-12 | Purinergic receptor P2Y, G-protein coupled, 14 | |||
Trib3 | 3.7 | 4.8E-15 | 8.1E-12 | Tribbles homolog 3 (Drosophila) | |||
Ifit3 | −3.2 | 6.4E-15 | 9.6E-12 | Interferon-induced protein with tetratricopeptide repeats 3 | |||
Ly6a | −3.9 | 7.4E-15 | 9.8E-12 | Lymphocyte antigen 6 complex, locus A | |||
Vegfa | 2.7 | 9.9E-15 | 1.2E-11 | Vascular endothelial growth factor A |
The fold change after cholesterol loading is represented as the β-coefficient for the fitted linear model.Positive log2 fold change, transcript expressed higher in response to cholesterol loading; negative log2 fold change, transcript expressed lower upon cholesterol loading.
FDR adjusted p-value based upon permutation.
A similar number of significantly differentially expressed transcripts were found in macrophages in response to cholesterol loading (3,308 transcripts; FDR adjusted p-value<0.01;
The cholesterol loading effect on gene expression was largely reproducible in both experiments, despite microarray platform differences. There were 2,475 cholesterol regulated transcripts with identical probes on the two array platforms, and of these, 1140 transcripts were significantly regulated by cholesterol loading in both experiments (
Transcripts that were significantly up-regulated in response to cholesterol loading in both experiments include: tribbles homolog 3 (
Eight pathways were significantly enriched in transcripts regulated by cholesterol loading in both experiments: lysosome, cytokine-cytokine receptor interaction, primary bile acid biosynthesis, allograft rejection, aminoacyl tRNA biosynthesis, autoimmune thyroid disease, hematopoietic cell lineage, and type I diabetes mellitus (permutation test p-value<0.0001,
Expt. 1 Gene Symbol | AKR Log2 Fold Change |
DBA Log2 Fold Change |
Adjusted P-Value | Expt. 2 Gene Symbol | AKR Log2 Fold Change |
DBA Log2 Fold Change |
Adjusted P-Value |
Hyal1 | 2.3 | 0.6 | 5.9E-16 | Hyal1 | 1.3 | 1.1 | 2.8E-07 |
Igf2r | 1.4 | 0.6 | 2.3E-12 | Atp6v1h | 0.8 | 0.7 | 1.0E-06 |
Ctsk | 1.3 | 0.4 | 9.6E-12 | Sort1 | 0.8 | 1.6 | 4.8E-05 |
Tcirg1 | 1.3 | 0.2 | 2.9E-12 | Gla | 0.7 | 0.0 | 5.9E-04 |
Ctsb | 0.9 | 0.1 | 1.1E-08 | Ctsl | 0.7 | 0.3 | 5.0E-04 |
Gla | 0.7 | 0.1 | 1.5E-06 | Ctsz | 0.7 | 0.3 | 9.3E-05 |
Naglu | 0.7 | 0.1 | 2.8E-08 | Igf2r | 0.6 | 1.3 | 2.2E-04 |
Gga2 | 0.7 | 0.3 | 1.8E-09 | Cltb | 0.6 | 0.3 | 1.2E-04 |
Cd68 | 0.6 | 0.1 | 8.6E-05 | Mcoln1 | 0.6 | 0.5 | 8.6E-05 |
Gnptab | 0.6 | 0.1 | 2.0E-07 | Slc11a1 | 0.6 | 0.5 | 1.2E-05 |
Slc17a5 | 0.6 | −0.1 | 1.5E-06 | Dnase2a | 0.6 | 0.5 | 8.2E-05 |
Cln5 | 0.6 | 0.3 | 6.8E-08 | Tcirg1 | 0.6 | 0.6 | 2.0E-05 |
Atp6v0b | 0.5 | 0.2 | 1.2E-06 | Cln5 | 0.6 | 0.3 | 4.1E-05 |
Ctsz | 0.5 | 0.2 | 6.8E-07 | Ctsa | 0.5 | 0.0 | 7.9E-03 |
Ctns | 0.5 | −0.1 | 6.9E-05 | Ap1s1 | 0.5 | 0.3 | 1.9E-03 |
Ap3d1 | 0.5 | −0.1 | 8.8E-06 | Atp6v0a2 | 0.5 | 0.1 | 1.1E-04 |
Neu1 | 0.4 | 0.1 | 2.9E-04 | Atp6v0d1 | 0.5 | 0.1 | 4.0E-04 |
Mcoln1 | 0.3 | 0.1 | 4.2E-04 | Cd68 | 0.5 | 0.6 | 5.0E-03 |
Ctsd | 0.3 | 0.1 | 9.3E-04 | Gaa | 0.4 | 0.3 | 5.8E-03 |
Abca2 | 0.3 | 0.1 | 5.9E-04 | Gba | 0.3 | 0.3 | 2.1E-03 |
Manba | 0.3 | 0.1 | 3.2E-03 | Psap | 0.3 | 0.4 | 2.3E-03 |
Dnase2a | 0.3 | 0.0 | 1.4E-03 | Glb1 | −0.3 | 0.1 | 3.4E-03 |
Ap1s1 | 0.3 | 0.4 | 1.1E-03 | Lgmn | −0.4 | −0.2 | 2.7E-03 |
Atp6v0a1 | 0.2 | 0.2 | 5.2E-03 | Man2b1 | −0.4 | 0.3 | 4.6E-04 |
Psap | 0.2 | 0.0 | 5.1E-03 | Ap4m1 | −0.4 | −0.3 | 5.0E-03 |
Acp2 | −0.3 | −0.1 | 2.1E-03 | Pla2g15 | −0.4 | −0.2 | 2.5E-04 |
Ap4m1 | −0.3 | 0.1 | 1.4E-03 | Lamp2 | −0.4 | −0.7 | 7.8E-04 |
Ap1b1 | −0.3 | −0.2 | 2.2E-03 | Ap3m2 | −0.4 | −0.3 | 4.1E-04 |
Ap3m2 | −0.3 | 0.2 | 2.1E-03 | Arsb | −0.4 | 0.1 | 6.8E-03 |
Ppt2 | −0.3 | 0.0 | 1.0E-04 | Smpd1 | −0.5 | −0.5 | 4.4E-03 |
Ctsh | −0.4 | −0.1 | 2.2E-05 | Acp2 | −0.5 | −1.0 | 2.5E-04 |
Sort1 | −0.4 | −0.3 | 5.6E-04 | Clta | −0.5 | −0.1 | 1.3E-05 |
Ap4s1 | −0.4 | 0.0 | 1.5E-05 | Ctss | −0.5 | 0.1 | 2.7E-03 |
Asah1 | −0.5 | −0.9 | 1.8E-03 | Ctsf | −0.5 | −0.2 | 4.6E-03 |
Clta | −0.5 | 0.3 | 1.6E-03 | Manba | −0.6 | 0.1 | 3.5E-05 |
Lamp2 | −0.5 | 0.0 | 4.6E-06 | Sgsh | −0.6 | 0.4 | 3.7E-03 |
Slc11a1 | −0.5 | −0.2 | 1.6E-06 | Gusb | −0.6 | −0.2 | 2.3E-04 |
Ap1s2 | −0.5 | 0.1 | 2.6E-06 | Ppt1 | −0.7 | −0.5 | 4.1E-05 |
Napsa | −0.5 | −0.1 | 3.1E-07 | Ctsc | −0.7 | −1.4 | 1.2E-04 |
Ctse | −0.7 | −0.4 | 7.7E-08 | Hgsnat | −0.8 | −0.4 | 5.4E-05 |
Ppt1 | −0.7 | −0.3 | 1.4E-08 | Ids | −0.8 | −0.5 | 6.7E-07 |
Laptm4a | −0.8 | −0.3 | 1.1E-08 | Ctse | −0.8 | −0.8 | 4.0E-07 |
Ctsc | −1.2 | −0.1 | 8.9E-13 | Asah1 | −0.9 | −1.1 | 6.9E-06 |
Napsa | −0.9 | −0.1 | 8.0E-06 | ||||
Ctsc | −1.2 | −1.6 | 1.7E-08 | ||||
Ap1b1 | −1.3 | −0.3 | 9.9E-10 |
The 25 overlapping genes between the two experiments are shown in bold.
Calculated as log2 of AKR loaded/AKR unloaded average expression, positive numbers higher in loaded, negative numbers higher in loaded.
Calculated as log2 of DBA loaded/DBA unloaded average expression
Most of the lysosome pathway genes regulated by cholesterol showed a larger fold change in macrophages from the atherosclerosis resistant AKR than the susceptible DBA/2 strain, and a systematic analysis of the strain-cholesterol loading interaction is provided below. These experimental-validated regulated transcripts include the following lysosomal acid hydrolases: 1) proteases represented by cathepsins (
Since GSEA for sequence motifs was not very informative for the cholesterol loading datasets (data not shown); we examined motifs for two well known sterol regulated transcription factors. The classical example of cholesterol regulation of gene expression involves the down regulation of genes containing the sterol responsive element (SRE). This regulation is mediated by sterol control of sterol regulatory element binding protein (SREBP) processing in the ER and Golgi, such that high sterols repress its processing and low sterols permit its processing into a positively acting transcription factor
The oxysterol activated transcription factor LXR heterodimerizes with RXR and binds to genes harboring LXR responsive elements, often leading to sterol mediated up-regulated gene expression, as demonstrated for
A fitted linear model using strain and loading as additive covariates was used to identify the transcripts with a significant cholesterol loading-strain interaction effect. This interactive effect identifies transcripts that have different directions or degrees of cholesterol regulation between the two strains, for example, a transcript that is up regulated in AKR and down regulated in DBA/2, or a transcript that is highly up regulated in AKR but only moderately up regulated in DBA/2 and vice-versa.
1,929 probes were identified with a significant loading-strain interaction effect at an FDR adjusted p-value<0.01, with several transcripts independently identified by multiple probes (
Slamf9 (A), Trib3 (B), and Dner (C) expression in unloaded and loaded macrophages. Values are expressed as mean±SD (N = 4). Different numbers above bars show p<0.01 (A), p<0.05 (B, C) by Newman-Keuls ANOVA posttest, while similar numbers above bars show no significant differences.
965 probes were identified with a significant loading-strain interaction effect at an FDR adjusted p-value<0.01, with several transcripts independently identified by multiple probes (
Ddit3 (A), Ccr5 (B), and Trib3(C) expression in unloaded and loaded macrophages. Values are expressed as mean±SD (N = 4). Different numbers above bars show p<0.05 (A), p<0.001 (B), or p<0.001 (C) by Newman-Keuls ANOVA posttest, while similar numbers above bars show no significant differences. (D) Proteins isolated from unloaded and cholesterol loaded AKR and DBA/2 BMM were subjected to Western blot analysis, showing TRIB3 band density normalized to GAPDH.
There were 213 probes with highly significant strain-loading interactions that were conserved in both experiments (presented in
ER stress, CHOP, and
To confirm the microarray data we performed quantitative real-time PCR for four selected transcripts: three highly regulated ones,
Linear regression analysis of microarray expression and qPCR data for Trib3(A), Abcg1 (B), Atf4 (C) and Cln5 (D) performed in experiment 1 unloaded and loaded AKR and DBA/2 macrophages. Microarray data were not log2 transformed for this analysis.
We compared Trib3 mRNA levels (microarray) and protein levels (Western blot) in unloaded and cholesterol loaded AKR and DBA/2 BMM (
Several studies have integrated genetics and genomics to identify plausible candidate genes for complex diseases. Previous studies in our lab have identified atherosclerosis QTLs from an AKRxDBA/2
Chromosome 2 |
Chromosome 15 |
Chromosome 17 |
|
Slc13a3 |
Capsl |
Tmem63b |
Srpk1 |
Sdc4 |
Myo10 |
C2 |
Gtpbp2 |
Mmp9 |
Oxct1 |
Tmem8 |
Nme4 |
Rnf114 |
Fam134b |
Ltb |
Cfb |
Gnas |
Rai14 |
Prss22 |
Nrm |
Ctsz |
Il7r |
Rpl7l1 |
Mapk14 |
Gnas |
Ank |
Enpp4 |
Angptl4 |
Cebpb |
Itpr3 |
Cdkn1a |
|
Rae1 |
Tnfrsf21 |
H2-T23 | |
Amdhd2 |
B430306N03Rik |
||
Fpr1 |
Brd2 |
||
Cul7 |
Cyp4f16 |
||
Fahd1 |
Rrp1b |
||
Emr1 |
Tcf19 |
||
Mrpl14 |
Aurka |
||
Dusp1 |
Pex6 |
||
Hagh |
Vars |
||
Ubxn6 |
Hmga1 |
||
Tubb5 |
Trip10 |
||
Fpr2 |
Klc4 |
||
Aif1 |
Plcl2 |
||
Itpr3 |
Cul7 |
||
Fgd2 |
Slc35b2 |
||
Ebi3 |
Mocs1 |
||
Tnfrsf12a |
Nfkbie |
||
H2-Eb1 |
Dpp9 |
||
9030025P20Rik |
Vegfa |
||
Ccnd3 |
Atp6v0e |
||
Uhrf1 |
Gpr108 |
||
Cnpy3 |
Alkbh7 |
||
Flywch2 |
Chaf1a |
||
D17H6S56E-5 |
2410011O22Rik |
||
Lemd2 |
Transcripts significant for strain effect
Transcripts significant for cholesterol loading effect
Transcripts significant for cholesterol loading-strain interaction effect
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A portion of this work was published as a meetings proceeding abstract in Arteriosclerosis, Thrombosis and Vascular Biology, Volume 32, Issue 5 Supplement; May 2012.