Atherosclerosis is a chronic multifactorial inflammatory disease with high prevalence worldwide and has become the leading cause of death [1
]. The principal clinical manifestations of atherosclerosis are represented by coronary heart disease, cerebral stroke, and peripheral vascular disease. Atherosclerosis represents a heterogeneous group of pathological phenomena that include endothelium damage, inflammation, metabolic disorder, cell proliferation, foam cell formation, and soft inflamed atherosclerotic plaque rupture [2
]. The molecular mechanisms underlying atherosclerosis remain unclear.
The mouse has been a powerful model in elucidating the genetic basis of atherogenesis. More than 80 genes have been confirmed to play a role in atherosclerosis by using gene-targeted mice [3
]. Apolipoprotein E (ApoE) gene has a central role in lipoprotein metabolism, where it is required for the efficient clearance of chylomicrons and very-low-density lipoproteins. Notably, the ApoE null mouse was the first model showing severe and rapid development of atherosclerotic plaques without dietary intervention [4
]. Using ApoE null mice fed with a high-fat diet, a previous study has determined the global gene expression profiles associated with arthrosclerosis using the microarray approach [5
RNA-sequencing (RNA-seq), which utilizes the latest massively parallel sequencing, has provided an alternative way to obtain mRNA expression profile at unprecedented sensitivity and accuracy [6
]. In contrast to the microarray, RNA-seq has several advantages. Firstly, it is an unbiased method which is not limited to detecting pre-designed sequences [6
]. Secondly, it does not suffer from cross-hybridization since DNA sequences can be mapped to unique regions of the genome [7
]. Lastly, it has no upper limit for quantification [8
]. In the present study, we took advantage of the RNA-seq approach to investigate the global gene expression changes in the aorta of ApoE null mice fed a high-fat diet in comparison to normal diet. Our study contributes to an increase in the knowledge on molecular mechanisms underlying atherosclerosis.
In the present study, we investigated the genome-wide gene expression profiles in the aorta of ApoE null mice fed a high-fat diet compared with a normal diet using RNA-seq. A total of 280 genes, including 163 upregulated and 117 downregulated genes, were identified to be differentially expressed between the two groups. Quantitative RT-PCR (qRT-PCR) analysis demonstrated that the expression trend of selected genes was consistent with RNA-seq, suggesting that our data were of high quality.
In order to explore the functions of these differentially expressed genes, we conducted gene ontology (GO) and pathway analysis. As expected, we found fatty acid metabolic process was the most enriched term under the biological process (BP) category of GO. Notably, inflammatory response was also enriched, although not among the top-ranked GO terms. In addition, we found that PPAR signaling pathway was the most enriched pathway in KEGG pathway analysis. PPAR signaling pathway plays a central role in fatty acid metabolic process and atherosclerosis [18
]. Network analysis was performed to identify seven hub genes: acetyl-coenzyme A carboxylase alpha (Acaca
), peroxisome proliferator activated receptor gamma (Pparg
), uncoupling protein 1 (Ucp1
), fatty acid binding protein 4 (Fabp4
), patatin-like phospholipase domain containing 2 (Pnpla2
), guanine nucleotide binding protein alpha transducing 3 (Gnat3
), and stearoyl-coenzyme A desaturase 1 (Scd1
). Acaca is an important enzyme involved in the synthesis of saturated fatty acids, which is a known risk factor for cardiovascular diseases. Pparg plays a crucial role in the expression of key genes involved in adipogenesis, and lipid and glucid metabolism. Conditional knockout of macrophage Pparg increases atherosclerosis in C57BL/6 and low-density lipoprotein receptor-deficient mice [19
]. Ucp1 is a key mitochondrial protein involved in thermogenesis in brown adipose tissue. Ucp1 over-expression in aortic smooth muscle cells causes hypertension and increases dietary atherosclerosis without affecting cholesterol levels [20
]. Fabp4 belongs to a family of intracellular lipid chaperones that is expressed in active lipid metabolic tissues. The impact of Fabp4 on atherosclerosis is mainly due to the role of this molecule in macrophages and dendritic cells [21
]. The pharmacological inhibition of Fabp4 significantly protected against atherosclerotic plaque formation in the ApoE-deficient animal model of atherosclerosis [22
]. The Pnpla2
gene encodes an enzyme called adipose triglyceride lipase (ATGL), which plays a role in breaking down triglycerides. Deficiency of ATGL in macrophages resulted in reduced atherosclerosis susceptibility [23
]. Gnat3 plays a prominent role in taste transduction. The role of Gnat3 in atherosclerosis is unknown. Scd1 is an endoplasmic reticulum enzyme that catalyzes the rate-limiting step in the formation of monounsaturated fatty acids. Scd1 inhibition reduces atherosclerosis in cholesterol-fed C57BL/6J mice exposed to chronic intermittent hypoxia (CIH) [24
]. These hub genes are likely more important than other genes due to their key positions in the network. According to GO annotations, all these hub genes except Gnat3
, are involved in fatty acid metabolic process. Thus, the network analysis once again highlights the role of fatty acid metabolic process in atherosclerosis.
Furthermore, we predicted the transcription factors which might drive the expression of differentially expressed genes by enrichment test. We found that the binding sites of LXR (M00766), SREBP-1 (M00749), and LBP-1 (M00644) were significantly over-represented among upregulated genes. LXR (liver X receptor) is known to modulate cholesterol and fatty acid homeostasis as well as inflammation in macrophages in the context of atherosclerosis [25
]. SREBP-1 (sterol regulatory element-binding transcription factor 1) belongs to a family of basic helix-loop-helix-leucine zipper (bHLHLZ) transcription factors [26
]. Known target genes of SREBP-1 are involved in cholesterol biosynthesis and transport [27
]. Interestingly, two hub genes for differentially expressed gene network, Ucp1
, are predicted target genes for SREBP-1 in our transcription factor analysis. This result indicates that SREBP-1 may be a crucial regulator for differentially expressed genes. LBP-1 (upstream binding protein 1) is a member of the NTF (neurogenic element binding transcription factor) family of transcription factors. Currently, little is known about the role of LBP-1 in regulating atherosclerosis. Only the NF-κB (M00052) binding sites were significantly over-represented among downregulated genes. NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls immune response, cytokine production, and cell survival [29
]. Previous studies have shown that NF-κB inhibition reduces foam cell formation and atherosclerotic plaque accumulation [30
]. Although nearly equal number of target genes can be activated and repressed by NF-κB respectively [32
], the studies on repressed target genes of NF-κB are sparse. Hence, these four transcription factors deserve further investigation.
Using the CMap, we were able to identify compounds with a negatively correlating gene expression profile to that of differentially expressed genes. The top 10 most promising repositioned compounds were: felodipine, tanespimycin, diethylstilbestrol, nifuroxazide, carisoprodol, nifurtimox, iopanoic acid, (+/‒)-catechin, colistin, and colecalciferol. Felodipine is a calcium channel blocker which is used to treat high blood pressure. We predicted that felodipine is able to reverse the expression of 24 upregulated genes and 13 downregulated genes. The top-ranked upregulated gene is Acaca. Acaca has been identified as a hub gene in our network analysis. Felodipine represses the expression of Acaca, raising the possibility that the anti-atherosclerotic role of felodipine may be exerted by reducing saturated fatty acid synthesis via Acaca. Thus, felodipine deserves further investigation. By acting as a potent HSP90 inhibitor, tanespimycin is an antibiotic being studied in the treatment of cancers. Diethylstilbestrol is a synthetic form of the female hormone estrogen. Nifuroxazide is a nitrofuran antibiotic and is used to treat colitis and diarrhea. Carisoprodol is a muscle relaxant. It may work by altering communication among nerves in parts of the brain that control the sensation of pain and in the spinal cord. Nifurtimox is a nitrofuran derivative with antiprotozoal and potential antineoplastic activities. Iopanoic acid is a potent inhibitor of thyroid hormone release from thyroid gland, as well as of peripheral conversion of thyroxine (T4) to triiodothyronine (T3). (+/‒)-catechin is a flavan-3-ol, a type of natural phenol and antioxidant. Colistin is an antibiotic produced by certain strains of the bacteria Paenibacillus polymyxa
, which is effective against most Gram-negative bacilli. Cholecalciferol, also known as vitamin D3, is a type of vitamin D found in food and used as a dietary supplement. These results demonstrate the validity of our CMap analysis, because estrogen and vitamin D are well-known to have beneficial effects on heart and vessels [33
Finally, our study has some limitations. One clear limitation is the complex cell-type composition of the aorta samples. There are many different cell types including endothelial cells, smooth muscle cells, and macrophages. It is undoubtedly that application of laser-capture microdissection (LCM) to dissect the atherosclerotic plaque will provide a better resolution of the atherosclerotic transcriptome. Recently, considerable progress in RNA-seq technologies makes it possible to study the transcriptome of a single cell [35
]. However, like LCM, the compromised sensitivity is the limiting factor for this approach at the moment [36
]. Another limitation of the study is that the whole aorta was used for RNA-seq. Atherosclerotic lesions do not occur at random sites [37
]. For example, the inner curvature of the aortic archa is an athero-susceptible site, whereas the descending thoracic aorta is an athero-protected region. This difference should be taken into consideration in further studies.
In conclusion, in the present study, using RNA-seq, we analyzed the transcriptomic differences in the aorta of ApoE null mice fed a high-fat diet compared with a normal diet. Our study provides a valuable resource for in-depth understanding of the molecular mechanisms underlying atherosclerosis.