1. GENES ASSOCIATED WITH CHOLESTEROL ACCUMULATION IN MACROPHAGES (TRANSCRIPTOME ANALYSIS)
Nikiforov NG1,2,8, Zhelankin AV1,2, Bukrinsky M3, Makeev VJ4, Foxx KK5, Kruth HS6, Oishi Y7, Orekhov AN8.
1 Laboratory of Angiopathology, Institute of General Pathology and Pathophysiology, Moscow, Russia; 2 Laboratory of Medical Genetics, A. N. Myasnikov Institute of Clinical Cardiology, Russian Cardiology Research and Production Complex, Ministry of Healthcare, Moscow, Russia;
3 GW School of Medicine and Health Sciences, George Washington University, Washington, DC, USA;
4 Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia;
5 Kalen Biomedical, LLC, Montgomery Village, MD, USA;
6 Experimental Atherosclerosis Section, Center for Molecular, National Heart, Lung, and Blood Institute , National Institutes of Health, Bethesda, MD, USA; 7 Department of Cellular and Molecular Medicine, Medical Research Institute, Tokyo Medical and Dental University, Tokyo , Japan. 
8 Institute for Atherosclerosis Research, Skolkovo Innovative Center, Moscow, Russia.
Aim
Cholesterol accumulation in arterial cells triggers atherogenesis at the cellular level. The aim of this work was to identify the genes associated and responsible for cholesterol accumulation caused by modified LDL
Methods
Monocytes were isolated from the blood of healthy individuals. Then monocytes were differentiated into macrophages in culture. Intracellular cholesterol accumulation was induced by incubation of cultured cells with native and modified LDL: oxidized and acetylated. Total RNA was isolated from macrophages and sequenced using Illumina HiSeq Then the transcriptome was analyzed.
Results
Modified LDL caused significant 1.5- to 3-fold increase in intracellular cholesterol level as compared with native LDL. Cholesterol accumulation was accompanied with changes in the activity of hundreds of macrophage genes. A comparison of the transcriptomes resulting from incubation with different forms of modified LDL selected forty genes encoding molecules with known functions. Many of these genes may be related to innate immunity. Integrated promoter-pathway analysis was performed to identify the master-regulators not only associated but also responsible for cholesterol accumulation. 6 master-regulators were identified. All of them are associated with inflammation.
Conclusions
We have identified genes associated and responsible for intracellular cholesterol accumulation caused by modified LDL. The next step will be analysis of identified master-regulators and their functional responsibility for cholesterol accumulation.
This work was supported by Russian Science Foundation (Grant # ).
Electrophoresis mobility of LDLs
Master-regulators:
IL-15
IL-7
IL-7R
Annexin A1
CXCL8
TIGIT
1 - Positive control
2 - Negative control
3 - Native LDL
4 - Oxidized LDL
5 - Acetylated LDL
Atherogenicity of modified LDLs
Upstream analysis
Differentially expressed genes
nucleophosmin 1 (nucleolar phosphoprotein B23, numatrin) pseudogene 12
C-type lectin domain family 9 member A
proteasome 26S subunit, non-ATPase, 10 pseudogene 2
annexin A3
EF-hand domain (C-terminal) containing 2
mitogen-activated protein kinase 10
ubiquitin conjugating enzyme E2N-like (gene/pseudogene)
diazepam binding inhibitor-like 5, pseudogene
nuclear receptor subfamily 2 group E member 3
major facilitator superfamily domain containing 2B
G protein-coupled receptor 137C
long intergenic non-protein coding RNA 1352
ACTN1 antisense RNA 1
heterogeneous nuclear ribonucleoprotein A1 pseudogene 1
fer (fps/fes related) tyrosine kinase (phosphoprotein NCP94) pseudogene 1
Signaling pathways
TGFbeta pathway
p53 pathway
E2F network
EGF pathway
HIF-1alpha pathway
Caspase network
cyclosome regulation
cyclosome regulatory network
G1 phase (Cdk2)
angiotensin
stress-associated pathways
Transcription factors
v-ets avian erythroblastosis virus E26 oncogene homolog 1
v-ets avian erythroblastosis virus E26 oncogene homolog 2
ELK4, ETS-domain protein (SRF accessory protein 1)
nuclear receptor subfamily 3 group C member 1
histone deacetylase 2
GA binding protein transcription factor, beta subunit 1
TATA-box binding protein associated factor 1
zinc finger and BTB domain containing 33